A method for separating and purifying high-purity gramicidin A from polypeptide fermentation broth

Abstract

The invention relates to the technical field of pharmaceutical chemical industry, and discloses a method for separating and purifying gramicidin A from polypeptide fermentation broth. The invention enriches and purifies the fermentation broth of gramicidin A, and adopts a process of macroporous adsorption resin enrichment, chromatography polymer filler purification and decolorization resin decolorization, and finally obtains high-purity gramicidin A, with a comprehensive yield of more than 91%, and a purity of gramicidin A of more than 99%, and large-scale production can be achieved. The invention uses a combination of polymer resins, and the chromatographic resin has high resolution and good impurity removal effect, so that the operation difficulty of the process is greatly reduced, and the recycling and repeated use of the polymer resin reduces the production cost.

Description

Technical Field

[0001] The invention relates to the technical field of pharmaceutical chemical industry, and in particular to a method for separating and purifying gramicidin A from polypeptide fermentation broth. Background Art

[0002] Gramicidin A is a peptide antibiotic isolated from soil actinomycetes. It is a linear peptide composed of 15 amino acids, 8 of which are L-amino acids and 7 are D-amino acids. It has a hydrophobic side chain. Two molecules together form a transmembrane channel, so it is a channel-forming ion carrier that can selectively pass monovalent cations through the membrane along the electrochemical gradient. It has broad-spectrum antibacterial activity against Gram-positive bacteria and certain pathogenic fungi. It easily turns yellow in the air and is easy to absorb moisture. It is easily soluble in pyridine and dimethyl sulfoxide, soluble in ethanol and acetone, and difficult to dissolve in water and petroleum ether.

[0003] At present, there are many defects in its extraction and purification process and technical means, for example, low yield, small output and low product purity, which are difficult to reach the level of industrial production. In the prior art, gramicidin A is suitable for small-scale preparation through cyclic peptide insurance resin synthesis method and linear peptide solid phase synthesis-liquid phase cyclization method. Although parallel synthesis or repeated synthesis can accumulate output, it causes a large amount of solvent waste on the one hand and increases preparation cost. On the other hand, the post-processing workload of the synthesis process is increased, and the purification method includes enzyme catalysis, silica gel and liquid-liquid extraction, etc., which are inefficient. However, the silica gel purification process often has a low processing capacity and is not suitable for industrial production. The extraction process has a large loss, resulting in low yield. Therefore, the purification technology of gramicidin A that can be industrialized has not been proposed in the existing disclosed technology.

[0004] Therefore, how to develop a method for extracting and purifying gramicidin A with good separation effect, large loading capacity, cost saving and suitable for industrial promotion is a technical problem that needs to be solved urgently in this field. Summary of the invention

[0005] In order to solve the problem of lack of industrializable purification technology of gramicidin A in the prior art, the present invention proposes a method for separating and purifying gramicidin A from polypeptide fermentation broth.

[0006] A method for separating and purifying gramicidin A from polypeptide fermentation broth comprises the following steps:

[0007] Step 1) loading the polypeptide fermentation liquid onto a macroporous resin column; then using a first mobile phase to wash the macroporous resin column; then using a second mobile phase to elute the macroporous resin column, and collecting the eluate to obtain a separation solution;

[0008] Step 2) loading the separated liquid onto a reverse phase column of a polymer chromatography packing material; then eluting the reverse phase column of the polymer chromatography packing material with a third mobile phase, and collecting the eluate to obtain a gramicidin A solution;

[0009] Step 3) loading the gramicidin A solution onto a decolorizing resin column, and collecting the effluent to obtain a purified gramicidin A solution;

[0010] The first mobile phase, the second mobile phase and the third mobile phase are all aqueous solutions containing alcohol and organic acid; the volume fraction of alcohol in the first mobile phase is 30% to 50%, and the volume fraction of alcohol in the second mobile phase is 50% to 80%; the volume fraction of alcohol in the third mobile phase is 10% to 30%; the organic acid concentrations in the first mobile phase, the second mobile phase and the third mobile phase are all 50mmol / L to 100mmol / L.

[0011] Optionally, the alcohol in the alcohol-containing solvent is a monohydric alcohol having a carbon number not greater than 4 and being miscible with water in any proportion.

[0012] Optionally, the alcohol comprises at least one of methanol, ethanol, n-propanol, isopropanol and butanol.

[0013] Optionally, the organic acid is at least one of trifluoroacetic acid, malonic acid, glycolic acid, succinic acid, glycine, and oxalic acid.

[0014] Optionally, the filler of the macroporous resin column is a polyacrylate-styrene-divinylbenzene cross-linked polymer, the mass of the polyacrylate in the polyacrylate-styrene-divinylbenzene cross-linked polymer accounts for 5-25% of the mass of the cross-linked polymer, and the degree of cross-linking is 10%-70%;

[0015] Optionally, the filler of the macroporous resin column is polyacrylate-styrene-divinylbenzene cross-linked polymer microspheres, the particle size of the polyacrylate-styrene-divinylbenzene cross-linked polymer microspheres is 0.75 mm to 1.25 mm, and the pore size is Specific surface area not less than 120m 2 / g;

[0016] Optionally, the macroporous resin column is prepared by wet filling.

[0017] Optionally, the macroporous resin column is prepared by the following method: the polyacrylate-styrene-divinylbenzene cross-linked polymer microspheres are mixed with 1 to 3 times the column volume of an alcohol aqueous solution and then packed into the column, with a filling height of not less than 60 cm; and the volume fraction of the alcohol-water solution is 30% to 50%.

[0018] Optionally, after the macroporous resin column is loaded and before the impurities are washed, 2 to 6 column volumes of a mixed aqueous solution of alcohol with a volume concentration of 5% to 30% and an organic acid reagent with a molar concentration of 10 mmol / L to 50 mmol / L is used to balance the macroporous resin column, and the linear flow rate of the effluent during the balancing process is not higher than 6 cm / min.

[0019] Optionally, the amount of the first mobile phase is 1 to 4 times the column volume; the linear velocity of the effluent during the washing process is not higher than 6 cm / min;

[0020] The amount of the second mobile phase B is 4 to 25 times the column volume; the linear velocity of the eluent flowing out during the elution process is not higher than 6 cm / min;

[0021] During the loading process of the macroporous resin column, the loading is stopped when the concentration of gramicidin A at the outlet of the macroporous resin column is detected to be less than 0.05 mg / mL, and the linear velocity of the outflow liquid during the loading process is not higher than 6 cm / min;

[0022] Optionally, in step 1), after collecting the eluate to obtain the separation liquid, the macroporous resin column is regenerated: 2 to 6 column volumes of an alcohol-water solution with a volume concentration of 80% to 90% are used to elute the macroporous resin column, and the linear velocity of the effluent during the regeneration process is not higher than 6 cm / min.

[0023] Optionally, the polymer chromatography filler in the reverse phase column of the polymer chromatography filler is at least one of C18 bonded silica gel, C12 bonded silica gel, C8 bonded silica gel, phenyl bonded silica gel, butyl bonded silica gel, a polymer reverse phase filler containing an ester group, butyl group, phenyl group, octyl group, dodecyl group, hexadecyl group, vinyl group, allyl benzene, vinyl benzene or divinyl benzene;

[0024] Optionally, the polymer chromatography filler reverse phase column is prepared by wet packing;

[0025] Optionally, the reverse phase column of the polymer chromatography filler is prepared by the following method: the reverse phase filler is mixed with 1 to 3 times the column volume and the volume concentration of 0% to 100% alcohol-water solution and then packed into the column, the packing height is not less than 60 cm, and the packing pressure is not higher than 10 MPa;

[0026] Optionally, after the polymer chromatography packing reverse phase column is loaded and before elution, 2 to 6 column volumes of a mixed aqueous solution of an alcohol with a volume concentration of 0% to 5% and an organic acid reagent with a molar concentration of 10 mmol / L to 50 mmol / L are used to balance the polymer chromatography packing reverse phase column, and the linear flow rate of the effluent during the balancing process is not higher than 6 cm / min;

[0027] Optionally, in the step 1), the separation liquid is evaporated and concentrated to remove alcohol in the separation liquid; in the step 2), the concentrated separation liquid is diluted to 35 g / L to 75 g / L and then loaded onto the reverse phase column;

[0028] Optionally, in the step 2), the concentrated separation liquid is diluted to 35 g / L to 75 g / L with an aqueous alcohol solution having a volume fraction of 0% to 5%, and then loaded onto the reverse phase column.

[0029] Optionally, in the process of loading the separated liquid onto the reverse phase column of the polymer chromatography filler in the step 2), the sample is loaded at a loading capacity of 45-90 mg / ml, and the linear velocity of the effluent during the loading process is not higher than 6 cm / min;

[0030] Optionally, step S2 further includes regenerating the reverse phase column after the elution process, which includes: eluting the reverse phase column with 2 to 8 column volumes of an alcohol-water solution with a volume concentration of 80% to 90%, wherein the linear velocity of the effluent during the regeneration process is not higher than 6 cm / min;

[0031] Optionally, in the step 2), before eluting the polymer chromatography filler reverse column, a fourth mobile phase is used, the washing volume is 4-15 times the column volume, and the linear velocity of the washing process is not higher than 6 cm / min; the fourth mobile phase is an aqueous solution containing alcohol and organic acid; the volume fraction of alcohol in the fourth mobile phase is 5% to 10%; the concentration of organic acid in the fourth mobile phase is 50mmol / L to 100mmol / L.

[0032] Optionally, the filler in the decolorizing resin column in step 3) is a non-ionic decolorizing resin with a styrene skeleton and a high molecular cross-linked stable structure.

[0033] Compared with the prior art, the technical solution of the present invention has the following advantages:

[0034] The present invention provides a method for extracting and purifying gramicidin A. The present invention adopts macroporous resin adsorption and desorption, reverse chromatography and decolorization resin decolorization, and finally obtains a comprehensive yield of gramicidin A of more than 96%, and the purity of gramicidin A is more than 99%. The present invention shields the electronegativity of polypeptide amino acids by adding an organic acid reagent, and makes the peptide chain structure have only hydrophobic forces, so that the adsorption amount of gramicidin A on the polymer filler is larger, the load is increased, the retention time is extended, the separation effect is improved, it is more conducive to industrialization, and the equipment investment cost is saved. The chromatographic separation load is large, the overall process yield is high, and the process can flexibly adjust the output according to production needs, and the scale is high. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the specific implementation of the present invention or the technical solution in the prior art, the drawings required for use in the specific implementation or the description of the prior art will be briefly introduced below.

[0036] Figure 1 This is the liquid phase detection spectrum of the crude polypeptide fermentation broth of Example 1 of the present invention.

[0037] Figure 2 This is the liquid phase detection spectrum of the macroporous resin adsorption and desorption step in Example 1 of the present invention.

[0038] Figure 3 It is the liquid phase detection spectrum of the reverse phase chromatography separation step in Example 1 of the present invention.

[0039] Figure 4 It is the liquid phase detection spectrum of the macroporous resin adsorption and desorption step in Example 2 of the present invention.

[0040] Figure 5 It is the liquid phase detection spectrum of the reverse phase chromatography separation step in Example 2 of the present invention.

[0041] Figure 6 It is the liquid phase detection spectrum of the macroporous resin adsorption and desorption step in Example 3 of the present invention.

[0042] Figure 7 It is the liquid phase detection spectrum of the reverse phase chromatography separation step in Example 3 of the present invention.

[0043] Figure 8 It is the liquid phase detection spectrum of the macroporous resin adsorption and desorption step in Example 4 of the present invention.

[0044] Fig. 9 It is the liquid phase detection spectrum of the reverse phase chromatography separation step in Example 4 of the present invention. DETAILED DESCRIPTION

[0045] Now, various exemplary embodiments of the present invention are described in detail, and this detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present invention. It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention.

[0046] In addition, for the numerical range in the present invention, it is understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0047] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0048] The raw materials, reagents and other materials used in the following examples are all commercially available products. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art in the art described in the present invention. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention.

[0049] The present invention provides a method for separating and purifying high-purity gramicidin A from a polypeptide fermentation broth, comprising the following steps:

[0050] S1, macroporous resin column separation

[0051] 1) 1 volume of macroporous resin plus 1-3 volumes of 30-60% (V / V) alcohol aqueous solution are wet packed into the column, and the filling height is not less than 60cm;

[0052] 2) using a mixed aqueous solution of 30% to 50% alcohol and an organic acid reagent with a molar concentration of 50 mmol / L to 100 mmol / L to balance the macroporous resin column for 2 to 6 times the column volume, and the linear flow rate of the equilibrium process is not higher than 6 cm / min;

[0053] 3) Loading the polypeptide fermentation broth onto a macroporous resin column, loading the sample in the forward direction, with a macroporous resin column loading capacity of 80-120 g / L (pure gramicidin A / macroporous resin volume), with a linear speed of no more than 6 cm / min during the loading process, and the concentration of gramicidin A detected in the effluent was less than 0.05 mg / ml;

[0054] 4) washing with a mixed aqueous solution of 30% to 50% alcohol and an organic acid reagent with a molar concentration of 50 mmol / L to 100 mmol / L, the washing volume is 1 to 5 times the column volume, and the linear speed of the washing process is not higher than 6 cm / min;

[0055] 5) Using a mixed aqueous solution of 50% to 80% alcohol and an organic acid reagent with a molar concentration of 50 mmol / L to 100 mmol / L as the mobile phase for desorption, the desorption volume is 3 to 20 times the column volume, and the linear velocity of the desorption process is not higher than 6 cm / min. The desorbed liquid is collected and concentrated to remove the alcohol therein, and then proceed to the next step;

[0056] 6) regenerating the macroporous resin using 80-90% alcohol aqueous solution, with a regeneration volume of 2-6 times the column volume and a linear speed of no more than 6 cm / min during the regeneration process;

[0057] 7) The next cycle can be entered, the macroporous resin balancing step;

[0058] S2, reverse phase column chromatography separation

[0059] 1) Use reverse phase filler as the stationary phase of the chromatographic column. Add 1-3 volumes of 0-100% alcohol aqueous solution to 1 volume of reverse phase filler, and wet pack the column. The column height should not be less than 60 cm, and the packing pressure should not be higher than 10 MPa.

[0060] 2) using a mixed aqueous solution of 5% to 10% alcohol and an organic acid reagent with a molar concentration of 50 mmol / L to 100 mmol / L to balance the chromatographic column for 2 to 6 times the column volume, and the linear flow rate of the equilibrium process is not higher than 6 cm / min;

[0061] 3) the concentrate in the above step is dissolved in a mixed aqueous solution of 0-5% alcohol and 50 mmol / L to 100 mmol / L organic acid reagent at a molar concentration of 35-75 g / L (gramicidin A mass / dissolved solution volume), and loaded onto a chromatographic column, in a forward loading state, with a loading capacity of 45-90 g / L (gramicidin A mass / reverse phase chromatographic column volume), and the linear speed of the loading process is not higher than 6 cm / min;

[0062] 4) using a mixed aqueous solution of 5% to 10% alcohol and an organic acid reagent with a molar concentration of 50 mmol / L to 100 mmol / L to wash the impurities, the washing volume is 4 to 15 times the column volume, and the linear speed of the washing process is not higher than 6 cm / min;

[0063] 5) using a mixed aqueous solution of 10% to 30% alcohol and an organic acid reagent with a molar concentration of 50 mmol / L to 100 mmol / L to desorb, the desorption volume is 4 to 15 times the column volume, the linear speed of the desorption process is not higher than 6 cm / min, collecting the gramicidin A desorption solution and temporarily storing it;

[0064] 6) regenerating the chromatographic column using an 80-90% alcohol-water solution, with a regeneration volume of 2-6 times the column volume and a linear velocity of no more than 6 cm / min during the regeneration process;

[0065] 7) The next cycle can be entered, the column equilibration step;

[0066] S3, decolorization resin decolorization

[0067] 1) 1 volume of decolorizing resin plus 1-3 volumes of 10-40% (V / V) alcohol aqueous solution are wet packed into the column, and the filling height is not less than 60 cm;

[0068] 2) using a mixed aqueous solution of 10% to 30% alcohol and an organic acid reagent with a molar concentration of 50 mmol / L to 100 mmol / L to balance the decolorizing resin column for 2 to 4 times the column volume, and the linear flow rate of the equilibrium process is not higher than 6 cm / min;

[0069] 3) The desorbed liquid from the chromatography in S2 is loaded onto the decolorizing resin column in a forward direction, and the linear speed of the loading process is not higher than 6 cm / min. The concentration of gramicidin A is detected in the effluent, and the decolorized gramicidin A solution is collected and concentrated to dryness to obtain a high-purity gramicidin A product.

[0070] 4) regenerating the macroporous resin using 80-90% alcohol aqueous solution, with a regeneration volume of 2-6 times the column volume and a linear speed of no more than 6 cm / min during the regeneration process;

[0071] 5) The next cycle can be entered, the macroporous resin balancing step;

[0072] The organic acid reagent is at least one of trifluoroacetic acid, malonic acid and glycolic acid.

[0073] In an optional embodiment, the filler of the macroporous resin column is resin microspheres.

[0074] In an optional embodiment, the particle size of the resin microspheres is 0.1 mm to 1.5 mm.

[0075] In an optional embodiment, the pore size of the resin microspheres is

[0076] In an optional embodiment, the specific surface area of ​​the resin microspheres is not less than 100 m 2 / g.

[0077] In an optional embodiment, the chemical structure of the resin microspheres is a cross-linked polymer of polyacrylate-styrene-divinylbenzene, and the mass of the polyacrylate accounts for 0 to 20% of the mass of the cross-linked polymer, and the degree of cross-linking is 15% to 80%.

[0078] In an optional embodiment, the alcohol in the alcohol-containing solvent is a monohydric alcohol having no more than 4 carbon atoms that is miscible with water in any proportion, preferably at least one of methanol, ethanol, n-propanol, isopropanol, and butanol.

[0079] In an optional embodiment, the reverse phase filler is at least one of C18 bonded silica gel, C12 bonded silica gel, C8 bonded silica gel, phenyl bonded silica gel, butyl bonded silica gel, and a polymer reverse phase filler containing an ester group, butyl, phenyl, octyl, dodecyl, hexadecyl, vinyl, allyl, vinylbenzene or divinylbenzene.

[0080] The present invention is further described in detail below in conjunction with specific embodiments.

[0081] Example 1

[0082] This embodiment provides a method for separating and purifying high-purity gramicidin A from a polypeptide fermentation broth, comprising the following steps: S1, separating the gramicidin A with a macroporous resin column;

[0083] 1) 10 ml of macroporous resin LX-T8 plus 10 ml of 30% (V / V) ethanol aqueous solution were wet packed into the column, with a filling height of 60 cm and a volume of 10 ml;

[0084] 2) using a mixed aqueous solution of 30% ethanol and 50 mmol / L trifluoroacetic acid reagent to balance the macroporous resin column, balancing 20 ml, and a linear flow rate of 6 cm / min during the balancing process;

[0085] 3) Load the polypeptide fermentation broth onto a macroporous resin column, with the loading capacity of the macroporous resin column at 80 g / L (pure gramicidin A / macroporous resin volume), load 800 mg of gramicidin A fermentation broth, and detect the concentration of gramicidin A in the effluent at 0.01 mg / ml;

[0086] 4) Wash with a mixed aqueous solution of 30% ethanol and 50 mmol / L trifluoroacetic acid, with a washing volume of 10 ml and a flow rate of 1 ml / min;

[0087] 5) Using a mixed aqueous solution of 50% ethanol and 50 mmol / L trifluoroacetic acid reagent as the mobile phase for desorption, the desorption volume is 30 ml, the flow rate of the desorption process is 10 ml / min, collecting the desorbed liquid, and concentrating it to remove the alcohol therein to obtain 770 mg of gramicidin A sample to proceed to the next step;

[0088] 6) Regenerate the macroporous resin using 80% ethanol aqueous solution, with a regeneration volume of 20 ml and a regeneration flow rate of 1 ml / min;

[0089] 7) The next cycle can be entered, the macroporous resin balancing step;

[0090] S2, reverse phase column chromatography separation

[0091] 1) Use reverse phase filler LX-21 as the stationary phase of the chromatographic column. 10 ml of reverse phase filler is homogenized with 10 ml of 15% ethanol aqueous solution, and wet packed into the column with a column height of 60 cm and a column pressure of 10 MPa;

[0092] 2) using a mixed aqueous solution of 5% ethanol and 50 mmol / L trifluoroacetic acid reagent to equilibrate the chromatographic column, 20 ml of the solution was equilibrated, and the flow rate during the equilibration process was 1 ml / min;

[0093] 3) The concentrate in the previous step was dissolved in a mixed aqueous solution of 5% ethanol and 50 mmol / L trifluoroacetic acid, with a concentration of 35 g / L (gramicidin A mass / dissolved solution volume), and a total of 12.8 ml of the solution was loaded onto the chromatographic column, with a forward loading capacity of 45 g / L (gramicidin A mass / reverse phase chromatography column volume), containing 450 mg of gramicidin A, and a flow rate of 1 ml / min during the loading process;

[0094] 4) Wash with a mixed aqueous solution of 5% ethanol and 50 mmol / L trifluoroacetic acid, the washing volume is 40 ml, and the flow rate of the washing process is 1 ml / min;

[0095] 5) Desorb using a mixed aqueous solution of 10% ethanol and 50 mmol / L trifluoroacetic acid, with a desorption volume of 40 ml and a flow rate of 1 ml / min during the desorption process; collect the gramicidin A desorption solution, and temporarily store 440 mg of gramicidin A;

[0096] 6) Regenerate the chromatographic column using 80% ethanol-water solution, with a regeneration volume of 20 ml and a regeneration process flow rate of 1 ml / min;

[0097] 7) The next cycle can be entered, the column equilibration step;

[0098] S3, decolorization resin decolorization

[0099] 1) 10 ml of decolorizing resin LSC-95 plus 10 ml of 10% (V / V) ethanol-water solution were wet packed into the column, with a filling height of 60 cm;

[0100] 2) Use a mixed aqueous solution of 10% ethanol and 50 mmol / L trifluoroacetic acid reagent to equilibrate the decolorizing resin column, equilibrate 20 ml, and the flow rate of the equilibration process is 1 ml / min;

[0101] 3) The desorbed liquid from the chromatography in S2 was loaded onto a decolorizing resin column in the forward direction at a flow rate of 1 ml / min; the concentration of gramicidin A was detected in the effluent, and the decolorized gramicidin A solution was collected and concentrated to dryness to obtain 430 mg of gramicidin A product with a purity of 99.2% and a comprehensive yield of 92%.

[0102] 4) The macroporous resin was regenerated using an 80% ethanol-water solution, with a regeneration volume of 20 ml and a regeneration flow rate of 1 ml / min;

[0103] 5) The next cycle can be entered, the macroporous resin balancing step.

[0104] Figure 1 This is the liquid phase detection spectrum of the crude polypeptide fermentation broth of Example 1. Figure 2 This is the liquid phase detection spectrum of the macroporous resin adsorption and desorption step in Example 1. Figure 3 It is the liquid phase detection spectrum of the reverse phase chromatography separation step in Example 1.

[0105] Example 2

[0106] This embodiment provides a method for separating and purifying high-purity gramicidin A from a polypeptide fermentation broth, comprising the following steps:

[0107] S1, macroporous resin column separation

[0108] 1) 10 ml of macroporous resin LX-T75 plus 20 ml of 40% (V / V) methanol aqueous solution was wet packed into the column, with a filling height of 65 cm;

[0109] 2) using a mixed aqueous solution of 40% methanol and 70 mmol / L malonic acid reagent to equilibrate the macroporous resin column, equilibration of 30 ml, and a flow rate of 0.8 ml / min during the equilibration process;

[0110] 3) Load the polypeptide fermentation broth onto a macroporous resin column, with the loading capacity of the macroporous resin column at 90 g / L (pure gramicidin A / macroporous resin volume), and the flow rate during the loading process containing 900 mg gramicidin A was 0.8 ml / min. The concentration of gramicidin A detected in the effluent was 0.03 mg / ml;

[0111] 4) washing with a mixed aqueous solution of 40% methanol and 70 mmol / L ortho-diacid, the washing volume was 20 ml, and the flow rate of the washing process was 0.8 ml / min;

[0112] 5) using 60% methanol and 70 mmol / L succinic acid mixed aqueous solution as the mobile phase for desorption, the desorption volume was 80 ml, the desorption process flow rate was 0.8 ml / min, the desorption liquid was collected, and concentrated to remove the alcohol therein, to obtain 870 mg of gramicidin A sample;

[0113] 6) Regenerating the macroporous resin with 85% methanol-water solution, the regeneration volume is 30 ml, and the regeneration process flow rate is 0.8 ml / min;

[0114] 7) The next cycle can be entered, the macroporous resin balancing step;

[0115] S2, reverse phase column chromatography separation

[0116] 1) Use reverse phase filler LX-22 as the stationary phase of the chromatographic column. Add 20 ml of 20% methanol-water solution to 10 ml of reverse phase filler, and wet pack the column. The column height is 65 cm and the packing pressure is 9 MPa.

[0117] 2) using 7% methanol and 70 mmol / L succinic acid mixed aqueous solution to equilibrate the chromatographic column, equilibrate 30 ml, and the flow rate of the equilibration process is 0.8 ml / min;

[0118] 3) The concentrate in the previous step was dissolved in a mixed aqueous solution of 2% methanol and 70 mmol / L succinic acid, with a concentration of 40 g / L (gramicidin A mass / dissolved solution volume), and 500 mg of gramicidin A was loaded onto the chromatographic column, with a forward loading capacity of 50 g / L (gramicidin A mass / reverse phase chromatography column volume), and a flow rate of 0.8 ml / min during the loading process;

[0119] 4) Wash with a mixed aqueous solution of 7% methanol and 70 mmol / L succinic acid, the washing volume is 60 ml, and the flow rate of the washing process is 0.8 ml / min;

[0120] 5) Desorption was performed using a mixed aqueous solution of 20% methanol and 70 mmol / L oxadiazine, the desorption volume was 80 ml, the desorption process flow rate was 0.8 ml / min, and the gramicidin A desorption solution was collected and temporarily stored;

[0121] 6) Regenerating the chromatographic column using 85% methanol-water solution, with a regeneration volume of 40 ml and a regeneration flow rate of 0.8 ml / min;

[0122] 7) The next cycle can be entered, the column equilibration step;

[0123] S3, decolorization resin decolorization

[0124] 1) 10 ml of decolorizing resin LSC-94 was added with 20 ml of 30% (V / V) methanol-water solution and wet packed into the column, with a filling height of 65 cm;

[0125] 2) using a mixed aqueous solution of 20% methanol and 70 mmol / L malonic acid reagent to equilibrate the decolorizing resin column, equilibrate 30 ml, and the flow rate of the equilibration process is 0.8 ml / min;

[0126] 3) The chromatographic desorption liquid of S2 was loaded onto the decolorizing resin column in the forward direction at a flow rate of 0.8 ml / min. The decolorized gramicidin A solution was collected and concentrated to dryness to obtain 470 mg of a high-purity gramicidin A product with a purity of 99.5% and a comprehensive yield of 93.7%.

[0127] 4) Regenerating the decolorizing resin using 85% methanol-water solution, with a regeneration volume of 40 ml and a regeneration flow rate of 0.8 ml / min;

[0128] 5) The next cycle can be entered, the macroporous resin balancing step.

[0129] Figure 4This is the liquid phase detection spectrum of the macroporous resin adsorption and desorption step in Example 2. Figure 5 It is the liquid phase detection spectrum of the reverse phase chromatography separation step in Example 2.

[0130] Example 3

[0131] This embodiment provides a method for separating and purifying high-purity gramicidin A from a polypeptide fermentation broth, comprising the following steps:

[0132] S1, macroporous resin column separation

[0133] 1) 10 ml of macroporous resin LX-T28 plus 30 ml of 50% (V / V) n-propanol-water solution were wet packed into the column, with a filling height of 67 cm;

[0134] 2) using a mixed aqueous solution of 50% n-propanol-water and 80 mmol / L glycolic acid to equilibrate the macroporous resin column, equilibration of 60 ml, and a flow rate of 0.7 ml / min during the equilibration process;

[0135] 3) The polypeptide fermentation broth was loaded onto a macroporous resin column, with the loading capacity of the macroporous resin column being 100 g / L (pure gramicidin A / macroporous resin volume), containing 100 mg gramicidin A, the flow rate during loading was 0.7 ml / min, and the concentration of gramicidin A detected in the effluent was 0.01 mg / ml;

[0136] 4) washing with a mixed aqueous solution of 50% n-propanol and 80 mmol / L glycolic acid, 30 ml of which was washed at a flow rate of 0.7 ml / min;

[0137] 5) using a mixed aqueous solution of 70% n-propanol and 80 mmol / L glycolic acid as the mobile phase for desorption, the desorption volume was 100 ml, the flow rate of the desorption process was 0.7 ml / min, the desorbed liquid was collected, and concentrated to remove the alcohol therein, to obtain 97 mg of a sample containing gramicidin A;

[0138] 6) The macroporous resin was regenerated using a 90% n-propanol-water solution, with a regeneration volume of 40 ml and a linear flow rate of 0.7 ml / min during the regeneration process;

[0139] 7) The next cycle can be entered, the macroporous resin balancing step;

[0140] S2, reverse phase column chromatography separation

[0141] 1) Use reverse phase filler LX-26 as the stationary phase of the chromatographic column. 10 ml of reverse phase filler was homogenized with 30 ml of 50% n-propanol aqueous solution, and wet packed into the column with a column height of 67 cm and a packing pressure of 9 MPa;

[0142] 2) using a 9% n-propanol and 80 mmol / L glycolic acid aqueous solution to balance the chromatographic column, balancing 40 ml, and a flow rate of 0.7 ml / min during the balancing process;

[0143] 3) The concentrate in the previous step was dissolved in a mixed aqueous solution of 5% n-propanol and 80 mmol / L glycolic acid at a molar concentration of 70 g / L (gramicidin A mass / dissolved solution volume), and 700 mg was loaded onto the chromatographic column in the forward direction with a loading capacity of 70 g / L (gramicidin A mass / reverse phase chromatography column volume), and the flow rate during the loading process was 0.7 ml / min;

[0144] 4) washing with a mixed aqueous solution of 9% n-propanol and 80 mmol / L glycolic acid, with a washing volume of 100 ml and a washing flow rate of 0.7 ml / min;

[0145] 5) Desorbing with a mixed aqueous solution of 27% n-propanol and 80 mmol / L glycolic acid reagent, the desorption volume is 100 ml, the desorption process flow rate is 0.7 ml / min, collecting the gramicidin A desorption solution and temporarily storing;

[0146] 6) Regenerating the chromatographic column using a 90% n-propanol-water solution, with a regeneration volume of 50 ml and a regeneration flow rate of 0.7 ml / min;

[0147] 7) You can proceed to the next cycle, the column equilibration step.

[0148] Figure 6 This is the liquid phase detection spectrum of the macroporous resin adsorption and desorption step in Example 3. Figure 7 It is the liquid phase detection spectrum of the reverse phase chromatography separation step in Example 3.

[0149] S3, decolorization resin decolorization

[0150] 1) 10 ml of decolorizing resin LSC-200 resin plus 30 ml of 40% (V / V) n-propanol aqueous solution was wet packed into the column, with a filling height of 67 cm;

[0151] 2) using a mixed aqueous solution of 28% n-propanol and an organic acid reagent with a molar concentration of 80 mmol / L to equilibrate the decolorizing resin column, equilibrate 40 ml, and the flow rate of the equilibration process is 0.7 ml / min;

[0152] 3) The desorbed liquid from the chromatography in S2 was loaded onto the decolorizing resin column in the forward direction at a flow rate of 0.7 ml / min. The decolorized gramicidin A solution was collected and concentrated to dryness to obtain 670 mg of a high-purity gramicidin A product with a purity of 92.7% and a comprehensive yield of 91%;

[0153] 4) Regenerating the decolorizing resin using a 90% n-propanol-water solution, with a regeneration volume of 60 ml and a regeneration flow rate of 0.7 ml / min;

[0154] 5) You can enter the next cycle and balance the decolorization resin step;

[0155] Example 4

[0156] This embodiment provides a method for separating and purifying high-purity gramicidin A from a polypeptide fermentation broth, comprising the following steps:

[0157] S1, macroporous resin column separation

[0158] 1) 10 ml of macroporous resin LX-T81 plus 30 ml of 60% (V / V) isopropanol is wet packed into the column, with the packing height not less than 90 cm;

[0159] 2) using a mixed aqueous solution of 50% isopropanol and 100 mmol / L acetic acid reagent to equilibrate the macroporous resin column, equilibrate 60 ml, and the flow rate of the equilibration process is 0.6 ml / min;

[0160] 3) The polypeptide fermentation broth was loaded onto a macroporous resin column in a positive loading state. The loading capacity of the macroporous resin column was 120 g / L (pure gramicidin A / macroporous resin volume). The flow rate during loading of the fermentation broth containing 1200 mg of gramicidin A was 0.6 ml / min. The concentration of gramicidin A detected in the effluent was 0.04 mg / ml.

[0161] 4) washing with a mixed aqueous solution of 50% isopropanol and 100 mmol / L acetic acid, the washing volume is 50 ml, and the flow rate of the washing process is 0.6 ml / min;

[0162] 5) Using a mixed aqueous solution of 80% isopropanol and 100 mmol / L acetic acid reagent as the mobile phase for desorption, the desorption volume is 200 ml, the desorption process flow rate is 0.6 ml / min, the desorbed liquid is collected, and concentrated to remove the alcohol therein;

[0163] 6) Regenerate the macroporous resin using 90% isopropanol, with a regeneration volume of 60 ml and a regeneration flow rate of 0.6 ml / min;

[0164] 7) The next cycle can be entered, the macroporous resin balancing step;

[0165] S2, reverse phase column chromatography separation

[0166] 1) Use reverse phase filler LX-261 as the stationary phase of the chromatographic column. Add 30 ml of 100% isopropanol slurry to 10 ml of reverse phase filler, and wet pack the column. The column height is 90 cm and the packing pressure is 7 MPa.

[0167] 2) using a mixed aqueous solution of 10% isopropanol and 100 mmol / L acetic acid reagent to equilibrate the chromatographic column, equilibrate 60 ml, and the flow rate of the equilibration process is 0.6 ml / min;

[0168] 3) The concentrate in the previous step was dissolved in a mixed aqueous solution of 5% isopropanol and 100 mmol / L acetic acid reagent at a molar concentration of 75 g / L (gramicidin A mass / dissolved solution volume), and 900 mg of gramicidin A was loaded onto the chromatographic column in the forward direction with a loading capacity of 90 g / L (gramicidin A mass / reverse phase chromatography column volume), and the flow rate during the loading process was 0.6 ml / min;

[0169] 4) Wash with a mixed aqueous solution of 10% isopropanol and 100 mmol / L acetic acid reagent, the washing volume is 150 ml, and the flow rate of the washing process is 0.6 ml / min;

[0170] 5) Desorbing with a mixed aqueous solution of 30% isopropanol and 100 mmol / L acetic acid reagent, the desorption volume is 150 ml, the desorption process flow rate is 0.6 ml / min, collecting the gramicidin A desorption solution and temporarily storing;

[0171] 6) Regenerating the chromatographic column using a 90% n-propanol-water solution, with a regeneration volume of 60 ml and a regeneration flow rate of 0.6 ml / min;

[0172] 7) The next cycle can be entered, the column equilibration step;

[0173] S3, decolorization resin decolorization

[0174] 1) 10 ml of decolorizing resin LSC-300 plus 30 ml of 40% (V / V) isopropanol is wet packed into the column, with the packing height not less than 90 cm;

[0175] 2) using a mixed aqueous solution of 30% isopropanol and 100 mmol / L acetic acid reagent to equilibrate the decolorizing resin column, equilibrate 40 ml, and the flow rate of the equilibration process is 0.6 ml / min;

[0176] 3) The chromatographic desorption liquid of S2 was loaded onto the decolorizing resin column, with the loading rate of 0.6 ml / min, and the decolorized gramicidin A solution was collected and concentrated to dryness to obtain 867 mg of high-purity gramicidin A product with a purity of 99.7% and a comprehensive yield of 93%.

[0177] 4) Regenerate the decolorizing resin using 90% isopropanol, with a regeneration volume of 60 ml and a regeneration flow rate of 0.6 ml / min;

[0178] 5) The next cycle can be entered, the macroporous resin balancing step.

[0179] Figure 8 This is the liquid phase detection spectrum of the macroporous resin adsorption and desorption step in Example 4. Fig. 9 It is the liquid phase detection spectrum of the reverse phase chromatography separation step of Example 4.

[0180] Comparative Example 1

[0181] Compared with Example 1, in this embodiment, no organic acid is added to the aqueous solution of alcohol, and the steps are specifically as follows:

[0182] S1, macroporous resin column separation

[0183] 1) 10 ml of macroporous resin LX-T8 plus 10 ml of 30% (V / V) ethanol aqueous solution were wet packed into the column, with a filling height of 60 cm and a volume of 10 ml;

[0184] 2) Use 30% ethanol aqueous solution to balance the macroporous resin column, balance 20 ml, and the linear flow rate of the balance process is 6 cm / min;

[0185] 3) Load the polypeptide fermentation broth onto a macroporous resin column, with the loading capacity of the macroporous resin column at 80 g / L (pure gramicidin A / macroporous resin volume), load 800 mg of gramicidin A fermentation broth, and detect the concentration of gramicidin A in the effluent at 0.01 mg / ml;

[0186] 4) Wash with 30% ethanol aqueous solution, the washing volume is 10 ml, and the flow rate is 1 ml / min;

[0187] 5) Use 50% ethanol aqueous solution as the mobile phase for desorption, the desorption volume is 30 ml, the desorption process flow rate is 10 ml / min, collect the desorbed liquid, and concentrate it to remove the alcohol therein to obtain 762 mg of a sample containing gramicidin A and proceed to the next step;

[0188] 6) Regenerate the macroporous resin using 80% ethanol aqueous solution, with a regeneration volume of 20 ml and a regeneration flow rate of 1 ml / min;

[0189] 7) The next cycle can be entered, the macroporous resin balancing step;

[0190] S2, reverse phase column chromatography separation

[0191] 1) Use reverse phase filler LX-21 as the stationary phase of the chromatographic column. 10 ml of reverse phase filler is homogenized with 10 ml of 15% ethanol aqueous solution, and wet packed into the column with a column height of 60 cm and a column pressure of 10 MPa;

[0192] 2) Use 5% ethanol aqueous solution to balance the chromatographic column, balance 20 ml, and the flow rate of the balance process is 1 ml / min;

[0193] 3) The concentrate in the previous step was dissolved in 5% ethanol aqueous solution to a concentration of 35 g / L (gramicidin A mass / dissolved solution volume), and a total of 12.8 ml of the solution was loaded onto the chromatographic column, with a forward loading capacity of 45 g / L (gramicidin A mass / reverse phase chromatography column volume), containing 450 mg of gramicidin A, and a flow rate of 1 ml / min during the loading process;

[0194] 4) Wash with 5% ethanol aqueous solution, the washing volume is 40 ml, and the flow rate of the washing process is 1 ml / min;

[0195] 5) Desorb using 10% ethanol aqueous solution, the desorption volume is 40 ml, and the desorption process flow rate is 1 ml / min; collect the gramicidin A desorption solution, and temporarily store 435 mg of gramicidin A;

[0196] 6) Regenerate the chromatographic column using 80% ethanol-water solution, with a regeneration volume of 20 ml and a regeneration process flow rate of 1 ml / min;

[0197] 7) The next cycle can be entered, the column equilibration step;

[0198] S3, decolorization resin decolorization

[0199] 1) 10 ml of decolorizing resin LSC-95 plus 10 ml of 10% (V / V) ethanol-water solution were wet packed into the column, with a filling height of 60 cm;

[0200] 2) Use 10% ethanol aqueous solution to balance the decolorizing resin column, balance 20ml, and the flow rate of the balance process is 1ml / min;

[0201] 3) The desorbed liquid from the chromatography in S2 was loaded onto the decolorizing resin column, with the loading process flow rate of 1 ml / min; the concentration of gramicidin A was detected in the effluent, and the decolorized gramicidin A solution was collected and concentrated to dryness to obtain 406 mg of gramicidin A product with a purity of 89.4% and a comprehensive yield of 85.94%.

[0202] 4) The macroporous resin was regenerated using an 80% ethanol-water solution, with a regeneration volume of 20 ml and a regeneration flow rate of 1 ml / min;

[0203] Comparative Example 2

[0204] Compared with Example 1, in this example, the concentration of organic acid in the aqueous solution of alcohol is increased to 150 mmol / L, and the specific steps are as follows:

[0205] S1, macroporous resin column separation

[0206] 1) 10 ml of macroporous resin LX-T8 plus 10 ml of 30% (V / V) ethanol aqueous solution were wet packed into the column, with a filling height of 60 cm and a volume of 10 ml;

[0207] 2) using a mixed aqueous solution of 30% ethanol and a trifluoroacetic acid reagent with a molar concentration of 150 mmol / L to equilibrate the macroporous resin column, 20 ml of the solution was balanced, and the linear flow rate of the equilibrium process was 6 cm / min;

[0208] 3) Load the polypeptide fermentation broth onto a macroporous resin column, with the loading capacity of the macroporous resin column at 80 g / L (pure gramicidin A / macroporous resin volume), load 800 mg of gramicidin A fermentation broth, and detect the concentration of gramicidin A in the effluent at 0.01 mg / ml;

[0209] 4) Wash with a mixed aqueous solution of 30% ethanol and 150 mmol / L trifluoroacetic acid, with a washing volume of 10 ml and a flow rate of 1 ml / min;

[0210] 5) Using a mixed aqueous solution of 50% ethanol and 150 mmol / L trifluoroacetic acid reagent as the mobile phase for desorption, the desorption volume is 30 ml, the flow rate of the desorption process is 10 ml / min, collecting the desorbed liquid, and concentrating it to remove the alcohol therein to obtain 740 mg of a sample containing gramicidin A and proceed to the next step;

[0211] 6) Regenerate the macroporous resin using 80% ethanol aqueous solution, with a regeneration volume of 20 ml and a regeneration flow rate of 1 ml / min;

[0212] 7) The next cycle can be entered, the macroporous resin balancing step;

[0213] S2, reverse phase column chromatography separation

[0214] 1) Use reverse phase filler LX-21 as the stationary phase of the chromatographic column. 10 ml of reverse phase filler is homogenized with 10 ml of 15% ethanol aqueous solution, and wet packed into the column with a column height of 60 cm and a column pressure of 10 MPa;

[0215] 2) using a mixed aqueous solution of 5% ethanol and 150 mmol / L trifluoroacetic acid reagent to equilibrate the chromatographic column, 20 ml of the solution was equilibrated, and the flow rate during the equilibration process was 1 ml / min;

[0216] 3) The concentrate in the previous step was dissolved in a mixed aqueous solution of 5% ethanol and 150 mmol / L trifluoroacetic acid, with a concentration of 35 g / L (gramicidin A mass / dissolved solution volume), and a total of 12.8 ml of the solution was loaded onto the chromatographic column, with a forward loading of 45 g / L (gramicidin A mass / reverse phase chromatography column volume), containing 440 mg gramicidin A, and a flow rate of 1 ml / min during the loading process;

[0217] 4) Wash with a mixed aqueous solution of 5% ethanol and 150 mmol / L trifluoroacetic acid, the washing volume is 40 ml, and the flow rate of the washing process is 1 ml / min;

[0218] 5) Desorb using a mixed aqueous solution of 10% ethanol and 150 mmol / L trifluoroacetic acid, with a desorption volume of 40 ml and a flow rate of 1 ml / min during the desorption process; collect the gramicidin A desorption solution and temporarily store 410 mg of gramicidin A;

[0219] 6) Regenerate the chromatographic column using 80% ethanol-water solution, with a regeneration volume of 20 ml and a regeneration process flow rate of 1 ml / min;

[0220] 7) The next cycle can be entered, the column equilibration step;

[0221] S3, decolorization resin decolorization

[0222] 1) 10 ml of decolorizing resin LSC-95 plus 10 ml of 10% (V / V) ethanol-water solution were wet packed into the column, with a filling height of 60 cm;

[0223] 2) Use a mixed aqueous solution of 10% ethanol and 150 mmol / L trifluoroacetic acid reagent to balance the decolorizing resin column, balancing 20 ml, and the flow rate of the balancing process is 1 ml / min;

[0224] 3) The desorbed liquid from the chromatography in S2 was loaded onto a decolorizing resin column in the forward direction at a flow rate of 1 ml / min; the concentration of gramicidin A was detected in the effluent, and the decolorized gramicidin A solution was collected and concentrated to dryness to obtain 390 mg of gramicidin A product with a purity of 88.3% and a comprehensive yield of 81.9%.

[0225] 4) The macroporous resin was regenerated using an 80% ethanol-water solution, with a regeneration volume of 20 ml and a regeneration flow rate of 1 ml / min.

[0226] According to the comparison of the experimental results of the above embodiment and the comparative example, it can be known that in the purification process of the present invention, by adding an appropriate amount of organic acid reagent to the mobile phase, the electronegativity of the polypeptide amino acid can be shielded, and the peptide chain structure has only hydrophobic interaction, so that the adsorption amount of gramicidin A on the polymer filler is larger, the loading capacity is increased, the retention time is extended, the separation effect is improved, and it is more conducive to industrialization and saves equipment investment costs. When an excessive amount of organic acid reagent is added, the separation effect will be reduced, and the product purity and yield will be reduced.

[0227] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for separating and purifying gramicidin A from a polypeptide fermentation broth, characterized in that: The steps include: Step 1) loading the polypeptide fermentation liquid onto a macroporous resin column; then using a first mobile phase to wash the macroporous resin column; then using a second mobile phase to elute the macroporous resin column, and collecting the eluate to obtain a separation solution; Step 2) loading the separated liquid onto a reverse phase column of a polymer chromatography packing material; then using a third mobile phase to elute the reverse phase column of the polymer chromatography packing material, and collecting the eluate to obtain a gramicidin A solution; Step 3) loading the gramicidin A solution onto a decolorizing resin column, and collecting the effluent to obtain a purified gramicidin A solution; The first mobile phase, the second mobile phase and the third mobile phase are all aqueous solutions containing alcohol and organic acid; the volume fraction of alcohol in the first mobile phase is 30% to 50%, and the volume fraction of alcohol in the second mobile phase is 50% to 80%; the volume fraction of alcohol in the third mobile phase is 10% to 30%; the organic acid concentrations in the first mobile phase, the second mobile phase and the third mobile phase are all 50mmol / L to 100mmol / L.

2. The method for separating and purifying gramicidin A from polypeptide fermentation broth according to claim 1, characterized in that: The alcohol in the alcohol-containing solvent is a monohydric alcohol having a carbon number not greater than 4 and being miscible with water in any proportion; Preferably, the alcohol comprises at least one of methanol, ethanol, n-propanol, isopropanol and butanol.

3. The method for separating and purifying gramicidin A from polypeptide fermentation broth according to claim 1, characterized in that: The organic acid is at least one of trifluoroacetic acid, malonic acid, glycolic acid, succinic acid, glycine and oxalic acid.

4. The method for separating and purifying gramicidin A from polypeptide fermentation broth according to claim 1, characterized in that: The filler of the macroporous resin column is a polyacrylate-styrene-divinylbenzene cross-linked polymer, the mass of the polyacrylate in the polyacrylate-styrene-divinylbenzene cross-linked polymer accounts for 5-25% of the mass of the cross-linked polymer, and the degree of cross-linking is 10%-70%; Preferably, the filler of the macroporous resin column is polyacrylate-styrene-divinylbenzene cross-linked polymer microspheres, the particle size of the polyacrylate-styrene-divinylbenzene cross-linked polymer microspheres is 0.75 mm to 1.25 mm, and the pore size is Specific surface area not less than 120m 2 / g; Preferably, the macroporous resin column is prepared by wet packing; Preferably, the macroporous resin column is prepared by the following method: the polyacrylate-styrene-divinylbenzene crosslinked polymer microspheres are mixed with 1 to 3 times the column volume of an alcohol aqueous solution and then packed into the column, the filling height is not less than 60 cm; and the volume fraction of the alcohol-water solution is 30% to 50%; Preferably, after the macroporous resin column is loaded and before the impurities are washed, 2 to 6 column volumes of a mixed aqueous solution of alcohol with a volume concentration of 5% to 30% and an organic acid reagent with a molar concentration of 10 mmol / L to 50 mmol / L is used to balance the macroporous resin column, and the linear flow rate of the effluent during the balancing process is not higher than 6 cm / min.

5. The method for separating and purifying gramicidin A from polypeptide fermentation broth according to claim 1, characterized in that: The amount of the first mobile phase is 1 to 4 times the column volume; the linear velocity of the effluent during the washing process is not higher than 6 cm / min; The amount of the second mobile phase B is 4 to 25 times the column volume; the linear velocity of the eluent flowing out during the elution process is not higher than 6 cm / min; During the loading process of the macroporous resin column, the loading is stopped when the concentration of gramicidin A at the outlet of the macroporous resin column is detected to be less than 0.05 mg / mL, and the linear velocity of the outflow liquid during the loading process is not higher than 6 cm / min.

6. The method for separating and purifying gramicidin A from polypeptide fermentation broth according to claim 1, characterized in that: In step 1), after collecting the eluent to obtain the separation liquid, the macroporous resin column is regenerated: 2 to 6 column volumes of an alcohol-water solution with a volume concentration of 80% to 90% are used to elute the macroporous resin column, and the linear velocity of the effluent during the regeneration process is not higher than 6 cm / min.

7. The method for separating and purifying gramicidin A from polypeptide fermentation broth according to claim 1, characterized in that: The polymer chromatography filler in the reverse phase column of the polymer chromatography filler is at least one of C18 bonded silica gel, C12 bonded silica gel, C8 bonded silica gel, phenyl bonded silica gel, butyl bonded silica gel, and a polymer reverse phase filler containing an ester group, butyl group, phenyl group, octyl group, dodecyl group, hexadecyl group, vinyl group, allyl benzene, vinyl benzene or divinyl benzene; Preferably, the polymer chromatography filler reverse phase column is prepared by wet packing; Preferably, the reverse phase column of the polymer chromatography filler is prepared by the following method: the reverse phase filler is mixed with 1 to 3 times the column volume and the volume concentration of 0% to 100% alcohol-water solution and then packed into the column, the packing height is not less than 60cm, and the packing pressure is not higher than 10MPa; Preferably, after the polymer chromatography filler reverse phase column is packed and before elution, 2 to 6 column volumes of a mixed aqueous solution of alcohol with a volume concentration of 0% to 5% and an organic acid reagent with a molar concentration of 10 mmol / L to 50 mmol / L are used to balance the polymer chromatography filler reverse phase column, and the linear flow rate of the effluent during the equilibration process is not higher than 6 cm / min.

8. The method for separating and purifying gramicidin A from polypeptide fermentation broth according to claim 1, characterized in that: In the step 1), the separation liquid is evaporated and concentrated to remove alcohol in the separation liquid; in the step 2), the concentrated separation liquid is diluted to 35 g / L to 75 g / L and then loaded onto the reverse phase column; Preferably, in the step 2), the concentrated separation liquid is diluted to 35 g / L to 75 g / L with an aqueous alcohol solution having a volume fraction of 0% to 5%, and then loaded onto the reverse phase column.

9. The method for separating and purifying gramicidin A from polypeptide fermentation broth according to claim 1, characterized in that: In the step 2), during the process of loading the separated liquid onto the reverse phase column of the polymer chromatography filler, the sample is loaded at a loading amount of 45-90 mg / ml, and the linear velocity of the effluent during the loading process is not higher than 6 cm / min; Preferably, the step 2) further comprises regenerating the reverse phase column after the elution process, comprising: eluting the reverse phase column with 2 to 8 column volumes of an alcohol-water solution with a volume concentration of 80% to 90%, wherein the linear velocity of the effluent during the regeneration process is not higher than 6 cm / min; Preferably, in the step 2), before eluting the polymer chromatography filler reverse column, a fourth mobile phase is used, the washing volume is 4-15 times the column volume, and the linear velocity of the washing process is not higher than 6 cm / min; the fourth mobile phase is an aqueous solution containing alcohol and organic acid; the volume fraction of alcohol in the fourth mobile phase is 5% to 10%; the concentration of organic acid in the fourth mobile phase is 50mmol / L to 100mmol / L.

10. The method for separating and purifying gramicidin A from polypeptide fermentation broth according to claim 1, characterized in that: The filler in the decolorizing resin column in the step 3) is a non-ionic decolorizing resin with a styrene skeleton.

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