CN116004430A - Lactobacillus plantarum NHE-LpE9 and application thereof - Google Patents
Lactobacillus plantarum NHE-LpE9 and application thereof Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention provides a lactobacillus plantarum strainLactobacillus plantarum) NHE-LpE belongs to the technical field of probiotics for livestock feeding. The strain is separated from piglet intestinal canal and preserved in China general microbiological culture Collection center (CGMCC), the address is the national academy of sciences of China, including No. 3, of North Chen West Lu, korea, beijing, the preservation number is CGMCC No.24433, and the preservation date is 2022, 2, 28 days. The strain has broad-spectrum killing effectThe bacterial activity can be used for preparing broad-spectrum bactericides.
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
At present, antibiotics are generally used for treating and preventing diseases in animal husbandry production, so that the aims of relieving stress and promoting growth are fulfilled.
However, the long-term use of antibiotics in large-scale farming can lead to pathogen resistance and can lead to veterinary drug residues in meat products and environmental pollution. There is therefore a need for new antibiotic substitutes to replace antibiotics.
Probiotics are one of the common alternatives to antibiotics, which have a positive effect on the growth performance of pigs and the control of diseases. The existing research shows that probiotics can effectively supplement the deficiency of microorganisms in intestinal tracts and improve the disease resistance of piglets. However, the existing strains have single functions and still have a plurality of defects, so that the development of more strains with excellent performance is still an urgent problem to be solved at present.
Disclosure of Invention
The invention aims to provide a lactobacillus plantarum NHE-LpE9 and application thereof.
In order to achieve the above purpose, the present invention provides the following technical solutions:
lactobacillus plantarum NHE-LpE9, classified and named Lactobacillus plantarum (Lactobacillus plantarum) NHE-LpE9, is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of 24433 and a preservation address of Beijing, china, for 2 months and 28 days.
Preferably, the lactobacillus plantarum NHE-LpE is lactobacillus plantarum isolated from intestinal contents of fresh piglets, and is determined to be lactobacillus plantarum by colony morphology observation, molecular biological 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: strain E9 can grow well on MRS flat plate, and cultivate for 48 hours to form circular colony, the diameter of colony is 2-3mm, and the colony is milky white, which shows that the colony is smooth, opaque, middle convex, edge is regular, colony morphology is shown in figure 1, microscopic morphology is shown in figure 2, 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 as follows:
collecting seed solution (viable bacteria concentration 10) of Lactobacillus plantarum NHE-LpE9 (preservation number is CGMCC No. 24433) 9 CFU/mL) 3mL, inoculating in 500mL shake flask fermentation medium for shake flask fermentation culture; fermentation tank for fermentation after shaking bottle fermentationFermenting and culturing, namely inoculating 1000mL of shake flask fermentation seed liquid into a fermentation medium in a 100L fermentation tank for fermenting and culturing, wherein the liquid amount of the 100L fermentation tank is 70L fermentation medium. After fermentation, detecting the viable count of the fermentation liquor to be 2.2X10 10 CFU/mL。
The shake flask fermentation medium consists of the following components: 0.5 to 4 percent of sucrose, 0.5 to 2.5 percent of glucose, 0.5 to 3.0 percent of yeast extract powder, 0.5 to 2.5 percent of soybean peptone, 0.01 to 0.5 percent of magnesium chloride, 0.01 to 1.0 percent of calcium carbonate, 0.01 to 0.5 percent of manganese sulfate and the balance of water.
Preferably, it is: sucrose 2%, glucose 0.5%, yeast extract 1%, soybean peptone 1%, magnesium chloride 0.1%, calcium carbonate 0.1%, manganese sulfate 0.02% and water in balance.
The shaking flask fermentation conditions are as follows: the inoculation amount is 1% (volume ratio), the fermentation temperature is 30 ℃, the initial pH is 6.8, the initial pH is 200r/min, and the fermentation time is 8h.
The culture medium components and the fermentation conditions of the 100L fermentation tank are as follows: 1.0 to 4.0 percent of soft white sugar, 0.2 to 2.0 percent of glucose, 0.5 to 3.5 percent of yeast extract, 0.5 to 2.5 percent of soybean peptone, 0.01 to 0.5 percent of magnesium chloride, 0.01 to 1.0 percent of calcium carbonate, 0.01 to 0.5 percent of manganese sulfate, 0.05 to 0.2 percent of tween-80 and the balance of water.
Preferably, it is: 1.0% of soft white sugar, 1.0% of glucose, 1.0% of yeast extract, 1.0% of soybean peptone, 0.2% of magnesium chloride, 0.2% of calcium carbonate, 0.05% of manganese sulfate, 0.2% of tween-80 and the balance of water.
The shaking flask fermentation conditions are as follows: the liquid amount of a 100L fermentation tank is 70L culture medium, the tank pressure is controlled to be 0.05-0.06MPa, the inoculation amount is 1L, the fermentation temperature is 30 ℃, the fermentation time is 14h, the pH value is 6.5, and the stirring rotating speed is 300r/min.
Application of lactobacillus plantarum NHE-LpE9 in preparing feed for improving growing performance of fattening pigs.
Application of lactobacillus plantarum NHE-LpE in preparing broad-spectrum bactericides.
Preferably, the broad spectrum fungicide has the following antimicrobial spectrum: erysipelas, pseudomonas aeruginosa, klebsiella pneumoniae, listeria monocytogenes, streptococcus pneumoniae, enteropathogenic escherichia coli, staphylococcus aureus, salmonella typhi, salmonella, bacillus perfringens, proteus peng, aeromonas hydrophila and vibrio parahaemolyticus.
Use of lactobacillus plantarum NHE-LpE9 in the preparation of a salmonella infection inhibitor.
Preferably, the inhibitor reduces the incidence of salmonella infection and delays the onset time after challenge.
Application of lactobacillus plantarum NHE-LpE in preparation of oral microecological preparation.
Application of lactobacillus plantarum NHE-LpE in preparing high-temperature resistant microecological preparations.
Preferably, the elevated temperature is in the range 55 ℃ to 75 ℃.
The beneficial effects of the invention are as follows:
the invention provides a novel lactobacillus plantarum NHE-LpE9 which has the properties of gastric acid resistance, intestinal juice resistance and bile salt resistance, so that the novel lactobacillus plantarum NHE-LpE can be orally taken in the intestinal tract to play a role;
secondly, the strain has excellent temperature resistance, broad-spectrum sterilization performance and a certain immunoprophylaxis effect on salmonella infection.
In addition, the microbial ecological agent fermented feed of the strain can obviously improve the growth performance of fattening pigs.
Drawings
FIG. 1 is a colony morphology of Lactobacillus plantarum NHE-LpE9 on MRS medium;
FIG. 2 is a gram stain of Lactobacillus plantarum NHE-LpE9 strain
Detailed Description
The examples are presented for better illustration of the invention, but the invention is not limited to the examples. Those skilled in the art will appreciate that various modifications and adaptations of the embodiments described above are possible in light of the above teachings and are intended to be within the scope of the invention.
Unless otherwise indicated, all chemical reagents used in the examples were conventional commercial reagents, and the technical means used in the examples were conventional means well known to those skilled in the art.
The percentage "%" referred to in the present invention refers to mass percent unless otherwise specified; however, the percentage of the solution, unless otherwise specified, refers to the grams of solute contained in 100mL of solution.
Example 1
Isolation, screening and identification of Lactobacillus plantarum NHE-LpE9
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 a sterile 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-fold ratio dilution method, and selecting three dilutions of 1 ten thousand times, 10 ten thousand times and 100 ten thousand times;
(2) Sucking 0.1mL, coating on an improved MRS agar plate, inversely culturing for 48 hours at 37 ℃ after the plate coating, picking out a colony of suspected lactobacillus with obvious calcium dissolving ring and larger than 5mm by using an inoculating loop, and carrying out streak separation culture on the MRS agar plate;
(3) After 48h of culture, picking out bacterial colony with better separation effect, transferring the bacterial colony onto MRS agar inclined plane by using an inoculating loop for pure culture, repeating the pure culture for 3 times, suspending bacterial cells in 20% glycerol solution, and preserving 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 the strains can be preliminarily determined to be lactobacillus and 182 strains are obtained. Activating the glycerol tube strain stored in the step 1 for 2-3 times by using an MRS agar plate, inoculating the glycerol tube strain into an MRS broth culture medium, performing shake culture for 18-20 hours at the constant temperature of 37 ℃ for 180r/min, taking a clean glass slide for gram staining, performing microscopic examination, observing the microscopic morphology of the strain, and selecting non-spore-producing bacteria with positive gram staining as standby.
3. Preparation of lactic acid bacteria suspension and fermentation broth
Culturing the lactobacillus obtained in the step 1 on an MRS agar plate at 35 ℃ for 48 hours, picking single colony from the plate, and shake culturing for 24 hours at 35 ℃ and 180r/min in 100mL MRS liquid culture medium to obtain lactobacillus suspension for later use; and continuing to shake-culture at 35 ℃ and 180r/min for 96 hours to obtain lactobacillus fermentation liquor for later use.
4. Screening of lactic acid bacteria for acid producing ability
Inoculating the lactobacillus suspension obtained in the step 3 into MRS liquid culture medium according to an inoculum size of 1% (volume ratio), culturing for 12 hours at 35 ℃, screening out lactobacillus with the pH of fermentation liquor being less than 4.0 for 12 hours, and obtaining 39 strains of lactobacillus altogether.
5. Screening of lysine degradation by lactic acid bacteria
Inoculating the lactobacillus suspension obtained in the step 3 into MRS liquid culture medium added with 1% of lysine according to an inoculum size of 1% (volume ratio), culturing for 24 hours at 35 ℃, measuring free lysine content every 4 hours, and selecting 26 strains of strains which do not degrade lysine in the fermentation process.
6. Screening of bacteriostatic lactic acid bacteria
(1) Collecting pathogenic bacteria (such as erysipelas, pseudomonas aeruginosa, klebsiella pneumoniae, listeria monocytogenes, streptococcus pneumoniae, enteropathogenic Escherichia coli, staphylococcus aureus, salmonella typhi, salmonella, bacillus perfringens, proteus pensonii, aeromonas hydrophila, and Vibrio parahaemolyticus) with a concentration of 10 9 2mL of CFU/mL bacterial suspension is added into a pathogenic bacteria culture medium which is filled with 200mL of bacterial suspension and cooled to about 45 ℃ after sterilization, then 10mL of non-solidified bacteria-carrying culture medium is sucked, and the bacteria-carrying culture medium is transferred onto a nutrient agar plate poured with 10mL of bottom plate, so that a plurality of pathogenic bacteria plates are prepared.
(2) Each pathogen nutrient agar plate on the ultra-clean workbench is clamped with 1 sterilized oxford cup (a round small tube with the inner diameter of 6mm, the outer diameter of 8mm and the height of 10mm, 200 mu L of liquid can be added into the tube, and two ends of the tube are smooth) and placed on the plate, so that the plate is in contact with a culture medium without gaps, after a few minutes, 200 mu L of suspected lactobacillus strain fermentation liquor (obtained in the step 3) is respectively sucked into the oxford cup, and the oxford cup is cultivated at the constant temperature of 37 ℃ for 24 hours. At least 3 replicates were made for each strain, and the size of the zone of inhibition was observed and measured, wherein 5 strains were identified as E5, E9, E15, E115, and E162, respectively, for the large zone of inhibition.
7. Identification of strain species
(1) The strain E9 is subjected to morphological and physiological biochemical identification, the strain E9 can well grow on an MRS plate and is cultured for 48 hours to form circular colonies, the diameters of the colonies are 2-3mm, the colonies are milky white, smooth and opaque, the middle of the colonies are convex, the edges of the colonies are regular, the colony morphology is as shown in figure 1, the micro morphology is as shown in figure 2, the micro morphology is as a rod shape, no spores exist, the bacteria are facultative anaerobism, the growth proper temperature 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.
(2) Extracting the genome DNA of the E9 strain by using a kit for extracting bacterial DNA. Sequencing the 16S rDNA gene fragment of the E9 strain by using the primers F and R, wherein the obtained sequence is shown as SEQ ID NO.1, and comparing the measured sequence with the 16S rDNA sequence in GenBank by BLAST analysis, so that the homology of the strain E9 and lactobacillus plantarum can be found to reach 99.86%.
(3) Strain E9 was identified as lactobacillus plantarum (Lactobacillus plantarum) by morphological characteristics of strain E9, 16S rDNA characteristics, and formally labeled NHE-LpE9.
8. Preservation of strains
The lactobacillus plantarum (Lactobacillus plantarum) NHE-LpE obtained by separation, purification and screening is preserved in China general microbiological culture Collection center (CGMCC for short, with the address of: the collection number is CGMCC No.24433, and the collection is classified and named as lactobacillus plantarum (Lactobacillus plantarum).
The composition of the improved MRS agar medium is as follows: 1.0% peptone, 0.5% sodium acetate, 1.0% beef extract, 2% glucose, 0.5% yeast extract, 0.1% Tween 80, K 2 HPO 4 0.2％，MgSO 4 0.058%, diammonium citrate 0.2%, mnSO 4 0.025%, agar 1.8%, calcium carbonate 1%, the balance being water, pH 7.0.+ -. 0.2.
MRS agar medium consists of: 1.0% peptone, 0.5% sodium acetate, 1.0% beef extract, 2% glucose, 0.5% yeast extract, 0.1% Tween 80, K 2 HPO 4 0.2％，MgSO 4 0.058%, diammonium citrate 0.2%, mnSO 4 0.025%, agar 1.8%, balance water, pH 7.0.+ -. 0.2.
MRS broth medium composition was: 1.0% peptone, 0.5% sodium acetate, 1.0% beef extract, 2% glucose, 0.5% yeast extract, 0.1% Tween 80, K 2 HPO 4 0.2％，MgSO 4 0.058%, diammonium citrate 0.2%, mnSO 4 0.025%, the balance being water, pH 7.0.+ -. 0.2.
The pathogen culture mediums are respectively as follows: the swine erysipelas, the listeria monocytogenes and the streptococcus pneumoniae are TSA+5% defibrinated sheep blood agar, the pseudomonas aeruginosa, the klebsiella pneumoniae, the enteropathogenic escherichia coli, the staphylococcus aureus, the salmonella typhi, the salmonella, the proteus peng and the aeromonas hydrophila are nutrient agar culture mediums, the vibrio parahaemolyticus is a TCBS culture medium, and the clostridium perfringens is a tryptone-sulfite-cycloserine agar culture medium.
TSA+5% defibrinated sheep blood agar composition was: 1.5% of tryptone, 0.5% of soyase, 0.5% of sodium chloride, 1.5% of agar, the balance of water, and pH 7.2+/-0.2, and 5% of defibrinated sheep blood is added when the mixture is cooled to 50 ℃ during use.
The nutrient agar medium consists of: 1% of peptone, 0.3% of beef extract, 2% of agar, 0.5% of NaCl and the balance of water, and the pH value is 7.2+/-0.2.
The TCBS agar medium consists of: yeast powder 0.5%, peptone 1%, sodium thiosulfate 1%, sodium citrate 1%, ox gall powder 0.5%, sodium taurocholate 0.3%, sucrose 2%, sodium chloride 1%, ferric citrate 0.1%, thymol blue 0.0004%, agar 1.5% and pH 8.6+ -0.1.
The composition of the tryptone-sulfite-cycloserine agar medium is as follows: 1.5% of tryptone, 0.5% of soyase, 0.5% of yeast powder, 0.1% of sodium metabisulfite, 0.1% of ferric ammonium citrate, 2% of agar and the balance of water, wherein pH 7.6+/-0.2 is adopted, and 20mL/250mL of 0.5% D-cycloserine solution which is sterilized by filtration is added when the solution is cooled to 50 ℃ during use.
EXAMPLE 2 preparation of Lactobacillus plantarum NHE-LpE9 fermentation broth
Collecting seed solution (viable bacteria concentration 10) of Lactobacillus plantarum NHE-LpE9 (preservation number is CGMCC No. 24433) 9 CFU/mL) 3mL, inoculating in 500mL shake flask fermentation medium for shake flask fermentation culture; after the shake flask fermentation is finished, fermenting and culturing in a fermentation tank, and inoculating 1000mL of shake flask fermentation seed liquid into a fermentation medium in a 100L fermentation tank for fermenting and culturing, wherein the liquid amount of the 100L fermentation tank is 70L fermentation medium. After fermentation, detecting the viable count of the fermentation liquor to be 2.2X10 10 CFU/mL。
The shake flask fermentation medium consists of the following components: sucrose 2%, glucose 0.5%, yeast extract 1%, soybean peptone 1%, magnesium chloride 0.1%, calcium carbonate 0.1%, manganese sulfate 0.02% and water in balance.
The shaking flask fermentation conditions are as follows: the inoculation amount is 1% (volume ratio), the fermentation temperature is 30 ℃, the initial pH is 6.8, the initial pH is 200r/min, and the fermentation time is 8h.
The culture medium components and the fermentation conditions of the 100L fermentation tank are as follows: 1.0% of soft white sugar, 1.0% of glucose, 1.0% of yeast extract, 1.0% of soybean peptone, 0.2% of magnesium chloride, 0.2% of calcium carbonate, 0.05% of manganese sulfate, tween-800.2% and the balance of water.
The shaking flask fermentation conditions are as follows: the liquid amount of a 100L fermentation tank is 70L culture medium, the tank pressure is controlled to be 0.05-0.06MPa, the inoculation amount is 1L, the fermentation temperature is 30 ℃, the fermentation time is 14h, the pH value is 6.5, and the stirring rotating speed is 300r/min.
EXAMPLE 3 probiotic verification of Lactobacillus plantarum NHE-LpE9
(1) Collecting pathogenic bacteria (such as erysipelas, pseudomonas aeruginosa, klebsiella pneumoniae, listeria monocytogenes, streptococcus pneumoniae, enteropathogenic Escherichia coli, staphylococcus aureus, salmonella typhi, salmonella, bacillus perfringens, proteus pensonii, aeromonas hydrophila, and Vibrio parahaemolyticus) with a concentration of 10 9 2mL of CFU/mL bacterial suspension is added into a pathogenic bacteria culture medium which is filled with 200mL of bacterial suspension and cooled to about 45 ℃ after sterilization, then 10mL of non-solidified bacteria-carrying culture medium is sucked, and the bacteria-carrying culture medium is transferred onto a nutrient agar plate poured with 10mL of bottom plate, so that a plurality of pathogenic bacteria plates are prepared.
(2) On an ultra-clean bench, each pathogen nutrient agar plate was clamped with 1 sterilized oxford cup (circular small tube with inner diameter of 6mm, outer diameter of 8mm and height of 10mm, 200. Mu.L of liquid was added to the tube, both ends of the tube were smooth) and placed on the plate, and after 10 minutes, 200. Mu.L of the fermentation broth prepared in example 2 was added dropwise to each small tube without overflowing, and incubated at 37℃for 12 hours, and then the diameter of the inhibition zone was measured. Three replicates per experiment were averaged and the results are shown in table 1.
The pathogen culture mediums are respectively as follows: the swine erysipelas, the listeria monocytogenes and the streptococcus pneumoniae are TSA+5% defibrinated sheep blood agar, the pseudomonas aeruginosa, the klebsiella pneumoniae, the enteropathogenic escherichia coli, the staphylococcus aureus, the salmonella typhi, the salmonella, the proteus peng and the aeromonas hydrophila are nutrient agar culture mediums, the vibrio parahaemolyticus is a TCBS culture medium, and the clostridium perfringens is a tryptone-sulfite-cycloserine agar culture medium.
TSA+5% defibrinated sheep blood agar composition was: 1.5% of tryptone, 0.5% of soyase, 0.5% of sodium chloride, 1.5% of agar, the balance of water, and pH 7.2+/-0.2, and 5% of defibrinated sheep blood is added when the mixture is cooled to 50 ℃ during use.
The nutrient agar medium consists of: 1% of peptone, 0.3% of beef extract, 2% of agar, 0.5% of NaCl and the balance of water, and the pH value is 7.2+/-0.2.
The TCBS agar medium consists of: yeast powder 0.5%, peptone 1%, sodium thiosulfate 1%, sodium citrate 1%, ox gall powder 0.5%, sodium taurocholate 0.3%, sucrose 2%, sodium chloride 1%, ferric citrate 0.1%, thymol blue 0.0004%, agar 1.5% and pH 8.6+ -0.1.
The composition of the tryptone-sulfite-cycloserine agar medium is as follows: 1.5% of tryptone, 0.5% of soyase, 0.5% of yeast powder, 0.1% of sodium metabisulfite, 0.1% of ferric ammonium citrate, 2% of agar and the balance of water, wherein pH 7.6+/-0.2 is adopted, and 20mL/250mL of 0.5% D-cycloserine solution which is sterilized by filtration is added when the solution is cooled to 50 ℃ during use.
TABLE 1 antibacterial Effect of Lactobacillus plantarum NHE-LpE9 on pathogenic bacteria
| Pathogenic bacteria | Antibacterial diameter (mm) |
| Erysipelas suis of erysipelothrix rhusiopathiae | 24.19 |
| Pseudomonas aeruginosa Pseudomonas aeruginosa | 14.23 |
| Klebsiella pneumoniae Klebsiella pneumoniae | 29.32 |
| Listeria monocytogenes Listeria ivanovii | 31.21 |
| Streptococcus pneumoniae Streptococcus pneumoniae | 18.54 |
| Intestinal pathogenic Escherichia coli | 17.98 |
| Staphylococcus aureus StBphylococcus Bureus | 33.48 |
| Salmonella typhi Salmonella typhimurium | 18.23 |
| Salmonella | 18.67 |
| Shigella Shigella Castellani | 21.32 |
| Clostridium perfringens Clostridium perfringen | 32.14 |
| Proteus penneri | 21.34 |
| Aeromonas hydrophila Aeromonas hydrophila | 15.61 |
| Vibrio parahaemolyticus Vibrio parahaemolyticus | 18.94 |
As can be seen from Table 1, lactobacillus plantarum NHE-LpE has inhibitory effect on erysipelas, pseudomonas aeruginosa, klebsiella pneumoniae, listeria monocytogenes, streptococcus pneumoniae, enterobacter, staphylococcus aureus, salmonella typhi, salmonella, bacillus perfringens, proteus pensonii, aeromonas hydrophila, and Vibrio parahaemolyticus. Among them, staphylococcus aureus has the best inhibitory effect.
EXAMPLE 4 stress resistance verification of Lactobacillus plantarum NHE-LpE9
1. Determination of Artificial gastric juice resistance
10mL of Lactobacillus plantarum NHE-LpE bacterial suspension (prepared according to the method described in example 1) is placed in 90mL (250 mL triangular flask) of prepared artificial gastric juice, and is subjected to constant-temperature shaking at 37 ℃ for 180min at 200 r/min; and after the shaking is finished, 10mL of sample solution is taken to adjust the pH value to 7.0, 90mL of physiological saline is added, the shaking is performed for 30min at a constant temperature of 200r/min at 37 ℃, and then the colony culture count of the dilution plate is performed. The results are shown in Table 2. As can be seen from Table 2, the survival rate of Lactobacillus plantarum NHE-LpE9 treated in artificial gastric juice (containing enzyme) with pH of 1.5, pH2.0 and pH2.5 respectively for 3 hours is over 90%, which shows that the strain NHE-LpE9 has higher acid resistance, can resist gastric acid and can smoothly reach intestinal tracts to play a role.
The preparation method of the artificial gastric juice comprises the following steps: preparation of artificial gastric juice referring to preparation method in the "pharmacopoeia of the people's republic of China" 2010 edition, 16.4mL of diluted hydrochloric acid is taken, about 800mL of water and 10g of pepsin are added, shaking is carried out, water is added to be weighed and released into 1000mL, pH values are respectively adjusted to 1.5, 2.0 and 2.5, and microporous filter membrane is sterilized (0.22 μm) for standby.
TABLE 2 survival of Lactobacillus plantarum NHE-LpE9 after 3h treatment in artificial gastric juice
| Treatment of | pH1.5 | pH2.0 | pH2.5 |
| Initial Activity CFU/mL | 2.2×10 10 | 2.2×10 10 | 2.2×10 10 |
| Post-treatment Activity CFU/mL | 1.8×10 10 | 2.0×10 10 | 2.2×10 10 |
| Survival after treatment% | 81.81 | 90.90 | 100.00 |
2. Artificial intestinal juice survival performance determination
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 juice, shaking is performed at a constant temperature of 37 ℃ for 5 hours at 200r/min, after shaking is completed, 1mL of sample liquid is added into 99mL of physiological saline, shaking is performed at a constant temperature of 37 ℃ for 30 minutes at 200r/min, and then dilution plate colony culture counting is performed. The result shows that the activity of the lactobacillus plantarum NHE-LpE9 in artificial intestinal juice is not reduced, and the survival rate is 100%, which shows that the strain can well survive in the intestinal juice and preserve the activity, thereby playing the probiotic effect.
Artificial intestinal juice preparation: preparation of artificial intestinal juice referring to preparation method in the "pharmacopoeia of the people's republic of China" 2010 edition, phosphate buffer solution (containing pancreatin) (pH 6.8), taking 6.8g of monopotassium phosphate, adding 500mL of water to dissolve, and adjusting pH value to 6.8 with 0.1mol/L sodium hydroxide solution; and dissolving 10g of pancreatin in a proper amount of water, mixing the two solutions, diluting the mixture to 1000mL by adding water, and filtering and sterilizing the mixture by using a microfiltration membrane with the thickness of 0.22 mu m.
3. Bile salt survival assay
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 solutions with different bile salt concentrations, wherein the bile salt concentrations are 0.15%, 0.3%, 1% and 1.5% respectively, then the solution is subjected to constant-temperature shaking at 37 ℃ and 200r/min for 120min, after shaking, 1mL of sample solution is taken, 99mL of physiological saline is added, the solution is subjected to constant-temperature shaking at 37 ℃ and 200r/min for 30min, and then the colony culture count of a dilution plate is carried out. The results are shown in Table 3. Lactobacillus plantarum NHE-LpE9 is treated in a solution with 0.3% bile salt solubility for 2 hours, and the survival rate is 95.45%, which shows that the strain has higher bile salt resistance, can resist bile salt in duodenal juice, and can reach intestinal tracts to play a role.
The preparation method of the bile salt solution with different concentrations comprises the following steps: respectively adding 9mL, 18mL, 60mL and 90mL of 5% bile salt solution into PBS solution with pH of 7.4, fixing volume to 300mL, and uniformly 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 100mLPBS solution to constant volume, and sterilizing at 121deg.C for 20min.
The preparation method of the PBS solution comprises the following steps: sodium chloride 0.8%, potassium chloride 0.02%, disodium hydrogen phosphate 0.363%, potassium dihydrogen phosphate 0.024%, the balance being water. Adjusting pH to 7.4 with 6mol/L HCl, sterilizing at 121deg.C for 20min, and keeping.
TABLE 3 survival of Lactobacillus plantarum NHE-LpE9 after 2h treatment in bile salts solutions of different concentrations
4. Temperature resistance measurement
10mL of lactobacillus plantarum NHE-LpE fermentation liquid prepared in example 2 is placed in a sterile test tube, then placed in a water bath kettle at 55, 65 and 75 ℃ for treatment for 30min, then the number of viable bacteria of the lactobacillus plantarum NHE-LpE after treatment is measured, and the survival rate is calculated according to the number of viable bacteria before treatment. The results are shown in Table 4. Lactobacillus plantarum NHE-LpE9 is treated for 30min at 55 ℃, and the survival rate is 90.90%, which shows that the strain has stronger temperature resistance.
TABLE 4 survival of Lactobacillus plantarum NHE-LpE9 after 30min treatment at different temperatures
EXAMPLE 5 acute toxicity test of Lactobacillus plantarum NHE-LpE9
The safety evaluation of lactobacillus plantarum is carried out by adopting an acute toxicity test and referring to the national standard GB15193.3-2003 maximum tolerance dosage method. 60 common Kunming mice are taken, the male and female mice are half, 18-20g are kept for 1 week conventionally, the mice are subjected to stomach irrigation three times a day, 0.25g/mL lactobacillus plantarum NHE-LpE bacterial liquid (equivalent to 15000mg/kg body weight) is continuously irrigated for 2 weeks, and whether the mice are poisoned or die is observed.
During the test, the mice have good mental state and no poisoning and death phenomena, so that the maximum tolerance 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 of a non-toxic grade according to the grading standard, and has higher safety.
Example 6 toxicity test in mice
(1) 60C 57 mice, female, 11-13g, were selected and routinely bred. The mice were randomized into three groups, each treatment was fed basal diet for 5 days to adapt as soon as possible. And starting a formal test, wherein the formal test is divided into two stages. In the first stage of growth performance observation test, lactobacillus plantarum fermentation liquor is added into drinking water in the concentration of 1 multiplied by 10 in an experimental group 7 CFU/mL. The control group and the negative control group were drunk with purified water without lactobacillus plantarum added and were continuously drunk for two weeks.
(2) The mice were weighed 1 time before and after the test, the growth of the mice was observed during the test, and the average growth rate of the weights of the groups was calculated, and the results are shown in Table 5.
(3) In the second stage salmonella challenge test, salmonella challenge is carried out on the negative control group and the test group, and a gastric lavage mode is adopted for the challenge, specifically, the gastric lavage concentration is 10 8 Salmonella in the order of magnitude of 0.5 mL/patient, and the control group was perfused with 0.5 mL/patient saline.
(4) After the toxicity attack, the mental state and death condition of the mice are observed, and the test group continuously drinks 1 multiplied by 10 after the toxicity attack 7 Lactobacillus plantarum NHE-LpE fermentation broth with CFU/mL concentration, control group and negative control group drink tap water normally. Mice are fed in separate cages in the same room during the test period, and are naturally illuminated and eat freely. The ambient temperature is controlled at 25+/-2 ℃ and the humidity is 60 percent.
The lactobacillus plantarum fermentation broth is the fermentation broth prepared in example 2.
After salmonella challenge, mice developed symptoms by daily observation: basically no food, inactivity, listlessness, curling together, no luster of hair, and back Mao Lingluan, congestion of eyes. After challenge, on day 1, the negative control group started to have symptoms, and 2 deaths occurred, with other surviving symptoms occurring to varying degrees. The test group died 1 on day 4, and the other mice were ill-conditioned, fed no food, and gradually returned to normal within 5 days.
The death time, death number and survival rate of each group of mice after the toxicity attack are shown in Table 6, and the poisoning phenomenon of the mice in the test group after the toxicity attack is not serious, which shows that lactobacillus plantarum NHE-LpE has a certain immunoprophylaxis effect on salmonella infection, thereby effectively reducing the morbidity of the mice when the mice are infected by salmonella, and delaying the morbidity time after the toxicity attack, and further effectively improving the survival rate.
Table 5 weight gain status of mice
| Grouping | Average initial weight (g/only) | Average final weight (g/only) | 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 | 11.45±0.36 | 27.21±0.31 | 137.64 a |
TABLE 6 death of mice before and after challenge
Example 7 Effect of Lactobacillus plantarum NHE-LpE9 on growing-finishing pig production Performance
(1) Healthy, about 20kg of body weight, consistent male-female proportion and similar day-old (Duroc X Changbai X Dabai) ternary commercial fattening pigs 80 are selected and completely randomly divided into 2 groups (control group and test group), 4 columns are arranged in each group, and 10 pigs are arranged in each column. Control group: test diet is taken as basic diet (refer to Chinese pig raising standard); test group was basal diet + lactobacillus plantarum NHE-LpE fermentation broth prepared in example 2, sprayed in feed at 5% before feeding.
(2) The test pigs adopt a closed colony house, are fed 3 times a day, eat and drink water freely, clean the colony house every day, and feed for 80 days. The feeding management and the immunization program are the same as the daily management of a pig farm, and the pigs are regularly disinfected and found to be ill and treated in time. The test results showed that the feed meat ratio of the test group was significantly lower than that of the control group, reduced by 0.25, and improved the growth performance (table 6).
TABLE 6 influence of Lactobacillus plantarum NHE-LpE9 on growth performance of fattening pigs
| Index (I) | Control group | Test group |
| Initial weight (kg) | 22.05±1.21 | 22.01±1.34 |
| Last weight (kg) | 101.34±6.90 b | 116.89±4.32 a |
| Average daily gain (kg/d) | 0.99±0.05 b | 1.19±0.08 a |
| Average daily feed intake (kg/d) | 2.38±0.31 b | 2.57±0.45 a |
| Feed to meat ratio | 2.40±0.11 b | 2.15±0.43 a |
Finally, it is noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and 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 and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered by the scope of the claims of the present invention.
Claims (9)
1. A strain of lactobacillus plantarum NHE-LpE9 is characterized in that the class of the lactobacillus plantarum NHE-LpE is named as lactobacillus plantarumLactobacillus plantarum) NHE-LpE, 2.2.2.2022The microbial strain is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.24433 and the preservation address of Beijing, china.
2. Use of lactobacillus plantarum NHE-LpE9 according to claim 1 for the preparation of a feed for improving the growth performance of fattening pigs.
3. Use of lactobacillus plantarum NHE-LpE9 as claimed in claim 1 for the preparation of a broad-spectrum fungicide.
4. Use according to claim 3, wherein the broad-spectrum fungicide has the following antimicrobial spectrum: erysipelas, pseudomonas aeruginosa, klebsiella pneumoniae, listeria monocytogenes, streptococcus pneumoniae, enteropathogenic escherichia coli, staphylococcus aureus, salmonella typhi, salmonella, bacillus perfringens, proteus peng, aeromonas hydrophila and vibrio parahaemolyticus.
5. Use of lactobacillus plantarum NHE-LpE9 as claimed in claim 1 for the preparation of a salmonella infection inhibitor.
6. The use of claim 5, wherein the inhibitor reduces the incidence of salmonella infection and delays the onset time after challenge.
7. Use of lactobacillus plantarum NHE-LpE9 as claimed in claim 1 for the preparation of an oral microecological formulation.
8. Use of lactobacillus plantarum NHE-LpE9 according to claim 1 for the preparation of a high temperature resistant microecological formulation.
9. The use according to claim 8, wherein the elevated temperature is in the range of 55 ℃ to 75 ℃.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211079820.5A CN116004430A (en) | 2022-09-05 | 2022-09-05 | Lactobacillus plantarum NHE-LpE9 and application thereof |
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| CN117448213A (en) * | 2023-10-24 | 2024-01-26 | 山东宝来利来生物工程股份有限公司 | Lactobacillus plantarum for inhibiting clostridium perfringens and its progeny and application |
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