Method for extracting and separating cannabinoid phenol from microbial fermentation thalli

Abstract

The invention relates to a method for extracting and separating cannabinoid phenol from microbial fermentation thalli, which comprises the following steps: crushing the microbial fermentation thalli, and mixing the crushed microbial fermentation thalli with an alcohol-water solution for extraction; mixing the extracting solution with a decolorizing agent for decolorizing treatment; carrying out cationic resin solid-phase extraction on the decolorized product, collecting an eluent, concentrating and drying to obtain an extract; redissolving the extract, filtering with silica gel, and collecting filtrate; and performing crystallization treatment on the filtrate. According to the method, cannabinoid phenol is extracted and separated from thalli for biosynthesis of cannabinoid phenol, microbial fermentation thalli serve as raw materials, and a cannabinoid phenol product with high yield and high purity can be obtained through organic solvent extraction, decoloration, cationic resin elution, silica gel filtration and crystallization in sequence. Compared with the prior art, the extraction and separation method has the advantages that the operation is simpler and quicker, special production equipment is not needed, the price of separation filler is lower, the production cost is lower, the used solvent is easy to recycle, and the method is more environment-friendly.

Description

Technical Field

[0001] The invention belongs to the field of purification of microbial fermentation products and relates to a method for extracting and separating cannabinoids from microbial fermentation cells. Background Art

[0002] Cannabigerol (CBG) is the precursor for the synthesis of all other cannabinoids, so it is often called the "mother of cannabinoids". The molecular formula of cannabigerol is C 21 H 32 O2, white powdery solid, melting point 49℃~50℃. It has no hallucinogenic side effects and has important application value in the fields of medicine, food, health care and cosmetics. CBG binds to human CB1 and CB2 receptors and exerts effects such as anti-tumor, anti-inflammatory, anti-oxidation, nerve protection, immune regulation and cancer treatment. At the same time, CBG also has a bactericidal effect on methicillin-resistant Staphylococcus aureus.

[0003] Cannabidiol is quickly converted into tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabichromene (CBC) in cannabis plants, so the content of cannabidiol in cannabis plants is low (<1%), making extraction difficult. Furthermore, since the properties of various cannabinoid compounds in industrial hemp are very similar, it is inevitable that a small amount of cannabidiol, tetrahydrocannabinol, etc. will be present in the extracted CBG, which places extremely high demands on the preparation process of high-purity CBG.

[0004] CN110878010A uses industrial hemp flowers and leaves as raw materials, extracts with ethanol and water solution, extracts and enriches cannabinol with organic solvent, decolorizes with activated carbon, purifies and enriches with polyamide resin column, neutral alumina and bonded silica gel column, and finally separates with polyamide filler to prepare chromatographic column, concentrates and crystallizes the obtained target segment to obtain cannabinol. This method requires multiple column separations, has complex process steps and high production costs.

[0005] Currently, CBG has been biosynthesized. Compared with traditional plant extraction methods, biosynthetic cannabinoids can effectively get rid of the influence of weather, geography, pests and other factors, save arable land, reduce resource waste, and quickly achieve large-scale production. It only takes a few weeks of fermentation cycle to replace months or even years of planting cycle.

[0006] Although biosynthetic bacteria can achieve efficient synthesis, the special structure and composition of microbial cells make the extraction process of CBG complicated and susceptible to interference. The cell wall of microorganisms is usually thick and difficult to lyse, resulting in low extraction efficiency. In addition, complex components in cells such as polysaccharides, proteins, fats, etc. will also interfere with extraction and purification. The existing process for extracting and separating CBG from microbial fermentation products has the disadvantages of complex operation, low yield, high price of separation fillers, and high production cost. Summary of the invention

[0007] In view of the shortcomings of the prior art, the object of the present invention is to provide a method for extracting and separating cannabinoids from microbial fermentation cells. The operation of this method is relatively simple, no special production equipment is required, the solvent used is easy to recycle, the production cost is lower, it is green and environmentally friendly, and the yield and purity of the separated cannabinoids can reach a high level.

[0008] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0009] The present invention provides a method for extracting and separating cannabinoids from microbial fermentation cells, the method comprising the following steps:

[0010] (1) crushing the microbial fermentation cells, mixing the crushed cells with an alcohol aqueous solution for extraction, and performing solid-liquid separation to obtain an extract;

[0011] (2) mixing the extract with a decolorizing agent for decolorization, and separating the solid and liquid to obtain a decolorized product;

[0012] (3) adjusting the pH of the decolorized product to acidic, then subjecting it to solid phase extraction with a cationic resin, eluting with a solvent, collecting the eluate, concentrating and drying it to obtain an extract;

[0013] (4) re-dissolving the extract with a solvent, filtering it with silica gel, and collecting the filtrate;

[0014] (5) The filtrate is subjected to crystallization treatment to obtain cannabinoids.

[0015] The method involved in the present invention is to extract and separate cannabidiol from the bacterial body of biosynthetic cannabidiol, and the microbial fermentation bacterial body is used as the raw material, and the cannabidiol product with high yield and high purity can be obtained by sequentially undergoing organic solvent extraction, decolorization, cationic resin elution, silica gel filtration, and crystallization. Compared with the prior art method, the extraction and separation method is simpler and faster to operate, does not require special production equipment, has a lower price for separation fillers, lower production costs, and the solvent used is easy to recycle, which is more environmentally friendly, and the yield and purity of the separated cannabidiol can reach a high level, which is suitable for industrial application.

[0016] Preferably, the powder in step (1) is crushed to a particle size of less than 20 mesh, more preferably less than 40 mesh.

[0017] Preferably, the volume fraction of alcohol in the alcohol-water solution in step (1) is 70-85%, for example, 70%, 72%, 73%, 75%, 78%, 80%, 82%, 85%, etc.

[0018] Preferably, the alcohol aqueous solution comprises an ethanol aqueous solution or a methanol aqueous solution.

[0019] Compared with other volume fractions of alcohol aqueous solutions, the specific selection of 70-85% alcohol aqueous solution in the present invention can achieve better extraction effect, and the final yield and purity of cannabinoids are further improved.

[0020] Preferably, the solid-liquid ratio of the bacteria to the alcohol aqueous solution in step (1) is 1:(5-12) kg / L, for example 1:5 kg / L, 1:6 kg / L, 1:7 kg / L, 1:8 kg / L, 1:9 kg / L, 1:10 kg / L, 1:12 kg / L, etc.

[0021] Preferably, the extraction in step (1) is carried out at 10-40°C, for example, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, etc.

[0022] Preferably, the extraction in step (1) is performed 1-3 times, for example, 1 time, 2 times or 3 times; the time for each extraction is 0.5-1.5 h, for example, 0.5 h, 0.7 h, 0.8 h, 1.0 h, 1.2 h, 1.3 h, 1.5 h, etc.

[0023] Preferably, the decolorizing agent in step (2) comprises activated carbon.

[0024] Preferably, the amount of the decolorizing agent added in step (2) is 0.5-3% of the extract, for example 0.5%, 0.8%, 1%, 1.2%, 1.5%, 1.8%, 2%, 2.2%, 2.5%, 2.8%, 3%, etc.

[0025] Preferably, the decolorization treatment in step (2) is stirred at 40-60°C (e.g., 40°C, 45°C, 50°C, 55°C, 60°C, etc.) for 10-90 min (e.g., 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 70 min, 80 min, 90 min, etc.).

[0026] Preferably, in step (3), the pH of the decolorized product is adjusted to 1.5-4.5, such as pH=1.5, pH=2.0, pH=2.5, pH=3.0, pH=3.5, pH=4.0, pH=4.5, etc. More preferably, pH=2-3.

[0027] Compared with other acidic pH ranges, the specific pH range of 1.5-4.5 of the present invention is more preferably pH=2-3, which can achieve better separation effect, and the final yield and purity of cannabinoids are further improved.

[0028] Preferably, the pH is adjusted in step (3) by adding an inorganic acid.

[0029] Preferably, the amount of the cationic resin in step (3) is 1-3 times the mass of the bacteria, for example, 1 time, 1.5 times, 2 times, 2.5 times, 3 times, etc.

[0030] Preferably, the cationic resin is a strongly acidic cation exchange resin comprising ES-4060, Amberlite IR-120, Dowex 50W, Lewatit S100, etc.

[0031] Compared with other types of cationic resins, when the present invention selects the above-mentioned strongly acidic cation exchange resin, the separation effect can be better, and the final yield and purity of cannabinoids are further improved.

[0032] Preferably, the solvent in step (3) is an alcohol aqueous solution with a volume fraction of 70-85% (e.g., 70%, 73%, 75%, 78%, 80%, 82%, 85%, etc.).

[0033] Preferably, the amount of solvent used for elution in step (3) is 0.5-2 column volumes, for example, 0.5 column volumes, 1 column volume, 1.5 column volumes, 2 column volumes, etc.

[0034] Preferably, the alcohol aqueous solution comprises an ethanol aqueous solution or a methanol aqueous solution.

[0035] Preferably, the solvent in step (4) is a C5-C8 alkane, more preferably n-heptane.

[0036] The C5-C8 alkanes include n-pentane, n-hexane, n-heptane, n-octane and the like.

[0037] Preferably, the amount of the solvent used in step (4) is 8-15 times the mass of the extract, for example, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, etc.

[0038] Preferably, the amount of silica gel used in step (4) is 1-3 times the mass of the extract, for example, 1 time, 1.5 times, 2 times, 2.5 times, 3 times, etc.

[0039] Preferably, the silica gel in step (4) is 100-200 mesh silica gel.

[0040] Preferably, the crystallization treatment in step (5) comprises:

[0041] First, the first crystallization is carried out at -20 to -5°C (for example, -20°C, -18°C, -15°C, -12°C, -10°C, -8°C, -5°C, etc.), the solid-liquid separation is carried out, the first crystallization product is taken for redissolution, and then the second crystallization is carried out at -10 to 0°C (for example, -20°C, -10°C, -8°C, -7°C, -6°C, -5°C, -2°C, 0°C, etc.), the solid-liquid separation is carried out, and cannabinol is obtained.

[0042] The preferred crystallization procedure of the present invention is the above-mentioned two-stage crystallization. By carrying out the crystallization treatment in this way, the cannabinoid product obtained has a higher purity.

[0043] Preferably, the solvent used for the re-dissolution is a C5-C8 alkane, more preferably n-heptane.

[0044] The C5-C8 alkanes include n-pentane, n-hexane, n-heptane, n-octane and the like.

[0045] Preferably, the ratio of the mass of the first crystallization product to the volume of n-heptane is 1:(10-20) g / mL, for example 1:10 g / mL, 1:12 g / mL, 1:14 g / mL, 1:16 g / mL, 1:18 g / mL, 1:20 g / mL, etc.

[0046] Preferably, the separated solid is rinsed after the solid-liquid separation.

[0047] Preferably, the crystallization treatment in step (5) is followed by a drying treatment, and the drying treatment is carried out under vacuum at 35-45°C (e.g., 35°C, 37°C, 38°C, 40°C, 42°C, 43°C, 45°C, etc.).

[0048] Other specific point values ​​within the above numerical ranges can be selected, all of which are within the protection scope of the present invention and will not be described in detail here.

[0049] Compared with the prior art, the present invention has the following beneficial effects:

[0050] The method involved in the present invention is to extract and separate cannabidiol from the bacterial body of biosynthetic cannabidiol, and the microbial fermentation bacterial body is used as the raw material, and the cannabidiol product with high yield and high purity can be obtained by sequentially undergoing organic solvent extraction, decolorization, cationic resin elution, silica gel filtration, and crystallization. Compared with the prior art method, the extraction and separation method is simpler and faster to operate, does not require special production equipment, has a lower price for separation fillers, lower production costs, and the solvent used is easy to recycle, which is more environmentally friendly, and the yield and purity of the separated cannabidiol can reach a high level, which is suitable for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1This is an appearance diagram of the cannabidiol product obtained in Example 1;

[0052] Figure 2 This is an appearance diagram of the cannabinoid product obtained in Example 2;

[0053] Figure 3 This is the appearance of the cannabinoid product obtained in Example 3. DETAILED DESCRIPTION

[0054] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0055] Some of the raw materials and material information involved in the following examples and comparative examples are as follows:

[0056] Cannabinol standards were obtained from Shanghai Yuanye Biotechnology Co., Ltd.; ES-4060 cationic resin comes from Kehaisi (Beijing) Technology Co., Ltd.; Amberlite IR-120 cationic resin comes from Shanghai Yuanye Biotechnology Co., Ltd.; Dowex 50W-X8 (NA) cationic resin comes from Shanghai Yuanye Biotechnology Co., Ltd.; LewatitS100 cationic resin comes from Germany's Lanxess Group; silica gel comes from the 100-200 mesh product of Qingdao Kangyexin Pharmaceutical Silica Gel Co., Ltd.; microbial fermentation cells come from Senruis Biotechnology (Shenzhen) Co., Ltd. (CBG content is not less than 5.0%).

[0057] The detection of the cannabinoid content involved in the following examples and comparative examples was carried out using high performance liquid chromatography, the instrument was an Agilent 1260 liquid chromatograph (Agilent, USA), octadecylsilane bonded silica gel was used as the filler; 0.1% phosphoric acid aqueous solution was used as the mobile phase A, 0.1% phosphoric acid acetonitrile solution was used as the mobile phase B, gradient elution, the elution program was as follows, and the detection wavelength was 220nm.

[0058]

[0059] The calculation formula for the yield of cannabinoids in each step is: n / W n-1 х100%(W n is the CBG quality in the product of the current step, W n-1 is the CBG quality in the product of the previous step);

[0060] The calculation formula of the total yield of cannabinoids is: 成 / W 始 х100%(W 成 is the quality of the final CBG product, W始 is the mass of CBG in the initial feed);

[0061] Example 1

[0062] This embodiment provides a method for extracting and isolating cannabigerol (CBG) from microbial fermentation cells, which specifically includes the following steps:

[0063] (1) Weigh 1.5 kg of microbial fermentation cells, crush them into 50 meshes with a crusher, add 15 L of 70% ethanol aqueous solution, stir and extract at 25° C. for 1.5 h, separate the solid and liquid after extraction, collect the separated extract, and the CBG yield is 90.3% after testing;

[0064] (2) adding 3% activated carbon to the extract, stirring in a water bath at 55° C. for 30 min, and obtaining a decolorized extract after solid-liquid separation. The CBG yield was 88.6% after testing;

[0065] (3) adding hydrochloric acid to adjust the pH of the decolorized extract to 2.5, using 1.8 kg of Dowex 50W-X8 (NA) cationic resin as the extraction filler, passing through a column, collecting the effluent, and eluting with 70% ethanol aqueous solution for 1 column volume, combining the effluent and the eluate, further concentrating and drying to obtain CBG extract, and the CBG yield was 93.1% after testing;

[0066] (4) Add 8 times the weight of n-heptane to the CBG extract, heat in a water bath at 60°C to dissolve the extract, weigh 1 times the weight of silica gel as much as the weight of the CBG extract, filter the n-heptane solution through silica gel, rinse the silica gel once with 0.3 L of n-heptane, combine the two solutions, and the CBG yield is 94.6% after testing;

[0067] (5) The collected solution was placed in a -10°C refrigerator for 12 hours for crystallization, filtered, and rinsed once with n-heptane to obtain a primary crystallization sample; the primary crystallization sample was added with n-heptane (1:10 g / mL) and heated at 50°C for redissolution, and then placed in a 0°C refrigerator for 12 hours for crystallization, filtered, and rinsed once with n-heptane to obtain a secondary crystallization sample;

[0068] (6) The secondary crystallized sample was placed in a vacuum drying oven at 40°C, evacuated to -0.08 MPa, and dried for 24 hours to obtain a final product having an appearance as shown in FIG. Figure 1 The total yield of the finished product was 50.3% and the product purity was 99.5%.

[0069] Example 2

[0070] This embodiment provides a method for extracting and isolating cannabigerol (CBG) from microbial fermentation cells, which specifically includes the following steps:

[0071] (1) Weigh 1.0 kg of microbial fermentation cells, crush them into 40 meshes with a crusher, add 8 L of 75% methanol aqueous solution, stir and extract at 30° C. for 1.0 h, separate the solid and liquid after extraction, collect the separated extract, take the separated solid, add 5 L of 75% methanol aqueous solution, stir and extract at 30° C. for 0.5 h, separate the solid and liquid after extraction, collect the separated secondary extract, combine the primary and secondary extracts to obtain the total extract, and the CBG yield is 95.6% after testing;

[0072] (2) adding 1% activated carbon to the extract, stirring in a water bath at 50° C. for 60 min, and obtaining a decolorized extract after solid-liquid separation. The CBG yield was 91.8% after testing;

[0073] (3) Add sulfuric acid solution to adjust the pH of the decolorized extract to 3.0, and add 1.0 kg ES-4060 cationic resin was used as the extraction filler, and the effluent was collected and eluted with 75% methanol aqueous solution for 1 column volume. The effluent and the eluate were combined, further concentrated and dried to obtain CBG extract. The CBG yield was 92.3% after testing.

[0074] (4) Add 10 times the weight of n-heptane to the CBG extract, heat in a water bath at 60°C to dissolve the extract, weigh 2 times the weight of silica gel as much as the weight of the CBG extract, filter the n-heptane solution through silica gel, rinse the silica gel once with 0.5L n-heptane, and combine them. The CBG yield was 92.1% after testing.

[0075] (5) The collected solution was placed in a -20°C refrigerator for 18 hours for crystallization, filtered, and rinsed once with n-heptane to obtain a primary crystallization sample; the primary crystallization sample was added with n-heptane (1:15 g / mL) and heated at 50°C for redissolution, and then placed in a -5°C refrigerator for 18 hours for crystallization, filtered, and rinsed once with n-heptane to obtain a secondary crystallization sample;

[0076] (6) The secondary crystallized sample was placed in a vacuum drying oven at 35°C, evacuated to -0.08 MPa, and dried for 24 hours to obtain a final product having an appearance as shown in FIG. Figure 2 The total yield of the finished product was 57.1% and the purity of the product was 99.6%.

[0077] Example 3

[0078] This embodiment provides a method for extracting and isolating cannabigerol (CBG) from microbial fermentation cells, which specifically includes the following steps:

[0079] (1) Weigh 2.0 kg of microbial fermentation cells, crush them into 30 meshes with a crusher, add 18 L of 85% ethanol aqueous solution, stir and extract at 35° C. for 1.5 h, separate the solid and liquid after extraction, collect the separated extract, and the CBG yield is 91.1% after testing;

[0080] (2) adding 2% activated carbon to the extract, stirring in a water bath at 45° C. for 45 min, and obtaining a decolorized extract after solid-liquid separation. The CBG yield was 90.3% after testing;

[0081] (3) adding hydrochloric acid to adjust the pH of the decolorized extract to 2.0, using 2.0 kg of Amberlite IR-120 cationic resin as the extraction filler, passing through a column, collecting the effluent, and eluting with 85% ethanol aqueous solution for 2 column volumes, combining the effluent and the eluate, further concentrating and drying to obtain CBG extract, and the CBG yield was 93.2% after testing;

[0082] (4) Add 12 times the weight of n-heptane to the CBG extract, heat in a water bath at 60°C to dissolve the extract, weigh 3 times the weight of silica gel as much as the weight of the CBG extract, filter the n-heptane solution through silica gel, rinse the silica gel once with 1.2 L of n-heptane, and combine them. The CBG yield was 91.8% after testing.

[0083] (5) The collected solution was placed in a -20°C refrigerator for 15 hours for crystallization, filtered, and rinsed once with n-heptane to obtain a primary crystallization sample; the primary crystallization sample was added with n-heptane (1:20 g / mL) and heated at 50°C for redissolution, and then placed in a -10°C refrigerator for 15 hours for crystallization, filtered, and rinsed once with n-heptane to obtain a secondary crystallization sample;

[0084] (6) The secondary crystallized sample was placed in a 45°C vacuum drying oven, evacuated to -0.08 MPa, and dried for 24 hours to obtain a final product having an appearance as shown in FIG. Figure 3 The total yield of the finished product was 51.7% and the product purity was 99.6%.

[0085] Example 4

[0086] This embodiment provides a method for extracting and isolating cannabinoids (CBG) from microbial fermentation cells. The specific steps are different from those of Example 1 only in that the 70% ethanol aqueous solution in step (1) is replaced with a 95% ethanol aqueous solution, and other conditions remain unchanged. The total yield of the finished product is 43.2% and the product purity is 99.2%.

[0087] Example 5

[0088] This embodiment provides a method for extracting and isolating cannabinoids (CBG) from microbial fermentation cells. The specific steps are different from those of Example 1 only in that the 70% ethanol aqueous solution in step (1) is replaced by a 60% ethanol aqueous solution, and other conditions remain unchanged. The finished product yield is 37.1% and the product purity is 99.0%.

[0089] Example 6

[0090] This embodiment provides a method for extracting and isolating cannabinoids (CBG) from microbial fermentation cells. The specific steps are different from those of Example 1 only in that hydrochloric acid is added in step (3) to adjust the pH of the decolorized extract to 4.5, and other conditions remain unchanged. The finished product yield is 51.3% and the product purity is 98.1%.

[0091] Example 7

[0092] This embodiment provides a method for extracting and isolating cannabinoids (CBG) from microbial fermentation cells. The specific steps are different from those of Example 1 only in that hydrochloric acid is added in step (3) to adjust the pH of the decolorized extract to 1.5, and other conditions remain unchanged. The total yield of the finished product is 49.4% and the product purity is 99.1%.

[0093] Example 8

[0094] This embodiment provides a method for extracting and isolating cannabinol (CBG) from microbial fermentation cells. The specific steps are different from those of Example 1 only in that in step (3), Dowex 50W-X8 (NA) strong acid cationic resin is replaced with Amberlite (r) irc-50 weak acid cationic resin in equal amounts, and other conditions remain unchanged. The total yield of the finished product is 50.4% and the product purity is 97.8%.

[0095] Example 9

[0096] This embodiment provides a method for extracting and isolating cannabinoids (CBG) from microbial fermentation cells. The specific steps are different from those of Example 1 only in that the crystallization procedure in step (5) is as follows: the collected liquid is placed in a -10°C refrigerator for 24 hours for crystallization, filtered, and rinsed once with n-heptane to obtain a crystal sample. Other conditions remain unchanged. The total yield of the finished product was 58.9% and the product purity was 97.2%.

[0097] Example 10

[0098] The present embodiment provides a method for extracting and separating cannabinoids (CBG) from microbial fermentation cells. The specific steps are different from those of Example 1 only in that the crystallization procedure in step (5) is as follows: the collected liquid is placed in a 0°C refrigerator, placed for 12 hours for crystallization, filtered, and rinsed once with n-heptane to obtain a primary crystallization sample; the primary crystallization sample is heated and dissolved at 50°C with n-heptane (1:10 g / mL), then placed at 25°C, placed for 12 hours for crystallization, filtered, and rinsed once with n-heptane to obtain a secondary crystallization sample. Other conditions remain unchanged. The total yield of the finished product was 46.1% and the product purity was 99.5%.

[0099] Comparative Example 1

[0100] This comparative example provides a method for extracting and isolating cannabinoids (CBG) from microbial fermentation cells. The specific steps thereof differ from those of Example 1 only in that the decolorization treatment in step (2) is omitted, and the extract obtained in step (1) is directly subjected to step (3). Other conditions remain unchanged. It is found that the product in step (5) cannot be crystallized.

[0101] Comparative Example 2

[0102] This comparative example provides a method for extracting and separating cannabinoids (CBG) from microbial fermentation cells. The specific steps are different from those of Example 1 only in that the silica gel filtration treatment in step (4) is missing. The CBD extract obtained in step (3) is added with 8 times the mass of n-heptane, heated in a water bath at 60°C to dissolve the extract, and then directly proceeds to step (5). Other conditions remain unchanged. It is found that the product in step (5) cannot be crystallized.

[0103] Comparative Example 3

[0104] This comparative example provides a method for extracting and separating cannabinoids (CBG) from microbial fermentation cells, which comprises the following steps:

[0105] (1) Weigh 1.5 kg of microbial fermentation cells, crush them into 50 meshes with a crusher, add 15 L of 70% ethanol aqueous solution, stir and extract at 25° C. for 1.5 h, separate the solid and liquid after extraction, collect the separated extract, and the CBG yield is 90.3% after testing;

[0106] (2) The extract was concentrated to an ethanol volume fraction of 50%, and then n-hexane in a volume twice that of the concentrated extract was added for extraction. The solution was separated into two layers, and the upper n-hexane layer was separated. The CBG content was determined, and the yield was only 10.3%.

[0107] The yield and purity results of the cannabinoid products obtained in Examples 1-10 are summarized in Table 1:

[0108] Table 1

[0109]

[0110]

[0111] It can be seen from the data results in Table 1 that the extraction and separation method involved in the present invention uses microbial fermentation cells as raw materials, and sequentially undergoes organic solvent extraction, decolorization, cationic resin elution, silica gel filtration, and crystallization to harvest high-yield and high-purity cannabinoid products.

[0112] The applicant declares that the present invention illustrates the technical solution of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0113] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0114] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A method for extracting and separating cannabinoids from microbial fermentation cells, characterized in that: The method comprises the following steps: (1) crushing the microbial fermentation cells, mixing the crushed cells with an alcohol aqueous solution for extraction, and performing solid-liquid separation to obtain an extract; (2) mixing the extract with a decolorizing agent for decolorization, and separating the solid and liquid to obtain a decolorized product; (3) adjusting the pH of the decolorized product to acidic, then subjecting it to solid phase extraction with a cationic resin, eluting with a solvent, collecting the eluate, concentrating and drying it to obtain an extract; (4) re-dissolving the extract with a solvent, filtering it with silica gel, and collecting the filtrate; (5) The filtrate is subjected to crystallization treatment to obtain cannabinoids.

2. The method according to claim 1, characterized in that The step (1) is to crush the bacteria to a particle size of less than 20 mesh, and more preferably less than 40 mesh.

3. The method according to claim 1 or 2, characterized in that: The volume fraction of alcohol in the alcohol-water solution in step (1) is 70-85%; Preferably, the alcohol aqueous solution comprises an ethanol aqueous solution or a methanol aqueous solution; Preferably, the solid-liquid ratio of the bacterial cells to the alcohol aqueous solution in step (1) is 1:(5-12) kg / L; Preferably, the extraction in step (1) is carried out at 10-40°C; Preferably, the extraction in step (1) is performed 1 to 3 times, and each extraction lasts 0.5 to 1.5 hours.

4. The method according to any one of claims 1 to 3, characterized in that The decolorizing agent in step (2) comprises activated carbon; Preferably, the amount of the decolorizing agent added in step (2) is 0.5-3% of the extract; Preferably, the decolorization treatment in step (2) is carried out by stirring at 40-60° C. for 10-90 min.

5. The method according to any one of claims 1 to 4, characterized in that Step (3) wherein the pH of the decolorized product is adjusted to 1.5-4.5; Preferably, the pH is adjusted in step (3) by adding an inorganic acid.

6. The method according to any one of claims 1 to 5, characterized in that The amount of the cationic resin in step (3) is 1-3 times the mass of the bacterial cells; Preferably, the solvent in step (3) is an alcohol aqueous solution with a volume fraction of 70-85%; Preferably, the alcohol aqueous solution comprises an ethanol aqueous solution or a methanol aqueous solution; Preferably, the amount of solvent used for elution in step (3) is 0.5-2 column volumes.

7. The method according to any one of claims 1 to 6, characterized in that The solvent in step (4) is a C5-C8 alkane, more preferably n-heptane; Preferably, the amount of the solvent used in step (4) is 8-15 times the mass of the extract; Preferably, the amount of silica gel used in step (4) is 1-3 times the mass of the extract.

8. The method according to any one of claims 1 to 7, characterized in that The crystallization treatment in step (5) includes: First, the first crystallization is carried out at -20 to -5°C, the solid-liquid separation is carried out, the first crystallization product is taken and redissolved, and then the second crystallization is carried out at -10 to 0°C, the solid-liquid separation is carried out, and cannabinol is obtained.

9. The method according to claim 8, characterized in that The solvent used for the re-dissolution is a C5-C8 alkane, more preferably n-heptane; Preferably, the ratio of the mass of the first crystallization product to the volume of n-heptane is 1:(10-20) g / mL; Preferably, the separated solid is rinsed after the solid-liquid separation.

10. The method according to any one of claims 1 to 9, characterized in that The crystallization treatment in step (5) is followed by a drying treatment, which is carried out at 35-45° C. under vacuum.

Images