Isolation of porcine intestinal Lactobacillus salivarius and antimicrobial potential of its biosurfactant

Isolation of porcine intestinal Lactobacillus salivarius and antimicrobial potential of its biosurfactant | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Isolation of porcine intestinal Lactobacillus salivarius and antimicrobial potential of its biosurfactant Xiaotian Zhou, Jing Ren, Chen Yuan, Feiyan Wang, Ligong Chen, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6524903/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Aug, 2025 Read the published version in BMC Veterinary Research → Version 1 posted 13 You are reading this latest preprint version Abstract Background Lactobacillus salivarius ( L. salivarius ) is a probiotic widely colonized in the gastrointestinal tracts of humans and animals. Its biosurfactant (BS) exhibits antibacterial and antiviral activities, along with advantages such as biodegradability, non-toxicity, and stability. Porcine circovirus type 2 (PCV2) is a major pathogen in swine farms, causing immunosuppression or immune dysfunction. Elevated susceptibility in PCV2-positive herds is accompanied by severe secondary bacterial infections. To explore novel antimicrobial biologics for enhancing swine immunity, this study isolated a biosurfactant-producing Lactobacillus strain from the intestines of PCV2-infected piglets using the calcium carbonate plate method, and analyzed the anti-microbial activity of the biosurfactant. Results A strain of biosurfactant-producing L. salivarius was successfully isolated from the ileum of PCV2-infected piglets. The extracted BS from the isolate demonstrated surface tension-reducing properties, forming spreading zones or dispersions on vegetable oil. The antimicrobial experiment showed that at the concentrations of 6.25–50 mg/mL, BS inhibited the growth of porcine E. coli and S. suis by 46.13% − 95.06% and 46.13% − 95.06%, respectively. Furthermore, BS of 6.25–12.5 mg/mL significantly inhibited PCV2 replication in PK-15 cells. These findings suggest that L. salivarius is a promising probiotic candidate for improving swine immune status and developing novel alternatives to conventional antimicrobial agents. Lactobacillus salivarius Isolation and identification Biosurfactants Antimicrobial activity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Antimicrobial agents play a crucial role in safeguarding the health of humans and animals, improving the economic benefits of farming, and ensuring food safety. With the rapid development of the economy and the increasing intensification of animal farming, the use of antibiotics has continued to rise. However, this has led to multiple challenges, including antimicrobial resistance (AMR), food safety issues, and environmental pollution [1, 2]. The World Health Organization (WHO) has listed AMR as one of the major threats to human health in the 21st century. As a result, the search for safe, effective, and alternatives to antibiotics has become a focal point in the development of new therapeutic approaches. Lactic Acid Bacteria (LAB), as probiotics, are important members of the normal microbial flora in specific regions of humans and animals, such as the oral cavity and intestines. By producing organic acids, specific enzymes, and bacteriocins, etc., LAB regulate intestinal microecological balance, inhibit the proliferation of pathogenic microorganisms, and enhance host immunity, and exhibit multiple activities, including antiviral, antitumor, and antistress, as well as the ability to improve intestinal barrier function [3–5]. Therefore, LAB are considered ideal alternatives to antibiotics [6]. Additionally, L. salivarius , as an important member of LAB, is widely present in the gastrointestinal mucosa of humans and animals, and can produce biosurfactant, various bacteriocins, antibiotics, antioxidants, and immunomodulators. These substances enable L. salivarius to balance and improve gut microbiota, combat microbial infections, regulate mucosal immunity, and enhance production performance [7, 8]. L. salivarius has emerged as a potential candidate for anti-infection therapy and immune function improvement. Biosurfactants (BS) are natural active substances produced by microorganisms, featuring an amphiphilic structure. They possess various biological activities, including antimicrobial, antiviral, and anti-adhesive properties. Additionally, BS offer advantages such as excellent biodegradability, low toxicity, and high stability [9, 10]. Therefore, biosurfactants are considered as potential candidates for replacing antibiotics. Compared to those produced by pathogenic bacteria, biosurfactants derived from probiotics, known as probiotic biosurfactants, are safer and have broad application prospects in the food, pharmaceutical, and agricultural industries [9–11]. Porcine circovirus type 2 (PCV2), a 17-nm diameter DNA virus belonging to the genus Circovirus of the family Circoviridae , is characterized by damaging porcine immune organs, causing immunosuppression and multisystemic diseases, and poses a serious threat to the development and economic benefits of pig farms [12, 13]. Common secondary or synergistic pathogens in PCV2-infected pigs include S. suis , E. coli , Pasteurella multocida , and Glaesserella parasuis , etc. The co-infections or secondary infections with these pathogens significantly exacerbate the pathogenic effects of PCV2 [14]. Consequently, PCV2 and associated secondary pathogens have become key targets for disease control in pig operations, making the development of effective preventive and therapeutic agents of paramount importance. In this study, a strain of L. salivarius was isolated from the ileum of piglets experimentally infected with PCV2, and the physicochemical properties and antimicrobial activity of the L. salivarius -derived biosurfactants were investigated, aiming to provide a scientific basis for promoting the application of probiotics and developing probiotic-based therapies and antibiotic alternatives. 2. Materials and methods 2.1. Bacterial or Viral Strains, and Cells The E. coli O141 strain and the S. suis SJZ2021 strain were preserved in our laboratory, the Laboratory of Animal Infectious Diseases, College of Veterinary Medicine, Hebei Agricultural University, Baoding, China. L. reuteri was purchased from Beijing Bio-BW Biotechnology Co., Ltd., Beijing, China. The PCV2 HBDX2018 strain (GenBank ID: MK585076), with a TCID 50 of 10 6.1 /mL, was preserved in our laboratory. PK-15 cells were cultured in RPMI-1640 medium (Sigma, USA) supplemented with 10% fetal bovine serum (FBS, Wisent Biotechnology, Nanjing, China) and 100 IU/mL each of penicillin and streptomycin. 2.2. Animal Infection and Sample Collection Three 28-day-old healthy piglets were purchased from Hebei Tangsheng Animal Husbandry Co., Ltd. (Baoding, China). The piglets tested negative for PCV2 nucleic acid and specific antibodies by PCR and ELISA. The piglets were infected with the PCV2 HBDX2018 strain via intranasal and intramuscular routes at a dose of 10 6.1 TCID 50 per piglet. Seven days post-infection, the piglets were anesthetized by intravenous injection of xylazine hydrochloride, and the ileum along with contents were aseptically collected for bacterial isolation and culture. This study was approved by the Animal Welfare and Ethics Committee of Hebei Agricultural University (China) (License No. 1820026). All experimental procedures were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals of Hebei Agricultural University and China. 2.3. Bacterial Isolation The ileal mucosa and intestinal contents were scraped using a sterile glass slide and transferred into a tube containing an equal volume of pH7.4 0.01 mol/L phosphate-buffered saline (PBS). The mixture was incubated anaerobically at 37℃ with shaking for 5 hours to enrich the bacteria. A 100 µL aliquot of the enriched bacterial suspension was serially diluted from 10 − 1 to 10 − 5 . After vortex mixing, 10 µL of each diluted suspension was spread evenly onto the surface of DeMan, Rogosa and Sharpe (MRS) solid medium (Luqiao Technology Co., Ltd., Beijing, China) and incubated anaerobically at 37℃ for 24 hours. Single colony exhibiting smooth, raised, and well-defined edges with a milky-white appearance were selected and streaked onto 1% CaCO 3 -MRS solid medium, followed by anaerobic incubation at 37℃ for 24 hours. Colonies showing clear calcium dissolution zones were selected and subjected to three consecutive rounds of purification to obtain pure strains. The purified strains were Gram-stained and examined under a microscope until all observed bacteria were Gram-positive rods. The isolated bacteria were then suspended in a preservation solution (60% glycerol in LB medium) and stored at -20℃. 2.4. 16S rDNA Amplification and Sequence Analysis Total DNA was extracted from the bacterial strain using the boiling lysis method. Briefly, 1.5 mL of bacterial culture was centrifuged at 8,000 r/min for 5 minutes. The bacterial pellet was resuspended in 500 µL of sterile water, boiled for 10 minutes, and centrifuged at 12,000 r/min for 10 minutes. The supernatant was collected as the DNA template for PCR amplification of the 16S rDNA sequence. The PCR reaction mixture (20 µL) consisted of 10 µL of 2× Es Taq Master Mix (Kangwei Century Biotechnology Co., Ltd., Jiangsu, China), 0.5 µL each of forward and reverse primers (25 µmol/L), 2 µL of DNA template, and 7 µL of ddH 2 O. The PCR conditions were as follows: initial denaturation at 94℃ for 3 minutes; 30 cycles of 94℃ for 45 seconds, 52℃ for 1 minute, and 72℃ for 1 minute; and a final extension at 72℃ for 10 minutes. The universal primers used for 16S rDNA amplification were F: 5ʹ-AGAGTTTGATCCTGGCTCAG-3ʹ and R: 5ʹ-GGTTACCTTGTTACGACTT-3ʹ. The PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The obtained sequences were compared with the reference sequences in the GenBank nucleotide database for homology analysis. A phylogenetic tree was constructed using the Neighbor-Joining (NJ) method in MEGA 7.0 software. 2.5. Determination of Growth Curve A single colony was inoculated into MRS broth and anaerobically cultured at 37℃ for 14 hours to prepare the seed culture. The seed culture was then inoculated into fresh MRS broth at a 1∶100 ratio and incubated under the same conditions. Samples were collected at 0, 8, 12, 14, 16, 18, and 24 hours, serially diluted, and used for colony counting. A triplicate was performed for each dilution. The bacterial concentration (colony-forming units per milliliter, CFU/mL) was calculated based on the colony counts. 2.6. Identification of Physicochemical Properties of the Isolate The biochemical characteristics of the isolate were identified by Bacterial identification micro-biochemical reaction tube (Hangzhou Binhe Microbial Reagent Co., Ltd., Hangzhou, China), and a standard Lactobacillus ( L. reuteri , Beijing Bio-BW Biotechnology Co., Ltd., Beijing, China) control was established simultaneously. The purified bacteria were inoculated into microtubes containing maltose, mannitol, sorbitol, sucrose, raffinose, lactose, peptone, and hydrogen sulfide according to the manufacturer’s instructions. The tubes then were anaerobically incubated at 37℃ for 24 hours, and the performance of the isolate in acid production and the generation of indole and hydrogen sulfide were observed. To clarify motility, the isolates were stab-inoculated into semi-solid (Hangzhou Binhe Microbial Reagent Co., Ltd., China) and anaerobically incubated at 37℃ for 24 hours. The motility of the bacteria was judged based on the spread of growth along the puncture line. In addition, after puncture inoculation, the gelatin cultures were placed in a refrigerator for 2 hours at 4℃ to observe the solidification of gelatin, which indicated whether the isolate produced gelatinase to liquefy gelatin. The catalase test was conducted furtherly. The colonies were picked up and placed on a clean glass slide and 3% H 2 O 2 were added. After dropping 3% H 2 O 2 , the production of abundant bubbles within 30 seconds indicated the isolate could generate catalase. Conversely, catalase was unable to be produce. 2.7. Detection of Biosurfactants Production To detect biosurfactant production, the isolated bacteria were inoculated into 300 mL of MRS broth at a 1∶100 ratio and cultured at 37℃ for 48 hours. The bacterial cells were harvested by centrifugation at 8000 r/min for 5 minutes at 4℃. The cells were washed twice with sterile deionized water and resuspended in 50 mL of pH 7.0 0.01 mol/L PBS. The suspension was gently stirred and incubated at room temperature for 2 hours to release the biosurfactants. After centrifugation to remove the cells, the supernatant was filtered through a membrane filter with pore size of 0.22 µm (Jinteng Experimental Equipment Co., Ltd., Tianjin, China). The filtrate was dried using a rotary evaporator (RE-52AA, Shanghai Yarong, China), and the dried extract was resuspended in PBS to a concentration of 10 mg/mL. The pH of the solution was adjusted to 2.0 using 1 mol/L HCl and incubated at 4℃ for 2 hours. The precipitate was collected by centrifugation at 9,000 r/min for 15 minutes at 4℃ and washed twice with sterile distilled water (pH 2.0). The precipitate was then dissolved in sterile distilled water, adjusted to pH 7.0 using 1 mol/L NaOH, filtered, and freeze-dried for the measurement of biosurfactant. Oil spreading test was implemented for measurement of the biosurfactant produced by the isolated bacteria. Briefly, one drop of vegetable oil was added to distilled water, followed by one drop of the prepared biosurfactant solution (100 mg/mL) onto the oil surface. One drop of distilled water was simultaneously applied to the oil surface as a negative control. The diameter of the oil displacement zone was measured to evaluate the surface tension-reducing activity of the prepared biosurfactant. Furthermore, the oil displacement zone diameters were measured for biosurfactant solutions at different concentrations (0, 50, 100, 150, and 200 mg/mL), and a standard curve was plotted to analyze the relationship between biosurfactant concentration and oil displacement zone diameter. Oil drop-collapse test was also employed to test the biosurfactant production. A drop of the prepared biosurfactant was applied to vegetable oil surface, and the spreading or collapse of the biosurfactant drop was observed after 2 minutes. A negative control of adding a drop of water on the vegetable oil surface was set up at the same time. If the solution contained biosurfactants, the interfacial tension between the droplet and the hydrophobic surface of the vegetable oil would be reduced, causing the droplet to spread and became flat. In contrast, if the solution lacked biosurfactants, the polar water molecules would be repelled from the hydrophobic surface, and the droplet would remain stable to form bead. 2.8. Examination of Antimicrobial Activity of Biosurfactants 2.8.1. Examination of Antibacterial Activity The antibacterial activity of the prepared biosurfactant, the common pathogenic bacteria E. coli and S. suis used as models, was determined using a modified microdilution method. Briefly, in a 96-well cell culture plate, 200 µL of sterile nutrient broth was added to the first well as a blank control. In wells 2 to 11, 100 µL of serially diluted biosurfactant was added to make final concentrations ranging from 50 mg/mL to 0.1 mg/mL, followed by the addition of 100 µL of E. coli or S. suis culture ( OD 600 = 0.5) to each well. The 12th well contained 100 µL of nutrient broth and an equal volume of bacterial culture as a bacterial control. Each treatment was performed in triplicate. The plate was incubated anaerobically at 37℃ for 24 hours. After incubation, the cultures from wells containing different concentrations of biosurfactants were subjected to colony counting to analyze the antibacterial activity of the biosurfactants. 2.8.2. Measurement of Antiviral Activity Using PCV2 as the model, the antiviral activity of the biosurfactants produced by L. salivarius was detected. Measurement of 50% Cytotoxic Concentration (CC 50 ) of biosurfactant was conducted firstly prior to determination of antiviral activity. The biosurfactant was diluted to concentrations of 6.25, 12.5, 25, 50, 100, and 200 mg/mL using RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS). The diluted biosurfactant were added to a 96-well cell culture plate at 50 µL per well. Then, 50 µL of PK-15 cell suspension (2 × 10 4 cells) was added to each well. Each concentration was tested in triplicate. Meanwhile, the cell control without biosurfactant and the medium control with medium-only without cells were established. The plate was placed at 37℃ in a 5% CO 2 incubator for 24 hours. According to the manufacturer’s instructions of the Cell Counting Kit-8 (CCK-8) (APExBIO, America), 10 µL of CCK-8 was put into each well. The plate was gently mixed on a microplate shaker (MH-2, QILNBEIER, Haimen, China) for 30 seconds and then incubated at 37℃ in a 5% CO 2 incubator for 1 hour. The optical density ( OD ) at 450 nm was measured using a microplate reader (Synergy HTX, Gene Company Limited, USA) and the cell viability was calculated based on the following formula (1). The CC 50 value was calculated according a nonlinear regression curve of BS concentration versus cell viability plotted using GraphPad Prism 8.0 software. Then Immunofluorescence assay (IFA) and Western blot were employed for analysis of anti-PCV2 activity of the biosurfactant (BS). \(\:\text{C}\text{e}\text{l}\text{l}\:\text{v}\text{i}\text{a}\text{b}\text{i}\text{l}\text{i}\text{t}\text{y}\:\left(\text{\%}\right)=\frac{OD\left(\text{B}\text{S}\:\text{w}\text{e}\text{l}\text{l}\right)-\:OD\left(\text{m}\text{e}\text{d}\text{i}\text{u}\text{m}\:\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}\right)}{OD\left(\text{c}\text{e}\text{l}\text{l}\:\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}\right)-\:OD\left(\text{m}\text{e}\text{d}\text{i}\text{u}\text{m}\:\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}\right)}\) ×100% (1) Immunofluorescence assay (IFA): PCV2 at a dose of 0.2 multiplicity of infection (MOI) was mixed with 12.5 mg/mL or 6.25 mg/mL of BS or an equal volume of RPMI-1640 medium supplemented with 3% FBS. The mixtures were incubated at 37℃ in a 5% CO 2 incubator for 1 hour. PK-15 cells in the logarithmic growth phase were digested with trypsin, centrifuged, and resuspended in medium. 100 µL cells were seeded into a 96-well cell culture plate containing 5 × 10 4 cells per well. The cells were divided into four groups: Blank control (NC), Positive control (PC), BS-12.5 group, and BS-6.25 group, with three replicates per group. NC group: 100 µL of RPMI-1640 medium containing 3% FBS was added to each well. PC group: 100 µL of pre-mixed solution containing RPMI-1640 medium and PCV2 was added to each well. BS-12.5 group and BS-6.25 group: 100 µL incubated mixture of PCV2 and 12.5 mg/mL or 6.25 mg/mL BS, respectively, was put into each well. The plate was incubated at 37℃ in a CO 2 incubator for 36 hours. After incubation, the cells were gently washed with PBS, and 50 µL of pre-cooled methanol was added to each well for fixation at -20℃ for 10 minutes. The cells were washed again, and 100 µL of 2% BSA-PBS was added to each well, followed by incubation at 37℃ for 1 hour. Subsequently, 50 µL of a 1∶500 dilution of PCV2 monoclonal antibody (prepared by the Laboratory of Animal Infectious Diseases, Hebei Agricultural University) was added to each well and incubated at 37℃ for 1 hour. After washing, 50 µL of a 1∶500 dilution of YF®594 goat anti-mouse IgG (Uelandy Biotechnology Co., Ltd., Suzhou, China) was added to each well and incubated at 37℃ for 45 minutes. The cells were washed again, and the cellular nucleus were stained with Bisbenzimide. Red fluorescence, indicating the specific binding of the monoclonal antibody to PCV2 antigen, was observed under a fluorescence microscope. Western blot: 1 mL mixture of 0.2 MOI PCV2 and 6.25 mg/mL or 3.125 mg/mL biosurfactant (BS) was added to each well of a 6-well cell culture plate, with three replicates per condition. The plate was incubated at 37℃ in a 5% CO 2 incubator for 1 hour. Controls included a viral control (Positive control, PC) without BS and a cell control (Negative control, NC). One milliliter of 1 × 10 6 PK-15 cells in the logarithmic growth phase was added to each well of above different treatments. The plates were transferred to a 5% CO 2 incubator at 37℃ for 24, 48, and 72 hours, and the cells were collected for Western blot analysis to detect viral protein and evaluate the inhibitory effect of BS on PCV2. For the Western blot assay, the PCV2 monoclonal antibody was diluted at 1∶50, and HRP-conjugated goat anti-mouse IgG (Biodragon Biotechnology Co., Ltd., Suzhou, China) was diluted at 1∶10,000. Chemiluminescent substrate (Yeasen Biotechnology Co., Ltd., Shanghai, China) was used for signal detection. 2.9. Statistical Analysis All data were presented as means ± standard deviations (SD) and statistical analysis were performed using an unpaired two-tailed Student’s t-test in GraphPad Prism 8.0. Graphs were generated using GraphPad Prism 8.0, and asterisk signs in the Figure indicate statistical significance (* P < 0.05, ** P < 0.01, and *** P < 0.001). 3. Results 3.1. Culture and Morphological Characteristics, 16S rDNA Analysis, and Growth Curve of the Isolated Strain The isolated strain was streaked onto MRS solid medium and incubated at 37°C for 24 h, forming medium-sized, convex, moist, and smooth-edged milky-white colonies. When cultured on 1% CaCO₃-MRS solid medium for 24 h, a clear calcium-dissolving zone was observed around the colonies (Fig. 1 A). Gram staining revealed Gram-positive, short rod-shaped bacteria, occurring in pairs or short chains under microscopy (Fig. 1 B). These cultural and morphological characteristics were consistent with those of Lactobacillus . The isolated strain was statically cultured at 37°C, and samples were collected every 2 h for colony counting. The results showed that the bacterial count peaked at 12 h, indicating that the logarithmic growth phase of this strain lasted 12 h (Fig. 1 C). For further identification, the bacterial 16S rDNA sequence was amplified by PCR. Agarose gel electrophoresis confirmed an amplified fragment of approximately 1400 bp (Fig. 1 D). Sequencing and alignment analysis revealed 99–100% homology with L. salivarius , including 100% homology with L. salivarius strain LGM8-7 (MG674666.1) from swine. In contrast, homology with L. johnsonii and L. reuteri was only 25–27%. Phylogenetic analysis using the Neighbor-Joining method in MEGA7.0 demonstrated that the isolated strain clustered within the L. salivarius branch, forming a subclade with L. salivarius LGM8-7, while L. johnsonii and L. reuteri were located in distinct branches (Fig. 1 E). These results confirmed that the isolated strain was L. salivarius , designated as pig-SJZ2023. 3.2. Physicochemical Characteristics of the Isolated strain The physicochemical characterization results of the isolated strain pig-SJZ2023 are presented in Table 1 . After anaerobic incubation at 37°C for 24 h in various culture media including maltose, mannitol, sorbitol, sucrose, raffinose, lactose, indole, and hydrogen sulfide, the color change of six carbohydrate fermentation tubes from purple to yellow indicated acid production capability. No color alterations were observed in either indole or hydrogen sulfide media, demonstrating the strain's inability to produce indole or hydrogen sulfide. The catalase test, performed by applying 3% hydrogen peroxide (H 2 O 2 ) to bacterial colonies, yielded no observable bubble formation, confirming the absence of catalase activity. In gelatin stab culture assays, bacterial growth was strictly confined to the inoculation line without any radial expansion or gelatin liquefaction, indicating both non-motile behavior and lack of gelatinolytic activity. These collective biochemical characteristics exhibit complete consistency with the standard phenotypic profile of Lactobacillus species, thereby providing additional validation for the taxonomic classification of strain pig-SJZ2023. Table 1 Physiological and biochemical characteristics of L. salivarius. pig-SJZ2023: the isolated strain; Item Lactobacillus Standard strain[15] pig-SJZ2023 Morphology Bacillus Bacillus Gram staining + + Moveability − − Gelatin liquefaction − − Sucrose + + Cottonseed sugar + + Lactose + + Maltose + + Mannitol + + Sorbitol + + Catalase − − Indole − − H 2 S − − * "+": positive; "−": negative. 3.3. Biosurfactant production ability of the isolated strain When the extract of L. salivarius isolates was dropped onto the surface of vegetable oil in water, an oil-displacement circle with a diameter of 2.7 cm was produced, while no such circle was observed in the water control (Fig. 2 A and 2 B). This result indicated that the isolated L. salivarius was capable of biosurfactant production. Furtherly, the diameter of the oil displacement zone increased along with rising biosurfactant concentrations, demonstrating a strong linear correlation (R 2 = 0.99) (Fig. 2 C). Meanwhile, upon application of the biosurfactant to the vegetable oil surface, immediate droplet spreading and flattening were observed (Fig. 2 E and 2 E). In contrast, the water control showed neither droplet expansion nor surface flattening. These results confirmed that the biosurfactant produced by the isolated L. salivarius strain possesses significant surface tension-reducing properties, which the activity exhibited clear concentration dependence. 3.4. Antimicrobial Activity of the Prepared Biosurfactant Different concentrations of the biosurfactant were co-cultured with E. coli or S. suis for 24 h, followed by colony counting. As shown in Fig. 3 , when the biosurfactant concentration ranged from 50 mg/mL to 6.25 mg/mL, the inhibition rates against E. coli were 95.06–46.13% (13/263–142/263), showing significant differences compared to the E. coli control without the biosurfactant ( P < 0.001). At a concentration of 3.125 mg/mL, the inhibition rate of biosurfactant against E. coli was 22.69% (203/263), an obviously difference compared to the control ( P < 0.05) (Fig. 3 A). For S. suis , when the concentrations of biosurfactant ranged from 50 mg/mL to 12.5 mg/mL, the inhibition rates were 81.80–49.60% (38/207–104/207), showing significant differences compared to the S. suis control without biosurfactants ( P < 0.01). When the concentration being 6.25 mg/mL and 3.125 mg/mL, the inhibition rates of the biosurfactant against S. suis were 41.06% (122/207) and 38.49% (127/207) respectively, showing differences compared to the control ( P 0.05). These experimental results demonstrated that the biosurfactant extracted from L. salivarius isolates has stark inhibitory effects against both E. coli and S. suis , with the inhibitory effect being dose-dependent. 3.5. Anti-PCV2 Activity of the Prepared Biosurfactant Different concentrations of biosurfactants were co-cultured with PK-15 cells for 24 hours. Based on the nonlinear regression curve of biosurfactants concentration versus cell viability, the CC 50 of biosurfactants was calculated to be 24.73 mg/mL (Fig. 4 ). Subsequently, when 12.5 or 6.25 mg/mL of biosurfactants was co-incubated with PCV2 for 1 hour before infecting PK-15 cells, immunofluorescence assay (IFA) at 36 hours post-infection revealed distinctly weaker PCV2 antigen-specific fluorescence signals in treated cells compared to the viral control cells (Fig. 5 ). The mixture obtained after incubating biosurfactants at 6.25 or 3.125 mg/mL with PCV2 for 1 hour was inoculated into PK-15 cells, and the proliferation of PCV2 was analyzed by Western blot at different times after infection. At 24 hours post-infection, there was no or almost no specific reaction band visible on the polyvinylidene fluoride (PVDF) membrane, which shown that the biosurfactant displayed distinct inhibitory effects on PCV2 proliferation. However, the antiviral effects of the biosurfactant markedly weakened between 48–72 hours post-infection, which the antiviral activity was positively correlated with the concentration of the biosurfactant (Fig. 6 ). 4. Discussion Probiotics primarily consist of bacteria from the genera Lactobacillus , Lactococcus , Bifidobacterium , Bacillus , Streptococcus , Propionibacteria , and Pediococcus , along with certain yeasts. These microorganisms play crucial roles in maintaining microbial balance, nutrient absorption, and immune function [16–18]. Among these, Lactobacillus spp. have been extensively studied. Through the production of biosurfactants, lactic acid, hydrogen peroxide, bacteriocins, and bacteriocin-like substances, they regulate intestinal microecological balance, inhibit pathogenic microorganism proliferation, mitigate stress responses, enhance intestinal barrier function, and improve immunity [19, 20]. Dietary or feed supplementation with lactic acid bacteria has been shown to enhance host immune function and promote growth [21]. L. salivarius , one of the particularly important probiotics, is capable of secreting organic acids (e.g., phenyllactic acid, clavaminic acid), antibiotics, and bacteriocins. These metabolites exhibit inhibitory effects against pathogens such as E. coli , Streptococcus , and Staphylococcus aureus, as well as viruses including herpesvirus, rotavirus, and Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) [8, 22–24]. Oral administration of L. salivarius is safe, non-toxic, and effective in treating mastitis in humans with low recurrence rates [25, 26]. In piglets, L. salivarius supplementation alleviates stress, enhances immune function, modulates gut microbiota, reduces diarrhea incidence, and improves growth [27]. PCV2 mainly damages porcine immune organs of pigs, resulting in immunosuppression or immune dysfunction [12, 13]. The virus causes diverse clinical manifestations, including post-weaning multisystemic wasting syndrome (PMWS), porcine dermatitis and nephropathy syndrome (PDNS), reproductive failure, respiratory disease complex, and enteric disease. These conditions are collectively termed porcine circovirus-associated diseases (PCVD). PCV2-positive herds exhibit increased susceptibility to secondary bacterial infections like S. suis , E. coli , and Glaesserella parasuis , leading to elevated medication costs, mortality rates, and exacerbated direct or indirect economic losses [14]. Although subunit and inactivated vaccines are available, PCVD emergence and the losses it causes still persist [28]. Enhancing herd immunity, reducing susceptibility, and mitigating secondary infections are critical strategies for PCVD control and economic sustainability. In previous experiments, we observed an increased abundance of L. salivarius in the small intestines of PCV2-infected pigs, suggesting its potential role in host resistance. Therefore, this study aimed to isolate L. salivarius from PCV2-infected porcine intestines for clinical application, with the goal of improving immune function and herd health. The probiotic efficacy of Lactobacillus is closely associated with their origin and may exhibit strain-specific effects, meaning a Lactobacillus strain beneficial for one animal species may not necessarily confer the same benefits to others [23, 29]. To ensure optimal probiotic effects, lactic acid bacteria should ideally be isolated from homologous sites, such as the gastrointestinal environment of the target species, as this enhances their survival and mucosal colonization capacity [29]. In this study, L. salivarius was isolated from the ileum of piglets, a homologous source that could promotes its colonization and probiotic function in the porcine intestinal tract. This homologous isolation strategy aligns with the principle of host adaptation, which maximizing the strain's potential to exert beneficial effects in swine. The biosurfactant production represents one of the key functional characteristics of probiotics. Various microorganisms including bacteria, yeasts, and fungi are capable of biosurfactant production, with bacteria being the predominant producers. Among bacterial species, the genera Pseudomonas , Acinetobacter , Bacillus , and Arthrobacter have been most frequently reported as efficient biosurfactant producers [30]. However, the application of biosurfactants in food and medical industries is limited when produced by potentially pathogenic bacteria [9]. In contrast, biosurfactants derived from probiotics like lactic acid bacteria have attracted significant attention due to their superior safety profile. These microbial surfactants not only exhibit valuable properties such as surface tension reduction, solubilization capacity, and anti-pathogenic microorganism activity, but also offer multiple advantages including biodegradability, non-toxicity, broad applicability, and maintenance of biological activity under extreme conditions, like high temperature, wide pH ranges, and high salinity [9]. Biosurfactants therefore exhibit extensive application prospects across diverse fields, including the food industry, agriculture, medicine, marine ecosystem protection, and petroleum extraction. Notably, biosurfactants exhibit both anti-inflammatory and antimicrobial activities, and previous studies have shown that biosurfactants possess strong inhibitory activity against a wide range of Gram-negative and Gram-positive bacteria, even against multidrug-resistant strains [9, 31, 32]. Similarly, biosurfactants can obviously inhibit replications of viruses such as SARS-CoV-2, HSV-1, and NDV [33–37]. These findings highlight biosurfactants as promising biotherapeutic agents and potential alternatives to conventional antimicrobial drugs. Clinical trials across multiple countries have proved the efficacy of biosurfactants as antiviral agents against acute respiratory distress syndrome (ARDS) [33]. At present, bacterial and viral infectious diseases are the major causes of economic losses in pig farms, which necessitates the isolation of porcine-derived probiotics and evaluation of their biosurfactants' antimicrobial activity. A crucial functional property of biosurfactants is their ability to reduce surface tension, which alters the surface properties of pathogenic microorganisms, inhibits biofilm formation and microbial adhesion, contributing to their antimicrobial effects [11, 38, 39]. L. salivarius strain pig-SJZ2023 isolated in this study possesses a feature of producing biosurfactant which exhibited significant surface tension-reducing properties in both oil-spreading and drop-collapse assays. Moreover, the surface activity increased proportionally with biosurfactant concentration. These findings suggest that L. salivarius -derived biosurfactants have immense potential serve as effective antibiotic alternatives for controlling swine pathogens. Immunosuppression and secondary infections represent hallmark features of porcine circovirus type 2 (PCV2) infection [40]. Accordingly, this study selected pathogenic strains of E. coli and S. suis which are common in pig farms, to evaluate the antimicrobial efficacy of biosurfactant produced by a porcine-derived L. salivarius isolate. We found that the L. salivarius -derived biosurfactant displayed significant antimicrobial activity, achieving inhibition rates of 95.06% against porcine E. coli and 81.80% against S. suis , along with notable anti-PCV2 effects. However, this antimicrobial ability displayed distinct dose- and time-dependent characteristics. The antimicrobial action increased with BS concentration, the inhibitory effects diminished or were lost with prolonged exposure time. These kinetic patterns align with previous observations of L. plantarum -derived biosurfactant against Newcastle disease virus (NDV) [36, 37]. The temporal limitation of biosurfactant efficacy underscores the necessity to develop effective drug encapsulation or delivery systems to prolong therapeutic outcome. Current evidence confirms that nanoliposome carriers can significantly enhance the antimicrobial performance of biosurfactants [10, 31]. In subsequent studies, we will investigate the antimicrobial activities of encapsulated delivery systems containing biosurfactants and associated metabolites derived from porcine-derived L. salivarius strain pig-SJZ2023, with the goal of facilitating clinical applications of L. salivarius -based therapeutic and immunomodulatory formulations. 5. Conclusions In this study, a strain of L. salivarius was successfully isolated and identified from the ileum of PCV2-infected piglets. The biosurfactant produced by this strain displayed significant inhibitory effects against E. coli , S. suis, and PCV2. These findings establish an important foundation for further development of L. salivarius and related antimicrobial products. Abbreviations PCV2 Porcine circovirus type 2 PK-15 cells Pig kidney cells AMR Antimicrobial resistance LAB Lactic acid bacteria BS Biosurfactants MRS DeMan, Rogosa and Sharpe. Declarations Authors′ contributions XZ participated in design of the study, analyzed the data and wrote original draft. JR, CY and FW carried out the experiments and data analysis. LC was involved in animal experiment and data processing. XW, LY and LF participated in experiment and made suggestions. QS designed, reviewed and edited the manuscript. All authors have read and approved the final manuscript. Ethics approval and consent to participate This study was ethically approved by Hebei Agricultural University (China) Animal Welfare and Ethical Review Board (Permit Number:1820026). The Hebei Agricultural University's and China guidelines for the Care and Use of Laboratory Animals were followed. Consent for publication Not applicable. Competing Interests The authors declare that they have no conflict of interest. Author details 1 College of Veterinary Medicine, Hebei Agricultural University, Baoding 071000, China. 2 Hebei Veterinary Biotechnology Innovation Center, Baoding 071000, China. 3 National Center of Technology Innovation for Pigs, Rongchang 402400, Chongqing, China. 4 Hebei Kexing Pharmaceutical Co., LTD, Shijiazhuang 050200, China Funding This work was supported by Research and Development Fund Program of Agricultural University of Hebei (Grant number: JY2022025). Author Contribution XZ participated in design of the study, analyzed the data and wrote original draft. JR, CY and FW carried out the experiments and data analysis. LC was involved in animal experiment and data processing. XW, LY and LF participated in experiment and made suggestions. QS designed, reviewed and edited the manuscript. All authors have read and approved the final manuscript. Acknowledgement We gratefully thank Ms. Yanan Zhang, Mr. Huaining Yue and Dr. Kai Su for his excellent technical assistance. Data Availability The datasets used and analyzed during the current study are available from the corresponding author on reasonable request. References 1. O'Neill L, Manzanilla EG, Ekhlas D, Leonard FC: Antimicrobial resistance in commensal escherichia coli of the porcine gastrointestinal tract. Antibiotics (Basel, Switzerland) 2023, 12(11):1–30. 2. Keenan K, Silva Corrêa J, Sringernyuang L, Nayiga S, Chandler CIR: The social burden of antimicrobial resistance: what is it, how can we measure it, and why does it matter? JAC-antimicrobial resistance 2025, 7(2):1–10. 3. Maragkoudakis PA, Chingwaru W, Gradisnik L, Tsakalidou E, Cencic A: Lactic acid bacteria efficiently protect human and animal intestinal epithelial and immune cells from enteric virus infection. International journal of food microbiology 2010, 141 Suppl 1:S91-97. 4. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6524903","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":457018201,"identity":"8e5f3eaf-e741-4b42-968d-8f7cf19ef264","order_by":0,"name":"Xiaotian Zhou","email":"","orcid":"","institution":"Hebei Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xiaotian","middleName":"","lastName":"Zhou","suffix":""},{"id":457018202,"identity":"4bdcabfd-9fb0-4c40-90f5-9f486c24d17b","order_by":1,"name":"Jing Ren","email":"","orcid":"","institution":"Hebei Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Ren","suffix":""},{"id":457018203,"identity":"295887f9-85cc-433c-8d55-42eb412d56fa","order_by":2,"name":"Chen Yuan","email":"","orcid":"","institution":"Hebei Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Chen","middleName":"","lastName":"Yuan","suffix":""},{"id":457018204,"identity":"0a00e6f0-e2c5-474f-b133-674f10be75f4","order_by":3,"name":"Feiyan Wang","email":"","orcid":"","institution":"Hebei Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Feiyan","middleName":"","lastName":"Wang","suffix":""},{"id":457018205,"identity":"197bb1c3-ff37-452f-8bad-d4341853a0a9","order_by":4,"name":"Ligong Chen","email":"","orcid":"","institution":"Hebei Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Ligong","middleName":"","lastName":"Chen","suffix":""},{"id":457018206,"identity":"3c64b2f5-d0ad-4593-ba18-0a41eb670bfc","order_by":5,"name":"Xiaobo Wang","email":"","orcid":"","institution":"Hebei Kexing Pharmaceutical Co., LTD","correspondingAuthor":false,"prefix":"","firstName":"Xiaobo","middleName":"","lastName":"Wang","suffix":""},{"id":457018207,"identity":"0f6a7d74-4cce-4621-b2b8-0eea6729bbea","order_by":6,"name":"Liu Yang","email":"","orcid":"","institution":"National Center of Technology Innovation for Pigs","correspondingAuthor":false,"prefix":"","firstName":"Liu","middleName":"","lastName":"Yang","suffix":""},{"id":457018209,"identity":"54c85eee-4d03-495d-915d-aa130ef59fe7","order_by":7,"name":"Lizhi Fu","email":"","orcid":"","institution":"National Center of Technology Innovation for Pigs","correspondingAuthor":false,"prefix":"","firstName":"Lizhi","middleName":"","lastName":"Fu","suffix":""},{"id":457018216,"identity":"b4663b6c-0c31-49b7-afcb-b4e72d6ee487","order_by":8,"name":"Qinye Song","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYNACAyBmb2x88IE0LTyHmw1nkGaTRHqbNAcxCuXbew+/eFNwJ7Ff8mGDNAODnZxuAyEnnTmXZjnH4FnizNmJDcYFDMnGZgcIaZHIMTPmMTicuOF2YkPyDIYDidsIaZGfAdWy/+bBhsM8xGhhuJFj/BhsiwRjYzNRWgzOnDFjnGNw2HjGmcRmxhkGRPhFvr3H+MObP4dl+9uPP//xocJOjqAWIGCT4GFgcGyAWEpYOQgwfwBqsSdO7SgYBaNgFIxIAACMD0f7h0or2gAAAABJRU5ErkJggg==","orcid":"","institution":"Hebei Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Qinye","middleName":"","lastName":"Song","suffix":""}],"badges":[],"createdAt":"2025-04-25 03:38:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6524903/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6524903/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12917-025-04978-4","type":"published","date":"2025-08-26T15:57:29+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":83047414,"identity":"f9ffc613-2253-4101-b7fb-e93933a9cbca","added_by":"auto","created_at":"2025-05-19 12:00:31","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2400960,"visible":true,"origin":"","legend":"\u003cp\u003eIsolation, growth curve and 16S rDNA sequence analysis of \u003cem\u003eL. salivarius\u003c/em\u003e (×1000). (\u003cstrong\u003eA\u003c/strong\u003e) Colony morphology on 1% CaCO3-MRS solid medium; (\u003cstrong\u003eB\u003c/strong\u003e) Gram stain. (\u003cstrong\u003eC\u003c/strong\u003e) Growth curve. (\u003cstrong\u003eD\u003c/strong\u003e) 16S rDNA amplification by PCR. M: DNA molecular weight standard DL2000; 1: The isolates (pig-SJZ2023); 2: Negative control. (\u003cstrong\u003eE\u003c/strong\u003e) Phylogenetic tree of the isolates (pig-SJZ2023) and reference strains in the GenBank based on 16S rRNA sequences.\u003c/p\u003e","description":"","filename":"Fig.1..jpg","url":"https://assets-eu.researchsquare.com/files/rs-6524903/v1/efdf27de2c65f419f0484b1f.jpg"},{"id":83047413,"identity":"afad3f97-ea5b-4965-8dfb-5d4c2366d888","added_by":"auto","created_at":"2025-05-19 12:00:31","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1311212,"visible":true,"origin":"","legend":"\u003cp\u003eDetection of biosurfactant production. Oil diffusion: Biosurfactant produced by \u003cem\u003eL. salivarius\u003c/em\u003eisolates (\u003cstrong\u003eA\u003c/strong\u003e), Water control (\u003cstrong\u003eB\u003c/strong\u003e), Correlation analysis between biosurfactant concentration and oil diffusion diameter (\u003cstrong\u003eC\u003c/strong\u003e). Oil droplet collapse: Biosurfactant produced by \u003cem\u003eL. salivarius\u003c/em\u003e isolates. (\u003cstrong\u003eD\u003c/strong\u003e), and Water control (\u003cstrong\u003eE\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"Fig.2..jpg","url":"https://assets-eu.researchsquare.com/files/rs-6524903/v1/450d24813146eaaeb21820f4.jpg"},{"id":83048223,"identity":"e2e8c12a-5c96-4cb2-9bc7-b9f09b4cd549","added_by":"auto","created_at":"2025-05-19 12:08:32","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1011561,"visible":true,"origin":"","legend":"\u003cp\u003eInhibition effect of biosurfactants on\u003cem\u003e E. coli \u003c/em\u003eand \u003cem\u003eS. suis. \u003c/em\u003eColony count results when the bacterial suspension was diluted 10\u003csup\u003e-5 \u003c/sup\u003etimes and three replicates were performed for virous concentrations of biosurfactants. *, **, and *** indicated significant difference between biosurfactant-bacterial co-culture and bacterial control with \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 or \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, respectively.\u003c/p\u003e","description":"","filename":"Fig.3..jpg","url":"https://assets-eu.researchsquare.com/files/rs-6524903/v1/3d068f2ef250bb047c6c56db.jpg"},{"id":83047422,"identity":"67db900a-822c-4a00-91dd-6df99578f694","added_by":"auto","created_at":"2025-05-19 12:00:32","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":521717,"visible":true,"origin":"","legend":"\u003cp\u003eCC\u003csub\u003e50\u003c/sub\u003e measurements of the biosurfactant from\u003cem\u003e L. salivarius\u003c/em\u003e pig-SJZ2023 strain on PK15 cells by CCK8 assay.\u003c/p\u003e","description":"","filename":"Fig.4..jpg","url":"https://assets-eu.researchsquare.com/files/rs-6524903/v1/0f20e6981ace766280cf2130.jpg"},{"id":83048225,"identity":"6c44f531-d510-4107-b9b7-1c63426bc13e","added_by":"auto","created_at":"2025-05-19 12:08:32","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3120368,"visible":true,"origin":"","legend":"\u003cp\u003eInhibitory effect of the biosurfactant from\u003cem\u003e \u003c/em\u003ethe isolated\u003cem\u003e L. salivarius \u003c/em\u003eon PCV2. PCV2 antigen was detected by IFA 36 h after the PK-15 cells were infected with the co-incubation mixture of biosurfactant with virus (\u003cstrong\u003eA\u003c/strong\u003e). NC: Blank control, PC: Positive control. Fluorescence intensity analysis (\u003cstrong\u003eB\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"Fig.5..jpg","url":"https://assets-eu.researchsquare.com/files/rs-6524903/v1/ecc8c53533544340c1b22ae2.jpg"},{"id":83048221,"identity":"1860d179-f6e9-4986-bf2f-b490f00a3786","added_by":"auto","created_at":"2025-05-19 12:08:32","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2270613,"visible":true,"origin":"","legend":"\u003cp\u003eDynamics detection of inhibition effect of the biosurfactant on PCV2. PK-15 cells were infected with the co-incubation solution of 6.25 mg/mL and 3.125 mg/mL biosurfactant and 0.2 MOI PCV2, respectively. After infection for 24 h, 48 h and 72 h, the virus antigen was detected by Western blot (\u003cstrong\u003eA\u003c/strong\u003e, \u003cstrong\u003eB\u003c/strong\u003e and \u003cstrong\u003eC\u003c/strong\u003e). Gray value analysis of antigen-antibody response bands based on Western blot results (\u003cstrong\u003eD\u003c/strong\u003e, \u003cstrong\u003eE\u003c/strong\u003e and \u003cstrong\u003eF\u003c/strong\u003e). NC: Negative control, PC: Positive control.\u003c/p\u003e","description":"","filename":"Fig.6..jpg","url":"https://assets-eu.researchsquare.com/files/rs-6524903/v1/d88592a948a9ca4d03ea13a1.jpg"},{"id":90344990,"identity":"79dbba3b-4094-4ace-b0f9-67ecf36d73ff","added_by":"auto","created_at":"2025-09-01 16:08:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11651087,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6524903/v1/e333527f-e18c-4ddd-9409-3d38a41a2794.pdf"},{"id":83048867,"identity":"35d7d5d2-6f7b-414d-9414-32cc8257aac0","added_by":"auto","created_at":"2025-05-19 12:16:32","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":200990,"visible":true,"origin":"","legend":"","description":"","filename":"25.5.15Originalversionofgelandblotimages.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6524903/v1/745780efddb64e244194adc4.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Isolation of porcine intestinal Lactobacillus salivarius and antimicrobial potential of its biosurfactant","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAntimicrobial agents play a crucial role in safeguarding the health of humans and animals, improving the economic benefits of farming, and ensuring food safety. With the rapid development of the economy and the increasing intensification of animal farming, the use of antibiotics has continued to rise. However, this has led to multiple challenges, including antimicrobial resistance (AMR), food safety issues, and environmental pollution [1, 2]. The World Health Organization (WHO) has listed AMR as one of the major threats to human health in the 21st century. As a result, the search for safe, effective, and alternatives to antibiotics has become a focal point in the development of new therapeutic approaches.\u003c/p\u003e \u003cp\u003eLactic Acid Bacteria (LAB), as probiotics, are important members of the normal microbial flora in specific regions of humans and animals, such as the oral cavity and intestines. By producing organic acids, specific enzymes, and bacteriocins, etc., LAB regulate intestinal microecological balance, inhibit the proliferation of pathogenic microorganisms, and enhance host immunity, and exhibit multiple activities, including antiviral, antitumor, and antistress, as well as the ability to improve intestinal barrier function [3\u0026ndash;5]. Therefore, LAB are considered ideal alternatives to antibiotics [6]. Additionally, \u003cem\u003eL. salivarius\u003c/em\u003e, as an important member of LAB, is widely present in the gastrointestinal mucosa of humans and animals, and can produce biosurfactant, various bacteriocins, antibiotics, antioxidants, and immunomodulators. These substances enable \u003cem\u003eL. salivarius\u003c/em\u003e to balance and improve gut microbiota, combat microbial infections, regulate mucosal immunity, and enhance production performance [7, 8]. \u003cem\u003eL. salivarius\u003c/em\u003e has emerged as a potential candidate for anti-infection therapy and immune function improvement.\u003c/p\u003e \u003cp\u003eBiosurfactants (BS) are natural active substances produced by microorganisms, featuring an amphiphilic structure. They possess various biological activities, including antimicrobial, antiviral, and anti-adhesive properties. Additionally, BS offer advantages such as excellent biodegradability, low toxicity, and high stability [9, 10]. Therefore, biosurfactants are considered as potential candidates for replacing antibiotics. Compared to those produced by pathogenic bacteria, biosurfactants derived from probiotics, known as probiotic biosurfactants, are safer and have broad application prospects in the food, pharmaceutical, and agricultural industries [9\u0026ndash;11].\u003c/p\u003e \u003cp\u003ePorcine circovirus type 2 (PCV2), a 17-nm diameter DNA virus belonging to the genus \u003cem\u003eCircovirus\u003c/em\u003e of the family \u003cem\u003eCircoviridae\u003c/em\u003e, is characterized by damaging porcine immune organs, causing immunosuppression and multisystemic diseases, and poses a serious threat to the development and economic benefits of pig farms [12, 13]. Common secondary or synergistic pathogens in PCV2-infected pigs include \u003cem\u003eS. suis\u003c/em\u003e, \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003ePasteurella multocida\u003c/em\u003e, and \u003cem\u003eGlaesserella parasuis\u003c/em\u003e, etc. The co-infections or secondary infections with these pathogens significantly exacerbate the pathogenic effects of PCV2 [14]. Consequently, PCV2 and associated secondary pathogens have become key targets for disease control in pig operations, making the development of effective preventive and therapeutic agents of paramount importance.\u003c/p\u003e \u003cp\u003eIn this study, a strain of \u003cem\u003eL. salivarius\u003c/em\u003e was isolated from the ileum of piglets experimentally infected with PCV2, and the physicochemical properties and antimicrobial activity of the \u003cem\u003eL. salivarius\u003c/em\u003e-derived biosurfactants were investigated, aiming to provide a scientific basis for promoting the application of probiotics and developing probiotic-based therapies and antibiotic alternatives.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Bacterial or Viral Strains, and Cells\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eE. coli\u003c/em\u003e O141 strain and the \u003cem\u003eS. suis\u003c/em\u003e SJZ2021 strain were preserved in our laboratory, the Laboratory of Animal Infectious Diseases, College of Veterinary Medicine, Hebei Agricultural University, Baoding, China. \u003cem\u003eL. reuteri\u003c/em\u003e was purchased from Beijing Bio-BW Biotechnology Co., Ltd., Beijing, China. The PCV2 HBDX2018 strain (GenBank ID: MK585076), with a TCID\u003csub\u003e50\u003c/sub\u003e of 10\u003csup\u003e6.1\u003c/sup\u003e/mL, was preserved in our laboratory. PK-15 cells were cultured in RPMI-1640 medium (Sigma, USA) supplemented with 10% fetal bovine serum (FBS, Wisent Biotechnology, Nanjing, China) and 100 IU/mL each of penicillin and streptomycin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Animal Infection and Sample Collection\u003c/h2\u003e \u003cp\u003eThree 28-day-old healthy piglets were purchased from Hebei Tangsheng Animal Husbandry Co., Ltd. (Baoding, China). The piglets tested negative for PCV2 nucleic acid and specific antibodies by PCR and ELISA. The piglets were infected with the PCV2 HBDX2018 strain via intranasal and intramuscular routes at a dose of 10\u003csup\u003e6.1\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e per piglet. Seven days post-infection, the piglets were anesthetized by intravenous injection of xylazine hydrochloride, and the ileum along with contents were aseptically collected for bacterial isolation and culture.\u003c/p\u003e \u003cp\u003e This study was approved by the Animal Welfare and Ethics Committee of Hebei Agricultural University (China) (License No. 1820026). All experimental procedures were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals of Hebei Agricultural University and China.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Bacterial Isolation\u003c/h2\u003e \u003cp\u003eThe ileal mucosa and intestinal contents were scraped using a sterile glass slide and transferred into a tube containing an equal volume of pH7.4 0.01 mol/L phosphate-buffered saline (PBS). The mixture was incubated anaerobically at 37℃ with shaking for 5 hours to enrich the bacteria. A 100 \u0026micro;L aliquot of the enriched bacterial suspension was serially diluted from 10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e. After vortex mixing, 10 \u0026micro;L of each diluted suspension was spread evenly onto the surface of DeMan, Rogosa and Sharpe (MRS) solid medium (Luqiao Technology Co., Ltd., Beijing, China) and incubated anaerobically at 37℃ for 24 hours. Single colony exhibiting smooth, raised, and well-defined edges with a milky-white appearance were selected and streaked onto 1% CaCO\u003csub\u003e3\u003c/sub\u003e-MRS solid medium, followed by anaerobic incubation at 37℃ for 24 hours. Colonies showing clear calcium dissolution zones were selected and subjected to three consecutive rounds of purification to obtain pure strains. The purified strains were Gram-stained and examined under a microscope until all observed bacteria were Gram-positive rods. The isolated bacteria were then suspended in a preservation solution (60% glycerol in LB medium) and stored at -20℃.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. 16S rDNA Amplification and Sequence Analysis\u003c/h2\u003e \u003cp\u003eTotal DNA was extracted from the bacterial strain using the boiling lysis method. Briefly, 1.5 mL of bacterial culture was centrifuged at 8,000 r/min for 5 minutes. The bacterial pellet was resuspended in 500 \u0026micro;L of sterile water, boiled for 10 minutes, and centrifuged at 12,000 r/min for 10 minutes. The supernatant was collected as the DNA template for PCR amplification of the 16S rDNA sequence. The PCR reaction mixture (20 \u0026micro;L) consisted of 10 \u0026micro;L of 2\u0026times; Es Taq Master Mix (Kangwei Century Biotechnology Co., Ltd., Jiangsu, China), 0.5 \u0026micro;L each of forward and reverse primers (25 \u0026micro;mol/L), 2 \u0026micro;L of DNA template, and 7 \u0026micro;L of ddH\u003csub\u003e2\u003c/sub\u003eO. The PCR conditions were as follows: initial denaturation at 94℃ for 3 minutes; 30 cycles of 94℃ for 45 seconds, 52℃ for 1 minute, and 72℃ for 1 minute; and a final extension at 72℃ for 10 minutes. The universal primers used for 16S rDNA amplification were F: 5ʹ-AGAGTTTGATCCTGGCTCAG-3ʹ and R: 5ʹ-GGTTACCTTGTTACGACTT-3ʹ. The PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The obtained sequences were compared with the reference sequences in the GenBank nucleotide database for homology analysis. A phylogenetic tree was constructed using the Neighbor-Joining (NJ) method in MEGA 7.0 software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Determination of Growth Curve\u003c/h2\u003e \u003cp\u003eA single colony was inoculated into MRS broth and anaerobically cultured at 37℃ for 14 hours to prepare the seed culture. The seed culture was then inoculated into fresh MRS broth at a 1∶100 ratio and incubated under the same conditions. Samples were collected at 0, 8, 12, 14, 16, 18, and 24 hours, serially diluted, and used for colony counting. A triplicate was performed for each dilution. The bacterial concentration (colony-forming units per milliliter, CFU/mL) was calculated based on the colony counts.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Identification of Physicochemical Properties of the Isolate\u003c/h2\u003e \u003cp\u003eThe biochemical characteristics of the isolate were identified by Bacterial identification micro-biochemical reaction tube (Hangzhou Binhe Microbial Reagent Co., Ltd., Hangzhou, China), and a standard \u003cem\u003eLactobacillus\u003c/em\u003e (\u003cem\u003eL. reuteri\u003c/em\u003e, Beijing Bio-BW Biotechnology Co., Ltd., Beijing, China) control was established simultaneously. The purified bacteria were inoculated into microtubes containing maltose, mannitol, sorbitol, sucrose, raffinose, lactose, peptone, and hydrogen sulfide according to the manufacturer\u0026rsquo;s instructions. The tubes then were anaerobically incubated at 37℃ for 24 hours, and the performance of the isolate in acid production and the generation of indole and hydrogen sulfide were observed.\u003c/p\u003e \u003cp\u003eTo clarify motility, the isolates were stab-inoculated into semi-solid (Hangzhou Binhe Microbial Reagent Co., Ltd., China) and anaerobically incubated at 37℃ for 24 hours. The motility of the bacteria was judged based on the spread of growth along the puncture line. In addition, after puncture inoculation, the gelatin cultures were placed in a refrigerator for 2 hours at 4℃ to observe the solidification of gelatin, which indicated whether the isolate produced gelatinase to liquefy gelatin.\u003c/p\u003e \u003cp\u003eThe catalase test was conducted furtherly. The colonies were picked up and placed on a clean glass slide and 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e were added. After dropping 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, the production of abundant bubbles within 30 seconds indicated the isolate could generate catalase. Conversely, catalase was unable to be produce.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Detection of Biosurfactants Production\u003c/h2\u003e \u003cp\u003eTo detect biosurfactant production, the isolated bacteria were inoculated into 300 mL of MRS broth at a 1∶100 ratio and cultured at 37℃ for 48 hours. The bacterial cells were harvested by centrifugation at 8000 r/min for 5 minutes at 4℃. The cells were washed twice with sterile deionized water and resuspended in 50 mL of pH 7.0 0.01 mol/L PBS. The suspension was gently stirred and incubated at room temperature for 2 hours to release the biosurfactants. After centrifugation to remove the cells, the supernatant was filtered through a membrane filter with pore size of 0.22 \u0026micro;m (Jinteng Experimental Equipment Co., Ltd., Tianjin, China). The filtrate was dried using a rotary evaporator (RE-52AA, Shanghai Yarong, China), and the dried extract was resuspended in PBS to a concentration of 10 mg/mL. The pH of the solution was adjusted to 2.0 using 1 mol/L HCl and incubated at 4℃ for 2 hours. The precipitate was collected by centrifugation at 9,000 r/min for 15 minutes at 4℃ and washed twice with sterile distilled water (pH 2.0). The precipitate was then dissolved in sterile distilled water, adjusted to pH 7.0 using 1 mol/L NaOH, filtered, and freeze-dried for the measurement of biosurfactant.\u003c/p\u003e \u003cp\u003eOil spreading test was implemented for measurement of the biosurfactant produced by the isolated bacteria. Briefly, one drop of vegetable oil was added to distilled water, followed by one drop of the prepared biosurfactant solution (100 mg/mL) onto the oil surface. One drop of distilled water was simultaneously applied to the oil surface as a negative control. The diameter of the oil displacement zone was measured to evaluate the surface tension-reducing activity of the prepared biosurfactant. Furthermore, the oil displacement zone diameters were measured for biosurfactant solutions at different concentrations (0, 50, 100, 150, and 200 mg/mL), and a standard curve was plotted to analyze the relationship between biosurfactant concentration and oil displacement zone diameter.\u003c/p\u003e \u003cp\u003eOil drop-collapse test was also employed to test the biosurfactant production. A drop of the prepared biosurfactant was applied to vegetable oil surface, and the spreading or collapse of the biosurfactant drop was observed after 2 minutes. A negative control of adding a drop of water on the vegetable oil surface was set up at the same time.\u003c/p\u003e \u003cp\u003eIf the solution contained biosurfactants, the interfacial tension between the droplet and the hydrophobic surface of the vegetable oil would be reduced, causing the droplet to spread and became flat. In contrast, if the solution lacked biosurfactants, the polar water molecules would be repelled from the hydrophobic surface, and the droplet would remain stable to form bead.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Examination of Antimicrobial Activity of Biosurfactants\u003c/h2\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.8.1. Examination of Antibacterial Activity\u003c/h2\u003e \u003cp\u003eThe antibacterial activity of the prepared biosurfactant, the common pathogenic bacteria \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. suis\u003c/em\u003e used as models, was determined using a modified microdilution method. Briefly, in a 96-well cell culture plate, 200 \u0026micro;L of sterile nutrient broth was added to the first well as a blank control. In wells 2 to 11, 100 \u0026micro;L of serially diluted biosurfactant was added to make final concentrations ranging from 50 mg/mL to 0.1 mg/mL, followed by the addition of 100 \u0026micro;L of \u003cem\u003eE. coli\u003c/em\u003e or \u003cem\u003eS. suis\u003c/em\u003e culture (\u003cem\u003eOD\u003c/em\u003e\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.5) to each well. The 12th well contained 100 \u0026micro;L of nutrient broth and an equal volume of bacterial culture as a bacterial control. Each treatment was performed in triplicate. The plate was incubated anaerobically at 37℃ for 24 hours. After incubation, the cultures from wells containing different concentrations of biosurfactants were subjected to colony counting to analyze the antibacterial activity of the biosurfactants.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.8.2. Measurement of Antiviral Activity\u003c/h2\u003e \u003cp\u003eUsing PCV2 as the model, the antiviral activity of the biosurfactants produced by \u003cem\u003eL. salivarius\u003c/em\u003e was detected. Measurement of 50% Cytotoxic Concentration (CC\u003csub\u003e50\u003c/sub\u003e) of biosurfactant was conducted firstly prior to determination of antiviral activity. The biosurfactant was diluted to concentrations of 6.25, 12.5, 25, 50, 100, and 200 mg/mL using RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS). The diluted biosurfactant were added to a 96-well cell culture plate at 50 \u0026micro;L per well. Then, 50 \u0026micro;L of PK-15 cell suspension (2 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells) was added to each well. Each concentration was tested in triplicate. Meanwhile, the cell control without biosurfactant and the medium control with medium-only without cells were established. The plate was placed at 37℃ in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator for 24 hours. According to the manufacturer\u0026rsquo;s instructions of the Cell Counting Kit-8 (CCK-8) (APExBIO, America), 10 \u0026micro;L of CCK-8 was put into each well. The plate was gently mixed on a microplate shaker (MH-2, QILNBEIER, Haimen, China) for 30 seconds and then incubated at 37℃ in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator for 1 hour. The optical density (\u003cem\u003eOD\u003c/em\u003e) at 450 nm was measured using a microplate reader (Synergy HTX, Gene Company Limited, USA) and the cell viability was calculated based on the following formula (1). The CC\u003csub\u003e50\u003c/sub\u003e value was calculated according a nonlinear regression curve of BS concentration versus cell viability plotted using GraphPad Prism 8.0 software. Then Immunofluorescence assay (IFA) and Western blot were employed for analysis of anti-PCV2 activity of the biosurfactant (BS).\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\:\\text{C}\\text{e}\\text{l}\\text{l}\\:\\text{v}\\text{i}\\text{a}\\text{b}\\text{i}\\text{l}\\text{i}\\text{t}\\text{y}\\:\\left(\\text{\\%}\\right)=\\frac{OD\\left(\\text{B}\\text{S}\\:\\text{w}\\text{e}\\text{l}\\text{l}\\right)-\\:OD\\left(\\text{m}\\text{e}\\text{d}\\text{i}\\text{u}\\text{m}\\:\\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}\\right)}{OD\\left(\\text{c}\\text{e}\\text{l}\\text{l}\\:\\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}\\right)-\\:OD\\left(\\text{m}\\text{e}\\text{d}\\text{i}\\text{u}\\text{m}\\:\\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}\\right)}\\)\u003c/span\u003e \u003c/span\u003e\u0026times;100% (1)\u003c/p\u003e \u003cp\u003eImmunofluorescence assay (IFA): PCV2 at a dose of 0.2 multiplicity of infection (MOI) was mixed with 12.5 mg/mL or 6.25 mg/mL of BS or an equal volume of RPMI-1640 medium supplemented with 3% FBS. The mixtures were incubated at 37℃ in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator for 1 hour. PK-15 cells in the logarithmic growth phase were digested with trypsin, centrifuged, and resuspended in medium. 100 \u0026micro;L cells were seeded into a 96-well cell culture plate containing 5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per well. The cells were divided into four groups: Blank control (NC), Positive control (PC), BS-12.5 group, and BS-6.25 group, with three replicates per group. NC group: 100 \u0026micro;L of RPMI-1640 medium containing 3% FBS was added to each well. PC group: 100 \u0026micro;L of pre-mixed solution containing RPMI-1640 medium and PCV2 was added to each well. BS-12.5 group and BS-6.25 group: 100 \u0026micro;L incubated mixture of PCV2 and 12.5 mg/mL or 6.25 mg/mL BS, respectively, was put into each well. The plate was incubated at 37℃ in a CO\u003csub\u003e2\u003c/sub\u003e incubator for 36 hours. After incubation, the cells were gently washed with PBS, and 50 \u0026micro;L of pre-cooled methanol was added to each well for fixation at -20℃ for 10 minutes. The cells were washed again, and 100 \u0026micro;L of 2% BSA-PBS was added to each well, followed by incubation at 37℃ for 1 hour. Subsequently, 50 \u0026micro;L of a 1∶500 dilution of PCV2 monoclonal antibody (prepared by the Laboratory of Animal Infectious Diseases, Hebei Agricultural University) was added to each well and incubated at 37℃ for 1 hour. After washing, 50 \u0026micro;L of a 1∶500 dilution of YF\u0026reg;594 goat anti-mouse IgG (Uelandy Biotechnology Co., Ltd., Suzhou, China) was added to each well and incubated at 37℃ for 45 minutes. The cells were washed again, and the cellular nucleus were stained with Bisbenzimide. Red fluorescence, indicating the specific binding of the monoclonal antibody to PCV2 antigen, was observed under a fluorescence microscope.\u003c/p\u003e \u003cp\u003eWestern blot: 1 mL mixture of 0.2 MOI PCV2 and 6.25 mg/mL or 3.125 mg/mL biosurfactant (BS) was added to each well of a 6-well cell culture plate, with three replicates per condition. The plate was incubated at 37℃ in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator for 1 hour. Controls included a viral control (Positive control, PC) without BS and a cell control (Negative control, NC). One milliliter of 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e PK-15 cells in the logarithmic growth phase was added to each well of above different treatments. The plates were transferred to a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator at 37℃ for 24, 48, and 72 hours, and the cells were collected for Western blot analysis to detect viral protein and evaluate the inhibitory effect of BS on PCV2. For the Western blot assay, the PCV2 monoclonal antibody was diluted at 1∶50, and HRP-conjugated goat anti-mouse IgG (Biodragon Biotechnology Co., Ltd., Suzhou, China) was diluted at 1∶10,000. Chemiluminescent substrate (Yeasen Biotechnology Co., Ltd., Shanghai, China) was used for signal detection.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Statistical Analysis\u003c/h2\u003e \u003cp\u003eAll data were presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviations (SD) and statistical analysis were performed using an unpaired two-tailed Student\u0026rsquo;s t-test in GraphPad Prism 8.0. Graphs were generated using GraphPad Prism 8.0, and asterisk signs in the Figure indicate statistical significance (*\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and ***\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Culture and Morphological Characteristics, 16S rDNA Analysis, and Growth Curve of the Isolated Strain\u003c/h2\u003e \u003cp\u003eThe isolated strain was streaked onto MRS solid medium and incubated at 37\u0026deg;C for 24 h, forming medium-sized, convex, moist, and smooth-edged milky-white colonies. When cultured on 1% CaCO₃-MRS solid medium for 24 h, a clear calcium-dissolving zone was observed around the colonies (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Gram staining revealed Gram-positive, short rod-shaped bacteria, occurring in pairs or short chains under microscopy (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). These cultural and morphological characteristics were consistent with those of \u003cem\u003eLactobacillus\u003c/em\u003e. The isolated strain was statically cultured at 37\u0026deg;C, and samples were collected every 2 h for colony counting. The results showed that the bacterial count peaked at 12 h, indicating that the logarithmic growth phase of this strain lasted 12 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFor further identification, the bacterial 16S rDNA sequence was amplified by PCR. Agarose gel electrophoresis confirmed an amplified fragment of approximately 1400 bp (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Sequencing and alignment analysis revealed 99\u0026ndash;100% homology with \u003cem\u003eL. salivarius\u003c/em\u003e, including 100% homology with \u003cem\u003eL. salivarius\u003c/em\u003e strain LGM8-7 (MG674666.1) from swine. In contrast, homology with \u003cem\u003eL. johnsonii\u003c/em\u003e and \u003cem\u003eL. reuteri\u003c/em\u003e was only 25\u0026ndash;27%. Phylogenetic analysis using the Neighbor-Joining method in MEGA7.0 demonstrated that the isolated strain clustered within the \u003cem\u003eL. salivarius\u003c/em\u003e branch, forming a subclade with \u003cem\u003eL. salivarius\u003c/em\u003e LGM8-7, while \u003cem\u003eL. johnsonii\u003c/em\u003e and \u003cem\u003eL. reuteri\u003c/em\u003e were located in distinct branches (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). These results confirmed that the isolated strain was \u003cem\u003eL. salivarius\u003c/em\u003e, designated as pig-SJZ2023.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Physicochemical Characteristics of the Isolated strain\u003c/h2\u003e \u003cp\u003eThe physicochemical characterization results of the isolated strain pig-SJZ2023 are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. After anaerobic incubation at 37\u0026deg;C for 24 h in various culture media including maltose, mannitol, sorbitol, sucrose, raffinose, lactose, indole, and hydrogen sulfide, the color change of six carbohydrate fermentation tubes from purple to yellow indicated acid production capability. No color alterations were observed in either indole or hydrogen sulfide media, demonstrating the strain's inability to produce indole or hydrogen sulfide. The catalase test, performed by applying 3% hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) to bacterial colonies, yielded no observable bubble formation, confirming the absence of catalase activity. In gelatin stab culture assays, bacterial growth was strictly confined to the inoculation line without any radial expansion or gelatin liquefaction, indicating both non-motile behavior and lack of gelatinolytic activity. These collective biochemical characteristics exhibit complete consistency with the standard phenotypic profile of \u003cem\u003eLactobacillus\u003c/em\u003e species, thereby providing additional validation for the taxonomic classification of strain pig-SJZ2023.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePhysiological and biochemical characteristics of\u0026nbsp;\u003cem\u003eL. salivarius.\u0026nbsp;\u003c/em\u003epig-SJZ2023: the isolated strain;\u003c/p\u003e\u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eItem\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eLactobacillus\u003c/em\u003e\u003c/p\u003e \u003cp\u003eStandard strain[15]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003epig-SJZ2023\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMorphology\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBacillus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBacillus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGram staining\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMoveability\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026minus;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026minus;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGelatin liquefaction\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026minus;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026minus;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSucrose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCottonseed sugar\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLactose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMaltose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMannitol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSorbitol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCatalase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026minus;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026minus;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIndole\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026minus;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026minus;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026minus;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026minus;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003e* \"+\": positive; \"\u0026minus;\": negative.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Biosurfactant production ability of the isolated strain\u003c/h2\u003e \u003cp\u003eWhen the extract of \u003cem\u003eL. salivarius\u003c/em\u003e isolates was dropped onto the surface of vegetable oil in water, an oil-displacement circle with a diameter of 2.7 cm was produced, while no such circle was observed in the water control (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). This result indicated that the isolated \u003cem\u003eL. salivarius\u003c/em\u003e was capable of biosurfactant production. Furtherly, the diameter of the oil displacement zone increased along with rising biosurfactant concentrations, demonstrating a strong linear correlation (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.99) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Meanwhile, upon application of the biosurfactant to the vegetable oil surface, immediate droplet spreading and flattening were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). In contrast, the water control showed neither droplet expansion nor surface flattening. These results confirmed that the biosurfactant produced by the isolated \u003cem\u003eL. salivarius\u003c/em\u003e strain possesses significant surface tension-reducing properties, which the activity exhibited clear concentration dependence.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Antimicrobial Activity of the Prepared Biosurfactant\u003c/h2\u003e \u003cp\u003eDifferent concentrations of the biosurfactant were co-cultured with \u003cem\u003eE. coli\u003c/em\u003e or \u003cem\u003eS. suis\u003c/em\u003e for 24 h, followed by colony counting. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, when the biosurfactant concentration ranged from 50 mg/mL to 6.25 mg/mL, the inhibition rates against \u003cem\u003eE. coli\u003c/em\u003e were 95.06\u0026ndash;46.13% (13/263\u0026ndash;142/263), showing significant differences compared to the \u003cem\u003eE. coli\u003c/em\u003e control without the biosurfactant (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). At a concentration of 3.125 mg/mL, the inhibition rate of biosurfactant against \u003cem\u003eE. coli\u003c/em\u003e was 22.69% (203/263), an obviously difference compared to the control (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). For \u003cem\u003eS. suis\u003c/em\u003e, when the concentrations of biosurfactant ranged from 50 mg/mL to 12.5 mg/mL, the inhibition rates were 81.80\u0026ndash;49.60% (38/207\u0026ndash;104/207), showing significant differences compared to the \u003cem\u003eS. suis\u003c/em\u003e control without biosurfactants (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). When the concentration being 6.25 mg/mL and 3.125 mg/mL, the inhibition rates of the biosurfactant against \u003cem\u003eS. suis\u003c/em\u003e were 41.06% (122/207) and 38.49% (127/207) respectively, showing differences compared to the control (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). No significant inhibitory effect was observed against either \u003cem\u003eE. coli\u003c/em\u003e or \u003cem\u003eS. suis\u003c/em\u003e at 1.56 mg/mL of biosurfactant (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). These experimental results demonstrated that the biosurfactant extracted from \u003cem\u003eL. salivarius\u003c/em\u003e isolates has stark inhibitory effects against both \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. suis\u003c/em\u003e, with the inhibitory effect being dose-dependent.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Anti-PCV2 Activity of the Prepared Biosurfactant\u003c/h2\u003e \u003cp\u003eDifferent concentrations of biosurfactants were co-cultured with PK-15 cells for 24 hours. Based on the nonlinear regression curve of biosurfactants concentration versus cell viability, the CC\u003csub\u003e50\u003c/sub\u003e of biosurfactants was calculated to be 24.73 mg/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Subsequently, when 12.5 or 6.25 mg/mL of biosurfactants was co-incubated with PCV2 for 1 hour before infecting PK-15 cells, immunofluorescence assay (IFA) at 36 hours post-infection revealed distinctly weaker PCV2 antigen-specific fluorescence signals in treated cells compared to the viral control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe mixture obtained after incubating biosurfactants at 6.25 or 3.125 mg/mL with PCV2 for 1 hour was inoculated into PK-15 cells, and the proliferation of PCV2 was analyzed by Western blot at different times after infection. At 24 hours post-infection, there was no or almost no specific reaction band visible on the polyvinylidene fluoride (PVDF) membrane, which shown that the biosurfactant displayed distinct inhibitory effects on PCV2 proliferation. However, the antiviral effects of the biosurfactant markedly weakened between 48\u0026ndash;72 hours post-infection, which the antiviral activity was positively correlated with the concentration of the biosurfactant (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eProbiotics primarily consist of bacteria from the genera \u003cem\u003eLactobacillus\u003c/em\u003e, \u003cem\u003eLactococcus\u003c/em\u003e, \u003cem\u003eBifidobacterium\u003c/em\u003e, \u003cem\u003eBacillus\u003c/em\u003e, \u003cem\u003eStreptococcus\u003c/em\u003e, \u003cem\u003ePropionibacteria\u003c/em\u003e, and \u003cem\u003ePediococcus\u003c/em\u003e, along with certain yeasts. These microorganisms play crucial roles in maintaining microbial balance, nutrient absorption, and immune function [16\u0026ndash;18]. Among these, \u003cem\u003eLactobacillus\u003c/em\u003e spp. have been extensively studied. Through the production of biosurfactants, lactic acid, hydrogen peroxide, bacteriocins, and bacteriocin-like substances, they regulate intestinal microecological balance, inhibit pathogenic microorganism proliferation, mitigate stress responses, enhance intestinal barrier function, and improve immunity [19, 20]. Dietary or feed supplementation with lactic acid bacteria has been shown to enhance host immune function and promote growth [21]. \u003cem\u003eL. salivarius\u003c/em\u003e, one of the particularly important probiotics, is capable of secreting organic acids (e.g., phenyllactic acid, clavaminic acid), antibiotics, and bacteriocins. These metabolites exhibit inhibitory effects against pathogens such as \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eStreptococcus\u003c/em\u003e, and \u003cem\u003eStaphylococcus\u003c/em\u003e aureus, as well as viruses including herpesvirus, rotavirus, and Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) [8, 22\u0026ndash;24]. Oral administration of \u003cem\u003eL. salivarius\u003c/em\u003e is safe, non-toxic, and effective in treating mastitis in humans with low recurrence rates [25, 26]. In piglets, \u003cem\u003eL. salivarius\u003c/em\u003e supplementation alleviates stress, enhances immune function, modulates gut microbiota, reduces diarrhea incidence, and improves growth [27].\u003c/p\u003e \u003cp\u003ePCV2 mainly damages porcine immune organs of pigs, resulting in immunosuppression or immune dysfunction [12, 13]. The virus causes diverse clinical manifestations, including post-weaning multisystemic wasting syndrome (PMWS), porcine dermatitis and nephropathy syndrome (PDNS), reproductive failure, respiratory disease complex, and enteric disease. These conditions are collectively termed porcine circovirus-associated diseases (PCVD). PCV2-positive herds exhibit increased susceptibility to secondary bacterial infections like \u003cem\u003eS. suis\u003c/em\u003e, \u003cem\u003eE. coli\u003c/em\u003e, and \u003cem\u003eGlaesserella parasuis\u003c/em\u003e, leading to elevated medication costs, mortality rates, and exacerbated direct or indirect economic losses [14]. Although subunit and inactivated vaccines are available, PCVD emergence and the losses it causes still persist [28]. Enhancing herd immunity, reducing susceptibility, and mitigating secondary infections are critical strategies for PCVD control and economic sustainability. In previous experiments, we observed an increased abundance of \u003cem\u003eL. salivarius\u003c/em\u003e in the small intestines of PCV2-infected pigs, suggesting its potential role in host resistance. Therefore, this study aimed to isolate \u003cem\u003eL. salivarius\u003c/em\u003e from PCV2-infected porcine intestines for clinical application, with the goal of improving immune function and herd health.\u003c/p\u003e \u003cp\u003eThe probiotic efficacy of \u003cem\u003eLactobacillus\u003c/em\u003e is closely associated with their origin and may exhibit strain-specific effects, meaning a \u003cem\u003eLactobacillus\u003c/em\u003e strain beneficial for one animal species may not necessarily confer the same benefits to others [23, 29]. To ensure optimal probiotic effects, lactic acid bacteria should ideally be isolated from homologous sites, such as the gastrointestinal environment of the target species, as this enhances their survival and mucosal colonization capacity [29]. In this study, \u003cem\u003eL. salivarius\u003c/em\u003e was isolated from the ileum of piglets, a homologous source that could promotes its colonization and probiotic function in the porcine intestinal tract. This homologous isolation strategy aligns with the principle of host adaptation, which maximizing the strain's potential to exert beneficial effects in swine.\u003c/p\u003e \u003cp\u003eThe biosurfactant production represents one of the key functional characteristics of probiotics. Various microorganisms including bacteria, yeasts, and fungi are capable of biosurfactant production, with bacteria being the predominant producers. Among bacterial species, the genera \u003cem\u003ePseudomonas\u003c/em\u003e, \u003cem\u003eAcinetobacter\u003c/em\u003e, \u003cem\u003eBacillus\u003c/em\u003e, and \u003cem\u003eArthrobacter\u003c/em\u003e have been most frequently reported as efficient biosurfactant producers [30]. However, the application of biosurfactants in food and medical industries is limited when produced by potentially pathogenic bacteria [9]. In contrast, biosurfactants derived from probiotics like lactic acid bacteria have attracted significant attention due to their superior safety profile. These microbial surfactants not only exhibit valuable properties such as surface tension reduction, solubilization capacity, and anti-pathogenic microorganism activity, but also offer multiple advantages including biodegradability, non-toxicity, broad applicability, and maintenance of biological activity under extreme conditions, like high temperature, wide pH ranges, and high salinity [9]. Biosurfactants therefore exhibit extensive application prospects across diverse fields, including the food industry, agriculture, medicine, marine ecosystem protection, and petroleum extraction. Notably, biosurfactants exhibit both anti-inflammatory and antimicrobial activities, and previous studies have shown that biosurfactants possess strong inhibitory activity against a wide range of Gram-negative and Gram-positive bacteria, even against multidrug-resistant strains [9, 31, 32]. Similarly, biosurfactants can obviously inhibit replications of viruses such as SARS-CoV-2, HSV-1, and NDV [33\u0026ndash;37]. These findings highlight biosurfactants as promising biotherapeutic agents and potential alternatives to conventional antimicrobial drugs. Clinical trials across multiple countries have proved the efficacy of biosurfactants as antiviral agents against acute respiratory distress syndrome (ARDS) [33]. At present, bacterial and viral infectious diseases are the major causes of economic losses in pig farms, which necessitates the isolation of porcine-derived probiotics and evaluation of their biosurfactants' antimicrobial activity.\u003c/p\u003e \u003cp\u003eA crucial functional property of biosurfactants is their ability to reduce surface tension, which alters the surface properties of pathogenic microorganisms, inhibits biofilm formation and microbial adhesion, contributing to their antimicrobial effects [11, 38, 39]. \u003cem\u003eL. salivarius\u003c/em\u003e strain pig-SJZ2023 isolated in this study possesses a feature of producing biosurfactant which exhibited significant surface tension-reducing properties in both oil-spreading and drop-collapse assays. Moreover, the surface activity increased proportionally with biosurfactant concentration. These findings suggest that \u003cem\u003eL. salivarius\u003c/em\u003e-derived biosurfactants have immense potential serve as effective antibiotic alternatives for controlling swine pathogens.\u003c/p\u003e \u003cp\u003eImmunosuppression and secondary infections represent hallmark features of porcine circovirus type 2 (PCV2) infection [40]. Accordingly, this study selected pathogenic strains of \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. suis\u003c/em\u003e which are common in pig farms, to evaluate the antimicrobial efficacy of biosurfactant produced by a porcine-derived \u003cem\u003eL. salivarius\u003c/em\u003e isolate. We found that the \u003cem\u003eL. salivarius\u003c/em\u003e-derived biosurfactant displayed significant antimicrobial activity, achieving inhibition rates of 95.06% against porcine \u003cem\u003eE. coli\u003c/em\u003e and 81.80% against \u003cem\u003eS. suis\u003c/em\u003e, along with notable anti-PCV2 effects. However, this antimicrobial ability displayed distinct dose- and time-dependent characteristics. The antimicrobial action increased with BS concentration, the inhibitory effects diminished or were lost with prolonged exposure time. These kinetic patterns align with previous observations of \u003cem\u003eL. plantarum\u003c/em\u003e-derived biosurfactant against Newcastle disease virus (NDV) [36, 37]. The temporal limitation of biosurfactant efficacy underscores the necessity to develop effective drug encapsulation or delivery systems to prolong therapeutic outcome. Current evidence confirms that nanoliposome carriers can significantly enhance the antimicrobial performance of biosurfactants [10, 31]. In subsequent studies, we will investigate the antimicrobial activities of encapsulated delivery systems containing biosurfactants and associated metabolites derived from porcine-derived \u003cem\u003eL. salivarius\u003c/em\u003e strain pig-SJZ2023, with the goal of facilitating clinical applications of \u003cem\u003eL. salivarius\u003c/em\u003e-based therapeutic and immunomodulatory formulations.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eIn this study, a strain of \u003cem\u003eL. salivarius\u003c/em\u003e was successfully isolated and identified from the ileum of PCV2-infected piglets. The biosurfactant produced by this strain displayed significant inhibitory effects against \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eS.\u003c/em\u003e suis, and PCV2. These findings establish an important foundation for further development of \u003cem\u003eL. salivarius\u003c/em\u003e and related antimicrobial products.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePCV2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePorcine circovirus type 2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePK-15 cells\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePig kidney cells\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAMR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAntimicrobial resistance\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLAB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLactic acid bacteria\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBiosurfactants\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMRS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDeMan, Rogosa and Sharpe.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eAuthors\u0026prime; contributions\u003c/h2\u003e \u003cp\u003eXZ participated in design of the study, analyzed the data and wrote original draft. JR, CY and FW carried out the experiments and data analysis. LC was involved in animal experiment and data processing. XW, LY and LF participated in experiment and made suggestions. QS designed, reviewed and edited the manuscript. All authors have read and approved the final manuscript.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e \u003cp\u003e This study was ethically approved by Hebei Agricultural University (China) Animal Welfare and Ethical Review Board (Permit Number:1820026). The Hebei Agricultural University's and China guidelines for the Care and Use of Laboratory Animals were followed.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting Interests\u003c/strong\u003e \u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eAuthor details\u003c/h2\u003e \u003cp\u003e \u003csup\u003e1\u003c/sup\u003eCollege of Veterinary Medicine, Hebei Agricultural University, Baoding 071000, China. \u003csup\u003e2\u003c/sup\u003eHebei Veterinary Biotechnology Innovation Center, Baoding 071000, China. \u003csup\u003e3\u003c/sup\u003eNational Center of Technology Innovation for Pigs, Rongchang 402400, Chongqing, China. \u003csup\u003e4\u003c/sup\u003eHebei Kexing Pharmaceutical Co., LTD, Shijiazhuang 050200, China\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by Research and Development Fund Program of Agricultural University of Hebei (Grant number: JY2022025).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eXZ participated in design of the study, analyzed the data and wrote original draft. JR, CY and FW carried out the experiments and data analysis. LC was involved in animal experiment and data processing. XW, LY and LF participated in experiment and made suggestions. QS designed, reviewed and edited the manuscript. All authors have read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe gratefully thank Ms. Yanan Zhang, Mr. Huaining Yue and Dr. Kai Su for his excellent technical assistance.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets used and analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003e1. O'Neill L, Manzanilla EG, Ekhlas D, Leonard FC: Antimicrobial resistance in commensal escherichia coli of the porcine gastrointestinal tract. 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Morais IMC, Cordeiro AL, Teixeira GS, Domingues VS, Nardi RMD, Monteiro AS, Alves RJ, Siqueira EP, Santos VL: Biological and physicochemical properties of biosurfactants produced by Lactobacillus jensenii P(6A) and Lactobacillus gasseri P(65). Microbial cell factories 2017, 16(1):1\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e40. Park SW, Park IB, Kang SJ, Bae J, Chun T: Interaction between host cell proteins and open reading frames of porcine circovirus type 2. Journal of animal science and technology 2023, 65(4):698\u0026ndash;719.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary Research","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Lactobacillus salivarius, Isolation and identification, Biosurfactants, Antimicrobial activity","lastPublishedDoi":"10.21203/rs.3.rs-6524903/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6524903/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eLactobacillus salivarius\u003c/em\u003e (\u003cem\u003eL. salivarius\u003c/em\u003e) is a probiotic widely colonized in the gastrointestinal tracts of humans and animals. Its biosurfactant (BS) exhibits antibacterial and antiviral activities, along with advantages such as biodegradability, non-toxicity, and stability. Porcine circovirus type 2 (PCV2) is a major pathogen in swine farms, causing immunosuppression or immune dysfunction. Elevated susceptibility in PCV2-positive herds is accompanied by severe secondary bacterial infections. To explore novel antimicrobial biologics for enhancing swine immunity, this study isolated a biosurfactant-producing \u003cem\u003eLactobacillus\u003c/em\u003e strain from the intestines of PCV2-infected piglets using the calcium carbonate plate method, and analyzed the anti-microbial activity of the biosurfactant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA strain of biosurfactant-producing \u003cem\u003eL. salivarius\u003c/em\u003e was successfully isolated from the ileum of PCV2-infected piglets. The extracted BS from the isolate demonstrated surface tension-reducing properties, forming spreading zones or dispersions on vegetable oil. The antimicrobial experiment showed that at the concentrations of 6.25–50 mg/mL, BS inhibited the growth of porcine \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. suis\u003c/em\u003e by 46.13% − 95.06% and 46.13% − 95.06%, respectively. Furthermore, BS of 6.25–12.5 mg/mL significantly inhibited PCV2 replication in PK-15 cells. These findings suggest that \u003cem\u003eL. salivarius\u003c/em\u003e is a promising probiotic candidate for improving swine immune status and developing novel alternatives to conventional antimicrobial agents.\u003c/p\u003e","manuscriptTitle":"Isolation of porcine intestinal Lactobacillus salivarius and antimicrobial potential of its biosurfactant","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-19 12:00:27","doi":"10.21203/rs.3.rs-6524903/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-17T16:53:10+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-14T02:36:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"281044092657878872037730293631418217013","date":"2025-06-26T15:06:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"206768085415146857883774087589886189344","date":"2025-06-02T19:10:20+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-02T00:34:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"14145843047889638316779472752581821453","date":"2025-05-26T14:03:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"244404352749054603572062378697234270077","date":"2025-05-26T12:20:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"34425034879629232876133829586820871717","date":"2025-05-15T10:18:03+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-15T09:42:49+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-15T09:39:55+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-05-15T09:35:50+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-15T09:20:03+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Veterinary Research","date":"2025-05-15T09:18:53+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary Research","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"53bdc66e-2be2-455e-a457-f7345652934e","owner":[],"postedDate":"May 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-09-01T16:04:35+00:00","versionOfRecord":{"articleIdentity":"rs-6524903","link":"https://doi.org/10.1186/s12917-025-04978-4","journal":{"identity":"bmc-veterinary-research","isVorOnly":false,"title":"BMC Veterinary Research"},"publishedOn":"2025-08-26 15:57:29","publishedOnDateReadable":"August 26th, 2025"},"versionCreatedAt":"2025-05-19 12:00:27","video":"","vorDoi":"10.1186/s12917-025-04978-4","vorDoiUrl":"https://doi.org/10.1186/s12917-025-04978-4","workflowStages":[]},"version":"v1","identity":"rs-6524903","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6524903","identity":"rs-6524903","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}