CN115745863A - Method for extracting prodigiosin from fermentation liquor containing prodigiosin - Google Patents
Method for extracting prodigiosin from fermentation liquor containing prodigiosin Download PDFInfo
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- CN115745863A CN115745863A CN202211431354.2A CN202211431354A CN115745863A CN 115745863 A CN115745863 A CN 115745863A CN 202211431354 A CN202211431354 A CN 202211431354A CN 115745863 A CN115745863 A CN 115745863A
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- prodigiosin
- eluent
- silica gel
- ethyl acetate
- chloroform
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- HCOLPNRPCMFHOH-UHFFFAOYSA-N Prodigiosin Natural products CCCCCC1C=C(C=C/2N=C(C=C2OC)c3ccc[nH]3)N=C1C HCOLPNRPCMFHOH-UHFFFAOYSA-N 0.000 title claims abstract description 134
- TWFGRJUTAULJPZ-USZBIXTISA-N prodigiosin Chemical compound N1=C(C)C(CCCCC)=C\C1=C/C1=NC(C=2[N]C=CC=2)=C[C]1OC TWFGRJUTAULJPZ-USZBIXTISA-N 0.000 title claims abstract description 134
- 238000000855 fermentation Methods 0.000 title claims abstract description 68
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- 238000000034 method Methods 0.000 title claims abstract description 44
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 98
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- 229910002027 silica gel Inorganic materials 0.000 claims abstract description 96
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- 238000000926 separation method Methods 0.000 claims abstract description 20
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 18
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 222
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 183
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 140
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 130
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- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 claims description 13
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- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
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Images
Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of extraction of prodigiosin, and provides a method for extracting prodigiosin from a fermentation broth containing prodigiosin. The method provided by the invention utilizes double silica gel column cascade chromatography to separate and purify prodigiosin, and simultaneously, an antioxidant is added in an auxiliary manner, so that a high-purity prodigiosin pure product is obtained, and the loss rate of prodigiosin in the separation process is greatly reduced. The data of the examples show that: the purity of the pure prodigiosin product separated and extracted from the fermentation liquor by adopting the method provided by the invention is more than or equal to 91.5 percent and even more than 99 percent; the yield is more than or equal to 86.8 percent and even reaches 98.5 percent; the method provided by the invention is simple to operate and suitable for large-scale industrial production.
Description
Technical Field
The invention relates to the technical field of extraction of prodigiosin, in particular to a method for extracting prodigiosin from a fermentation broth containing prodigiosin.
Background
Prodigiosin (prodigiosin) is a class of microbial pigments, a secondary metabolite produced by a variety of bacteria. At present, there are two main types of prodigiosin reported in the literature: one is a compound containing linear alkyl side chains, such as Prodigiosin and undecycylprodigiosin, and the other is a cyclic compound, such as cyclicoprodigiosin, metacylindropterosin, prodigiosin R1 and streptorubicin B.
Prodigiosin is a common red pigment and is widely used for textile printing and dyeing. In addition, the prodigiosin also has obvious effect in the process of inducing apoptosis of colorectal cancer cells, and has the potential of being used as a novel anti-cancer drug for treating tumors. Since prodigiosin has the color change characteristic of pH response, in the field of material science, recently reports prove that pH response color change performance and a biodegradable color change film have wide application prospects in the aspects of marking, packaging, base materials and the like; recent reports show that the prodigiosin has natural bacteriostatic performance, has obvious inhibitory effect on escherichia coli under the condition of higher than Minimum Inhibitory Concentration (MIC), and has no obvious DNA damage and cytoplasmic membrane disintegration.
At present, prodigiosin is obtained by separating and purifying from fermentation liquor, but the purity of the prodigiosin obtained by separation and purification is low at present.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for extracting prodigiosin from a fermentation broth containing prodigiosin. The prodigiosin obtained by the method provided by the invention has high purity.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for extracting prodigiosin from a prodigiosin-containing fermentation broth, which comprises the following steps:
carrying out first solid-liquid separation on fermentation liquor containing prodigiosin to obtain thallus precipitates and fermentation liquor supernatant;
extracting the supernatant of the fermentation liquor to obtain an extract phase;
mixing the extract phase with an antioxidant, and carrying out first reduced pressure distillation on the obtained extract phase system to obtain a first prodigiosin crude product;
mixing the thallus precipitate with an acidic alcohol solvent, and sequentially carrying out ultrasonic treatment, leaching and second solid-liquid separation to obtain a thallus supernatant;
mixing the thallus supernatant with an antioxidant, and carrying out second reduced pressure distillation on an obtained thallus supernatant system to obtain a second prodigiosin crude product;
mixing the first prodigiosin crude product and the second prodigiosin crude product, performing double silica gel column cascade chromatography, and performing gradient elution by using an eluent to obtain a prodigiosin eluent;
concentrating the prodigiosin eluent under reduced pressure to obtain a prodigiosin pure product;
the silica gel column of the double silica gel column cascade chromatography comprises an upper silica gel column and a lower silica gel column which are connected in series;
in the process of gradient elution, the eluents are a first eluent, a second eluent, a third eluent, a fourth eluent, a fifth eluent, a sixth eluent and a seventh eluent in sequence;
the first eluent is petroleum ether, ethyl acetate, acetone and chloroform with the volume ratio of 7:2:0.5:0.5 of a mixed solvent;
the second eluent is petroleum ether, ethyl acetate, acetone and chloroform, and the volume ratio of the petroleum ether to the ethyl acetate to the acetone to the chloroform is 6:3:0.5:0.5 of a mixed solvent;
the third eluent is petroleum ether, ethyl acetate, acetone and chloroform with the volume ratio of 5:4:0.5:0.5 of a mixed solvent;
the fourth eluent is petroleum ether, ethyl acetate, acetone and chloroform, and the volume ratio of the petroleum ether to the ethyl acetate to the acetone to the chloroform is 4:5:0.5:0.5 of a mixed solvent;
the fifth eluent is petroleum ether, ethyl acetate, acetone and chloroform, and the volume ratio of the petroleum ether to the ethyl acetate to the acetone to the chloroform is 3:6:0.5:0.5 of a mixed solvent;
the sixth eluent is petroleum ether, ethyl acetate, acetone and chloroform, and the volume ratio of the petroleum ether to the ethyl acetate to the acetone to the chloroform is 2:7:0.5:0.5 of a mixed solvent;
the seventh eluent is petroleum ether, ethyl acetate, acetone and chloroform with the volume ratio of 1:8:0.5:0.5 of a mixed solvent.
Preferably, the volume ratio of the upper silica gel column to the lower silica gel column is 1: 1.5-1: 2; the ratio of the silica gel filling volume in the upper silica gel column to the silica gel filling volume in the lower silica gel column is 1:3 to 1:4.
preferably, the first, second, third, fourth, fifth, sixth and seventh eluents are used in an amount of 1 time the volume of the silica gel column.
Preferably, the fermentation liquor containing prodigiosin is a serratia marcescens fermentation liquor; the preparation method of the serratia marcescens fermentation liquor comprises the following steps:
inoculating the plate-activated serratia marcescens into a seed culture medium, and obtaining an inoculated strain when the strain grows to a logarithmic phase;
inoculating the inoculated strain to an antibiotic-free LB culture medium, and culturing to obtain the serratia marcescens fermentation liquor;
the volume percentage content of the inoculated strain in the nonreactive LB culture medium is 1-2%.
Preferably, the temperature of the culture is 25-27 ℃, and the time is 24-48 h; the culture is carried out under the condition of shaking, and the speed of shaking is 150-200 rpm.
Preferably, the extractive extractant comprises ethyl acetate.
Preferably, the antioxidant comprises vitamin C and/or tetrahydrofuran.
Preferably, the concentration of the antioxidant in the extraction phase system and the bacterial supernatant system is 0.01-0.05 g/L independently.
Preferably, the acidic alcohol solvent has a pH of 3; the acidic alcohol solvent comprises methanol and an inorganic acid; the inorganic acid includes hydrochloric acid, sulfuric acid and nitric acid.
Preferably, the temperature of the first reduced pressure distillation is 40-45 ℃, and the temperature of the second reduced pressure distillation is 30-35 ℃; the first reduced pressure distillation and the second reduced pressure distillation are carried out under the condition of avoiding light.
The invention provides a method for extracting prodigiosin from a prodigiosin-containing fermentation broth, which comprises the following steps: carrying out first solid-liquid separation on fermentation liquor containing prodigiosin to obtain thallus precipitates and fermentation liquor supernatant; extracting the supernatant of the fermentation liquor to obtain an extract phase; mixing the extract phase with an antioxidant, and carrying out first reduced pressure distillation on the obtained extract phase system to obtain a first prodigiosin crude product; mixing the thallus precipitate with an acidic alcohol solvent, and sequentially carrying out ultrasonic treatment, leaching and second solid-liquid separation to obtain a thallus supernatant; mixing the thallus supernatant with an antioxidant, and carrying out second reduced pressure distillation on an obtained thallus supernatant system to obtain a second prodigiosin crude product; mixing the first prodigiosin crude product and the second prodigiosin crude product, performing double silica gel column cascade chromatography, and performing gradient elution by using an eluent to obtain a prodigiosin eluent; carrying out reduced pressure concentration on the prodigiosin eluent to obtain a prodigiosin pure product; the silica gel column of the double silica gel column cascade chromatography comprises an upper silica gel column and a lower silica gel column which are connected in series; in the process of gradient elution, the eluents are a first eluent, a second eluent, a third eluent, a fourth eluent, a fifth eluent, a sixth eluent and a seventh eluent in sequence; the first eluent is petroleum ether, ethyl acetate, acetone and chloroform, and the volume ratio of the petroleum ether to the ethyl acetate to the acetone to the chloroform is 7:2:0.5:0.5 of a mixed solvent; the second eluent is petroleum ether, ethyl acetate, acetone and chloroform, and the volume ratio of the petroleum ether to the ethyl acetate to the acetone to the chloroform is 6:3:0.5:0.5 of a mixed solvent; the third eluent is petroleum ether, ethyl acetate, acetone and chloroform, and the volume ratio of the petroleum ether to the ethyl acetate to the acetone to the chloroform is 5:4:0.5:0.5 of a mixed solvent; the fourth eluent is petroleum ether, ethyl acetate, acetone and chloroform, and the volume ratio of the petroleum ether to the ethyl acetate to the acetone to the chloroform is 4:5:0.5:0.5 of a mixed solvent; the fifth eluent is petroleum ether, ethyl acetate, acetone and chloroform, and the volume ratio of the petroleum ether to the ethyl acetate to the acetone to the chloroform is 3:6:0.5:0.5 of a mixed solvent; the sixth eluent is petroleum ether, ethyl acetate, acetone and chloroform, and the volume ratio of the petroleum ether to the ethyl acetate to the acetone to the chloroform is 2:7:0.5:0.5 of a mixed solvent; the seventh eluent is petroleum ether, ethyl acetate, acetone and chloroform with the volume ratio of 1:8:0.5:0.5 of a mixed solvent. The method provided by the invention utilizes double silica gel column cascade chromatography to separate and purify prodigiosin, and simultaneously, an antioxidant is added in an auxiliary manner, so that a high-purity prodigiosin pure product is obtained, and the loss rate in the separation process is greatly reduced. The data of the examples show that: the purity of the pure prodigiosin product separated and extracted from the fermentation liquor by adopting the method provided by the invention is more than or equal to 91.5 percent and even more than 99 percent; the yield is more than or equal to 86.8 percent and even reaches 98.5 percent; the method provided by the invention is simple to operate and suitable for large-scale industrial production.
Further, the fermentation liquor containing prodigiosin is a serratia marcescens fermentation liquor; the preparation method of the serratia marcescens fermentation liquor comprises the following steps: inoculating the plate-activated serratia marcescens into a seed culture medium, and obtaining an inoculated strain when the strain grows to a logarithmic phase; inoculating the inoculated strain to an LB culture medium without an antibody, and culturing to obtain the serratia marcescens fermentation liquor; the volume percentage content of the inoculated strain in the nonreactive LB culture medium is 1-2%. The preparation method of the serratia marcescens fermentation liquid provided by the invention is suitable for large production systems, namely 10L fermentation can be expanded.
Drawings
FIG. 1 is a photograph showing a fermentation broth of Serratia marcescens obtained in example 1;
FIG. 2 is a diagram of a silica gel plate obtained by thin layer chromatography using five developers according to example 1;
FIG. 3 is a flow chart of the double silica gel column cascade chromatography in example 1;
FIG. 4 is a graph of eluents of different colors collected at different time periods in the double silica gel column cascade chromatography of example 1;
FIG. 5 shows the results of LC-MS detection of the red eluate obtained in example 1;
FIG. 6 shows the results of LC-MS detection of pink eluate obtained in example 1;
FIG. 7 shows the results of liquid chromatography-mass spectrometry (LC-MS) detection of the yellow eluate obtained in example 1;
FIG. 8 is a photograph showing a pure prodigiosin obtained in example 1;
FIG. 9 is a graph showing the results of Mass Spectrometry (MS) of the pure prodigiosin obtained in example 1;
FIG. 10 is a graph showing the results of Ultraviolet (UV) spectroscopy on pure prodigiosin obtained in example 1;
FIG. 11 is a graph showing the results of infrared spectroscopy (IR) detection of a pure prodigiosin obtained in example 1;
FIG. 12 is a graph showing the results of nuclear magnetic resonance hydrogen (NMR) measurement of a pure prodigiosin obtained in example 1;
FIG. 13 is a High Performance Liquid Chromatography (HPLC) result chart of the pure prodigiosin obtained in example 1;
FIG. 14 is a standard curve drawn by measuring OD535 of pure prodigiosin obtained in example 1 by UV spectrophotometry.
Detailed Description
The invention provides a method for extracting prodigiosin from a prodigiosin-containing fermentation broth, which comprises the following steps:
carrying out first solid-liquid separation on fermentation liquor containing prodigiosin to obtain thallus precipitates and fermentation liquor supernatant;
extracting the supernatant of the fermentation liquor to obtain an extract phase;
mixing the extract phase with an antioxidant, and carrying out first reduced pressure distillation on an obtained extract phase system to obtain a first prodigiosin crude product;
mixing the thallus precipitate with an acidic alcohol solvent, and sequentially carrying out ultrasonic treatment, leaching and second solid-liquid separation to obtain a thallus supernatant;
mixing the thallus supernatant with an antioxidant, and carrying out second reduced pressure distillation on an obtained thallus supernatant system to obtain a second prodigiosin crude product;
mixing the first prodigiosin crude product and the second prodigiosin crude product, performing double silica gel column cascade chromatography, and performing gradient elution by using an eluent to obtain a prodigiosin eluent;
carrying out reduced pressure concentration on the prodigiosin eluent to obtain a prodigiosin pure product;
the silica gel column of the double silica gel column cascade chromatography comprises an upper silica gel column and a lower silica gel column which are connected in series;
in the process of gradient elution, the eluents are a first eluent, a second eluent, a third eluent, a fourth eluent, a fifth eluent, a sixth eluent and a seventh eluent in sequence;
the first eluent is petroleum ether, ethyl acetate, acetone and chloroform with the volume ratio of 7:2:0.5:0.5 of a mixed solvent;
the second eluent is petroleum ether, ethyl acetate, acetone and chloroform, and the volume ratio of the petroleum ether to the ethyl acetate to the acetone to the chloroform is 6:3:0.5:0.5 of a mixed solvent;
the third eluent is petroleum ether, ethyl acetate, acetone and chloroform, and the volume ratio of the petroleum ether to the ethyl acetate to the acetone to the chloroform is 5:4:0.5:0.5 of a mixed solvent;
the fourth eluent is petroleum ether, ethyl acetate, acetone and chloroform, and the volume ratio of the petroleum ether to the ethyl acetate to the acetone to the chloroform is 4:5:0.5:0.5 of a mixed solvent;
the fifth eluent is petroleum ether, ethyl acetate, acetone and chloroform, and the volume ratio of the petroleum ether to the ethyl acetate to the acetone to the chloroform is 3:6:0.5:0.5 of a mixed solvent;
the sixth eluent is petroleum ether, ethyl acetate, acetone and chloroform, and the volume ratio of the petroleum ether to the ethyl acetate to the acetone to the chloroform is 2:7:0.5:0.5 of a mixed solvent;
the seventh eluent is petroleum ether, ethyl acetate, acetone and chloroform with the volume ratio of 1:8:0.5:0.5 of a mixed solvent.
In the present invention, the starting materials used in the present invention are preferably commercially available products unless otherwise specified.
The invention carries out first solid-liquid separation on fermentation liquor containing prodigiosin to obtain thallus sediment and fermentation liquor supernatant.
In the present invention, the fermentation broth containing prodigiosin is preferably a serratia marcescens fermentation broth. In the present invention, the preparation method of the serratia marcescens fermentation liquid preferably comprises the following steps:
inoculating the plate-activated serratia marcescens into a seed culture medium, and obtaining an inoculated strain when the strain grows to a logarithmic phase;
and inoculating the inoculated strain to an antibiotic-free LB culture medium, and culturing to obtain the serratia marcescens fermentation liquor.
The invention inoculates the flat activated serratia marcescens into the seed culture medium, and obtains the inoculated strain when the strain grows to logarithmic phase. The method for obtaining the plate-activated serratia marcescens is not particularly limited, and the method can be performed by the operation known by those skilled in the art. In the present invention, the seed culture medium preferably includes: tryptone 10g/L, yeast powder 5g/L, naCl 10g/L and natural pH.
After the inoculated strain is obtained, the inoculated strain is inoculated in an LB culture medium without an antibody for culture, and the serratia marcescens fermentation liquor is obtained. In the invention, the volume percentage content of the inoculated strain in the LB culture medium without the antibody is 1-2%.
In the present invention, the non-resistant LB medium is preferably a slant/plate LB medium or a dual-carbon-source fermentation medium, and more preferably a dual-carbon-source fermentation medium. In the present invention, the slant/plate LB medium preferably comprises: 10g/L tryptone, 5g/L, naCl g/L yeast powder, 20g/L agar powder and natural pH. In the present invention, the dual-carbon source fermentation medium preferably includes: 5g/L of peanut powder, 20g/L of soybean peptone, 20g/L of glycerol and 10g/L, KH of sucrose 2 PO 4 3.5g/L、Na 2 HPO 4 ·2H 2 O 15g/L、MgSO 4 ·2H 2 O 0.5g/L、ZnSO 4 ·7H 2 O1 g/L, pH is natural.
In the invention, the temperature of the culture is preferably 25-27 ℃, and the time is preferably 24-48 h; the culture is preferably carried out under the condition of shaking, and the speed of shaking is preferably 150-200 rpm.
The preparation method of the serratia marcescens fermentation liquid provided by the invention is suitable for large production systems, namely 10L fermentation can be expanded.
In the present invention, the first solid-liquid separation method preferably includes centrifugation or filtration, and more preferably centrifugation. In the invention, the rotating speed of the centrifugation is preferably 8000-12000 r/min, and the time is preferably 10-40 min.
After the supernatant of the fermentation liquor is obtained, the supernatant of the fermentation liquor is extracted to obtain an extract phase. In the present invention, the extractant for the extraction preferably comprises ethyl acetate. In the present invention, the time for the extraction is preferably 36 to 60 hours.
After an extract phase is obtained, the extract phase is mixed with an antioxidant, and the obtained extract phase system is subjected to first reduced pressure distillation to obtain a first prodigiosin crude product. In the present invention, the antioxidant preferably includes vitamin C and/or tetrahydrofuran, and more preferably vitamin C. In the present invention, the concentration of the antioxidant in the extract phase system is preferably 0.01 to 0.05g/L, and more preferably 0.02 to 0.04g/L. In the present invention, the temperature of the first reduced pressure distillation is preferably 40 to 45 ℃; the first reduced pressure distillation is preferably carried out under exclusion of light.
After the thallus precipitate is obtained, the thallus precipitate and the acidic alcohol solvent are mixed, and ultrasonic treatment, leaching and second solid-liquid separation are sequentially carried out to obtain a thallus supernatant. In the present invention, the pH of the acidic alcohol solvent is preferably 3. In the present invention, the acidic alcohol solvent preferably comprises methanol and a mineral acid; the inorganic acid preferably includes hydrochloric acid, sulfuric acid and nitric acid, and further preferably hydrochloric acid. In the present invention, the concentration of the inorganic acid is preferably 1mol/L. The volume ratio of the inorganic acid to the methanol is not particularly limited in the present invention, as long as the pH of the acidic alcohol solvent can be adjusted to 3.
In the present invention, the power of the ultrasound is preferably 30% of the rated power; the time of the ultrasound is preferably 10min.
In the present invention, the leaching is preferably a standing leaching; the leaching time is preferably 1 day.
After the thallus supernatant is obtained, the thallus supernatant is mixed with an antioxidant, and the obtained thallus supernatant system is subjected to second reduced pressure distillation to obtain a second prodigiosin crude product. In the present invention, the antioxidant preferably includes vitamin C and/or tetrahydrofuran, and more preferably vitamin C. In the present invention, the concentration of the antioxidant in the cell supernatant system is preferably 0.01 to 0.05g/L, and more preferably 0.02 to 0.04g/L. In the present invention, the temperature of the second reduced pressure distillation is preferably 30 to 35 ℃, and the second reduced pressure distillation is preferably performed under a condition of being protected from light.
After a first prodigiosin crude product and a second prodigiosin crude product are obtained, the first prodigiosin crude product and the second prodigiosin crude product are mixed and then subjected to double silica gel column cascade chromatography, and an eluent is utilized for gradient elution to obtain a prodigiosin eluent.
In the present invention, the silica gel column of the double silica gel column cascade chromatography comprises an upper silica gel column and a lower silica gel column connected in series. In the present invention, the volume ratio of the upper silica gel column and the lower silica gel column is preferably 1: 1.5-1: 2. in the present invention, the ratio of the silica gel packing volume in the upper silica gel column to the silica gel packing volume in the lower silica gel column is preferably 1:3 to 1:4. in the specific embodiment of the present invention, the size of the silica gel column on the column is preferably 60mm × 200mm or 80mm × 200mm, and more preferably 60mm × 200mm; the silica gel filling height is preferably 80mm or 100mm, and more preferably 80mm; the specification of the lower silica gel column is preferably 60mm × 300mm or 80mm × 300mm, and more preferably 60mm × 300mm; the silica gel packing height is preferably 250mm or 147.5mm, and more preferably 250mm. In the present invention, the upper silica gel column and the lower silica gel column are preferably connected through # 24/29.
In the invention, after the first prodigiosin crude product and the second prodigiosin crude product are mixed, the sample loading mode is preferably wet-process sample loading; the wet loading preferably comprises the steps of: and mixing and dissolving the first prodigiosin crude product and the second prodigiosin crude product, and loading the obtained solution dissolved with the prodigiosin crude product into an upper silica gel column and a lower silica gel column which are connected in series. In the present invention, the dissolved reagent preferably comprises methanol.
Before the gradient elution with the eluent, the method preferably further comprises the step of carrying out equilibrium; the agent for equilibration preferably comprises petroleum ether. The operation of the balancing is not particularly limited in the present invention, and may be performed by a balancing operation known to those skilled in the art.
In the present invention, in the process of gradient elution, the eluents are a first eluent, a second eluent, a third eluent, a fourth eluent, a fifth eluent, a sixth eluent and a seventh eluent in sequence.
In the present invention, the first eluent is petroleum ether, ethyl acetate, acetone and chloroform at a volume ratio of 7:2:0.5:0.5 of a mixed solvent.
In the invention, the second eluent is petroleum ether, ethyl acetate, acetone and chloroform, and the volume ratio of the petroleum ether to the ethyl acetate to the acetone to the chloroform is 6:3:0.5:0.5 of a mixed solvent.
In the present invention, the third eluent is petroleum ether, ethyl acetate, acetone and chloroform in a volume ratio of 5:4:0.5:0.5 of a mixed solvent.
In the invention, the fourth eluent is petroleum ether, ethyl acetate, acetone and chloroform, and the volume ratio of the petroleum ether to the ethyl acetate to the acetone to the chloroform is 4:5:0.5:0.5 of mixed solvent.
In the invention, the fifth eluent is petroleum ether, ethyl acetate, acetone and chloroform, and the volume ratio of the petroleum ether to the ethyl acetate to the acetone to the chloroform is 3:6:0.5:0.5 of a mixed solvent.
In the invention, the sixth eluent is petroleum ether, ethyl acetate, acetone and chloroform, and the volume ratio of the petroleum ether to the ethyl acetate to the acetone to the chloroform is 2:7:0.5:0.5 of a mixed solvent.
In the invention, the seventh eluent is petroleum ether, ethyl acetate, acetone and chloroform with the volume ratio of 1:8:0.5:0.5 of a mixed solvent.
In the present invention, the first eluent, the second eluent, the third eluent, the fourth eluent, the fifth eluent, the sixth eluent and the seventh eluent are all preferably used in an amount of 1 time of the volume of the silica gel column.
After obtaining the prodigiosin eluent, the invention carries out reduced pressure concentration on the prodigiosin eluent to obtain a prodigiosin pure product. The parameters of the reduced pressure concentration are not particularly limited, and the solvent in the prodigiosin eluent can be removed completely. After the concentration under reduced pressure, the present invention preferably further comprises drying.
After obtaining the pure prodigiosin product, the invention preferably also comprises the step of detecting the pure prodigiosin product; the detection method preferably comprises nuclear magnetic resonance detection (NMR), ultraviolet absorption spectroscopy detection (UV), infrared absorption spectroscopy detection (IR), high performance liquid chromatography detection (HPLC), mass spectrometry detection (MS) and ultraviolet spectroscopy detection.
The method for extracting prodigiosin from a fermentation broth containing prodigiosin according to the present invention will be described in detail with reference to the following examples, but they should not be construed as limiting the scope of the present invention.
Example 1
The preparation method of the serratia marcescens fermentation liquor comprises the following steps:
streaking on an LB (Langmuir-Blodgett) plate, culturing in an incubator at 30 ℃ for 24h, and selecting a single colony which is smooth and uniform and plump as plate-activated serratia marcescens; placing the plate-activated serratia marcescens into a test tube filled with 10mL of seed culture medium (the seed culture medium comprises 10g/L of tryptone, 5g/L, naCl g/L of yeast powder and natural pH), and culturing at 30 ℃ until the post-logarithmic growth period to obtain an inoculated strain; inoculating the inoculated strain to 10L double carbon source fermentation medium (the double carbon source fermentation medium comprises peanut powder 5g/L, soybean peptone 20g/L, glycerol 20g/L, sucrose 10g/L, KH) 2 PO 4 3.5g/L、Na 2 HPO 4 ·2H 2 O 15g/L、MgSO 4 ·2H 2 O 0.5g/L、ZnSO 4 ·7H 2 O1 g/L, pH natural), fermenting for 48h at 30 ℃, controlling the pH to be 7.0 by ammonia water, coupling the dissolved oxygen and the rotating speed to be constant by 20 percent, and performing solid-liquid separation for pretreatment before separation and purification. And after the carbon source in the initial culture medium is exhausted, the dissolved oxygen is suddenly increased, feeding is started at the moment, the feeding rate is adjusted by a DO-STAT method to maintain the dissolved oxygen in a proper range, and the fermentation is finished after 48h of culture, so that the serratia marcescens fermentation liquid is obtained.
FIG. 1 is a photograph showing the result of fermentation of Serratia marcescens.
The establishment experiment of the method for separating and extracting prodigiosin from the serratia marcescens fermentation liquor is repeated three times to obtain an average value, and the repeated steps are as follows:
1. taking the serratia marcescens fermentation liquor, centrifuging for 10min at 8000r/min, and respectively collecting thallus precipitate and fermentation liquor supernatant.
2. Adding 500g of thallus precipitate and 4.5L of acidic methanol (pH value is 3, inorganic acid in the acidic methanol is 1mol/L hydrochloric acid) into a 5L beaker, uniformly mixing, carrying out auxiliary crushing for 10min by an ultrasonic crusher (power is 30% of rated power), soaking for 1 day, and collecting thallus supernatant; adding 0.05g/L vitamin C into the supernatant, and rotary steaming at 35 deg.C under reduced pressure in the dark to obtain prodigiosin crude product.
3. And (2) adding 2L of the supernatant of the fermentation liquor obtained in the step (1) and 3L of ethyl acetate into a 5L separating funnel, mixing uniformly, performing liquid separation extraction for 48 hours, separating the extract by using the separating funnel, draining the extract with almost no color on the lower layer after the extraction is finished, reserving the ethyl acetate phase on the upper layer, adding 0.05g/L of vitamin C into the ethyl acetate phase, and performing reduced pressure rotary evaporation at 45 ℃ under the condition of keeping out of the sun to obtain a prodigiosin crude product.
4. Mixing the prodigiosin crude products obtained in the step 2 and the step 3, and carrying out thin-layer chromatography to determine an optimal eluent; the instrument and the thin layer chromatography process are as follows:
the silica gel plate for thin layer chromatography is a silica gel plate with the specification of 200mm × 10mm produced by Qingdao ocean chemical plants, the proportion of five developing agents is tried, the developing agent 1 (the volume ratio of petroleum ether: ethyl acetate: acetone: chloroform is 7; as can be seen from fig. 2: the separation effect of the developing solvent 2 is the best, and the sample spot aggregation is not tailing, so that the developing solvent 2 is selected as the eluent.
5. Separating and purifying by double silica gel column cascade chromatography, wherein the specification of an upper silica gel column is 60mm multiplied by 200mm, the filling height of silica gel is 80mm, the specification of a lower silica gel column is 60mm multiplied by 300mm, and the filling height of silica gel is 250mm; the upper and lower silica gel columns were connected via a # 24/29 port.
6. And (3) wet column packing, soaking the activated silica gel in petroleum ether, uniformly stirring, placing the silica gel column on a chromatographic column by using a glass funnel with a larger caliber, injecting petroleum ether with the height of 1/2 onto the silica gel column, opening a piston of a lower silica gel column, and then pouring the silica gel into the funnel uniformly as much as possible, so that a fault can be prevented from being formed in the chromatographic column.
7. And (3) mixing the prodigiosin crude products obtained in the steps (2) and (3), dissolving the prodigiosin crude products in 50mL of methanol, and loading the methanol solution in which the prodigiosin crude products are dissolved onto a silica gel column. After the column equilibration was completed, 1 time of the column volume of the first eluent (a mixed solvent of petroleum ether, ethyl acetate, acetone and chloroform in a volume ratio of 7: 0.5; by analogy, a third eluent (a mixed solvent of petroleum ether, ethyl acetate, acetone and chloroform at a volume ratio of 5.
As can be seen from fig. 4: the color difference of the eluent is large, the eluent turns red gradually from the initial white eluent, and then turns pink eluent, and the final effluent liquid of the eluent turns yellow gradually.
8. Performing LC-MS detection on the white eluate, the red eluate, the yellow eluate and the pink eluate, wherein the results are shown in FIGS. 5-7, and FIG. 5 is the LC-MS detection result of the red eluate obtained in example 1; FIG. 6 shows the results of LC-MS detection of pink eluate obtained in example 1; FIG. 7 shows the results of LC-MS detection of the yellow eluate obtained in example 1. As can be seen from fig. 5 to 7: the red eluate is an eluate containing prodigiosin, and has only a single peak, and the molecular weight is 324 corresponding to the molecular weight of prodigiosin.
9. Adding 0.025g/L vitamin C into the red eluate, performing reduced pressure rotary evaporation under the condition of keeping out of the sun, and vacuum drying the rotary evaporation product to obtain 103.5g pure prodigiosin, wherein the yield is 98.6%, and FIG. 8 is a photograph of the obtained pure prodigiosin.
10. The Mass Spectrum (MS) of the prepared pure prodigiosin product is detected, and the result is shown in figure 9, and the following results can be seen from figure 9: the peak result is consistent with the peak result of each group of prodigiosin predicted by chemdraw software.
11. The ultraviolet absorption spectrum (UV) detection result of the prepared pure prodigiosin product is shown in fig. 10, and it can be seen from fig. 10 that: because the compound is dissolved in acetonitrile solution, under the condition of slight alkali, an obvious absorption peak exists at a position of 520-530 nm, and a certain blue shift phenomenon is reported in the literature.
12. The infrared absorption spectrum (IR) detection of the prepared pure prodigiosin product is shown in fig. 11, and the results are shown in fig. 11: there was a clear peak of pyrrole groups.
13. The Nuclear Magnetic Resonance (NMR) detection of the prepared pure prodigiosin product showed that the results are shown in fig. 12, and it can be seen from fig. 12 that: there was a clear peak of pyrrole groups.
14. The prepared pure prodigiosin product was subjected to High Performance Liquid Chromatography (HPLC) and carried out by using 100nm to 900nm full-wavelength high performance liquid chromatography, and the results are shown in fig. 13, as can be seen from fig. 13: the peak is single, and no obvious impurity peak exists.
15. The prepared pure prodigiosin product is compared with a prodigiosin standard product (purity is more than 99%) purchased from an MCS official website by using an ultraviolet spectroscopy method, and the results are shown in Table 1.
TABLE 1 Standard Curve profiles
As can be seen from table 1: the pure prodigiosin product obtained by the method is basically consistent with the MCS official website, and the purity is more than 99 percent; the concentration of the standard substance prepared by the invention can reach the purity of the standard substance sold by MCS.
Fig. 14 is a standard curve drawn by detecting OD535 of pure prodigiosin by ultraviolet spectrophotometry, and it can be seen from fig. 14 that: the linear correlation is better, the marking curve y =169.23x +0.0347 can be drawn, and R 2 ＝0.9992。
Example 2
The preparation method of the Serratia marcescens fermentation liquid is the same as that of example 1.
The difference from the example 1 is that the specification of the silica gel column on the silica gel used in the example is 80mm multiplied by 200mm, and the height of the silica gel is 100mm; the specification of the lower silica gel column is 80mm multiplied by 300mm, and the height is 147.5mm; the upper and lower silica gel columns were connected via a # 24/29 port. It should be noted that the volumes of the filled silica gel in the two embodiments are the same.
1. Taking the serratia marcescens fermentation liquor, centrifuging for 10min at 8000r/min, and respectively collecting thallus precipitate and fermentation liquor supernatant.
2. Adding 500g of thallus precipitate and 4.5L of acidic methanol (pH value is 3, inorganic acid in the acidic methanol is 1mol/L hydrochloric acid) into a 5L beaker, uniformly mixing, carrying out auxiliary crushing for 10min by an ultrasonic crusher (power is 30% of rated power), soaking for 1 day, and collecting thallus supernatant; adding 0.05g/L vitamin C into the supernatant, and rotary steaming at 35 deg.C under reduced pressure in the dark to obtain prodigiosin crude product.
3. And (2) adding 2L of the supernatant of the fermentation liquor obtained in the step (1) and 3L of ethyl acetate into a 5L separating funnel, mixing uniformly, performing liquid separation extraction for 48 hours, separating the extract by using the separating funnel, draining the extract with almost no color on the lower layer after the extraction is finished, reserving the ethyl acetate phase on the upper layer, adding 0.05g/L of vitamin C into the ethyl acetate phase, and performing reduced pressure rotary evaporation at 45 ℃ under the condition of keeping out of the sun to obtain a prodigiosin crude product.
4. Mixing the prodigiosin crude products obtained in the step 2 and the step 3, and carrying out thin-layer chromatography to determine an optimal eluent; the instrument and the thin layer chromatography process are as follows:
the silica gel plate for thin layer chromatography is a silica gel plate with the specification of 200mm × 10mm produced by Qingdao ocean chemical plants, the proportion of five developing agents is tried, the developing agent 1 (the volume ratio of petroleum ether: ethyl acetate: acetone: chloroform is 7; as can be seen from fig. 2: the separation effect of the developer 2 is the best, and the sample spot is gathered without tailing, so the developer 2 is selected as the eluent.
5. Separating and purifying by double silica gel column cascade chromatography, wherein the specification of the upper silica gel column is 80mm multiplied by 200mm, the height of the silica gel is 100mm, and the specification of the lower silica gel column is 80mm multiplied by 300mm, and the height is 147.5mm; the upper and lower silica gel columns were connected via a # 24/29 port.
6. And (3) wet column packing, soaking the activated silica gel in petroleum ether, uniformly stirring, placing the silica gel column on a chromatographic column by using a glass funnel with a larger caliber, injecting petroleum ether with the height of 1/2 onto the silica gel column, opening a piston of a lower silica gel column, and then pouring the silica gel into the funnel uniformly as much as possible, so that a fault can be prevented from being formed in the chromatographic column.
7. And (3) mixing the prodigiosin crude products obtained in the steps 2 and 3, dissolving the mixture in 50mL of methanol, and loading the methanol solution dissolved with the prodigiosin crude products to a silica gel column. After the column equilibration was completed, 1-fold column volume of the first eluent (a mixed solvent of petroleum ether, ethyl acetate, acetone and chloroform at a volume ratio of 7; by analogy, a third eluent (a mixed solvent of petroleum ether, ethyl acetate, acetone and chloroform at a volume ratio of 5.
8. Adding 0.025g/L vitamin C into the red eluate, performing rotary evaporation under reduced pressure in the dark, and vacuum drying the rotary evaporation product to obtain 91.2g pure prodigiosin with a yield of 86.8%.
9. The prepared pure prodigiosin product has a purity of 91.5% compared with a standard product by using an ultraviolet spectroscopy method.
Comparative example 1
The differences from example 1 are: the double silica gel column in the double silica gel column cascade chromatography in example 1 was replaced with an upper silica gel column having a size of 60mm × 200mm and a height of 80 mm.
The results were: the purity of the obtained pure prodigiosin product is 98%, and the yield is 65%.
Comparative example 2
The differences from example 1 are: the double silica gel column in the double silica gel column cascade chromatography in example 1 was replaced with a lower silica gel column of 60mm × 300mm specification and 250mm height.
The results were: the purity of the obtained pure prodigiosin product is 98%, and the yield is 80%.
Comparative example 3
The differences from example 1 are: in the process of reduced pressure distillation, antioxidant vitamin C is not added
The results were: the purity of the obtained pure prodigiosin product is 86%, and the yield is 54%.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A method for extracting prodigiosin from a fermentation broth containing prodigiosin is characterized by comprising the following steps:
carrying out first solid-liquid separation on fermentation liquor containing prodigiosin to obtain thallus precipitates and fermentation liquor supernatant;
extracting the supernatant of the fermentation liquor to obtain an extract phase;
mixing the extract phase with an antioxidant, and carrying out first reduced pressure distillation on the obtained extract phase system to obtain a first prodigiosin crude product;
mixing the thallus precipitate with an acidic alcohol solvent, and sequentially performing ultrasonic treatment, leaching and second solid-liquid separation to obtain a thallus supernatant;
mixing the thallus supernatant with an antioxidant, and carrying out second reduced pressure distillation on an obtained thallus supernatant system to obtain a second prodigiosin crude product;
mixing the first prodigiosin crude product and the second prodigiosin crude product, performing double silica gel column cascade chromatography, and performing gradient elution by using an eluent to obtain a prodigiosin eluent;
carrying out reduced pressure concentration on the prodigiosin eluent to obtain a prodigiosin pure product;
the silica gel column of the double silica gel column cascade chromatography comprises an upper silica gel column and a lower silica gel column which are connected in series;
in the process of gradient elution, the eluents are a first eluent, a second eluent, a third eluent, a fourth eluent, a fifth eluent, a sixth eluent and a seventh eluent in sequence;
the first eluent is petroleum ether, ethyl acetate, acetone and chloroform, and the volume ratio of the petroleum ether to the ethyl acetate to the acetone to the chloroform is 7:2:0.5:0.5 of a mixed solvent;
the second eluent is petroleum ether, ethyl acetate, acetone and chloroform, and the volume ratio of the petroleum ether to the ethyl acetate to the acetone to the chloroform is 6:3:0.5:0.5 of a mixed solvent;
the third eluent is petroleum ether, ethyl acetate, acetone and chloroform with the volume ratio of 5:4:0.5:0.5 of a mixed solvent;
the fourth eluent is petroleum ether, ethyl acetate, acetone and chloroform, and the volume ratio of the petroleum ether to the ethyl acetate to the acetone to the chloroform is 4:5:0.5:0.5 of a mixed solvent;
the fifth eluent is petroleum ether, ethyl acetate, acetone and chloroform, and the volume ratio of the petroleum ether to the ethyl acetate to the acetone to the chloroform is 3:6:0.5:0.5 of a mixed solvent;
the sixth eluent is petroleum ether, ethyl acetate, acetone and chloroform, and the volume ratio of the petroleum ether to the ethyl acetate to the acetone to the chloroform is 2:7:0.5:0.5 of a mixed solvent;
the seventh eluent is petroleum ether, ethyl acetate, acetone and chloroform with the volume ratio of 1:8:0.5:0.5 of mixed solvent.
2. The process as claimed in claim 1, wherein the volume ratio of the upper silica gel column to the lower silica gel column is 1: 1.5-1: 2; the ratio of the silica gel filling volume in the upper silica gel column to the silica gel filling volume in the lower silica gel column is 1:3 to 1:4.
3. a method as claimed in claim 1, wherein the first, second, third, fourth, fifth, sixth and seventh eluents are all used in 1 x the volume of the silica gel column on top.
4. The method according to claim 1, wherein the prodigiosin-containing fermentation broth is a serratia marcescens fermentation broth; the preparation method of the serratia marcescens fermentation liquid comprises the following steps:
inoculating the plate-activated serratia marcescens into a seed culture medium, and obtaining an inoculated strain when the strain grows to a logarithmic phase;
inoculating the inoculated strain to an LB culture medium without an antibody, and culturing to obtain the serratia marcescens fermentation liquor;
the volume percentage content of the inoculated strain in the nonreactive LB culture medium is 1-2%.
5. The method according to claim 4, wherein the temperature of the culture is 25-27 ℃ and the time is 24-48 h; the culture is carried out under the condition of shaking, and the speed of shaking is 150-200 rpm.
6. The method of claim 1, wherein the extracted extractant comprises ethyl acetate.
7. The method of claim 1, wherein the antioxidant comprises vitamin C and/or tetrahydrofuran.
8. The method of claim 1 or 7, wherein the concentration of the antioxidant in the extract phase system and the cell supernatant system is independently 0.01 to 0.05g/L.
9. The process according to claim 1, characterized in that the acidic alcoholic solvent has a pH value of 3; the acidic alcohol solvent comprises methanol and inorganic acid; the inorganic acid includes hydrochloric acid, sulfuric acid and nitric acid.
10. The method according to claim 1, wherein the temperature of the first reduced pressure distillation is 40 to 45 ℃ and the temperature of the second reduced pressure distillation is 30 to 35 ℃; the first reduced pressure distillation and the second reduced pressure distillation are carried out under the condition of avoiding light.
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