Salivaria lactobacillus of pig intestinal tract with antimicrobial activity and application thereof

Abstract

The application discloses a pig intestinal lactobacillus salivarius with antimicrobial activity and application thereof, relates to the technical field of microorganisms, and is named pig-LS2023. The strain can produce specific surfactants, the surfactants have antibacterial and antiviral activities, and have the advantages of biodegradability, non-toxicity and stability. The results of antimicrobial experiments show that the inhibition rates of the produced surfactants on pig escherichia coli and streptococcus are 95.06% and 81.80% respectively, and the surfactants have obvious inhibitory effect on PCV2. The research results show that the pig-LS2023 strain is expected to become a candidate probiotic strain for improving the immune state of pigs and producing a new type of anti-infection drug substitute.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a porcine intestinal lactobacillus with antimicrobial activity and its application. Background Technology

[0002] Antimicrobial drugs play a crucial role in protecting human and animal health, improving the economic efficiency of livestock farming, and ensuring food safety. With rapid economic development and increasing intensification of livestock farming, the use of antibiotics is constantly increasing, and multiple challenges such as antimicrobial resistance (AMR), food safety, and environmental pollution urgently need to be addressed. The World Health Organization (WHO) has listed AMR as one of the major threats to human health in the 21st century. Finding safe, effective antibiotic alternatives that are less likely to induce resistance has become a hot topic in current research on new treatment methods.

[0003] Lactic acid bacteria (LAB), as a type of probiotic, are important members of the normal microbiota in specific areas of the human and animal oral cavity and intestines. By producing organic acids, specific enzyme systems, and bacteriocins, they can regulate the intestinal microecological balance, inhibit the proliferation of pathogenic microorganisms, and enhance host immunity. Therefore, lactic acid bacteria are considered an ideal alternative to antibiotics. Lactic acid bacteria also possess various probiotic properties, including antiviral, antitumor, anti-stress, and intestinal barrier function improvement. Lactobacillus salivarius (L. salivarius), as an important member of lactic acid bacteria, has attracted much attention due to its unique probiotic properties. Widely distributed in the gastrointestinal mucosa of humans and animals, L. salivarius can produce biosurfactants, various bacteriocins, antibiotics, antioxidants, and immunomodulators, playing a role in balancing and improving the intestinal flora, resisting microbial infections, regulating mucosal immunity, and improving production performance. L. salivarius has become a potential candidate strain for anti-infective therapy and improving immune function.

[0004] Biosurfactants (BS) are naturally occurring active substances produced by microorganisms. They possess an amphiphilic structure, reduce liquid surface tension, and exhibit various biological activities such as antibacterial, antiviral, and anti-adhesion properties. Furthermore, they offer advantages such as good biodegradability, non-toxicity, and stability. Therefore, biosurfactants are considered potential candidates for antimicrobial drug alternatives. Compared to pathogenic bacteria, probiotic-produced surfactants, namely probiotic BS, are safer and have broad application prospects in the food, pharmaceutical, and agricultural fields.

[0005] Porcine circovirus type 2 (PCV2) is a DNA virus approximately 17 nm in diameter, belonging to the genus *Circovirus* within the family Circoviridae. This virus is characterized by damaging the immune organs of pigs, causing immunosuppression and multisystemic diseases, seriously threatening the development and economic benefits of pig farms. Streptococci, *Escherichia coli*, *Pasteurella multocida*, and *Haemophilus parasuis* are common synergistic or secondary pathogens in PCV2-infected pigs, and these synergistic and secondary infections exacerbate the severity of PCV2 infection. Therefore, PCV2 and its common synergistic or secondary pathogens are key targets for prevention and control in pig farms, and research into relevant preventative and control agents is of great significance. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide a porcine intestinal Lactobacillus with antimicrobial activity and its application. This invention isolates a strain of L. salivarius from the ileum of piglets experimentally infected with porcine circovirus type 2 (PCV2), and studies the physicochemical properties and antimicrobial activity of the surfactant it produces, aiming to provide a scientific basis for promoting the application of probiotics and developing antibiotic alternatives.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows:

[0008] In a first aspect, the present invention provides a porcine intestinal Lactobacillus salivarius with antimicrobial activity, named pig-LS2023, which is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC No: M 20251406 and deposit date June 17, 2025.

[0009] It should be noted that *Lactobacillus salivarius* (L. salivarius) is a probiotic widely found in the gastrointestinal tract of humans and animals. Its biosurfactant (BS) possesses antibacterial and antiviral activity, and exhibits advantages such as biodegradability, non-toxicity, and stability. Porcine circovirus type 2 (PCV2) is a common and important pathogen in pig farms, causing immunosuppression or immune dysfunction in pigs. Susceptibility to PCV2-positive pig herds is increased, and secondary bacterial infections are severe. To explore novel biological agents with antimicrobial activity to improve pig immunity, this invention used the calcium carbonate plate method to isolate and identify a strain of *Lactobacillus* from the small intestine of PCV2-infected pigs. The isolate was identified through 16S rRNA gene sequence analysis and physiological and biochemical tests. Furthermore, the bacterial BS was extracted, and its surface activity was detected using oil diffusion and oil droplet collapse experiments. Finally, the in vitro antibacterial activity of BS against Escherichia coli and Streptococcus was analyzed by colony counting method, and the anti-PCV2 activity of BS was analyzed by Western blot and indirect immunofluorescence (IFA) assay.

[0010] The results showed that a strain of *L. salivarius* was isolated from the ileum of PCV2-infected piglets. The BS produced by this strain could form an oil diffusion ring or spread on the surface of vegetable oil, indicating that the extracted BS has surface tension-reducing properties. Antimicrobial experiments showed that at concentrations of 6.25–50 mg / mL, BS could inhibit the growth of *Escherichia coli* and *Streptococcus suis* by 46.13%–95.06% and 41.06–81.80%, respectively, and that BS at concentrations of 6.25–12.5 mg / mL significantly inhibited the proliferation of PCV2 on PK-15 cells. These results suggest that *L. salivarius* is a promising candidate probiotic strain for improving the immune status of pigs and producing novel alternatives to anti-infective drugs.

[0011] Furthermore, the sequence of the porcine intestinal Lactobacillus salivarius is SEQ ID NO: 3.

[0012] In a second aspect, the present invention provides the use of the above-described porcine intestinal lactobacillus in the preparation of a drug for regulating intestinal infections in animals.

[0013] Furthermore, the animal's intestinal infection is at least one of Escherichia coli, Streptococcus, and porcine circovirus infection, and the animal is a pig.

[0014] In a third aspect, the present invention provides a surfactant produced by the aforementioned *Lactobacillus salivarius* from the swine intestinal tract.

[0015] In a fourth aspect, the present invention provides the use of the surfactant described above in the preparation of antibacterial agents.

[0016] Furthermore, the antibacterial agent is an agent that is effective against Escherichia coli, Streptococcus, or porcine circovirus infection.

[0017] A fifth aspect of the present invention provides a method for preparing the surfactant described above, comprising the following steps:

[0018] (1) The porcine intestinal lactobacillus described in the present invention was inoculated into MRS broth, cultured at 37°C for 48 h, and then centrifuged to collect the bacterial cells.

[0019] (2) Wash the bacterial cells obtained in step (1) with sterile deionized water, suspend the bacterial cells in PBS buffer, and incubate at room temperature for 2 hours with stirring to release biosurfactants. Remove the cells by centrifugation and filter with a filter. Dry the supernatant using a rotary evaporator to obtain the suspension extract.

[0020] (3) The extract was suspended in PBS to a concentration of 10 mg / mL. The pH was adjusted to 2.0 with HCl and then incubated at 4°C for 2 h. The precipitate was collected by centrifugation, washed with sterile distilled water, dissolved in sterile distilled water, and the pH was adjusted to 7.0 with NaOH. The precipitate was then filtered through a filter membrane and freeze-dried.

[0021] The present invention also provides a sustained-release formulation, wherein the surfactant described above is the active ingredient.

[0022] Furthermore, the sustained-release formulation includes solid sustained-release formulations and liquid sustained-release formulations. The solid sustained-release formulation is a sustained-release microsphere or sustained-release tablet, and the liquid sustained-release formulation includes nanoliposomes, sustained-release microspheres, or sustained-release emulsions.

[0023] The beneficial effects of this invention include at least the following:

[0024] (1) This invention provides a new antibiotic alternative for the preparation of drugs against porcine intestinal infections - porcine intestinal lactobacillus; the surfactant produced by it has an inhibition rate of 95.06% and 81.80% against porcine Escherichia coli and Streptococcus, respectively, and also has a significant inhibitory effect on PCV2;

[0025] (2) This invention provides a new option for preparing drugs against porcine intestinal infections. Attached Figure Description

[0026] Figure 1For the isolation, growth curve and 16S rDNA sequence analysis of Lactobacillus salivarius. (A) Colony morphology on 1% CaCO3-MRS solid medium; (B) Gram staining; (C) Growth curve; (D) 16S rDNA amplification by PCR, M: DL2000 DNA molecular weight standard, 1: isolate (pig-LS2023), 2: negative control.

[0027] Figure 2 The results show the detection of biosurfactants. Oil diffusion experiment: biosurfactants produced by *Lactobacillus salivarius* isolate (A), water blank control (B), correlation analysis of biosurfactant concentration and oil diffusion diameter (C). Oil droplet collapse experiment: biosurfactants produced by *Lactobacillus salivarius* isolate (D) and water blank control (E).

[0028] Figure 3 The inhibitory effect of biosurfactants on *Escherichia coli* (A) and *Streptococcus suis* (B) was measured. The bacterial solution was diluted 10... -5 Colony counts were performed in triplicate for each biosurfactant (BS) concentration. *, **, and *** indicate significant differences in BS-bacterial co-culture compared to the bacterial control group at P<0.05, P<0.01, or P<0.001 levels, respectively.

[0029] Figure 4 To determine the effect of biosurfactants from *Lactobacillus salivatus* strain pig-LS2023 on the CCK8 assay in PK15 cells. 50 Measured value.

[0030] Figure 5 The inhibitory effect of biosurfactants produced by *Lactobacillus salivarius* on PCV2 was investigated. After PK-15 cells were infected with the virus and co-incubated with the biosurfactant solution for 36 hours, PCV2 antigen was detected by immunofluorescence assay (IFA).

[0031] Figure 6 This study investigated the kinetics of the inhibitory effect of biosurfactants on PCV2. PK-15 cells were co-incubated with 0.2 MOI of PCV2 containing 6.25 mg / mL and 3.125 mg / mL biosurfactants, respectively. Viral antigens were detected by Western blot (A, B, and C) at 24, 48, and 72 hours after infection. Gray-scale analysis of the antigen-antibody reaction bands was performed based on the Western blot results (D, E, and F).

[0032] Figure 7 The inhibitory effect of Lactobacillus salivarius (LS) on PCV2 is shown. LS: Lactobacillus salivarius; LR: Lactobacillus reuteri control; PC: virus control.

[0033] Figure 8 For LS( Figure 8 A) and LR( Figure 8 B) Results of IPEC cytotoxicity (CC50) assay. Detailed Implementation

[0034] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0035] Furthermore, the technical solutions of the various embodiments of the present invention can be combined with each other, but only if they are feasible for those skilled in the art. If the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0036] The materials and methods used in this invention are as follows:

[0037] 1. Bacterial strains, viral strains, and cells

[0038] Escherichia coli O 141 (E. coli O) 141 Streptococcus suis (S. suis) strain SJZ2021, a porcine streptococcus, was preserved by the Animal Infectious Disease Laboratory of the College of Veterinary Medicine, Hebei Agricultural University. Lactobacillus reuteri was purchased from Beijing Bio-BW Biotechnology Co., Ltd.; PCV2 strain HBDX2018 (GenBank ID: MK585076), TCID... 50 10 6.1 / mL, preserved by the Animal Infectious Disease Laboratory of Hebei Agricultural University. The culture medium for PK-15 cells was DMEM medium (Gibco, USA) containing 10% FBS (Fetalbovine serum), 100 IU each of penicillin and streptomycin.

[0039] 2. Animal infection and sample collection

[0040] Three healthy 28-day-old piglets were purchased from Hebei Tangsheng Animal Husbandry Co., Ltd. PCR and ELISA tests showed negative results for both porcine circovirus type 2 (PCV2) nucleic acid and antibodies. The three piglets were inoculated with PCV2 HBDX2018 strain via nasal and intramuscular routes, with each piglet receiving 10 mg of the strain. 6.1 TCID 50 / mL. Seven days post-infection, pigs were anesthetized by intravenous injection of xylazine hydrochloride, and the ileum and its contents were aseptically collected for bacterial isolation and culture.

[0041] This invention has received ethical approval from the Animal Welfare and Ethics Review Committee of Hebei Agricultural University (China) (License No.: 1820026). The entire experimental process was conducted in accordance with the "Guidelines for the Care and Use of Laboratory Animals" of Hebei Agricultural University.

[0042] 3. Isolation, culture and purification of bacteria

[0043] Scrape porcine ileal mucosa and intestinal contents from sterile glass slides and place them in test tubes containing the same volume of PBS. Incubate anaerobically at 37°C on a shaker for 5 hours to enrich bacteria. Take 100 μL of the enriched bacterial suspension for 10... -1 ~10 -5 Serial dilutions were performed; after vortexing and mixing, 10 μL of each diluted bacterial suspension was taken and evenly spread onto the surface of DeMan, Rogosa and Sharpe (MRS) solid medium (Beijing Luqiao Technology Co., Ltd.), and anaerobically incubated at 37℃ for 24 h. Single colonies with uniform, smooth, raised surfaces and neat edges (milky white) were selected and streaked onto 1% CaCO3-MRS solid medium, and anaerobically incubated at 37℃ for 24 h. Single colonies with calcification zones were selected and purified three times consecutively to obtain purified strains. The purified strains were Gram-stained and examined under a microscope until all bacteria in the field of view were Gram-positive rod-shaped strains. The isolated bacteria were placed in preservation solution (60% glycerol LB) and frozen at -20℃.

[0044] 4. Amplification and Sequence Analysis of 16S rDNA

[0045] Total bacterial DNA was extracted using a boiling lysis method. 1.5 mL of bacterial culture was centrifuged at 8000 rpm for 5 min, and the bacterial cells were collected. The cells were resuspended in 500 μL of sterile water, boiled for 10 min, and centrifuged at 12000 rpm for 10 min. The supernatant was collected as a DNA template for PCR amplification of the 16S rDNA sequence. The PCR reaction system (20 μL) consisted of: 10 μL of 2×Es Taq Master Mix (Jiangsu Kangwei Century Biotechnology Co., Ltd.), 0.5 μL each of forward and reverse primers (25 μmol / L), 2 μL of DNA template, and 7 μL of ddH2O. PCR reaction conditions: 94℃ for 3 min; 94℃ for 45 s, 52℃ for 1 min, 72℃ for 1 min, 30 cycles; 72℃ for 10 min. The universal primer F for amplifying 16S rDNA was 5. ′ -AGAGTTTGATCCTGGCTCAG-3 ′ (SEQ ID NO.1) and R:5′ -GGTTACCTTGTTACGACTT-3 ′ (SEQ ID NO.2). The PCR product was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were compared with the reference sequence in the GenBank nucleic acid database for homology, and a phylogenetic tree was constructed using MEGA 7.0 software and the Neighbor-Joining (NJ) method.

[0046] 5. Determination of growth curve

[0047] Single colonies were picked and inoculated into MRS broth and anaerobically cultured at 37°C for 14 h to serve as seed culture. Seed culture was then inoculated into MRS broth at a 1:100 inoculum and cultured. Samples were taken at 0 h, 8 h, 12 h, 14 h, 16 h, 18 h, and 24 h, and serially diluted for colony counting. Each dilution was performed in triplicate. The bacterial concentration (colony forming units / mL) was calculated based on the colony count.

[0048] 6. Identification of physicochemical properties

[0049] The biochemical characteristics of the isolated strains were identified using a micro-method, while a standard Lactobacillus control was established. Following the manufacturer's recommended instructions, purified bacteria were picked and inoculated into micro-reaction tubes containing maltose, mannitol, sorbitol, sucrose, raffinose, lactose, indole, and hydrogen sulfide (Hangzhou Binhe Microbial Reagent Co., Ltd.). The tubes were anaerobically cultured at 37℃ for 24 hours, and the fermentation performance of the isolated strains in producing acid, indole, and hydrogen sulfide was observed.

[0050] Bacteria were inoculated into semi-solid or gelatin culture media via stab inoculation and anaerobically cultured at 37°C for 24 hours. The motility of the bacteria was determined based on their spread and growth along the stab line. The gelatin culture was then placed in a 4°C refrigerator for 2 hours, and the gelatin coagulation was observed to determine whether the strain possessed the ability to produce gelatin liquefying enzymes and liquefy gelatin.

[0051] Then, the isolated bacteria were subjected to a catalase test. A single colony was picked and placed on a clean glass slide, and 3% H2O2 was added. If a large number of bubbles were produced within 30 seconds, it indicated that the isolated strain could produce catalase and was catalase positive; otherwise, it could not produce catalase.

[0052] 7. Preparation and Detection of Biosurfactants

[0053] (1) Preparation of surfactants

[0054] The isolated bacterial strain was inoculated into 300 mL of MRS broth at a ratio of 1:100 and cultured at 37°C for 48 h. The cells were then harvested by centrifugation at 10,000 g for 5 min at 4°C. The cells were washed twice with sterile deionized water and resuspended in 50 mL of PBS buffer (pH 7.0, 0.01 mol / L). The suspension was gently stirred and incubated at room temperature for 2 h to release the biosurfactant. Cells were removed by centrifugation and filtered through a 0.22 μm filter (Tianjin Jinteng Experimental Equipment Co., Ltd.). The supernatant was dried in a rotary evaporator (RE-52AA). The extract was resuspended in PBS (pH 7.0, 0.01 mol / L) to a concentration of 10 mg / mL. The pH was adjusted to 2.0 with 1 mol / L HCl and incubated at 4°C for 2 h. The precipitate was collected by centrifugation at 10,000 g for 15 min at 4°C and washed twice with acid-adjusted sterile distilled water (pH 2.0). The precipitate was dissolved in sterile distilled water, and the pH was adjusted to 7.0 with 1 mol / L NaOH. After filtration through a 0.22 μm filter membrane, it was lyophilized for the next step of the experiment.

[0055] (2) Oil diffusion test

[0056] One drop of vegetable oil was added to distilled water, followed by one drop of the prepared surfactant (100 mg / mL) on the surface of the vegetable oil. Simultaneously, one drop of distilled water was added to the surface of the vegetable oil as a negative control. The diameter of the oil expansion ring was measured, and the effect of the surfactant in reducing surface tension was determined based on the diameter of the oil expansion ring. The diameter of the oil expansion ring was also measured for different concentrations (0, 50, 100, 150, 200, 250 mg / mL) of surfactant, and a standard curve was plotted to analyze the relationship between surfactant concentration and the diameter of the oil expansion ring.

[0057] (3) Oil droplet collapse test

[0058] Vegetable oil was dropped into a petri dish, followed by an equal volume of surfactant. After 2 minutes, the collapse of the droplets on the surface of the vegetable oil was observed. A negative control was also set up with an equal volume of water added. If the liquid contains surfactant, the interaction force between the droplet and the hydrophobic surface of the vegetable oil decreases, the droplet diffuses, and the surface becomes flat (droplet collapse). If the droplet does not contain surfactant, the polar water molecules will repel the hydrophobic surface, the droplet will remain stable, and the surface will bulge.

[0059] 8. Inhibitory effect of Lactobacillus salivarius on PCV2

[0060] The half-maximal cytotoxic concentration (CC50) was determined as follows: *Lactobacillus salivarius* (LS) was inoculated into MRS liquid medium at a ratio of 1:100 and incubated at 37°C for 12 hours. The bacterial cells were then harvested by centrifugation. The bacterial concentration was adjusted to 5 × 10⁻⁶. 9CFU / mL, inactivated at 80℃ for 30 min; centrifuged the inactivated bacterial solution at 4℃ and 4000 r / min for 20 min, collected the supernatant, and filtered through a 0.22 μm filter membrane to obtain the inactivated LS or LR sample. Simultaneously, *Lactobacillus reuteri* (LR) was used as a probiotic control. IPEC-J2 was inoculated into 96-well cell culture plates (4 × 10⁻⁶). 4 Cells / well), place the cell culture plate in a 37°C, 5% CO2 incubator; when the cells reach 90% confluence, discard the liquid in the wells. Add 100 μL of inactivated LS or LR (10) to each well. 8 ~10 4 Cell proliferation and cytotoxicity assay kits were prepared, with three replicates per group. The wells were set up as follows: negative control wells (containing 100 μL of DMEM nutrient solution with 10% FBS) and blank control wells (containing only 100 μL of DMEM nutrient solution with 10% FBS, without cell seeding). All wells were incubated at 37°C for 24 h in a 5% CO2 incubator. Following the instructions for the CCK-8 cell proliferation-cytotoxicity assay kit, 10 μL of CCK-8 solution was added to each well, and the wells were incubated at 37°C for 2 h in a 5% CO2 incubator. OD values ​​were read using a microplate reader. 450 nm value. Calculate and analyze: Cell viability (%) = (OD 样品孔 -OD 空白对照 ) / (OD 阴性对照 -OD 空白对照 )×100%.

[0061] Effects of LR or LS on PCV2 proliferation: IPEC-J2 cells in good growth condition were used at a concentration of 1.2 × 10⁻⁶. 5 Seeds were placed in 24-well plates at 5 × 10⁶ cells / well. When the cells reached 90% confluence, the liquid in the wells was discarded. 1 mL of inactivated *Lactobacillus salivarius* (5 × 10⁶ cells / well) was added to each well. 6 CFU / mL, cultured in a 37℃ 5% CO2 incubator for 6h, 12h, 18h, and 24h, then infected IPEC-J2 cells with 0.2 MOI PCV2 and incubated for 1h in a 37℃ 5% CO2 incubator. The liquid in the wells was discarded, and 1mL of maintenance medium containing the appropriate concentration of inactivated *Lactobacillus salivarius* was added. The cells were then incubated in a 37℃ 5% CO2 incubator for 72h. Simultaneously, *Lactobacillus reuteri* (LR) control, cell control (DMEM nutrient medium with only 3% FBS), and positive (virus) control (inoculated with only 0.2 MOI PCV2) were set up, with three replicates for each time point. The cultures were harvested, repeatedly frozen and thawed, centrifuged, and the supernatant was collected for PCV2 detection using qPCR.

[0062] 9. Detection of antimicrobial activity of surfactants

[0063] (1) Detection of anti-Escherichia coli and anti-streptococcal activity

[0064] To detect the antibacterial activity of the surfactant extracted from *Lactobacillus salivarius* isolates, this invention employed a modified microdilution method to determine the antibacterial activity of the prepared surfactant against *E. coli* and *S. suis*. 200 μL of sterile nutrient broth was added to well 1 of a 96-well cell culture plate as a blank control. In wells 2-8, 100 μL of serially diluted surfactant was first added to each well, resulting in final concentrations of 0, 1.5625, 3.125, 6.25, 12.5, 25, and 50 mg / mL, respectively. Then, 100 μL of OD200 was added to each well. 600 Prepare 0.5% E. coli or S. suis culture medium and mix well. Add 100 μL of nutrient broth and an equal volume of bacterial culture to well 9 as a bacterial control. Perform three replicates per well. Incubate the culture plate anaerobically at 37°C for 24 hours. Take bacterial cultures with different concentrations of BS and count colonies according to the bacterial dilution method to analyze the antibacterial activity of BS.

[0065] (2) Determination of anti-PCV2 activity

[0066] The half-maximal concentration of cytotoxicity (CC50%) 50 Assay: To determine the anti-PCV2 activity of BS, the effect of BS on CC in PK-15 cells was first measured. 50 BS was diluted to 6.25, 12.5, 25, 50, 100, and 200 mg / mL using DMEM medium containing 10% FBS, and then seeded into 96-well cell culture plates at 50 μL / well. 50 μL of PK-15 cell suspension (2 × 10⁻⁶ cells / well) was added to each well. 4 Add 10 μL of CCK-8 solution to each well, mix well, and repeat the process three times for each concentration. Also include cell control wells without BS and nutrient solution control wells. Incubate the cell culture plates at 37°C with 5% CO2 for 24 hours. Following the Cell Counting Kit-8 (CCK-8) instructions (APExBIO), add 10 μL of CCK-8 solution to each well and incubate at 37°C with 5% CO2 for 1 hour. Read the OD values ​​using a microplate reader (Synergy HTX, Gene Company Limited, USA). 450 Cell viability is calculated using the following formula:

[0067] Cell proliferation activity % = (OD BS孔 -OD 营养液对照 ) / (OD 细胞对照 -OD 营养液对照 )×100%.

[0068] Using GraphPad 8.0 software, a nonlinear fitting curve of BS concentration versus cell proliferation activity was plotted, and CC was calculated. 50 Then, the inhibitory effect of BS on PCV2 was detected by indirect immunofluorescence assay (IFA) and Western blot assay, respectively.

[0069] IFA: PCV2 at an infectious dose of 0.2 MOI was mixed with 12.5 or 6.25 mg / mL BS, or an equal volume of DMEM culture medium containing 3% FBS, and incubated at 37°C for 1 h in a 5% CO2 incubator. Logarithmically growing PK-15 cells were digested with trypsin, centrifuged to collect the cells, resuspended in DMEM culture medium containing 3% FBS, and seeded into 96-well cell culture plates at 100 μL per well containing 5 × 10⁶ cells / well. 4 Cells were divided into four groups: a cell control group (NC), a virus control group (PC), a BS-12.5 group, and a BS-6.25 group, with three replicates per group. For the NC group, 100 μL of DMEM culture medium containing 3% FBS was added to each well; for the PC group, 100 μL of premixed culture medium co-incubated with PCV2 was added to each well; for the BS-12.5 and BS-6.25 groups, 100 μL of premixed BS and PCV2 co-incubated at different concentrations were added to each well, respectively. Cell culture plates were placed in a 37℃ CO2 incubator and cultured for 36 h. Cells were gently washed with PBS, and 50 μL of pre-chilled methanol was added to each well. The cells were fixed at -20℃ for 10 min. After washing the cells, 100 μL of 2% BSA-PBS was added to each well and incubated at 37℃ for 1 h. 50 μL of 1:500 diluted PCV2 monoclonal antibody (prepared by the Animal Infectious Diseases Laboratory of Hebei Agricultural University) was added to each well and incubated at 37℃ for 1 h. After washing, 50 μL of 1:500 diluted... 594 goat anti-mouse IgG (Suzhou Youyilandi Biotechnology Co., Ltd.) was incubated at 37℃ for 45 min; after washing, the cell nuclei were stained with Bisbenzimde and the red fluorescence was observed under a fluorescence microscope to analyze the specific binding of the monoclonal antibody to the PCV2 antigen.

[0070] Western Blot: To investigate the duration of the inhibitory effect of BS on PCV2 proliferation, 0.2 MOI of PCV2 mixed with a mixture of 6.25 mg / mL or 3.125 mg / mL BS was added to 6-well cell culture plates, 1 mL / well, with a repeat of 3 wells; cells were incubated at 37°C in a 5% CO2 incubator for 1 h. A virus control and a cell control without BS were also included. Vigorously growing PK-15 cells were digested with trypsin, and the cell concentration was adjusted to 1×10⁻⁶ cells / well with 3% FBS 1×1640 medium. 6Cells / mL were added to each well containing either PCV2-BS mixture or PCV2 virus solution. Cells were incubated at 37℃ for 24h, 48h, and 72h. Western blot was performed to detect viral protein antigens and analyze the inhibitory effect of BS on PCV2. For the Western blot experiment, the PCV2 monoclonal antibody was diluted 1:500, HRP-goat anti-mouse IgG (Boaolong Biotechnology Co., Ltd. (Suzhou)) was diluted 1:10000, and chemiluminescent reagent (Shanghai Yisen Biotechnology Co., Ltd.) was used.

[0071] 10. Statistical Analysis

[0072] All data are expressed as the mean ± labeled error of three trials. Statistical analysis was performed using the t-test. A p-value < 0.05 indicates a significant difference between groups (*); a p-value < 0.01 or 0.001 indicates an extremely significant difference between groups (** or ***). Graphs were plotted using GraphPad Prism 8.0 software.

[0073] 11. Results

[0074] (1) Culture and morphological characteristics of the isolated strain, 16S rDNA analysis and growth curve

[0075] The isolated strain was streaked onto MRS solid medium and incubated at 37°C for 24 hours. Medium-sized, raised, moist, and well-defined milky-white circular colonies were observed. After incubation on 1% CaCO3-MRS solid medium for 24 hours, a clear zone of calcium dissolution was visible around the colonies. Figure 1 A). After Gram staining, Gram-positive bacilli can be seen under a microscope, appearing as short rods, arranged in pairs or short chains. Figure 1 B). It is evident that the above-mentioned culture characteristics and morphological features are consistent with those of *Lactobacillus*. The isolated strain was incubated statically at 37°C, with samples collected every 2 hours. The bacterial growth curve was analyzed using colony counting. The results showed that the highest colony count was observed after 12 hours of incubation, indicating that the logarithmic growth phase of this strain is 12 hours. Figure 1 C).

[0076] To further identify the isolated lactobacilli, the bacterial 16S rDNA sequence was amplified by PCR. Agarose gel electrophoresis showed that the amplified sequence was approximately 1400 bp. Figure 1

[0077] Based on sequence homology analysis, the phylogenetic tree constructed using the Neighbor-Joining method with MEGA 7.0 software showed that the isolated strain was on the same branch as Lactobacillus salivarius. The results indicated that the isolated strain was Lactobacillus salivarius, named pig-LS2023. This strain was deposited at the China Center for Type Culture Collection on June 17, 2025, with the microbial accession number CCTCC No: M 20251406.

[0078] (2)Physical and chemical properties

[0079] The physicochemical characteristics of the isolated strain pig-LS2023 are shown in Table 1. When pig-LS2023 was inoculated into maltose, mannitol, sorbitol, sucrose, raffinose, lactose, indole, and hydrogen sulfide culture media and anaerobically cultured at 37℃ for 24 h, the culture media in the six sugar fermentation tubes changed from purple to yellow, indicating that the bacteria can produce acid. No color change occurred in the indole and hydrogen sulfide media, indicating that the isolated strain does not produce indole or hydrogen sulfide. When 3% H2O2 was added to the colonies, no bubbles were produced, indicating that the strain does not produce hydrogen peroxide. After pig-LS2023 was stab-inoculated into gelatin culture medium, the bacteria grew only at the stab line and did not spread to the surrounding area; the gelatin did not liquefy, indicating that the strain is non-motile and does not liquefy gelatin. These characteristics are consistent with those of standard lactobacillus.

[0080] Table 1 Physiological and biochemical characteristics of Lactobacillus salivarius

[0081] project Standard strains of lactic acid bacteria pig-LS2023 morphology Bacillus Bacillus Gram staining + + athleticism - - Gelatin liquefaction - - Sucrose fermentation experiment + + Raffinose fermentation experiment + + lactose fermentation experiment + + Maltose fermentation experiment + + Mannitol fermentation experiment + + Sorbitol fermentation experiment + + Catalase test - - Indole test - - Hydrogen sulfide test - -

[0082] * "+" indicates positive; "-" indicates negative.

[0083] (3) The isolated strains have the ability to produce biosurfactants.

[0084] When an extract of *Lactobacillus salivarius* isolate was added to the surface of vegetable oil in water, an oil-removing ring with a diameter of 2.7 cm was produced, while no oil-removing ring was observed in the water control. Figure 2 A and Figure 2 B). As the surfactant concentration increases, the diameter of the oil diffusion ring increases, and the two show a linear relationship (R² = 0.99). Figure 2 C). When a surfactant is added to the surface of vegetable oil, the droplets diffuse and the surface becomes flat, unlike the water control which shows no droplet amplification or flattening. Figure 2 D and Figure 2 E). These experimental results indicate that the surfactants produced by isolated Lactobacillus salivarius have the property of reducing surface tension, and the surface tension-reducing effect of surfactants is concentration-dependent.

[0085] (4) The prepared biosurfactant has antibacterial activity.

[0086] Different concentrations of surfactant were co-cultured with common clinical pathogens, *Escherichia coli* and *Streptococcus suis*, for 24 hours, followed by colony counting. Results are as follows: Figure 3 As shown, when the concentration of BS was 50 mg / mL to 6.25 mg / mL, the inhibition rate against porcine Escherichia coli was 95.06% to 46.01% (13 / 263 to 142 / 263), which was significantly different from the control group of porcine Escherichia coli without BS (P<0.001); when the concentration of BS was 3.125 mg / mL, the inhibition rate against porcine Escherichia coli was 22.81% (203 / 263), which was significantly different from the control group of porcine Escherichia coli (P<0.05). When the concentration of BS was 50 mg / mL to 12.5 mg / mL, the inhibition rate against *Streptococcus suis* was 81.64%–49.76% (38 / 207–104 / 207), which was significantly different from the control group without BS (P<0.01). However, when the concentration of BS was 6.25 mg / mL and 3.125 mg / mL, the inhibition rates against *Streptococcus suis* were 41.06% (122 / 207) and 38.65% (127 / 207), respectively, which were significantly different from the control group (P<0.05). When the concentration of BS was 1.56 mg / mL, there was no significant inhibitory effect on *Escherichia coli* or *Streptococcus suis* (P>0.05). These experimental results indicate that BS extracted from *Lactobacillus salivarius* isolates has a significant inhibitory effect on *Escherichia coli* and *Streptococcus suis*, and this inhibitory effect is dose-dependent.

[0087] (5) The prepared biosurfactant has anti-PCV2 activity.

[0088] Different concentrations of BS were co-cultured with PK-15 cells for 24 hours. The CC of BS was calculated based on the nonlinear fitting curve of BS concentration versus cell viability. 50 It is 24.73 mg / mL ( Figure 4 Then, PK-15 cells were infected after co-incubating PCV2 with 12.5 or 6.25 mg / mL BS for 1 h. PCV2 antigen was detected using IFA 36 h post-infection. It was observed that the intracellular PCV2 antigen-specific fluorescence signal was significantly weaker than the fluorescence signal in the virus control cells. Figure 5 ).

[0089] A mixture of 6.25 or 3.125 mg / mL BS and PCV2, co-incubated for 1 h, was seeded into PK-15 cells. PCV2 levels were detected by Western blot at different post-infection time points. At 24 h post-infection, BS significantly inhibited PCV2; however, this inhibitory effect weakened significantly between 48 and 72 h post-infection, and higher BS concentrations showed stronger inhibitory effects on the virus. Figure 6 ).

[0090] (6) Lactobacillus salivarius has anti-PCV2 activity

[0091] IPEC-J2 cells were treated with *Lactobacillus salivarius* for 6, 12, 18, and 24 hours, then infected with PCV2 and cultured for another 72 hours. The PCV2 nucleic acid level was detected by qPCR. Figure 7 As shown, compared with the positive control (PC), IPEC-J2 cells treated with *Lactobacillus salivarius* or *Lactobacillus reuteri* showed a significant decrease in viral nucleic acid levels after PCV2 infection (P < 0.05). *Lactobacillus salivarius* exhibited a stronger inhibitory effect on PCV2 compared to *Lactobacillus reuteri*. These results indicate that *Lactobacillus salivarius* has a significant inhibitory effect on PCV2 proliferation, and its inhibitory effect is higher than that of the probiotic *Lactobacillus reuteri*.

[0092] Through half-cytotoxicity (CC) 50 The experiment tested the toxicity of inactivated Lactobacillus salivarius or Lactobacillus reuteri to IPEC-J2 cells, and the results were as follows: Figure 8 As shown, when 5×10 6 When Lactobacillus salivarius and Lactobacillus reuteri were inoculated onto IPEC-J2 at CFU / mL, cell growth was not affected, indicating that the Lactobacillus salivarius isolated by this invention has good safety.

[0093] Probiotics mainly consist of bacteria belonging to the genera *Lactobacillus*, *Lactococcus*, *Bifidobacterium*, *Bacillus*, *Streptococcus*, *Propionibacteria*, and *Pediococcus*, as well as some yeasts. They play an important role in maintaining the body's microbial balance, nutrient absorption, and immune function. *Lactobacillus* is one of the most studied probiotics. By producing biosurfactants, lactic acid, hydrogen peroxide, bacteriocins, and bacteriocin-like substances, it can regulate the intestinal microecological balance, inhibiting the reproduction of pathogenic microorganisms, resisting stress, improving intestinal barrier function, and enhancing immunity. Adding lactic acid bacteria to feed can improve the host's immune function and promote growth. L. salivarius is an important probiotic that secretes organic acids such as phenyllactic acid and clavaminic acid, as well as antibiotics and bacteriocins. It has inhibitory effects on pathogens such as Escherichia coli, Streptococcus, and Staphylococcus aureus, as well as herpesviruses, rotaviruses, and HIV. Oral administration of L. salivarius is safe and has no toxic side effects. It has a good therapeutic effect on human mastitis, and the inflammation is less likely to recur. Oral administration of L. salivarius to piglets can relieve stress, improve immune function, regulate the intestinal flora of weaned piglets, reduce diarrhea, and promote growth.

[0094] Porcine circovirus type 2 (PCV2) primarily damages the immune organs of pigs, causing immunosuppression or immune dysfunction. PCV2 can cause a variety of clinical symptoms, including post-weaning multisystemic wasting syndrome, dermatitis-nephropathy syndrome, reproductive disorders, respiratory disease syndrome, and diarrhea syndrome; these diseases are collectively known as porcine circovirus-associated diseases (PCV2-associated diseases, PCVD). PCV2-positive pig herds have increased susceptibility and higher rates of secondary infections by bacteria such as Streptococcus, Escherichia coli, and Haemophilus parasuis, leading to increased drug costs and mortality, thus exacerbating the direct and indirect economic losses caused by PCV2 to pig farms. Although subunit or inactivated vaccines are currently available for PCV2 prevention, PCVD still occurs frequently. Improving herd immunity and reducing susceptibility and secondary bacterial infections are crucial measures for controlling PCVD and improving the economic efficiency of pig farms. Previous experiments have shown that the proportion of Lactobacillus salivarius in the small intestine of PCV2-infected pigs increases, suggesting that L. salivarius is beneficial for the anti-infection effect of PCV2-infected pigs. Therefore, this invention attempts to isolate Lactobacillus salivarius from the small intestine of PCV2-infected pigs for clinical use, aiming to improve the immune function of pigs and promote the health of the pig herd.

[0095] The probiotic effect of lactobacilli is closely related to their source, and even exhibits strain specificity; that is, lactobacilli that have a probiotic effect on one animal may not have a probiotic effect on other animals. To provide beneficial effects to animals, lactic acid bacteria must be isolated from their homologous sites, such as the gastrointestinal environment, to facilitate their survival and colonization of the gastrointestinal mucosa. The *Lactobacillus salivarius* isolated in this invention originated from the ileum of piglets. This homologous isolation strategy helps ensure the colonization of this strain in the pig intestine, thereby enabling it to exert its probiotic effect.

[0096] The production of biosurfactants is one of the key characteristics of probiotics in exerting their beneficial effects. Bacteria, yeasts, and fungi can all produce biosurfactants, with bacteria being the primary producers. *Acinetobacter*, *Bacillus*, and *Arthrobacter* are the most frequently reported producers of biosurfactants. However, due to the pathogenicity of some bacteria, the application of their produced surfactants in the food industry and medical fields is limited. Biosurfactants produced by probiotics, primarily lactic acid bacteria, have become a focus of attention due to their safety. Biosurfactants not only possess activities such as reducing surface tension, solubilizing, and inhibiting pathogenic microorganisms, but also have advantages such as easy degradation, non-toxicity, wide applicability, and high bioactivity under extreme temperatures, pH values, and salt concentrations. Therefore, biosurfactants have broad application prospects in the food industry, agriculture, medicine, marine ecosystem protection, and oil extraction. It is known that biosurfactants have anti-inflammatory and pathogenic microorganism-inhibiting activities. Existing studies have shown that biosurfactants have strong inhibitory activity against a variety of Gram-negative and Gram-positive bacteria, and also have a significant inhibitory effect on multidrug-resistant bacteria. Similarly, biosurfactants also exhibit significant inhibitory effects against various viruses, including HIV, HSV-1, and NDV. These findings suggest that biosurfactants are promising biotherapeutic agents and potential alternatives to antimicrobial drugs. Clinical trials in multiple countries have demonstrated that biosurfactants can be used as antiviral agents for acute respiratory distress syndrome (ARDS). Currently, bacterial and viral infectious diseases are major causes of economic losses in pig farms, necessitating the isolation of porcine probiotics and the preparation and evaluation of their antimicrobial activity against surfactants.

[0097] Reducing surface tension is one of the key activities of surfactants. This activity can alter the surface properties of pathogenic microorganisms, inhibiting their biofilm formation and adhesion, thereby achieving antimicrobial function. The surfactant extracted from *L. salivarius* in this invention exhibits the ability to alter interfacial surface tension in oil diffusion and droplet collapse experiments, and the effect of reducing surface tension increases with increasing surfactant concentration. These findings suggest that *Lactobacillus salivarius*-derived biosurfactants have great potential as an effective antibiotic alternative for controlling porcine pathogens.

[0098] Immunosuppression and secondary infection are key characteristics of PCV2 infection. Therefore, this invention selected common pathogenic porcine Escherichia coli and Streptococcus suis from pig farms to evaluate the antimicrobial efficacy of surfactants produced by porcine L. salivarius isolates. This invention found that the surfactants produced by porcine L. salivarius isolates exhibited inhibition rates of 95.06% and 81.80% against porcine Escherichia coli and Streptococcus suis, respectively, and also showed significant inhibitory effects on PCV2.

[0099] This invention identified a strain of *L. salivarius* from the ileum of PCV2-infected piglets. The biosurfactant produced by this strain significantly inhibited the proliferation of *Escherichia coli*, *Streptococcus*, and porcine circovirus. These findings lay an important foundation for further research and development of *L. salivarius* and related products.

[0100] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0101] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0102] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A Lactobacillus salivarius of porcine intestinal tract with antimicrobial activity, named pig-LS2023, deposited in China Center for Type Culture Collection, Wuhan, Wuhan University, China, with the microbial accession number CCTCC No: M 20251406 on June 17, 2025. L. salivarius ​ 2. The use of the Lactobacillus salivarius of claim 1 in the preparation of a medicine for resisting animal intestinal infection. The animal intestinal infection is at least one of Escherichia coli, Streptococcus, and porcine circovirus type 2 (PCV2) infection, and the animal is a pig.

3. A method for preparing a surfactant, characterized by, The method comprises the following steps: (1) inoculate the Lactobacillus salivarius of claim 1 in MRS broth, and incubate at 37°C for 48 h, then centrifuge to collect the bacterial cells; (2) wash the bacterial cells obtained in step (1) with sterilized deionized water, suspend the bacterial cells in PBS buffer, and incubate at room temperature for 2 h under stirring to release the biosurfactant, centrifuge to remove the cells, filter with a filter, dry the supernatant with a rotary evaporator to obtain an extract; (3) suspend the extract in PBS to a concentration of 10 mg / mL, adjust the pH to 2.0 with HCl, then centrifuge to collect the precipitate, wash the precipitate with sterile distilled water, dissolve the precipitate in sterile distilled water, adjust the pH to 7.0 with NaOH, filter with a filter membrane, and freeze-dry.

4. The use of the biosurfactant prepared in claim 3 in the preparation of an antibacterial preparation, which is a preparation for resisting Escherichia coli, Streptococcus, or porcine circovirus type 2 (PCV2) infection in pigs.

5. Use according to claim 4, characterized in that, The antibacterial preparation comprises a controlled-release preparation.

6. Use according to claim 5, characterized in that, The controlled-release preparation comprises a solid controlled-release preparation, which is a controlled-release pellet or a controlled-release tablet, and a liquid controlled-release preparation, which comprises nano-liposomes, controlled-release microspheres, or controlled-release emulsions.

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