Lactobacillus plantarum NX69 and application thereof

Abstract

The invention provides lactobacillus plantarum NX69 and application thereof, the lactobacillus plantarum NX69 is lactobacillus plantarum NX69, the lactobacillus plantarum NX69 is preserved in the Guangdong Microbial Culture Collection Center on November 21, 2024, the address is No.59 building, No.100 courtyard, Xianlie Middle Road, Guangzhou, and the preservation number is GDMCC No.65517; the lactobacillus plantarum (Lactobacillus plantarum) NX69 is used for preventing and treating the abalone vibriosis, and the lactobacillus plantarum (Lactobacillus plantarum) NX69 can be used for preventing and treating the abalone vibriosis. The strain disclosed by the invention has relatively strong inhibition capability on abalone vibriosis, relatively strong tolerance on acid, good cholate resistance, good automatic agglutination capability and hydrophobicity, and is beneficial to colonization at parts such as intestinal mucosa and the like.

Description

[Technical field]

[0001] The present invention relates to the field of microorganisms, and in particular to a Lactobacillus plantarum NX69 and an application thereof. [Background technology]

[0002] Abalone farming, as an important part of marine fisheries, plays an important role in the development of coastal fishery economy. However, due to the presence of a large number of pathogenic microorganisms in the ocean, it poses a major threat to the health of abalone. Among them, Vibrio in the ocean is one of the main pathogens of abalone. Vibrio is widely present in the marine environment, easily invades the body of abalone, colonizes in the intestines of abalone and reproduces rapidly, causing an imbalance in intestinal flora and causing abalone disease. Sick abalone grow slowly, lose weight, and even die on a large scale. In order to prevent and control Vibrio disease in abalone, farmers mainly use antibiotics or antibacterial drugs. The use of these drugs not only increases the cost of farming, but also easily causes damage to the marine ecosystem. Therefore, in order to promote the sustainable development of abalone farming and maintain the stability of the marine ecosystem, strengthening the ecological prevention and control of abalone bacterial diseases has become the key to the healthy development of abalone farming.

[0003] The metabolites of probiotics, such as organic acids, exopolysaccharides, hydrogen peroxide, fatty acids, and bacteriocins, all have certain antibacterial and antimicrobial activities. In addition, probiotics can improve the health of animals and their growth performance by regulating intestinal flora. Among them, Lactobacillus plantarum, as one of the important probiotics, plays a positive role in maintaining animal health. On the one hand, it can stimulate the intestinal immune system to activate intestinal-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, exopolysaccharides, and bacteriocins produced by Lactobacillus plantarum all have certain antibacterial and antibacterial activities, and are the main substances that replace antibiotics in beneficial bacteria. Previous studies have found that when Lactobacillus plantarum is made into a feed additive and fed to abalone, it can easily colonize in the abalone intestine, activate its digestive enzyme activity, and improve the feed conversion rate. [Summary of the invention]

[0004] The technical problem to be solved by the present invention is to provide a Lactobacillus plantarum NX69 and an application thereof. The strain has a strong inhibitory ability against abalone Vibrio disease, a strong tolerance to acid, and a good bile salt resistance, and at the same time has good autoagglutination ability and hydrophobicity, which is conducive to colonization in the intestinal mucosa and other parts.

[0005] The present invention is achieved in that:

[0006] A Lactobacillus plantarum NX69, wherein the Lactobacillus plantarum NX69 is Lactobacillus plantarum (Lactiplantibacillus plantarum) NX69, which has been deposited in Guangdong Provincial Microbiological Culture Collection Center on November 21, 2024, with the address being Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number being GDMCC No.65517.

[0007] Furthermore, the Lactobacillus plantarum NX69 is used to prevent and treat Vibrio disease in abalone.

[0008] Furthermore, the Lactobacillus plantarum NX69 is used to prepare a bacterial agent product for preventing and treating abalone Vibrio disease.

[0009] Furthermore, the Lactobacillus plantarum NX69 is used to prepare abalone feed.

[0010] The present invention has the following advantages:

[0011] The plant lactobacillus (Lactiplantibacillus plantarum) NX69 of the present invention has a strong inhibitory ability against abalone vibriosis, has a strong tolerance to acid, has a good bile salt tolerance, and has a good autoagglutination ability and hydrophobicity, which is conducive to colonization in the intestinal mucosa and other parts; it is sensitive to antibiotics and can reduce the risk of bacterial antibiotic resistance transmission. The plant lactobacillus (Lactiplantibacillus plantarum) NX69 of the present invention can be used to regulate the microecological balance in the intestine of abalone, has the effect of enhancing nonspecific immune function to prevent diseases, and can also provide nutritional factors, promote the digestion and absorption of nutrients, promote animal growth and improve feed conversion rate.

Brief Description of the Drawings

[0012] The present invention will be further described below in conjunction with embodiments with reference to the accompanying drawings.

[0013] Figure 1 The figure is a Gram staining oil immersion microscopy result (100×) of an embodiment of the present invention, wherein A: NX69-1; B: NX69-2; C: NX69-3; D: NX69.

[0014] Figure 2 Schematic diagram of phylogenetic analysis of isolated strains according to an embodiment of the present invention.

[0015] Figure 3Schematic diagram of the in vitro antibacterial results of the isolated strains of the present invention, wherein A: NX69-1; B: NX69-2; C: NX69-3; D: NX69; E: negative control.

[0016] Figure 4 Schematic diagram of the in vitro antibacterial results of the isolated strains after acid excretion in an embodiment of the present invention, wherein A: NX69-1; B: NX69-2; C: NX69-3; D: NX69.

[0017] Figure 5 Schematic diagram of the determination of the minimum inhibitory concentration of MIC in an embodiment of the present invention, wherein A: NX69-1; B: NX69-2; C: NX69-3; D: NX69.

[0018] Figure 6 Schematic diagram of hemolytic activity of isolated strains according to an embodiment of the present invention, wherein A: NX69-1; B: NX69-2; C: NX69-3; and D: NX69.

[0019] Figure 7 Schematic diagram of biofilm identification of isolated strains according to an embodiment of the present invention, wherein A: NX69-1; B: NX69-2; C: NX69-3; D: NX69.

[0020] Figure 8 It is a schematic diagram of the automatic agglutination ability of 4 isolated bacteria in the examples of the present invention.

[0021] Fig. 9 Schematic diagram of the hydrophobicity of 4 isolated bacteria in the examples of the present invention, wherein 1: xylene; 2: chloroform; 3: ethyl acetate.

[0022] Fig.10 1 is a graph showing the mortality rate of abalone in three different treatment groups according to an embodiment of the present invention, wherein CK: control group; GD: challenge group; LP: treatment group. [Specific implementation method]

[0023] The present invention relates to a Lactobacillus plantarum NX69. The Lactobacillus plantarum NX69 is Lactobacillus plantarum NX69, which has been preserved in Guangdong Provincial Microbiological Culture Collection Center on November 21, 2024, with the address of Building 59, No. 100 Xianlie Middle Road, Guangzhou City, and the preservation number is GDMCC No.65517.

[0024] The Lactobacillus plantarum NX69 is used for preventing and treating abalone Vibrio disease.

[0025] The Lactobacillus plantarum NX69 is used for preparing abalone Vibrio disease prevention and treatment bacterial agent products.

[0026] The Lactobacillus plantarum NX69 is used for preparing abalone feed.

[0027] The following will be combined with the attached Figure 1-10 The technical scheme of the present invention is clearly and completely described in detail with specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0028] 1 Materials and methods

[0029] 1.1 Sample

[0030] Rice wine lees were purchased from a market in Ningde, Fujian Province; V. parahaemolyticus V-25, V. alginolyticus V-3, and V. natriegens V-103 were isolated, identified, and preserved from the intestinal contents of infected abalone in Pingtan, Fujian by the Intestinal Microbiology Research Center of Fujian Academy of Agricultural Sciences. The strains obtained from rice wine in Ningde, Fujian Province were identified as Lactobacillus plantarum.

[0031] 1.2 Reagents and instruments

[0032] 2216E culture medium (Hibio HB0132-1), MRS broth medium (Solarbio Cat#M8540); bacterial genomic DNA rapid extraction kit (Tiangen Biotechnology); nucleic acid dyes and Gram staining reagents (Qingdao Hibio); antibiotic susceptibility tablets (Hangzhou Microbiological Reagent Co., Ltd.); multi-function microplate reader (SynergyH1 M); nucleic acid gel imager (BG-gdsAUTO520), PCR instrument (Bio-Rad ETC811, USA).

[0033] 1.3 Isolation of Lactobacillus plantarum

[0034] The collected rice wine samples were sealed and stored to avoid contamination by other bacteria in the environment. Using a sterile operating table, filter the lees sample to remove larger impurities. Take 1 mL of lees sample and add 9 mL of sterile saline (0.85% NaCl solution) to obtain a 10-fold dilution. Perform continuous liquid gradient dilution to reduce the interference of miscellaneous bacteria and improve separation efficiency. Then evenly spread it on MRS solid culture medium; culture at 35°C for 24 hours, pick a single colony, add glycerol, and store it in a -80°C refrigerator.

[0035] 1.4 Purification and cultivation of isolated strains

[0036] The preserved samples were inoculated into MRS liquid culture medium under a sterile environment and placed in a constant temperature shaking box at 35°C for incubation for 4 hours for pre-proliferation; the isolated bacterial solution after gradient dilution was then streaked onto an MRS agar plate, cultured at 35°C for more than 24 hours, a single colony was picked and inoculated again into MRS liquid culture medium for expansion culture, and then the isolated strains were repeatedly streaked and purified (under the same conditions as before) to obtain purified single colonies; the four plant lactobacillus strains were numbered NX69-1, NX69-2, NX69-3 and NX69, respectively.

[0037] 1.5 Morphological observation

[0038] The four strains of Lactobacillus plantarum were stained in turn using conventional Gram staining, and their bacterial morphology was observed using an optical microscope for preliminary identification of the strains.

[0039] 1.6 Analysis of physiological and biochemical characteristics

[0040] According to the method described in "Lactic Acid Bacteria Classification and Identification and Experimental Methods", the physiological and biochemical characteristics of the strains were determined. Use the bacterial microbiochemical identification tube according to the instructions, record and determine the results. 1.716S rDNA molecular biological identification

[0041] Bacterial DNA was extracted using a bacterial genomic DNA extraction kit for PCR amplification. Referring to the bacterial genome sequence, the universal primers for bacterial PCR identification: 27F (5'-AGA GTT TGA TCC TGG CTCAG-3') (as shown in SEQ ID NO.1) and reverse primer 1492R (5'-TAC GGC TAC CTT GTTACGACT T-3') (as shown in SEQ ID NO.2) were used to synthesize at Boshang Biotechnology Co., Ltd. The parameters of the PCR reaction were set as follows: 95°C pre-denaturation for 5min, 95°C for 30s (denaturation), 52°C for 30s (annealing) and 72°C for 5min (extension), and stored at -4°C. The amplified product was purified by 1% gel electrophoresis and gel recovery, and sent to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing. The sequencing results were compared with the NCBI database, and then the Neighbor-joining method was selected by MEGA11 software to construct the phylogenetic tree of the strain. In addition, the whole genome of NX69 was sequenced, and the 16SrDNA gene sequence in the whole genome was extracted and compared with the reference sequence in the public database NCBI. By calculating the sequence homology, it can be determined that the strain is Lactobacillus plantarum.

[0042] 1.8 Evaluation of probiotic properties of isolated strains

[0043] 1.8.1 Antibacterial test of isolated strains

[0044] The concentrations of the four isolated bacteria were 3.5×10 9 CFU / mL, 2.7×10 9 CFU / mL, 8.2×10 8 CFU / mL and 4.2×10 9 CFU / mL was used to separate the bacterial supernatant, and the agar diffusion test was used to separate the strains for antibacterial test, and the antibacterial effect of Lactobacillus plantarum on three common harmful bacteria in the abalone intestine, Vibrio parahaemolyticus (V-25), Vibrio alginolyticus (V-3) and Vibrio natrii (V-103) was detected. The pathogenic bacteria were spread on a 2216E agar plate, and then the agar plate was punched with a puncher. The centers of the holes were separated by more than 25 mm and separated by more than 15 mm from the edge of the culture dish. 200 μl of supernatant was taken from each of the four strains of Lactobacillus plantarum, placed in the hole, and incubated in a constant temperature incubator at 28°C for 12 hours, and then the diameter of the inhibition zone was measured and recorded.

[0045] 1.8.2 MIC minimum inhibitory concentration determination

[0046] The concentration of the supernatant of the four isolated bacteria was the same as in 1.8.1, and the indicator bacteria were tested for antibacterial activity. The steps are as follows: Take a 96-well plate, add 200 μl of the supernatant to be tested in the first column (A1-H1), and add 100 μl of sterile MRS culture medium to each column from 2 to 12. Use a pipette to dilute the supernatant in the first column by 2 times in sequence, and set the 11th column and the 12th column as the positive control (only the indicator bacteria are added) and the blank control (only the culture medium is added), respectively. Before the bacterial culture begins, place the 96-well plate in a microplate reader, read and record the initial absorbance value (OD600initial) of each well. Place the 96-well plate in an incubator and record again (OD600final) after 24 hours of static culture at 28°C. Calculate the change in absorbance value (ΔOD600) of each well, that is, the final absorbance value minus the initial absorbance value.

[0047] ΔOD600=OD600final-OD600initial

[0048] 1.8.3 Determination of hemolytic activity of isolated strains

[0049] Take 4 plant lactobacillus isolated strains, streak inoculate on MRS agar plate, and after multiple purification, streak inoculate the bacterial solution on blood plate (Blood Agar Plate, BAP), place it in a 35°C incubator and incubate it for 24 hours, and observe the changes in the bacterial growth area and the surrounding blood red blood cells on the blood plate to determine the type of bacterial hemolysis. Judgment criteria: Bacteria form green or brown discolored areas on the culture medium, and red blood cells are partially destroyed but not completely dissolved, which is α hemolysis (partial hemolysis). Bacteria form a transparent clear area on the culture medium, and red blood cells are completely dissolved, which is β hemolysis (complete hemolysis). γ hemolysis (no hemolysis) There is no obvious hemolysis zone around the bacterial growth, and the color of the culture medium remains unchanged.

[0050] 1.8.4 Biofilm assay of isolated strains

[0051] Four strains of Lactobacillus plantarum were streaked onto MRS agar plates containing 0.8 g / L Congo red and incubated at 35°C for 36 h before observing the colony morphology. Judgment criteria: Bacteria that form biofilms usually form red or black colonies under the action of Congo red dye, while bacteria that do not form biofilms appear as pink or white colonies.

[0052] 1.8.5 Determination of drug susceptibility of isolated strains

[0053] The sensitivity of the four Lactobacillus plantarum isolates to 20 common antibiotics was detected using drug sensitivity sheets. The four Lactobacillus plantarum isolates were evenly spread on MRS agar plates, and then the drug sensitivity sheets were attached to the surface of the plates with a gap of 2 cm between the sheets. The plates were cultured in a 35°C incubator for 24 hours to observe whether an inhibition zone was produced.

[0054] 1.8.6 Bile salt tolerance test

[0055] The isolated strains were inoculated into MRS liquid culture medium containing 0.1%, 0.3% and 0.5% bile salt by mass fraction after gradient dilution, and cultured at 100 r / min and 35° C. for 4 h, and then the plate counts were performed.

[0056] 1.8.7 Acid resistance test

[0057] The isolated strains were inoculated into MRS liquid culture medium with pH values ​​of 3, 7, and 8 after gradient dilution, and cultured at 100 r / min and 35°C for 4 h before plate counting.

[0058] 1.8.8 High temperature resistance test

[0059] The isolated strains were inoculated into MRS liquid culture medium after gradient dilution, and the bacterial liquid was water bathed at 35℃, 45℃ and 75℃ for 30min before plate counting.

[0060] 1.9 Determination of adhesion of isolated strains

[0061] 1.9.1 Self-agglutination

[0062] The detailed steps are as follows: Cultivate the bacterial solution to be tested for 12 hours to the logarithmic growth phase, centrifuge at 4000rpm / min for 10 minutes to collect the bacterial precipitate, and wash the bacterial precipitate twice with sterile PBS to remove the culture medium residue. Resuspend the bacteria with PBS and measure its OD600 value (initial absorbance) using a spectrophotometer. Vortex and oscillate for 30 seconds, and then take the bacterial solution to measure the OD600 value (absorbance change) at 35°C for 1 hour, 2 hours, 3 hours, and 4 hours, and calculate the percentage of self-agglutination.

[0063]

[0064] 1.9.2 Hydrophobicity

[0065] Bacterial hydrophobicity determination: The bacterial solution to be tested was cultured for 12 hours to the logarithmic growth phase, centrifuged at 4000rpm for 10 minutes to collect the bacterial precipitate, and the bacterial precipitate was washed twice with sterile PBS to remove the culture medium residue. The bacteria were resuspended in PBS and measured at OD600 value (initial absorbance) using a spectrophotometer. Equal volumes of xylene, chloroform, and ethyl acetate were added respectively, vortexed for 2 minutes to ensure full contact between the two phases, and allowed to stand at room temperature for 15-30 minutes. After stratification, the aqueous phase was taken, and the OD600 value (final absorbance) was measured to calculate the hydrophobicity.

[0066]

[0067] 1.10 Animal Pathogenicity Test

[0068] 1.10.1 Breeding conditions

[0069] The abalone used in this experiment were all taken from the offshore abalone breeding base in Xiapu County, Ningde City, Fujian Province. The experiment selected abalone seedlings from the same batch with good vitality and normal appearance, with similar weight specifications (19±1.5g). They were placed in the breeding box, and the incubator was placed separately in the breeding pond to ensure environmental safety.

[0070] 1.10.2 Preparation of bacterial solution

[0071] In this experiment, Vibrio parahaemolyticus (V-25), which is highly pathogenic to abalone, was selected as the test challenge strain. The strain was derived from the intestinal tissue of the diseased Haliotis discus, and was isolated, identified and purified using the 2216E selective medium. The purified Vibrio parahaemolyticus was then stored at -80°C for later use. When preparing the bacterial solution for the challenge test, the stored Vibrio parahaemolyticus strain was thawed at room temperature and cultured at 28°C for 8 hours. Subsequently, the concentrations of the Vibrio parahaemolyticus and Lactobacillus plantarum (NX69) solutions were adjusted to 5×10 7 CFU / mL, and after centrifugation at 8000r / min, rinsed twice with PBS water to remove impurities in the culture medium.

[0072] 1.10.3 Vibrio parahaemolyticus stress test

[0073] The volume of each layer of the breeding cage in this experiment was 40*30*12 (cm), and one group was raised on each layer, with 20 abalones in each group (n=20), and the water volume was 15L. The abalones were divided into 3 groups, with 3 replicates in each group (a total of 9 test boxes, 180 abalones), namely the control group CK: injected with 50μl of PBS sterile water, the challenge group GD: injected with a concentration of 5×10 7 CFU / mL centrifuged and rinsed bacterial solution 50μl, treatment group LP: after the abalone was challenged with poison, 50μl of 5×10 7 The centrifuged and rinsed Lactobacillus plantarum liquid with CFU / mL was injected at the same site. The injection position and depth of each abalone were ensured to be similar. After the injection operation was completed, the abalone was placed in a breeding box and observed at 3, 6, 12, 24, 36, 48, and 72 hours, and the abalone mortality rate was recorded and analyzed.

[0074] 1.11 Statistical analysis

[0075] All data were calculated from 3 parallel experiments and analyzed using SPSS Statistics 22 and Graphpad Prims 2021, respectively, and presented as mean ± standard deviation. Duncan's multiple comparison method was used to compare and analyze the significance of differences (***P≤0.001, **P≤0.01, *P≤0.05, ns P>0.05).

[0076] 2 Results

[0077] 2.1 Bacterial isolation and identification

[0078] The samples were spread on the MRS plate, and four strains of plant lactobacillus with high homology were selected after separation, purification and 16s rDNA sequencing. After Gram staining, the colonies were observed to be purple and round, with smooth surfaces and flat edges; the bacteria were rod-shaped or short rod-shaped positive bacteria, such as Figure 1 .

[0079] 2.2 Identification of isolated strains

[0080] 2.2.1 Physiological and biochemical identification

[0081] The biochemical identification results of the four strains of Lactobacillus plantarum are shown in Table 1. The four strains were positive for esculin, maltose, mannitol, salicylic acid, cellobiose, lactose, raffinose and inulin, NX69-1 was negative for sorbitol, and the other three strains were positive.

[0082] Table 1 Biochemical characterization of isolated strains

[0083]

[0084] Note: +, positive; -, negative

[0085] 2.2.2 PCR identification

[0086] The 16S rDNA gene of the target strain was amplified by universal primers, and the product was about 1500bp. The sequencing results were compared by Blast in the NCBI database, and four target strains were selected and numbered NX69-1, NX69-2, NX69-3 and NX69, and their corresponding homologous strains were all Lactobacillus plantarum. In the NCBI comparison results, bacteria with higher similarity to the target strain were selected to draw a phylogenetic tree of the four Lactobacillus plantarum strains ( Figure 2). The results showed that NX69-1 was distantly related to the other three isolates, while NX69-2 and NX69 were closely related. In addition, the whole genome sequencing results of NX69 confirmed that it was Lactobacillus plantarum and uploaded to NCBI. (Accession: PRJNA1166953)

[0087] 2.3 In vitro antibacterial test

[0088] 2.3.1 In vitro bacterial supernatant antibacterial test

[0089] The results of in vitro inhibition test of beneficial bacteria on Vibrio parahaemolyticus V-25, Vibrio alginolyticus V-3 and Vibrio natrii V-103 are as follows Figure 3 As shown: the four strains of Lactobacillus plantarum isolates had inhibitory effects on the three strains of Vibrio (V-25, V-3 and V-103), and the diameters of their inhibition zones were 13mm±0.52mm, 20mm±0.52mm, 18mm±0.52mm and 13mm±0.52mm, respectively.

[0090] 2.3.2 Antibacterial test of bacterial supernatant under different pH conditions

[0091] Adjust the pH of the Lactobacillus plantarum supernatant to above 5, and remove the effect of acid on the indicator bacteria. The inhibition zone will shrink or disappear. Figure 4 shown.

[0092] 2.3.3 MIC minimum inhibitory concentration determination

[0093] The supernatant of four plant lactobacillus strains was taken at 8000r / min and the antibacterial test was performed on the indicator bacteria. Figure 5 As shown, the results showed that after four times of two-fold dilution (supernatant concentration was 0.0625), the supernatants of the four plant lactobacillus strains significantly inhibited the indicator bacteria, and after five dilutions, except for the NX69 supernatant that had an inhibitory effect on the indicator bacteria, the indicator bacteria treated with the other supernatants grew rapidly, indicating that the MICs of NX69-1, NX69-2, and NX69-3 were 0.0625, and the MIC of NX69 was 0.3125.

[0094] 2.4 Identification of hemolytic activity of isolated strains

[0095] Four strains of Lactobacillus plantarum were inoculated into blood plates and cultured for 24 hours. The colonies were observed to be grayish white with no hemolytic ring around them, and they were judged to be γ-hemolytic bacteria, i.e., non-hemolytic (e.g. Figure 6 shown).

[0096] 2.5 Biofilm assay of isolated strains

[0097] like Figure 7As shown, after the four strains of Lactobacillus plantarum were inoculated on Congo red MRS plates and cultured for 24 hours, milky white round colonies appeared, and no black colonies were seen, indicating that the four strains of Lactobacillus plantarum did not produce biofilms.

[0098] 2.6 Determination of drug sensitivity of isolated strains

[0099] In this study, 20 common clinical antibiotics were selected to conduct multiple tests on 4 strains of Lactobacillus plantarum. The results are shown in Table 2. Most of the isolated strains were sensitive to cephalosporin antibiotics (except cephalexin). The 4 isolated strains were resistant to tetracycline, neomycin, kanamycin, cephalexin and oxacillin, and were sensitive to cefuroxime, piperacillin, carbenicillin and ampicillin.

[0100] Table 2 Sensitivity test of isolated strains to different drugs

[0101]

[0102]

[0103] Note: The diameter of the inhibition zone is greater than 1.0 cm, which is sensitive (S), and less than 1.0 cm, which is resistant (R).

[0104] 2.7 Stress resistance results of isolated strains

[0105] 2.7.1 Bile Salt Tolerance

[0106] The bile salt tolerance test results of the four isolated strains are shown in Table 3. As the bile salt concentration in the culture medium increases, the concentration of viable bacteria gradually decreases. When the bile salt concentration increases to 0.3%, the isolated strains can still grow, indicating that the four plant lactobacillus strains have strong bile salt tolerance.

[0107] Table 3 Tolerance of isolated strains to different concentrations of bile salts

[0108]

[0109] 2.7.2 pH tolerance

[0110] The results of the acid resistance test of the four plant lactobacillus strains are shown in Table 4. After being treated in the medium with pH values ​​of 7 and 8 for 4 h, the viable bacterial concentrations of the four strains could reach 5.78×10 4 When the pH value was 3, the proliferation of the four plant lactobacillus strains decreased slightly, and their viable bacterial concentration decreased to 5.43×10 5 Below, the results show that the four Lactobacillus plantarum strains have good acid resistance.

[0111] Table 4 Tolerance of four Lactobacillus plantarum strains to different acidities

[0112]

[0113] 2.7.3 High temperature tolerance

[0114] After the four strains of Lactobacillus plantarum were treated at different temperatures for 30 minutes, the plate count test was performed. The results are shown in Table 5. After the bacterial solution was treated at 35°C and 55°C, the four strains of Lactobacillus plantarum were able to grow; after the treatment at 75°C, the four strains of Lactobacillus plantarum did not grow. The results show that high temperature treatment can affect the proliferation of the four strains of Lactobacillus plantarum to varying degrees. The experiment showed that the optimal growth temperature of Lactobacillus plantarum is around 35°C, and it can grow at a high temperature of around 55°C, but the growth rate and survival rate will be affected. At a high temperature of 75°C, long-term treatment is not suitable for its survival.

[0115] Table 5 Tolerance of isolated strains to different temperatures

[0116]

[0117] 2.8 Determination of adhesion of isolated strains

[0118] 2.8.1 Autoagglutination ability

[0119] The autoagglutination ability is closely related to the adhesion performance of bacteria. The autoagglutination ability test results of four strains of Lactobacillus plantarum are as follows: Figure 8 As shown: the autoagglutination rate of the four plant lactobacillus strains gradually increased over time, among which NX69-1 increased from 6.05% to 22.34%, and NX69-2 increased from 55.29% to 65.94%; NX69-3 and NX69 had little difference, increasing from 2.66, 5.67 to 7.77 and 11.47 respectively. The autoagglutination ability of the four plant lactobacillus strains generally showed an upward trend within 4 hours, among which NX69-2 had a stronger agglutination ability, significantly higher than the other three strains of bacteria. There were significant differences between NX69-1 at 2h, NX69-3 at 1h and 2h, and NX69 at 1h and 4h (P<0.05), and there was no statistical difference in the autoagglutination ability at other time points (P>0.05).

[0120] 2.8.2 Surface hydrophobicity

[0121] The surface hydrophobicity test results of the four Lactobacillus plantarum strains are as follows: Fig. 9 As shown in the results, the hydrophobicity of NX69-1 and NX69 in the non-polar solvent xylene was as high as 95%, and NX69-3 also reached 94.31%, followed by the alkaline solvent ethyl acetate, and the lowest in the acidic solvent chloroform; NX69 had a better hydrophobicity in chloroform; NX69-3 had a higher hydrophobicity in ethyl acetate, and there was no significant difference with xylene overall. The hydrophobicity of the four Lactobacillus plantarum strains in xylene and ethyl acetate was significantly different (P<0.05).

[0122] 2.9 Pathogenicity test results

[0123] After the V-25 isolate was inoculated into abalone, all of them showed symptoms such as decreased vitality and decreased appetite. Deaths began to occur 3 hours after inoculation. Fig.10 :After 3 days of V-25 infection, the mortality rates of the sterile control group, infection group and treatment group were 15%, 70% and 50%, respectively. This showed that the survival rate of abalone after treatment was significantly higher than that of the infection group (P<0.05). The above results showed that NX69 could resist Vibrio parahaemolyticus in abalone and reduce the mortality rate of Vibrio parahaemolyticus.

[0124] 3 Discussions

[0125] As we all know, most probiotics are taken orally. Therefore, a probiotic should be able to tolerate environmental changes in pH in the animal intestine and remain relatively stable. Previous studies have also shown that probiotics mostly exert their beneficial effects through the gastrointestinal route. Therefore, stability and stress resistance in a certain acid-base environment (pH3-8) are the key to evaluating probiotics. The results of the acid resistance, bile salt resistance and temperature resistance tests of four plant lactobacillus strains in this study showed that all four plant lactobacillus strains could proliferate in MRS broth culture at pH 3 and had strong tolerance, indicating that they all had strong tolerance to acid, and at pH 7-8, there was no significant difference in bacterial proliferation. Bile salts have a destructive effect on bacterial membrane lipids. They can destroy bacterial membranes, denature proteins, chelate iron and calcium, and cause oxidative damage to DNA. In order to ensure that potential probiotics can survive in extreme conditions in the gastrointestinal tract, the resistance of probiotics to bile salts is also a criterion for testing their potential probiotic effects. The results of this study showed that when the bile salt concentration increased to 0.3%, the isolated strains were still able to grow, indicating that the four strains of Lactobacillus plantarum were resistant to bile salts. Secondly, this study also investigated the effect of temperature fluctuations on the proliferation activity of probiotics. The results showed that the proliferation activity of the four strains of Lactobacillus plantarum was close to 0 CFU / mL after 30 minutes of high temperature stress at 75°C in a water bath. The results of this test showed that the four strains of Lactobacillus plantarum were extremely sensitive to temperature, and the most suitable growth temperature was around 35°C. Therefore, when handling such strains, pay attention to temperature changes and try to avoid exposure to high temperatures.

[0126] Self-aggregation and hydrophobicity are the key factors to measure the colonization ability of probiotics in the intestinal mucosa of animals. Self-aggregation refers to the ability of bacteria to spontaneously aggregate into clumps, which is related to self-recognition and non-self-recognition, surface colonization and the formation of biofilms. Hydrophobicity is an important factor affecting bacterial adhesion. The hydrophobicity of bacteria affects their ability to adhere to different surfaces. Bacteria with strong hydrophobicity are more likely to adhere to hydrophobic surfaces, and there is a mucosal hydrophobic layer in the gastrointestinal tract of humans or animals, so the hydrophobicity of bacteria affects their colonization in the intestinal mucosa and other parts.

[0127] The hemolytic ability of bacteria can reflect the interaction mechanism between pathogens and hosts after bacteria enter the gastrointestinal tract of animals. The hemolysin produced by bacteria may damage the red blood cell membrane and cause hemolysis, posing a threat to the health of the host. In this study, none of the four plant lactobacillus strains produced hemolysis circles in the blood plate, indicating that none of the four plant lactobacillus strains produced hemolysin, which is consistent with the probiotic properties of probiotics.

[0128] Bacteria secrete extracellular polysaccharides and other substances to produce biofilms, which have the ability to resist antibiotics and the environment, providing bacteria with a relatively stable living environment. None of the four plant lactobacillus strains in this study produced biofilms, which met the standards of the food industry. In order to prevent gastrointestinal disorders during antibiotic treatment, probiotics, as a tool for regulating intestinal function, sometimes need to survive with the use of some antibiotics, but the spread of antibiotic resistance is also closely related to it. Therefore, the antibiotic resistance of probiotics has become an important criterion for screening and selecting probiotics. The four strains isolated in this study are sensitive to most antibiotics and meet the antibiotic resistance standards for probiotics in aquaculture. When used, a good antibiotic management strategy should be used to reduce the risk of bacterial antibiotic resistance transmission.

[0129] In this study, we evaluated the protective effect of NX69 strain on H. discus under Vibrio parahaemolyticus stress through abalone challenge test. The test results showed that after abalone was inoculated with Vibrio parahaemolyticus V-25 isolate, the 70% mortality rate of the challenge group showed the severity of the infection and showed strong pathogenicity, indicating that the pathogenicity of the bacteria was strong, close to the pathogenicity of Vibrio harveyi previously reported. The mortality rate of abalone in the NX69 treatment group decreased by 20%, indicating that the NX69 strain has a certain anti-Vibrio parahaemolyticus V-25 effect and can reduce the lethality of Vibrio parahaemolyticus V-25 infection.

[0130] 4 Conclusion

[0131] The present invention isolates 4 target strains from the fermentation products of cereal crops in Ningde area, and the results are all plant lactobacillus after PCR reaction amplification and 16S rDNA sequencing and physiological and biochemical identification. These strains show high stress resistance and strong adhesion ability, and show different degrees of tolerance to multiple antibiotics. The supernatants of these strains all show obvious in vitro inhibitory effects on abalone pathogenic Vibrio, especially the NX69 strain is most effective in inhibiting the activity of pathogenic Vibrio. Further in vivo research results show that NX69 has a significant preventive and therapeutic effect on abalone Vibrio parahaemolyticus V-25 infection. Therefore, the NX69 strain can be used as a candidate strain for preventing and treating abalone vibrio disease, and plays an important role in future abalone farming.

[0132] Although the specific implementation modes of the present invention are described above, those skilled in the art should understand that the specific implementation modes described are only illustrative and are not intended to limit the scope of the present invention. Equivalent modifications and changes made by those skilled in the art in accordance with the spirit of the present invention should be included in the scope of protection of the claims of the present invention.

Claims

1. A Lactobacillus plantarum NX69, characterized in that: The Lactobacillus plantarum NX69 is Lactobacillus plantarum NX69, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on November 21, 2024, with the address being Building 59, No. 100, Xianlie Middle Road, Guangzhou, and the deposit number being GDMCC No. 65517.

2. A Lactobacillus plantarum NX69 according to claim 1, characterized in that: The Lactobacillus plantarum NX69 is used for preventing and treating abalone Vibrio disease.

3. A Lactobacillus plantarum NX69 according to claim 1, characterized in that: The Lactobacillus plantarum NX69 is used for preparing abalone Vibrio disease prevention and treatment bacterial agent products.

4. A Lactobacillus plantarum NX69 according to claim 1, characterized in that: The Lactobacillus plantarum NX69 is used for preparing abalone feed.

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