CN114181050A - Extraction method of fermented thallus cannabidiol - Google Patents
Extraction method of fermented thallus cannabidiol Download PDFInfo
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Abstract
The application relates to the field of cannabidiol extraction, in particular to a cannabidiol treatment method of fermentation thalli, which comprises the following steps: pretreating thalli: treating the fermentation thallus into thallus particles; subcritical extraction: under the pressurization state, extracting the thallus particles by a solvent A to obtain an extracting solution and mushroom dregs; the step can be carried out any time of extracting solution treatment: removing the solvent from the extracting solution under reduced pressure to obtain an extract; and (3) extract treatment: decarboxylating the extract, and further separating to obtain cannabidiol; wherein, the solvent A is any one of pentane, hexane, heptane, methanol and ethanol, or a mixed system of any two or more than two. In the technical scheme, the subcritical extraction method is adopted, and the solvent is selected, so that the yield and the extraction rate are high, the content of impurities in the extract is reduced, and the application prospect is good.
Description
Technical Field
The application relates to the field of cannabidiol extraction, in particular to a method for treating fermentation thallus cannabidiol.
Background
Cannabidiol is a cannabinoids compound and has high use value. The extraction method of cannabidiol mainly comprises three methods of plant flower and leaf tissue extraction, chemical synthesis and microbial fermentation, and the specific extraction method mainly comprises static intermittent method of an extraction tank and supercritical fluid extraction.
In the microbial fermentation method, because a microbial fermentation thallus system is complex, the extraction yield is lower when a common solvent is adopted for static intermittent extraction in the extraction process of thallus. And by adopting supercritical fluid extraction, although the yield is high, the single treatment capacity is small, the equipment cost is high, and the method is not suitable for large-scale production.
Disclosure of Invention
In order to provide a cannabidiol extraction method with high yield and low equipment cost, the application provides a fermentation thallus cannabidiol treatment method.
In the application, the method for treating the fermentation thallus cannabidiol specifically comprises the following steps:
pretreating thalli: treating the fermentation thallus into thallus particles;
subcritical extraction: under the pressurization state, extracting the thallus particles by a solvent A to obtain an extracting solution and mushroom dregs; this step can be carried out any number of times
Treating an extracting solution: removing the solvent from the extracting solution under reduced pressure to obtain an extract;
and (3) extract treatment: decarboxylating the extract, and further separating to obtain cannabidiol;
wherein, the solvent A is any one of pentane, hexane, heptane, methanol and ethanol, or a mixed system of any two or more than two.
In the technical scheme, the subcritical extraction method is adopted, compared with supercritical extraction, the subcritical extraction condition is milder, the requirement on equipment is low, the subcritical extraction can be directly carried out through pressurization treatment by virtue of the closed extraction tank, liquid in the extraction tank after extraction can be directly pressurized and conveyed to other equipment by virtue of high-pressure gas, the whole process is simple, and the method is suitable for industrial large-scale production.
Compared with the method of directly extracting by using a solvent, the method of extracting by using the subcritical extraction method can extract at a lower temperature on one hand, has a higher extraction effect on the other hand, and has a higher yield compared with the method of directly extracting.
In the application, any number of pentane, hexane, heptane, methanol and ethanol are used as solvents for extraction, the solvents have low viscosity and low surface tension, the diffusion coefficient of the solvents is high, rapid extraction can be achieved, and meanwhile, the extraction time is shortened, the required extraction temperature is reduced, damage and loss of cannabidiol can be reduced, and the yield is further improved.
Optionally, subcritical extraction is carried out each time, and the mass-volume ratio of the thalli to the solvent A is 1g to (1-8 mL).
By adopting the solute ratio, the cannabidiol component in the thallus can be extracted as much as possible in the extraction times of not more than six times, and the requirement on subsequent treatment is low. Within the above range of solute ratio, it is the most economical practical method.
Optionally, the pressure is 0.2-1.2 MPa in subcritical extraction.
Within the pressure range, the requirement on equipment is low, the implementation is easy, and the fluidity and the diffusivity of the fluid within the range are both good, so that the method is favorable for fully extracting the cannabidiol in the thallus particles.
Optionally, in the subcritical extraction, the time for each extraction is 10-60 min.
In the technical scheme, the extraction is carried out for 10-60 min, the dissolution balance of single extraction can be basically realized, and meanwhile, the cannabidiol is not easy to lose in the extraction process due to short extraction time control.
Optionally, the extraction temperature is 20-50 ℃.
Within the temperature range, the cannabidiol can be fully dissolved in the solvent, the loss of the cannabidiol caused by heating is reduced, and the yield of the cannabidiol is further improved.
Optionally, the method further comprises the following steps:
and (3) treatment of mushroom dregs: and (3) carrying out decompression desolventizing on the fungus dregs obtained after subcritical extraction, wherein the pressure of the decompression desolventizing is not higher than-0.085 MPa.
According to the technical scheme, the mushroom dregs are treated, the solvent can be recovered after the mushroom dregs are desolventized, and meanwhile, the mushroom dregs can be continuously used as a raw material of a fertilizer or a feed, so that cost saving of enterprises is facilitated.
Optionally, the step of processing the extract comprises the following sub-steps:
concentrating the extracting solution under reduced pressure to obtain a crude extracting solution;
performing winterization impurity removal on the crude liquid to obtain winterization mixed liquid;
and (4) further performing negative pressure desolventizing on the winterization mixed liquor to obtain an extract.
In the technical scheme, the extracting solution is subjected to reduced pressure concentration, and then winterization and impurity removal are carried out, and then negative pressure desolventizing is further carried out. In the process, after the vacuum concentration and winterization impurity removal, residual fat components in the extracting solution can be removed, meanwhile, cannabidiol is basically not affected, and further desolventizing is carried out to obtain an extract, so that the cannabidiol content in the extract is higher, the impurities are less, and the subsequent further separation is facilitated.
Optionally, the temperature of the extracting solution is controlled to be 40-60 ℃ in the whole process of decompression and concentration of the extracting solution.
Within the temperature range, the solvent can be quickly removed, the influence on cannabidiol is small, and optionally after the extracting solution is subjected to reduced pressure concentration, the mass fraction of solids in the crude body fluid is 1-5%.
And (3) keeping 1-5% of solid components by mass fraction, and entering a winterization step, so that the winterization impurity removal effect is improved, and meanwhile, the loss of cannabidiol in the winterization impurity removal process is ensured to be as small as possible.
Optionally, in the step of thallus pretreatment, the particle size of thallus particles is controlled to be 10-120 meshes.
The thallus particles in the particle size range are easy to realize, have good dissolving performance, have low requirements on equipment due to large integral particle size, and are not easy to cause cannabidiol loss in the crushing process.
In general, the present application includes at least one of the following benefits:
1. in the application, cannabidiol in the fermentation thalli is extracted by a subcritical extraction method, and pentane, hexane, heptane, methanol and ethanol are used, so that the yield is high, and the equipment cost is low.
2. In the application, the extract is concentrated and winterized, and then is further desolventized to obtain the extract, so that the content of cannabidiol in the extract is increased, and a good foundation is laid for subsequent further separation.
Detailed Description
The present application will be described in further detail with reference to examples.
In this example, the cells used were obtained by fermentation of a single batch, and the fermentation strain was Penicillium.
The determination of the cannabidiol is carried out by liquid chromatography, a sample to be determined is crushed and fully dissolved to prepare a solution, a standard solution is prepared to draw a standard curve, and then the standard solution is substituted into a regression equation to determine the cannabidiol concentration in the solution, and further the cannabidiol content in the extract and the cannabidiol extraction rate are calculated. The mobile phase used for liquid chromatography is a mixed phase prepared by acetonitrile and water in a mass ratio of 1: 1.
Example 1-1, extraction of cannabidiol from bacterial cells using heptane as solvent, comprises the following steps: pretreating thalli: weighing 1000g of cannabidiol thallus sample (containing 16.3mg/g of cannabidiol), crushing by a crusher, and screening components with the granularity of 10-120 meshes to obtain thallus particles;
subcritical extraction: and (2) placing the thallus particles in a closed extraction tank, vacuumizing, adding a heptane solvent, controlling the temperature fluctuation range to be 20-25 ℃ and the pressure to be 0.85-0.88 MPa, mixing and stirring, wherein the solvent dosage, the extraction times and the extraction time are shown in table 1.
Treating an extracting solution: pumping the extracting solution into an evaporating pot through compressed air, controlling the temperature in the evaporating pot to fluctuate within the range of 40-45 ℃, starting a vacuum pump, controlling the air pressure to be lower than-0.085 MPa, evaporating, and concentrating until the solid content is 1.5 wt% to obtain crude body fluid; cooling the crude liquid to-65 deg.C at room temperature for winterization for 8h, filtering to obtain winterization liquid, desolventizing at negative pressure below-0.085 MPa, and removing solvent to obtain fructus Cannabis extract.
In examples 1-2 to 1-7, the extraction time, the amount of solvent used, and the number of times of extraction were adjusted on the basis of example 1-1, and specific extraction parameters are shown in table 1.
TABLE 1, parameter tables of examples 1-1 to 1-7
Examples 1 to 8 to 1-12, the temperature and pressure of supercritical extraction were adjusted in the same manner as in example 1-3 using heptane as a solvent, and the results are shown in Table 2.
TABLE 2 adjustment of the different subcritical conditions in examples 1-8 to 1-12
In addition to the above examples, the following examples were obtained by adjusting the treatment of the extract.
Examples 1 to 13 are different from examples 1 to 3 in that the solid content of the concentrated crude body fluid is 3%, the cannabidiol mass fraction in the extract is 49.2%, and the extraction rate is 96.8%.
Examples 1 to 14 are different from examples 1 to 3 in that the solid content of the concentrated crude body fluid is 5%, the cannabidiol mass fraction in the extract is 49.0%, and the extraction rate is 96.9%.
Examples 1 to 15 differ from examples 1 to 3 in that the solid content of the concentrated crude body fluid was 8%, the cannabidiol mass fraction in the extract was 45.1%, and the extraction rate was 95.8%.
Examples 1 to 16 are different from examples 1 to 3 in that the solid content of the concentrated crude body fluid is 1%, the cannabidiol mass fraction in the extract is 49.2%, and the extraction rate is 96.9%.
Examples 1 to 17 are different from examples 1 to 3 in that, when the crude liquid is concentrated under reduced pressure, the temperature is controlled to be 55 to 60 ℃, the mass fraction of cannabidiol in the extract is 48.9%, and the extraction rate is 96.9%.
Example 2, cannabidiol in the cells was extracted using n-hexane, the specific steps were as follows:
pretreating thalli: weighing 1000g of cannabidiol thallus sample (containing 16.3mg/g of cannabidiol), crushing by a crusher, and screening components with the granularity of 10-120 meshes to obtain thallus particles;
subcritical extraction: placing the thallus particles in a closed extraction tank, vacuumizing, adding n-hexane solvent, extracting for three times, wherein the extraction time is 30min each time, the dosage of the solvent is 3L each time, mixing and stirring, and the specific extraction parameters are shown in Table 3.
Treating an extracting solution: pumping the extracting solution into an evaporating pot through compressed air, controlling the temperature in the evaporating pot to fluctuate within the range of 40-45 ℃, starting a vacuum pump, controlling the air pressure to be lower than-0.085 MPa, evaporating, and concentrating until the solid content is 1.5 wt% to obtain crude body fluid; cooling the crude liquid to-65 deg.C at room temperature for winterization for 8h, filtering to obtain winterization liquid, desolventizing at negative pressure below-0.085 MPa, and removing solvent to obtain fructus Cannabis extract.
TABLE 3 adjustment of the different subcritical conditions in examples 2-1 to 2-6
Example 3, cannabidiol in the cells was extracted using n-pentane, which specifically included the following steps:
pretreating thalli: weighing 1000g of cannabidiol thallus sample (containing 16.3mg/g of cannabidiol), crushing by a crusher, and screening components with the granularity of 10-120 meshes to obtain thallus particles;
subcritical extraction: placing the thallus particles in a closed extraction tank, vacuumizing, adding n-pentane solvent, extracting for three times, wherein the extraction time is 30min each time, the dosage of the solvent is 3L each time, mixing and stirring, and the specific extraction parameters are shown in Table 4.
Treating an extracting solution: pumping the extracting solution into an evaporating pot through compressed air, controlling the temperature in the evaporating pot to fluctuate within the range of 40-45 ℃, starting a vacuum pump, controlling the air pressure to be lower than-0.085 MPa, evaporating, and concentrating until the solid content is 1.5 wt% to obtain crude body fluid; cooling the crude liquid to-65 deg.C at room temperature for winterization for 8h, filtering to obtain winterization liquid, desolventizing at negative pressure below-0.085 MPa, and removing solvent to obtain fructus Cannabis extract.
TABLE 4 adjustment of the different subcritical conditions in examples 3-1 to 3-6
Example 4, cannabidiol in the cells was extracted with methanol, the specific steps were as follows:
pretreating thalli: weighing 1000g of cannabidiol thallus sample (containing 16.3mg/g of cannabidiol), crushing by a crusher, and screening components with the granularity of 10-120 meshes to obtain thallus particles;
subcritical extraction: placing thallus particles in a closed extraction tank, vacuumizing, adding methanol solvent, extracting for three times, wherein the extraction time is 30min each time, the dosage of the solvent is 3L each time, mixing and stirring, and the specific extraction parameters are shown in Table 5.
Treating an extracting solution: pumping the extracting solution into an evaporating pot through compressed air, controlling the temperature in the evaporating pot to fluctuate within the range of 40-45 ℃, starting a vacuum pump, controlling the air pressure to be lower than-0.085 MPa, evaporating, and concentrating until the solid content is 1.5 wt% to obtain crude body fluid; cooling the crude liquid to-65 deg.C at room temperature for winterization for 8h, filtering to obtain winterization liquid, desolventizing at negative pressure below-0.085 MPa, and removing solvent to obtain fructus Cannabis extract.
TABLE 5 adjustment of the different subcritical conditions in examples 4-1 to 4-6
Example 5, cannabidiol in the cells was extracted with ethanol, the specific steps were as follows:
pretreating thalli: weighing 1000g of cannabidiol thallus sample (containing 16.3mg/g of cannabidiol), crushing by a crusher, and screening components with the granularity of 10-120 meshes to obtain thallus particles;
subcritical extraction: placing thallus particles in a closed extraction tank, vacuumizing, adding ethanol solvent, extracting for three times, wherein the extraction time is 30min each time, the dosage of the solvent is 3L each time, mixing and stirring, and the specific extraction parameters are shown in Table 6.
Treating an extracting solution: pumping the extracting solution into an evaporating pot through compressed air, controlling the temperature in the evaporating pot to fluctuate within the range of 40-45 ℃, starting a vacuum pump, controlling the air pressure to be lower than-0.085 MPa, evaporating, and concentrating until the solid content is 1.5 wt% to obtain crude body fluid; cooling the crude liquid to-65 deg.C at room temperature for winterization for 8h, filtering to obtain winterization liquid, desolventizing at negative pressure below-0.085 MPa, and removing solvent to obtain fructus Cannabis extract.
TABLE 6 adjustment of the different subcritical conditions in examples 5-1 to 5-6
For the above examples, comparative examples were set as follows.
Comparative example 1, cannabidiol in the cells was extracted with petroleum ether, the specific steps were as follows:
pretreating thalli: weighing 1000g of cannabidiol thallus sample (containing 16.3mg/g of cannabidiol), crushing by a crusher, and screening components with the granularity of 10-120 meshes to obtain thallus particles;
subcritical extraction: placing the thallus particles in a closed extraction tank, vacuumizing, adding petroleum ether solvent, extracting for three times, wherein the extraction time is 30min each time, the dosage of the solvent is 3L each time, mixing and stirring, and the specific extraction parameters are shown in Table 7.
Treating an extracting solution: pumping the extracting solution into an evaporating pot through compressed air, controlling the temperature in the evaporating pot to fluctuate within the range of 40-45 ℃, starting a vacuum pump, controlling the air pressure to be lower than-0.085 MPa, evaporating, and concentrating until the solid content is 1.5 wt% to obtain crude body fluid; cooling the crude liquid to-65 deg.C at room temperature for winterization for 8h, filtering to obtain winterization liquid, desolventizing at negative pressure below-0.085 MPa, and removing solvent to obtain fructus Cannabis extract.
TABLE 7 adjustment of different subcritical conditions in comparative examples 1-1 to 1-6
Comparative example 2, cannabidiol in the cells was extracted with acetone, the specific steps were as follows:
pretreating thalli: weighing 1000g of cannabidiol thallus sample (containing 16.3mg/g of cannabidiol), crushing by a crusher, and screening components with the granularity of 10-120 meshes to obtain thallus particles;
subcritical extraction: placing thallus particles in a closed extraction tank, vacuumizing, adding acetone solvent, extracting for three times, wherein the extraction time is 30min each time, the dosage of the solvent is 3L each time, mixing and stirring, and the specific extraction parameters are shown in Table 8.
Treating an extracting solution: pumping the extracting solution into an evaporating pot through compressed air, controlling the temperature in the evaporating pot to fluctuate within the range of 40-45 ℃, starting a vacuum pump, controlling the air pressure to be lower than-0.085 MPa, evaporating, and concentrating until the solid content is 1.5 wt% to obtain crude body fluid; cooling the crude liquid to-65 deg.C at room temperature for winterization for 8h, filtering to obtain winterization liquid, desolventizing at negative pressure below-0.085 MPa, and removing solvent to obtain fructus Cannabis extract.
TABLE 8 adjustment of different subcritical conditions in comparative examples 2-1 to 2-6
Comparative examples 3-6, the subcritical extraction step was replaced with normal pressure extraction, the solvents were n-heptane, n-hexane, methanol and ethanol, respectively, the extraction time, temperature, times and solvent dosage remained unchanged, and the specific results are shown in table 10.
Table 10, extraction results of comparative examples 3 to 6
Through comparison between the above examples and comparative examples, it can be easily found that in the present application, the subcritical extraction method is adopted to effectively improve the extraction rate and the cannabidiol mass fraction in the extract, and heptane, hexane, pentane, methanol and ethanol are selected to effectively improve the extraction rate and the cannabidiol content in the extract. Although the acetone has higher extraction rate, the solubility of the acetone is too good, and the acetone has stronger dissolution to other components, so that the obtained hemp extract contains more irrelevant components, and causes greater difficulty to subsequent treatment.
On the whole, the extraction temperature is preferably 25-30 ℃, and in experiments, the screened thallus particles have better extraction effect when the particle size is in the range of 40-80 meshes, the extraction time and the dosage of the solvent can be reduced, but the extraction effectiveness can also be realized in the range of 10-120 meshes. By comprehensive consideration, the particles with 10-120 meshes are also feasible to be taken into the raw materials.
The present embodiment is only for explaining the present application, and it is not limited to the present application, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present application.
Claims (10)
1. The extraction method of the zymophyte cannabidiol is characterized by comprising the following steps:
pretreating thalli: treating the fermentation thallus into thallus particles;
subcritical extraction: under the pressurization state, extracting the thallus particles by a solvent A to obtain an extracting solution and mushroom dregs; this step can be carried out any number of times
Treating an extracting solution: removing the solvent from the extracting solution under reduced pressure to obtain an extract;
and (3) extract treatment: decarboxylating the extract, and further separating to obtain cannabidiol;
wherein, the solvent A is any one of pentane, hexane, heptane, methanol and ethanol, or a mixed system of any two or more than two.
2. The method for extracting cannabidiol from fermented bacteria according to claim 1, wherein the subcritical extraction is performed each time, and the mass-to-volume ratio of the bacteria to the solvent A is 1g to (1-8 mL).
3. The method for extracting cannabidiol from fermented bacteria according to claim 1, wherein the pressure is 0.2 to 1.2MPa in the subcritical extraction.
4. The method for extracting cannabidiol from fermented bacteria according to claim 3, wherein the extraction time is 10-60 min per time of subcritical extraction.
5. The method for extracting cannabidiol from fermented bacteria according to claim 1, wherein the extraction temperature is 20 to 50 ℃.
6. The method for extracting cannabidiol from fermented bacteria according to claim 1, further comprising the steps of:
and (3) treatment of mushroom dregs: and (3) carrying out decompression desolventizing on the fungus dregs obtained after subcritical extraction, wherein the pressure of the decompression desolventizing is not higher than-0.085 MPa.
7. The method for extracting cannabidiol from fermented bacteria according to claim 1, wherein the step of processing the extract comprises the following sub-steps:
concentrating the extracting solution under reduced pressure to obtain a crude extracting solution;
performing winterization impurity removal on the crude liquid to obtain winterization mixed liquid;
and (4) further performing negative pressure desolventizing on the winterization mixed liquor to obtain an extract.
8. The method for extracting cannabidiol from fermented bacteria according to claim 4, wherein the temperature of the extract is controlled to 40-60 ℃ during the whole process of vacuum concentration.
9. The method for extracting cannabidiol from fermented bacteria according to claim 4, wherein the mass fraction of solids in the crude body fluid is 1-5% after the extract is concentrated under reduced pressure.
10. The method for extracting cannabidiol from fermented bacteria according to claim 1, wherein the particle size of the bacteria particles is controlled to 10 to 120 mesh in the step of pretreating the bacteria.
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