CN113387861B - Method for preparing pyrrole-2-carboxylic acid by using pepper endogenous bacillus cereus and application - Google Patents
Method for preparing pyrrole-2-carboxylic acid by using pepper endogenous bacillus cereus and application Download PDFInfo
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Abstract
The method for preparing pyrrole-2-carboxylic acid by fermenting the pepper endophytic bacillus cereus has the advantages of simple steps, convenience in operation, high product purity, high product yield and easiness in large-scale production.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for preparing pyrrole-2-carboxylic acid by using pepper endophytic bacillus cereus and application thereof.
Background
The pepper is an important spice and medicinal economic crop, and the chemical components of the pepper mainly comprise volatile oil, alkaloid, flavone, phenolic substances, amide substances and the like, and the pepper has various biological activities of oxidation resistance, inflammation resistance, pain relief and the like. Endophytes are a class of microorganisms that colonize plant intercellular spaces or cells and establish a harmonious symbiotic relationship with the host plant. The endophyte forms a specific metabolism and defense system in the process of adapting to a special living environment, and can generate natural secondary metabolites with various structures and biological activities. The method for searching and developing a new natural active compound from the zanthoxylum bungeanum endophyte has important scientific significance and application value.
The traditional pyrrole-2-carboxylic acid is used for hydrolyzing raw materials containing protein by acid, alkali or enzyme, but the operation is inconvenient, the product purity is low and the product yield is low. Therefore, a method for preparing pyrrole-2-carboxylic acid by using the xanthoxylum bungeanum endophytic bacillus cereus and application thereof are needed.
Disclosure of Invention
The invention provides a method for preparing pyrrole-2-carboxylic acid by utilizing pepper endophytic bacillus cereus, which is simple and efficient to operate and has small compound loss, and an application thereof.
The invention comprises the following steps:
(1) Strain activation: carrying out constant-temperature culture and activation on the zanthoxylum bungeanum endophytic bacillus cereus strain at 37 ℃;
(2) Fermenting the strain: inoculating the pepper endophytic bacillus cereus strain to a liquid culture medium, and placing the liquid culture medium in a water bath for constant-temperature shaking culture for 36-72 hours to obtain a fermentation liquid;
(3) Processing the fermentation liquor by a high-speed centrifuge, collecting supernatant, processing the fermentation liquor by the high-speed centrifuge at the rotating speed of 8000-15000 r/min for 5-10 min, concentrating under reduced pressure, and freeze-drying to obtain a secondary metabolite crude sample;
(4) Separation and purification of secondary metabolites: and separating the secondary metabolite crude sample by using a chromatographic column, and then separating and purifying by using high performance liquid chromatography to obtain a secondary metabolite pyrrole-2-carboxylic acid.
Further, the time for constant-temperature culture activation is 24-72 h.
Further, in the strain fermentation step, the liquid medium carbon source and the liquid medium nitrogen source are glycerol and peptone respectively; the concentration of the glycerol is 0.3 to 1.0 percent; the concentration of the peptone is 0.5-2.0%; the initial pH of the medium is 6.5 to 7.2.
Further, the chromatographic column is a macroporous adsorption resin HP-20 chromatographic column, and the separation conditions of the macroporous adsorption resin HP-20 chromatographic column are as follows: the volume of the ultrapure water eluent is 800-1000 mL; the mass ratio of the secondary metabolite crude sample to the HP-20 filler is 1:20 to 1:25; the sample loading amount of the secondary metabolite crude sample is 15-20% of the column volume; the flow rate of ultrapure water isocratic elution is 2.0-3.0 mL/min.
Further, the operating conditions of the high performance liquid chromatography preparation are as follows: the flow rate is 0.5-1.2 mL/min; the column temperature is 25-30 ℃; detection wavelength, 254nm.
Further, the molecular structural formula of the secondary metabolite pyrrole-2-carboxylic acid is:
an application of pyrrole-2-carboxylic acid prepared from fructus Zanthoxyli endophytic Bacillus cereus in inhibiting food-borne pathogenic bacteria is provided.
The invention has the beneficial effects that:
the method for preparing pyrrole-2-carboxylic acid by using the pepper endophytic bacillus cereus has the advantages of simple steps, convenient operation, high product purity, high product yield and easy large-scale production, and in addition, the pyrrole-2-carboxylic acid prepared by the method has a good inhibition effect on various food-borne pathogenic bacteria and can be applied to the field of preparation of antibacterial agents.
Drawings
FIG. 1 is an electrospray ionization mass spectrum (ESI-MS) of pyrrole-2-carboxylic acid;
FIG. 2 shows a hydrogen spectrum of pyrrole-2-carboxylic acid ( 1 H-NMR)；
FIG. 3 shows pyrrole-2-of carboxylic acids carbon spectrum diagram ( 13 C-NMR)。
Detailed Description
The principles and features of this invention are described below in conjunction with examples which are set forth to illustrate, but are not to be construed to limit the scope of the invention.
The features and properties of the present invention are further described in detail below with reference to examples.
The preservation address of the biological strain material sample zanthoxylum bungeanum endophytic Bacillus cereus ZBE adopted in the technical scheme is in China center for type culture Collection, wuhan university, china, the preservation date is 2021 year, 5 month, 27 day, and the preservation number is M2021566.
Example 1
(1) And (2) taking a glycerol cryopreservation tube of the pepper endophytic bacillus cereus preserved in an ultralow-temperature refrigerator at the temperature of minus 80 ℃, placing the glycerol cryopreservation tube on an ultraclean workbench, taking a proper amount of bacterial suspension, diluting the bacterial suspension with a proper amount of basic broth culture medium, sucking 100 mu L of diluted bacterial suspension by using a micropipettor, coating the bacterial suspension on a basic agar culture medium plate, and placing the plate in an electrothermal constant-temperature incubator at the temperature of 37 ℃ for culture and activation for 24 hours for later use.
(2) In a clean bench, a burned and sterilized inoculating loop is used for selecting a bacillus cereus single colony from a strain activation plate, and the bacillus cereus single colony is inoculated to an optimized liquid culture medium, wherein a carbon source and a nitrogen source of the culture medium are 0.5% of glycerol and 1.5% of peptone respectively, and the initial pH of the culture medium is 6.8. And then placing the culture medium in a 37.2 ℃ water bath constant temperature oscillator, and carrying out oscillation culture at 120r/min for 48h to obtain fermentation liquor. Centrifuging the fermentation liquid at room temperature at 8000r/min for 5min with high speed centrifuge, collecting supernatant, concentrating under reduced pressure with rotary evaporator, freeze drying to obtain crude secondary metabolite, and storing at 4 deg.C.
(3) And (3) carrying out coarse sampling on the secondary metabolite according to the mass ratio of 1: and (3) loading 20 samples to a macroporous adsorption resin HP-20 chromatographic column, wherein the loading amount is 15% of the column volume. Isocratically eluting with 800mL of ultrapure water, wherein the flow rate of an eluent is 3.0mL/min; collecting eluent to obtain 4 components of F1, F2, F3 and F4, taking staphylococcus aureus ATCC 6538 as a tested bacterium, detecting the bacteriostatic activity of each component on the tested bacterium, determining that the component F2 has the bacteriostatic activity, passing the component F2 through a 0.22 mu m polyether sulfone filter membrane, and separating, purifying and preparing by using a high performance liquid chromatograph to obtain a chromatographic peak of 11.22min to obtain a secondary metabolite pyrrole-2-carboxylic acid, wherein the preparative high performance liquid chromatograph selects an ODS-3 chromatographic column (20 multiplied by 250 nm); the operating conditions of the preparative high performance liquid chromatography are as follows: flow rate, 1.0mL/min; column temperature, 25 ℃; detection wavelength, 254nm; ODS-3 chromatographic column (4.6 × 250 nm) is selected for analytical high performance liquid chromatography; the operating conditions of the analytical high performance liquid chromatography are as follows: flow rate, 1.0mL/min; column temperature, 25 ℃; the purity of pyrrole-2-carboxylic acid reaches more than 98% and the yield is 149.33mg/L when the detection wavelength is 254nm.
Example 2
(1) Placing a glycerol cryopreservation tube of the pepper endophytic bacillus cereus preserved in an ultra-low temperature refrigerator at minus 80 ℃ on an ultra-clean workbench, taking a proper amount of bacterial suspension, diluting the bacterial suspension with a proper amount of basic broth culture medium, sucking 100 mu L of the diluted bacterial suspension by using a micropipette, coating the bacterial suspension on a basic agar culture medium plate, and placing the plate in an electric heating constant temperature incubator at 37 ℃ for culturing and activating for 24 hours for later use.
(2) Selecting a bacillus cereus single colony from a strain activation plate by using an inoculating loop after burning sterilization in a super clean workbench, inoculating the bacillus cereus single colony to an optimized liquid culture medium, wherein a carbon source and a nitrogen source of the culture medium are respectively 0.8% of glycerol and 1.0% of peptone, the initial pH of the culture medium is 7.0, then placing the culture medium in a 37.2 ℃ water bath constant temperature oscillator, carrying out shaking culture at 110r/min for 45h to obtain a fermentation liquid, centrifuging the fermentation liquid by using a high-speed centrifuge at room temperature by 10000 r/min for 8min, collecting a supernatant, carrying out reduced pressure concentration by using a rotary evaporator, carrying out freeze drying to obtain a secondary metabolite crude sample, and storing at 4 ℃ for later use.
(3) And (3) carrying out mass ratio of the secondary metabolite crude sample to the primary metabolite crude sample of 1: loading 25 sample to macroporous adsorption resin HP-20 chromatographic column, wherein the loading amount is 20% of the column volume, and isocratic eluting with 1000mL ultrapure water, and the eluent flow rate is 2.0mL/min; collecting eluent to obtain 4 components of F1, F2, F3 and F4, taking staphylococcus aureus ATCC 6538 as a tested bacterium, detecting the bacteriostatic activity of each component on the tested bacterium, determining that the component F2 has the bacteriostatic activity, passing the component F2 through a 0.22 mu m polyether sulfone filter membrane, and separating, purifying and preparing by using a high performance liquid chromatograph to obtain a chromatographic peak of 11.23min to obtain a secondary metabolite pyrrole-2-carboxylic acid, wherein the preparative high performance liquid chromatograph selects an ODS-3 chromatographic column (20 multiplied by 250 nm); the operating conditions of the preparative high performance liquid chromatography are as follows: flow rate, 1.2mL/min; column temperature, 25 ℃; detection wavelength, 254nm; ODS-3 chromatographic column (4.6 × 250 nm) is selected for analytical high performance liquid chromatography; the operating conditions of the analytical high performance liquid chromatography are as follows: flow rate, 1.0mL/min; column temperature, 25 ℃; the purity of the pyrrole-2-carboxylic acid reaches more than 98 percent and the yield is 149.63mg/L under the detection wavelength of 254nm.
Example 3
(1) Placing a glycerol cryopreservation tube of the pepper endophytic bacillus cereus preserved in an ultra-low temperature refrigerator at minus 80 ℃ on an ultra-clean workbench, taking a proper amount of bacterial suspension, diluting the bacterial suspension with a proper amount of basic broth culture medium, sucking 100 mu L of the diluted bacterial suspension by using a micropipette, coating the bacterial suspension on a basic agar culture medium plate, and placing the plate in an electric heating constant temperature incubator at 37 ℃ for culturing and activating for 24 hours for later use.
(2) Selecting a bacillus cereus single colony from a strain activation plate by using an inoculating loop after burning sterilization in a super clean workbench, inoculating the bacillus cereus single colony to an optimized liquid culture medium, wherein a carbon source and a nitrogen source of the culture medium are respectively 0.7% of glycerol and 1.0% of peptone, the initial pH of the culture medium is 7.0, then placing the culture medium in a 37.2 ℃ water bath constant temperature oscillator, carrying out oscillation culture at 100r/min for 45h to obtain a fermentation liquid, carrying out centrifugation for 10min by using a high-speed centrifuge under the normal temperature condition of 12000 r/min, collecting a supernatant, carrying out reduced pressure concentration by using a rotary evaporator, carrying out freeze drying to obtain a secondary metabolite crude sample, and storing at 4 ℃ for later use.
(3) And (3) carrying out mass ratio of the secondary metabolite crude sample to the primary metabolite crude sample of 1: loading 25 sample to macroporous adsorption resin HP-20 chromatographic column, wherein the loading amount is 20% of the column volume, and isocratic eluting with 1000mL ultrapure water, and the eluent flow rate is 2.5mL/min; collecting eluent to obtain 4 components of F1, F2, F3 and F4, taking staphylococcus aureus ATCC 6538 as a tested bacterium, detecting the bacteriostatic activity of each component on the tested bacterium, determining that the component F2 has the bacteriostatic activity, passing the component F2 through a 0.22 mu m polyether sulfone filter membrane, and separating, purifying and preparing by using a high performance liquid chromatograph to obtain a chromatographic peak of 11.20min to obtain a secondary metabolite pyrrole-2-carboxylic acid, wherein the preparative high performance liquid chromatograph selects an ODS-3 chromatographic column (20 multiplied by 250 nm); the operating conditions of the preparative high performance liquid chromatography are as follows: flow rate, 1.5mL/min; column temperature, 25 ℃; detection wavelength, 254nm; ODS-3 chromatographic column (4.6 × 250 nm) is selected for analytical high performance liquid chromatography; the operating conditions of the analytical high performance liquid chromatography are as follows: flow rate, 1.0mL/min; column temperature, 25 ℃; the purity of pyrrole-2-carboxylic acid reaches more than 98% and the yield is 149.72mg/L when the detection wavelength is 254nm.
Application example 1
The method for measuring the minimum inhibitory concentration of the pyrrole-2-carboxylic acid on the food-borne pathogenic bacteria prepared in the embodiment comprises the following steps:
(1) Inoculating the tested strain to nutrient broth liquid culture medium, culturing at 37 deg.C and 120r/min for 10h, centrifuging to collect strain in logarithmic growth phase, correcting bacterial suspension to 0.5 McLeod turbidity with normal saline, and diluting with nutrient broth 100 times to obtain bacterial concentration of 1 × 10 6 CFU/mL of bacterial liquid to be tested, gram-positive bacteria to be tested: listeria monocytogenes (ATCC 19111), staphylococcus aureus (ATCC 29247), bacillus subtilis (BNCC 109047), bacillus cereus (CICC 23828); gram-negative bacteria tested: pseudomonas aeruginosa (ATCC 27853), escherichia coli (ATCC 25922), salmonella (ATCC 14028), shigella (CMCC 51572).
(2) Dissolving pyrrole-2-carboxylic acid with a proper amount of DMSO (dimethylsulfoxide), diluting the pyrrole-2-carboxylic acid to 80mg/mL with sterile water, sterilizing with a 0.22 mu m polyethersulfone filter membrane to obtain a drug stock solution, diluting the drug stock solution to 48mg/mL with nutrient broth, sequentially diluting in a double manner until the concentration of the drug stock solution is 24mg/mL, 12mg/mL, 6mg/mL, 3mg/mL, 1.5mg/mL, 0.75mg/mL and 0.375mg/mL, taking 100 mu L of pyrrole-2-carboxylic acid with different concentrations by using a micropipette, sequentially adding the pyrrole-2-carboxylic acid into a sterile 96-well plate, wherein each concentration is 3 in parallel, and simultaneously using the nutrient broth as a growth control well.
(3) Taking 100 mu L of the bacterial liquid to be detected in the step (1) by using a micropipettor, adding the bacterial liquid to be detected into the holes of the pyrrole-2-carboxylic acid with different concentrations in the step (2) to obtain the final inoculated bacterial liquid with the concentration of 5 multiplied by 10 5 CFU/mL, the tested concentration of the drug is 12-0.1875 mg/mL, after a 96-well plate is covered and placed in a constant-temperature incubator at 37 ℃ for culturing for 24h, the result is observed, and the Minimum Inhibitory Concentration (MIC) is the Minimum drug concentration for inhibiting the growth of bacteria.
(4) The test uses benzoic acid as positive control, and the MIC value of the benzoic acid to the tested strain is determined by the same method as the steps for determining the MIC value of pyrrole-2-carboxylic acid to gram-positive bacteria and gram-negative bacteria.
(5) The MIC result of pyrrole-2-carboxylic acid on each tested strain is shown in Table 1, and the experimental result shows that the pyrrole-2-carboxylic acid prepared by the bacillus cereus endophytic in the pepper has better performance in inhibiting food-borne pathogenic bacteria, the prepared pyrrole-2-carboxylic acid has good inhibition effect on gram-positive bacteria and gram-negative bacteria, and the inhibition effect on the gram-positive bacteria is better than that of the gram-negative bacteria.
TABLE 1
In summary, the embodiments of the present invention provide a method for preparing pyrrole-2-carboxylic acid from secondary metabolites of b.cereus endogenous to zanthoxylum bungeanum. The method for preparing pyrrole-2-carboxylic acid provided by the invention has the advantages of simple steps, convenience in operation, high product purity, high product yield, easiness in large-scale production and the like. The secondary metabolite obtained by separation and purification was confirmed to be pyrrole-2-carboxylic acid by electrospray ionization mass spectrometry (ESI-MS). In addition, the pyrrole-2-carboxylic acid prepared by the embodiment of the invention has good inhibition effect on gram-positive bacteria and gram-negative bacteria, can be applied to the field of antibacterial agent preparation, and has higher economic value and wide development and application prospects.
The method comprises the steps of carrying out preliminary separation on a liquid fermentation product of xanthoxylum endophytic Cereus ZBE (preserved in China center for type culture collection with the preservation number of CCTCC M2021566) through a Sephadex LH-20 chromatographic column, carrying out High Performance Liquid Chromatography (HPLC) analysis, further separating and purifying the fermentation product, carrying out chromatographic column separation on a secondary metabolite crude sample, carrying out High Performance Liquid Chromatography (HPLC) separation and purification to obtain a secondary metabolite pyrrole-2-carboxylic acid, and screening the obtained product by taking the inhibitory activity of staphylococcus aureus as an indication, wherein the product has an inhibitory effect on both gram-positive bacteria and gram-negative bacteria.
The molecular structural formula of pyrrole-2-carboxylic acid obtained by the preparation of the example.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (4)
1. A method for preparing pyrrole-2-carboxylic acid by using pepper endophytic bacillus cereus is characterized by comprising the following steps:
(1) Strain activation: carrying out constant-temperature culture and activation on the zanthoxylum bungeanum endophytic bacillus cereus strain at 37 ℃;
(2) Fermenting the strain: inoculating the pepper endophytic bacillus cereus strain to a liquid culture medium, and placing the strain in a water bath for constant-temperature shaking culture for 36-72 hours to obtain a fermentation liquid;
(3) Processing the fermentation liquor by a high-speed centrifuge, collecting supernatant, processing the fermentation liquor by the high-speed centrifuge at the rotating speed of 8000-15000 r/min for 5-10 min, concentrating under reduced pressure, and freeze-drying to obtain a secondary metabolite crude sample;
(4) Separation and purification of secondary metabolites: separating the secondary metabolite crude sample by a chromatographic column, and then separating and purifying by high performance liquid chromatography to obtain a secondary metabolite pyrrole-2-carboxylic acid;
the chromatographic column is a macroporous adsorption resin HP-20 chromatographic column, and the separation conditions of the macroporous adsorption resin HP-20 chromatographic column are as follows: the volume of the ultrapure water eluent is 800-1000 mL; the mass ratio of the secondary metabolite crude sample to the HP-20 filler is 1:20 to 1:25; the sample loading amount of the secondary metabolite crude sample is 15-20% of the column volume; the flow rate of the ultrapure water isocratic elution is 2.0-3.0 mL/min;
the preparation operating conditions of the high performance liquid chromatography are as follows: the flow rate is 0.5-1.2 mL/min; the column temperature is 25-30 ℃; detection wavelength, 254nm.
2. The method for preparing pyrrole-2-carboxylic acid by using the pepper endophytic bacillus cereus as claimed in claim 1, wherein the time of the constant-temperature culture activation is 24-72 h.
3. The method for preparing pyrrole-2-carboxylic acid by using Bacillus cereus endophytic in Zanthoxylum bungeanum according to claim 1, wherein in the strain fermentation step, the liquid medium carbon source and nitrogen source are glycerol and peptone, respectively;
the concentration of the glycerol is 0.3 to 1.0 percent; the concentration of the peptone is 0.5 to 2.0 percent; the initial pH of the medium is 6.5 to 7.2.
4. The method for preparing pyrrole-2-carboxylic acid by using the pepper endophytic bacillus cereus as claimed in claim 1, wherein the operating conditions of the high performance liquid chromatography are as follows: the flow rate is 0.5-1.2 mL/min; the column temperature is 25-30 ℃; detection wavelength, 254nm.
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