CN116003221A - Method for enriching and purifying sclareol from sclareol microbial fermentation liquor - Google Patents
Method for enriching and purifying sclareol from sclareol microbial fermentation liquor Download PDFInfo
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- CN116003221A CN116003221A CN202211431986.9A CN202211431986A CN116003221A CN 116003221 A CN116003221 A CN 116003221A CN 202211431986 A CN202211431986 A CN 202211431986A CN 116003221 A CN116003221 A CN 116003221A
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- XVULBTBTFGYVRC-HHUCQEJWSA-N sclareol Chemical compound CC1(C)CCC[C@]2(C)[C@@H](CC[C@](O)(C)C=C)[C@](C)(O)CC[C@H]21 XVULBTBTFGYVRC-HHUCQEJWSA-N 0.000 title claims abstract description 190
- XVULBTBTFGYVRC-UHFFFAOYSA-N Episclareol Natural products CC1(C)CCCC2(C)C(CCC(O)(C)C=C)C(C)(O)CCC21 XVULBTBTFGYVRC-UHFFFAOYSA-N 0.000 title claims abstract description 95
- LAEIZWJAQRGPDA-UHFFFAOYSA-N Manoyloxid Natural products CC1(C)CCCC2(C)C3CC=C(C)OC3(C)CCC21 LAEIZWJAQRGPDA-UHFFFAOYSA-N 0.000 title claims abstract description 95
- 238000000855 fermentation Methods 0.000 title claims abstract description 35
- 230000004151 fermentation Effects 0.000 title claims abstract description 35
- 238000000034 method Methods 0.000 title claims abstract description 24
- 230000000813 microbial effect Effects 0.000 title claims description 4
- 239000000047 product Substances 0.000 claims abstract description 43
- 238000002425 crystallisation Methods 0.000 claims abstract description 42
- 230000008025 crystallization Effects 0.000 claims abstract description 42
- 239000002904 solvent Substances 0.000 claims abstract description 34
- 239000012452 mother liquor Substances 0.000 claims abstract description 21
- 239000006228 supernatant Substances 0.000 claims abstract description 12
- 238000000605 extraction Methods 0.000 claims abstract description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 55
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 38
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 33
- 239000000284 extract Substances 0.000 claims description 23
- 239000007787 solid Substances 0.000 claims description 22
- 239000000706 filtrate Substances 0.000 claims description 21
- 238000001914 filtration Methods 0.000 claims description 21
- 239000002244 precipitate Substances 0.000 claims description 21
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 20
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 17
- 239000000741 silica gel Substances 0.000 claims description 17
- 229910002027 silica gel Inorganic materials 0.000 claims description 17
- 238000010438 heat treatment Methods 0.000 claims description 14
- 239000007788 liquid Substances 0.000 claims description 11
- 239000003208 petroleum Substances 0.000 claims description 10
- 238000003756 stirring Methods 0.000 claims description 10
- 239000000126 substance Substances 0.000 claims description 10
- 238000011068 loading method Methods 0.000 claims description 2
- 244000005700 microbiome Species 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 238000001556 precipitation Methods 0.000 claims description 2
- 239000000203 mixture Substances 0.000 abstract description 7
- 238000000746 purification Methods 0.000 abstract description 4
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- 239000013078 crystal Substances 0.000 description 35
- 235000019441 ethanol Nutrition 0.000 description 15
- 238000000926 separation method Methods 0.000 description 13
- 238000005406 washing Methods 0.000 description 12
- 238000004458 analytical method Methods 0.000 description 11
- 238000001035 drying Methods 0.000 description 11
- 239000000243 solution Substances 0.000 description 10
- 239000012535 impurity Substances 0.000 description 9
- 238000004811 liquid chromatography Methods 0.000 description 7
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
- 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 6
- 239000008103 glucose Substances 0.000 description 6
- 238000004128 high performance liquid chromatography Methods 0.000 description 6
- 238000002347 injection Methods 0.000 description 6
- 239000007924 injection Substances 0.000 description 6
- 239000001963 growth medium Substances 0.000 description 5
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 4
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 4
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 4
- 230000006835 compression Effects 0.000 description 4
- 238000007906 compression Methods 0.000 description 4
- 229910052760 oxygen Inorganic materials 0.000 description 4
- 239000001301 oxygen Substances 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
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- 238000005516 engineering process Methods 0.000 description 2
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- 235000019319 peptone Nutrition 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000012807 shake-flask culturing Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
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- -1 diterpenoid compound Chemical class 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical group O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
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Abstract
The invention discloses a method for enriching and purifying sclareol from sclareol production strain fermentation liquor. The method comprises (1) centrifuging fermentation broth to obtain thallus and sclareol mixture; (2) Dissolving sclareol in the mixture with an organic solvent, and centrifuging to obtain a supernatant; (3) And (3) after the upper part is steamed to dryness, adding a solvent for crystallization to obtain a sclareol product I. The sclareol content of the product is more than 97%, and the yield is more than 70%; the obtained crystallization mother liquor can be combined with crystallization means by chromatography to obtain sclareol product II, wherein the sclareol content of the product is more than 95 percent, and the yield is more than 65 percent; the total yield of the products I and II can reach more than 90 percent. The method has the advantages of few steps in the extraction and purification process, high yield, simple operation, recycling of the used solvent and environmental friendliness.
Description
Technical Field
The invention discloses a method for enriching and purifying sclareol from fermentation liquor. Belongs to the field of biological functional molecule extraction and purification.
Background
Sclareol (Scalareol) is diterpenoid compound, and has the structure shown below
Is that the growth, development and general metabolism of plants are not necessaryThe product can be used in medicine, cosmetics, health products, essence, spice and pesticide fields.
At present, a lot of reports are about extracting and separating natural sclareol from sclareol inflorescences. With the continuous development of synthetic biology, technology for producing sclareol by using a synthetic biological method is continuously mature. However, there are few reports on downstream work in the microbial fermentation process for producing sclareol, i.e., enrichment and purification of sclareol from fermentation broth. Patent (application number 202210267083.5) discloses a method for extracting and purifying sclareol from sclareol fermentation broth, however, the separation object of the process is sclareol generated in cells, and for the fermentation broth of most of the microorganisms producing sclareol in cells, the facing impurities to be separated are more complex due to the complexity of the components of the fermentation broth, and the corresponding enrichment and purification technology is lacking at present.
Disclosure of Invention
The invention aims at the defects and provides a method for enriching and purifying sclareol from fermentation liquor. The concrete contents are as follows:
a method for enriching and purifying sclareol from sclareol fermentation liquor mainly comprises the following steps:
(1) Centrifuging the fermentation broth at 3000rpm-15000rpm for 5-30min, removing supernatant, and collecting lower precipitate;
(2) Adding alcohol solvent into the precipitate, stirring at 35-40deg.C for 2-4 hr for sclareol extraction, centrifuging the extractive solution, separating solid from liquid, centrifuging the precipitate again with solvent, repeatedly extracting and centrifuging, extracting and centrifuging for 3-5 times, mixing the supernatant, and concentrating to dry to obtain extract;
(3) Adding solvent into the extract, heating at 50-60deg.C for dissolving, filtering to remove insoluble substances, crystallizing the filtrate, and separating solid from liquid to obtain crystallization mother liquor and purified sclareol product I;
(4) The obtained crystallization mother liquor is directly passed through a silica gel chromatographic column and eluted with n-hexane and ethyl acetate in sequence, n-hexane (volume ml): silica gel (mass g) =5-8:1, ethyl acetate (volume ml): silica gel (mass g) =5-8:1, collecting sclareol-containing component, concentrating to dryness;
(5) And adding a solvent into the dried sample containing sclareol components, heating and dissolving at 50-60 ℃, filtering to remove insoluble substances, and crystallizing the filtrate to obtain a purified sclareol product II.
Wherein, the sediment in the step (1) mainly contains thalli, fermentation liquor including sclareol and water-insoluble compounds in metabolites, and sclareol can be rapidly enriched by a centrifugal means; the purpose of step (2) is to use the difference in solubility to dissolve sclareol in the precipitate in the solvent as much as possible and to prevent dissolution of impurities. The solvent is ethanol water solution with the ethanol volume fraction of 50-100%, preferably 70-90% ethanol, and most preferably 80-85% ethanol; the solvent addition is precipitation: solvent (volume ratio) =1:1 to 1:5, preferably 1:1.5 to 1:3, most preferably 1:2 to 1:2.5.
The purpose of the step (3) is to obtain a sclareol product with high purity by means of crystallization. Wherein the crystallization solvent is one or two of petroleum ether or n-hexane, preferably n-hexane; the solvent is added in the following amount: the solvent (mass volume ratio) is 1:1.5-1:5, preferably 1:1.5-1:3, more preferably 1:2-1:2.5; the crystallization temperature is-5-20 ℃, preferably 5-15 ℃, more preferably 11-12 ℃; the crystallization time is 1 to 20 hours, preferably 5 to 15 hours, more preferably 10 to 12 hours.
In the step (4), the chromatographic column filler is silica gel, two mobile phases are adopted for elution, the mobile phase I is n-hexane which is used for eluting impurities, and the n-hexane (volume ml): silica gel (mass g) =5-8:1, less than 5 times, incomplete impurity elution, more than 8 times, and solvent waste; mobile phase II was ethyl acetate, used for elution of the target, ethyl acetate (volume ml): silica gel (mass g) =5-8:1, less than 5 times, sclareol can remain in the column, more than 8 times, wasting solvent. The loading amount in the step (4) is that a sample (solid mass in crystallization mother liquor): silica gel (mass ratio, g/g) =1:3 to 1:10, preferably 1:4 to 1:8, more preferably 1:5 to 1:6.
The solvent in the step (5) is one of petroleum ether or normal hexane, preferably normal hexane; the solvent is added in the following amount: the solvent (mass volume ratio) is 1:1.5-1:5, preferably 1:1.5-1:3, more preferably 1:2-1:2.5; the crystallization temperature is-5-20 ℃, preferably 5-15 ℃, more preferably 11-12 ℃; the crystallization time is 1 to 20 hours, preferably 5 to 15 hours, more preferably 10 to 12 hours. The crystallization mother liquor obtained in the step can be separated and purified again according to the processes of the steps (4) and (5), and the steps are repeatedly used until all the crystallization mother liquor is separated.
After the fermentation liquor is enriched and purified by the method, the products I and II are respectively determined by liquid chromatography analysis, and the analysis result shows that the content of sclareol in the product I is more than 97 percent and the yield is more than 70 percent; the content of sclareol in the product II is more than 95 percent, and the yield is more than 65 percent; the total yield of the products I and II can reach more than 90 percent.
The invention has the positive effects that:
the method for enriching and purifying sclareol from the sclareol fermentation broth supernatant is few in technical process steps, simple to operate, simple in used solvent, low in toxicity, recyclable and environment-friendly. Meanwhile, the yield is high, the total recovery rate of sclareol in fermentation liquor can reach more than 90%, the production cost is greatly reduced, and the industrialization of the process route for producing sclareol by utilizing a synthetic biological means is possible.
Drawings
FIG. 1 is a liquid chromatograph of the fermentation broth, product I and product II of example 2 (fermentation broth, product II, product I, 97.0% by mass of standard in this order from top to bottom).
Detailed Description
In order to make the objects, technical solutions and technical effects of the present invention more apparent, specific embodiments of the present invention will be described below.
Example 1
The fermentation of sclareol is carried out by using a Saccharomyces cerevisiae strain S7 reported in the document "high-density culture production of sclareol by Saccharomyces cerevisiae engineering bacteria" (journal of bioengineering, 2015, 31 (1): 147-151). The specific process is as follows: inoculating S7 single colony into 1L shake flask (containing shake flask culture medium 400 ml), culturing at 200r/min and 30deg.C for 96 hr to obtain seed solution, and inoculating into 50L fermenter. The initial volume of the culture medium of the fermentation tank in the fermentation tank is 20L, the temperature is 30 ℃, the air flow is 100L/h, the culture medium p H of the fermentation tank is controlled to be about 5.0 by adopting 3mol/L NaOH solution, and the dissolved oxygen is controlled to be more than 20 percent by adjusting the stirring revolution. After the consumption of glucose and ethanol produced in the fermentation broth, 800g/L glucose aqueous solution is used for feeding, so that the final concentration of glucose reaches 20g/L. When the OD600 reaches about 50, feeding is performed in a feeding-dissolved oxygen linkage mode, when the dissolved oxygen is higher than 30%, feeding is started, and when the dissolved oxygen reaches 35%, feeding is stopped, wherein the feeding solution is 800g/L glucose aqueous solution. Discharging the tank after culturing for 196h, stirring evenly, and sampling and detecting that the sclareol content in the fermentation liquor is 820-1200mg/L.
Wherein the shake flask culture medium comprises the following components: glucose 20g/L, peptone 20g/L, yeast powder 10g/L, and water as a solvent;
the components of the culture medium of the fermentation tank are as follows: glucose 20g/L, peptone 20g/L, yeast powder 10g/L, potassium dihydrogen phosphate 3g/L, and water as a solvent.
Example 2
Step 1) 30L of the fermentation broth of example 1 was centrifuged at 5000rpm for 15 minutes and the supernatant was removed to obtain 3000mL of a lower precipitate.
Step 2) extracting the precipitate with 95% ethanol for 4 times, wherein the dosage of the precipitate is 3000mL each time, adding 95% ethanol each time, stirring for 4 hours at 35 ℃, and then centrifuging for solid-liquid separation; the extracts were combined 4 times and concentrated to dryness to give 44.4g of extract (sclareol mass content 55.4%).
Step 3) adding 66.6mL of normal hexane into the extract, heating and dissolving at 55 ℃, filtering to remove insoluble substances, standing the filtrate at 20 ℃ for crystallization, filtering the crystal after 20 hours, washing with about 30mL-10 ℃ of cold normal hexane, drying the crystal to obtain a purified sclareol product I18.3g (white needle-shaped crystal), and analyzing by high performance liquid chromatography, wherein the mass purity is 97.1%, and the single-step mass yield reaches 72.2%. The filtrate and the washing solution were simultaneously combined to obtain 80ml of a mother liquor for crystallization.
Step 4) the above-mentioned crystallization mother liquor (solid mass 25.0g, sclareol-containing 6.84 g) was subjected to column separation. The column is a glass column, 75g of 200 mesh silica gel is filled, after sample injection is finished, 375ml of normal hexane is used for eluting nonpolar impurities, 375ml of ethyl acetate is used for eluting sclareol, the components containing sclareol are combined, and the mixture is concentrated to dryness, so that 8.4g of solid is obtained.
Step 5) adding 12.6mL of normal hexane to the solid, heating and dissolving at 55 ℃, standing the filtrate at 12 ℃ for crystallization, filtering the crystals and drying the crystals after 12 hours to obtain 4.74g (white needle-like crystals) of a purified sclareol product II, wherein the mass purity is 95.8% through liquid chromatography analysis, and the single-step mass yield reaches 66.4%. The total yield of the products I and II is up to 90.7%.
Example 3
Step 1) 30L of the fermentation broth of example 1 was centrifuged at 3000rpm for 30 minutes and the supernatant was removed to obtain 3000mL of a lower precipitate.
Step 2) extracting the precipitate with 50% ethanol for 5 times, wherein the dosage of the precipitate is 13.5L each time, adding 50% ethanol each time, stirring for 2 hours at 40 ℃, and then centrifuging for solid-liquid separation; the extracts were combined 5 times and concentrated to dryness to give 51.4g of extract (sclareol content 46.7%).
Step 3) adding 257mL petroleum ether into the extract, heating and dissolving at 50 ℃, filtering to remove insoluble substances, standing the filtrate at-5 ℃ for crystallization, filtering the crystal after 1h, washing with about 30mL-10 ℃ cold petroleum ether, drying the crystal to obtain a purified sclareol product I17.4g (white needle-like crystal), and carrying out high performance liquid chromatography analysis to obtain the sclareol product I17.4g with the quality purity of 97.0%, wherein the single-step quality yield reaches 70.0%. The filtrate and washings were combined simultaneously to give 260mL of a mother liquor for crystallization.
Step 4) the above-mentioned crystallization mother liquor (solid mass 33.2g, sclareol-containing 7.21 g) was subjected to column separation. The column is a glass column, 332g of 200 mesh silica gel is filled, after sample injection is finished, 2656ml of normal hexane is used for eluting nonpolar impurities, 2656ml of ethyl acetate is used for eluting sclareol, the components containing sclareol are combined, and the mixture is concentrated to dryness, so that 8.1g of solid is obtained.
Step 5) adding 40.5ml petroleum ether to the solid, heating and dissolving at 50 ℃, standing the filtrate at 20 ℃ for crystallization, filtering the crystals and drying the crystals after 20 hours to obtain 4.85g (white needle-shaped crystals) of a purified sclareol product II, wherein the mass purity is 97.2% by liquid chromatography analysis, and the single-step mass yield reaches 65.4%. The total yield of the products I and II reaches 90.0 percent.
Example 4
Step 1) 30L of the fermentation broth of example 1 was centrifuged at 15000rpm for 5 minutes, and the supernatant was removed to obtain 2500ml of a lower precipitate.
Step 2) extracting the precipitate with 90% ethanol for 3 times, wherein the dosage is 10L each time, adding 90% ethanol each time, stirring for 3 hours at 38 ℃, and then centrifuging for solid-liquid separation; the extracts were combined 3 times and concentrated to dryness to give 46.5g of extract (sclareol content 50.8%).
Step 3) adding 200mL petroleum ether into the extract, heating and dissolving at 55 ℃, filtering to remove insoluble substances, standing the filtrate at 4 ℃ for crystallization, filtering the crystal after 5 hours, washing the crystal with about 30mL-10 ℃ cold petroleum ether, drying the crystal to obtain a purified sclareol product I17.5g (white needle-like crystal), and carrying out high performance liquid chromatography analysis to obtain the sclareol product I17.5g with the mass purity of 97.4%, wherein the single-step mass yield reaches 72.1%. The filtrate and the washing solution were simultaneously combined to obtain 210ml of a mother liquor for crystallization.
Step 4) the above-mentioned crystallization mother liquor (solid mass 28.0g, sclareol-containing 6.58 g) was subjected to column separation. The column is a dynamic axial compression column, 84g of 400 mesh silica gel is filled, after sample injection is finished, 420ml of normal hexane is used for eluting nonpolar impurities, 420ml of ethyl acetate is used for eluting sclareol, the sclareol-containing components are combined, and the mixture is concentrated to dryness, so that 8.8g of solid is obtained.
Step 5) adding 25ml petroleum ether to the solid, heating and dissolving at 60 ℃, standing the filtrate at-5 ℃ for crystallization, filtering the crystals after 1h, and drying to obtain 4.40g (white needle-like crystals) of a purified sclareol product II, wherein the mass purity is 97.5% through liquid chromatography analysis, and the single-step mass yield reaches 65.1%. The total yield of the products I and II is up to 90.3%.
Example 5 (optimal conditions)
Step 1) 30L of the fermentation broth of example 1 was centrifuged at 5000rpm for 15 minutes and the supernatant was removed to obtain 3000ml of a lower precipitate.
Step 2) extracting the precipitate with 85% ethanol for 4 times, wherein the dosage is 6L each time, adding 85% ethanol each time, stirring for 4 hours at 35 ℃, and then centrifuging for solid-liquid separation; the extracts were combined 4 times and concentrated to dryness to give 45.2g of extract (sclareol content 54.4%).
Step 3) adding 95ml of normal hexane into the extract, heating and dissolving at 55 ℃, filtering to remove insoluble substances, standing the filtrate at 12 ℃ for crystallization, filtering the crystal after 12 hours, washing the crystal with cold normal hexane at-10 ℃, drying the crystal to obtain a purified sclareol product I18.4g (white needle-shaped crystal), and carrying out high performance liquid chromatography analysis to obtain the sclareol product I with the mass purity of 98.2% and the single-step mass yield of 73.4%. The filtrate and the washing solution were simultaneously combined to obtain 105ml of a mother liquor for crystallization.
Step 4) the above-mentioned crystallization mother liquor (solid mass 26.2g, sclareol-containing 6.51 g) was subjected to column separation. The column is a dynamic axial compression column, 150g of 400-mesh silica gel is filled, after sample injection is finished, 900ml of normal hexane is used for eluting nonpolar impurities, 900ml of ethyl acetate is used for eluting sclareol, the sclareol-containing components are combined, and the mixture is concentrated to be dry, so that 6.8g of solid is obtained.
Step 5) adding 15ml of normal hexane into the solid, heating and dissolving at 55 ℃, standing the filtrate at 11 ℃ for crystallization, filtering the crystal after 11 hours, and drying to obtain 4.67g (white needle-shaped crystal) of a purified sclareol product II, wherein the mass purity is 97.8% through liquid chromatography analysis, and the single-step mass yield reaches 70.1%. The total yield of the products I and II reaches 91.9 percent.
Comparative example 1
Step 1) 30L of the fermentation broth of example 1 was centrifuged at 5000rpm for 15 minutes and the supernatant was removed to obtain 3000mL of a lower precipitate.
Step 2), extracting the precipitate with absolute ethyl alcohol for 4 times, wherein the dosage is 6L each time, adding absolute ethyl alcohol each time, stirring for 5 hours at 30 ℃, and then centrifuging for solid-liquid separation; the extracts were combined 4 times and concentrated to dryness to give 40.3g of extract (sclareol content 53.1%).
Step 3) adding 160ml of acetone into the extract, heating and dissolving at 55 ℃, filtering to remove insoluble substances, standing the filtrate at 4 ℃ for crystallization, filtering the crystals after 4 hours, washing the crystals with about 30ml-10 ℃ of cold acetone, drying the crystals to obtain a purified sclareol product I14.2 (white needle-like crystals) g, and carrying out high performance liquid chromatography analysis to obtain the sclareol product I14.2 with the purity of 95.5% and the single-step yield of 63.4%. The filtrate and washings were combined simultaneously to give 175ml of mother liquor for crystallization.
Step 4) the above-mentioned crystallization mother liquor (solid mass 26g, sclareol-containing 7.83 g) was taken and subjected to column separation. The column is a dynamic axial compression column, 150g of 400-mesh silica gel is filled, after sample injection is finished, 1.5L of solvent is used for eluting, and the eluting solvent is 120 # solvent: petroleum ether: acetone=3:1:1, combining sclareol-containing fractions, and concentrating to dryness to give 4.41g (pale yellow needle crystals) of purified sclareol product II, which was analyzed by liquid chromatography to a purity of 89.8% and a single step yield of 50.5%. The total yield of products I and II was 81.9%.
Comparative example 2
Step 1) 30L of the fermentation broth of example 1 was centrifuged at 5000rpm for 15 minutes and the supernatant was removed to obtain 3000ml of a lower precipitate.
Step 2) extracting the precipitate with 45% ethanol for 5 times, wherein the dosage is 3L each time, adding 45% ethanol each time, stirring for 2 hours at 40 ℃, and then centrifuging for solid-liquid separation; the extracts were combined 5 times and concentrated to dryness to give 62.4g of extract (sclareol content 38.7%).
Step 3) adding 350ml of normal hexane into the extract, heating and dissolving at 50 ℃, filtering to remove insoluble substances, standing the filtrate at 25 ℃ for crystallization, filtering the crystal after 48 hours, washing the crystal with about 30ml-10 ℃ of cold normal hexane, drying the crystal to obtain 12.2g (white needle-like crystals) of purified sclareol product I, and analyzing the purified sclareol product I by high performance liquid chromatography, wherein the purity is 97.1%, and the single-step yield reaches 49.0%. The filtrate and the washing solution were simultaneously combined to obtain 380ml of a mother liquor for crystallization.
Step 4) the above-mentioned crystallization mother liquor (solid mass 40g, sclareol-containing 12.3 g) was subjected to column separation. The column is a dynamic axial compression column, 100g of 400-mesh silica gel is filled, after sample injection is finished, nonpolar impurities are eluted by 500ml of hexane, sclareol is eluted by 500ml of ethyl acetate, the sclareol-containing components are combined, and the mixture is concentrated to dryness, so that 15.8g of solid is obtained.
Step 5) adding 88ml of normal hexane into the solid for dissolution, standing the filtrate at-6 ℃ for crystallization, filtering the crystal after 0.5h, and drying to obtain 6.62g (pale yellow needle-shaped crystal) of a purified sclareol product II, wherein the purity is 85.8% through liquid chromatography analysis, and the single-step yield reaches 46.2%. The total yield of the products I and II was 72.6%.
Example parameter summary table
Claims (8)
1. The method for enriching and purifying sclareol from sclareol microbial fermentation liquor is characterized by comprising the following main steps of:
1) Centrifuging sclareol microorganism fermentation broth at 3000-15000 rpm for 5-30min, removing supernatant, and collecting lower precipitate;
2) Adding alcohol solvent into the precipitate, stirring at 35-40deg.C for 2-4 hr for sclareol extraction, centrifuging the extractive solution, separating solid from liquid, centrifuging the precipitate again with alcohol solvent, repeatedly extracting and centrifuging, extracting and centrifuging for 3-5 times, mixing the supernatant, and concentrating to dry to obtain extract;
3) Adding solvent into the extract, heating at 50-60deg.C for dissolving, filtering to remove insoluble substances, crystallizing the filtrate, and separating solid from liquid to obtain crystallization mother liquor and purified sclareol product I;
4) The obtained crystallization mother liquor is directly passed through a silica gel chromatographic column and eluted with n-hexane and ethyl acetate in sequence, n-hexane (volume ml): silica gel (mass g) =5-8:1, ethyl acetate (volume ml): silica gel (mass g) =5-8:1, collecting sclareol-containing component, concentrating to dryness;
5) And adding a solvent into the dried sample containing sclareol components, heating and dissolving at 50-60 ℃, filtering to remove insoluble substances, and crystallizing the filtrate to obtain a purified sclareol product II.
2. The method according to claim 1, wherein the alcohol solvent used in step (2) is 50-95% ethanol by volume, preferably 70-90% ethanol by volume, more preferably 75-85% ethanol by volume.
3. The method according to claim 1 or 2, characterized in that: the single alcohol solvent addition in step (2) is precipitation: solvent (volume ratio) =1:1 to 1:4.5, preferably 1:1.5 to 1:3, more preferably 1:2 to 1:2.5.
4. The method according to claim 1, wherein the solvent in step (3) and step (5) is one or both of petroleum ether or n-hexane, respectively, preferably n-hexane.
5. The method according to claim 1, wherein the solvent is added in the amounts of the extracts in the step (3) and the step (5), respectively: solvent (mass to volume ratio, g/ml) =1:1.5 to 1:5, preferably 1:1.5 to 1:3, more preferably 1:2 to 1:2.5.
6. The process according to claim 1, 4 or 5, wherein the crystallization temperature in step (3) and step (5), respectively, is-5 to 20 ℃, preferably 5 to 15 ℃, more preferably 11 to 12 ℃.
7. The process according to claim 6, wherein the crystallization time in step (3) and step (5) is 1 to 20 hours, preferably 5 to 15 hours, more preferably 10 to 12 hours, respectively.
8. The method for enriching and purifying sclareol from sclareol fermentation broth according to claim 1, wherein the loading in step (4) is a sample (solid mass in crystallization mother liquor): silica gel (mass ratio, g/g) =1:3 to 1:10, preferably 1:4 to 1:8, more preferably 1:5 to 1:6.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR19980071950A (en) * | 1998-07-07 | 1998-10-26 | 노경호 | Extraction and Separation of Peryl alcohol and D-limonene from Korean Tangerine Peel |
| US20060134083A1 (en) * | 2004-12-17 | 2006-06-22 | David Peele | Compositions comprising sclareol or derivatives thereof and uses thereof |
| CN113502306A (en) * | 2021-07-12 | 2021-10-15 | 江南大学 | Method for producing sclareolide by catalyzing sclareol |
| CN114573421A (en) * | 2022-03-17 | 2022-06-03 | 北京理工大学 | Method for extracting and purifying sclareol from sclareol fermentation liquor |
| CN114988983A (en) * | 2022-06-09 | 2022-09-02 | 新疆师范大学 | Method for separating sclareol from sclareol extract |
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- 2022-11-16 CN CN202211431986.9A patent/CN116003221A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR19980071950A (en) * | 1998-07-07 | 1998-10-26 | 노경호 | Extraction and Separation of Peryl alcohol and D-limonene from Korean Tangerine Peel |
| US20060134083A1 (en) * | 2004-12-17 | 2006-06-22 | David Peele | Compositions comprising sclareol or derivatives thereof and uses thereof |
| CN113502306A (en) * | 2021-07-12 | 2021-10-15 | 江南大学 | Method for producing sclareolide by catalyzing sclareol |
| CN114573421A (en) * | 2022-03-17 | 2022-06-03 | 北京理工大学 | Method for extracting and purifying sclareol from sclareol fermentation liquor |
| CN114988983A (en) * | 2022-06-09 | 2022-09-02 | 新疆师范大学 | Method for separating sclareol from sclareol extract |
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