CN113897406B - Method for extracting and purifying salidroside from rhodiola rosea powder - Google Patents
Method for extracting and purifying salidroside from rhodiola rosea powder Download PDFInfo
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- CN113897406B CN113897406B CN202111150297.6A CN202111150297A CN113897406B CN 113897406 B CN113897406 B CN 113897406B CN 202111150297 A CN202111150297 A CN 202111150297A CN 113897406 B CN113897406 B CN 113897406B
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- ILRCGYURZSFMEG-UHFFFAOYSA-N Salidroside Natural products OC1C(O)C(O)C(CO)OC1OCCC1=CC=C(O)C=C1 ILRCGYURZSFMEG-UHFFFAOYSA-N 0.000 title claims abstract description 131
- ILRCGYURZSFMEG-RQICVUQASA-N salidroside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)OC1OCCC1=CC=C(O)C=C1 ILRCGYURZSFMEG-RQICVUQASA-N 0.000 title claims abstract description 131
- 244000042430 Rhodiola rosea Species 0.000 title claims abstract description 113
- 235000003713 Rhodiola rosea Nutrition 0.000 title claims abstract description 112
- 239000000843 powder Substances 0.000 title claims abstract description 79
- 238000000034 method Methods 0.000 title claims abstract description 50
- 238000000746 purification Methods 0.000 claims abstract description 51
- 239000007788 liquid Substances 0.000 claims abstract description 49
- 238000000855 fermentation Methods 0.000 claims abstract description 40
- 230000004151 fermentation Effects 0.000 claims abstract description 40
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 39
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims abstract description 28
- 108090000790 Enzymes Proteins 0.000 claims abstract description 26
- 102000004190 Enzymes Human genes 0.000 claims abstract description 26
- 239000000287 crude extract Substances 0.000 claims abstract description 22
- 241000235342 Saccharomycetes Species 0.000 claims abstract description 21
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims abstract description 14
- 238000002156 mixing Methods 0.000 claims abstract description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 58
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims description 38
- 239000012535 impurity Substances 0.000 claims description 29
- 229940088598 enzyme Drugs 0.000 claims description 25
- 238000012258 culturing Methods 0.000 claims description 21
- 239000006228 supernatant Substances 0.000 claims description 21
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- 229920005989 resin Polymers 0.000 claims description 19
- 101001065065 Aspergillus awamori Feruloyl esterase A Proteins 0.000 claims description 18
- 108010059892 Cellulase Proteins 0.000 claims description 18
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- 239000007787 solid Substances 0.000 claims description 14
- 238000001179 sorption measurement Methods 0.000 claims description 14
- 101100313763 Arabidopsis thaliana TIM22-2 gene Proteins 0.000 claims description 12
- 238000004587 chromatography analysis Methods 0.000 claims description 12
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- 241001138401 Kluyveromyces lactis Species 0.000 claims description 11
- 239000012528 membrane Substances 0.000 claims description 11
- 230000004913 activation Effects 0.000 claims description 9
- 235000014663 Kluyveromyces fragilis Nutrition 0.000 claims description 8
- 235000018368 Saccharomyces fragilis Nutrition 0.000 claims description 8
- 229940031154 kluyveromyces marxianus Drugs 0.000 claims description 8
- 239000002609 medium Substances 0.000 claims description 7
- 238000010438 heat treatment Methods 0.000 claims description 6
- 239000003960 organic solvent Substances 0.000 claims description 5
- 238000010025 steaming Methods 0.000 claims description 5
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- 238000011081 inoculation Methods 0.000 claims description 2
- 238000001728 nano-filtration Methods 0.000 claims description 2
- 238000011218 seed culture Methods 0.000 claims description 2
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- 230000006872 improvement Effects 0.000 abstract description 3
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- 238000004128 high performance liquid chromatography Methods 0.000 description 38
- 230000000052 comparative effect Effects 0.000 description 36
- 238000001514 detection method Methods 0.000 description 31
- 229910001868 water Inorganic materials 0.000 description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 16
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 15
- 229930182470 glycoside Natural products 0.000 description 13
- 239000002994 raw material Substances 0.000 description 13
- 150000002338 glycosides Chemical class 0.000 description 12
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- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 241000083902 Rhodiola sachalinensis Species 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000001816 cooling Methods 0.000 description 3
- HQVFCQRVQFYGRJ-UHFFFAOYSA-N formic acid;hydrate Chemical compound O.OC=O HQVFCQRVQFYGRJ-UHFFFAOYSA-N 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000012071 phase Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 239000012086 standard solution Substances 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 2
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 2
- KDLHZDBZIXYQEI-UHFFFAOYSA-N Palladium Chemical compound [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 239000004480 active ingredient Substances 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
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- 230000000813 microbial effect Effects 0.000 description 2
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 229910021642 ultra pure water Inorganic materials 0.000 description 2
- 239000012498 ultrapure water Substances 0.000 description 2
- YCCILVSKPBXVIP-UHFFFAOYSA-N 2-(4-Hydroxyphenyl)ethanol Natural products OCCC1=CC=C(O)C=C1 YCCILVSKPBXVIP-UHFFFAOYSA-N 0.000 description 1
- 108010013043 Acetylesterase Proteins 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- DBLDQZASZZMNSL-QMMMGPOBSA-N L-tyrosinol Natural products OC[C@@H](N)CC1=CC=C(O)C=C1 DBLDQZASZZMNSL-QMMMGPOBSA-N 0.000 description 1
- 241000997135 Rhodiola crenulata Species 0.000 description 1
- ILRCGYURZSFMEG-RKQHYHRCSA-N Salidroside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OCCC1=CC=C(O)C=C1 ILRCGYURZSFMEG-RKQHYHRCSA-N 0.000 description 1
- DZGWFCGJZKJUFP-UHFFFAOYSA-N Tyramine Natural products NCCC1=CC=C(O)C=C1 DZGWFCGJZKJUFP-UHFFFAOYSA-N 0.000 description 1
- UAOKXEHOENRFMP-ZJIFWQFVSA-N [(2r,3r,4s,5r)-2,3,4,5-tetraacetyloxy-6-oxohexyl] acetate Chemical compound CC(=O)OC[C@@H](OC(C)=O)[C@@H](OC(C)=O)[C@H](OC(C)=O)[C@@H](OC(C)=O)C=O UAOKXEHOENRFMP-ZJIFWQFVSA-N 0.000 description 1
- 230000021736 acetylation Effects 0.000 description 1
- 238000006640 acetylation reaction Methods 0.000 description 1
- 238000005903 acid hydrolysis reaction Methods 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000003712 anti-aging effect Effects 0.000 description 1
- 230000002929 anti-fatigue Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000003262 anti-osteoporosis Effects 0.000 description 1
- 230000003064 anti-oxidating effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 238000011097 chromatography purification Methods 0.000 description 1
- 239000013065 commercial product Substances 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 230000006196 deacetylation Effects 0.000 description 1
- 238000003381 deacetylation reaction Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000004134 energy conservation Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 230000012447 hatching Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 241000411851 herbal medicine Species 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- 229910052763 palladium Inorganic materials 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 238000010587 phase diagram Methods 0.000 description 1
- 238000000053 physical method Methods 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000011403 purification operation Methods 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 238000012807 shake-flask culturing Methods 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 235000012239 silicon dioxide Nutrition 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 230000035882 stress Effects 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 229960003732 tyramine Drugs 0.000 description 1
- DZGWFCGJZKJUFP-UHFFFAOYSA-O tyraminium Chemical compound [NH3+]CCC1=CC=C(O)C=C1 DZGWFCGJZKJUFP-UHFFFAOYSA-O 0.000 description 1
- 235000004330 tyrosol Nutrition 0.000 description 1
- -1 tyrosol glycoside compound Chemical class 0.000 description 1
- 238000000825 ultraviolet detection Methods 0.000 description 1
- 229940089401 xylon Drugs 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
- C07H1/08—Separation; Purification from natural products
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/18—Acyclic radicals, substituted by carbocyclic rings
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- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
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- Chemical Kinetics & Catalysis (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to a method for extracting and purifying salidroside from rhodiola rosea powder, which comprises the following steps: (1) pretreatment: mixing radix Rhodiolae powder with phosphoric acid solution, heat treating, adjusting pH, adding complex enzyme, and performing enzymolysis to obtain radix Rhodiolae crude extract; (2) preparing a yeast seed solution; (3) fermenting by using saccharomycetes to obtain rhodiola rosea fermentation liquor; (4) separating and purifying to obtain the salidroside purification liquid. According to the method disclosed by the invention, after enzymolysis, fermentation extraction and purification, the extraction rate of the salidroside reaches 90.8% -92.3%, the purity is also improved from 2.8% to 50-55%, and the great improvement of the extraction yield and purity of the salidroside is realized. The method has the characteristics of low cost, simple operation, high efficiency, high extraction rate and continuous production, and has good application prospect for producing high-purity salidroside on a large scale.
Description
Technical Field
The invention belongs to the technical field of natural product extraction, and particularly relates to a method for extracting and purifying salidroside from rhodiola rosea powder.
Background
The salidroside, namely 2- (4-hydroxyphenyl) ethyl-beta-D-glucopyranoside (C 14H20O7), is a tyrosol glycoside compound separated and identified from various natural rhodiola plants, such as rhodiola sachalinensis (Rhodiolacrenulata), rhodiola rosea (Rhodiolarosea L) and rhodiola sachalinensis (Rhodiolasachalinensis), and is one of the most main and effective active ingredients in the rhodiola plants, and is used as a traditional Chinese medicine at the earliest. Salidroside has been proved to have good physiological effects of anti-altitude stress, anti-inflammatory, anti-oxidation, anti-fatigue, improving energy, anti-osteoporosis, anti-aging and the like. In recent years, researchers have also found that it also exerts a remarkable curative effect in the treatment of certain cancers and tumors, and also exhibits a certain therapeutic effect on diabetes. The salidroside attracts more and more attention due to the outstanding pharmacological and health care effects, and the salidroside is widely applied to industries such as medicines, cosmetics, special medical foods and the like as a functional component, so that the market demand is gradually expanded, the research on the extraction and purification method of the active ingredient in the rhodiola rosea is important, and the extraction and purification method of the salidroside with low production cost, simple operation and easy large-scale industrial production needs to be developed.
At present, the prior art for extracting and purifying salidroside mainly comprises the following steps: firstly, the salidroside is separated and extracted according to the solubility difference of different components in a solvent by using a similar compatibility principle. Usually, the dried rhodiola root and stem materials are crushed and ground into powder, and then the salidroside is extracted by adopting water or ethanol. Wang Wei respectively steaming and boiling with water and ethanol to extract the effective component salidroside in radix Rhodiolae, wherein the water and boiling conditions are as follows: steaming with water for 30min for two times; the ethanol digestion conditions are as follows: steaming with 70% ethanol twice for 30min each time; the yields of the two are 0.16% and 0.14% (Wang Wei, liu Chuan, xylon, gaoshan rhodioside extraction technique [ J ]. Chinese herbal medicine 1999,30 (11): 824-826.). And the second method is combined with resin method by organic solvent, which usually uses ethanol, diethyl ether and other steps to extract salidroside, and then combines with macroporous resin extraction to improve the purity, for example, the yield of patent CN201610688538.5 is 1.6-2.1%. In another patent CN201910168702.3, salidroside is extracted from the rhodiola rosea pretreated powder by ethanol reflux, and is finally adsorbed and purified by macroporous adsorption resin, but the extraction rate and purity value of the salidroside are not provided. The leaching method and the organic reagent combined resin purification method have relatively poor separation and selection effects, large loss of target components, relatively easy low extraction rate and low purity, and relatively difficult post-treatment process, and high alcohol consumption, thus leading to higher production cost. Thirdly, the physical method is combined with resin, and after the rhodiola rosea is crushed in the invention patents CN201810718980.7, CN201610918396.7, CN201310463903.9 and CN201510273811.3, the crude extract is pretreated by physical ultrasonic wave or microwave auxiliary ethanol, and then is purified by alcohol precipitation or macroporous adsorption resin chromatography; in addition, the invention patent CN201710694349.3 also discloses a method for separating salidroside by activating the rhodiola powder by using a high-frequency magnetic field and then adding solvents such as ethanol, acetone, ethyl acetate, n-hexane and the like; and the latest invention patent CN202110049505.7 provides a process for extracting salidroside by means of magnetic particle materials, wherein the magnetic particles are prepared by fixing nickel or palladium complex on the surfaces of magnetic silicon dioxide particles through tyramine, then mixing and hatching with rhodiola powder, placing the mixture in a magnetic field for attraction and separation, immersing the obtained magnetic particles in eluent, placing the magnetic particles in the magnetic field again for attraction and separation, then carrying out acetylation on the obtained sample liquid, reacting with pentaacetyl glucose under the catalysis of the magnetic particles, and obtaining the salidroside after the deacetylation treatment of reactants. Besides the complex separation and purification operation process, the method has high technical requirements on operators and high production cost (including equipment or materials and the like), is basically in a laboratory exploration stage at present, and is not beneficial to industrial application.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for extracting and purifying salidroside from rhodiola rosea powder, which mainly comprises the steps of obtaining a salidroside product with high extraction rate and higher purity by fermenting a natural yeast strain after enzymolysis and then carrying out resin chromatography, and has the advantages of simple purification process, energy conservation and environmental protection. The biological extraction and purification of salidroside have not been reported yet.
The technical scheme of the invention is as follows:
a method for extracting and purifying salidroside from radix Rhodiolae powder comprises the following steps:
(1) Pretreatment: mixing rhodiola rosea powder with a phosphoric acid solution, performing heat treatment, adjusting the pH to 4.5-5.8, and adding a complex enzyme, wherein the complex enzyme consists of glucanase, feruloyl esterase and cellulase; performing enzymolysis for 9-12 h at 30-60 ℃ to obtain rhodiola rosea crude extract;
(2) Preparing a saccharomycete seed solution: activating saccharomycetes, inoculating the activated saccharomycetes into a seed culture medium, and culturing to obtain saccharomycetes seed liquid;
(3) Fermentation: inoculating the yeast seed liquid in the step (2) into the rhodiola rosea crude extract in the step (1), and standing and fermenting for 24-72 h at 28-40 ℃ to obtain rhodiola rosea fermentation liquor;
(4) And (3) separating and purifying: centrifuging the rhodiola rosea fermentation liquor obtained in the step (3) to obtain a supernatant, and purifying to obtain a salidroside purification liquor.
According to the invention, the rhodiola rosea powder in the step (1) is a rhodiola rosea powder obtained by directly crushing dried rhodiola rosea roots or a rhodiola rosea extract powder obtained by extracting with an organic solvent.
According to a preferred embodiment of the invention, the concentration of the phosphoric acid solution in step (1) is 0.01 to 0.1wt%.
According to the invention, the ratio of the mass volume of the rhodiola rosea powder to the phosphoric acid solution in the step (1) is preferably 1-3:1, and the unit g/mL.
According to a preferred embodiment of the present invention, the heat treatment in step (1) comprises: heat treatment is carried out for 14-45 minutes at 121-160 ℃, and then cooling is carried out to 50-60 ℃.
According to the invention, the mass ratio of glucanase, feruloyl esterase and cellulase in the complex enzyme in the step (1) is 1:1:2.
According to the invention, preferably, the feeding mass ratio of the complex enzyme to the rhodiola rosea powder in the step (1) is (0.05-0.1): (10-50).
According to a preferred embodiment of the present invention, the complex enzyme in step (1) is in the form of a solid powder.
According to a preferred embodiment of the invention, the yeast in step (2) is Saccharomyces cerevisiae, kluyveromyces marxianus or Kluyveromyces lactis.
According to a preferred embodiment of the present invention, the activation in the step (2) is performed by inoculating yeast into YPD solid medium and culturing at 20-37 ℃.
According to a preferred embodiment of the present invention, the seed medium in step (2) is YPD liquid medium.
According to the present invention, the culture conditions in the step (2) are preferably 20 to 37℃and 100 to 200rpm, and the culture is carried out until the yeast seed liquid OD 600 is 0.6 to 0.8.
According to the invention, the inoculation amount of the saccharomycete seed liquid in the step (3) is preferably 5-10% by mass.
According to the present invention, the centrifugation conditions in the step (4) are preferably 8000 to 12000rpm for 10 to 20 minutes.
According to the invention, the purification in the step (4) is carried out by filtering the supernatant, decolorizing and removing impurities by a 400kDa nano-membrane, purifying by HP20 macroporous adsorption resin chromatography, eluting with 2% ethanol, and rotary steaming to obtain the salidroside purification liquid.
According to the invention, preferably, the salidroside purification liquid in the step (4) can be dried to obtain the salidroside purification powder.
In the invention, the purity of the rhodiola rosea powder raw material is 2.8 percent through HPLC-UV detection after the rhodiola rosea powder raw material is dissolved by adding water; according to the method, after the dissolution of the phosphoric acid solution, enzymolysis of the compound enzyme, microbial fermentation, centrifugation, nanofiltration and chromatography purification of the HP20 macroporous adsorption resin, the extraction rate of the salidroside reaches 90.8-92.3%, the purity is also improved from 2.8% to 50-55%, and the great improvement of the extraction yield and purity of the salidroside is realized.
The beneficial effects are that:
1. According to the invention, three enzymes of glucanase, feruloyl esterase and cellulase (mass ratio of 1:1:2) are utilized for the first time to cooperatively pretreat the rhodiola rosea powder raw material, so that the rhodiola rosea glycoside in the raw material is dissolved out to the maximum extent, the enzyme reaction condition is mild, the biological activity of the rhodiola rosea glycoside is protected, and the extraction rate is ensured to be obviously improved.
2. The invention uses Saccharomyces cerevisiae, kluyveromyces marxianus and Kluyveromyces lactis to ferment the rhodiola rosea powder and then purify the rhodiola rosea powder to obtain the salidroside purified liquid, thereby remarkably improving the extraction rate and purity of the rhodiola rosea. The rhodiola rosea powder sold in the market at present is usually obtained by grinding into powder or simply extracting with an organic reagent (methanol, ethanol and the like), so that the extraction rate and purity of the rhodiola rosea glycoside are low. According to the method, after acidolysis and enzymolysis pretreatment of rhodiola rosea powder are utilized, the salidroside is extracted by adopting a microbial fermentation method, so that the extraction rate is greatly improved, the purity is also obviously improved, and the method has the advantages of mild conditions, easiness in product separation, short production period, easiness in regulation and control and the like, and the method has the advantages of few organic reagents, greatly protects the edible and medicinal values of the salidroside, and is beneficial to large-scale industrial production popularization.
3. The invention can solve the problems of low extraction efficiency, low product purity and complex separation and purification process of rhodiola rosea powder prepared by directly crushing rhodiola rosea plants or extracting rhodiola rosea glycoside by an organic solvent method. According to the method disclosed by the invention, after enzymolysis, fermentation extraction and purification, the extraction rate of the salidroside reaches 90.8% -92.3%, the purity is also improved from 2.8% to 50-55%, and the great improvement of the extraction yield and purity of the salidroside is realized. In conclusion, the method has the characteristics of low cost, simple operation, high efficiency and continuous production, and has good application prospect for producing high-purity salidroside on a large scale.
Drawings
FIG. 1 is an HPLC chart after dissolving the raw material rhodiola root powder in pure water.
FIG. 2 is an HPLC chart of the purified solution of salidroside in example 1.
FIG. 3 is an HPLC chart of the purified solution of salidroside of example 2.
FIG. 4 is an HPLC chart of the purified solution of salidroside of example 3.
FIG. 5 is an HPLC chart of the purified solution of salidroside of comparative example 1.
FIG. 6 is an HPLC chart of the purified solution of salidroside of comparative example 2.
FIG. 7 is an HPLC chart of the purified solution of salidroside of comparative example 3.
FIG. 8 is an HPLC chart of the purified solution of salidroside of comparative example 4.
FIG. 9 is an HPLC chart of the purified solution of salidroside of comparative example 5.
Detailed Description
The technical aspects of the present invention will be described in further detail with reference to the following examples, comparative examples and drawings, which are provided to illustrate the present invention, but the scope of the present invention is not limited thereto.
The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise specified in the present invention.
The rhodiola rosea powder used in the invention is rhodiola rosea extract powder (purity 3%) which is obtained by crude extraction by an organic solvent, and is a common commercial product; the salidroside standard, dextranase, feruloyl esterase, cellulase and HP20 macroporous adsorption resin are all common commercial products; saccharomyces cerevisiae, kluyveromyces marxianus, kluyveromyces lactis are also common commercial products available from Santa Clara and chemical industry Co., ltd., shanghai Yu Biotech Co., ltd., or Feng Shou (Shanghai) Biotech Co., ltd.
YPD liquid medium: 2% of tryptone, 1% of yeast extract and 2% of glucose; the solid culture medium is additionally added with 1.5 percent of agar powder, which is the mass percent.
Preparing a salidroside standard substance gradient solution: adding 0.1mg of salidroside standard substance into 100 μl of ultrapure water, mixing to obtain 1mg/mL of primary standard substance solution, and standing at-20deg.C for storage; taking 10 mu L of primary standard solution, adding 90 mu L of ultrapure water to obtain standard solution with the concentration of 100mg/L, and respectively diluting the standard solution into different concentrations step by step to obtain standard gradient solution of salidroside; then, each gradient standard was detected using High Performance Liquid Chromatography (HPLC). The detection condition is that the wavelength is 280nm, the flow rate is 0.5mL/min, the mobile phase is acetonitrile-0.1% formic acid water, and the ultraviolet detector is used. According to the detection result, a standard curve is drawn, wherein y= 2295997x-34651, R 2 =1, the linear relation is good at 0.01-2mg/mL, and the salidroside peak-out time is 10.080-13.100min.
Detecting salidroside in the raw material rhodiola rosea powder by adopting high performance liquid chromatography: dissolving 10mg of rhodiola rosea powder in 10mL of pure water, centrifuging at 12000rpm for 20min, collecting supernatant, filtering with a 0.22 μm filter membrane, and performing HPLC detection under the following conditions: the wavelength is 280nm, the flow rate is 0.5mL/min, the mobile phase is acetonitrile-0.1% formic acid water, and the ultraviolet detector is used for detecting the ultraviolet radiation. The detection results are shown in FIG. 1.
From the liquid phase diagram in fig. 1, it can be seen that the raw material rhodiola rosea powder has more impurity peaks, and the concentration of rhodiola rosea glycoside in the raw material rhodiola rosea powder is 0.028mg/mL calculated by combining the standard curve and the HPLC diagram, the content of rhodiola rosea glycoside is 28mg/g rhodiola rosea powder, and the purity of rhodiola rosea glycoside is 2.8% calculated by using the formula of rhodiola rosea glycoside purity=rhodiola rosea glycoside mass/rhodiola rosea powder mass x 100%.
Example 1
A method for extracting and purifying salidroside from radix Rhodiolae powder comprises the following steps:
(1) Pretreatment: 10g of rhodiola rosea powder is taken and placed in a triangular flask, 10mL of 0.1wt% phosphoric acid solution is added, the mixture is treated for 30 minutes at the constant temperature of 121 ℃, the mixture is kept stand and cooled to 60 ℃, then the pH is adjusted to 5.8 by sodium hydroxide solution, 100mg of complex enzyme consisting of dextranase, feruloyl esterase and cellulase is added, the mass ratio of the dextranase to the feruloyl esterase to the cellulase is 1:1:2, then pure water is added to the mixture to fix the volume to 500mL, and the mixture is placed in a shaking table, and is subjected to enzymolysis for 12 hours at 150rpm at 50 ℃ to prepare rhodiola rosea crude extract;
(2) Preparing a saccharomycete seed solution: inoculating Saccharomyces cerevisiae into YPD solid culture medium, performing activation culture in a constant temperature incubator at 30deg.C, inoculating activated Saccharomyces cerevisiae into 1L YPD liquid culture medium, placing in a shaking table at 30deg.C, and shake-culturing at 200rpm until OD 600 is 0.8 to obtain Saccharomyces cerevisiae seed solution;
(3) Fermentation: inoculating the saccharomyces cerevisiae seed liquid obtained in the step (2) into the rhodiola rosea crude extract obtained in the step (1) according to the inoculum size of 5wt%, and standing, fermenting and culturing for 48 hours at 37 ℃ to obtain rhodiola rosea fermentation liquid;
(4) And (3) separating and purifying: centrifuging the rhodiola rosea fermentation liquor obtained in the step (3) at 12000rpm for 20min to obtain a supernatant, carrying out suction filtration on the centrifuged supernatant, decolorizing and removing impurities through a 400kDa nano membrane, purifying through HP20 macroporous adsorption resin chromatography, eluting with 2% ethanol, and carrying out rotary evaporation to obtain a salidroside purification liquor.
Detecting the obtained salidroside purification liquid by using High Performance Liquid Chromatography (HPLC), wherein the detection conditions are as follows: the wavelength is 280nm, the flow rate is 0.5mL/min, the mobile phase is acetonitrile-0.1% formic acid water, and the ultraviolet detector is consistent with the detection method of the rhodiola rosea standard substance. The detection results are shown in FIG. 2.
By combining a standard curve and an HPLC detection map, the salidroside concentration of the salidroside purification liquid obtained in the embodiment is calculated to be 0.50mg/mL, the quality is 254.5mg, and the extraction rate of the salidroside is 90.9%.
The salidroside purification liquid obtained in the embodiment is dried to obtain high-purity salidroside powder with the purity of 50.0%.
Example 2
A method for extracting and purifying salidroside from radix Rhodiolae powder comprises the following steps:
(1) Pretreatment: adding 15g of rhodiola rosea powder into a triangular flask, adding 6mL of 0.01wt% phosphoric acid solution, carrying out constant temperature treatment for 30 minutes at 155 ℃, standing and cooling to 50 ℃, regulating the pH to 4.5 by using sodium hydroxide solution, adding 50mg of complex enzyme consisting of dextranase, feruloyl esterase and cellulase, wherein the mass ratio of the dextranase to the feruloyl esterase to the cellulase is 1:1:2, adding pure water to 500mL, placing into a shaking table, and carrying out enzymolysis for 9 hours at 200rpm at 50 ℃ to prepare rhodiola rosea crude extract;
(2) Preparing a saccharomycete seed solution: inoculating Kluyveromyces marxianus into YPD solid culture medium, activating and culturing in a constant temperature incubator at 30deg.C, inoculating activated Kluyveromyces marxianus into 1.5L YPD liquid culture medium, placing in a shaking table at 25deg.C at 200rpm, and shake-culturing until OD 600 is 0.6 to obtain Kluyveromyces marxianus seed solution;
(3) Fermentation: inoculating the Kluyveromyces marxianus seed solution obtained in the step (2) into the rhodiola rosea crude extract obtained in the step (1) according to the inoculum size of 10wt%, and standing, fermenting and culturing for 56 hours at the temperature of 30 ℃ to obtain rhodiola rosea fermentation liquor;
(4) And (3) separating and purifying: centrifuging the rhodiola rosea fermentation liquor obtained in the step (3) at 8000rpm for 10min to obtain a supernatant, carrying out suction filtration on the centrifuged supernatant, decolorizing and removing impurities through a 400kDa nano membrane, purifying through HP20 macroporous adsorption resin chromatography, eluting with 2% ethanol, and carrying out rotary evaporation to obtain a salidroside purification liquor.
The obtained salidroside purification liquid was detected by High Performance Liquid Chromatography (HPLC), and the detection conditions were the same as in example 1. The detection results are shown in FIG. 3.
By combining a standard curve and an HPLC detection map, the salidroside concentration of the salidroside purification liquid obtained in the embodiment is calculated to be 0.52mg/mL, the quality is 382.7mg, and the extraction rate of the salidroside is 91.1%.
The salidroside purification liquid obtained in the embodiment is dried to obtain high-purity salidroside powder with the purity of 51.9%.
Example 3
A method for extracting and purifying salidroside from radix Rhodiolae powder comprises the following steps:
(1) Pretreatment: adding 20g of rhodiola rosea powder into a triangular flask, adding 10mL of 0.01wt% phosphoric acid solution, carrying out constant temperature treatment for 20 minutes at 140 ℃, standing and cooling to 55 ℃, then adjusting the pH to 5.0 by using sodium hydroxide solution, adding 80mg of complex enzyme consisting of dextranase, feruloyl esterase and cellulase, wherein the mass ratio of the dextranase to the feruloyl esterase to the cellulase is 1:1:2, adding pure water to 500mL, placing into a shaking table, and carrying out enzymolysis for 10 hours at 120rpm at 40 ℃ to prepare rhodiola rosea crude extract;
(2) Preparing a saccharomycete seed solution: inoculating kluyveromyces lactis into YPD solid culture medium, performing activation culture in a constant temperature incubator at 30 ℃, then inoculating activated kluyveromyces lactis into 2L YPD liquid culture medium, placing in a shaking table at 37 ℃ and performing shake flask culture at 200rpm until OD 600 is 0.7, and obtaining kluyveromyces lactis seed liquid;
(3) Fermentation: inoculating the Kluyveromyces lactis seed liquid obtained in the step (2) into the rhodiola rosea crude extract obtained in the step (1) according to the inoculum size of 8wt%, and standing, fermenting and culturing for 64h at 35 ℃ to obtain rhodiola rosea fermentation liquid;
(4) And (3) separating and purifying: centrifuging the rhodiola rosea fermentation liquor obtained in the step (3) at 10000rpm for 15min, taking supernatant, carrying out suction filtration on the centrifuged supernatant, decolorizing and removing impurities through a 400kDa nano membrane, purifying through HP20 macroporous adsorption resin chromatography, eluting with 2% ethanol, and carrying out rotary evaporation to obtain a salidroside purification liquor.
The obtained salidroside purification liquid was detected by High Performance Liquid Chromatography (HPLC), and the detection conditions were the same as in example 1. The detection results are shown in FIG. 4.
By combining a standard curve and an HPLC detection map, the salidroside concentration of the salidroside purification liquid obtained in the embodiment is calculated to be 0.54mg/mL, the quality is 516.7mg, and the extraction rate of the salidroside is 92.3%.
The salidroside purification liquid obtained in the embodiment is dried to obtain high-purity salidroside powder with the purity of 54.1 percent.
Comparative example 1
A method for extracting and purifying salidroside from radix Rhodiolae powder comprises the following steps:
(1) Pretreatment: taking 10g of rhodiola rosea powder in a triangular flask, directly adding pure water to a constant volume of 500mL, placing in a shaking table, shaking uniformly at 150rpm for 12 hours at 50 ℃ to prepare a rhodiola rosea crude extract;
(2) Preparing a saccharomycete seed solution: inoculating Saccharomyces cerevisiae into YPD solid culture medium, performing activation culture in a constant temperature incubator at 30deg.C, inoculating activated Saccharomyces cerevisiae into 1L YPD liquid culture medium, placing in a shaking table at 30deg.C, and shake-culturing at 200rpm until OD 600 is 0.8 to obtain Saccharomyces cerevisiae seed solution;
(3) Fermentation: inoculating the saccharomyces cerevisiae seed liquid obtained in the step (2) into the rhodiola rosea crude extract obtained in the step (1) according to the inoculum size of 5wt%, and standing, fermenting and culturing for 48 hours at 37 ℃ to obtain rhodiola rosea fermentation liquid;
(4) And (3) separating and purifying: centrifuging the rhodiola rosea fermentation liquor obtained in the step (3) at 12000rpm for 20min to obtain a supernatant, carrying out suction filtration on the centrifuged supernatant, decolorizing and removing impurities through a 400kDa nano membrane, purifying through HP20 macroporous adsorption resin chromatography, eluting with 2% ethanol, and carrying out rotary evaporation to obtain a salidroside purification liquor.
The obtained salidroside purification liquid was detected by High Performance Liquid Chromatography (HPLC), and the detection conditions were the same as in example 1. The detection results are shown in FIG. 5.
Combining a standard curve and an HPLC detection map, calculating to obtain the salidroside concentration of the salidroside purification liquid obtained in the comparative example to be 0.29mg/mL, the quality to be 174.3mg, and the extraction rate of the salidroside to be 62.3%; and drying to obtain salidroside powder with purity of 29.0%. The HPLC detection spectrum shows that the impurity peaks are more, and the extraction rate and purity of the rhodiola rosea are reduced, which indicates that after the rhodiola rosea powder which is not pretreated by dilute acid and enzymolysis is directly fermented and chromatographed, some impurity substances cannot be degraded by yeast, and the purity is reduced. In addition, a part of salidroside may be lost with the solid impurities of the suction filtration, resulting in a decrease in the extraction rate.
Comparative example 2
A method for extracting and purifying salidroside from radix Rhodiolae powder comprises the following steps:
(1) Pretreatment: 10g of rhodiola rosea powder is taken and added into a triangular flask, 10mL of pure water is added, the constant temperature treatment is carried out for 30 minutes at 121 ℃, the mixture is kept stand and cooled to 60 ℃, then the pH is regulated to 5.8 by a sodium hydroxide solution, 100mg of complex enzyme consisting of dextranase, feruloyl esterase and cellulase is added, the mass ratio of the dextranase to the feruloyl esterase to the cellulase is 1:1:2, the volume is fixed to 500mL by pure water, the mixture is placed into a shaking table, and shaking is carried out for 12 hours at 150rpm at 50 ℃ to prepare rhodiola rosea crude extract;
(2) Preparing a saccharomycete seed solution: inoculating Saccharomyces cerevisiae into YPD solid culture medium, performing activation culture in a constant temperature incubator at 30deg.C, inoculating activated Saccharomyces cerevisiae into 1L YPD liquid culture medium, placing in a shaking table at 30deg.C, and shake-culturing at 200rpm until OD 600 is 0.8 to obtain Saccharomyces cerevisiae seed solution;
(3) Fermentation: inoculating the saccharomyces cerevisiae seed liquid obtained in the step (2) into the rhodiola rosea crude extract obtained in the step (1) according to the inoculum size of 5wt%, and standing, fermenting and culturing for 48 hours at 37 ℃ to obtain rhodiola rosea fermentation liquid;
(4) And (3) separating and purifying: centrifuging the rhodiola rosea fermentation liquor obtained in the step (3) at 12000rpm for 20min to obtain a supernatant, carrying out suction filtration on the centrifuged supernatant, decolorizing and removing impurities through a 400kDa nano membrane, purifying through HP20 macroporous adsorption resin chromatography, eluting with 2% ethanol, and carrying out rotary evaporation to obtain a salidroside purification liquor.
The obtained salidroside purification liquid was detected by High Performance Liquid Chromatography (HPLC), and the detection conditions were the same as in example 1. The detection results are shown in FIG. 6.
Combining a standard curve and an HPLC detection map, calculating to obtain the salidroside concentration of the salidroside purification liquid obtained in the comparative example to be 0.36mg/mL, the quality to be 196.3mg, and the extraction rate of the salidroside to be 70.1%; drying to obtain salidroside powder with purity of 35.8%.
Compared with the embodiment 1, the extraction rate and the extraction purity of the salidroside in the comparative example 2 are obviously reduced, and the impurity peaks in the HPLC map are more; compared with comparative example 1, the HPLC chromatogram of comparative example 2 has slightly less impurity peaks, and the extraction rate and purity of the obtained salidroside are improved. The results show that after the rhodiola rosea raw material powder is treated by the compound enzyme, the influence of some cellulose impurities on the fermentation extraction and purification of target products is reduced, but the acid hydrolysis treatment is not carried out, so that some impurity substances cannot be utilized by saccharomycetes, and the extraction and purification of salidroside are influenced.
Comparative example 3
A method for extracting and purifying salidroside from radix Rhodiolae powder comprises the following steps:
(1) Pretreatment: 10g of rhodiola rosea powder is taken and placed in a triangular flask, 10mL of 0.1wt% phosphoric acid solution is added, the mixture is treated for 30 minutes at the constant temperature of 121 ℃, after the mixture is stood and cooled to 60 ℃, the pH is adjusted to 5.8 by sodium hydroxide solution, only 100mg of glucanase is added, the mixture is then added to the constant volume of 500mL by pure water, the mixture is placed in a shaking table, and shaking is carried out for 12 hours at 150rpm at 50 ℃ to prepare rhodiola rosea crude extract;
(2) Preparing a saccharomycete seed solution: inoculating Saccharomyces cerevisiae into YPD solid culture medium, performing activation culture in a constant temperature incubator at 30deg.C, inoculating activated Saccharomyces cerevisiae into 1L YPD liquid culture medium, placing in a shaking table at 30deg.C, and shake-culturing at 200rpm until OD 600 is 0.8 to obtain Saccharomyces cerevisiae seed solution;
(3) Fermentation: inoculating the saccharomyces cerevisiae seed liquid obtained in the step (2) into the rhodiola rosea crude extract obtained in the step (1) according to the inoculum size of 5wt%, and standing, fermenting and culturing for 48 hours at 37 ℃ to obtain rhodiola rosea fermentation liquid;
(4) And (3) separating and purifying: centrifuging the rhodiola rosea fermentation liquor obtained in the step (3) at 12000rpm for 20min to obtain a supernatant, carrying out suction filtration on the centrifuged supernatant, decolorizing and removing impurities through a 400kDa nano membrane, purifying through HP20 macroporous adsorption resin chromatography, eluting with 2% ethanol, and carrying out rotary evaporation to obtain a salidroside purification liquor.
The obtained salidroside purification liquid was detected by High Performance Liquid Chromatography (HPLC), and the detection conditions were the same as in example 1. The detection results are shown in FIG. 7.
Combining a standard curve and an HPLC detection map, calculating to obtain the salidroside concentration of the salidroside purification liquid obtained in the comparative example to be 0.41mg/mL, the quality to be 225.1mg, and the extraction rate of the salidroside to be 80.4%; drying to obtain salidroside powder with purity of 40.9%.
Compared with the embodiment 1, the extraction rate and the extraction purity of the salidroside in the comparative example 3 are reduced, and the impurity peaks in the HPLC map are more; compared with comparative example 1, the HPLC chromatogram of the comparative example has few impurity peaks, and the extraction rate and purity of the obtained salidroside are improved. The results show that after the rhodiola rosea raw material powder is treated by acid and glucanase, the influence of impurities such as cellulose on the fermentation extraction and purification of target products is reduced, but the enzymolysis effect is inferior to that of complex enzyme only by adopting the glucanase.
Comparative example 4
A method for extracting and purifying salidroside from radix Rhodiolae powder comprises the following steps:
(1) Pretreatment: 10g of rhodiola rosea powder is taken and placed in a triangular flask, 10mL of 0.1wt% phosphoric acid solution is added, the mixture is treated for 30 minutes at the constant temperature of 121 ℃, the mixture is kept stand and cooled to 60 ℃, the pH is adjusted to 5.8, only 100mg of feruloyl esterase is added, the mixture is then subjected to constant volume to 500mL by pure water, and the mixture is placed in a shaking table, and shaking is carried out for 12 hours at 150rpm at 50 ℃ to prepare rhodiola rosea crude extract;
(2) Preparing a saccharomycete seed solution: inoculating Saccharomyces cerevisiae into YPD solid culture medium, performing activation culture in a constant temperature incubator at 30deg.C, inoculating activated Saccharomyces cerevisiae into 1L YPD liquid culture medium, placing in a shaking table at 30deg.C, and shake-culturing at 200rpm until OD 600 is 0.8 to obtain Saccharomyces cerevisiae seed solution;
(3) Fermentation: inoculating the saccharomyces cerevisiae seed liquid obtained in the step (2) into the rhodiola rosea crude extract obtained in the step (1) according to the inoculum size of 5wt%, and standing, fermenting and culturing for 48 hours at 37 ℃ to obtain rhodiola rosea fermentation liquid;
(4) And (3) separating and purifying: centrifuging the rhodiola rosea fermentation liquor obtained in the step (3) at 12000rpm for 20min to obtain a supernatant, carrying out suction filtration on the centrifuged supernatant, decolorizing and removing impurities through a 400kDa nano membrane, purifying through HP20 macroporous adsorption resin chromatography, eluting with 2% ethanol, and carrying out rotary evaporation to obtain a salidroside purification liquor.
The obtained salidroside purification liquid was detected by High Performance Liquid Chromatography (HPLC), and the detection conditions were the same as in example 1. The detection results are shown in FIG. 8.
Combining a standard curve and an HPLC detection map, calculating to obtain the salidroside concentration of the salidroside purification liquid obtained in the comparative example to be 0.45mg/mL, the quality to be 228.2mg, and the extraction rate of the salidroside to be 81.5%; drying to obtain salidroside powder with purity of 45.2%.
Compared with the embodiment 1, the extraction rate and the extraction purity of the salidroside in the comparative example 4 are reduced, and the impurity peaks in the HPLC map are more; compared with comparative example 1, the HPLC chromatogram of the comparative example has few impurity peaks, and the extraction rate and purity of the obtained salidroside are improved. The results show that after the rhodiola rosea raw material powder is treated by acid and feruloyl esterase, the influence of impurities such as cellulose on the fermentation, extraction and purification of target products is reduced, but the enzymolysis effect is inferior to that of complex enzyme only by using feruloyl esterase.
Comparative example 5
A method for extracting and purifying salidroside from radix Rhodiolae powder comprises the following steps:
(1) Pretreatment: 10g of rhodiola rosea powder is taken and placed in a triangular flask, 10mL of 0.1wt% phosphoric acid solution is added, the mixture is treated for 30 minutes at the constant temperature of 121 ℃, after the mixture is stood and cooled to 60 ℃, the pH is adjusted to 5.8, only 100mg of cellulase is added, the mixture is then added to the constant volume of 500mL of pure water, the mixture is placed in a shaking table, and shaking is carried out for 12 hours at 150rpm at 50 ℃ to prepare rhodiola rosea crude extract;
(2) Preparing a saccharomycete seed solution: inoculating Saccharomyces cerevisiae into YPD solid culture medium, performing activation culture in a constant temperature incubator at 30deg.C, inoculating activated Saccharomyces cerevisiae into 1L YPD liquid culture medium, placing in a shaking table at 30deg.C, and shake-culturing at 200rpm until OD 600 is 0.8 to obtain Saccharomyces cerevisiae seed solution;
(3) Fermentation: inoculating the saccharomyces cerevisiae seed liquid obtained in the step (2) into the rhodiola rosea crude extract obtained in the step (1) according to the inoculum size of 5wt%, and standing, fermenting and culturing for 48 hours at 37 ℃ to obtain rhodiola rosea fermentation liquid;
(4) And (3) separating and purifying: centrifuging the rhodiola rosea fermentation liquor obtained in the step (3) at 12000rpm for 20min to obtain a supernatant, carrying out suction filtration on the centrifuged supernatant, decolorizing and removing impurities through a 400kDa nano membrane, purifying through HP20 macroporous adsorption resin chromatography, eluting with 2% ethanol, and carrying out rotary evaporation to obtain a salidroside purification liquor.
The obtained salidroside purification liquid was detected by High Performance Liquid Chromatography (HPLC), and the detection conditions were the same as in example 1. The detection results are shown in FIG. 9.
Combining a standard curve and an HPLC detection map, calculating to obtain the salidroside concentration of the salidroside purification liquid obtained in the comparative example to be 0.48mg/mL, the quality to be 241.6mg, and the extraction rate of the salidroside to be 86.3%; drying to obtain salidroside powder with purity of 47.7%.
Compared with the embodiment 1, the extraction rate and the extraction purity of the salidroside in the comparative example 5 are reduced, and the impurity peaks in the HPLC map are more; compared with comparative example 1, the HPLC chromatogram of the comparative example has few impurity peaks, and the extraction rate and purity of the obtained salidroside are improved. The results show that after the rhodiola rosea raw material powder is treated by acid and cellulase, the influence of impurities such as cellulose on the fermentation, extraction and purification of target products is reduced, but the enzymolysis effect is inferior to that of complex enzyme by only adopting cellulase.
The results of analysis of the produced salidroside of examples 1 to 3 and comparative examples 1 to 5 are shown in Table 1.
TABLE 1 detection analysis results of inventive examples 1 to 3 and comparative examples 1 to 5
| Rhodiola rosea glycoside extraction rate | Purification of salidroside powder | |
| Example 1 | 90.8％ | 50％ |
| Example 2 | 91.1％ | 51.9％ |
| Example 3 | 92.3％ | 54.1％ |
| Comparative example 1 | 62.3％ | 29.0％ |
| Comparative example 2 | 70.1％ | 35.8％ |
| Comparative example 3 | 80.4％ | 40.9％ |
| Comparative example 4 | 81.5％ | 45.2％ |
| Comparative example 5 | 86.3％ | 47.7％ |
In conclusion, the method of the invention is adopted, after acidolysis, complex enzyme enzymolysis and saccharomycete fermentation, the extraction rate and the product purity of the purified salidroside are greatly improved, the extraction rate of the salidroside reaches 90.8-92.3%, and the purity is 50-55%. In the rhodiola rosea powder raw material, cellulose and other impurities bind the release of the rhodiola rosea glycoside, which is not beneficial to the extraction and purification of the rhodiola rosea glycoside. In addition, the complex enzyme adopted by the invention is prepared by mixing dextranase, feruloyl esterase and cellulase in a mass ratio of 1:1:2, and compared with the comparison results of comparative examples 3-5 and example 1, the effect of using the complex enzyme for enzymolysis is optimal compared with the single enzyme for enzymolysis, and the effects of the three enzymes complement each other to jointly promote the degradation of cellulose and other impurities in rhodiola rosea powder.
Claims (12)
1. A method for extracting and purifying salidroside from rhodiola rosea powder, which is characterized by comprising the following steps:
(1) Pretreatment: mixing rhodiola rosea powder with a phosphoric acid solution, performing heat treatment, adjusting the pH to 4.5-5.8, and adding a complex enzyme, wherein the complex enzyme consists of glucanase, feruloyl esterase and cellulase; performing enzymolysis for 9-12 h at 30-60 ℃ to obtain rhodiola rosea crude extract;
the mass volume ratio of the rhodiola rosea powder to the phosphoric acid solution is 1-3:1, and the unit g/mL;
The heat treatment method comprises the following steps: heat treatment is carried out for 14 to 45 minutes at the temperature of 121 to 160 ℃, and then the mixture is cooled to 50 to 60 ℃;
The mass ratio of dextranase, feruloyl esterase and cellulase in the complex enzyme is 1:1:2;
(2) Preparing a saccharomycete seed solution: activating saccharomycetes, inoculating the activated saccharomycetes into a seed culture medium, and culturing to obtain saccharomycetes seed liquid;
The saccharomycete is Saccharomyces cerevisiae, kluyveromyces marxianus or Kluyveromyces lactis;
(3) Fermentation: inoculating the yeast seed liquid in the step (2) into the rhodiola rosea crude extract in the step (1), and standing and fermenting for 24-72 h at 28-40 ℃ to obtain rhodiola rosea fermentation liquor;
(4) And (3) separating and purifying: centrifuging the rhodiola rosea fermentation liquor obtained in the step (3) to obtain a supernatant, and purifying to obtain a salidroside purification liquor.
2. The method according to claim 1, wherein the rhodiola rosea powder in the step (1) is a rhodiola rosea powder obtained by directly pulverizing dried rhodiola rosea roots or a rhodiola rosea extract powder obtained by extracting with an organic solvent.
3. The method of claim 1, wherein the phosphoric acid solution in step (1) has a concentration of 0.01 to 0.1wt%.
4. The method according to claim 1, wherein the feeding mass ratio of the complex enzyme to the rhodiola rosea powder in the step (1) is (0.05-0.1): (10-50).
5. The method of claim 1, wherein the complex enzyme in step (1) is in the form of a solid powder.
6. The method according to claim 1, wherein the activation in the step (2) is performed by inoculating yeast into YPD solid medium and culturing at 20-37 ℃.
7. The method of claim 1, wherein the seed medium in step (2) is YPD liquid medium.
8. The method according to claim 1, wherein the culturing in the step (2) is carried out at a temperature of 20 to 37℃and a speed of 100 to 200rpm until the yeast seed liquid OD 600 is 0.6 to 0.8.
9. The method of claim 1, wherein the inoculation amount of the yeast seed solution in the step (3) is 5-10% by mass.
10. The method according to claim 1, wherein the centrifugation conditions in step (4) are 8000 to 12000rpm for 10 to 20 minutes.
11. The method of claim 1, wherein the purifying in step (4) is performed by filtering the supernatant, decolorizing and removing impurities through a 400kDa nanofiltration membrane, purifying by chromatography with HP20 macroporous adsorption resin, eluting with 2% ethanol, and steaming with spin to obtain the salidroside purified solution.
12. The method of claim 1, wherein the salidroside purification solution in step (4) is dried to obtain a salidroside purification powder.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2697073A1 (en) * | 2007-08-27 | 2009-03-05 | Iogen Energy Corporation | Method for the production of a fermentation product from a pretreated lignocellulosic feedstock |
| CN104800110A (en) * | 2015-02-23 | 2015-07-29 | 福建师范大学泉港石化研究院 | Method for preparing total sapindus saponins through microbial fermentation |
| CN105543253A (en) * | 2016-02-15 | 2016-05-04 | 江南大学 | Esterase and application thereof |
| CN107857784A (en) * | 2017-12-18 | 2018-03-30 | 四川合盛生物科技有限公司 | A kind of method that rhodioside is extracted from rhodiola root |
| CN109929002A (en) * | 2019-03-06 | 2019-06-25 | 广州翰江生物科技有限公司 | Preparation method of salidroside |
| CN110772460A (en) * | 2019-11-29 | 2020-02-11 | 华熙生物科技股份有限公司 | Preparation method and application of rhodiola rosea fermentation extract |
-
2021
- 2021-09-29 CN CN202111150297.6A patent/CN113897406B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2697073A1 (en) * | 2007-08-27 | 2009-03-05 | Iogen Energy Corporation | Method for the production of a fermentation product from a pretreated lignocellulosic feedstock |
| CN104800110A (en) * | 2015-02-23 | 2015-07-29 | 福建师范大学泉港石化研究院 | Method for preparing total sapindus saponins through microbial fermentation |
| CN105543253A (en) * | 2016-02-15 | 2016-05-04 | 江南大学 | Esterase and application thereof |
| CN107857784A (en) * | 2017-12-18 | 2018-03-30 | 四川合盛生物科技有限公司 | A kind of method that rhodioside is extracted from rhodiola root |
| CN109929002A (en) * | 2019-03-06 | 2019-06-25 | 广州翰江生物科技有限公司 | Preparation method of salidroside |
| CN110772460A (en) * | 2019-11-29 | 2020-02-11 | 华熙生物科技股份有限公司 | Preparation method and application of rhodiola rosea fermentation extract |
Non-Patent Citations (2)
| Title |
|---|
| 常景玲等."预处理对作物秸秆纤维素降解的影响".《江苏农业科学》.2006,(第4期),1.2.4预处理原理和3小结. * |
| 许朵霞.《脂肪替物及其制备新技术》.陕西科学技术出版社,2021,(第1版),第37页. * |
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