Lactobacillus johnsonii with function of resisting multiple drug-resistant bacterium biofilms and anti-inflammatory activity and application of lactobacillus johnsonii

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to lactobacillus johnsonii with a function of resisting multiple drug-resistant bacterium biological membranes and anti-inflammatory activity and application of the lactobacillus johnsonii. The lactobacillus johnsonii A25041 provided by the invention has strong inhibition and anti-biofilm activity on multi-drug-resistant gram-negative bacteria (including escherichia coli, pseudomonas aeruginosa and acinetobacter baumannii) and methicillin-resistant staphylococcus aureus, and the lactobacillus johnsonii A25041 has good acid-base tolerance and gastrointestinal fluid tolerance, so that the lactobacillus johnsonii A25041 can be used for preparing medicines for preventing and treating various diseases. The composition shows excellent anti-inflammatory activity and oxidation resistance, does not have hemolysis capacity, is good in safety, has natural drug resistance to gentamicin, tetracycline, ciprofloxacin, lincomycin and compound sulfamethoxazole, can be used together with antibiotics at the same time or in sequence, maintains intestinal microecological balance and relieves antibiotic burden while exerting the sterilization effect of the antibiotics, and has a good application prospect. The method can be widely applied to functional food development, and has important application value in the field of biological medicine and daily chemical products.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Lactobacillus johnsonii with biofilm function against multidrug-resistant bacteria and anti-inflammatory activity, and its application. Background Technology

[0002] The widespread use and abuse of broad-spectrum antibiotics and antimicrobial drugs have led to a surge in drug-resistant bacteria. On February 27, 2017, the World Health Organization (WHO) listed 12 drug-resistant bacteria, nine of which are Gram-negative and three are Gram-positive. Among the listed Gram-negative bacteria, carbapenem-resistant Acinetobacter baumannii is the most resistant. Of the three listed Gram-positive bacteria, methicillin-resistant Staphylococcus aureus exhibits the most severe drug resistance globally.

[0003] Probiotics, as a class of live microorganisms that can regulate the host microbiome, enhance barrier function, and inhibit pathogen colonization, have shown unique potential in the prevention and adjunctive treatment of infectious diseases, and are considered a promising direction for antibiotic replacement or supplemental therapy. Among the many probiotic species, Lactobacillus is widely studied and applied due to its good safety profile and diverse probiotic functions. Lactobacillus johnsonii (… Lactobacillus johnsonii As an important member of the Lactobacillus genus, *Lactobacillus johnsonii* has been shown in studies to have significant effects in regulating intestinal flora balance, enhancing intestinal barrier function, and modulating immune responses. However, there are still significant limitations in the current technology: most published strains of *Lactobacillus johnsonii* have relatively single functions, especially lacking the ability to inhibit biofilm infections caused by multidrug-resistant bacteria, which are clinically challenging; and the low survival rate of conventional probiotics in the acidic environment of the stomach seriously affects their intestinal colonization and actual efficacy; in addition, multifunctional probiotic strains with strong antibacterial, anti-inflammatory, antioxidant, and high safety properties are still scarce. Summary of the Invention

[0004] The purpose of this invention is to provide a strain of Lactobacillus johnsonii that combines potent inhibition of multidrug-resistant bacteria, anti-inflammatory, antioxidant, high survival rate and high safety.

[0005] This invention provides a strain of Lactobacillus johnsonii A25041, with accession number GDMCC No:67373.

[0006] This invention provides a microbial agent, wherein the active ingredients of the microbial agent include one or more of the following: (1) The live Lactobacillus johnsonii A25041 described in the above technical solution; (2) The inactivated Lactobacillus johnsonii A25041 cells described in the above technical solution; (3) The culture of Lactobacillus johnsonii A25041 described in the above technical solution; (4) The extract of Lactobacillus johnsonii A25041 described in the above technical solution.

[0007] Preferably, the culture comprises one or more of a fermentation plate, a fermentation broth, and a fermentation supernatant; the extract comprises a fermentation broth extract.

[0008] This invention provides the application of Lactobacillus johnsonii A25041 or the bacterial agent described in the above-mentioned technical solutions in one or more of the following: (1) Prepare antibacterial products; (2) Prepare anti-biofilm products; (3) Prepare drugs for the prevention and / or treatment of pathogenic bacterial infections; (4) Prepare anti-inflammatory products; (5) Prepare antioxidant products; (6) Prepare products for regulating intestinal flora.

[0009] Preferably, the antimicrobial product targets Gram-negative bacteria and / or Gram-positive bacteria; The anti-biofilm product is used to break down the membranes of Gram-negative and / or Gram-positive bacteria. The pathogenic bacteria include Gram-negative bacteria and / or Gram-positive bacteria; The anti-inflammatory product is used to inhibit the expression of IL-6 and / or TNF-α; The antioxidant product is used to scavenge reactive oxygen free radicals and / or increase superoxide dismutase activity.

[0010] Preferably, the Gram-negative bacteria include one or more of drug-resistant Escherichia coli, hemorrhagic Escherichia coli, drug-resistant Acinetobacter baumannii, and drug-resistant Pseudomonas aeruginosa; The Gram-positive bacteria include drug-resistant Staphylococcus aureus; The reactive oxygen species include one or more of hydroxyl radicals, superoxide anions, and DPPH radicals; The anti-biofilm product is used to inhibit the formation of biofilms.

[0011] Preferably, the drug-resistant Escherichia coli includes Escherichia coli resistant to β-lactam antibiotics; The drug-resistant Acinetobacter baumannii includes imipenem-resistant Acinetobacter baumannii; The drug-resistant Pseudomonas aeruginosa includes one or more of the following: β-lactam antibiotics, polypeptide antibiotics, aminoglycoside antibiotics, and fluoroquinolone antibiotics. The drug-resistant Staphylococcus aureus includes methicillin-resistant Staphylococcus aureus.

[0012] Preferably, the product is a functional food, health product, daily chemical product, or pharmaceutical; The dosage forms of the product include lyophilized powder or capsules.

[0013] This invention provides a product that inhibits multidrug-resistant bacteria and has anti-biofilm function, anti-inflammatory activity and antioxidant capacity, wherein the drug includes active ingredients and excipients; The active ingredient includes Lactobacillus johnsonii A25041 or the bacterial agent described in the above technical solution.

[0014] Preferably, the active ingredient further includes antibiotics; the antibiotics include one or more of gentamicin, tetracycline, ciprofloxacin, lincomycin, and trimethoprim-sulfamethoxazole.

[0015] Beneficial effects: This invention provides a strain of *Lactobacillus johnsonii* A25041, with accession number GDMCC No:67373. The *Lactobacillus johnsonii* A25041 described in this invention possesses broad-spectrum antibacterial activity, exhibiting potent inhibition and anti-biofilm activity against multidrug-resistant Gram-negative bacteria (including *Escherichia coli*, *Pseudomonas aeruginosa*, and *Acinetobacter baumannii*) and methicillin-resistant *Staphylococcus aureus*, demonstrating significant potential in addressing the problem of drug-resistant bacterial infections. Furthermore, the *Lactobacillus johnsonii* A25041 provided in this invention exhibits good acid-base tolerance and gastrointestinal fluid tolerance, displaying excellent anti-inflammatory and antioxidant activity. It has no hemolytic ability, good safety profile, and exhibits natural resistance to gentamicin, tetracycline, ciprofloxacin, lincomycin, and trimethoprim-sulfamethoxazole. It can be used simultaneously or sequentially with antibiotics, maintaining intestinal microecological balance and reducing antibiotic burden while exerting the bactericidal effect of antibiotics. It can be widely applied in the development of functional foods and has significant application value in the biopharmaceutical and daily chemical products fields.

[0016] Biological Preservation Information Lactobacillus johnsonii A25041, classified as Lactobacillus johnsonii Lactobacillus johnsonii It was deposited on November 27, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong, China, with accession number GDMCC No:67373. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0018] Figure 1 The colony morphology of Lactobacillus johnsonii A25041; Figure 2 Phylogenetic tree of Lactobacillus johnsonii A25041; Figure 3 The growth curves and acid production curves of Lactobacillus johnsonii A25041 are shown. Figure 4 The results of the pH tolerance test for Lactobacillus johnsonii A25041; Figure 5 Determination of the antibacterial activity of Lactobacillus johnsonii A25041 during plate fermentation; Figure 6 Determination of the antibacterial activity of Lactobacillus johnsonii A25041 during liquid fermentation; Figure 7 Results of hemolytic activity evaluation for Lactobacillus johnsonii A25041; Figure 8 The results are from the antibiotic susceptibility assay for Lactobacillus johnsonii A25041. Detailed Implementation

[0019] This invention provides a strain of Lactobacillus johnsonii A25041, with accession number GDMCC No:67373.

[0020]

[0021] The present invention provides a microbial agent, wherein the active ingredients of the microbial agent include one or more of the following: (1) live bacteria of Lactobacillus johnsonii A25041 as described in the above technical solution; (2) inactivated bacteria of Lactobacillus johnsonii A25041 as described in the above technical solution; (3) culture of Lactobacillus johnsonii A25041 as described in the above technical solution; (4) extract of Lactobacillus johnsonii A25041 as described in the above technical solution.

[0022] In one embodiment, the microbial agent of the present invention is a solid microbial agent or a liquid microbial agent.

[0023] In one embodiment, the culture of the present invention includes one or more of a fermentation plate, a fermentation broth, and a fermentation supernatant. In another embodiment, the extract of the present invention includes a fermentation broth extract.

[0024] As one embodiment, the method for preparing the fermentation broth of the present invention includes: inoculating activated Lactobacillus johnsonii A25041 into MRS liquid medium for seed culture, obtaining seed broth, and then inoculating it into fresh MRS liquid medium for fermentation culture to obtain fermentation broth.

[0025] In one embodiment, the inoculum size for seed culture in this invention is 1-3%; in another embodiment, the inoculum size is 3%. In one embodiment, the seed culture is anaerobic culture at a temperature of 36-38°C; in another embodiment, the anaerobic culture temperature is 37°C. In one embodiment, the inoculum size for fermentation culture in this invention is 3-5%; in another embodiment, the inoculum size is 3%. In one embodiment, the fermentation culture is anaerobic culture at a temperature of 36-38°C; in another embodiment, the anaerobic culture temperature is 37°C.

[0026] In one embodiment, the method for preparing the fermentation supernatant of the present invention includes: performing solid-liquid separation on the fermentation broth and collecting the liquid portion. In one embodiment, the solid-liquid separation method of the present invention includes centrifugation. In one embodiment, the centrifugation speed of the present invention is 10000~12000 rpm; in another embodiment, the centrifugation speed of the present invention is 10000 rpm. In one embodiment, the centrifugation time of the present invention is 5~10 min; in another embodiment, the centrifugation time of the present invention is 5 min.

[0027] In one embodiment, the preparation method of the fermentation plate of the present invention includes: coating the seed liquid onto MRS solid culture medium for anaerobic culture, and obtaining the fermentation plate after the bacterial strain has grown confluently. In one embodiment, the culture temperature for anaerobic culture of the present invention is 36~38℃; in another embodiment, the culture temperature for anaerobic culture of the present invention is 37℃.

[0028] The fermentation broth, fermentation supernatant, and fermentation plate described in this invention can all inhibit the growth of multidrug-resistant bacteria. Compared with the fermentation plate, the fermentation broth and fermentation supernatant have stronger antibacterial effects.

[0029] As one embodiment, the viable count of Lactobacillus johnsonii A25041 in the bacterial agent of the present invention is 1 × 10⁻⁶. 7 ~1×10 9 CFU / mL; As another embodiment, the viable count of Lactobacillus johnsonii A25041 in the bacterial agent of the present invention is 1×10⁻⁶. 8 CFU / mL.

[0030] This invention investigated the antibacterial activity of *Lactobacillus johnsonii* A25041 against pathogenic bacteria. The results showed that *Lactobacillus johnsonii* A25041 possesses broad-spectrum antibacterial activity, exhibiting potent inhibitory activity against multidrug-resistant Gram-negative bacteria (including *Escherichia coli*, *Pseudomonas aeruginosa*, and *Acinetobacter baumannii*) and methicillin-resistant *Staphylococcus aureus*, demonstrating significant potential in addressing drug-resistant bacterial infections. Furthermore, compared to fermentation plates (solid inoculum), the fermentation supernatant (liquid inoculum) showed a significant and strong inhibitory effect on all tested pathogenic bacteria, indicating that the full utilization of the antibacterial activity of *Lactobacillus johnsonii* A25041 depends on liquid culture.

[0031] The present invention also conducted a biofilm detection test on the anti-biofilm ability of the Lactobacillus johnsonii A25041 against pathogenic bacteria. The results showed that the Lactobacillus johnsonii A25041, especially its fermentation supernatant, has broad-spectrum and highly efficient anti-biofilm activity, especially with a near-complete inhibitory effect on biofilms of multidrug-resistant Gram-negative bacteria, demonstrating good application potential in the prevention and control of related biofilm infections.

[0032] The present invention also conducted an antioxidant capacity test on the Lactobacillus johnsonii A25041. The results showed that Lactobacillus johnsonii A25041, especially its fermentation supernatant, exhibited highly efficient scavenging ability against hydroxyl radicals, superoxide anions, and DPPH radicals, and could also enhance the activity of superoxide dismutase (SOD), thus having a significant antioxidant effect.

[0033] The present invention also conducted an anti-inflammatory activity assay of the Lactobacillus johnsonii A25041. The results showed that Lactobacillus johnsonii A25041, especially its fermentation supernatant, had a significant inhibitory effect on the expression of IL-6 and TNF-α in LPS-induced mouse Raw267.4 cells, and could effectively inhibit the expression of pro-inflammatory factors IL-6 and TNF-α, thus playing a role in related inflammation.

[0034] The present invention also conducted hemolytic activity and gastrointestinal tolerance tests on the Lactobacillus johnsonii A25041. The results showed that Lactobacillus johnsonii A25041, especially its fermentation supernatant, does not destroy red blood cells in vitro and exhibits rare gastric acid tolerance and good intestinal colonization potential in simulated gastric juice (pH 3.0) and intestinal juice (pH 8.0).

[0035] In the specific implementation process of this invention, the effects of different Lactobacillus johnsonii were compared. Compared with Lactobacillus johnsonii A20083, the newly discovered Lactobacillus johnsonii A25041 exhibited excellent anti-biofilm ability, anti-inflammatory activity, antioxidant capacity, antibiotic resistance, and gastrointestinal fluid resistance.

[0036] In view of the effects of Lactobacillus johnsonii A25041 provided by the present invention, the present invention provides the application of Lactobacillus johnsonii A25041 or the culture or the bacterial agent described in the above technical solution in one or more of the following: (1) preparing antimicrobial products; (2) preparing anti-biofilm products; (3) preparing drugs for preventing and / or treating pathogenic bacterial infections; (4) preparing anti-inflammatory products; (5) preparing antioxidant products; (6) preparing products for regulating intestinal flora.

[0037] In one embodiment, the antibacterial product of the present invention targets Gram-negative bacteria and / or Gram-positive bacteria. In one embodiment, the anti-biofilm product of the present invention is used to disrupt the biofilm of Gram-negative bacteria and / or Gram-positive bacteria. In one embodiment, the pathogenic bacteria of the present invention include Gram-negative bacteria and / or Gram-positive bacteria. In one embodiment, the anti-inflammatory product of the present invention is used to inhibit the expression of IL-6 and / or TNF-α. In one embodiment, the antioxidant product of the present invention is used to scavenge reactive oxygen species and / or increase superoxide dismutase activity.

[0038] In one embodiment, the Gram-negative bacteria of the present invention include one or more of drug-resistant Escherichia coli, hemorrhagic Escherichia coli, drug-resistant Acinetobacter baumannii, and drug-resistant Pseudomonas aeruginosa. In another embodiment, the Gram-positive bacteria of the present invention include drug-resistant Staphylococcus aureus. In another embodiment, the reactive oxygen species of the present invention include one or more of hydroxyl radicals, superoxide anions, and DPPH radicals. In another embodiment, the anti-biofilm product of the present invention is used to inhibit biofilm formation.

[0039] In one embodiment, the drug-resistant *Escherichia coli* of the present invention includes *Escherichia coli* resistant to β-lactam antibiotics. In one embodiment, the drug-resistant *Acinetobacter baumannii* of the present invention includes *Acinetobacter baumannii* resistant to imipenem antibiotics. In one embodiment, the drug-resistant *Pseudomonas aeruginosa* of the present invention includes *Pseudomonas aeruginosa* resistant to one or more of β-lactam antibiotics, polypeptide antibiotics, aminoglycoside antibiotics, and fluoroquinolone antibiotics. In one embodiment, the drug-resistant *Staphylococcus aureus* of the present invention includes methicillin-resistant *Staphylococcus aureus*.

[0040] In one embodiment, the product of this invention is a functional food, health product, daily chemical product, or pharmaceutical. In one embodiment, the functional food of this invention includes fermented milk beverages and / or functional yogurt. In one embodiment, the health product of this invention includes intestinal flora regulators. In one embodiment, the daily chemical product of this invention includes oral health products and / or antibacterial wound dressings.

[0041] As one implementation method, the dosage form of the product of the present invention includes lyophilized powder or capsules.

[0042] This invention provides a product that inhibits multidrug-resistant bacteria and has anti-biofilm function, anti-inflammatory activity, and antioxidant capacity. The product includes an active ingredient and excipients; the active ingredient includes Lactobacillus johnsonii A25041 or the bacterial agent described in the above-mentioned technical solution.

[0043] In one embodiment, the active ingredient of the present invention further includes antibiotics. In another embodiment, the antibiotics of the present invention include one or more of gentamicin, tetracycline, ciprofloxacin, lincomycin, and trimethoprim-sulfamethoxazole. The present invention also conducted antibiotic susceptibility testing on *Lactobacillus johnsonii* A25041. The results showed that *Lactobacillus johnsonii* A25041, especially its fermentation supernatant, exhibits natural resistance or insensitivity to gentamicin, tetracycline, ciprofloxacin, lincomycin, and trimethoprim-sulfamethoxazole. It can be used simultaneously or sequentially with gentamicin, tetracycline, ciprofloxacin, lincomycin, and trimethoprim-sulfamethoxazole to exert antibiotic bactericidal effects while maintaining intestinal microecological balance, alleviating antibiotic-associated diarrhea, and enhancing overall therapeutic efficacy.

[0044] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes a strain of Lactobacillus johnsonii with biofilm function against multidrug-resistant bacteria and anti-inflammatory activity, and its applications. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0045] The culture medium used in this invention is as follows: MRS liquid culture medium (g / L): glucose 20.0 g, beef extract 10.0 g, peptone 10.0 g, yeast extract 5.0 g, sodium acetate 5.0 g, dipotassium hydrogen phosphate 2.0 g, diammonium citrate 2.0 g, magnesium sulfate 0.2 g, manganese sulfate 0.05 g, Tween 80 1.0 g, distilled water 1000 mL, pH 5.7±0.2, autoclaved at 121℃ for 15 min.

[0046] MRS solid culture medium (g / L): glucose 20.0 g, beef extract 10.0 g, peptone 10.0 g, yeast extract 5.0 g, sodium acetate 5.0 g, dipotassium hydrogen phosphate 2.0 g, diammonium citrate 2.0 g, magnesium sulfate 0.2 g, manganese sulfate 0.05 g, Tween 80 1.0 g, agar 15.0 g, distilled water 1000 mL, pH 6.2±0.2, autoclaved at 121℃ for 15 min.

[0047] LB liquid medium (g / L): 10 g tryptone, 5 g yeast extract, 10 g NaCl, 1000 mL distilled water, autoclaved at 121℃ for 15 min.

[0048] LB solid medium (g / L): 10 g tryptone, 5 g yeast extract, 10 g NaCl, 15 g agar, 1000 mL distilled water, autoclaved at 121℃ for 15 min.

[0049] Columbia blood culture medium (g / L): 29 g of commercial Columbia culture medium, 17 g of agar, 1000 mL of distilled water, autoclaved at 121℃ for 15 min, and then added at approximately 50℃ with sterile defibrinated sheep blood at a dosage of 5%.

[0050] Example 1 Isolation, purification and identification of strains 1. Using fecal samples from healthy centenarians, the samples were serially diluted to 10-10. -5 10 -6 and 10 -7Subsequently, 100 μL of each sample was plated onto MRS solid medium and anaerobic incubated at 37°C for 2 days. Characteristic colonies were selected for multiple isolation and purification processes, and a strain was screened out. Its growth morphology on the plate is as follows: Figure 1 As shown, the colonies are milky white, opaque, with irregular edges, a dry and rough surface, and are easy to pick up.

[0051] 2. Use a toothpick to pick a single colony and prepare a bacterial suspension in 30 μL PBS as a PCR template for amplification; the upstream primer sequence for PCR amplification is 27F, and the nucleotide sequence is shown in SEQ ID NO:2: 5'-AGAGTTTGATCCTGGCTCAG-3'; the downstream primer is 1492R, and the nucleotide sequence is shown in SEQ ID NO:3: 5'-CTACGGCTACCTTGTTACGA-3'. PCR reaction system (25μL): 12.5μL 2×Tag PCR Mix, 1μL DNA template, 0.5μL upstream primer, 0.5μL downstream primer, 1.5μL DMSO, and 9μL sterile water.

[0052] PCR reaction program: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 1 min, 56℃ annealing for 45 s, 72℃ extension for 1 min 30 s, 30 cycles, 72℃ extension for 5 min.

[0053] 3. The PCR product, approximately 1500 bp in length, was amplified and sent to Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results are shown in SEQ ID NO:1. BLAST alignment was performed in the NCBI database (www.ncbi.nlm.nih.gov), and a phylogenetic tree analysis was conducted on the bacterium to determine its closest phylogenetic relationship with *Lactobacillus johnsonii*. The phylogenetic tree is shown below. Figure 2 As shown. The strain was identified as *Lactobacillus johnsonii* (…). Lactobacillusjohnsonii The culture was named A25041 and biologically preserved. After purification, a single colony was picked and cultured overnight in MRS liquid medium at 37°C under anaerobic conditions. The culture was then preserved in a laboratory culture bank after being mixed with 40% glycerol in equal proportions.

[0054] Example 2 Growth and metabolic characteristics of Lactobacillus johnsonii A25041 1. Basic growth and acid production kinetics Lactobacillus johnsonii A25041 seed culture was inoculated into fresh MRS liquid medium at a 3% inoculum. The absorbance and pH of the culture medium were measured every 2 hours, with three replicates per group. The values ​​were plotted on the x-axis as time and on the OD value of the culture medium. 600 The growth and acid production curves of *Lactobacillus johnsonii* A25041 were plotted with pH values ​​on the ordinate and pH value on the ordinate. The results are as follows: Figure 3 As shown, the growth curve and pH change curve of this strain show a clear correspondence: during the logarithmic growth phase (2-12 h), the cells proliferate rapidly and have a strong acid production capacity, and the pH value drops linearly; after entering the stationary phase (after 12 h), cell growth and acid production metabolism tend to be stable, and the pH value tends to be stable.

[0055] 2. Tolerance and adaptability to different environments Lactobacillus johnsonii A25041 bacterial suspension, activated and cultured for 24 hours, was inoculated at a rate of 3.0% into MRS liquid medium with initial pH values ​​of 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0, and cultured anaerobically at 37°C. OD values ​​were measured every 2 hours. 600 Values, with 3 parallels per group. Results are as follows. Figure 4 As shown, this strain grows fastest and most vigorously in an environment with pH 6-7 (neutral to slightly acidic). Under pH 4-5 (acidic) conditions, the strain can still grow, but at a slow rate, with growth and death occurring in parallel, indicating its acid tolerance and ability to survive and slowly multiply in acidic environments. No effective growth was observed at pH 3 (strongly acidic) and below, indicating significant inhibition of growth. Under pH 8-9 (alkaline) conditions, rapid growth was observed in the first 2-10 hours, followed by a peak growth curve after 10 hours, indicating that the strain rapidly enters the stationary and death phases in alkaline environments, possibly due to the accumulation of metabolic byproducts or unsuitable environmental conditions leading to rapid cell lysis or loss of activity. Although the growth rate decreased later under pH 8-9 conditions, the OD value ultimately remained higher than the stable value under pH 4-5 conditions within 10-22 hours. This demonstrates that bacterial biomass can accumulate rapidly even under short-term (<10 hours) alkaline culture.

[0056] Example 3 Antimicrobial activity test of Lactobacillus johnsonii A25041 against clinically resistant pathogens - agar block method 1. Test strain: Lactobacillus johnsonii A25041.

[0057] 2. Clinically resistant drug-causing bacteria: Pseudomonas aeruginosa ( Pseudomonas aeruginosa PA01: Resistant to β-lactam antibiotics, published in: Yang Fang. Evaluation and mechanism study of quorum sensing activity of quinolinone compounds against Pseudomonas aeruginosa [D]. Hebei Agricultural University, 2023. DOI:10.27109 / d.cnki.ghbnu.2023.000394. Acinetobacter baumannii ( Acinetobacter baumanniiHRAB85: Imipenem-resistant antibiotic, published in: Isolation and Whole-genome Sequence Analysis of the ImipenemHeteroresistant Acinetobacter baumannii Clinical Isolate HRAB-85 DOI:10.1016 / j.ijid.2017.07.005; Enterohemorrhagic Escherichia coli E. coli O157: Published in Zhou Yan, Wan Qiyang, Zhu Shujiao, et al. Whole genome sequencing and virulence and drug resistance gene analysis of a porcine enterohemorrhagic Escherichia coli strain O157:H7 [J]. Guangdong Agricultural Sciences, 2025, 52(02):58-70. DOI:10.16768 / j.issn.1004-874X.2025.02.005. Drug-resistant Escherichia coli (DRC) Escherichia coli 4-1: Resistance to β-lactam antibiotics, published in: Epidemiology of extended-spectrum beta-lactamase (ESBL) producing Escherichiacoli in Japan: Characteristics of community-associated versus healthcare-associated ESBL E. coli. Journal of Infection and Chemotherapy, DOI: 10.1016 / j.jiac.2016.08.010; Golden grape balls ( Staphylococcus aureus MRSA1-1: Methicillin-resistant, published in: Yang Yancheng. Molecular typing and drug resistance analysis of clinical isolates of methicillin-sensitive Staphylococcus aureus [D]. Third Military Medical University, 2016.

[0058] 3. Cultivation of *Lactobacillus johnsonii* A25041: After activating *Lactobacillus johnsonii* A25041 on MRS solid plates, single colonies were picked and cultured in MRS liquid medium until the logarithmic growth phase. Then, a 3% inoculum was transferred to fresh MRS liquid medium and cultured until the logarithmic growth phase, yielding a *Lactobacillus johnsonii* A25041 seed culture. 100 μL of this seed culture was spread onto an MRS solid plate and anaerobically cultured at 37°C for 2 days to allow the strain to fully colonize the plate, obtaining a probiotic fermentation plate.

[0059] 4. Culture of clinically resistant pathogens: Clinically resistant pathogens were activated using LB solid medium, and single colonies were picked and cultured overnight in LB liquid medium. Then, they were transferred to new LB liquid medium at an inoculum of 0.5% and cultured until the logarithmic growth phase to obtain the pathogen seed culture.

[0060] 5. Antibacterial effect of Lactobacillus johnsonii A25041 against pathogenic bacteria (1) After autoclaving the LB medium containing agar, place it in an environment of about 50°C. Add the seed liquid of clinical drug-resistant pathogens cultured in step 4 to the medium at an inoculation rate of 0.5% (v / v). After mixing evenly, pour it into a square plate and let it dry to obtain indicator bacteria plates.

[0061] (2) Using a sterile blue pipette tip (7.5 mm), scoop out an agar block from the Lactobacillus johnsonii A25041 fermentation plate and place it upright on the indicator plate. After overnight anaerobic incubation at 37°C, observe the antibacterial activity and record the size of the inhibition zone (mm). The inhibition zone is the actual size after removing the diameter of the blue pipette tip. The results are as follows: Figure 5 As shown in Table 1, Lactobacillus johnsonii A25041 exhibits inhibitory effects against five pathogenic bacteria: Pseudomonas aeruginosa PA01, Acinetobacter baumannii HRAB85, Escherichia coli O157, drug-resistant Escherichia coli 4-1, and Staphylococcus aureus MRSA1-1. Among these, the inhibitory effect against Acinetobacter baumannii is the best (8.67 mm), while it has significant inhibitory effects against Pseudomonas aeruginosa (6.67 mm) and Staphylococcus aureus MRSA (5.33 mm). It also has a weak inhibitory effect against Escherichia coli and drug-resistant Escherichia coli.

[0062] Table 1. Inhibition zone diameter (mm) of Lactobacillus johnsonii A25041 against clinically resistant pathogens - agar block method

[0063] Example 4 Antimicrobial activity test of Lactobacillus johnsonii A25041 against clinically resistant pathogens - liquid method 1. Seed culture of Lactobacillus johnsonii A25041 and seed culture of five clinically resistant pathogens were prepared according to the method in Example 3. The seed culture of Lactobacillus johnsonii A25041 was centrifuged at 10,000 rpm for 5 min, and the supernatant was collected and filtered through a 0.22 μm filter membrane for later use.

[0064] 2. Antibacterial effect of Lactobacillus johnsonii A25041 against pathogenic bacteria (1) After autoclaving the LB medium containing agar, place it in an environment of about 50°C. Add the cultured clinical drug-resistant pathogenic bacteria seed liquid to the medium at an inoculation rate of 0.5% (v / v), mix well, pour into a square plate and air dry to obtain indicator bacteria plates.

[0065] (2) Using a sterile punch, make holes (7.5 mm) sequentially on the indicator plate. Add 200 μL of Lactobacillus johnsonii A25041 supernatant to each hole and incubate overnight at 37°C under anaerobic conditions. Observe the antibacterial effect and record the size of the inhibition zone (mm). The recording method is the same as in Example 3. The results are as follows: Figure 6 As shown in Table 2, after liquid culture, Lactobacillus johnsonii A25041 metabolites exhibited strong and balanced broad-spectrum antibacterial activity. The strongest inhibitory effects were observed against Pseudomonas aeruginosa PA01 and Acinetobacter baumannii HRAB85 (inhibition zone diameter greater than 13 mm). Strong inhibitory effects were also observed against drug-resistant Escherichia coli 4-1 and Staphylococcus aureus MRSA1-1 (inhibition zone diameter greater than 10 mm). Even against the relatively insensitive hemorrhagic Escherichia coli O157, a significant inhibition zone (8.17 mm) was produced.

[0066] Table 2. Inhibition zone diameter (mm) of Lactobacillus johnsonii A25041 against clinically resistant pathogens - liquid method

[0067] Based on the data from Example 3, the culture method of *Lactobacillus johnsonii* A25041 has a decisive influence on its antibacterial effect. When cultured using the liquid method, the metabolites produced by the strain showed significant and strong inhibitory effects on all tested clinically resistant pathogens, indicating that the full utilization of the strain's antibacterial ability depends on liquid culture. Combining the two methods, *Lactobacillus johnsonii* A25041 exhibits stable and highly effective inhibitory characteristics against clinically important drug-resistant bacteria, especially *Acinetobacter baumannii* and *Pseudomonas aeruginosa*, highlighting its excellent application value.

[0068] Example 5 Determination of the anti-biofilm ability of Lactobacillus johnsonii A25041 1. Test strain: Lactobacillus johnsonii A25041.

[0069] 2. Pathogens to be tested: Pseudomonas aeruginosa PAO1, Acinetobacter baumannii HRAB85, Hemorrhagic Escherichia coli O157, Drug-resistant Escherichia coli 4-1, and Staphylococcus aureus MRSA1-1.

[0070] 3. The inhibitory effect of the supernatant of the strain on the formation of biofilm by pathogenic bacteria was quantitatively evaluated by crystal violet staining. After the pathogenic bacteria and the strain to be tested were activated and cultured on LB solid medium and MRS solid medium, respectively, single colonies were picked and transferred to the corresponding liquid medium for culture. Lactobacillus johnsonii A25041 was centrifuged at 8000 rpm for 5 min and sterilized with a 0.22 μm filter membrane to obtain the fermentation supernatant of Lactobacillus johnsonii for the determination of anti-biofilm ability. A total of 4 groups were set up in the experiment, with 5 replicates in each group. The grouping is as follows: (1) Experimental group: 100 μL of pathogenic bacteria and 100 μL of fermentation supernatant; (2) Positive control: 100 μL of pathogenic bacteria and 100 μL of LB liquid medium; (3) Blank control: 200 μL of LB liquid medium; (4) Supernatant background control: 100 μL of fermentation supernatant and 100 μL of LB liquid medium.

[0071] After mixing, the mixture was incubated at 37°C for 24 hours to allow bacteria to form a biofilm at the bottom of the wells. The airborne bacterial culture was discarded, and the plate was gently washed three times with PBS, leaving only the adhered biofilm. 100 μL of methanol was added for fixation for 15 min. The fixative was discarded, and the plate was air-dried before staining with 1% crystal violet solution (100 μL / well) for 15 min. Excess dye was then gently washed away three times with PBS until the water was clear. After drying at 37°C, 200 μL of 33% glacial acetic acid solution was added to dissolve the crystal violet bound to the biofilm. The solution was transferred to a new 96-well plate, and the absorbance (OD) was measured at 570 nm using a microplate reader. A higher OD value indicates a greater biofilm quantity. The biofilm inhibition rate (%) was calculated using the following formula, and the results are shown in Table 3.

[0072] Biofilm inhibition rate (%) = (1 - Corrected OD of experimental group / Corrected OD of positive control) × 100%; Corrected OD in experimental group = OD in experimental group - OD in supernatant background control; Positive control correction OD = OD positive control - OD blank control.

[0073] Table 3. Anti-biofilm ability of Lactobacillus johnsonii A25041 against pathogenic bacteria

[0074] As shown in Table 3, the fermentation supernatant of *Lactobacillus johnsonii* A25041 exhibits inhibitory effects on biofilm formation against five clinically important pathogenic bacteria. The most significant inhibitory effects were observed against *Pseudomonas aeruginosa* PAO1 and *Acinetobacter baumannii* HRAB85, with inhibition rates exceeding 93%, indicating the presence of key substances capable of effectively disrupting or blocking the early formation of these complex biofilms. Strong inhibitory activity was also observed against *Escherichia coli* O157 and drug-resistant *Escherichia coli* 4-1, but the effects were weaker, with inhibition rates of 80.03% and 63.29%, respectively. The inhibition rate against *Staphylococcus aureus* MRSA1-1 was relatively low, at 50.38%. The fermentation supernatant of *Lactobacillus johnsonii* A25041 demonstrates broad-spectrum and highly effective anti-biofilm activity, particularly showing near-complete inhibition against biofilms of multidrug-resistant Gram-negative bacteria, demonstrating promising application potential in the prevention and control of related biofilm infections.

[0075] Example 6 Anti-inflammatory activity assay of Lactobacillus johnsonii A25041 1. Reagents for testing Dual antibiotics: Prepare a mixed antibiotic solution of 80 U / mL penicillin and 0.08 mg / mL streptomycin using sterile water; Cell culture medium: DMEM medium with 10% fetal bovine serum and 1% dual antibiotics added.

[0076] Lipopolysaccharide (LPS) working solution: Prepare a 50 ng / mL LPS solution using DMEM culture medium containing 1% dual antibiotics. After dissolving, filter the solution through a 0.22 μm filter membrane for sterilization. Prepare and use immediately.

[0077] 2. After activating *Lactobacillus johnsonii* A25041 on MRS solid plates, single colonies were picked and cultured in MRS liquid medium. After re-transplantation, the culture was anaerobic at 37°C for 24 h to obtain *Lactobacillus johnsonii* A25041 fermentation broth. The fermentation broth was centrifuged at 10,000 rpm for 5 min, and the fermentation supernatant was collected for the determination of anti-inflammatory capacity. Frozen RAW264.7 mouse macrophages were resuscitated, washed with PBS, resuspended in complete medium, transferred to T25 culture flasks, and cultured routinely in a 37°C, 5% CO2 incubator. Cell status was observed regularly, and when the cells reached a suitable density, they were digested and passaged to ensure that the cells used in the experiment were in the logarithmic growth phase and had normal morphology. Cells were collected by centrifugation at 800 rpm for 5 min and counted. The cell density was adjusted to 500,000 cells / mL and then evenly seeded into each well of a culture plate. The plates were then incubated in a constant temperature incubator until the cells were fully adherent. The original medium was then removed, and the cells were gently washed with PBS buffer. Three groups were set up for the experiment, with six replicates in each group. Specifically, the groups included: (1) blank control group: cell culture medium treatment; (2) lipopolysaccharide (LPS) stimulation group; and (3) bacterial strain supernatant intervention group: LPS stimulation + Lactobacillus johnsonii A25041 fermentation broth. The corresponding treatment solutions were added under the dark according to the experimental protocol. After further culture, the inflammatory factors were detected using an IL-6 and TNF-α ELISA kit (purchased from Shanghai Jianglai Biotechnology Co., Ltd.). The detection steps were strictly followed according to the kit operation steps. The inhibition rate was calculated according to the formula (inhibition rate (%) = (LPS stimulation group - bacterial strain supernatant intervention group) / LPS stimulation group) × 100%. The results are shown in Table 4.

[0078] Table 4. Inhibition rate of Lactobacillus johnsonii A25041 on IL-6 and TNF-α expression in Raw267.4 cells.

[0079] As shown in Table 4, treatment with Lactobacillus johnsonii A25041 significantly reduced the expression levels of IL-6 and TNF-α in LPS-induced mouse Raw267.4 cells, with inhibition rates reaching 97.35% and 99.74%, respectively. This indicates that the supernatant of this strain can effectively inhibit the expression of pro-inflammatory factors IL-6 and TNF-α and play a role in related inflammation.

[0080] Example 7 Antioxidant capacity determination of Lactobacillus johnsonii A25041 Lactobacillus johnsonii A25041 was activated on MRS solid plates, and after subculturing, single colonies were picked and inoculated into MRS liquid medium. The culture was subcultured once at an inoculum of 2% (v / v) and cultured until mid-logarithmic growth. After adjusting the OD value to 0.6±0.05, the culture was centrifuged at 8000 rpm for 5 min, and the cell-free supernatant was collected as the sample for antioxidant activity testing. The in vitro antioxidant capacity of the supernatant was determined according to the instructions of the corresponding detection kit from Jianglai Biotechnology. The measured indicators included hydroxyl radical scavenging capacity, superoxide anion scavenging rate, DPPH radical scavenging rate, and superoxide dismutase (SOD) activity. The results are shown in Table 5.

[0081] Table 5 Antioxidant capacity of Lactobacillus johnsonii A25041

[0082] As shown in Table 5, the fermentation supernatant of *Lactobacillus johnsonii* A25041 exhibited highly efficient scavenging capabilities against all three major reactive oxygen species (ROS), with a hydroxyl radical scavenging rate of 79.24%. The scavenging rates of superoxide anion and DPPH radicals also exceeded 70%, and SOD activity was detected at 68.02 U / mL. These data indicate that the metabolites of this strain can effectively exert antioxidant effects through two mechanisms: direct free radical scavenging and the provision of antioxidant enzyme activity. This demonstrates significant in vitro antioxidant activity and provides experimental evidence for its application in antioxidant-related functional products.

[0083] Example 8 Detection of hemolytic activity of Lactobacillus johnsonii A25041 To assess the biosafety of *Lactobacillus johnsonii* A25041, hemolytic activity was detected using Columbia blood agar plates. After activation of *Lactobacillus johnsonii* A25041 on MRS solid plates, single colonies were picked and cultured in MRS liquid medium. After anaerobic incubation at 37°C for 24 h, 5 μL of the bacterial culture was dropped onto Columbia blood agar plates. After anaerobic incubation at 37°C for 24 h, the presence of hemolytic zones on the blood agar plates was observed. Results are as follows: Figure 7 As shown, no clear zone was observed around the colony of *Lactobacillus johnsonii* A25041, indicating that it has no hemolytic activity. This demonstrates that this strain does not damage red blood cells in vitro, meeting the safety requirements for probiotics and providing important safety evidence for its further application in the food and medical fields.

[0084] Example 9 Gastrointestinal tolerance assessment of Lactobacillus johnsonii A25041 To evaluate the survival potential of Lactobacillus johnsonii A25041 after oral administration across the gastrointestinal tract, a simulated gastrointestinal fluid tolerance experiment was conducted. Purified Lactobacillus johnsonii A25041 was activated on MRS solid medium, then anaerobically expanded on MRS liquid medium at 37°C before being used in the experiment.

[0085] 1. Artificial gastric fluid tolerance phase: Mix 1 mL of bacterial culture with artificial gastric fluid (pH=3.0) to a total volume of 10 mL and incubate anaerobically at 37°C. Take samples at 0 h and 3 h, dilute appropriately, spread on MRS agar plates, and incubate at 37°C for 48 h. Calculate the viable cell count.

[0086] 2. Artificial intestinal fluid tolerance phase: 1 mL of the bacterial culture treated with gastric juice for 3 hours was transferred to artificial intestinal fluid at pH 8.0, and the volume was adjusted to 10 mL. Anaerobic culture was continued at 37°C. Samples were taken at 0, 2, 4, 6, and 8 hours for dilution, spread, and incubation. The viable bacterial count was calculated at each time point.

[0087] 3. Survival rate calculation: The number of viable bacteria at 0 h in each stage is used as the baseline (set as 100%). The survival rate at each subsequent time point is calculated according to the following formula: Survival rate (%) = (number of viable bacteria at this time point / number of viable bacteria at the corresponding stage 0 h) × 100%. This is used to dynamically evaluate the survival stability of the strain in the simulated gastrointestinal environment. The results are shown in Table 6.

[0088] Table 6. Gastrointestinal fluid tolerance test of Lactobacillus johnsonii A25041

[0089] As shown in Table 6, the viable count of *Lactobacillus johnsonii* A25041 in simulated gastric fluid showed an increasing trend after 3 hours, with a survival rate of 125%. This suggests that the strain may not have fully entered dormancy in the gastric environment, but rather maintained a low level of metabolic and proliferative activity. Furthermore, its cellular structure (such as the cell membrane and cell wall) and internal pH homeostasis system may effectively resist the killing effect of gastric acid. In simulated intestinal fluid, the survival rate of *Lactobacillus johnsonii* A25041 was as high as 92.7% within 2 hours. Afterward, the viable count fluctuated and decreased over time, but after 8 hours of exposure in intestinal fluid, more than 40% of the strain still survived. This survival rate ensures that a sufficient number of viable bacteria can reach the intestines and potentially colonize, thereby exerting probiotic functions. This indicates that the application prospect of this strain as an oral live bacteria preparation is very clear and reliable.

[0090] Example 10 Antibiotic susceptibility testing of Lactobacillus johnsonii A25041 To assess the potential drug resistance risk of *Lactobacillus johnsonii* A25041, the susceptibility of this bacterium to 10 commonly used clinical antibiotics was determined using the disk diffusion method. *Lactobacillus johnsonii* A25041 was activated on MRS solid medium, transferred to MRS liquid medium, and after culturing, inoculated into fresh MRS liquid medium at a 3% (v / v) inoculation rate. The culture was then anaerobically cultured at 37°C for 24 h to obtain the bacterial suspension. MRS solid medium was prepared, and when the temperature dropped to approximately 50°C, the bacterial suspension was added to the medium at a 1% inoculation rate, mixed, and poured into square plates for drying. Antimicrobial susceptibility testing discs (6 mm) were affixed to the square plates containing the bacterium, and anaerobically cultured at 37°C for 24 h. The size of the inhibition zone was observed, and the results are as follows: Figure 8 As shown in Table 7.

[0091] Table 7. Antibiotic susceptibility testing of Lactobacillus johnsonii A25041

[0092] according to Figure 8 As shown in Table 7, *Lactobacillus johnsonii* A25041 produced inhibition zones against penicillin, ampicillin, ceftriaxone, erythromycin, and chloramphenicol. The inhibition zones against chloramphenicol and ceftriaxone were the largest, indicating potentially the highest sensitivity. No inhibition zones were produced against gentamicin, tetracycline, ciprofloxacin, lincomycin, and trimethoprim-sulfamethoxazole, suggesting that this strain may possess natural resistance or insensitivity to these antibiotics. This characteristic may make it suitable for combined use with these antibiotics. In clinical or veterinary treatment, live preparations, inactivated cells, or metabolites of *Lactobacillus johnsonii* A25041 can be used simultaneously or sequentially with the aforementioned insensitive antibiotics to maintain intestinal microecological balance, alleviate antibiotic-associated diarrhea, and potentially enhance overall therapeutic efficacy through microbial regulation while exerting the bactericidal effect of antibiotics. Therefore, *Lactobacillus johnsonii* A25041 can be used to prepare compound microecological therapeutic agents or adjuvants for combined use with antibiotics.

[0093] As can be seen from the above, the Lactobacillus johnsonii strain A25041 provided by this invention has the functions of inhibiting multidrug-resistant bacteria and anti-biofilm, has good acid and alkali tolerance and gastrointestinal fluid tolerance, exhibits excellent anti-inflammatory activity and antioxidant capacity, has no hemolytic ability, has good safety, and exhibits natural resistance to gentamicin, tetracycline, ciprofloxacin, lincomycin and compound sulfamethoxazole.

[0094] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Lactobacillus johnsonii ( Lactobacillus johnsonii A25041, with accession number GDMCC No:67373.

2. An inoculant characterized in that, The active ingredient of the bacterial agent includes one or more of the following; (1) live Lactobacillus johnsonii A25041 according to claim 1; (2) inactivated Lactobacillus johnsonii A25041 according to claim 1; (3) culture of Lactobacillus johnsonii A25041 according to claim 1; (4) extract of Lactobacillus johnsonii A25041 according to claim 1.

3. The bacterial agent of claim 2, wherein The culture includes one or more of fermentation plate, fermentation broth and fermentation supernatant; the extract includes fermentation broth extract.

4. Lactobacillus johnsonii A25041 according to claim 1 or the bacterial agent according to claim 2 or 3 for use in one or more of the following; (1) preparation of antibacterial products; (2) preparation of anti-biofilm products; (3) preparation of drugs for preventing and / or treating pathogenic bacteria infection; (4) preparation of anti-inflammatory products; (5) preparation of anti-oxidation products; (6) preparation of products for regulating intestinal flora.

5. Use according to claim 4, characterized in that, The antibacterial products are against Gram-negative bacteria and / or Gram-positive bacteria; The anti-biofilm products are for breaking the membrane of Gram-negative bacteria and / or Gram-positive bacteria; The pathogenic bacteria include Gram-negative bacteria and / or Gram-positive bacteria; The anti-inflammatory products are for inhibiting the expression of IL-6 and / or TNF-α; The anti-oxidation products are for scavenging reactive oxygen radicals and / or increasing superoxide dismutase activity.

6. Use according to claim 5, characterized in that, The Gram-negative bacteria include drug-resistant Escherichia coli (DEC). Escherichia coli ), Enterohemorrhagic Escherichia coli Escherichia coli ), drug-resistant Acinetobacter baumannii ( Acinetobacter baumnnii ) and drug-resistant Pseudomonas aeruginosa ( Pseudomonas aeruginosa One or more of the following; The gram-positive bacteria include drug-resistant Staphylococcus aureus (MRSA) Staphylococcus aureus ) ; The reactive oxygen radicals include one or more of hydroxyl radical, superoxide anion and DPPH radical; The anti-biofilm products are for inhibiting the formation of biofilm.

7. Use according to claim 6, characterized in that, The drug-resistant E. coli includes β-lactam antibiotic-resistant E. coli; The drug-resistant A. baumannii includes imipenem antibiotic-resistant A. baumannii; The drug-resistant P. aeruginosa includes one or more of β-lactam antibiotic-resistant, polypeptide antibiotic-resistant, aminoglycoside antibiotic-resistant and fluoroquinolone antibiotic-resistant P. aeruginosa; The drug-resistant S. aureus includes methicillin-resistant S. aureus.

8. Use according to claim 4, characterized in that, The products are functional foods, health products, daily chemical products or drugs; The dosage form of the products includes lyophilized powder or capsule.

9. A product for inhibiting multi-drug resistant bacteria, having an antibiofilm function, anti-inflammatory activity, and antioxidant capacity, characterized by, The drugs include active ingredients and excipients; The active ingredients include Lactobacillus johnsonii A25041 according to claim 1 or the bacterial agent according to claim 2 or 3.

10. The product of claim 9, wherein, The active ingredients further include antibiotics; the antibiotics include one or more of gentamicin, tetracycline, ciprofloxacin, lincomycin and cotrimoxazole.

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