Lactobacillus plantarum OBM7 and application thereof

Abstract

The invention discloses lactobacillus plantarum OBM7 and application thereof, and belongs to the technical field of biology. The strain is preserved in the Guangdong Microbial Culture Collection Center (GDMCC) on November 12, 2025, the preservation address is the 5th floor of the building 59, No.100 Courtyard, Xianlie Middle Road, Guangzhou, and the preservation number is GDMCC NO. 67282; and the probiotic preparation does not contain antibiotics or drug residues, and meets the development requirements of green and healthy breeding. The probiotic preparation can specifically prevent and control lamb diarrhea, significantly reduce the fatality rate of lamb diarrhea, effectively improve the survival rate of lambs, and provide targeted protection for lamb intestinal health; the lamb feed can regulate lamb gastrointestinal tract micro-ecological balance, improve digestion and absorption functions, significantly improve feed intake and feed conversion efficiency, and promote lamb growth performance improvement. Reliable technical support is provided for efficient, green and healthy breeding of lambs, high practicability and wide industrial application prospects are achieved, and the method has great significance in promoting green transformation of the livestock breeding industry.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to a strain of Lactobacillus plantarum OBM7 and its applications. Background Technology

[0002] Lamb diarrhea is a common and prevalent disease in large-scale meat and wool sheep farming. Susceptible lambs are those aged 1-6 weeks, but can also be affected at other ages. As a global challenge in livestock farming, its morbidity and mortality rates vary significantly depending on region, season, and feeding management practices. Specific data shows that in China, the morbidity rate of lamb diarrhea reaches 30% and the mortality rate 20% during winter and spring; the morbidity rate climbs to 85% in the mid-to-late stages of lambing, with lambs born to primiparous ewes facing nearly 100% risk of developing the disease; and the morbidity rate in lambs under 2 weeks of age is as high as 65%, with a mortality rate of approximately 30% under traditional treatment methods. This has become a core bottleneck restricting the improvement of the efficiency of the sheep farming industry.

[0003] The pathogenesis of this disease is multifactorial and complex, and can be induced by single or synergistic effects of pathogenic bacteria (such as Escherichia coli), viruses (such as rotavirus), parasites, feed mutations, and environmental stress. After onset, lambs exhibit symptoms such as stunted growth, impaired development, and decreased production performance. They may also experience secondary infections due to weakened immunity, such as respiratory diseases and gastrointestinal mucosal inflammation. Furthermore, early immunosuppression in young lambs can directly lead to failure of the immune response after vaccination, creating a vicious cycle of "onset-immune impairment-multiple infections." Simultaneously, the physiological characteristics of lambs further exacerbate the difficulty of diarrhea control: their digestive and immune systems are not yet fully developed, digestive enzyme secretion is insufficient, the intestinal physical barrier function is fragile, and the balance of the gastrointestinal flora is easily disturbed by fluctuations in environmental temperature and humidity, and adjustments in stocking density. Moreover, problems such as imbalanced dietary nutrition or sudden changes in feeding methods during the weaning transition period can easily lead to digestive disorders and induce non-infectious diarrhea.

[0004] In current large-scale or free-range farming, antibiotics remain the primary means of controlling lamb diarrhea. However, this approach has inherent and unavoidable drawbacks: First, long-term or inappropriate use of antibiotics can induce drug resistance genes in pathogenic microorganisms, leading to poor efficacy or even ineffectiveness. Furthermore, drug resistance can be transferred laterally through the food chain or environment, posing a potential threat to public health. Second, the broad-spectrum antibacterial properties of antibiotics can disrupt the normal microecological balance in the lamb's gut, leading to dysbiosis. Under the global policy guidance and technological demands for "antibiotic reduction and replacement" in the livestock industry, the development of safe, efficient, and drug-residue-free technologies for controlling lamb diarrhea is urgently needed. Probiotics, as ideal antibiotic reduction and replacement products or effective supplements, are increasingly important in promoting lamb growth and disease prevention. The World Health Organization (WHO) defines probiotics as: "live microorganisms that, when ingested in adequate amounts, produce beneficial effects on the health of the host." Among them, Lactobacillus (Lactobacillus spp.) Lactobacillus Bifidobacterium spp. Bifidobacterium Strains such as *Lactobacillus plantarum* have been widely used in medicine, food, and animal husbandry due to their proven probiotic effects. Lactiplantibacillus plantarum As one of the most widely used species in the genus *Lactobacillus*, *Lactobacillus plantarum* is widely found in the gastrointestinal tract of animals and fermented foods. Its safety has been certified as "Generally Recognized As Safe" (GRAS) by the US Food and Drug Administration (FDA) and "Qualified Presumption of Safety" (QPS) by the European Food Safety Authority (EFSA), providing a reliable foundation for industrial application. The probiotic mechanism of *Lactobacillus plantarum* is closely related to its metabolic characteristics: the organic acids (lactic acid, acetic acid, etc.), hydrogen peroxide, bacteriocins, and other active substances produced by its metabolism have broad-spectrum antibacterial activity, regulating the intestinal microecological balance by lowering intestinal pH and competitively inhibiting the attachment of pathogenic microorganisms. Simultaneously, this species can enhance the intestinal mucosal barrier function, regulate the body's immune response, and improve antioxidant capacity, thereby improving animal production performance. It has been proven that it can be used in livestock production to improve intestinal health, enhance immunity, promote growth, and prepare high-quality silage.

[0005] However, existing probiotic strains used in lamb farming generally suffer from weak targeted antibacterial activity and insufficient stress resistance (such as poor resistance to gastric acid and bile, making it difficult to reach the intestines and exert their effects), failing to effectively adapt to the unique physiological environment of the lamb's intestines and the complex pathogenic mechanisms of diarrhea. Therefore, screening for a *Lactobacillus plantarum* strain with high stress resistance and strong targeted antibacterial ability against lamb diarrhea, and developing a dedicated probiotic preparation based on this strain, is of significant practical importance and industrial value in overcoming the current predicament of lamb diarrhea prevention and control and promoting the green and healthy development of the sheep farming industry. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a strain of Lactobacillus plantarum OBM7 and its applications to solve the problems existing in the prior art.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows: In a first aspect, the present invention provides a strain of *Lactobacillus plantarum*. Lactiplantibacillus plantarum OBM7, the strain in question, was deposited on November 12, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Courtyard 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC NO. 67282.

[0008] In a second aspect, the present invention provides a probiotic preparation comprising the above-described *Lactobacillus plantarum* and / or the metabolites of the strain.

[0009] In a third aspect, the present invention provides a feed additive comprising the microbial agent as described above.

[0010] In a fourth aspect, the present invention provides a composition for preventing and treating diarrhea in lambs, the composition comprising Bacillus subtilis, Lactobacillus paracasei, and Lactobacillus plantarum OBM7 as described above.

[0011] Furthermore, the ratio of *Lactobacillus plantarum* OBM7, *Bacillus subtilis*, and *Lactobacillus paracasei* is 1:1~2:1~2.

[0012] In a fifth aspect, the present invention provides the use of the aforementioned *Lactobacillus plantarum* OBM7, or the aforementioned microbial agent, or the aforementioned feed additive, or the aforementioned composition in any of the following: (1) Application in the preparation of products that inhibit the listed pathogenic bacteria; the pathogenic bacteria include at least one of various Escherichia coli, Proteus vulgaris, Shigella boydii, Yersinia enterocolitica, Salmonella typhimurium, Salmonella enterica, Clostridium perfringens, Campylobacter jejuni, Mycoplasma bovis, Mycoplasma capsulatum, Staphylococcus aureus, Klebsiella pneumoniae and Pseudomonas aeruginosa; (2) Application in the preparation of products for the prevention and treatment of animal diarrhea; (3) Application in the preparation of products that increase animal feed intake.

[0013] Furthermore, the animal in question is a lamb.

[0014] The probiotics provided by this invention can specifically prevent and control lamb diarrhea, significantly reduce lamb mortality due to diarrhea, effectively improve lamb survival rate, and provide targeted protection for lamb intestinal health. They can also regulate the microecological balance of the lamb's gastrointestinal tract, improve digestive and absorptive functions, significantly increase feed intake and feed conversion efficiency, and promote lamb growth performance. This invention provides reliable technical support for efficient, green, and healthy lamb farming, possessing strong practicality and broad prospects for industrial application, and is of great significance for promoting the green transformation of the livestock industry.

[0015] The beneficial effects of this invention include at least the following: (1) The *Lactobacillus plantarum* OBM7 provided by this invention has been verified by hemolysis test and in vivo safety test in mice to have no hemolytic toxicity, no damage to host organs, and no induction of intestinal inflammation. The prepared product does not contain antibiotics and has no drug residues, thus avoiding the problems of drug resistance and dysbiosis caused by traditional antibiotic control, which meets the needs of green and healthy aquaculture development.

[0016] (2) The strain has outstanding stress resistance and can tolerate gastrointestinal stress in lambs and successfully colonize. It has a high adhesion rate and can stably bind to intestinal epithelial cells, competitively inhibiting the attachment of pathogenic bacteria. At the same time, it can tolerate 0-3 mmol / L H2O2 and has antioxidant capacity, which can provide multiple protections for intestinal health.

[0017] (3) The strain has broad-spectrum antibacterial activity against 30 common pathogens causing diarrhea in lambs, including Escherichia coli, Salmonella typhimurium, and Clostridium perfringens. The antibacterial effect is particularly significant against Campylobacter jejuni ZJ1, Proteus vulgaris standard strain ATCC29905, and Escherichia coli NQ4.

[0018] (4) The strain can regulate the gastrointestinal function of lambs and effectively increase the lambs’ feed intake; it can effectively prevent the occurrence of lamb diarrhea, reduce the incidence of lamb diarrhea, and improve the production performance of sheep. Attached Figure Description

[0019] Figure 1 This is a colony morphology diagram of Lactobacillus plantarum OBM7.

[0020] Figure 2 Gram staining image of Lactobacillus plantarum OBM7.

[0021] Figure 3 This is a scanning electron microscope image of Lactobacillus plantarum OBM7.

[0022] Figure 4 This is a diagram of the hemolysis test of Lactobacillus plantarum OBM7.

[0023] Figure 5 A visual representation of the internal organs (liver, kidneys, and spleen) of a mouse.

[0024] Figure 6 Image of mouse colon stained with HE.

[0025] Figure 7 Image of PAS staining in mouse colon.

[0026] Figure 8 Analysis of α-diversity of mouse gut microbiota.

[0027] Figure 9 Analysis of β-diversity of mouse gut microbiota.

[0028] Figure 10 Analysis of gut microbiota in mice.

[0029] Figure 11 This is a TEM electron micrograph of the cell-free supernatant of Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 25922, and Lactobacillus plantarum OBM7 after incubation.

[0030] Figure 12 The OD600 of Lactobacillus plantarum OBM7 after incubation with different concentrations of H2O2.

[0031] Figure 13 This is the complete genome map of Lactobacillus plantarum OBM7.

[0032] Figure 14 Annotation results for Lactobacillus plantarum OBM7 in the Nr database.

[0033] Figure 15 The results of the GO database analysis for Lactobacillus plantarum OBM7 are shown.

[0034] Figure 16 Annotation results of the KEGG pathway for Lactobacillus plantarum OBM7.

[0035] Figure 17 The results of eggNOG annotation for Lactobacillus plantarum OBM7.

[0036] Figure 18 Annotation results for Lactobacillus plantarum OBM7 in the CAZy database. Detailed Implementation

[0037] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0038] Furthermore, the technical solutions of the various embodiments of the present invention can be combined with each other, but only if they are feasible for those skilled in the art. If the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0039] The culture medium used in the embodiments of the present invention includes: Sterile PBS solution: Weigh 8.0 g NaCl, 0.2 g KCl, 0.27 g KH2PO4, and 1.42 g Na2HPO4 and dissolve them in 800 mL of deionized water. After thorough dissolution, add hydrochloric acid to adjust the pH to 7.2, then add deionized water to bring the volume to 1 L. Autoclave at 121℃ for 20 min and store at room temperature for later use.

[0040] MRS liquid culture medium: 10.0 g peptone, 5.0 g yeast extract, 20.0 g glucose, 2.0 g K2HPO4, 2.0 g triammonium citrate, 5.0 g anhydrous sodium acetate, 0.058 g MnSO4, 0.29 g MgSO4, 1.0 mL Tween 80. Dissolve the above components sequentially in 800 mL deionized water, adjust the pH to 7.2, and bring the volume to 1000 mL. Autoclave at 121°C for 20 min.

[0041] MRS solid medium: Add 1.5% agar powder to the MRS liquid medium and autoclave.

[0042] The following specific embodiments illustrate the solution proposed in this invention: Example 1: Isolation and Identification of Strains 1.1 Isolation and culture of strains The strain OBM7 of this invention was isolated from yogurt traditionally brewed by herders in Qinghai Province and identified by molecular identification as *Lactobacillus plantarum* OBM7. Lactiplantibacillus plantarum OBM7).

[0043] The specific isolation and purification method for OBM7 is as follows: Take 10 mL of yogurt made by herdsmen in Maduo County, Qinghai Province, into a centrifuge tube containing 90 mL of PBS, mix thoroughly, and then serially dilute to a suitable concentration. Take 100 μL of the mixture and place it on a solid medium plate containing 1% calcium carbonate-MRS, and anaerobically culture at 37℃ for 24 h ~ 48 h. Select suspected lactic acid bacteria colonies, gently touch the colonies with a sterile inoculation loop, and gently mix them in a centrifuge tube containing 500 μL of PBS. Streak the mixture onto an MRS solid medium plate. Following the above steps, purify for three generations or more, perform Gram staining to confirm the morphology of the strain. After each isolate is purified, pick a single colony from each isolate and anaerobically culture it in MRS for 24 h, then centrifuge and discard the supernatant. Preserve the strain in liquid nitrogen containing 15% glycerol-MRS preservation solution for later use.

[0044] 1.2 Morphological observation of the strain On MRS plates, strain OBM7 appears milky white, with round colonies that are smooth, moist, and have regular, raised edges. Figure 1 Under an oil immersion microscope, strain OBM7 was observed to be a Gram-positive bacillus without spores. Figure 2 Scanning electron microscopy revealed that the OBM7 strain exhibited a smooth, rod-like morphology. Figure 3 ).

[0045] 1.3 Molecular biological identification of strains The genome of the OBM7 isolate was extracted and used as a template for PCR amplification. PCR amplification was performed using universal primers 27F and 1492R for bacterial identification of 16S rDNA, with a reaction volume of 25 μL. The PCR amplification program was as follows: pre-denaturation at 94℃ for 3 min; 94℃ for 45 s; 55℃ for 45 s; 72℃ for 70 s; repeated for 30 cycles; 72℃ for 8 min. The amplified product was identified by 1.5% agarose gel electrophoresis, and the product size was approximately 1500 bp. The PCR product was sent to Qingke Zexi Biotechnology Co., Ltd. for sequencing, and the sequencing results were submitted to NCBI for BLAST comparison analysis. Based on the comparison results, strain OBM7 was named *Lactobacillus plantarum* OBM7. This strain was submitted and deposited at the Guangdong Provincial Microbial Culture Collection Center on November 12, 2025, with accession number GDMCC NO. 67282.

[0046] Example 2: Safety evaluation of Lactobacillus plantarum OBM7 2.1 Hemolytic test Lactobacillus plantarum OBM7 was passaged three times in MRS liquid medium. A small amount of culture medium was streaked onto Columbia blood agar medium using an inoculation loop. After anaerobic incubation at 37°C for 24 h, the presence of a hemolytic zone around the colony was observed. Bacillus cereus ZJTP2 (… Bacillus cereusA hemolytic strain was used as a positive control. Based on the bacteria's ability to lyse red blood cells, hemolysis can be classified into three types and phenomena: α-hemolysis: produces hemolysin, incomplete hemolysis, with a narrow, translucent, grass-green hemolytic ring of 1-2 mm around the colony on blood agar plates; β-hemolysis: complete hemolysis, with a 2-4 mm wide, well-defined, colorless, transparent hemolytic ring around the colony on blood agar plates; γ-hemolysis: does not produce hemolysin, with no hemolytic ring around the colony on blood agar plates. The results showed that *Lactobacillus plantarum* OBM7 did not exhibit hemolysis (γ-hemolysis) on blood agar plates, and strain OBM7 was preliminarily considered a safe strain. Figure 4 ).

[0047] 2.2 Drug susceptibility testing The drug susceptibility of strain OBM7 was determined using the Kirby-Bauer test (KB method) on paper disk agar diffusion. A sterile cotton swab was used to apply a suspension of OBM7 diluted to an appropriate concentration evenly onto an MRS agar plate, which was then incubated at room temperature for 10-20 minutes. Using sterile forceps, the drug susceptibility plate was placed on the MRS agar plate and anaerobically incubated at 37°C for 24 hours. The inhibition zone of the drug susceptibility plate was then measured using calipers. The susceptibility was determined using *Escherichia coli* (E. coli). Escherichia coli ATCC25922 was used as a quality control strain. The drug susceptibility results were determined according to the standards of the Clinical Laboratory Standards Institute (CIL) in the United States, and the results were: sensitive (S), moderately sensitive (I), and resistant (R). The antibiotics used in this study included 11 antibiotics from 8 classes, including amoxicillin (20 μg / tablet), β-lactams ampicillin (10 μg / tablet) and cefotaxime sodium (30 μg / tablet); quinolones norfloxacin (10 μg / tablet); macrolides erythromycin (15 μg / tablet); aminoglycosides streptomycin (10 μg / tablet) and kanamycin (30 μg / tablet); chloramphenicol (30 μg / tablet); tetracycline (30 μg / tablet); lincosamides clindamycin (10 μg / tablet); and glycopeptides vancomycin (30 μg / tablet). The results showed that strain OBM7 was sensitive to ampicillin, amoxicillin, erythromycin, norfloxacin, and chloramphenicol; but resistant to kanamycin, streptomycin, and vancomycin (Table 1).

[0048] Table 1 Results of antibiotic susceptibility testing for OBM7

[0049] 2.3 In vivo safety studies, immunomodulation, and effects on gut microbiota Twelve healthy male C57BL / 6J mice aged 35 days were selected and acclimatized for one week with free access to food and water. They were randomly divided into two groups of six mice each, including a normal control group and an experimental group. Mice in both experimental groups were fed a basal diet. The control group received 0.2 mL / mouse / day of PBS via gavage, while the experimental group received the *Lactobacillus plantarum* strain OBM7 of this invention via gavage. Specific gavage dosages are shown in Table 2. The experiment lasted for 14 days, and the mice's mental state, growth, coat, and feces were observed daily. After the experiment, the weight gain rate was calculated, and organs were dissected for lesions. Mouse colons were fixed in 5% paraformaldehyde, prepared into paraffin sections, dewaxed, and then stained with hematoxylin and eosin (HE) and periodic acid-Schiff (PAS). HE staining was used to assess basic pathological changes in the colonic tissue, such as inflammatory cell infiltration, mucosal structural damage, and epithelial injury. PAS staining was used to specifically visualize mucus secretion within goblet cells to assess intestinal barrier function. After the experiment, mouse serum was collected to detect the effect of OBM7 on inflammatory factors in mouse serum. Mouse colon contents were collected, and the effect of OBM7 on the mouse gut microbiota was evaluated using 16S rRNA microbiome technology.

[0050] Table 2. Mouse safety study design

[0051] The results are shown in Table 3. During the experiment, the mice in each group had normal food intake, mental state, behavior, and stool, with no significant differences, indicating that the strain of this invention had no effect on the growth of mice. Careful observation of the organs of each group of mice after dissection revealed no visible lesions in the heart, liver, spleen, kidneys, and lungs, indicating that *Lactobacillus plantarum* OBM7 of this invention is safe for mice. Figure 5 HE staining of mouse colon sections ( Figure 6 ) and PAS staining ( Figure 7 The results showed that the *Lactobacillus plantarum* OBM7 strain of this invention possesses both biosafety and intestinal homeostasis regulation functions, has no damaging effect on mouse intestinal mucosa and villi, and can significantly maintain intestinal epithelial cell homeostasis.

[0052] Table 3. Mouse weight gain rate (%)

[0053] Enzyme-linked immunosorbent assay (ELISA) analysis showed that the levels of five pro-inflammatory factors (TNF-α, IL-1β, IL-6, iNOS, and MPO) in the intestinal tissue of mice treated with strain OBM7 were not significantly different from those in the control group. While the expression levels of anti-inflammatory factors IL-4 and IL-10 showed an increasing trend, this did not reach statistical significance. These results indicate that strain OBM7 not only does not induce intestinal inflammation in mice but also enhances the body's immune function.

[0054] Intestinal contents microbiome analysis results showed that the gut microbiome α-diversity of mice orally administered Lactobacillus plantarum OBM7 was significantly higher than that of mice in this study. Figure 8 ) and β diversity ( Figure 9 The abundance of OBM7 showed an upward trend, but there was no significant difference compared to the control group. This indicates that OBM7 can increase the abundance of gut microbiota in mice and is safe for mice. The biota of the colon contents in both groups were mainly composed of Firmicutes, Bacteroides, and Verrucomicrobiota. OBM7 altered the structural composition of the colonic microbiota in mice. OBM7 gavage reduced the abundance of Firmicutes and Bacteroides, and increased the abundance of Verrucomicrobiota and Desulfobacterota. Figure 10 It is generally believed that an increase in the abundance of Verrucomicrobiota and Dethiobacterium can positively regulate the balance of the host's gut microbiota, thereby having a positive effect on the body's health.

[0055] Example 3: Study on the probiotic properties of the strain 3.1 Simulated Gastric Fluid and Bile Salt Tolerance Test The OBM7 strain was activated by three consecutive subcultures and inoculated at a 2% inoculum into artificial gastric fluid at pH 3.0. After vortexing and mixing, the mixture was anaerobically cultured at 37°C for 3 h. Samples were taken at 0 h and 3 h, diluted to an appropriate gradient, and counted on MRS plates. The plates were then anaerobically cultured at 37°C for 24 h.

[0056] The formula for calculating strain tolerance is: Survival rate = N3 / N0 × 100% In the formula: N3 is the number of viable bacteria in 3 h; N0 is the number of viable bacteria in 0 h; the number of viable bacteria is expressed in CFU / mL.

[0057] Similarly, strain OBM7 was activated and inoculated at a 2% inoculum into MRS medium containing 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, and 0.02% protectant, respectively. The medium was then anaerobic at 37°C for 24 h, and the absorbance at OD600 was measured at 0 h and 24 h.

[0058] The formula for calculating strain tolerance is:

[0059] Experimental results showed that strain OBM7 had a high survival rate in acidic and bile salt environments. Specifically, the 3-hour survival rate of OBM7 in simulated gastric fluid (pH=3) was 83.81±1.90%, and the 24-hour growth rate in a 0.3% bile salt environment was 40.24±1.48%. These experimental data further validated that strain OBM7 possesses excellent acid and bile salt tolerance and can withstand host gastrointestinal environmental stress.

[0060] 3.2 Antibacterial test The fresh suspension of *Lactobacillus plantarum* OBM7 was centrifuged (4℃, 6000 rpm, 10 min), and the supernatant was filtered through a 0.22 μm filter. The 30 pathogenic bacteria strains in this example were: *Escherichia coli* ETEC K88, *Escherichia coli* ATCC25922, *Escherichia coli* (O157:H7) ATCC 43888, *Escherichia coli* MY1 (bovine), *Escherichia coli* NX2 (bovine), *Escherichia coli* NQ4 (sheep), *Escherichia coli* GS2 (sheep), *Escherichia coli* ZJ1 (bovine), *Escherichia coli* ZJ3 (goat), and *Salmonella typhimurium* ATCC. 14028, *Salmonella Typhimurium* NQ1 (goat source), *Salmonella Enterica* ZJ2 (goat source), *Salmonella Typhimurium* ZJ4 (sheep source), *Salmonella Typhimurium* MD1 (sheep source), *Clostridium perfringens* MQ1 (sheep source), *Clostridium perfringens* MQ4 (sheep source), *Clostridium perfringens* MY6 (sheep source), *Clostridium perfringens* XH2 (yak source), *Campylobacter jejuni* TZ1 (yak source), *Campylobacter jejuni* ZJ1 (sheep source), *Shigella boydii* ATCC 9207, *Yersinia enterocolitica* ATCC 23715, *Mycoplasma bovis* 08m, *Mycoplasma bovis* NX1, *Mycoplasma caprineis* ZJ1, *Mycoplasma caprineis* ZJ6, *Staphylococcus aureus* ATCC 6538, *Proteus vulgaris* ATCC 29905, *Klebsiella pneumoniae* ZJ3 and *Pseudomonas aeruginosa* ATCC 27853 (Table 4). The concentrations of the nine pathogenic bacteria were adjusted to 1×10⁻⁶ using sterile PBS. 6CFU / mL was evenly spread onto NA agar plates using a sterile cotton swab. After standing at room temperature for 15 min, 200 μL of CFS was punched into each well. The plates were then incubated at room temperature for 2 h and then at 37°C. The plates were removed when a clear inhibition zone appeared (approximately 18 h). The diameter of the inhibition zone (mm) was measured using calipers, and the size of the inhibition zone indicated the antibacterial activity of the probiotics.

[0061] The results of this study showed that strain OBM7 possessed broad-spectrum antibacterial activity against 30 common pathogenic bacteria. This strain effectively inhibited the growth and reproduction of various pathogens, including enterotoxic Escherichia coli, Escherichia coli, Salmonella typhimurium, Salmonella enterica, Clostridium perfringens, Campylobacter jejuni, Shigella boydii, Yersinia enterocolitica, Mycoplasma bovis, Mycoplasma caprineis, Staphylococcus aureus, Proteus vulgaris, Klebsiella pneumoniae, and Pseudomonas aeruginosa. The antibacterial effect was particularly significant against sheep-derived Campylobacter jejuni ZJ1, the standard strain of Proteus vulgaris ATCC 29905, and sheep-derived Escherichia coli NQ4, with inhibition zone diameters reaching 25.44±0.26 mm, 24.49±0.21 mm, and 23.84±0.15 mm, respectively (Table 4).

[0062] Table 4. Determination of the antibacterial activity of Lactobacillus plantarum OBM7 (mm)

[0063] 3.3 Co-culture of OBM7 cell-free supernatant with pathogenic bacteria Adjust the concentrations of Staphylococcus aureus ATCC 6538 and Escherichia coli ATCC 25922 to 1×10⁻⁶. 6 CFU / mL was added to 10 mL of cell-free supernatant from OBM7 and co-cultured for 24 h. The surface morphology of the strains was observed by scanning electron microscopy. The results showed that the cell-free supernatant of *Lactobacillus plantarum* OBM7 could disrupt the cell structure of *Staphylococcus aureus* ATCC 6538 and *Escherichia coli* ATCC 25922, causing significant damage to the cell surface of these two strains. Figure 11 ).

[0064] 3.4 Determination of the Adhesion of OBM7 Caco-2 cell culture and passage: Caco-2 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (V / V), 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C in a 5% CO2 incubator. When adherent cells reached 80% confluence, they were digested with trypsin and passaged at a 1:5 ratio. The medium was changed after 1 day of growth, and passaged again after 3 days. The cell concentration was adjusted to 5 × 10⁶ cells / mL before the adhesion assay. 5Cells / mL were inoculated into 12-well culture plates and cultured in complete medium without antibiotics.

[0065] The test strain OBM7 was centrifuged (4℃, 6000×g, 5 min), washed three times with sterile PBS, and the concentration was adjusted to 1×10⁻⁶. 8 CFU / mL, observe the cells in the 12-well plate until a monolayer is formed (approximately 36 h after inoculation), then discard the cell culture medium and wash the cells with sterile PBS; set up test wells and control wells; add 0.5 mL of the above bacterial suspension to the test wells and incubate at 37℃ and 5% CO2 for 2 h; remove the 12-well plate and discard the culture medium, wash 3 times with PBS to remove unattached strains; add 0.1 mL of trypsin to each well to digest the cells for 1 min; after the cells are scattered like snowflakes under the microscope, add 0.4 mL of PBS and pipette, collect the cells into 1.5 mL EP tubes, serially dilute and count.

[0066] Strain adhesion rate (%) = 2h bacteria count / 0h bacteria count × 100 Probiotics need to adhere to intestinal epithelial cells or the mucus layer to remain in the intestine for a longer period and exert their beneficial effects for a longer time. Some probiotics can also compete with pathogenic bacteria for intestinal epithelial adhesion sites to inhibit pathogenic bacteria. The results showed that the adhesion rate of strain OBM7 was 10 ± 0.10%.

[0067] 3.5 Surface property determination Hydrophobicity: After overnight culture of strain OBM7, the bacterial culture was washed twice with PBS, and the OD600 was adjusted to 0.5±0.02, recorded as S0. Then, an equal volume of xylene was added to the bacterial culture and vortexed for 1 min. The culture was then incubated at 37℃ for 2 h. The absorbance of the upper aqueous phase was measured at 600 nm and recorded as S1. Three replicate tests were performed.

[0068] Hydrophobicity (%) is expressed as: (S0-S1) / S0×100.

[0069] Self-aggregation: After overnight culture of strain OBM7, the OD was adjusted by washing twice with PBS. 600 The result was 0.5 ± 0.02, recorded as Z0; 5 mL of bacterial culture was incubated at 37℃ for 24 h, and the absorbance of the supernatant was measured at 600 nm and recorded as Z1; the experiment was repeated three times.

[0070] The self-polymerization rate (%) is expressed as: (Z0-Z1) / Z0×100.

[0071] After probiotics reach the intestines, their interaction with the host is closely related to the surface properties of the strain. Experimental results showed that strain OBM7 had a self-aggregation rate of 73.31±0.18% and a hydrophobicity of 91.01±0.24% to xylene.

[0072] 3.6 H2O2 tolerance test The concentration of the OBM7 bacterial suspension cultured for 16-24 h was adjusted to 10. 8 CFU / mL, and then inoculated at a 2% inoculum into MRS broth medium with initial H2O2 concentrations of 0, 1, 2, and 3 mmol / L, respectively, and incubated at 37℃ for 16 h. OD was then measured at different H2O2 concentrations. 600 value.

[0073] The results showed that OBM7 could grow normally under conditions of 0-3 mmol H2O2, and its growth ability was improved under conditions of 1 mmol and 2 mmol H2O2. This indicates that OBM7 has good stress resistance and antioxidant capacity, and may have the ability to produce antioxidant substances. Figure 12 ).

[0074] 3.7 Whole Genome Analysis The OBM7 genome was extracted and sent to Biomarker Biotechnology Co., Ltd. for sequencing using a nanopore sequencer. The whole genome data of strain OBM7 has been uploaded to NCBI (CP179939-CP179940). The genome length of strain OBM7 is 3,230,636 bp, including a circular chromosome of 3,197,760 bp and a circular plasmid of 32,876 bp, with an average GC content of 44.54%. Figure 13 The genome contains 3007 coding sequences (CDS). In addition, 16 rRNAs were identified, including 5 23S rRNAs, 6 5S rRNAs, and 5 16S rRNAs, as well as 71 tRNA genes. The genome of strain OBM7 contains 11 CRISPR sequences, 8 gene islands, 1 prophage sequence, and 5 secondary metabolic gene clusters. The genome of strain OBM7 was annotated using 7 general databases (Table 5). Nr database annotation showed that 53.13% of the proteins in OBM7 were annotated as... Lactiplantibacillus plantarum ( Figure 14 According to the GO database ( Figure 15 Gene function is mainly divided into three major areas: cellular components, molecular functions, and biological processes. KEGG pathway annotation results are as follows: Figure 16 The coding sequence (CDS) of OBM7 is primarily enriched in the following three KEGG secondary pathway categories: amino acid biosynthesis, ABC transporters, and carbon metabolism. EggNOG annotation analysis shows that ( Figure 17 The OBM7 genome contains a large number of protein-coding genes with unknown functions (Class S, 471), indicating that this strain has the potential to produce a variety of unique proteins. CAZy database annotations show that 129 genes in the OBM7 genome have been identified as carbohydrate enzyme genes. Figure 18 Of the 49 glycoside hydrolases, 37.98% were glycoside hydrolases. Drug resistance analysis showed that strain OBM7 possessed a drug resistance gene, PoxtA, which was not located on transferable elements such as plasmids, posing no risk of drug resistance gene transfer.

[0075] Bacterial secondary metabolites are important natural sources of antimicrobial agents and other bioactive compounds. In this study, analysis using antiSMASH software predicted five gene clusters for the biosynthesis of secondary metabolites on the chromosome of the target strain. These clusters were annotated and classified into RiPP-like, terpene-precursor, type III polyketide synthase (T3PKS), terpene, and cyclic-lactone-autoinducer types, respectively. The first gene cluster was located in the 373,057–385,207 bp region of chromosome 373,057, which contains the genes ACMATP_01730 and ACMATP_01735 for the synthesis of the dipeptide bacteriocinolactam plantaricin EF. plnE and plnF The gene-encoded plant lactobacillus EF is an important effector molecule in which *Lactobacillus plantarum* exerts its antibacterial activity. Its mechanism of action involves disrupting the cell membrane integrity of pathogenic bacteria, leading to leakage of intracellular substances and ultimately causing bacterial lysis and death. Studies have confirmed that plant lactobacillus EF has significant inhibitory effects on various foodborne pathogens, including *Listeria monocytogenes*, *Salmonella*, and *Staphylococcus*. Furthermore, this bacteriocin can be degraded into amino acids by proteases, without inducing drug resistance in target strains, demonstrating its potential as a substitute for traditional antibiotics.

[0076] Table 5. Statistics of annotations in general databases

[0077] Example 4: Application of Lactobacillus plantarum OBM7 in the treatment of lamb diarrhea 4.1 Test Methods This experiment was conducted at a Tibetan sheep farm in Haibei Prefecture, Qinghai Province, where 70% of the lambs exhibited varying degrees of diarrhea. Fecal PCR testing and pathogen isolation revealed varying degrees of infection with pathogenic Escherichia coli, Salmonella enterica, and Campylobacter jejuni. Clostridium perfringens was detected and isolated from the intestines of some lambs with bloody stools. Affected lambs exhibited yellowish-white or grayish-green loose stools, watery stools, bloody stools in some cases, emaciation, and lethargy. 120 lambs with diarrhea were randomly selected and divided into four groups: a control group, an OBM7 group, a compound probiotic group, and a gentamicin treatment group, with 30 lambs in each group and numbered. The control group was fed a normal diet; the OBM7 group received the same diet as the control group, supplemented with Lactobacillus plantarum OBM7 (6×10⁻⁶). 9 CFU / animal); the compound group, in addition to the control group, received a compound probiotic preparation (6×10 CFU each of Lactobacillus plantarum OBM7, Bacillus subtilis BM3, and Lactobacillus paracasei BM2). 9 CFU / lamb; Gentamicin group, with an injection dose of 4 mg / kg body weight. The experiment lasted for 7 days. The dryness and wetness of the lambs around the anus, the form of their feces, their feed intake, and their mental state were observed daily. During the experiment, all groups had the same basal diet and free access to water.

[0078] 4.2 Treatment Results The statistical results of the treatment effects on lamb diarrhea are shown in Table 6. In the control group, the mortality rate of 30 lambs was 16.7% (5 / 30), with all deaths occurring within the experimental period. Three lambs died on day 3 and two on day 4. No lambs recovered in this group. All affected lambs exhibited significant weight loss, lethargy, and a continuous worsening of clinical symptoms. The survival rate of lambs in the OBM7 group was 100%, and the diarrhea cure rate was 83.3%. During the experiment, the feed intake of all lambs in the group increased, and their mental state improved significantly. The remaining five lambs that still had loose stools after day 5 recovered after continuous feeding with OBM7 until day 8. The survival rate of lambs in the compound probiotic group was also 100%, and the diarrhea cure rate was as high as 93.03%. All tested lambs showed a significant improvement in feed intake and their mental state fully returned to normal. The remaining two lambs that still had loose stools after day 5 recovered after continuous feeding with OBM7 until day 6. The mortality rate of lambs in the gentamicin treatment group was 3.3% (1 / 30), with deaths occurring on day 3 of the experiment. The cure rate for diarrhea in this group was 83.3%. Of the remaining 4 lambs with loose stools after day 5, 2 showed significant improvement in their mental state, while the other 2 continued to exhibit mild depression, lying down and unwilling to move, with a significant decrease in feed intake.

[0079] In summary, both the single-dose Lactobacillus plantarum OBM7 preparation and the compound probiotic preparation have significant therapeutic effects on lamb diarrhea, and the compound probiotic preparation is significantly more effective than the single-dose OBM7 preparation.

[0080] Table 6 Results of probiotic preparations in treating lamb diarrhea

[0081] Note: 1 * This indicates that the lamb had watery stool before it died; (17) 5 * This indicates that the five lambs had watery stools before they died.

[0082] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0083] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0084] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A strain of Lactobacillus plantarum Lactiplantibacillus plantarum OBM7, characterized in that, The strain was deposited on November 12, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Courtyard 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC NO. 67282.

2. A probiotic preparation, characterized in that, Includes the Lactobacillus plantarum as described in claim 1 and / or the metabolites of the strain.

3. A feed additive, characterized in that, The ingredients include the microbial agent as described in claim 2.

4. A composition for preventing and treating diarrhea in lambs, characterized in that, The composition comprises Bacillus subtilis, Lactobacillus paracasei, and Lactobacillus plantarum OBM7 as described in claim 1.

5. The composition according to claim 4, characterized in that, The ratio of *Lactobacillus plantarum* OBM7, *Bacillus subtilis*, and *Lactobacillus paracasei* is 1:1~2:1~2.

6. The use of Lactobacillus plantarum OBM7 according to claim 1, or the microbial agent according to claim 2, or the feed additive according to claim 3, or the composition according to claim 4 or 5, in any of the following: (1) Application in the preparation of products that inhibit pathogenic bacteria; among which, The pathogenic bacteria include at least one of the following: Escherichia coli, Proteus vulgaris, Shigella boydii, Yersinia enterocolitica, Salmonella typhimurium, Salmonella enterica, Clostridium perfringens, Campylobacter jejuni, Mycoplasma bovis, Mycoplasma capsulatum, Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa. (2) Application in the preparation of products for the prevention and treatment of animal diarrhea; (3) Application in the preparation of products that increase animal feed intake.

7. The application according to claim 6, characterized in that, The animal in question is a lamb.

Images