CN115851494A - Lactobacillus plantarum NHE-LpE and application thereof - Google Patents
Lactobacillus plantarum NHE-LpE and application thereof Download PDFInfo
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- CN115851494A CN115851494A CN202211081870.7A CN202211081870A CN115851494A CN 115851494 A CN115851494 A CN 115851494A CN 202211081870 A CN202211081870 A CN 202211081870A CN 115851494 A CN115851494 A CN 115851494A
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Abstract
The invention provides a lactobacillus plantarum (Lactobacillus plantarum) NHE-LpE relates to the technical field of probiotics for livestock feeding. The strain is separated from intestinal tracts of piglets, is preserved in China general microbiological culture Collection center (CGMCC for short,the address is as follows: the microbiological research institute of western road 1, 3, national academy of sciences, north-kyo, chaoyang, the postal code: 100101 CGMCC NO.24432 and the preservation date of 2022, 2 months and 28 days.
Description
Technical Field
The invention relates to the technical field of probiotics for livestock feeding, in particular to a probiotic lactobacillus plantarum NHE-LpE and application thereof.
Background
The cell immunity and the immune antibody level of the matrix of the weaned piglets are reduced, so that pathogenic bacteria are easy to grow in the intestinal tracts of the weaned piglets, and the healthy growth of the piglets is influenced. Currently, in actual production, antibiotics are usually used to treat and prevent autonomic diseases, so as to achieve the purposes of reducing stress and promoting growth.
However, the long-term use of antibiotics in large-scale farming can lead to pathogen resistance, as well as veterinary drug residues in meat products and environmental pollution. New antibiotic substitutes are therefore needed to replace antibiotics.
Probiotics are one of the common alternatives to antibiotics, which have a positive effect on the growth performance and the prevention and treatment of diseases in pigs. The existing research shows that the probiotics can effectively supplement the shortage of microorganisms in intestinal tracts and improve the disease resistance of piglets. However, the existing strains have more and more single functions and still have more defects, so that the development of more strains with excellent performance is still a problem to be solved urgently at present.
Disclosure of Invention
The invention aims to provide lactobacillus plantarum NHE-LpE and application thereof.
In order to achieve the purpose, the invention provides the following technical scheme:
the Lactobacillus plantarum NHE-LpE is classified and named as Lactobacillus plantarum NHE-LpE, is preserved in China general microbiological culture collection center (CGMCC) at 28 months at 2022, has the preservation number of CGMCC NO.24432 and the preservation address of Beijing China.
Preferably, the lactobacillus plantarum NHE-LpE is lactobacillus plantarum separated from intestinal contents of a fresh piglet, determined to be lactobacillus plantarum through colony morphology observation, molecular biology identification and the like, and the 16SrDNA sequence of the lactobacillus plantarum is shown as SEQ ID No. 1.
Preferably, the lactobacillus plantarum NHE-LpE has the following microbiological properties: the strain E5 can well grow on an MRS plate, is cultured for 48 hours to form a circular bacterial colony, the diameter of the bacterial colony is 1-2mm, the bacterial colony is milky white, the surface is smooth and opaque, the middle part is convex, the edge is neat, the bacterial colony is gram-positive, rod-shaped, spore-free, facultative anaerobic, the proper growth temperature range is 15-40 ℃, the optimal growth temperature is 25-35 ℃, the growth pH is 3.0-8.0, and the optimal pH is 5.0-7.0.
Preferably, the lactobacillus plantarum NHE-LpE is cultured by the following method:
taking seed liquid (viable bacteria concentration is 10) of lactobacillus plantarum NHE-LpE (preservation number is CGMCC NO. 24432) seed liquid 9 CFU/mL) 3mL, inoculating the mixture into 300mL of shake flask fermentation medium for shake flask fermentation culture; after the shake flask fermentation is finished, fermentation culture is carried out in a fermentation tank, 1.2L of shake flask fermentation seed liquid is inoculated into a fermentation culture medium in a 100L fermentation tank for fermentation culture, and the liquid loading capacity of the 100L fermentation tank is 70L of the fermentation culture medium. After the fermentation is finished, detecting that the number of viable bacteria in the fermentation liquor is 3.5 multiplied by 10 10 CFU/mL。
The shake flask fermentation medium consists of the following components: 0.5-4% of cane sugar, 0.5-2.5% of glucose, 0.5-3.0% of yeast extract powder, 0.5-2.5% of soybean peptone, 0.01-0.5% of magnesium chloride, 0.01-1.0% of calcium carbonate, 0.01-0.5% of manganese sulfate and the balance of water.
Preferably: 1% of sucrose, 1% of glucose, 0.5% of yeast extract powder, 1.2% of soybean peptone, 0.1% of magnesium chloride, 0.1% of calcium carbonate, 0.04% of manganese sulfate and the balance of water.
The shake flask fermentation conditions are as follows: the inoculation amount is 1 percent (volume ratio), the fermentation temperature is 32 ℃, the initial pH value is 6.5, 200r/min, and the fermentation time is 8h.
The culture medium components and fermentation conditions of the 100L fermentation tank are as follows: 1.0-5.0% of soft sugar, 0.5-3.5% of yeast extract, 0.5-2.5% of corn dry powder, 0.01-0.5% of magnesium chloride, 0.01-1.0% of calcium carbonate, 0.01-0.5% of manganese sulfate, 0.05-0.2% of tween-80 and the balance of water.
Preferably: 2.0% of soft sugar, 0.5% of yeast extract, 1% of corn dry powder, 0.2% of magnesium chloride, 0.1% of calcium carbonate, 0.04% of manganese sulfate, 0.1% of tween-80 and the balance of water.
The shake flask fermentation conditions are as follows: the liquid loading capacity of a 100L fermentation tank is 70L of culture medium, the tank pressure is controlled to be 0.05-0.06MPa, the inoculation amount is 1200mL, the fermentation temperature is 30 ℃, the fermentation time is 8h, the pH value is 6.2, and the stirring speed is 300r/min.
Secondly, the invention provides a spectrum antibacterial agent, which comprises lactobacillus plantarum NHE-LpE as described in claim 1 and a pharmaceutically acceptable carrier.
Preferably, the antibacterial spectrum of the spectrum antibacterial agent comprises the following strains: erysipelothrix suis, pseudomonas aeruginosa, klebsiella pneumoniae, listeria monocytogenes, streptococcus pneumoniae, enteropathogenic escherichia coli, staphylococcus aureus, salmonella typhi, salmonella, clostridium perfringens, proteus penonii, aeromonas hydrophila and vibrio parahaemolyticus.
Thirdly, the invention provides the application of lactobacillus plantarum NHE-LpE5 in preparing a spectrum antibacterial agent.
Preferably, the antimicrobial spectrum of the broad-spectrum antimicrobial agent comprises the following strains:
erysipelothrix suis, pseudomonas aeruginosa, klebsiella pneumoniae, listeria monocytogenes, streptococcus pneumoniae, enteropathogenic escherichia coli, staphylococcus aureus, salmonella typhi, salmonella, clostridium perfringens, proteus penonii, aeromonas hydrophila and vibrio parahaemolyticus.
Fourthly, the invention provides application of lactobacillus plantarum NHE-LpE in preparation of oral microecological preparations.
Preferably, the number of effective viable bacteria in the oral microecological preparation is more than or equal to 1.2 multiplied by 10 11 CFU/g。
Fifthly, the invention provides application of a microecological preparation prepared from lactobacillus plantarum NHE-LpE in improving the growth performance of weaned pigs.
Sixth, the invention provides an application of lactobacillus plantarum NHE-LpE5 in preparation of liquid fermented feed for improving growth performance of weaned pigs.
The invention further provides an application of lactobacillus plantarum NHE-LpE in preparation of the liquid fermented feed for reducing diarrhea of weaned piglets.
The invention has the beneficial effects that:
(1) The invention provides a new lactobacillus plantarum NHE-LpE which has broad-spectrum bactericidal activity and can inhibit erysipelothrix rhusiopathiae, pseudomonas aeruginosa, klebsiella pneumoniae, listeria exubertii, streptococcus pneumoniae, enteropathogenic escherichia coli, staphylococcus aureus, salmonella typhi, salmonella, clostridium perfringens, proteus pengiensis, aeromonas hydrophila and vibrio parahaemolyticus.
(2) The lactobacillus plantarum NHE-LpE provided by the invention can resist gastric acid, intestinal juice and bile salt and is suitable for oral administration.
(3) Lactobacillus plantarum NHE-LpE has a certain immunoprophylaxis effect on Salmonella infection.
(4) When the liquid fermentation feed prepared by the lactobacillus plantarum NHE-LpE is used for feeding weaned piglets, the feed-meat ratio can be reduced, and the diarrhea rate of the piglets is remarkably reduced.
Drawings
FIG. 1 is a colony morphology of Lactobacillus plantarum NHE-LpE on MRS medium.
FIG. 2 is a gram stain of Lactobacillus plantarum NHE-LpE strain.
Detailed Description
The examples are given for the purpose of better illustration of the invention, but the invention is not limited to the examples. Therefore, those skilled in the art should make insubstantial modifications and adaptations to the embodiments of the present invention in light of the above teachings and remain within the scope of the invention.
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.
The percent in the present invention means mass percent unless otherwise specified; but the percent of the solution, unless otherwise specified, refers to the grams of solute contained in 100mL of the solution.
Example 1 isolation, screening and characterization of Lactobacillus plantarum NHE-LpE5
1. Separation and purification of lactic acid bacteria
(1) Taking the collected fresh piglet intestinal contents back to a laboratory, weighing 10g of a sample under an aseptic condition, placing the sample in a triangular flask containing 90mL of sterilized normal saline, vibrating at a constant temperature of 37 ℃ for 1h, sequentially diluting to 100 ten thousand times by adopting a 10-time dilution method, and selecting three dilutions of 1 ten thousand times, 10 ten thousand times and 100 ten thousand times
(2) Sucking 0.1mL of the suspension, coating the suspension on an improved MRS agar plate, performing inverted culture at 37 ℃ for 48h after coating the plate, picking out a colony of suspected lactobacillus with obvious calcium-dissolving ring and larger than 5mm by using an inoculating loop, and performing streak isolation culture on the MRS agar plate
(3) After 48h of culture, selecting a colony with a good separation effect, inoculating the colony on an MRS agar slant for pure culture by using an inoculating loop, repeatedly carrying out subculture for 3 times, suspending the strain cells in a 20% glycerol solution, and storing the suspension in a refrigerator at-80 ℃ for later use.
2. Observation of colony morphology
According to the size of the calcium dissolving ring in the step 1, the strains with strong acid production capacity are preliminarily screened out and can be preliminarily determined to be lactic acid bacteria, and 182 strains are selected. Activating the glycerol tube strain preserved in the step 1 for 2-3 times by using an MRS agar plate, then inoculating the glycerol tube strain into an MRS broth culture medium, carrying out shake culture at the constant temperature of 37 ℃ for 18-20h at constant temperature of 180r/min, taking a clean glass slide for gram staining, carrying out microscopic examination, observing the microscopic morphology of the strain, and selecting the bacillus-producing-non-bacteria with gram staining as positive for later use.
3. Preparation of lactic acid bacteria suspension and fermentation broth
Streaking the lactobacillus obtained in the step 1 on an MRS agar plate, culturing for 48h at 35 ℃, picking a single colony from the plate, culturing for 24h in 100mL of MRS liquid culture medium at 35 ℃ and 180r/min in a shaking way, and obtaining lactobacillus suspension for later use; and continuing shaking culture at 35 ℃ and 180r/min for 96h to obtain lactobacillus fermentation liquor for later use.
4. Screening of acid-producing ability of lactic acid bacteria
Inoculating the lactobacillus suspension obtained in the step 3 into an MRS liquid culture medium according to the inoculation amount of 1% (volume ratio), culturing for 12h at 35 ℃, screening out lactobacillus which is used for 12h and enables the pH of fermentation liquor to be below 4.0, and obtaining 39 strains of lactobacillus in total.
5. Screening of lysine degraded by lactic acid bacteria
Inoculating the lactobacillus suspension obtained in the step 3 into an MRS liquid culture medium added with 1% of lysine according to the inoculation amount of 1% (volume ratio), culturing for 24h at 35 ℃, measuring the content of free lysine every 4h, and selecting 26 strains which do not degrade the lysine in the fermentation process.
6. Screening of bacteriostatic lactic acid bacteria
(1) Taking pathogenic bacteria (including Erysipelothrix rhusiopathiae, pseudomonas aeruginosa, klebsiella pneumoniae, listeria monocytogenes, streptococcus pneumoniae, enteropathogenic Escherichia coli, staphylococcus aureus, salmonella typhi, salmonella, clostridium perfringens, proteus pengpenii, aeromonas hydrophila, and Vibrio parahaemolyticus) with concentration of 10 9 2mL of CFU/mL of bacterial suspension is added into a pathogenic bacterium culture medium filled with 200mL of sterilized pathogenic bacterium and cooled to about 45 ℃;
(2) Then 10mL of non-solidified culture medium with bacteria is sucked and transferred to a nutrient agar plate with 10mL of bottom plate, and a plurality of pathogenic bacteria plates are prepared.
(3) Clamping 1 sterilized Oxford cup (a circular small tube with the inner diameter of 6mm, the outer diameter of 8mm and the height of 10mm, wherein 200 mu L of liquid can be added into the tube, and the two ends of the tube are required to be smooth) on an ultra-clean workbench by using sterile forceps, and placing the sterilized Oxford cup on a plate to ensure that the sterilized Oxford cup is in contact with a culture medium without a gap;
(4) After several minutes, respectively sucking 200. Mu.L of suspected lactobacillus strain fermentation broth (obtained in step 3) into an Oxford cup, and culturing at constant temperature of 37 ℃ for 24 hours. And (3) repeating each strain at least, observing and measuring the size of the inhibition zone, wherein the number of the strains with the large inhibition zone is 5, and the strains are respectively marked as E5, E9, E15, E115 and E162.
7. Identification of the Strain species
The strain E5 is subjected to morphological and physiological biochemical identification, the strain E5 can grow well on an MRS plate, a circular colony is formed after being cultured for 48 hours, the diameter of the colony is 1-2mm, the colony is milky white, smooth and opaque, the middle of the colony is convex, the edge of the colony is neat, the colony morphology is shown in a figure 1, the microscopic morphology is shown in a figure 2, the colony is gram-positive, rod-shaped, spore-free and facultative anaerobic, the growth suitable temperature range is 15-40 ℃, the optimal growth temperature is 25-35 ℃, the growth pH is 3.0-8.0, and the optimal pH is 5.0-7.0. And extracting E5 strain genome DNA by using a kit for extracting bacterial DNA. Sequencing the 16S rDNA gene segment of the E5 strain through the primers F and R to obtain a sequence shown as SEQ ID NO.1, and comparing the determined sequence with the 16S rDNA sequence in GenBank by BLAST analysis, which shows that the homology of the E5 strain and the lactobacillus plantarum reaches 100%. The strain E5 is determined to be Lactobacillus plantarum (Lactobacillus plantarum) through morphological characteristics and 16S rDNA characteristics of the strain E5, and is formally marked as NHE-LpE.
8. Strain preservation
The Lactobacillus plantarum (Lactobacillus plantarum) NHE-LpE obtained by separation, purification and screening is already preserved in China general microbiological culture Collection center (CGMCC for short, address: 100101, institute of microbiology, china academy of sciences, no. 3, north Cheng West Lu 1 institute of Western No.1, beijing, and the address: P.japonicum) in 2022.28.s.2022.A collection number is CGMCC NO.24432, and the Lactobacillus plantarum (Lactobacillus plantarum) is classified and named as Lactobacillus plantarum.
The improved MRS agar medium comprises the following components: peptone 1.0%, sodium acetate 0.5%, beef extract 1.0%, glucose 2%, yeast extract 0.5%, tween 80.1%, and K 2 HPO 4 0.2 percent, mgSO4 0.058 percent, diammonium citrate 0.2 percent and MnSO 4 0.025 percent, agar 1.8 percent, calcium carbonate 1 percent and the balance of water, and the pH value is 7.0 +/-0.2.
The MRS agar medium comprises the following components: peptone 1.0%, sodium acetate 0.5%, beef extract 1.0%, glucose 2%, yeast extract 0.5%, tween 80.1%, and K 2 HPO 4 0.2 percent, mgSO4 0.058 percent, diammonium citrate 0.2 percent and MnSO 4 0.025 percent, agar 1.8 percent and the balance of water, and the pH value is 7.0 +/-0.2.
The MRS broth culture medium comprises: peptone 1.0%, sodium acetate 0.5%, beef extract 1.0%, glucose 2%, yeast extract 0.5%, tween 80.1%, and K 2 HPO 4 0.2％，MgSO 4 0.058 percent of ammonium citrate, 0.2 percent of diammonium citrate and MnSO 4 0.025 percent, and the balance of water, and the pH value is 7.0 +/-0.2.
The pathogenic bacteria culture medium is respectively as follows: the erysipelothrix rhusiopathiae, listeria monocytogenes and streptococcus pneumoniae are TSA +5% defibrinated sheep blood agar, pseudomonas aeruginosa, klebsiella pneumoniae, enteropathogenic escherichia coli, staphylococcus aureus, salmonella typhi, salmonella, proteus pengpenii and aeromonas hydrophila are nutrient agar culture media, the vibrio parahaemolyticus is TCBS culture medium, and the clostridium perfringens is tryptone-sulfite-cycloserine agar culture medium.
TSA +5% defibrinated sheep blood agar consists of: tryptone 1.5%, soytone 0.5%, sodium chloride 0.5%, agar 1.5%, and water in balance, pH 7.2 + -0.2, cooling to 50 deg.C, and adding 5% defibrinated sheep blood.
The nutrient agar culture medium comprises the following components: peptone 1%, beef extract 0.3%, agar 2%, naCl 0.5%, and water in balance, and pH 7.2 + -0.2.
The TCBS agar medium consists of: 0.5% of yeast powder, 1% of peptone, 1% of sodium thiosulfate, 1% of sodium citrate, 0.5% of bile powder, 0.3% of sodium taurocholate, 2% of sucrose, 1% of sodium chloride, 0.1% of ferric citrate, 0.0004% of thymol blue, 1.5% of agar and pH 8.6 +/-0.1.
The tryptone-sulfite-cycloserine agar medium comprises the following components: tryptone 1.5%, soytone 0.5%, yeast powder 0.5%, sodium metabisulfite 0.1%, ferric ammonium citrate 0.1%, agar 2%, balance water, pH7.6 + -0.2, and filtering sterilized 0.5% D-cycloserine solution 20mL/250mL when used while cooling to 50 ℃.
EXAMPLE 2 preparation of Lactobacillus plantarum NHE-LpE fermentation broth
Taking seed liquid (viable bacteria concentration is 10) of lactobacillus plantarum NHE-LpE (preservation number is CGMCC NO. 24432) seed liquid 9 CFU/mL) 3mL, inoculating the mixture into 300mL of shake flask fermentation medium for shake flask fermentation culture; after the shake flask fermentation is finished, fermentation culture is carried out in a fermentation tank, 1.2L of shake flask fermentation seed liquid is inoculated into a fermentation culture medium in a 100L fermentation tank for fermentation culture, and the liquid loading capacity of the 100L fermentation tank is 70L of the fermentation culture medium. After the fermentation is finished, detecting that the number of viable bacteria in the fermentation liquor is 3.5 multiplied by 10 10 CFU/mL。
The shake flask fermentation medium consists of the following components: 1% of sucrose, 1% of glucose, 0.5% of yeast extract powder, 1.2% of soybean peptone, 0.1% of magnesium chloride, 0.1% of calcium carbonate, 0.04% of manganese sulfate and the balance of water.
The shake flask fermentation conditions were: the inoculation amount is 1 percent (volume ratio), the fermentation temperature is 32 ℃, the initial pH6.5, 200r/min, and the fermentation time is 8h.
The medium components and fermentation conditions of the 100L fermentation tank were: 2.0% of soft sugar, 0.5% of yeast extract, 1% of corn dry powder, 0.2% of magnesium chloride, 0.1% of calcium carbonate, 0.04% of manganese sulfate, 0.1% of tween-80 and the balance of water.
The shake flask fermentation conditions were: the liquid loading capacity of a 100L fermentation tank is 70L of culture medium, the tank pressure is controlled to be 0.05-0.06MPa, the inoculation amount is 1200mL, the fermentation temperature is 30 ℃, the fermentation time is 8h, the pH value is 6.2, and the stirring speed is 300r/min.
EXAMPLE 3 preparation of Lactobacillus plantarum NHE-LpE5 Microecological preparation
(1) The lactobacillus plantarum NHE-LpE (preservation number is CGMCC NO. 24432) is used for preparing NHE-LpE fermentation liquor according to the method of example 2, the fermentation liquor is centrifuged at 13000r/min for 10min to obtain lactobacillus plantarum NHE-LpE active bacterial sludge, and then the lactobacillus plantarum active bacterial sludge and water are uniformly mixed according to the weight proportion of 1:1 to obtain high-concentration bacterial suspension.
(2) Uniformly mixing the lactobacillus plantarum NHE-LpE thallus suspension and a protective agent according to the weight ratio of 1:2.2 to prepare microcapsule wet powder, preparing the microcapsule wet powder into wet particles through a granulator, uniformly mixing the microcapsule wet particles with a coating agent solution, putting the mixture into a fluidized drying bed, and drying and coating to obtain the preparation of the NHE-LpE microecological preparation, wherein the effective viable count reaches 1.2 multiplied by 10, and the microbial preparation is prepared by using the lactobacillus plantarum NHE-LpE thallus suspension and the protective agent in a weight ratio of 1 11 CFU/g。
The protective agent is: 40% of corn starch, 2% of sodium carboxymethylcellulose, 2% of glucan, 2.4% of inulin, 15% of skimmed milk powder, 10% of gelatinized modified starch, 5% of sucrose, 2% of peptone, 2% of trehalose, 5% of glycerol, 5% of microcrystalline cellulose and the balance of water.
The coating agent solution is: 10% of sodium alginate, 6.5% of hydroxymethyl cellulose, 8.5% of chitosan, 4% of mannan and the balance of water.
Example 4 Lactobacillus plantarum NHE-LpE probiotic validation
(1) Taking pathogenic bacteria (including Erysipelothrix rhusiopathiae, pseudomonas aeruginosa, klebsiella pneumoniae, listeria monocytogenes, streptococcus pneumoniae, enteropathogenic Escherichia coli, staphylococcus aureus, salmonella typhi, salmonella, clostridium perfringens, proteus pengpenii, aeromonas hydrophila, and Vibrio parahaemolyticus) with concentration of 10 9 2mL of CFU/mL bacterial suspension is added with 200mL of sterilized bacterial suspensionCooling to about 45 ℃ of pathogenic bacteria culture medium, then sucking 10mL of unset bacteria-carrying culture medium, transferring the culture medium to a nutrient agar plate poured with 10mL of bottom plate, and preparing a plurality of pathogenic bacteria plates.
(2) Clamping 1 sterilized Oxford cup (a circular small tube with the inner diameter of 6mm, the outer diameter of 8mm and the height of 10mm, wherein 200 mu L of liquid can be added into the tube, and the two ends of the tube are required to be smooth) on an ultra-clean workbench by using sterile forceps, and placing the sterilized Oxford cup on a plate to ensure that the sterilized Oxford cup is in contact with a culture medium without a gap;
(3) After 10 minutes, 200. Mu.L of the preserved fermentation liquid prepared in example 2 was dropped into each vial without overflowing, and cultured at 37 ℃ for 12 hours, and then the zone diameter was measured. Three replicates of each experiment were averaged and the results are shown in table 1.
The pathogenic bacteria culture medium is respectively as follows: the erysipelothrix rhusiopathiae, listeria monocytogenes and streptococcus pneumoniae are TSA +5% defibrinated sheep blood agar, pseudomonas aeruginosa, klebsiella pneumoniae, enteropathogenic escherichia coli, staphylococcus aureus, salmonella typhi, salmonella, proteus pengpenii and aeromonas hydrophila are nutrient agar culture media, the vibrio parahaemolyticus is TCBS culture medium, and the clostridium perfringens is tryptone-sulfite-cycloserine agar culture medium.
TSA +5% defibrinated sheep blood agar consists of: tryptone 1.5%, soytone 0.5%, sodium chloride 0.5%, agar 1.5%, and water in balance, pH 7.2 + -0.2, cooling to 50 deg.C, and adding 5% defibrinated sheep blood.
The nutrient agar culture medium comprises: peptone 1%, beef extract 0.3%, agar 2%, naCl 0.5%, and water in balance, and pH 7.2 + -0.2.
The TCBS agar medium consists of: 0.5% of yeast powder, 1% of peptone, 1% of sodium thiosulfate, 1% of sodium citrate, 0.5% of bile powder, 0.3% of sodium taurocholate, 2% of sucrose, 1% of sodium chloride, 0.1% of ferric citrate, 0.0004% of thymol blue, 1.5% of agar and pH 8.6 +/-0.1.
The tryptone-sulfite-cycloserine agar medium comprises the following components: tryptone 1.5%, soytone 0.5%, yeast powder 0.5%, sodium metabisulfite 0.1%, ferric ammonium citrate 0.1%, agar 2%, balance water, pH7.6 + -0.2, and filtering sterilized 0.5% D-cycloserine solution 20mL/250mL when used while cooling to 50 ℃.
TABLE 1 bacteriostatic effect of Lactobacillus plantarum NHE-LpE on pathogenic bacteria
| Pathogenic bacteria | Bacteriostatic diameter (mm) |
| Erysipelas suis Erysipelas suis | 31.67 |
| Pseudomonas aeruginosa | 28.34 |
| Klebsiella pneumoniae pUnoniae | 26.78 |
| Listeria ivalis ivanovvii | 27.43 |
| Streptococcus pneumoniae pneumococcus pneumniae | 22.43 |
| Intestinal pathogenic Escherichia coli | 28.97 |
| Staphylococcus aureus StBphylococcus Bureus | 33.41 |
| Salmonella typhosaSalmonella typhimurium | 21.21 |
| Salmonella | 22.13 |
| Shigella Shigella Castellani | 18.92 |
| Clostridium perfringens Clostridium perfringens perfringen | 31.24 |
| Proteus penneri | 18.32 |
| Aeromonas hydrophila Aeromonas hydrophila | 17.01 |
| Vibrio parahaemolyticus | 14.23 |
Example 5 Lactobacillus plantarum NHE-LpE stress resistance validation
1. Determination of resistance to Artificial gastric juice
Placing 10mL of lactobacillus plantarum NHE-LpE bacterial suspension (prepared according to the method described in example 1) in 90mL of artificial gastric juice (250 mL triangular flask) and shaking at the constant temperature of 200r/min at 37 ℃ for 180min; after shaking, 10mL of sample solution is taken to adjust the pH value to 7.0, 90mL of normal saline is added, shaking is carried out at the constant temperature of 37 ℃ and 200r/min for 30min, and then dilution plate colony culture counting is carried out. The results are shown in Table 2. As can be seen from Table 2, the survival rate of Lactobacillus plantarum NHE-LpE treated in artificial gastric juice (containing enzymes) with pH1.5, pH2.0 and pH2.5 for 3h is more than 97%, which indicates that the strain NHE-LpE has high acid resistance, can resist gastric acid and can successfully reach the intestinal tract to play a role.
The preparation method of the artificial gastric juice comprises the following steps: according to the preparation method in the pharmacopoeia of the people's republic of China 2010, 16.4mL of dilute hydrochloric acid is taken, about 800mL of water and 10g of pepsin are added, the mixture is uniformly shaken, then the diluted mixture is diluted into 1000mL of water, the pH values are adjusted to be 1.5, 2.0 and 2.5 respectively, and a microporous filter membrane is sterilized (0.22 mu m) for later use.
TABLE 2 survival of Lactobacillus plantarum NHE-LpE5 after 3h treatment in artificial gastric juice
| Treatment of | pH1.5 | pH2.0 | pH2.5 |
| Initial Activity CFU/mL | 3.5×10 10 | 3.5×10 10 | 3.5×10 10 |
| Activity CFU/mL after treatment | 3.4×10 10 | 3.5×10 10 | 3.5×10 10 |
| Survival rate after treatment% | 97.14 | 100 | 100 |
2. Determination of survival Performance of Artificial intestinal juice
1mL of lactobacillus plantarum NHE-LpE bacterial suspension (prepared according to the method described in example 1) is placed in 99mL (250 mL triangular flask) of artificial intestinal fluid, shaking is carried out at the constant temperature of 37 ℃ and 200r/min for 5 hours, 1mL of sample solution is added with 99mL of normal saline after shaking is finished, shaking is carried out at the constant temperature of 37 ℃ and 200r/min for 30 minutes, and then dilution plate bacterial colony culture counting is carried out. The result shows that the activity of the lactobacillus plantarum NHE-LpE in the artificial intestinal juice is not reduced, the survival rate is 100 percent, and the strain can well survive and preserve the activity in the intestinal juice, thereby exerting the probiotic effect.
Preparing artificial intestinal juice: the preparation of the artificial intestinal juice refers to a preparation method in pharmacopoeia of the people's republic of China 2010, phosphate buffer (containing pancreatin) (pH6.8), 6.8g of monopotassium phosphate is taken, 500mL of water is added for dissolving, and the pH value is adjusted to 6.8 by 0.1mol/L of sodium hydroxide solution; dissolving pancreatin 10g in water, mixing the two solutions, diluting with water to 1000mL, and filtering with 0.22 μm microfiltration membrane for sterilization.
3. Bile salt survival Performance assay
1mL of lactobacillus plantarum NHE-LpE bacterial suspension (prepared according to the method described in example 1) is placed in 99mL of solution (250 mL of triangular flask) with different concentrations of bile salts, wherein the concentrations of the bile salts are 0.15%, 0.3%, 1% and 1.5%, the mixture is vibrated at constant temperature of 200r/min at 37 ℃ for 120min, 1mL of sample solution is added with 99mL of normal saline after vibration is finished, the mixture is vibrated at constant temperature of 200r/min at 37 ℃ for 30min, and then dilution plate colony culture counting is carried out.
The results are shown in Table 3. The lactobacillus plantarum NHE-LpE is treated in a 0.3% bile salt solubility solution for 2 hours, and the survival rate is 85.71%, which shows that the strain has high bile salt resistance, can resist bile salt in duodenal juice, and can reach the intestinal tract to exert the function.
The preparation method of the bile salt solution with different concentrations comprises the following steps: adding 9mL, 18mL, 60mL and 90mL of 5% bile salt solution into PBS solution with pH7.4, diluting to 300mL, and mixing to obtain PBS solution containing 0.15%, 0.30%, 1% and 1.5% bile salt.
The preparation method of the 5% bile salt solution comprises the following steps: accurately weighing 5.0g of bile salt, dissolving with 100ml of LPBS solution to constant volume, and sterilizing at 121 deg.C for 20min.
The preparation method of the PBS solution comprises the following steps: 0.8% of sodium chloride, 0.02% of potassium chloride, 0.363% of disodium hydrogen phosphate, 0.024% of potassium dihydrogen phosphate and the balance of water. Adjusting pH to 7.4 with 6mol/L HCl, and sterilizing at 121 deg.C for 20min.
TABLE 3 survival of Lactobacillus plantarum NHE-LpE after 2h treatment in bile salt solutions of different concentrations
Example 6 Lactobacillus plantarum NHE-LpE acute toxicity test
The safety evaluation of the lactobacillus plantarum is carried out by adopting an acute toxicity test according to the national standard GB15193.3-2003 maximum tolerated dose method. Taking 60 common Kunming mice, each of which is 18-20g in female and male, feeding the mice for 1 week conventionally, and then, carrying out intragastric administration on the mice three times a day, continuously administrating the bacteria liquid of lactobacillus plantarum NHE-LpE (equivalent to 15000mg/kg of body weight) 0.25g/mL for 2 weeks, and observing whether the mice have poisoning or death phenomenon.
During the test, the mouse is in good mental state and has no poisoning and death phenomena, so the maximum tolerated dose MTD of the strain of the invention in the acute toxicity test is more than 15000mg/kg, and the strain can be determined to be non-toxic according to the classification standard and has higher safety.
Example 7 mouse challenge test
(1) 60C 57 mice, female, 11-13g were selected and bred conventionally. The mice are randomly divided into three groups, and each group is fed with basic ration for 5 days, so that the mice can adapt as soon as possible. The formal test is started and divided into two stages. The first stage of growth performance observation test, experimental group added with lactobacillus plantarum fermentation liquor with the concentration of 1 × 10 7 CFU/mL。
(2) The control group and the negative control group were drinking purified water without lactobacillus plantarum for two weeks. Before testingThen weighing for 1 time, observing the growth condition of the mice during the test period, and calculating the average growth rate of the body weight of each group, wherein the results are shown in a table 4. Performing salmonella challenge test in the second stage, wherein the negative control group and the test group are subjected to salmonella challenge in a gastric lavage manner, specifically, the gastric lavage concentration is 10 8 The order of magnitude of Salmonella is 0.5 mL/mouse, and the control group is intragastrically administered with 0.5 mL/mouse of physiological saline.
(3) Observing mental state and death condition of mice after challenge, and continuing drinking 1 × 10 7 The lactobacillus plantarum NHE-LpE fermentation broth with the concentration of CFU/mL, and the control group and the negative control group normally drink tap water. During the test period, mice are raised in cages in the same room, naturally illuminated and freely fed. The ambient temperature is controlled at 25 +/-2 ℃ and the humidity is 60 percent.
The lactobacillus plantarum broth was the broth prepared in example 2.
The experimental results show that after salmonella is attacked, the mice have the disease symptoms by daily observation: basically, the people do not eat, are inactive, are cachectic and do not curl together, the hair color is not glossy, the back hair is messy, and the eyes are congested. After challenge, on day 1, the negative control group began to have symptoms and died 2, with other surviving symptoms appearing to varying degrees. The test group died 2 mice on day 3, and other mice were not wonderful, did not eat food for 5 days, and then gradually returned to normal.
The death time, death number and survival rate of each group after challenge are shown in table 5, and the poisoning phenomenon of the mice in the test group after challenge is not serious, which shows that the lactobacillus plantarum NHE-LpE has a certain immunoprophylaxis effect on salmonella infection, the morbidity of the mice infected by salmonella is effectively reduced, the morbidity time after challenge is delayed, and the survival rate is effectively improved.
TABLE 4 weight gain in mice
| Grouping | Average initial weight (g/only) | Average final weight (g/piece) | Average growth rate (%) |
| Control group | 11.25±0.16 | 22.14±0.78 | 96.80 b |
| Negative control group | 11.43±0.24 | 22.42±0.49 | 96.15 b |
| Test group | 11.21±0.36 | 26.48±0.31 | 136.21 a |
TABLE 5 mice mortality before and after challenge
Example 8 Lactobacillus plantarum NHE-LpE Microecological formulation effects on growth Performance of weaned piglets
(1) The test selects three-element hybrid weaned piglets (half of male and female) with same birth times and similar weight (Duroc, long white and big white), 200 weaned piglets are randomly divided into two groups (a control group and a test group) after 28 days, 10 piglets are treated for each column, and the feeding test is carried out for 45 days under the same environment.
(2) The control group feed is basic feed (referring to Chinese pig feeding standard), and the test group feed is basic daily feed added with 1 × 10 7 CFU/g lactobacillus plantarum microecological preparation.
(3) Before the test began, the piggery was thoroughly disinfected. The test group calculates the addition of the micro-ecological preparation according to the daily food intake, and then drinks the micro-ecological preparation by dissolving water for 2 times to ensure that the micro-ecological preparation is drunk up each time. The feed amount and the health condition are recorded every day, the fur brightness and the excrement condition of the pigs are observed regularly, and other feeding management is the same. Compared with a control group, the liquid fermented feed group can obviously improve the daily gain of piglets in a test group, obviously reduce the feed conversion ratio by 0.2 and obviously reduce the diarrhea rate. (Table 5)
TABLE 5 influence of Lactobacillus plantarum microecologics on growth performance of weaned piglets
| Item | Control group | Test group |
| Initial weight (kg) | 6.41±0.17 | 6.38±0.23 |
| Terminal weight (kg) | 20.20±1.02 b | 23.82±2.21 a |
| Average daily gain (g/d) | 306±23.56 b | 387.55±31.24 a |
| Average daily food intake (kg/d) | 563±56.43 b | 632±21.34 a |
| Meat ratio of materials | 1.83±0.14 a | 1.63±0.04 b |
| Rate of diarrhea | 7.14±0.14 A | 1.46±0.05 B |
Note: the data in the same row are marked with different lower case letters in shoulder notation to show significant difference (p < 0.05), and marked with the same lower case letters or no letters in shoulder notation to show insignificant difference (p > 0.05).
Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.
Claims (10)
1. Lactobacillus plantarum NHE-LpE and classified name of lactobacillus plantarum (A)Lactobacillus plantarum) NHE-LpE, which was deposited in China general microbiological culture Collection center (CGMCC) at 28.2.2022, with the collection number of CGMCC NO.24432 and the collection address of Beijing, china.
2. A broad-spectrum antibacterial agent comprising lactobacillus plantarum NHE-LpE according to claim 1 and a pharmaceutically acceptable carrier.
3. The broad-spectrum antimicrobial agent of claim 2, wherein the antimicrobial spectrum of the broad-spectrum antimicrobial agent comprises the following strains: erysipelothrix suis, pseudomonas aeruginosa, klebsiella pneumoniae, listeria monocytogenes, streptococcus pneumoniae, enteropathogenic escherichia coli, staphylococcus aureus, salmonella typhi, salmonella, clostridium perfringens, proteus penonii, aeromonas hydrophila and vibrio parahaemolyticus.
4. Use of lactobacillus plantarum NHE-LpE as described in claim 1 for preparation of broad-spectrum antibacterial agent.
5. The use according to claim 4, characterized in that the antibiogram of said broad-spectrum antibacterial agent comprises the following strains:
erysipelothrix suis, pseudomonas aeruginosa, klebsiella pneumoniae, listeria monocytogenes, streptococcus pneumoniae, enteropathogenic escherichia coli, staphylococcus aureus, salmonella typhi, salmonella, clostridium perfringens, proteus penonii, aeromonas hydrophila and vibrio parahaemolyticus.
6. Use of lactobacillus plantarum NHE-LpE as described in claim 1 for the preparation of an oral probiotic.
7. The use according to claim 6, wherein the oral probiotic formulation has an effective viable count of 1.2 x 10 or more 11 CFU/g。
8. Use of a probiotic prepared from lactobacillus plantarum NHE-LpE as described in claim 1 for improving the growth performance of weaned pigs.
9. Use of lactobacillus plantarum NHE-LpE5 according to claim 1 for preparation of liquid fermented feed for improving growth performance of weaned pigs.
10. Use of lactobacillus plantarum NHE-LpE5 according to claim 1 for the preparation of a liquid fermented feed for reducing diarrhea in weaned piglets.
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