A strain of Lactobacillus plantarum NHE-LpE5 and its application

Abstract

The present invention provides a plant lactobacillus ( Lactobacillus plantarum )NHE‑LpE5, relates to the technical field of probiotics for animal husbandry. The bacteria were isolated from the intestines of piglets and deposited in the General Microbiology Center of China Microbiological Culture Collection (referred to as CGMCC, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101), with the deposit number CGMCC NO.24432 and the deposit date February 28, 2022.

Description

Technical Field

[0001] The invention relates to the technical field of probiotics for animal husbandry and feeding, and in particular to a probiotic plant lactobacillus NHE-LpE5 and an application thereof. Background Art

[0002] Since the cellular immunity and immune antibody levels of weaned piglets have decreased, pathogens are easy to grow in their intestines, so the healthy growth of piglets will be affected. At present, in actual production, antibiotics are usually used to treat and prevent autonomous diseases, so as to achieve the purpose of relieving stress and promoting growth.

[0003] However, the long-term use of antibiotics in large-scale farming can lead to drug resistance in pathogens, and cause veterinary drug residues in meat products and environmental pollution. Therefore, new antibiotic alternatives are needed to replace antibiotics.

[0004] Probiotics are one of the common alternatives to antibiotics, and they have a positive effect on the growth performance and disease prevention of pigs. Existing studies have shown that probiotics can effectively supplement the deficiencies of intestinal microorganisms and improve the disease resistance of piglets. However, the existing strains are mostly single in function and still have many deficiencies. Therefore, the development of more strains with excellent performance is still an urgent problem to be solved. Summary of the invention

[0005] The invention aims at a plant lactobacillus NHE-LpE5 and its application.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] First, the present invention provides a strain of Lactobacillus plantarum NHE-LpE5, which is classified and named Lactobacillus plantarum (Lactobacillus plantarum) NHE-LpE5, and was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on February 28, 2022. Its deposit number is CGMCC NO.24432, and the deposit address is Beijing, China.

[0008] Preferably, the Lactobacillus plantarum NHE-LpE5 is Lactobacillus plantarum isolated from the intestinal contents of fresh piglets, and is confirmed to be Lactobacillus plantarum by colony morphology observation, molecular biological identification, etc., and its 16SrDNA sequence is shown in SEQ ID NO.1.

[0009] Preferably, the plant lactobacillus NHE-LpE5 has the following microbiological characteristics: strain E5 can grow well on an MRS plate, culture for 48 hours, form round colonies with a colony diameter of 1-2 mm, milky white, smooth surface, opaque, convex in the middle, neat edges, Gram-positive, rod-shaped, spore-free, facultative anaerobic, suitable growth temperature range of 15°C-40°C, optimal growth temperature of 25°C-35°C, growth pH of 3.0-8.0, and optimum pH of 5.0-7.0.

[0010] Preferably, the culture method of the Lactobacillus plantarum NHE-LpE5 is as follows:

[0011] Take Lactobacillus plantarum NHE-LpE5 (preservation number CGMCC NO.24432) seed liquid (live bacteria concentration 10 9 CFU / mL) was inoculated into 300mL shake flask fermentation medium for shake flask fermentation culture; after the shake flask fermentation, fermentation culture was carried out in a fermenter, and 1.2L of shake flask fermentation seed liquid was inoculated into the fermentation medium in a 100L fermenter for fermentation culture. The 100L fermenter was filled with 70L fermentation medium. After the fermentation was completed, the number of viable bacteria in the fermentation liquid was detected to be 3.5×10 10 CFU / mL.

[0012] The shaking flask fermentation medium is composed of the following components: 0.5-4% sucrose, 0.5-2.5% glucose, 0.5-3.0% yeast extract powder, 0.5-2.5% soy peptone, 0.01-0.5% magnesium chloride, 0.01-1.0% calcium carbonate, 0.01-0.5% manganese sulfate, and the balance is water.

[0013] Preferably, the composition comprises: 1% sucrose, 1% glucose, 0.5% yeast extract powder, 1.2% soy peptone, 0.1% magnesium chloride, 0.1% calcium carbonate, 0.04% manganese sulfate, and the balance is water.

[0014] The shake flask fermentation conditions are: inoculation amount 1% (volume ratio), fermentation temperature 32° C., initial pH 6.5, 200 r / min, and fermentation time 8 h.

[0015] The culture medium components and fermentation conditions of the 100L fermentation tank are as follows: 1.0-5.0% soft sugar, 0.5-3.5% yeast extract, 0.5-2.5% corn powder, 0.01-0.5% magnesium chloride, 0.01-1.0% calcium carbonate, 0.01-0.5% manganese sulfate, 0.05-0.2% Tween-80, and the balance is water.

[0016] Preferably, the composition comprises: 2.0% soft sugar, 0.5% yeast extract, 1% corn powder, 0.2% magnesium chloride, 0.1% calcium carbonate, 0.04% manganese sulfate, 0.1% Tween-80, and the balance is water.

[0017] The shake flask fermentation conditions are as follows: 70 L of culture medium is loaded in a 100 L fermentation tank, the tank pressure is controlled at 0.05-0.06 MPa, the inoculation amount is 1200 mL, the fermentation temperature is 30° C., the fermentation time is 8 h, the pH value is 6.2, and the stirring speed is 300 r / min.

[0018] Secondly, the present invention provides a broad-spectrum antibacterial agent, which comprises the Lactobacillus plantarum NHE-LpE5 as claimed in claim 1 and a pharmaceutically acceptable carrier.

[0019] Preferably, the antibacterial spectrum of the broad-spectrum antibacterial agent includes the following strains: Erysipelothrix rhusiopathiae, Pseudomonas aeruginosa, Klebsiella pneumoniae, Listeria monocytogenes, Streptococcus pneumoniae, enteropathogenic Escherichia coli, Staphylococcus aureus, Salmonella typhi, Salmonella, Clostridium perfringens, Proteus pennisetum, Aeromonas hydrophila, and Vibrio parahaemolyticus.

[0020] Thirdly, the present invention provides the use of Lactobacillus plantarum NHE-LpE5 in the preparation of a broad-spectrum antibacterial agent.

[0021] Preferably, the antibacterial spectrum of the broad-spectrum antibacterial agent includes the following strains:

[0022] Erysipelothrix rhizoctonia solani, Pseudomonas aeruginosa, Klebsiella pneumoniae, Listeria monocytogenes, Streptococcus pneumoniae, enteropathogenic Escherichia coli, Staphylococcus aureus, Salmonella typhi, Salmonella, Clostridium perfringens, Proteus pennisetum, Aeromonas hydrophila, Vibrio parahaemolyticus.

[0023] Fourthly, the present invention provides the use of Lactobacillus plantarum NHE-LpE5 in the preparation of oral microecological preparations.

[0024] Preferably, the effective viable bacteria count in the oral probiotic preparation is greater than or equal to 1.2×10 11 CFU / g.

[0025] Fifthly, the present invention provides the use of a probiotic preparation prepared by Lactobacillus plantarum NHE-LpE5 in improving the growth performance of weaned piglets.

[0026] Sixth, the present invention provides the use of Lactobacillus plantarum NHE-LpE5 in the preparation of liquid fermented feed for improving the growth performance of weaned piglets.

[0027] Seventhly, the present invention provides the use of Lactobacillus plantarum NHE-LpE5 in the preparation of liquid fermented feed for reducing diarrhea in weaned piglets.

[0028] The beneficial effects of the present invention are:

[0029] (1) The present invention provides a novel Lactobacillus plantarum NHE-LpE5, which has a broad-spectrum bactericidal activity and can inhibit Erysipelothrix rhizogenes, Pseudomonas aeruginosa, Klebsiella pneumoniae, Listeria monocytogenes, Streptococcus pneumoniae, enteropathogenic Escherichia coli, Staphylococcus aureus, Salmonella typhi, Salmonella, Clostridium perfringens, Proteus pennisetum, Aeromonas hydrophila, and Vibrio parahaemolyticus.

[0030] (2) The Lactobacillus plantarum NHE-LpE5 provided by the present invention is resistant to gastric acid, intestinal fluid, and bile salts and is suitable for oral administration.

[0031] (3) Lactobacillus plantarum NHE-LpE5 has a certain immune preventive effect on Salmonella infection.

[0032] (4) When liquid fermentation prepared by Lactobacillus plantarum NHE-LpE5 is used to feed weaned piglets, it can reduce the feed-to-meat ratio and significantly reduce the diarrhea rate of piglets. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the colony morphology of Lactobacillus plantarum NHE-LpE5 on MRS medium.

[0034] Figure 2 is the Gram staining image of Lactobacillus plantarum NHE-LpE5 strain. DETAILED DESCRIPTION

[0035] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0036] Unless otherwise specified, the chemical reagents used in the examples are conventional commercially available reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.

[0037] The percentage sign "%" involved in the present invention, unless otherwise specified, refers to mass percentage; however, the percentage of a solution, unless otherwise specified, refers to the number of grams of a solute contained in 100 mL of the solution.

[0038] Example 1 Isolation, screening and identification of Lactobacillus plantarum NHE-LpE5

[0039] 1. Isolation and purification of lactic acid bacteria

[0040] (1) The collected fresh piglet intestinal contents were brought back to the laboratory. 10 g of the sample was weighed under sterile conditions and placed in a conical flask containing 90 mL of sterile saline. After constant temperature shaking at 37°C for 1 h, the sample was diluted to 1 million times using a 10-fold dilution method. Three dilutions of 10,000 times, 100,000 times, and 1 million times were selected.

[0041] (2) Pipette 0.1 mL onto a modified MRS agar plate, incubate the plate upside down at 37°C for 48 h, and use an inoculation loop to pick colonies of suspected lactic acid bacteria with obvious calcium dissolution rings larger than 5 mm and perform streak isolation and culture on the MRS agar plate.

[0042] (3) After 48 h of culture, pick the colonies with better isolation effect and transfer them to MRS agar slant with an inoculation loop for pure culture. Repeat the pure culture three times, and then suspend the bacterial cells in 20% glycerol solution and store them in a -80°C refrigerator for future use.

[0043] 2. Observation of Colony Morphology

[0044] According to the size of the calcium dissolving circle in step 1, the strains with strong acid production ability were preliminarily screened out and preliminarily determined to be lactic acid bacteria, totaling 182 strains. The glycerol tube strains preserved in step 1 were activated 2-3 times on MRS agar plates, then inoculated into MRS broth medium, cultured at 37°C with shaking at 180 rpm for 18-20h, and clean slides were taken for Gram staining and microscopic examination to observe the microscopic morphology of the strains, and Gram-positive non-spore-producing bacteria were selected as spares.

[0045] 3. Preparation of lactic acid bacteria suspension and fermentation broth

[0046] The lactobacillus obtained in step 1 was streaked on an MRS agar plate and cultured at 35°C for 48h. A single colony was picked from the plate and placed in 100mL MRS liquid culture medium, and cultured at 35°C, 180r / min, and shaking for 24h to obtain a lactobacillus suspension for use; the culture was continued at 35°C, 180r / min, and shaking for 96h to obtain a lactic acid bacteria fermentation liquid for use.

[0047] 4. Screening of lactic acid bacteria for acid production

[0048] The lactic acid bacteria suspension obtained in step 3 was inoculated into MRS liquid culture medium at an inoculation rate of 1% (volume ratio), cultured at 35°C for 12 hours, and lactic acid bacteria that reduced the pH of the fermentation liquid to below 4.0 within 12 hours were screened out, and a total of 39 lactic acid bacteria were obtained.

[0049] 5. Screening of lactic acid bacteria for lysine degradation

[0050] The lactic acid bacteria suspension obtained in step 3 was inoculated into MRS liquid culture medium supplemented with 1% lysine at an inoculum size of 1% (volume ratio), cultured at 35° C. for 24 hours, the free lysine content was measured every 4 hours, and 26 strains that did not degrade lysine during the fermentation process were selected.

[0051] 6. Screening of antibacterial lactic acid bacteria

[0052] (1) Take the concentration of pathogenic bacteria (Erysipelothrix rhizogenes, Pseudomonas aeruginosa, Klebsiella pneumoniae, Listeria monocytogenes, Streptococcus pneumoniae, enteropathogenic Escherichia coli, Staphylococcus aureus, Salmonella typhi, Salmonella, Clostridium perfringens, Proteus pennis, Aeromonas hydrophila, Vibrio parahaemolyticus) at 10 9 2 mL of bacterial suspension with a CFU / mL was added to 200 mL of pathogenic bacteria culture medium that had been sterilized and cooled to about 45°C;

[0053] (2) Then, take 10 mL of the unsolidified bacterial culture medium and transfer it to the nutrient agar plate with 10 mL of the bottom plate to prepare several pathogenic bacteria plates.

[0054] (3) On the clean bench, use sterile tweezers to pick up a sterilized Oxford cup (a small round tube with an inner diameter of 6 mm, an outer diameter of 8 mm, and a height of 10 mm. 200 μL of liquid can be added to the tube. Both ends of the tube should be smooth) from each pathogen nutrient agar plate and place it on the plate so that it is in contact with the culture medium without any gaps.

[0055] (4) After a few minutes, take 200 μL of the fermentation liquid of the suspected lactic acid strain (obtained in step 3) into an Oxford cup and culture at 37°C for 24 h. Perform at least 3 replicates for each strain, observe and measure the size of the inhibition zone, and there are 5 strains with large inhibition zones, which are labeled E5, E9, E15, E115, and E162 respectively.

[0056] 7. Identification of strain species

[0057] The strain E5 was identified morphologically and physiologically. The strain E5 grew well on the MRS plate and formed round colonies after 48 hours of culture. The colonies were 1-2 mm in diameter and milky white in color. They were smooth, opaque, convex in the middle, and had neat edges. Figure 1 , microscopic morphology Figure 2, Gram-positive, rod-shaped, non-spore-forming, facultative anaerobic, suitable growth temperature range of 15°C-40°C, optimal growth temperature of 25°C-35°C, growth pH of 3.0-8.0, and optimal pH of 5.0-7.0. The genomic DNA of strain E5 was extracted using a kit for bacterial DNA extraction. The 16S rDNA gene fragment of strain E5 was sequenced by primers F and R, and the obtained sequence was shown in SEQ ID NO.1. The measured sequence was compared with the 16S rDNA sequence in GenBank by BLAST analysis, and it can be seen that the homology between strain E5 and Lactobacillus plantarum reached 100%. According to the morphological characteristics and 16SrDNA characteristics of strain E5, strain E5 was determined to be Lactobacillus plantarum, and was officially labeled as NHE-LpE5.

[0058] 8. Strain preservation

[0059] The above-mentioned Lactobacillus plantarum NHE-LpE5 obtained by separation, purification and screening was deposited in the General Microbiology Center of China Microorganism Culture Collection Committee (referred to as CGMCC, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101) on February 28, 2022, with the deposit number CGMCC NO.24432 and the classification name Lactobacillus plantarum.

[0060] The composition of the modified MRS agar medium was as follows: peptone 1.0%, sodium acetate 0.5%, beef extract 1.0%, glucose 2%, yeast extract 0.5%, Tween 80 0.1%, K 2 HPO 4 0.2%, MgSO4 0.058%, diammonium citrate 0.2%, MnSO 4 0.025%, agar 1.8%, calcium carbonate 1%, the balance is water, pH 7.0±0.2.

[0061] MRS agar medium composition: peptone 1.0%, sodium acetate 0.5%, beef extract 1.0%, glucose 2%, yeast extract 0.5%, Tween 80 0.1%, K 2 HPO 4 0.2%, MgSO4 0.058%, diammonium citrate 0.2%, MnSO 4 0.025%, agar 1.8%, the balance is water, pH 7.0±0.2.

[0062] The composition of MRS broth medium is: peptone 1.0%, sodium acetate 0.5%, beef extract 1.0%, glucose 2%, yeast extract 0.5%, Tween 80 0.1%, K 2 HPO 4 0.2%,MgSO 4 0.058%, diammonium citrate 0.2%, MnSO 4 0.025%, the balance is water, pH 7.0±0.2.

[0063] The culture media for pathogenic bacteria are: TSA+5% defiberized sheep blood agar for Erysipelothrix rhusiopathiae, Listeria monocytogenes and Streptococcus pneumoniae, nutrient agar medium for Pseudomonas aeruginosa, Klebsiella pneumoniae, enteropathogenic Escherichia coli, Staphylococcus aureus, Salmonella typhi, Salmonella enterica, Proteus peneloyeri and Aeromonas hydrophila, TCBS medium for Vibrio parahaemolyticus, and tryptone-sulfite-cycloserine agar medium for Clostridium perfringens.

[0064] The composition of TSA+5% defibrinated sheep blood agar is: 1.5% tryptone, 0.5% soytone, 0.5% sodium chloride, 1.5% agar, and the balance is water, pH 7.2±0.2. When used, add 5% defibrinated sheep blood when cooled to 50℃.

[0065] The composition of the nutrient agar medium is: 1% peptone, 0.3% beef extract, 2% agar, 0.5% NaCl, and the balance is water, pH 7.2±0.2.

[0066] The composition of TCBS agar medium is: 0.5% yeast powder, 1% peptone, 1% sodium thiosulfate, 1% sodium citrate, 0.5% ox bile powder, 0.3% sodium taurocholate, 2% sucrose, 1% sodium chloride, 0.1% ferric citrate, 0.0004% thymol blue, 1.5% agar, pH 8.6±0.1.

[0067] The composition of the tryptone-sulfite-cycloserine agar medium is: tryptone 1.5%, soytone 0.5%, yeast powder 0.5%, sodium pyrosulfite 0.1%, ammonium ferric citrate 0.1%, agar 2%, and the balance is water, pH 7.6±0.2. When used, cool to 50°C and add 20ml / 250ml of filter-sterilized 0.5% D-cycloserine solution.

[0068] Example 2 Preparation of Lactobacillus plantarum NHE-LpE5 fermentation broth

[0069] Take Lactobacillus plantarum NHE-LpE5 (preservation number CGMCC NO.24432) seed liquid (live bacteria concentration 10 9CFU / mL) was inoculated into 300mL shake flask fermentation medium for shake flask fermentation culture; after the shake flask fermentation, fermentation culture was carried out in a fermenter, and 1.2L of shake flask fermentation seed liquid was inoculated into the fermentation medium in a 100L fermenter for fermentation culture. The 100L fermenter was filled with 70L fermentation medium. After the fermentation was completed, the number of viable bacteria in the fermentation liquid was detected to be 3.5×10 10 CFU / mL.

[0070] The shake flask fermentation medium consists of the following components: 1% sucrose, 1% glucose, 0.5% yeast extract powder, 1.2% soy peptone, 0.1% magnesium chloride, 0.1% calcium carbonate, 0.04% manganese sulfate, and the balance is water.

[0071] The shake flask fermentation conditions were as follows: inoculation amount 1% (volume ratio), fermentation temperature 32°C, initial pH 6.5, 200 r / min, and fermentation time 8 h.

[0072] The culture medium composition and fermentation conditions of the 100L fermentation tank are: 2.0% soft sugar, 0.5% yeast extract, 1% corn powder, 0.2% magnesium chloride, 0.1% calcium carbonate, 0.04% manganese sulfate, 0.1% Tween-80, and the balance is water.

[0073] The shake flask fermentation conditions were as follows: 70 L of culture medium was loaded into a 100 L fermentation tank, the tank pressure was controlled at 0.05-0.06 MPa, the inoculation volume was 1200 mL, the fermentation temperature was 30 °C, the fermentation time was 8 h, the pH value was 6.2, and the stirring speed was 300 r / min.

[0074] Example 3 Preparation of Lactobacillus plantarum NHE-LpE5 probiotic preparation

[0075] (1) Lactobacillus plantarum NHE-LpE5 (deposit number: CGMCC NO.24432) was used to prepare NHE-LpE5 fermentation broth according to the method of Example 2, and the fermentation broth was centrifuged at 13000 r / min for 10 min to obtain Lactobacillus plantarum NHE-LpE5 active bacterial slurry, and then the Lactobacillus plantarum active bacterial slurry and water were uniformly mixed in a weight ratio of 1:1 to obtain a high-concentration bacterial suspension.

[0076] (2) The plant lactobacillus NHE-LpE5 bacterial suspension and the protective agent were mixed uniformly in a weight ratio of 1:2.2 to prepare microcapsule wet powder, which was prepared into wet granules by a granulator, and then mixed uniformly with the coating agent solution, put into a fluidized drying bed, and dried and coated to obtain the preparation of NHE-LpE5 microecological preparation, with an effective viable count of 1.2×10 11 CFU / g.

[0077] The protective agent is: corn starch 40%, sodium carboxymethyl cellulose 2%, dextran 2%, inulin 2.4%, skimmed milk powder 15%, gelatinized modified starch 10%, sucrose 5%, peptone 2%, trehalose 2%, glycerol 5%, microcrystalline cellulose 5%, and the balance is water.

[0078] The coating agent solution is: 10% sodium alginate, 6.5% hydroxymethyl cellulose, 8.5% chitosan, 4% mannan, and the balance is water.

[0079] Example 4 Probiotic Verification of Lactobacillus plantarum NHE-LpE5

[0080] (1) Take the concentration of pathogenic bacteria (Erysipelothrix rhizogenes, Pseudomonas aeruginosa, Klebsiella pneumoniae, Listeria monocytogenes, Streptococcus pneumoniae, enteropathogenic Escherichia coli, Staphylococcus aureus, Salmonella typhi, Salmonella, Clostridium perfringens, Proteus pennis, Aeromonas hydrophila, Vibrio parahaemolyticus) at 10 9 2 mL of bacterial suspension with a CFU / mL was added to 200 mL of sterilized pathogenic bacteria culture medium cooled to about 45°C, and then 10 mL of unsolidified bacterial culture medium was aspirated and transferred to a nutrient agar plate with 10 mL of bottom plate to prepare several pathogenic bacteria plates.

[0081] (2) On the clean bench, use sterile tweezers to pick up a sterilized Oxford cup (a small round tube with an inner diameter of 6 mm, an outer diameter of 8 mm, and a height of 10 mm. 200 μL of liquid can be added to the tube. Both ends of the tube should be smooth) from each pathogen nutrient agar plate and place it on the plate so that it is in contact with the culture medium without any gaps.

[0082] (3) After 10 minutes, 200 μL of the fermentation liquid prepared in Example 2 was added to each tube, and the fermentation liquid was cultured at 37° C. for 12 h, and then the diameter of the inhibition zone was measured. Each experiment was repeated three times, and the average value was taken. The results are shown in Table 1.

[0083] The culture media for pathogenic bacteria are: TSA+5% defiberized sheep blood agar for Erysipelothrix rhusiopathiae, Listeria monocytogenes and Streptococcus pneumoniae, nutrient agar medium for Pseudomonas aeruginosa, Klebsiella pneumoniae, enteropathogenic Escherichia coli, Staphylococcus aureus, Salmonella typhi, Salmonella enterica, Proteus peneloyeri and Aeromonas hydrophila, TCBS medium for Vibrio parahaemolyticus, and tryptone-sulfite-cycloserine agar medium for Clostridium perfringens.

[0084] The composition of TSA+5% defibrinated sheep blood agar is: 1.5% tryptone, 0.5% soytone, 0.5% sodium chloride, 1.5% agar, and the balance is water, pH 7.2±0.2. When used, add 5% defibrinated sheep blood when cooled to 50℃.

[0085] The composition of the nutrient agar medium is: 1% peptone, 0.3% beef extract, 2% agar, 0.5% NaCl, and the balance is water, pH 7.2±0.2.

[0086] The composition of TCBS agar medium is: 0.5% yeast powder, 1% peptone, 1% sodium thiosulfate, 1% sodium citrate, 0.5% ox bile powder, 0.3% sodium taurocholate, 2% sucrose, 1% sodium chloride, 0.1% ferric citrate, 0.0004% thymol blue, 1.5% agar, pH 8.6±0.1.

[0087] The composition of the tryptone-sulfite-cycloserine agar medium is: tryptone 1.5%, soytone 0.5%, yeast powder 0.5%, sodium pyrosulfite 0.1%, ammonium ferric citrate 0.1%, agar 2%, and the balance is water, pH 7.6±0.2. When used, cool to 50°C and add 20ml / 250ml of filter-sterilized 0.5% D-cycloserine solution.

[0088] Table 1 Antibacterial effect of Lactobacillus plantarum NHE-LpE5 on pathogens

[0089] Pathogens Antibacterial diameter (mm) Erysipelas suis 31.67 Pseudomonas aeruginosa 28.34 Klebsiella pneumoniae 26.78 Listeria ivanovii 27.43 Streptococcus pneumoniae 22.43 Enteropathogenic Escherichia coli 28.97 Staphylococcus aureus 33.41 Salmonella typhimurium 21.21 Salmonella 22.13 Shigella Castellani 18.92 Clostridium perfringen 31.24 Proteus penneri 18.32 Aeromonas hydrophila 17.01 Vibrio parahaemolyticus 14.23

[0090] Example 5 Verification of stress resistance of Lactobacillus plantarum NHE-LpE5

[0091] 1. Determination of resistance to artificial gastric juice

[0092] Take 10mL of plant lactobacillus NHE-LpE5 bacterial suspension (prepared according to the method described in Example 1) and place it in artificial gastric juice configured by 90mL (250mL triangular flask), 37°C, 200r / min constant temperature oscillation for 180min; after the oscillation is completed, take 10mL of sample liquid to adjust the pH value to 7.0, add 90mL of normal saline, 37°C, 200r / min constant temperature oscillation for 30min, and then dilute the plate colony culture count. The results are shown in Table 2. As can be seen from Table 2, the survival rate of plant lactobacillus NHE-LpE5 in artificial gastric juice (containing enzyme) of pH1.5, pH2.0, and pH2.5 is more than 97% when treated for 3h, indicating that the strain NHE-LpE5 has a higher acid resistance, can tolerate gastric acid, and can smoothly reach the intestine to play a role.

[0093] Preparation method of artificial gastric juice: The preparation of artificial gastric juice refers to the preparation method in the 2010 edition of the Pharmacopoeia of the People's Republic of China. Take 16.4 mL of dilute hydrochloric acid, add about 800 mL of water and 10 g of pepsin, shake well, add water and weigh to 1000 mL, adjust the pH values ​​to 1.5, 2.0 and 2.5 respectively, and sterilize the microporous filter membrane (0.22 μm) for use.

[0094] Table 2 Survival of Lactobacillus plantarum NHE-LpE5 after treatment in artificial gastric juice for 3 h

[0095] deal with pH1.5 pH 2.0 pH 2.5 Initial activity CFU / mL <![CDATA[3.5×10 10 ]]> <![CDATA[3.5×10 10 ]]> <![CDATA[3.5×10 10 ]]> Active CFU / mL after treatment <![CDATA[3.4×10 10 ]]> <![CDATA[3.5×10 10 ]]> <![CDATA[3.5×10 10 ]]> Survival rate after treatment 97.14 100 100

[0096] 2. Determination of survival performance in artificial intestinal fluid

[0097] Take 1mL of plant lactobacillus NHE-LpE5 bacterial suspension (prepared according to the method described in Example 1) and place it in 99mL (250mL triangular flask) artificial intestinal fluid, 37°C, 200r / min constant temperature shock for 5h, take 1mL of sample liquid after the shock is completed, add 99mL normal saline, 37°C, 200r / min constant temperature shock for 30min, then dilute the plate colony culture count. The results show that the activity of plant lactobacillus NHE-LpE5 does not decrease in artificial intestinal fluid, and the survival rate is 100%, indicating that the strain can survive and preserve vitality in intestinal fluid well, thereby exerting its probiotic effect.

[0098] Preparation of artificial intestinal fluid: The preparation of artificial intestinal fluid refers to the preparation method in the 2010 edition of the Pharmacopoeia of the People's Republic of China, phosphate buffer (including pancreatic enzyme) (pH 6.8), take 6.8 g of potassium dihydrogen phosphate, add 500 mL of water to dissolve, and adjust the pH value to 6.8 with 0.1 mol / L sodium hydroxide solution; take another 10 g of pancreatic enzyme, add appropriate amount of water to dissolve, mix the two liquids, dilute with water to 1000 mL, and then filter and sterilize with a 0.22 μm microfiltration membrane to obtain.

[0099] 3. Bile Salt Survival Performance Assay

[0100] Take 1mL of Lactobacillus plantarum NHE-LpE5 bacterial suspension (prepared according to the method described in Example 1) and place it in 99mL (250mL Erlenmeyer flask) in solutions with different bile salt concentrations, the bile salt concentrations are 0.15%, 0.3%, 1%, and 1.5%, respectively, and then 37°C, 200r / min constant temperature shaking for 120min, after the shaking is completed, take 1mL of the sample solution and add 99mL of normal saline, 37°C, 200r / min constant temperature shaking for 30min, and then dilute the plate colony culture count.

[0101] The results are shown in Table 3. The survival rate of Lactobacillus plantarum NHE-LpE5 treated in a solution with 0.3% bile salt concentration for 2 hours was 85.71%, indicating that the strain has a high bile salt tolerance and can tolerate bile salts in duodenal fluid, allowing the strain to reach the intestine to play a role.

[0102] Preparation method of bile salt solutions of different concentrations: take 9mL, 18mL, 60mL, and 90mL of 5% bile salt solution respectively, add them to PBS solution of pH 7.4, make up to 300mL, and mix evenly. This is PBS solution containing 0.15%, 0.30%, 1%, and 1.5% bile salt.

[0103] Preparation method of 5% bile salt solution: Accurately weigh 5.0 g of bile salt, dissolve it in 100 mL of PBS solution, and sterilize it at 121°C for 20 min.

[0104] Preparation method of PBS solution: 0.8% sodium chloride, 0.02% potassium chloride, 0.363% disodium hydrogen phosphate, 0.024% potassium dihydrogen phosphate, and the balance is water. Adjust the pH to 7.4 with 6 mol / L HCl, sterilize at 121℃ for 20 minutes, and set aside.

[0105] Table 3 Survival of Lactobacillus plantarum NHE-LpE5 after treatment with bile salt solutions of different concentrations for 2 h

[0106]

[0107] Example 6 Acute toxicity test of Lactobacillus plantarum NHE-LpE5

[0108] The safety evaluation of Lactobacillus plantarum was conducted by acute toxicity test, referring to the maximum tolerated dose method of GB15193.3-2003. 60 ordinary Kunming mice, half male and half female, 18-20g, were taken. After one week of conventional feeding, the mice were gavaged three times a day with 0.25g / mL Lactobacillus plantarum NHE-LpE5 bacterial solution (equivalent to 15000mg / kg body weight) for two consecutive weeks to observe whether the mice were poisoned or died.

[0109] During the test, the mice were in good mental state, without poisoning or death. Therefore, the maximum tolerated dose (MTD) of the acute toxicity test of the strain of the present invention is greater than 15000 mg / kg. According to the grading standard, the strain can be determined to be non-toxic and has high safety.

[0110] Example 7: Mouse challenge test

[0111] (1) 60 female C57 mice, weighing 11-13 g, were selected and fed in a conventional manner. They were randomly divided into three groups. Each treatment was fed a basic diet for 5 days to allow the mice to adapt as quickly as possible. The formal experiment began. The formal experiment was divided into two stages. In the first stage, the growth performance observation test was conducted. The experimental group added Lactobacillus plantarum fermentation liquid to the drinking water at a concentration of 1×10 7 CFU / mL.

[0112] (2) The control group and the negative control group drank pure water without plant lactobacillus for two weeks. The mice were weighed once before and after the test. The growth of the mice was observed during the test, and the average weight growth rate of each group was calculated. The results are shown in Table 4. In the second stage of the Salmonella challenge test, the negative control group and the test group were challenged with Salmonella by gavage, specifically, the concentration of 10 8 The control group was gavaged with 0.5 mL / mouse Salmonella, while the control group was gavaged with 0.5 mL / mouse normal saline.

[0113] (3) After the challenge, the mental state and death of the mice were observed. The experimental group continued to drink 1×10 7 CFU / mL concentration of Lactobacillus plantarum NHE-LpE5 fermentation liquid, the control group and negative control group drink normal tap water. During the experiment, the mice were housed in separate cages in the same room, exposed to natural light, and fed freely. The ambient temperature was controlled at 25±2℃ and the humidity was 60%.

[0114] The Lactobacillus plantarum fermentation broth was the fermentation broth prepared in Example 2.

[0115] The experimental results showed that after the Salmonella challenge, the mice showed symptoms of illness through daily observation: basically no food intake, inactivity, listlessness, curling up, dull fur, messy back hair, and bloodshot eyes. On the first day after the challenge, the negative control group began to show symptoms of illness, and 2 mice died. The other survivors also showed symptoms of illness to varying degrees. Two mice in the experimental group died on the third day, and the other mice were in poor spirits and did not eat for 5 days, and then gradually returned to normal.

[0116] The death time, number of deaths and survival rate of each group after the virus attack are shown in Table 5. The poisoning phenomenon of mice in the experimental group after the virus attack was not serious, indicating that Lactobacillus plantarum NHE-LpE5 has a certain immune preventive effect on Salmonella infection, effectively reducing the incidence rate of mice infected with Salmonella, delaying the onset time after the virus attack, thereby effectively improving the survival rate.

[0117] Table 4 Weight gain of mice

[0118] Grouping Average initial weight (g / piece) Average final weight (g / piece) Average growth rate (%) Control group 11.25±0.16 22.14±0.78 <![CDATA[96.80 b ]]> Negative control group 11.43±0.24 22.42±0.49 <![CDATA[96.15 b ]]> Experimental Group 11.21±0.36 26.48±0.31 <![CDATA[136.21 a ]]>

[0119] Table 5 Death of mice before and after infection

[0120]

[0121]

[0122] Example 8 Effect of Lactobacillus plantarum NHE-LpE5 probiotic preparation on growth performance of weaned piglets

[0123] (1) The experiment selected weaned pigs (half male and half female) of the same parity and similar weight (Duroc×Landrace×Large White). A total of 200 weaned pigs at 28 days old were randomly divided into two groups (control group and experimental group). Each treatment group had 10 pens with 10 pigs in each pen. The experiment lasted 45 days and the feeding experiment was carried out under the same environment.

[0124] (2) The control group was fed a basic diet (referring to the Chinese pig feeding standard), and the experimental group was fed a basic diet supplemented with 1×10 7 CFU / g of Lactobacillus plantarum microecological preparation.

[0125] (3) Before the start of the experiment, the pig house was thoroughly disinfected. The experimental group calculated the amount of microecological preparations to be added according to the daily feed intake, and then dissolved it in water for drinking, divided into 2 times to ensure that each drink was completed. The feed intake and health status were recorded every day, and the brightness of the pigs' fur and feces were observed regularly. Other feeding and management were the same. Compared with the control group, the liquid fermented feed group can significantly increase the daily weight gain of the experimental group piglets, significantly reduce the feed-to-meat ratio by 0.2, and significantly reduce the diarrhea rate. (Table 5)

[0126] Table 5 Effects of Lactobacillus plantarum probiotic preparations on growth performance of weaned piglets

[0127] project Control group Experimental Group Initial weight (kg) 6.41±0.17 6.38±0.23 Final weight(kg) <![CDATA[20.20±1.02 b ]]> <![CDATA[23.82±2.21 a ]]> Average daily weight gain (g / d) <![CDATA[306±23.56 b ]]> <![CDATA[387.55±31.24 a ]]> Average daily feed intake (kg / d) <![CDATA[563±56.43 b ]]> <![CDATA[632±21.34 a <!-- 9 -->]]> Feed to meat ratio <![CDATA[1.83±0.14 a ]]> <![CDATA[1.63±0.04 b ]]> Diarrhea rate <![CDATA[7.14±0.14 A ]]> <![CDATA[1.46±0.05 B ]]>

[0128] Note: Data in the same row with different lowercase letters on their shoulders indicate significant differences (p<0.05), while data with the same lowercase letters on their shoulders or no letters on their shoulders indicate no significant differences (p>0.05).

[0129] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A strain of Lactobacillus plantarum ( Lactobacillus plantarum ) NHE-LpE5, characterized in that The Lactobacillus plantarum NHE-LpE5 was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on February 28, 2022, with a deposit number of CGMCC NO.24432 and a deposit address of Beijing, China.

2. A broad-spectrum antibacterial agent, characterized in that: The broad-spectrum antibacterial agent comprises the Lactobacillus plantarum NHE-LpE5 as claimed in claim 1 and a pharmaceutically acceptable carrier; The antibacterial spectrum of the broad-spectrum antibacterial agent is the following strains: Erysipelothrix rhizogenes, Pseudomonas aeruginosa, Klebsiella pneumoniae, Listeria monocytogenes, Streptococcus pneumoniae, enteropathogenic Escherichia coli, Staphylococcus aureus, Salmonella typhi, Salmonella, Clostridium perfringens, Proteus pennisetum, Aeromonas hydrophila, and Vibrio parahaemolyticus.

3. The use of Lactobacillus plantarum NHE-LpE5 as claimed in claim 1 in the preparation of a broad-spectrum antibacterial agent, characterized in that: The antibacterial spectrum of the broad-spectrum antibacterial agent is the following strains: Erysipelothrix rhizoctonia solani, Pseudomonas aeruginosa, Klebsiella pneumoniae, Listeria monocytogenes, Streptococcus pneumoniae, enteropathogenic Escherichia coli, Staphylococcus aureus, Salmonella typhi, Salmonella, Clostridium perfringens, Proteus pennisetum, Aeromonas hydrophila, Vibrio parahaemolyticus.

4. Use of Lactobacillus plantarum NHE-LpE5 as claimed in claim 1 in preparing oral probiotic preparations.

5. The use according to claim 4, characterized in that: The number of effective live bacteria in the oral microecological preparation is greater than or equal to 1.2×10 11 CFU / g.

6. Application of the probiotic preparation prepared by Lactobacillus plantarum NHE-LpE5 as claimed in claim 1 in improving the growth performance of weaned piglets.

7. Use of the Lactobacillus plantarum NHE-LpE5 as claimed in claim 1 in preparing a liquid fermented feed for improving the growth performance of weaned piglets.

8. Use of the Lactobacillus plantarum NHE-LpE5 according to claim 1 in preparing a liquid fermented feed for reducing diarrhea in weaned piglets.

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