CN115745863B - 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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- CN115745863B CN115745863B CN202211431354.2A CN202211431354A CN115745863B CN 115745863 B CN115745863 B CN 115745863B CN 202211431354 A CN202211431354 A CN 202211431354A CN 115745863 B CN115745863 B CN 115745863B
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- prodigiosin
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- ethyl acetate
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- 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 137
- 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 136
- 238000000855 fermentation Methods 0.000 title claims abstract description 69
- 230000004151 fermentation Effects 0.000 title claims abstract description 69
- 238000000034 method Methods 0.000 title claims abstract description 40
- 239000000741 silica gel Substances 0.000 claims abstract description 106
- 229910002027 silica gel Inorganic materials 0.000 claims abstract description 106
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 104
- 239000003963 antioxidant agent Substances 0.000 claims abstract description 21
- 230000003078 antioxidant effect Effects 0.000 claims abstract description 21
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 19
- 238000000926 separation method Methods 0.000 claims abstract description 19
- 238000000605 extraction Methods 0.000 claims abstract description 17
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 204
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 170
- 239000003480 eluent Substances 0.000 claims description 138
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 132
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 118
- 229960001866 silicon dioxide Drugs 0.000 claims description 105
- 239000003208 petroleum Substances 0.000 claims description 66
- 239000006228 supernatant Substances 0.000 claims description 40
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical group OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 39
- 239000012046 mixed solvent Substances 0.000 claims description 38
- 241000607715 Serratia marcescens Species 0.000 claims description 31
- 239000000047 product Substances 0.000 claims description 30
- 239000003795 chemical substances by application Substances 0.000 claims description 28
- 239000007788 liquid Substances 0.000 claims description 27
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 26
- 230000001580 bacterial effect Effects 0.000 claims description 26
- 238000002156 mixing Methods 0.000 claims description 25
- 238000004821 distillation Methods 0.000 claims description 23
- 235000006708 antioxidants Nutrition 0.000 claims description 20
- 239000001963 growth medium Substances 0.000 claims description 19
- 230000002378 acidificating effect Effects 0.000 claims description 16
- 239000012043 crude product Substances 0.000 claims description 16
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 claims description 13
- 229930003268 Vitamin C Natural products 0.000 claims description 13
- 239000002904 solvent Substances 0.000 claims description 13
- 235000019154 vitamin C Nutrition 0.000 claims description 13
- 239000011718 vitamin C Substances 0.000 claims description 13
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 12
- 238000010828 elution Methods 0.000 claims description 12
- 239000000284 extract Substances 0.000 claims description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 11
- 238000011049 filling Methods 0.000 claims description 10
- 150000007522 mineralic acids Chemical class 0.000 claims description 10
- 239000002244 precipitate Substances 0.000 claims description 10
- 230000008569 process Effects 0.000 claims description 9
- 238000002386 leaching Methods 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 7
- 238000011218 seed culture Methods 0.000 claims description 7
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 6
- 238000012258 culturing Methods 0.000 claims description 6
- 230000003698 anagen phase Effects 0.000 claims description 5
- 238000009210 therapy by ultrasound Methods 0.000 claims description 5
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 claims description 3
- 229910017604 nitric acid Inorganic materials 0.000 claims description 3
- 230000001476 alcoholic effect Effects 0.000 claims 1
- 238000009776 industrial production Methods 0.000 abstract description 2
- 238000001514 detection method Methods 0.000 description 16
- 238000004809 thin layer chromatography Methods 0.000 description 9
- 239000002609 medium Substances 0.000 description 7
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 238000005481 NMR spectroscopy Methods 0.000 description 6
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 6
- 229910052799 carbon Inorganic materials 0.000 description 6
- 238000011068 loading method Methods 0.000 description 6
- 239000000843 powder Substances 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 5
- 238000004128 high performance liquid chromatography Methods 0.000 description 5
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 4
- 238000000862 absorption spectrum Methods 0.000 description 4
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 4
- 238000004949 mass spectrometry Methods 0.000 description 4
- 239000012264 purified product Substances 0.000 description 4
- 238000002390 rotary evaporation Methods 0.000 description 4
- 239000013049 sediment Substances 0.000 description 4
- 238000010025 steaming Methods 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- DSHIIBUGOWQFSO-LICLKQGHSA-N Cycloprodigiosin Natural products COC1=CC(=N\C1=C\c1[nH]c(C)c2CCCC(C)c12)c1ccc[nH]1 DSHIIBUGOWQFSO-LICLKQGHSA-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
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- DSHIIBUGOWQFSO-YVLHZVERSA-N cycloprodigiosin Chemical compound N=1\C(=C/C2=C3C(C)CCCC3=C(C)N2)C(OC)=CC=1C1=CC=CN1 DSHIIBUGOWQFSO-YVLHZVERSA-N 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 229910052760 oxygen Inorganic materials 0.000 description 3
- 239000001301 oxygen Substances 0.000 description 3
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 3
- 239000012137 tryptone Substances 0.000 description 3
- 235000017060 Arachis glabrata Nutrition 0.000 description 2
- 244000105624 Arachis hypogaea Species 0.000 description 2
- 235000010777 Arachis hypogaea Nutrition 0.000 description 2
- 235000018262 Arachis monticola Nutrition 0.000 description 2
- 244000068988 Glycine max Species 0.000 description 2
- 235000010469 Glycine max Nutrition 0.000 description 2
- 239000007836 KH2PO4 Substances 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- HIYSWASSDOXZLC-UHFFFAOYSA-N Undecylprodigiosin Natural products CCCCCCCCCCCc1ccc(C=C2N=C(C=C2OC)c2ccc[nH]2)[nH]1 HIYSWASSDOXZLC-UHFFFAOYSA-N 0.000 description 2
- 238000004220 aggregation Methods 0.000 description 2
- 230000002776 aggregation Effects 0.000 description 2
- 239000003086 colorant Substances 0.000 description 2
- 230000006837 decompression Effects 0.000 description 2
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 2
- 229910000397 disodium phosphate Inorganic materials 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- 238000012856 packing Methods 0.000 description 2
- 235000020232 peanut Nutrition 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 2
- 125000000168 pyrrolyl group Chemical group 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 238000002791 soaking Methods 0.000 description 2
- 238000002798 spectrophotometry method Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 238000002604 ultrasonography Methods 0.000 description 2
- HIYSWASSDOXZLC-HKOYGPOVSA-N undecylprodigiosin Chemical compound N1C(CCCCCCCCCCC)=CC=C1\C=C\1C(OC)=CC(C=2NC=CC=2)=N/1 HIYSWASSDOXZLC-HKOYGPOVSA-N 0.000 description 2
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 2
- 229910000368 zinc sulfate Inorganic materials 0.000 description 2
- 239000011686 zinc sulphate Substances 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- 230000005778 DNA damage Effects 0.000 description 1
- 231100000277 DNA damage Toxicity 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- TXFXBRICRNFFRR-UHFFFAOYSA-N Streptorubin B Natural products CCCCC1CCCCCCc2cc1c(C=C1N=C(C=C1OC)c1ccc[nH]1)[nH]2 TXFXBRICRNFFRR-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 125000000217 alkyl group Chemical group 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 230000003385 bacteriostatic effect Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- TXFXBRICRNFFRR-JLPGSUDCSA-N chembl1812867 Chemical compound CCCCC1CCCCCCC(N2)=CC1=C2\C=C(C(=C1)OC)/N=C1C1=CC=CN1 TXFXBRICRNFFRR-JLPGSUDCSA-N 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 150000001923 cyclic compounds Chemical group 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 125000004435 hydrogen atom Chemical class [H]* 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- XEPVVPTUOMXJRW-SFHVURJKSA-N metacycloprodiginine Natural products CC[C@H]1CCCCCCCCc2cc1c(C=C3N=C(C=C3OC)c4ccc[nH]4)[nH]2 XEPVVPTUOMXJRW-SFHVURJKSA-N 0.000 description 1
- XEPVVPTUOMXJRW-JLPGSUDCSA-N metacycloprodigiosin Chemical compound CCC1CCCCCCCCC(N2)=CC1=C2\C=C(C(=C1)OC)/N=C1C1=CC=CN1 XEPVVPTUOMXJRW-JLPGSUDCSA-N 0.000 description 1
- XEPVVPTUOMXJRW-UHFFFAOYSA-N metacycloprodigiosin Natural products CCC1CCCCCCCCc2cc1c(C=C3/N=C(C=C3OC)c4ccc[nH]4)[nH]2 XEPVVPTUOMXJRW-UHFFFAOYSA-N 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000001054 red pigment Substances 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 239000004753 textile Substances 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
Classifications
-
- 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
Landscapes
- 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 fermentation liquor containing prodigiosin. According to the method provided by the invention, the prodigiosin is separated and purified by utilizing double silica gel column cascade chromatography, and simultaneously, an antioxidant is added in an auxiliary way, so that a prodigiosin pure product with high purity is obtained, and the prodigiosin loss rate in the separation process is greatly reduced. The data of the examples show that: the purity of the prodigiosin pure product separated and extracted from the fermentation liquor by adopting the method provided by the invention is more than or equal to 91.5%, even more than 99%; the yield is more than or equal to 86.8 percent, even 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 fermentation liquor containing prodigiosin.
Background
Prodigiosin (prodigiosin) is a type of microbial pigment that is a secondary metabolite produced by a variety of bacteria. Currently, there are two main types of prodigiosins reported in the literature: one is a compound containing a linear alkyl side chain, such as prodigiosin (prodigiosin) and undecyl prodigiosin (undecylprodigiosin) and the other is a cyclic compound, such as cycloprodigiosin (cycloprodigiosin), cycloprodigiosin (metacycloprodigiosin), prodigiosin R1 (Prodigiosin R1) and streptavidin B (streptorubin B).
Prodigiosin is used as a common red pigment and is widely used for textile printing and dyeing. In addition, prodigiosin also has a very remarkable effect in inducing apoptosis of colorectal cancer cells, and has potential as a novel anticancer drug for tumor treatment. Since prodigiosin has pH response color-changing characteristics, in the field of material science, recent reports prove that the pH response color-changing performance and the biodegradable color-changing film have wide application prospects in the aspects of marking, packaging, base materials and the like; recent reports indicate that prodigiosin has natural bacteriostatic properties, has a significant inhibitory effect on escherichia coli above a Minimum Inhibitory Concentration (MIC), and no significant DNA damage and cytoplasmic membrane disintegration are observed.
At present, the prodigiosin is obtained by separating and purifying the fermentation broth, but the purity of the prodigiosin obtained by separating and purifying is low.
Disclosure of Invention
In view of the above, an object of the present invention is 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 fermentation liquor containing prodigiosin, which comprises the following steps:
Performing first solid-liquid separation on the fermentation liquor containing prodigiosin to obtain bacterial precipitate and fermentation liquor supernatant;
Extracting the supernatant of the fermentation liquor to obtain an extraction phase;
Mixing the extract phase with an antioxidant, and performing first reduced pressure distillation on the obtained extract phase system to obtain a first prodigiosin crude product;
Mixing the bacterial precipitate with an acidic alcohol solvent, and sequentially carrying out ultrasonic treatment, leaching and second solid-liquid separation to obtain bacterial supernatant;
Mixing the thallus supernatant with an antioxidant, and performing second reduced pressure distillation on the obtained thallus supernatant system to obtain a second prodigiosin crude product;
Mixing the first crude prodigiosin and the second crude prodigiosin, performing double-silica-gel column cascade chromatography, and performing gradient elution by using an eluent to obtain 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 gradient elution process, the eluents are sequentially a first eluent, a second eluent, a third eluent, a fourth eluent, a fifth eluent, a sixth eluent and a seventh eluent;
The first eluent is petroleum ether, ethyl acetate, acetone and chloroform, and the volume ratio is 7:2:0.5:0.5 of a mixed solvent;
The second eluent is petroleum ether, ethyl acetate, acetone and chloroform with the volume ratio of 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 with the volume ratio of 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 is 3:6:0.5:0.5 of a mixed solvent;
The sixth eluent is petroleum ether, ethyl acetate, acetone and chloroform with the volume ratio of 2:7:0.5:0.5 of a mixed solvent;
the seventh eluent is petroleum ether, ethyl acetate, acetone and chloroform, and the volume ratio is 1:8:0.5: 0.5.
Preferably, the volume ratio of the upper silica gel column to the lower silica gel column is 1:1.5 to 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: 3-1: 4.
Preferably, the first, second, third, fourth, fifth, sixth and seventh eluents are used in an amount of 1 time the volume of the upper silica gel column.
Preferably, the fermentation broth containing prodigiosin is Serratia marcescens fermentation broth; the preparation method of the Serratia marcescens fermentation liquid comprises the following steps:
inoculating Serratia marcescens activated by a flat plate into a seed culture medium, and obtaining an inoculated strain after the strain grows to a logarithmic growth phase;
Inoculating the inoculated strain to an antibiotic-free LB (LB) culture medium, and culturing to obtain the serratia marcescens fermentation broth;
the volume percentage of the inoculated strain in the LB culture medium without resistance 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 the shaking is 150-200 rpm.
Preferably, the extracted extractant comprises ethyl acetate.
Preferably, the antioxidant comprises vitamin C and/or tetrahydrofuran.
Preferably, the concentration of the antioxidant in the extract phase system and the bacterial supernatant system is independently 0.01-0.05 g/L.
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 performed under light-shielding conditions.
The invention provides a method for extracting prodigiosin from fermentation liquor containing prodigiosin, which comprises the following steps: performing first solid-liquid separation on the fermentation liquor containing prodigiosin to obtain bacterial precipitate and fermentation liquor supernatant; extracting the supernatant of the fermentation liquor to obtain an extraction phase; mixing the extract phase with an antioxidant, and performing first reduced pressure distillation on the obtained extract phase system to obtain a first prodigiosin crude product; mixing the bacterial precipitate with an acidic alcohol solvent, and sequentially carrying out ultrasonic treatment, leaching and second solid-liquid separation to obtain bacterial supernatant; mixing the thallus supernatant with an antioxidant, and performing second reduced pressure distillation on the obtained thallus supernatant system to obtain a second prodigiosin crude product; mixing the first crude prodigiosin and the second crude prodigiosin, performing double-silica-gel column cascade chromatography, and performing gradient elution by using an eluent to obtain 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 gradient elution process, the eluents are sequentially a first eluent, a second eluent, a third eluent, a fourth eluent, a fifth eluent, a sixth eluent and a seventh eluent; the first eluent is petroleum ether, ethyl acetate, acetone and chloroform, and the volume ratio is 7:2:0.5:0.5 of a mixed solvent; the second eluent is petroleum ether, ethyl acetate, acetone and chloroform with the volume ratio of 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 with the volume ratio of 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 is 3:6:0.5:0.5 of a mixed solvent; the sixth eluent is petroleum ether, ethyl acetate, acetone and chloroform with the volume ratio of 2:7:0.5:0.5 of a mixed solvent; the seventh eluent is petroleum ether, ethyl acetate, acetone and chloroform, and the volume ratio is 1:8:0.5: 0.5. According to the method provided by the invention, the prodigiosin is separated and purified by utilizing double silica gel column cascade chromatography, and simultaneously, an antioxidant is added in an auxiliary way, so that the prodigiosin pure product with high purity is obtained, and the loss rate in the separation process is greatly reduced. The data of the examples show that: the purity of the prodigiosin pure product separated and extracted from the fermentation liquor by adopting the method provided by the invention is more than or equal to 91.5%, even more than 99%; the yield is more than or equal to 86.8 percent, even 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 serratia marcescens fermentation liquor; the preparation method of the Serratia marcescens fermentation liquid comprises the following steps: inoculating Serratia marcescens activated by a flat plate into a seed culture medium, and obtaining an inoculated strain after the strain grows to a logarithmic growth phase; inoculating the inoculated strain to an antibiotic-free LB (LB) culture medium, and culturing to obtain the serratia marcescens fermentation broth; the volume percentage of the inoculated strain in the LB culture medium without resistance is 1-2%. The preparation method of Serratia marcescens fermentation liquid is suitable for a large production system, namely, the fermentation can be expanded to 10L.
Drawings
FIG. 1 is a photograph showing the Serratia marcescens fermentation broth obtained in example 1;
FIG. 2 is a chart of silica gel plates obtained by thin layer chromatography of five developing agents in example 1;
FIG. 3 is a flow chart of a double silica gel column cascade chromatography in example 1;
FIG. 4 is a graph of eluents of different colors collected during different time periods in a double silica gel column cascade of example 1;
FIG. 5 shows the results of a liquid chromatography-mass spectrometer (LC-MS) test of the red eluate obtained in example 1;
FIG. 6 shows the results of a liquid chromatography-mass spectrometer (LC-MS) test of the pink eluate obtained in example 1;
FIG. 7 shows the results of a liquid chromatography-mass spectrometer (LC-MS) test of the yellow eluent obtained in example 1;
FIG. 8 is a photograph of the prodigiosin purified product obtained in example 1;
FIG. 9 is a diagram showing the results of Mass Spectrometry (MS) detection of the prodigiosin purified product obtained in example 1;
FIG. 10 is a graph showing the results of Ultraviolet (UV) detection of pure prodigiosin obtained in example 1;
FIG. 11 is a graph showing the results of Infrared (IR) detection of the prodigiosin purified product obtained in example 1;
FIG. 12 is a graph showing the results of nuclear magnetic resonance hydrogen (NMR) analysis of the prodigiosin purified product obtained in example 1;
FIG. 13 is a graph showing the results of High Performance Liquid Chromatography (HPLC) detection of the pure prodigiosin product obtained in example 1;
FIG. 14 is a standard curve drawn by ultraviolet spectrophotometry to detect OD535 of the prodigiosin pure obtained in example 1.
Detailed Description
The invention provides a method for extracting prodigiosin from fermentation liquor containing prodigiosin, which comprises the following steps:
Performing first solid-liquid separation on the fermentation liquor containing prodigiosin to obtain bacterial precipitate and fermentation liquor supernatant;
Extracting the supernatant of the fermentation liquor to obtain an extraction phase;
Mixing the extract phase with an antioxidant, and performing first reduced pressure distillation on the obtained extract phase system to obtain a first prodigiosin crude product;
Mixing the bacterial precipitate with an acidic alcohol solvent, and sequentially carrying out ultrasonic treatment, leaching and second solid-liquid separation to obtain bacterial supernatant;
Mixing the thallus supernatant with an antioxidant, and performing second reduced pressure distillation on the obtained thallus supernatant system to obtain a second prodigiosin crude product;
Mixing the first crude prodigiosin and the second crude prodigiosin, performing double-silica-gel column cascade chromatography, and performing gradient elution by using an eluent to obtain 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 gradient elution process, the eluents are sequentially a first eluent, a second eluent, a third eluent, a fourth eluent, a fifth eluent, a sixth eluent and a seventh eluent;
The first eluent is petroleum ether, ethyl acetate, acetone and chloroform, and the volume ratio is 7:2:0.5:0.5 of a mixed solvent;
The second eluent is petroleum ether, ethyl acetate, acetone and chloroform with the volume ratio of 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 with the volume ratio of 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 is 3:6:0.5:0.5 of a mixed solvent;
The sixth eluent is petroleum ether, ethyl acetate, acetone and chloroform with the volume ratio of 2:7:0.5:0.5 of a mixed solvent;
the seventh eluent is petroleum ether, ethyl acetate, acetone and chloroform, and the volume ratio is 1:8:0.5: 0.5.
In the present invention, the raw 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 bacterial precipitation and fermentation liquor supernatant.
In the present invention, the prodigiosin-containing fermentation broth is preferably Serratia marcescens fermentation broth. In the invention, the preparation method of the Serratia marcescens fermentation liquid preferably comprises the following steps of:
inoculating Serratia marcescens activated by a flat plate into a seed culture medium, and obtaining an inoculated strain after the strain grows to a logarithmic growth phase;
And inoculating the inoculated strain to a non-resistant LB (LB) culture medium, and culturing to obtain the Serratia marcescens fermentation broth.
The invention inoculates Serratia marcescens activated by the flat plate into a seed culture medium, and the inoculated strain is obtained after the strain grows to the logarithmic phase. The method for obtaining Serratia marcescens activated by the plate is not particularly limited, and can be performed by operations well known to those skilled in the art. In the present invention, the seed medium preferably includes: 10g/L tryptone, 5g/L, naCl g/L yeast powder and natural pH.
After the inoculated strain is obtained, the inoculated strain is inoculated in an antibiotic-free LB culture medium for culture, and the serratia marcescens fermentation broth is obtained. In the invention, the volume percentage of the inoculated strain in the LB culture medium without the antibody is 1-2%.
In the present invention, the antibiotic-free LB medium is preferably a slant/plate LB medium or a two-carbon source fermentation medium, and more preferably a two-carbon source fermentation medium. In the present invention, the slant/plate LB medium preferably comprises: 10g/L of tryptone, 5g/L, naCl g/L of yeast powder, 20g/L of agar powder and natural pH. In the present invention, the two-carbon source fermentation medium preferably includes: peanut powder 5g/L, soybean peptone 20g/L, glycerol 20g/L, sucrose 10g/L、KH2PO4 3.5g/L、Na2HPO4·2H2O 15g/L、MgSO4·2H2O 0.5g/L、ZnSO4·7H2O 1g/L、pH g/L.
In the present invention, the temperature of the culture is preferably 25 to 27℃and the time is preferably 24 to 48 hours; the cultivation is preferably carried out under shaking, preferably at a speed of 150 to 200rpm.
The preparation method of Serratia marcescens fermentation liquid is suitable for a large production system, namely, the fermentation can be expanded to 10L.
In the present invention, the first solid-liquid separation means preferably includes centrifugation or filtration, and more preferably centrifugation. In the invention, the rotation 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 invention extracts the supernatant of the fermentation liquor to obtain an extraction phase. In the present invention, the extracting agent for extraction preferably comprises ethyl acetate. In the present invention, the extraction time is preferably 36 to 60 hours.
After an extraction phase is obtained, the extraction phase is mixed with an antioxidant, and the obtained extraction 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, 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 light-protected conditions.
After the bacterial cell sediment is obtained, the bacterial cell sediment and an acidic alcohol solvent are mixed, and ultrasonic treatment, leaching and second solid-liquid separation are sequentially carried out to obtain bacterial cell supernatant. In the present invention, the pH of the acidic alcohol solvent is preferably 3. In the present invention, the acidic alcohol solvent preferably includes methanol and an inorganic acid; the inorganic acid preferably includes hydrochloric acid, sulfuric acid and nitric acid, and more 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 3.
In the present invention, the power of the ultrasound is preferably 30% of the rated power; the time of the ultrasound is preferably 10 minutes.
In the present invention, the leaching is preferably a stationary leaching; the time of the leaching is preferably 1 day.
After obtaining a bacterial supernatant, the invention mixes the bacterial supernatant with an antioxidant, and carries out second reduced pressure distillation on the obtained bacterial supernatant system 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, 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 dark condition.
After the first prodigiosin crude product and the second prodigiosin crude product are obtained, the first prodigiosin crude product and the second prodigiosin crude product are mixed and subjected to double silica gel column cascade chromatography, and gradient elution is carried out by using an eluent to obtain prodigiosin eluent.
In the 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 which are connected in series. In the present invention, the volume ratio of the upper silica gel column to the lower silica gel column is preferably 1:1.5 to 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: 3-1: 4. in a specific embodiment of the present invention, the specification of the upper silica gel column is preferably 60mm×200mm or 80mm×200mm, and more preferably 60mm×200mm; the filling height of the silica gel 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 filling height is preferably 250mm or 147.5mm, more preferably 250mm. In the present invention, the upper silica gel column and the lower silica gel column are preferably connected through a 24/29# port.
In the invention, after the first prodigiosin crude product and the second prodigiosin crude product are mixed, the loading mode is preferably wet loading; the wet loading preferably comprises the steps of: and mixing the first crude prodigiosin with the second crude prodigiosin, dissolving, and loading the obtained solution with the crude prodigiosin 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 includes methanol.
The invention preferably further comprises performing equilibration prior to said gradient elution with the eluent; the balancing agent preferably comprises petroleum ether. The operation of the balancing is not particularly limited in the present invention, and a balancing operation well known to those skilled in the art may be employed.
In the gradient elution process, the eluent is 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 invention, the first eluent is petroleum ether, ethyl acetate, acetone and chloroform with the volume ratio of 7:2:0.5: 0.5.
In the invention, the second eluent is petroleum ether, ethyl acetate, acetone and chloroform with the volume ratio of 6:3:0.5: 0.5.
In the invention, the third eluent is petroleum ether, ethyl acetate, acetone and chloroform with the volume ratio of 5:4:0.5: 0.5.
In the invention, the fourth eluent is petroleum ether, ethyl acetate, acetone and chloroform with the volume ratio of 4:5:0.5: 0.5.
In the invention, the fifth eluent is petroleum ether, ethyl acetate, acetone and chloroform with the volume ratio of 3:6:0.5: 0.5.
In the invention, the sixth eluent is petroleum ether, ethyl acetate, acetone and chloroform with the volume ratio of 2:7:0.5: 0.5.
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.
In the present invention, the amounts of 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 1 time the volume of the upper silica gel column.
After the prodigiosin eluent is obtained, the prodigiosin eluent is decompressed and concentrated to obtain a prodigiosin pure product. The parameters of the reduced pressure concentration are not particularly limited, so long as the solvent in the prodigiosin eluent can be removed cleanly. After the concentration under reduced pressure, the present invention preferably further comprises drying.
After the pure prodigiosin is obtained, the method preferably further comprises detecting the pure prodigiosin; the detection method preferably includes nuclear magnetic resonance detection (NMR), ultraviolet absorption spectrum detection (UV), infrared absorption spectrum detection (IR), high performance liquid chromatography detection (HPLC), mass spectrometry detection (MS) and ultraviolet spectroscopic detection.
The method for extracting prodigiosin from a prodigiosin-containing fermentation broth according to the present invention will be described in detail with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
The preparation method of Serratia marcescens fermentation broth comprises the following steps:
Streaking on an LB plate, culturing in a 30 ℃ incubator for 24 hours, and picking a single colony with smooth colony and even and full colony as a flat-plate activated serratia marcescens; placing flat activated Serratia marcescens in a test tube filled with 10mL of seed culture medium (the seed culture medium comprises 10g/L tryptone, 5g/L, naCl g/L yeast powder and natural pH), and culturing at 30deg.C to the late logarithmic growth phase to obtain inoculated strain; inoculating the inoculated strain into 10L of double-carbon source fermentation culture medium (the double-carbon source fermentation culture medium comprises 5g/L of peanut powder, 20g/L of soybean peptone, 20g/L of glycerol and 10g/L、KH2PO4 3.5g/L、Na2HPO4·2H2O 15g/L、MgSO4·2H2O 0.5g/L、ZnSO4·7H2O 1g/L、pH% of sucrose naturally) at an inoculum size of 1% by volume, fermenting at 30 ℃ for 48h, controlling pH to 7.0 by ammonia water, coupling dissolved oxygen with rotating speed to be constant by 20%, and performing solid-liquid separation for pretreatment before separation and purification. When the fermentation liquid is used up, dissolved oxygen can suddenly rise after the carbon source in the initial culture medium is exhausted, feeding is started at the moment, the feeding rate is adjusted through the DO-STAT method, the dissolved oxygen is maintained in a proper range, and fermentation is finished after 48 hours of culture, so that Serratia marcescens fermentation liquid is obtained.
FIG. 1 is a photograph of a Serratia marcescens fermentation broth.
The establishment experiment of the method for separating and extracting prodigiosin from Serratia marcescens fermentation broth is repeated three times to obtain an average value, and the repeated steps are as follows:
1. Taking Serratia marcescens fermentation liquor, centrifuging at 8000r/min for 10min, and respectively collecting bacterial precipitate and fermentation liquor supernatant.
2. Taking a 5L beaker, adding 500g of thallus sediment and 4.5L of acidic methanol (pH value is 3, inorganic acid in the acidic methanol is 1mol/L hydrochloric acid), uniformly mixing, performing auxiliary crushing for 10min by using an ultrasonic crusher (the power is 30% of rated power), soaking for 1 day, and collecting thallus supernatant; after 0.05g/L vitamin C is added into the bacterial supernatant, the bacterial supernatant is subjected to reduced pressure rotary evaporation at 35 ℃ under the dark condition to obtain a crude prodigiosin product.
3. And (3) taking a 5L separating funnel, adding 2L of the fermentation liquor supernatant obtained in the step (1) and 3L of ethyl acetate, uniformly mixing, carrying out liquid-liquid separation extraction for 48h, separating an extracting solution by using the separating funnel, draining the extracting solution with almost no color at the lower layer after extraction, retaining an upper ethyl acetate phase, adding 0.05g/L of vitamin C into the ethyl acetate phase, and carrying out reduced pressure rotary evaporation at 45 ℃ under the dark condition to obtain a crude prodigiosin product.
4. Mixing the crude prodigiosin 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 with the specification of 200mm multiplied by 10mm produced by Qingdao ocean chemical factory is selected as the silica gel plate for thin layer chromatography, the proportion of five developing agents is tried, developing agent 1 (petroleum ether: ethyl acetate: acetone: chloroform volume ratio of 7:3:0:0), developing agent 2 (petroleum ether: ethyl acetate: acetone: chloroform volume ratio of 7:2:0.5:0.5), developing agent 3 (petroleum ether: ethyl acetate: acetone: chloroform volume ratio of 5:4:0.5:0.5), developing agent 4 (petroleum ether: ethyl acetate: acetone: chloroform volume ratio of 3:6:0.5:0.5), developing agent 5 (petroleum ether: ethyl acetate: acetone: chloroform volume ratio of 1:8:0.5:0.5), RF values are respectively 0.45, 0.74, 0.84, 0.98, and the five developing agents are subjected to thin layer chromatography to obtain the silica gel plate, as shown in FIG. 2, ① is developing agent 1, ② is developing agent 2, ③, and ③ is developing agent 1:34; as can be seen from fig. 2: the separation effect of the developing agent 2 is best, and the sample spot aggregation is not tailing, so the developing agent 2 is selected as the eluent.
5. Separating and purifying by using double silica gel column cascade chromatography, wherein the specification of the upper silica gel column is 60mm multiplied by 200mm, the silica gel filling height is 80mm, the specification of the lower silica gel column is 60mm multiplied by 300mm, and the silica gel filling height is 250mm; the upper silica gel column and the lower silica gel column are connected by a 24/29# port.
6. The wet method is used for filling the column, the activated silica gel is soaked in petroleum ether and stirred uniformly, a glass funnel with larger caliber is placed on a chromatographic column, the silica gel column is filled with 1/2 of petroleum ether, a lower silica gel column piston is opened, and then the silica gel is poured into the funnel as uniformly as possible, so that faults can be prevented from being formed in the chromatographic column.
7. And (3) mixing the crude prodigiosin obtained in the steps (2) and (3), dissolving the mixture in 50mL of methanol, and loading the methanol solution dissolved with the crude prodigiosin to a silica gel column. Using petroleum ether to balance two silica gel columns, adding a first eluent (mixed solvent of petroleum ether, ethyl acetate, acetone and chloroform with the volume ratio of 7:2:0.5:0.5) with the volume of the silica gel column being 1 times after the column balancing is finished, and starting adding a second eluent (mixed solvent of petroleum ether, ethyl acetate, acetone and chloroform with the volume ratio of 6:3:0.5:0.5) with the volume of the silica gel column being 1 times after the red prodigiosin sample point migrates to the position of 1/2 of the silica gel volume; similarly, a third eluent (mixed solvent of petroleum ether, ethyl acetate, acetone and chloroform with the volume ratio of 5:4:0.5:0.5), a fourth eluent (mixed solvent of petroleum ether, ethyl acetate, acetone and chloroform with the volume ratio of 4:5:0.5:0.5), a fifth eluent (mixed solvent of petroleum ether, ethyl acetate, acetone and chloroform with the volume ratio of 3:6:0.5:0.5), a sixth eluent (mixed solvent of petroleum ether, ethyl acetate, acetone and chloroform with the volume ratio of 2:7:0.5:0.5) and a seventh eluent (mixed solvent of petroleum ether, ethyl acetate, acetone and chloroform with the volume ratio of 1:8:0.5:0.5) are sequentially added, and each eluent is collected, wherein fig. 3 is a flow chart of double-column cascade chromatography, and fig. 4 is a flow chart of eluent with different colors collected in different time periods of double-column cascade chromatography.
As can be seen from fig. 4: the color difference of the eluent is larger, the eluent gradually turns red from the initial white eluent, the eluent is pink, and the final effluent of elution turns yellow.
8. The white eluent, the red eluent, the yellow eluent and the pink eluent are detected by a liquid chromatography-mass spectrometry (LC-MS) and the results are shown in figures 5-7, wherein figure 5 shows the detection result of the liquid chromatography-mass spectrometry (LC-MS) of the red eluent obtained in example 1; FIG. 6 shows the results of a liquid chromatography-mass spectrometer (LC-MS) test of the pink eluate obtained in example 1; FIG. 7 shows the results of a liquid chromatography-mass spectrometer (LC-MS) test of the yellow eluent obtained in example 1. As can be seen from fig. 5 to 7: the red eluent is an eluent containing prodigiosin, has only a single peak, and has a molecular weight of 324 corresponding to the molecular weight of prodigiosin.
9. After 0.025g/L vitamin C is added into the red eluent, decompression rotary steaming is carried out under the condition of light shielding, and then the rotary steaming product is dried in vacuum, thus obtaining 103.5g pure prodigiosin product, the yield is 98.6%, and figure 8 is a physical photograph of the obtained pure prodigiosin product.
10. The prepared prodigiosin pure product is subjected to Mass Spectrometry (MS) and the result is shown in figure 9, and can be seen from figure 9: consistent with the peak results predicted by chemdraw software for prodigiosin groups.
11. The result of ultraviolet absorption spectrum (UV) detection of the prepared prodigiosin pure product is shown in fig. 10, and it can be seen from fig. 10: because of being dissolved in acetonitrile solution, the polymer has obvious absorption peak at 520-530 nm under the slightly alkaline environment, and has a certain blue shift phenomenon reported in literature.
12. The prepared prodigiosin pure product was subjected to infrared absorption spectrum (IR) detection, and the result is shown in FIG. 11, and it can be seen from FIG. 11: there is a distinct peak of pyrrole groups.
13. The prepared prodigiosin pure product was subjected to Nuclear Magnetic Resonance (NMR) detection, and the result is shown in fig. 12, and it can be seen from fig. 12: there is a distinct peak of pyrrole groups.
14. The prepared prodigiosin pure product was subjected to High Performance Liquid Chromatography (HPLC) using a full-wavelength high performance liquid chromatography of 100nm to 900nm, and the results are shown in FIG. 13, and it can be seen from FIG. 13: the peak is single, and no obvious impurity peak exists.
15. The prepared pure prodigiosin was subjected to ultraviolet spectroscopic comparison with a prodigiosin standard (purity > 99%) purchased from MCS officials network, and the results are shown in table 1.
TABLE 1 Standard Curve Condition
As can be seen from table 1: the prodigiosin pure product obtained by the invention is basically consistent with MCS official net, and the purity is more than 99%; the concentration of the standard prepared by the method can be determined to reach the purity of the standard sold by MCS.
Fig. 14 is a standard curve drawn by uv spectrophotometry detection of OD535 for prodigiosin purity, as can be seen from fig. 14: the linear correlation is better, the standard curve y=169.23x+0.0347 can be plotted, and R 2 = 0.9992.
Example 2
Serratia marcescens fermentation broth was prepared in the same manner as in example 1.
The difference from example 1 is that the upper silica gel column used in this example has a specification of 80mm×200mm and a silica gel height of 100mm; the specification of the lower silica gel column is 80mm multiplied by 300mm, and the height is 147.5mm; the upper silica gel column and the lower silica gel column are connected by a 24/29# port. It should be noted that the volumes of the filled silica gel in both examples are the same.
1. Taking Serratia marcescens fermentation liquor, centrifuging at 8000r/min for 10min, and respectively collecting bacterial precipitate and fermentation liquor supernatant.
2. Taking a 5L beaker, adding 500g of thallus sediment and 4.5L of acidic methanol (pH value is 3, inorganic acid in the acidic methanol is 1mol/L hydrochloric acid), uniformly mixing, performing auxiliary crushing for 10min by using an ultrasonic crusher (the power is 30% of rated power), soaking for 1 day, and collecting thallus supernatant; after 0.05g/L vitamin C is added into the bacterial supernatant, the bacterial supernatant is subjected to reduced pressure rotary evaporation at 35 ℃ under the dark condition to obtain a crude prodigiosin product.
3. And (3) taking a 5L separating funnel, adding 2L of the fermentation liquor supernatant obtained in the step (1) and 3L of ethyl acetate, uniformly mixing, carrying out liquid-liquid separation extraction for 48h, separating an extracting solution by using the separating funnel, draining the extracting solution with almost no color at the lower layer after extraction, retaining an upper ethyl acetate phase, adding 0.05g/L of vitamin C into the ethyl acetate phase, and carrying out reduced pressure rotary evaporation at 45 ℃ under the dark condition to obtain a crude prodigiosin product.
4. Mixing the crude prodigiosin 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 with the specification of 200mm multiplied by 10mm produced by Qingdao ocean chemical factory is selected as the silica gel plate for thin layer chromatography, the proportion of five developing agents is tried, developing agent 1 (petroleum ether: ethyl acetate: acetone: chloroform volume ratio of 7:3:0:0), developing agent 2 (petroleum ether: ethyl acetate: acetone: chloroform volume ratio of 7:2:0.5:0.5), developing agent 3 (petroleum ether: ethyl acetate: acetone: chloroform volume ratio of 5:4:0.5:0.5), developing agent 4 (petroleum ether: ethyl acetate: acetone: chloroform volume ratio of 3:6:0.5:0.5), developing agent 5 (petroleum ether: ethyl acetate: acetone: chloroform volume ratio of 1:8:0.5:0.5), RF values are respectively 0.45, 0.74, 0.84, 0.98, and the five developing agents are subjected to thin layer chromatography to obtain the silica gel plate, as shown in FIG. 2, ① is developing agent 1, ② is developing agent 2, ③, and ③ is developing agent 1:34; as can be seen from fig. 2: the separation effect of the developing agent 2 is best, and the sample spot aggregation is not tailing, so the developing agent 2 is selected as the eluent.
5. Separating and purifying by using double silica gel column cascade chromatography, wherein the specification of the upper silica gel column is 80mm×200mm, the silica gel height is 100mm, the specification of the lower silica gel column is 80mm×300mm, and the height is 147.5mm; the upper silica gel column and the lower silica gel column are connected by a 24/29# port.
6. The wet method is used for filling the column, the activated silica gel is soaked in petroleum ether and stirred uniformly, a glass funnel with larger caliber is placed on a chromatographic column, the silica gel column is filled with 1/2 of petroleum ether, a lower silica gel column piston is opened, and then the silica gel is poured into the funnel as uniformly as possible, so that faults can be prevented from being formed in the chromatographic column.
7. And (3) mixing the crude prodigiosin obtained in the steps (2) and (3), dissolving the mixture in 50mL of methanol, and loading the methanol solution dissolved with the crude prodigiosin to a silica gel column. Using petroleum ether to balance two silica gel columns, adding a first eluent (mixed solvent of petroleum ether, ethyl acetate, acetone and chloroform with the volume ratio of 7:2:0.5:0.5) with the volume of the silica gel column being 1 times after the column balancing is finished, and starting adding a second eluent (mixed solvent of petroleum ether, ethyl acetate, acetone and chloroform with the volume ratio of 6:3:0.5:0.5) with the volume of the silica gel column being 1 times after the red prodigiosin sample point migrates to the position of 1/2 of the silica gel volume; and the like, adding a third eluent (mixed solvent of petroleum ether, ethyl acetate, acetone and chloroform in a volume ratio of 5:4:0.5:0.5), a fourth eluent (mixed solvent of petroleum ether, ethyl acetate, acetone and chloroform in a volume ratio of 4:5:0.5:0.5), a fifth eluent (mixed solvent of petroleum ether, ethyl acetate, acetone and chloroform in a volume ratio of 3:6:0.5:0.5), a sixth eluent (mixed solvent of petroleum ether, ethyl acetate, acetone and chloroform in a volume ratio of 2:7:0.5:0.5) and a seventh eluent (mixed solvent of petroleum ether, ethyl acetate, acetone and chloroform in a volume ratio of 1:8:0.5:0.5) in sequence.
8. After 0.025g/L vitamin C is added into the red eluent, decompression rotary steaming is carried out under the condition of light shielding, and then the rotary steaming product is dried in vacuum, thus obtaining 91.2g prodigiosin pure product with the yield of 86.8 percent.
9. The prepared prodigiosin pure product is compared with a standard product by an ultraviolet spectrophotometry, and the purity of the prodigiosin pure product is 91.5 percent.
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 specification of 60mm×200mm and a height of 80 mm.
The results were: the purity of the obtained prodigiosin pure product is 98 percent, and the yield is 65 percent.
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 having a specification of 60mm×300mm and a height of 250 mm.
The results were: the purity of the obtained prodigiosin pure product is 98 percent, and the yield is 80 percent.
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 prodigiosin pure product is 86%, and the yield is 54%.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (9)
1. A method for extracting prodigiosin from a fermentation broth containing prodigiosin, comprising the steps of:
Performing first solid-liquid separation on the fermentation liquor containing prodigiosin to obtain bacterial precipitate and fermentation liquor supernatant;
extracting the supernatant of the fermentation liquor to obtain an extraction phase; the extracting agent for extraction comprises ethyl acetate;
Mixing the extract phase with an antioxidant, and performing first reduced pressure distillation on the obtained extract phase system to obtain a first prodigiosin crude product;
Mixing the bacterial precipitate with an acidic alcohol solvent, and sequentially carrying out ultrasonic treatment, leaching and second solid-liquid separation to obtain bacterial supernatant;
Mixing the thallus supernatant with an antioxidant, and performing second reduced pressure distillation on the obtained thallus supernatant system to obtain a second prodigiosin crude product;
Mixing the first crude prodigiosin and the second crude prodigiosin, performing double-silica-gel column cascade chromatography, and performing gradient elution by using an eluent to obtain 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 gradient elution process, the eluents are sequentially a first eluent, a second eluent, a third eluent, a fourth eluent, a fifth eluent, a sixth eluent and a seventh eluent;
The first eluent is petroleum ether, ethyl acetate, acetone and chloroform, and the volume ratio is 7:2:0.5:0.5 of a mixed solvent;
The second eluent is petroleum ether, ethyl acetate, acetone and chloroform with the volume ratio of 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 with the volume ratio of 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 is 3:6:0.5:0.5 of a mixed solvent;
The sixth eluent is petroleum ether, ethyl acetate, acetone and chloroform with the volume ratio of 2:7:0.5:0.5 of a mixed solvent;
the seventh eluent is petroleum ether, ethyl acetate, acetone and chloroform, and the volume ratio is 1:8:0.5: 0.5.
2. The method according to claim 1, wherein the volume ratio of the upper silica gel column to the lower silica gel column is 1:1.5 to 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: 3-1: 4.
3. The method of claim 1, wherein the first, second, third, fourth, fifth, sixth and seventh eluents are each present in an amount of 1 times the volume of the upper silica gel column.
4. The method of claim 1, wherein the prodigiosin-containing fermentation broth is serratia marcescens fermentation broth; the preparation method of the Serratia marcescens fermentation liquid comprises the following steps:
inoculating Serratia marcescens activated by a flat plate into a seed culture medium, and obtaining an inoculated strain after the strain grows to a logarithmic growth phase;
Inoculating the inoculated strain to an antibiotic-free LB (LB) culture medium, and culturing to obtain the serratia marcescens fermentation broth;
the volume percentage of the inoculated strain in the LB culture medium without resistance is 1-2%.
5. The method according to claim 4, wherein the temperature of the cultivation is 25-27 ℃ for 24-48 hours; the culture is carried out under the condition of shaking, and the speed of the shaking is 150-200 rpm.
6. The method of claim 1, wherein the antioxidant is vitamin C.
7. The process according to claim 1 or 6, wherein the concentration of antioxidant in the extract phase system and the cell supernatant system is independently 0.01 to 0.05g/L.
8. The method according to claim 1, wherein the acidic alcoholic solvent has a pH of 3; the acidic alcohol solvent is methanol and inorganic acid; the inorganic acid is hydrochloric acid, sulfuric acid or nitric acid.
9. 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 performed under light-shielding conditions.
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