Lactobacillus plantarum NX87 and application thereof

Abstract

The invention provides lactobacillus plantarum NX87 and application thereof, the lactobacillus plantarum NX87 is lactobacillus plantarum NX87, the lactobacillus plantarum NX87 is preserved in the Guangdong Microbial Culture Collection Center on April 23, 2025, the address is No.59 building, No.100 courtyard, Xianlie Middle Road, Guangzhou, and the preservation number is GDMCC No. 66203; the lactobacillus plantarum NX87 is used for preventing and treating the streptococcus suis, the escherichia coli and the balloon mycosis, and the lactobacillus plantarum NX87 is used for preventing and treating the streptococcus suis, the escherichia coli and the balloon mycosis. The strain disclosed by the invention has relatively strong inhibition capability on streptococcus suis, escherichia coli and balloon mycosis, has relatively strong tolerance to acid, has good cholate tolerance, and is beneficial to colonization at parts such as intestinal mucosa and the like.

Description

Technical Field

[0001] This invention relates to the field of microbiology, specifically to a Lactobacillus plantarum NX87 and its applications. Background Technology

[0002] Antibiotics, used extensively in livestock farming for the prevention and treatment of bacterial diseases, have led to serious problems such as the spread of drug-resistant genes, gut microbiota dysbiosis, and environmental pollution, while improving production efficiency in the short term. In the global pig farming sector, the long-term widespread use and even abuse of antibiotics is common. This has not only resulted in the proliferation of drug-resistant strains in farms but also frequent drug residue problems, seriously threatening the quality and safety of pork products and consumer health. Against this backdrop, antibiotic alternatives such as probiotics, enzymes, plant extracts, acidifiers, and antimicrobial peptides are gradually being applied. Among these, antibiotic alternative strategies centered on probiotics and their metabolites have become a current research hotspot. Probiotics, as beneficial live microorganisms, are widely used in feed additives, mainly including genera such as Bifidobacterium, Lactobacillus, and Bacillus.

[0003] Probiotics and their metabolites (such as short-chain fatty acids and bacteriocins) exert their effects through multiple mechanisms, including participating in digestive reactions, regulating immune function, promoting intestinal morphology development, and maintaining the balance of the gastrointestinal microbiota. They can reduce the colonization rate of pathogenic bacteria and have functions such as enhancing immunity and optimizing metabolism. Among them, *Lactobacillus plantarum* (… 植物乳杆菌 As an important probiotic, *Lactobacillus plantarum* plays a positive role in maintaining animal health. On the one hand, it can stimulate the intestinal immune system to activate gut-associated lymphoid tissue, enhance the function of the innate and humoral immune systems, and improve the host's disease resistance. On the other hand, the organic acids, extracellular polysaccharides, and bacteriocins produced by *Lactobacillus plantarum* all have certain antibacterial and antimicrobial activities, and are the main substances that replace antibiotics among beneficial bacteria. Previous studies have found that feeding pigs with feed additives made from *Lactobacillus plantarum* allows it to easily colonize the pig's intestines, activate its digestive enzyme activity, improve feed conversion rate, and enhance the growth performance of pigs. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a strain of Lactobacillus plantarum NX87 and its application. This strain has a strong inhibitory effect on swine diseases, a strong tolerance to acid, and a good tolerance to bile salts, which is beneficial for colonization in the intestinal mucosa and other parts of the body.

[0005] This invention is implemented as follows: A type of Lactobacillus plantarum NX87, wherein Lactobacillus plantarum NX87 is Lactobacillus plantarum ( 乳酸植物杆菌属 植物NX87 was deposited on April 23, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No. 66203.

[0006] Furthermore, the Lactobacillus plantarum ( 植物乳杆菌 NX87 is used to prevent and treat diseases caused by Streptococcus suis, Escherichia coli, or Polygonococcus suis.

[0007] Furthermore, the Lactobacillus plantarum ( 植物乳杆菌 NX87 is used to prepare antimicrobial agents for the prevention and treatment of streptococcal, swine coli, or swine balloon bacteria diseases.

[0008] Furthermore, the Lactobacillus plantarum ( 植物乳杆菌 NX87 is used to prepare fermented feed with beneficial bacteria for pigs.

[0009] The present invention has the following advantages: The present invention contains Lactobacillus plantarum ( 植物乳杆菌 NX87 exhibits strong inhibitory activity against Streptococcus suis, Escherichia coli, and Lactobacillus suis, demonstrates strong acid tolerance, and possesses good bile salt tolerance, which is beneficial for colonization in the intestinal mucosa and other sites. This invention relates to *Lactobacillus plantarum* (…). 植物乳杆菌 NX87 can be used to regulate the intestinal microecological balance of pigs, enhance non-specific immune function to prevent diseases, and also provide nutritional factors, promote the digestion and absorption of nutrients, promote animal growth and improve feed conversion rate. Attached Figure Description

[0010] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0011] Figure 1 The image shows the Gram staining oil immersion microscopic examination results (100×) of an embodiment of the present invention, where A: NX87 colony morphology, and B: NX87 Gram staining microscopic examination strain morphology.

[0012] Figure 2 This is a schematic diagram illustrating the phylogenetic analysis of the isolated strains in an embodiment of the present invention.

[0013] Figure 3 This is a schematic diagram illustrating the in vitro antibacterial results of the isolated strains in an embodiment of the present invention.

[0014] Figure 4 This is a schematic diagram of the in vitro antibacterial results of the isolated strain after acid removal in an embodiment of the present invention. Detailed Implementation

[0015] This invention relates to a *Lactobacillus plantarum* NX87, wherein *Lactobacillus plantarum* NX87 is *Lactobacillus plantarum* (… 植物乳杆菌NX87 was deposited on April 23, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No. 66203.

[0016] The plant lactobacillus ( 植物乳杆菌 NX87 is used to prevent and treat diseases caused by Streptococcus suis, Escherichia coli, or Polygonococcus suis in pigs.

[0017] The plant lactobacillus ( 植物乳杆菌 NX87 is used to prepare products containing Streptococcus suis, Escherichia coli, and Polysaccharidosis suis, or for the prevention and treatment of diseases.

[0018] The plant lactobacillus ( 植物乳杆菌 NX87 is used to prepare fermented feed with beneficial bacteria for pigs.

[0019] The following will be combined with the appendix Figures 1-4 The technical solution of the present invention will be clearly and completely described in detail with specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0020] 1. Materials and Methods 1.1 Sample The pickled vegetables were purchased from a market in Ningxia Hui Autonomous Region; Escherichia coli, Streptococcus, and Lactobacillus were isolated, identified, and preserved from the intestinal contents of pigs in Zhangzhou, Fujian Province, by the Intestinal Microbiology Research Center of the Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Sciences. The strains obtained from the Ningxia yogurt were identified as all being Lactobacillus plantarum.

[0021] 1.2 Reagents and Instruments LB medium (Solarbio Cat#M8540), MRS broth medium (Solarbio Cat#M8540); bacterial genomic DNA rapid extraction kit (Tiangen Biotech); nucleic acid dyes and Gram staining reagents (Qingdao Haibo); antibiotic susceptibility testing tablets (Hangzhou Microbial Reagent Co., Ltd.); multi-functional microplate reader (SynergyH1 M); nucleic acid gel imaging system (BG-gdsAUTO520); PCR instrument (Bio-Rad Laboratories ETC811).

[0022] 1.3 Isolation of Lactobacillus plantarum Yogurt samples collected from Guyuan City, Ningxia Hui Autonomous Region, were sealed and preserved to avoid contamination from other microorganisms in the environment. Using an aseptic workbench, kimchi tissue samples were taken and added to 10 mL of MRS broth medium to obtain a yogurt lactic acid bacteria enrichment solution. Continuous liquid gradient dilution was performed to reduce interference from other microorganisms and improve isolation efficiency. The solution was then evenly spread on MRS solid medium and incubated at 35 ℃ for 24 h. Single colonies were picked, glycerol was added, and the samples were stored at -80 ℃.

[0023] 1.4 Purification and culture of isolated strains The preserved samples were inoculated into MRS liquid culture medium under sterile conditions and incubated in a 35 ℃ constant temperature shaking incubator for 4 h for pre-proliferation. Then, the separated bacterial suspensions after serial dilution were streaked onto MRS agar plates and incubated at 35 ℃ for more than 24 h. Single colonies were picked and inoculated again into MRS liquid culture medium for expansion culture. The isolated strains were then subjected to repeated streak purification culture (under the same conditions) to obtain purified single colonies. One plant lactobacillus strain was numbered NX87.

[0024] 1.5 Morphological observation Using the conventional Gram staining method, one strain of *Lactobacillus* was stained sequentially, and its cell morphology was observed using an optical microscope to make a preliminary identification of the strain.

[0025] 1.6 Physiological and Biochemical Characteristics Analysis The physiological and biochemical characteristics of the strains were determined according to the methods described in "Classification, Identification and Experimental Methods of Lactic Acid Bacteria". Microbial identification tubes were used in accordance with the instruction manual, and the results were recorded and interpreted.

[0026] 1.7 Molecular biological identification of 16S rDNA Bacterial DNA was extracted using a bacterial genomic DNA extraction kit for PCR amplification. Based on the bacterial genome sequence, universal primers for bacterial PCR identification were used: 27F (5'-AGA GTT TGA TCC TGG CTC AG-3') (as shown in SEQ ID NO. 1) and reverse primer 1492R (5'-TAC GGC TAC CTT GTT ACG ACT T-3') (as shown in SEQ ID NO. 2), synthesized by Platinum Biotech Co., Ltd. PCR reaction parameters were set as follows: 95 ℃ pre-denaturation for 5 min, 95 ℃ for 30 s (denaturation), 52 ℃ for 30 s (annealing), and 72 ℃ for 5 min (extension), followed by storage at -4 ℃. The amplified products were purified by 1% gel electrophoresis and gel recovery, and then sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. Sequencing results were compared with the NCBI database, and a phylogenetic tree of the strains was constructed using the Neighbor-joining method with MEGA11 software. In addition, whole-genome sequencing was performed on NX87, and the 16S rDNA gene sequence was extracted from the whole genome and compared with the reference sequence in the public database NCBI. By calculating sequence homology, the strain was identified as Lactobacillus plantarum.

[0027] 1.8 Evaluation of the beneficial characteristics of isolated strains 1.8.1 Antibacterial test of isolated strains After reviving the NX87 isolate, the supernatant was collected by centrifugation. An agar diffusion assay was used to test the antibacterial activity of the isolated strain against three common harmful bacteria in the pig intestine: Streptococcus suis, Escherichia coli, and Polygonococcus suis. The pathogenic bacteria were spread onto LB agar plates, which were then perforated with wells spaced at least 25 mm apart and at least 15 mm from the edge of the plate. 200 μl of the supernatant from each NX87 isolate was added to the well, and the plates were incubated at 28°C for 6 hours. The diameter of the inhibition zone was then measured and recorded.

[0028] 1.8.2 Bile salt tolerance test After serial dilution, the isolated strains were inoculated into MRS liquid medium containing 0.1%, 0.3%, and 0.5% bile salts by mass, and incubated at 100 r / min and 35 ℃ for 4 h before plate counting.

[0029] 1.8.3 Acid resistance test After serial dilution, the isolated strains were inoculated into MRS liquid medium with pH values ​​of 3, 7, and 8, and incubated at 100 r / min and 35 ℃ for 4 h before plate counting.

[0030] 1.8.4 High Temperature Resistance Test The isolated strains were serially diluted and inoculated into MRS liquid medium. The bacterial suspensions were incubated in water baths at 35 ℃, 45 ℃, and 75 ℃ for 30 min before plate counting.

[0031] 1.9 Statistical Analysis All data were calculated from the average of three parallel experiments. Data analysis and plotting were performed using SPSS Statistics 22 and Graphpad Prims 2021, and expressed as mean ± standard deviation. Duncan's multiple comparison method was used to compare the significance of differences (***). P ≤ 0.001, ** P ≤ 0.01, * P≤ 0.05, ns P> 0.05).

[0032] 2 Results 2.1 Bacterial Isolation and Identification Samples were plated on MRS plates, and after isolation, purification, and 16S rDNA sequencing, a strain of *Lactobacillus plantarum* with high homology was selected. Gram staining revealed that the colonies were purple, round, smooth, and with even edges; the bacteria were rod-shaped or short rod-shaped positive bacteria. Figure 1 .

[0033] 2.2 Identification of isolated strains The 16S rDNA gene of the target strain was amplified using universal primers, yielding a product of approximately 1200 bp. The sequencing results were then compared against the NCBI database using BLAST. The target strain was selected and numbered NX87, with all corresponding homologous strains being *Lactobacillus plantarum*. Bacteria with high similarity to the target strain were selected from the NCBI comparison results to construct a phylogenetic tree for *Lactobacillus plantarum* strain NX87. Figure 2 The results showed that NX87 was distantly related to other Lactiplantibacillus species, but closely related to Lactiplantibacillus plantarum strain TMPC 3M325. Furthermore, whole-genome sequencing of NX87 confirmed it to be a Lactiplantibacillus species. 植物乳杆菌 (SAMN49831394) and uploaded it to NCBI. 2.3 In vitro antibacterial test

[0034] 2.3.1 In vitro bacterial supernatant antibacterial test The in vitro inhibition test results of Lactobacillus plantarum NX87 are as follows: Figure 3As shown, the NX87 isolate had an inhibitory effect on all three strains of bacteria (Streptococcus S1, Escherichia coli E1, and Balloonella A1), with inhibition zone diameters of 22 mm ± 0.52 mm, 21 mm ± 0.52 mm, and 20 mm ± 0.52 mm, respectively.

[0035] 2.3.2 Antibacterial test of adjusting the pH of NX87 supernatant After adjusting the pH of the *Lactobacillus plantarum* NX87 supernatant to above 5 to remove the influence of acid on the indicator bacteria, the antibacterial effect of the inhibition zone of NX87 was slightly reduced, but it still had a certain antibacterial effect. The diameters of the inhibition zones were 18 mm ± 0.52 mm, 17 mm ± 0.52 mm, and 16 mm ± 0.52 mm, respectively. Figure 4 As shown.

[0036] 2.4 Bile salt tolerance The results of the bile salt tolerance test for NX87 isolates are shown in Table 1. As the bile salt concentration in the culture medium increased, the viable bacterial concentration gradually decreased. When the bile salt concentration increased to 0.3%, the isolates were still able to grow, indicating that *Lactobacillus plantarum* NX87 has a strong bile salt tolerance.

[0037] Table 1. Tolerance of isolated strains to different concentrations of bile salts

[0038] 2.5 pH tolerance The acid tolerance test results of Lactobacillus plantarum NX87 are shown in Table 2. After treatment in culture media with pH 7 and 8 for 4 h, the viable cell concentration reached 5.78 × 10⁻⁶ cells / mL in both media, as determined by plate count. 5 The above indicates that at pH 3, the proliferation of *Lactobacillus plantarum* strain NX87 decreased slightly, with its viable cell concentration dropping to 7.48 × 10⁻⁶. 4 The results show that the plant Lactobacillus strain NX87 has good acid resistance.

[0039] Table 2. Tolerance of Lactobacillus plantarum strain NX87 to different acidities.

[0040] 2.6 High Temperature Resistance After being treated at different temperatures for 30 min, *Lactobacillus plantarum* NX87 was analyzed by plate counting experiments. The results are shown in Table 3. NX87 grew after treatment at 35 ℃ and 55 ℃, but no growth was observed after treatment at 75 ℃. This result indicates that high-temperature treatment can affect the proliferation of NX87 to varying degrees. The experiment shows that the optimal growth temperature for *Lactobacillus plantarum* is around 35 ℃, and it can grow at temperatures around 55 ℃, but the growth rate and survival rate will be affected. Prolonged treatment at 75 ℃ is not suitable for its survival.

[0041] Table 3. Tolerance of isolated strains to different temperatures

[0042] 3. Discussion As is well known, most probiotics are ingested orally. Therefore, a probiotic strain should possess the ability to tolerate and maintain relative stability in the pH environment of the animal's gut. Previous studies have also shown that probiotics mostly exert their beneficial effects through the gastrointestinal tract; therefore, stability and resistance to stress within a certain acid-base environment (pH 3-8) are key to evaluating probiotics. The acid and heat resistance tests on the *Lactobacillus plantarum* strain NX87 in this study showed that it could proliferate and exhibit strong tolerance in MRS broth culture at pH 3, indicating strong acid tolerance. Furthermore, there was no significant difference in bacterial proliferation at pH 7-8. Bile salts have a destructive effect on bacterial membrane lipids, damaging bacterial membranes, denaturing proteins, chelating iron and calcium, and causing oxidative damage to DNA. To ensure the survival of potential probiotics under the extreme conditions of the gastrointestinal tract, the resistance of probiotics to bile salts is also a standard for evaluating their potential probiotic effects. The results of this study indicate that the isolated strain could still grow when the bile salt concentration increased to 0.3%, demonstrating that NX87 possesses bile salt tolerance. Secondly, this study also investigated the effect of temperature fluctuations on the proliferative activity of the probiotic. The results showed that after 30 minutes of high-temperature stress at 75℃ in a water bath, the proliferative activity of NX87 was close to 0 CFU / mL. These experimental results indicate that the four *Lactobacillus plantarum* strains are extremely sensitive to temperature, with the optimal growth temperature being around 35℃. Therefore, when handling these strains, attention should be paid to temperature changes, and exposure to high-temperature environments should be avoided as much as possible.

[0043] In this embodiment of the invention, the protective effect of the NX87 strain on pigs under harmful bacterial stress was evaluated by in vitro antibacterial test. The results showed that NX87 had significant antibacterial effects against Streptococcus suis, Escherichia coli, and Stella ovalis.

[0044] 4. Conclusion This invention isolated a target strain from yogurt fermentation products in Ningxia Hui Autonomous Region. After PCR amplification, 16S rDNA sequencing, and physiological and biochemical identification, the strain was confirmed to be *Lactobacillus plantarum*. The strain exhibited high antibacterial activity and strong tolerance, demonstrating varying degrees of resistance to different environmental conditions. The supernatant of these strains showed significant in vitro inhibitory effects against *Streptococcus suis*, *Escherichia coli*, and *Gastroenterobacter*, with strain NX87 showing the most significant effect against *Streptococcus*. Therefore, strain NX87 can serve as a candidate strain for the prevention and control of swine diseases and may play an important role in future swine farming.

[0045] While specific embodiments of the present invention have been described above, those skilled in the art should understand that the specific embodiments described are merely illustrative and not intended to limit the scope of the present invention. Equivalent modifications and variations made by those skilled in the art in accordance with the spirit of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A type of Lactobacillus plantarum NX87, characterized in that: The *Lactobacillus plantarum* NX87 is *Lactobacillus plantarum* (… Lactiplantibacillus plantarum NX87 was deposited on April 23, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No. 66203.

2. The *Lactobacillus plantarum* NX87 according to claim 1, characterized in that: The plant lactobacillus ( Lactiplantibacillus plantarum NX87 is used to prevent and treat diseases caused by Streptococcus suis, Escherichia coli, or Polygonococcus suis.

3. The *Lactobacillus plantarum* NX87 according to claim 1, characterized in that: The plant lactobacillus ( Lactiplantibacillus plantarum NX87 is used to prepare antimicrobial agents for the prevention and treatment of Streptococcus suis, Escherichia coli, or Polysacchariformis suis.

4. The *Lactobacillus plantarum* NX87 according to claim 1, characterized in that: The plant lactobacillus ( Lactiplantibacillus plantarum NX87 is used to prepare fermented feed for pigs with beneficial bacteria.

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