CN113773989B - Application of extracellular polysaccharide produced by lactobacillus plantarum Y12 in relieving dysentery caused by shigella flexneri - Google Patents
Application of extracellular polysaccharide produced by lactobacillus plantarum Y12 in relieving dysentery caused by shigella flexneri Download PDFInfo
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- CN113773989B CN113773989B CN202111006449.5A CN202111006449A CN113773989B CN 113773989 B CN113773989 B CN 113773989B CN 202111006449 A CN202111006449 A CN 202111006449A CN 113773989 B CN113773989 B CN 113773989B
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- lactobacillus plantarum
- shigella flexneri
- supernatant
- extracellular polysaccharide
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Abstract
The invention discloses an application of extracellular polysaccharide produced by lactobacillus plantarum Y12 in preparing a medicament for relieving dysentery caused by shigella flexneri, and belongs to the technical field of microbial application. The lactobacillus plantarum (Lactobacillus plantarum) Y12 is preserved in China center for type culture collection (China center for type culture collection) in the 8 th month of 2018 and has a strain preservation number of CCTCC NO: m2018556. The lactobacillus plantarum Y12 extracellular polysaccharide provided by the invention can inhibit the formation of a biological film of shigella flexneri; inhibiting adhesion and invasion of shigella flexneri to intestinal epithelial cells; relieving inflammatory response of intestinal epithelial cells caused by shigella flexneri; the dosage of the antibiotics for relieving dysentery caused by shigella flexneri is reduced.
Description
Technical Field
The invention relates to an application of extracellular polysaccharide produced by lactobacillus plantarum Y12 in relieving dysentery caused by shigella flexneri, and belongs to the technical field of microorganisms.
Background
More and more studies have shown that shigella flexneri can form biofilms that can cause prolonged pathogenic bacterial infections and are difficult to remove thoroughly. Bacteria in the biological film are protected by the self-secreted extracellular polymeric matrix, so that the resistance of the bacteria to the external adverse environment is obviously improved. Antibiotic resistance is also increasingly developed in the environment where antibiotics are widely used. This problem is a serious safety issue both in the medical field and in the food processing field. Exopolysaccharide secreted in the growth and metabolism process of microorganisms can effectively inhibit the formation of a broad-spectrum pathogenic bacteria biological film and the expression of virulence genes on the premise of not having an antibacterial or bactericidal effect, and can reduce the antibiotic resistance of pathogenic bacteria, so that the exopolysaccharide is widely paid attention to.
Disclosure of Invention
The invention provides lactobacillus plantarum (Lactobacillus plantarum) Y12, wherein the lactobacillus plantarum (Lactobacillus plantarum) Y12 is preserved in China Center for Type Culture Collection (CCTCC) in the 8 th month of 2018 and 21 th day, and the strain preservation number is CCTCC NO: m2018556.
The invention also provides extracellular polysaccharide produced by lactobacillus plantarum (Lactobacillus plantarum) Y12, lactobacillus plantarum (Lactobacillus plantarum) Y12 is inoculated in a fresh MRS liquid culture medium for culture, fermentation liquor is taken for centrifugation, and thalli are removed to keep supernatant; concentrating the supernatant by using a rotary evaporator; adding trichloroacetic acid into the concentrated solution, shaking uniformly at room temperature, and precipitating protein; centrifuging, removing protein, and keeping supernatant; adding cold absolute ethyl alcohol into the supernatant, standing and precipitating polysaccharide; centrifuging, discarding supernatant to retain polysaccharide precipitate, and dissolving with deionized water; dialyzing, and lyophilizing.
Further, in the technical scheme, the inoculation amount of the lactobacillus plantarum (Lactobacillus plantarum) Y12 is 1% -2% (V/V); concentrating the supernatant to 1/5-1/4 of the original volume; adding trichloroacetic acid into the concentrated solution to a final concentration of 4% -7%, and shaking uniformly at room temperature for 30-60min; the volume ratio of the supernatant to the cold absolute ethyl alcohol is 2-3, and the conditions of placing and precipitating the polysaccharide are 2-8 ℃ for 12-16 h; the condition of dialysis is 2-8deg.C, and dialysis is performed for 36-48h.
The invention also provides application of the extracellular polysaccharide produced by lactobacillus plantarum (Lactobacillus plantarum) Y12 in preparation of medicines or health-care products for relieving dysentery caused by shigella flexneri.
Furthermore, in the technical scheme, the extracellular polysaccharide produced by the lactobacillus plantarum (Lactobacillus plantarum) Y12 can reduce the minimum action concentration of antibiotics for removing the shigella flexneri biomembrane; the concentration ratio of extracellular polysaccharide to antibiotics produced by the lactobacillus plantarum (Lactobacillus plantarum) Y12 is 1:18-1:20.
Further, in the above technical solution, the antibiotic includes levofloxacin, ciprofloxacin, cefazolin or tetracycline.
Furthermore, in the technical scheme, extracellular polysaccharide produced by lactobacillus plantarum (Lactobacillus plantarum) Y12 can inhibit shigella flexneri from forming a biological film when the action concentration is 0.01-0.200 mug/mu L. The preferred concentration of action is 0.01, 0.025, 0.050, 0.100, 0.200. Mu.g/. Mu.L.
Furthermore, in the technical scheme, when the action concentration of the extracellular polysaccharide produced by the lactobacillus plantarum (Lactobacillus plantarum) Y12 is 0.050-0.200 mug/mu L, adhesion and invasion of shigella flexneri to human intestinal epithelial cells are inhibited. The preferable concentration is 0.050, 0.100, 0.200. Mu.g/. Mu.L.
Furthermore, in the above technical scheme, the extracellular polysaccharide produced by the lactobacillus plantarum (Lactobacillus plantarum) Y12 can relieve inflammatory reaction of human intestinal epithelial cells caused by shigella flexneri when the action concentration is 0.050-0.200 mug/mu L. The preferable concentration is 0.050, 0.100, 0.200. Mu.g/. Mu.L.
The extracellular polysaccharide produced by the lactobacillus plantarum (Lactobacillus plantarum) Y12 reduces the minimum acting concentration of the antibiotic for removing the shigella flexneri biomembrane when the acting concentration is 0.200 mu g/mu L, the levofloxacin is reduced from 1 mu g/mu L to 0.25 mu g/mu L, the ciprofloxacin is reduced from 128 mu g/mu L to 32 mu g/mu L, the cefazolin is reduced from 512 mu g/mu L to 64 mu g/mu L, and the tetracycline is reduced from 512 mu g/mu L to 246 mu g/mu L.
The beneficial effects of the invention are as follows:
(1) The lactobacillus plantarum Y12 extracellular polysaccharide can inhibit the formation of shigella flexneri biofilm;
(2) The lactobacillus plantarum Y12 extracellular polysaccharide can inhibit adhesion and invasion of shigella flexneri on the surface of colon intestinal epithelial cells;
(3) The lactobacillus plantarum Y12 extracellular polysaccharide can relieve inflammatory response of colon intestinal epithelial cells caused by shigella flexneri;
(4) The lactobacillus plantarum Y12 extracellular polysaccharide can reduce the minimum acting concentration of antibiotics for removing the shigella flexneri biofilm.
Drawings
FIG. 1 is a scanning electron microscope image (. Times.30000) of Lactobacillus plantarum Y12.
FIG. 2 is a photograph of Lactobacillus plantarum Y12 exopolysaccharide.
FIG. 3 shows that Lactobacillus plantarum Y12 exopolysaccharide inhibits Shigella flexneri biofilm formation.
FIG. 4 shows the effect of Lactobacillus plantarum Y12 exopolysaccharide on Shigella flexneri growth.
Fig. 5 shows that the extracellular polysaccharide of lactobacillus plantarum Y12 inhibits adhesion of shigella flexneri on the surface of a cover slip by scanning electron microscopy.
FIG. 6 is a fluorescent microscope observation of Lactobacillus plantarum Y12 exopolysaccharide inhibiting the adhesion of Shigella flexneri on the surface of cell culture plates.
FIG. 7 shows that Lactobacillus plantarum Y12 exopolysaccharide inhibits adhesion (A) and invasion (B) of Shigella flexneri to HT-29 cells.
FIG. 8 shows that Lactobacillus plantarum Y12 exopolysaccharide affects HT-29 cytokine secretion under shigella flexneri stimulation, IL-8 (A), IL-10 (B), TNF- α (C).
FIG. 9 shows HPLC analysis of monosaccharide composition of Lactobacillus plantarum Y12 exopolysaccharide.
Detailed Description
The following non-limiting examples will enable those of ordinary skill in the art to more fully understand the invention and are not intended to limit the invention in any way.
EXAMPLE 1 Strain culture
The lactobacillus plantarum Y12 is cultured and activated in an MRS culture medium, and comprises the following specific steps:
a. strain culture method
(1) MRS Medium preparation
The preparation method comprises the following steps: 20g/L glucose, 10g/L peptone, 5g/L yeast extract, 10g/L beef extract, 1mL Tween-80, 2g/L dipotassium hydrogen phosphate, 2g/L diammonium citrate, 5g/L sodium acetate, 0.58g/L magnesium sulfate heptahydrate and 0.25g/L manganese sulfate tetrahydrate are weighed and uniformly mixed, the obtained mixture is mixed and dissolved in 1L deionized water, and the obtained solution is sterilized in a high-pressure steam sterilizing pot for 20min at the temperature of 121 ℃ to obtain the MRS liquid culture medium; 2wt% agar was added to the above formulation, and sterilized under the same conditions to obtain MRS solid medium.
(2) Culture of Lactobacillus plantarum Y12
Lactobacillus plantarum (Lactobacillus plantarum) Y12 is a probiotic isolated from Dalian traditional fermented fish food. Lactobacillus plantarum Y12 is streaked and inoculated in the MRS solid culture medium in the step (1), cultured for 48-72 hours at the temperature of 37 ℃, single bacterial colony is selected, and the single bacterial colony is identified as the contact enzyme negative gram positive rod-shaped bacteria through a hydrogen peroxide contact enzyme test and gram staining. It was identified as lactobacillus plantarum by 16S rDNA sequencing (Lactobacillus plantarum) and designated Y12. Inoculating in 5mL of the liquid culture medium in the step (1), culturing for 18-24h at 37 ℃, and carrying out subculture with 1% (V/V) inoculum size, wherein all operations of the transfer of the microbial strain are strict aseptic operations under aseptic environment.
(3) LB Medium preparation
The preparation method comprises the following steps: weighing 10g/L of tryptone, 5g/L of yeast extract and 10g/L of sodium chloride, uniformly mixing, mixing the obtained mixture into 1L of deionized water, and sterilizing the obtained solution at the temperature of 121 ℃ for 20min to obtain the LB liquid culture medium; 2wt% agar was added to the above formulation, and sterilized under the same conditions to obtain LB solid medium. .
(4) Activation of shigella flexneri
The shigella flexneri is streaked and inoculated in the LB solid medium in the step (3), cultured for 24-48 hours at the temperature of 37 ℃, single colony is selected, inoculated in the liquid medium in the step (3) with the concentration of 5mL, cultured for 18-24 hours at the temperature of 37 ℃, and then subcultured with the inoculum size of 1% (V/V), and all operations of transferring microbial strains are strictly aseptic in an aseptic environment.
(5) Preparation of shigella flexneri suspension
The activated shigella flexneri is serially diluted by 0.9% physiological saline with ten-fold gradient, and is coated in the LB solid medium in the step (3) and is cultured for 24 hours at the temperature of 37 ℃. Calculating the concentration of shigella flexneri thallus in the LB liquid medium in the step (3) by counting the colony number on the surface of the flat plate. Regulating the concentration of the shigella flexneri suspension to be 2 multiplied by 10 by using the liquid culture medium in the step (3) 6 CFU/mL。
b. Preservation of strains
The strain is preserved in China center for type culture collection (CCTCC, address: wuchang Lopa of Wuhan, china center for type culture collection, post code 430072). The strain collection number of the lactobacillus plantarum (Lactobacillus plantarum) Y12 is CCTCC NO: m2018556, date of preservation 2018, month 8 and 21, classified and named Lactobacillus plantarum (Lactobacillus plantarum) and strain Y12.
Lactobacillus plantarum (Lactobacillus plantarum) Y12 (hereinafter referred to as Y12) was inoculated into MRS culture medium and cultured at 37℃for 18-24 hours, followed by 3 serial passages. The activated Y12 is inoculated into fresh MRS liquid culture medium at 37 ℃ for 18-24h at 1-2% (V/V). Centrifuging (10000×g,10min, 4deg.C) the fermentation broth after culturing, removing thallus, and retaining supernatant; concentrating the supernatant to 1/5-1/4 of the original volume by using a rotary evaporator; concentrating supernatant, adding trichloroacetic acid (TCA) to a final concentration of 4-7%, shaking at room temperature for 30-60min, and precipitating protein; centrifuging (10000 Xg, 15min,4 ℃), removing protein, and retaining supernatant; adding 2-3 times volume of cold absolute ethyl alcohol into the supernatant, standing at 4 ℃ for 12-16h, and precipitating polysaccharide; centrifuging (10000 Xg, 15min,4 ℃), discarding supernatant to retain polysaccharide precipitate, and dissolving with appropriate amount of deionized water; dialyse at 4℃for 48h. Lyophilizing to obtain L-EPS sample. And the content of polysaccharide is 60% -70% by adopting a sulfuric acid phenol method.
EXAMPLE 3 Lactobacillus plantarum Y12L-EPS inhibits the formation of Shigella flexneri biofilm
The effect of Y12L-EPS on the formation of Shigella flexneri biofilm was determined by means of crystal violet staining. Y12L-EPS was dissolved in deionized water, sterilized by a 0.22 μm sterile filter, and formulated as a 0.2,0.5,1.0,2.0,4.0mg/mL gradient L-EPS solution. 1% (v/v) of activated shigella flexneri is inoculated in an LB liquid culture medium and evenly mixed; mu.L of bacterial liquid is taken and added into the wells of a 96-well plate, and 5 mu.L of L-EPS solutions with different concentrations are respectively added into the wells of different bacteria-containing liquids (the control well is added with 95 mu.L of bacterial liquid and 5 mu.L of LB liquid culture medium). The 96-well plate was incubated at 37℃for 24 hours. After the cultivation is finished, each well is washed 3 times by PBS, and then 200 mu L of 95% methanol is added to fix shigella flexneri cells adhered in the well for 15min; the plate is emptied and dried, and 200 mu L of 2% crystal violet is added for dyeing for 5min; excess dye was washed off with distilled water, and the dye stained in the cells was redissolved in 160. Mu.L of 33% glacial acetic acid for 30min, 125. Mu.L was added to a new 96-well plate and absorbance was measured at OD590 nm. Biofilm formation rate = OD1/OD2, wherein: OD1 is the absorbance value of the L-EPS treated group, and OD2 is the absorbance value of the control group.
When the method described in the specification is adopted for detection, FIG. 3 shows that the formation rate of the Shigella flexneri biological film measured in the example is obviously reduced under the influence of the extracellular polysaccharide (L-EPS) of lactobacillus plantarum Y12, and compared with a control group, the formation rate of the biological film can be reduced by 50 percent at most.
Example 4 Effect of Lactobacillus plantarum Y12L-EPS on Shigella flexneri growth
The growth curve of Shigella flexneri after addition of L-EPS was determined using a microbiological growth curve analyzer FP-1100-C (Thermo, finland). Briefly, 190. Mu.L of Shigella flexneri suspension (2X 10) was added to Honeycomb 2Sterilized culture plate (Oy Growth Curves Ab Ltd, finland) 6 CFU/mL) and 10. Mu.L of L-EPS solution, and 190. Mu.L of Shigella flexneri suspension (2X 10) was added to the control group 6 CFU/mL) and 10. Mu.mL fresh LB broth without L-EPS. Incubation was carried out at 37℃for 24h, once every 2h.
When the method described in the specification is adopted for detection, FIG. 4 shows that Shigella flexneri detected in the example can normally grow under the influence of Lactobacillus plantarum Y12 extracellular polysaccharide (L-EPS), and the four periods of the growth curve have no significant difference.
Example 5 scanning electron microscope observation
In a sterile plate
To this solution was added 9.5mL of Shigella flexneri suspension and 0.5mL of L-EPS solution. Cover slips (1X 1 cm) were placed in a plate and grown for 24h to allow Shigella flexneri to form a biofilm on the cover slips surface. After the incubation was completed, the slides were washed with PBS at pH 7.3 to remove all non-adherent shigella flexneri. The glass plates were fixed with 2.5% glutaraldehyde in PBS pH 7.3 for 2h and washed with 0.1M sodium acetate buffer (pH 7.3). Subsequently, the samples were washed in distilled water and dehydrated in ethanol (20%, 50%, 70%, 90% and 100%) of different concentrations for 5 minutes. Dehydrated with ethanol and then air-dried at room temperature. After metal spraying, the metal is observed and photographed by a scanning electron microscope under a magnification of 5000 times.
By adopting the method described in the specification to detect, FIG. 5 shows that the Lactobacillus plantarum Y12 extracellular polysaccharide (L-EPS) measured in the example can significantly inhibit the adhesion and aggregation of Shigella flexneri on the surface of a cover slip.
Example 6 fluorescent microscope observation
Taking activated Shigella flexneri suspension (2×10) 6 CFU/mL) 1.9mL, and 100. Mu.L of the L-EPS solution were inoculated into a cell culture plate (Costar 3506, corning, U.S. Pat. No.), and cultured at 37℃for 24 hours to form a biofilm on the surface of the culture plate. After the incubation was completed, each well was washed with PBS pH 7.3 to wash away non-adherent shigella flexneri. Each well was then stained with fluorescent whitening agent and an equal amount of 10% potassium hydroxide, excess dye was discarded after 1min, and observed and photographed using a fluorescent microscope (Olympus IX73, japan) at 200 and 400 x magnification.
As measured by the method described in the present specification, fig. 6 shows that lactobacillus plantarum Y12 extracellular polysaccharide (L-EPS) measured in this example can significantly inhibit adhesion and aggregation of shigella flexneri on the surface of polystyrene material cell culture plates.
EXAMPLE 7 Lactobacillus plantarum Y12L-EPS inhibits adhesion and invasion of Shigella flexneri to HT-29 cells
The human colon cancer cell line HT-29 is derived from Shanghai cell Bank of China academy of sciences (Shanghai, china). HT-29 cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated (56 ℃ C., 30 min) Fetal Bovine Serum (FBS), 1% penicillin and streptomycin, 37 ℃,90% humidity, 5% CO 2 Culturing in an incubator.
Cell adhesion experiments HT-29 cells were first treated at 5X 10 5 Cell/well density was seeded on 12-well cell culture plates (Costar 3513, corning, us). Culture plates were incubated at 37℃with 5% CO 2 And 95% air in an incubator until a confluent monolayer of HT-29 cells is obtained. The activated shigella flexneri is inoculated into LB liquid medium at 1 percent and cultured for 24 hours at 37 ℃. After the completion of the culture, the cell pellet was obtained by centrifugation at 10,000Xg at 4℃for 5min, and the cells were washed 3 times with sterile 0.1M PBS (pH 7.3) and resuspended in RPMI-1640 cell culture medium (containing no antibiotics and FBS) to a cell concentration of about 2.0X10 8 CFU/mL. L-EPS was dissolved in RPMI-1640 cell culture medium (without antibiotics and FBS), and culture solutions containing different concentrations of L-EPS (1.0, 2.0,4.0 mg/mL) were prepared and filtered with a 0.22 μm sterile filter membrane for use. The cultured HT-29 monolayer cells were washed twice with 0.1M PBS (pH 7.3). Each well was added 800. Mu.L of fresh RPMI-1640 medium, 100. Mu.L of bacterial suspension, and 100. Mu.L of L-EPS suspension. Culture plates were incubated at 37℃with 5% CO 2 And 95% air for 2 hours in an incubator. After incubation, the supernatant from each well was collected to determine cytokines. Each well was washed 3 times with 0.1M PBS (pH 7.3) to remove non-adherent cells. 1mL of 0.5% Triton X-100 ice bath was added to each well and the suspension was collected and counted on LB agar dilution plates. After 24h incubation at 37℃the colony count on LB agar is the adhesion count of Shigella flexneri on HT-29 cell monolayers. Adhesion number calculationThe formula is: adhesion number/cell number = colony number per well/HT-29 cell number.
The invasive experiment was based on the adhesion experiment and the incubation time was prolonged to 4h. After incubation, each well plate was washed three times with 0.1MPBS (pH 7.3) to remove non-adherent cells. 10mg/mL gentamicin was added to each well and treated at room temperature for 20min to kill HT-29 cell surface adhering bacteria. Cells were washed 3 times per well to remove excess antibiotic, and lysed by addition of 1mL of 0.5% Triton X-100 ice bath for 10min. The suspensions were collected per well and counted on LB agar dilution plates. After 24h incubation at 37℃the colony count on LB agar is the number of attacks of Shigella flexneri on HT-29 cell monolayers.
As measured by the method described in this specification, fig. 7 shows that lactobacillus plantarum Y12 exopolysaccharide measured in this example is capable of significantly inhibiting the adhesion and invasion of shigella flexneri to HT-29 monolayer cells.
Example 8 Lactobacillus plantarum Y12 EPS relieves the inflammatory response of HT-29 cells caused by Shigella flexneri
The suspension collected in the adhesion experiment of example 7 was centrifuged (4 ℃,8,000Xg, 5 min) to obtain a supernatant. Cytokine interleukins 8, 10 (IL-8, IL-10) and tumor necrosis factor alpha (TNF-alpha) were detected in the supernatant using an enzyme-linked immunosorbent assay kit (ELISA kit) (Nanjing, china).
As measured by the method described in the specification, FIG. 8 shows that the Lactobacillus plantarum Y12 exopolysaccharide measured in the example can significantly inhibit the secretion of HT-29 monolayer cell pro-inflammatory factors caused by Shigella flexneri and significantly promote the secretion of HT-29 cell anti-inflammatory factors.
Example 9 Effect of Lactobacillus plantarum Y12 EPS on the sensitivity of Shigella flexneri biofilm antibiotics
Determination of Minimum Inhibitory Concentration (MIC), minimum Bactericidal Concentration (MBC) and Minimum Biofilm Elimination Concentration (MBEC) of 8 antibiotics (levofloxacin, ciprofloxacin, amoxicillin, gentamicin, tobramycin, cefazolin, tetracycline, ampicillin) by microplate dilution method, determination of Minimum Inhibitory Concentration (MIC) and Minimum Biofilm Elimination Concentration (MBEC) of Shigella flexneri antibiotics, quality control of antibiotics by E.coli ATCC25922. The antibiotics were diluted from 1024. Mu.g/mL to 0.002. Mu.g/mL in a 2-fold gradient using MH medium. Inoculating activated Shigella flexneri to final concentration of about 2×10 6 CFU/mL. After 24h incubation at 37 ℃, OD600nm was detected using Multiskan GO 1510 to assess bacterial growth. The culture solution of the holes with obvious inhibition effect on shigella flexneri is dipped by an inoculating loop, lines are drawn on LB agar medium, colony growth is observed after incubation for 24 hours at 37 ℃, and MBC is measured.
MBEC assay Shigella flexneri (2X 10) 6 CFU/mL) was incubated in 96-well plates at 37 ℃ for 24h to form biofilms. After biofilm formation on the walls of the wells, each well was washed with PBS (pH 7.3) to remove non-adherent cells. The antibiotic-containing MH medium was then serially diluted 2-fold (when detecting the effect of EPS on MBEC, both antibiotic-containing MH medium and L-EPS were added simultaneously to the wells, incubated at 37℃for 24h, residual antibiotics were washed 2 times with PBS (pH 7.3), fresh LB medium was added to each well, sonicated in a water bath sonicator for 5min to detach adherent bacteria from the surface of the plates, which were incubated at 37℃for 24h, and the growth of Shigella flexneri in the surviving biofilms was observed.
By adopting the method described in the specification to detect, the drug resistance of the shigella flexneri biomembrane can be improved to 256 times compared with the drug resistance in a floating state, and the concentration of the required antibiotics such as levofloxacin, ciprofloxacin, cefazolin, tetracycline and the like can be obviously reduced when the Lactobacillus plantarum Y12 extracellular polysaccharide is treated together with the antibiotics.
TABLE 1 antibiotic sensitivity of flexneri
TABLE 2 IL-Effect of EPS (4 mg/mL) on S.flexneri biofilm resistance
EXAMPLE 10 monosaccharide composition analysis of Lactobacillus plantarum Y12 EPS
The monosaccharide composition of the EPS component was determined by high performance liquid chromatography. TFA was removed by rotary evaporation 3 times with methanol, using 2M trifluoroacetic acid (TFA) at 120℃for 4h. Redissolving the hydrolyzed EPS in ultrapure water. The sample solution was mixed with 0.6M NaOH and 0.5M 1-phenyl-3-methyl-5-pyrazolone (PMP) methanol solution (sample: naOH: pmp=1:1:2, v/v) to conduct the derivatization process. The derivatization method of monosaccharide standard (mannose, rhamnose, glucuronic acid, galacturonic acid, galactosamine, glucose, galactose, xylose, arabinose, fructose) is the same. Samples were analyzed on a Waters e2695 high performance liquid chromatography system. The analytical column was a Waters C18 column (250 mm. Times.4.6 mm,5 μm). The ultraviolet detection wavelength is 245nm. The sample loading was 20. Mu.L. The elution temperature was 30℃and the flow rate was 1.0mL/min. The mobile phase was 0.1M PBS (pH 7.0) -acetonitrile (83:17, v/v).
As measured by the method described in the present specification, FIG. 9 shows that the Lactobacillus plantarum Y12 exopolysaccharide measured in this example consists of mannose, glucuronic acid, galactosamine, glucose, galactose, xylose in a molar ratio of 32.26:0.99:1.79:5.63:0.05:4.07.
TABLE 3L EPS monosaccharide composition
The invention discloses an application of extracellular polysaccharide produced by lactobacillus plantarum Y12 in preparing a medicament for relieving dysentery caused by shigella flexneri, and belongs to the technical field of microbial application. The lactobacillus plantarum (Lactobacillus plantarum) Y12 is preserved in China center for type culture collection (China center for type culture collection) in the 8 th month of 2018 and has a strain preservation number of CCTCC NO: m2018556. The lactobacillus plantarum Y12 extracellular polysaccharide provided by the invention can inhibit the formation of a biological film of shigella flexneri; inhibiting adhesion and invasion of shigella flexneri to intestinal epithelial cells; relieving inflammatory response of intestinal epithelial cells caused by shigella flexneri; the dosage of the antibiotics for relieving dysentery caused by shigella flexneri is reduced.
SEQUENCE LISTING
<110> university of Dalian industry
Application of exopolysaccharide produced by lactobacillus plantarum Y12 in relieving dysentery caused by shigella flexneri
<130> 2021
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1481
<212> DNA
<213> Lactobacillus plantarum (Lactobacillus plantarum)
<400> 1
acgctggcgg cgtgcctaat acatgcaagt cgaacgaact ctggtattga ttggtgcttg 60
catcatgatt tacatttgag tgagtggcga actggtgagt aacacgtggg aaacctgccc 120
agaagcgggg gataacacct ggaaacagat gctaataccg cataacaact tggaccgcat 180
ggtccgagtt tgaaagatgg cttcggctat cacttttgga tggtcccgcg gcgtattagc 240
tagatggtgg ggtaacggct caccatggca atgatacgta gccgacctga gagggtaatc 300
ggccacattg ggactgagac acggcccaaa ctcctacggg aggcagcagt agggaatctt 360
ccacaatgga cgaaagtctg atggagcaac gccgcgtgag tgaagaaggg tttcggctcg 420
taaaactctg ttgttaaaga agaacatatc tgagagtaac tgttcaggta ttgacggtat 480
ttaaccagaa agccacggct aactacgtgc cagcagccgc ggtaatacgt aggtggcaag 540
cgttgtccgg atttattggg cgtaaagcga gcgcaggcgg ttttttaagt ctgatgtgaa 600
agccttcggc tcaaccgaag aagtgcatcg gaaactggga aacttgagtg cagaagagga 660
cagtggaact ccatgtgtag cggtgaaatg cgtagatata tggaagaaca ccagtggcga 720
aggcggctgt ctggtctgta actgacgctg aggctcgaaa gtatgggtag caaacaggat 780
tagataccct ggtagtccat accgtaaacg atgaatgcta agtgttggag ggtttccgcc 840
cttcagtgct gcagctaacg cattaagcat tccgcctggg gagtacggcc gcaaggctga 900
aactcaaagg aattgacggg ggcccgcaca agcggtggag catgtggttt aattcgaagc 960
tacgcgaaga accttaccag gtcttgacat actatgcaaa tctaagagat tagacgttcc 1020
cttcggggac atggatacag gtggtgcatg gttgtcgtca gctcgtgtcg tgagatgttg 1080
ggttaagtcc cgcaacgagc gcaaccctta ttatcagttg ccagcattaa gttgggcact 1140
ctggtgagac tgccggtgac aaaccggagg aaggtgggga tgacgtcaaa tcatcatgcc 1200
ccttatgacc tgggctacac acgtgctaca atggatggta caacgagttg cgaactcgcg 1260
agagtaagct aatctcttaa agccattctc agttcggatt gtaggctgca actcgcctac 1320
atgaagtcgg aatcgctagt aatcgcggat cagcatgccg cggtgaatac gttcccgggc 1380
cttgtacaca ccgcccgtca caccatgaga gtttgtaaca cccaaagtcg gtggggtaac 1440
cttttaggaa ccagccgcct aaggtgggac agatgattag g 1481
Claims (6)
1. Use of exopolysaccharide produced by lactobacillus plantarum (Lactobacillus plantarum) Y12 in combination with an antibiotic for the manufacture of a medicament for alleviating a diarrhea condition caused by shigella flexneri;
the preparation process of the extracellular polysaccharide produced by the lactobacillus plantarum (Lactobacillus plantarum) Y12 comprises the following steps: inoculating Lactobacillus plantarum (Lactobacillus plantarum) Y12 into fresh MRS liquid culture medium for culture, taking fermentation liquor for centrifugation, removing thalli and reserving supernatant; concentrating the supernatant by using a rotary evaporator; adding trichloroacetic acid into the concentrated solution, shaking uniformly at room temperature, and precipitating protein; centrifuging, removing protein, and keeping supernatant; adding cold absolute ethyl alcohol into the supernatant, standing and precipitating polysaccharide; centrifuging, discarding supernatant to retain polysaccharide precipitate, and dissolving with deionized water; dialyzing, and lyophilizing to obtain the final product;
the lactobacillus plantarum (Lactobacillus plantarum) Y12 is preserved in China center for type culture collection (China center for type culture collection) at 8-month and 21-day 2018, and the strain preservation number is CCTCC NO: m2018556.
2. Use according to claim 1, characterized in that lactobacillus plantarum (Lactobacillus plantarum) Y12 is inoculated in an amount of 1% -2% (V/V); concentrating the supernatant to 1/5-1/4 of the original volume; adding trichloroacetic acid into the concentrated solution to a final concentration of 4% -7%, and shaking uniformly at room temperature for 30-60min; the volume ratio of the cold absolute ethyl alcohol to the supernatant is 2-3, and the conditions of placing and precipitating the polysaccharide are 2-8 ℃ for 12-16 h; the condition of dialysis is 2-8deg.C, and dialysis is performed for 36-48h.
3. The use according to claim 1, wherein the antibiotic is selected from levofloxacin, ciprofloxacin, cefazolin or tetracycline.
4. The use according to claim 1, wherein the extracellular polysaccharide produced by lactobacillus plantarum (Lactobacillus plantarum) Y12 inhibits the formation of a biofilm by shigella flexneri at an action concentration of 0.01-0.200 μg/μl.
5. The use according to claim 1, wherein the extracellular polysaccharide produced by lactobacillus plantarum (Lactobacillus plantarum) Y12 inhibits the adhesion and invasion of shigella flexneri on human intestinal epithelial cells at an action concentration of 0.050-0.200 μg/μl.
6. The use according to claim 1, wherein the extracellular polysaccharide produced by lactobacillus plantarum (Lactobacillus plantarum) Y12 at an action concentration of 0.050 to 0.200 μg/μl, alleviates inflammatory response of human intestinal epithelial cells caused by shigella flexneri.
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| TWI401086B (en) * | 2010-07-20 | 2013-07-11 | Univ China Medical | Lactobacillus plantarum and uses thereof |
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| CN104818232B (en) * | 2015-05-08 | 2016-07-06 | 内蒙古农业大学 | One strain has Lactobacillus plantarum AB-3 and the application thereof of bacteriostatic activity |
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| CN107058422B (en) * | 2017-05-17 | 2020-03-17 | 华南农业大学 | Method for high yield of exopolysaccharide by lactobacillus plantarum |
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