Method for separating and purifying cannabinoid phenol from microbial fermentation thalli

Abstract

The invention relates to a method for separating and purifying 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; mixing the destaining solution with silica gel, carrying out solid-phase dispersion, and removing a solvent to obtain a solid-phase dispersion sample; loading the solid-phase dispersed sample to the top end of a silica gel column, eluting with a solvent, collecting eluent, concentrating and drying to obtain a crude product; and redissolving the crude product with a solvent, and performing crystallization treatment to obtain cannabinoid phenol. According to the method, microbial fermentation thalli are used as raw materials, and the cannabinoid phenol product with high yield and high purity can be obtained through organic solvent extraction, decoloration, solid-phase dispersion of a sample with a small amount of silica gel, silica gel column elution and crystallization in sequence.

Description

Technical Field

[0001] The invention belongs to the field of purification of microbial fermentation products and relates to a method for separating and purifying 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] 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.

[0005] Although biosynthetic bacteria can achieve efficient synthesis, it is difficult to extract and purify high-quality cannabinoids from microbial cells because the cell walls of microorganisms are usually thick and difficult to break down, and the complex components in the cells, such as polysaccharides, proteins, and fats, will also interfere with extraction and purification.

[0006] CN114478195A uses microbial fermentation cells as raw materials, uses ethanol extraction, removes the cells after extraction, and concentrates the liquid after removing the cells, then adds n-hexane for multiple extractions, collects the n-hexane layer, and separates it by high-performance liquid chromatography after concentration, obtains a cannabidiol extract sample after concentration, and finally dissolves the obtained cannabidiol extract sample with n-hexane, and recrystallizes it to obtain cannabidiol crystals. This method has cumbersome process steps, low extraction yield, and requires a dedicated industrial preparation chromatography, high filler and equipment prices, limited sample loading, high production costs, and is not suitable for large-scale production.

[0007] Therefore, it is very meaningful to develop a method for separating and purifying cannabinoids from microbial fermentation cells that is simple to operate, low in production cost, and environmentally friendly and suitable for industrial-scale production. Summary of the invention

[0008] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a method for separating and purifying cannabinoids from microbial fermentation cells. Compared with the prior art methods, the separation and purification method is simpler and faster to operate, greatly reduces production costs, is green and environmentally friendly, and can obtain high-yield and high-purity cannabinoid products.

[0009] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0010] The present invention provides a method for separating and purifying cannabinoids from microbial fermentation cells, the method comprising the following steps:

[0011] (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;

[0012] (2) mixing the extract with a decolorizing agent for decolorization, and separating the solid from the liquid to obtain a decolorized solution;

[0013] (3) mixing the decolorizing solution with silica gel for solid phase dispersion, or concentrating and / or drying the decolorizing solution and re-dissolving it with an alcohol aqueous solution and then mixing it with silica gel for solid phase dispersion, removing the solvent, and obtaining a solid phase dispersed sample;

[0014] (4) loading the solid phase dispersed sample onto the top of a silica gel column, eluting with a solvent, collecting the eluate, concentrating and / or drying it to obtain a crude product;

[0015] (5) The crude product is redissolved in a solvent and subjected to crystallization to obtain cannabinoids.

[0016] The method involved in the present invention is to extract and separate cannabinoid phenol from the bacterial body of biosynthetic cannabinoid phenol, using microbial fermentation bacterial body as raw material, sequentially undergoing organic solvent extraction, decolorization, solid phase dispersion of the sample with a small amount of silica gel, and then silica gel column elution and crystallization, so as to obtain a high-yield and high-purity cannabinoid phenol product. 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 green and environmentally friendly, and the yield and purity of the separated cannabinoid phenol can reach a high level, which is suitable for industrial application.

[0017] Preferably, the powder in step (1) is crushed to a particle size of less than 20 mesh, more preferably less than 40 mesh.

[0018] Preferably, the volume fraction of the alcohol aqueous solution in step (1) is 70-85%, for example, 70%, 72%, 73%, 75%, 78%, 80%, 82%, 85%, etc.

[0019] Preferably, the alcohol aqueous solution in step (1) comprises methanol aqueous solution and / or ethanol aqueous solution.

[0020] Compared with other volume fractions of methanol or ethanol aqueous solutions, the specific selection of 70-85% methanol or ethanol aqueous solutions in the present invention can achieve better extraction effects, and the final yield and purity of cannabinoids are further improved.

[0021] Compared with other volume fractions of methanol or ethanol aqueous solutions, the specific selection of 70-85% methanol or ethanol aqueous solutions in the present invention can achieve better extraction effects, and the final yield and purity of cannabinoids are further improved.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] Preferably, the decolorizing agent in step (2) comprises activated carbon.

[0026] 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.

[0027] 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.).

[0028] Preferably, after the solid-liquid separation in step (2), the separated solid is rinsed.

[0029] The decolorization treatment in step (2) can help improve the effect of subsequent silica gel elution, further improving the final yield and purity of cannabinoids.

[0030] Preferably, the silica gel in step (3) is 100-300 mesh silica gel, such as 100 mesh, 120 mesh, 150 mesh, 180 mesh, 200 mesh, 220 mesh, 250 mesh, 270 mesh, 300 mesh, etc.

[0031] Preferably, the volume fraction of alcohol in the alcohol aqueous solution in step (3) is 95%-100%, for example, 95%, 96%, 97%, 98%, 99%, 100%, etc.

[0032] Preferably, the alcohol aqueous solution in step (3) comprises methanol aqueous solution and / or ethanol aqueous solution.

[0033] In step (3), the decolorizing solution is first concentrated and / or dried and then re-dissolved with an alcohol-water solution and then mixed with silica gel for solid phase dispersion. Compared with directly mixing the decolorizing solution with silica gel for solid phase dispersion, it is more conducive to the subsequent volatilization and removal of the solvent and improves the efficiency.

[0034] Preferably, the method of removing the solvent in step (3) includes natural evaporation.

[0035] Preferably, the silica gel column in step (4) contains 100-300 mesh silica gel, such as 100 mesh, 120 mesh, 150 mesh, 180 mesh, 200 mesh, 220 mesh, 250 mesh, 270 mesh, 300 mesh, etc.

[0036] Preferably, the total amount of silica gel in the silica gel column in step (4) is 0.5-4 times that of the solid phase dispersed sample, for example, 0.5 times, 1 times, 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, etc.

[0037] Preferably, the solvent in step (4) is a mixture of n-heptane and ethyl acetate.

[0038] Compared with other elution solvents, the present invention finds that when a mixture of n-heptane and ethyl acetate is used as the elution solvent, the impurity removal effect of cannabidiol through the silica gel column elution can be better, and the yield and purity of the final cannabidiol product are further improved.

[0039] Preferably, the volume ratio of n-heptane to ethyl acetate is (4-9):1, for example 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, etc.

[0040] Preferably, the solvent elution in step (4) is performed for a total of 5-10 column volumes, for example, 5 column volumes, 6 column volumes, 7 column volumes, 8 column volumes, 9 column volumes or 10 column volumes.

[0041] Preferably, the solvent used for the re-dissolution in step (5) is a C5-C8 alkane, more preferably n-heptane.

[0042] The C5-C8 alkanes include n-pentane, n-hexane, n-heptane, n-octane and the like.

[0043] Compared with other C5-C8 alkane solvents, the present invention finds that the use of n-heptane as the redissolution solvent can make the subsequent crystallization effect of cannabidiol better, and the yield and purity of the final cannabidiol product are further improved.

[0044] Preferably, the ratio of the amount of the solvent used in step (5) to the mass of the crude product is 1:(8-15) g / mL, for example 1:8 g / mL, 1:9 g / mL, 1:10 g / mL, 1:12 g / mL, 1:14 g / mL, 1:15 g / mL, etc.

[0045] Preferably, the crystallization treatment in step (5) comprises:

[0046] 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.

[0047] 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.

[0048] Preferably, the solvent used for the re-dissolution is a C5-C8 alkane, more preferably n-heptane.

[0049] The C5-C8 alkanes include n-pentane, n-hexane, n-heptane, n-octane and the like.

[0050] Preferably, the ratio of the mass of the first crystallization product to the volume of the re-dissolution solvent 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.

[0051] Preferably, the separated solid is rinsed after the solid-liquid separation.

[0052] 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.).

[0053] 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.

[0054] Compared with the prior art, the present invention has the following beneficial effects:

[0055] The method involved in the present invention is to extract and separate cannabinoid phenol from the bacterial body of biosynthetic cannabinoid phenol, using microbial fermentation bacterial body as raw material, sequentially undergoing organic solvent extraction, decolorization, solid phase dispersion of the sample with a small amount of silica gel, and then silica gel column elution and crystallization, so as to obtain a high-yield and high-purity cannabinoid phenol product. 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 green and environmentally friendly, and the yield and purity of the separated cannabinoid phenol can reach a high level, which is suitable for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 This is an appearance diagram of the cannabidiol product obtained in Example 1;

[0057] Figure 2 This is an appearance diagram of the cannabinoid product obtained in Example 2;

[0058] Figure 3 This is the appearance of the cannabinoid product obtained in Example 3. DETAILED DESCRIPTION

[0059] 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.

[0060] Some of the raw materials and material information involved in the following examples and comparative examples are as follows:

[0061] The cannabinoid standard was sourced from Shanghai Yuanye Biotechnology Co., Ltd.; the silica gel was sourced from the 100-200 mesh products and 200-300 mesh products of Qingdao Kangyexin Medicinal Silica Gel Co., Ltd.; the microbial fermentation cells were sourced from Senruis Biotechnology (Shenzhen) Co., Ltd. (CBG content was not less than 5.0%).

[0062] 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.

[0063]

[0064] 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);

[0065] 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).

[0066] Example 1

[0067] This embodiment provides a method for separating and purifying cannabinoids (CBG) from microbial fermentation cells, which specifically includes the following steps:

[0068] (1) Weigh 0.5 kg of microbial fermentation cells, crush them into 20 meshes with a crusher, add 5 L of 70% ethanol aqueous solution, stir at 25° C. for 1.5 h, separate the solid and liquid after extraction, collect the first extract after separation, take the separated solid, add 3 L of 70% ethanol aqueous solution, stir at 25° C. for 1 h, separate the solid and liquid after extraction, collect the second extract after separation, combine the first and second extracts to obtain the total extract, and the CBG yield is 97.1%;

[0069] (2) adding 2% activated carbon to the extract, stirring in a water bath at 55°C for 20 min, obtaining a decolorized extract after solid-liquid separation, and rinsing with 2L of 80% ethanol aqueous solution, combining the decolorized collected solution and the rinsing solution to obtain a decolorized solution, with a CBG yield of 96.2%;

[0070] (3) Concentrating the decolorized liquid to obtain a CBG extract, adding 0.75 times the weight of 95% ethanol to the CBG extract, heating in a 60° C. water bath to disperse and dissolve, mixing and dispersing with 100-200 mesh silica gel 1.2 times the weight of the CBG extract, and evaporating the solvent to obtain a solid phase dispersion sample;

[0071] (4) Weigh twice the weight of the solid phase dispersed sample and load it into a 100-200 mesh silica gel column. Load the solid phase dispersed sample onto the top of the silica gel column and elute 7 BV with a mixed solution of n-heptane:ethyl acetate = 4:1 (V / V). Collect the eluate and concentrate it to obtain the CBG extract after chromatography. The CBG yield is 95.3%;

[0072] (5) According to the ratio of extract: n-heptane = 1:8 (g / mL), add n-heptane to heat and dissolve all the extract, place the solution in a -10°C refrigerator, leave it for 12 hours, filter it at 4°C, and rinse it with n-heptane to obtain a primary crystallization sample; take the primary crystallization sample, add n-heptane (1:15 g / mL) to heat and dissolve it again, then place it in a 0°C refrigerator, leave it for 12 hours, filter it at 4°C, and rinse it with n-heptane to obtain a secondary crystallization sample;

[0073] (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 As shown, the total yield is 63.9% and the product purity is 99.5%.

[0074] Example 2

[0075] This embodiment provides a method for separating and purifying cannabinoids (CBG) from microbial fermentation cells, which specifically includes the following steps:

[0076] (1) Weigh 0.3 kg of microbial fermentation cells, crush them into 40 meshes with a crusher, add 2 L of 75% methanol aqueous solution, stir at 30° C. for 1 h, separate the solid and liquid after extraction, collect the first extract after separation, take the separated solid, add 1.5 L of 75% methanol aqueous solution, stir at 30° C. for 0.5 h, separate the solid and liquid after extraction, collect the second extract after separation, combine the first and second extracts to obtain the total extract, and the CBG yield is 95.6%;

[0077] (2) Add 3% activated carbon to the extract, stir in a water bath at 50°C for 45 minutes, separate the solid and liquid to obtain a decolorized extract, rinse with 1.5L of 85% methanol aqueous solution, combine the decolorized collected solution and the rinse solution to obtain a decolorized solution, with a CBG yield of 93.7%;

[0078] (3) Concentrating the decolorized liquid to obtain a CBG extract, adding 1 times the weight of methanol to the CBG extract, heating in a water bath at 60° C. to disperse and dissolve, mixing and dispersing with 0.8 times the weight of the CBG extract of 200-300 mesh silica gel, and evaporating the solvent to obtain a solid phase dispersion sample;

[0079] (4) Weigh twice the weight of the solid phase dispersed sample and load it into a 200-300 mesh silica gel column. Load the solid phase dispersed sample onto the top of the silica gel column and elute 5 BV with a mixed solution of n-heptane:ethyl acetate=85:15 (V / V). Collect the eluate and concentrate it to obtain the CBG extract after chromatography. The CBG yield is 96.7%;

[0080] (5) According to the ratio of extract: n-heptane = 1:15 (g / mL), add n-heptane to heat and dissolve all the extract, place the solution in a -20°C refrigerator, place it for 12 hours, filter it at 4°C, and rinse it with n-heptane to obtain a primary crystallization sample; take the primary crystallization sample, add n-heptane (1:20 g / mL) to heat and dissolve it, then place it in a -10°C refrigerator, place it for 12 hours, filter it at 4°C, and rinse it with n-heptane to obtain a secondary crystallization sample;

[0081] (6) The secondary crystallized sample was placed in a 43°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 2 As shown, the total yield is 59.6% and the product purity is 99.6%.

[0082] Example 3

[0083] This embodiment provides a method for separating and purifying cannabinoids (CBG) from microbial fermentation cells, which specifically includes the following steps:

[0084] (1) Weigh 2.9 kg of microbial fermentation cells, crush them into 30 meshes with a crusher, add 23.3 L of 85% ethanol aqueous solution, stir at 30° C. for 1 hour, separate the solid and liquid after extraction, collect the first extract after separation, take the separated solid, add 20.3 L of 85% ethanol aqueous solution, stir at 30° C. for 1 hour, separate the solid and liquid after extraction, collect the second extract after separation, combine the first and second extracts to obtain the total extract, and the CBG yield is 96.8%;

[0085] (2) adding 0.5% activated carbon to the extract, stirring in a water bath at 45°C for 60 min, obtaining a decolorized extract after solid-liquid separation, and rinsing with 2.5L of 85% ethanol aqueous solution, combining the decolorized collection liquid and the rinsing liquid to obtain a decolorized liquid, with a CBG yield of 95.6%;

[0086] (3) Concentrating the decolorized liquid to obtain CBG extract, adding 0.5 times the weight of 95% ethanol to the CBG extract, heating in a 60° C. water bath to disperse and dissolve, mixing and dispersing with 1 times the weight of CBG extract of 200-300 mesh silica gel, and evaporating the solvent to obtain a solid phase dispersion sample;

[0087] (4) Weigh 3 times the weight of the solid phase dispersed sample and load it into a 200-300 mesh silica gel column. Load the solid phase dispersed sample onto the top of the silica gel column and elute 10 BV with a mixed solution of n-heptane:ethyl acetate = 90:10 (V / V). Collect the eluate and concentrate it to obtain the CBG extract after chromatography. The CBG yield is 94.1%;

[0088] (5) According to the ratio of extract: n-heptane = 1:12 (g / mL), add n-heptane to heat and dissolve all the extract, place the solution in a -5°C refrigerator, leave it for 12 hours, filter it at 4°C, and rinse it with n-heptane to obtain a primary crystallization sample; take the primary crystallization sample, add n-heptane (1:10 g / mL) to heat and dissolve it again, then place it in a 0°C refrigerator, leave it for 12 hours, filter it at 4°C, and rinse it with n-heptane to obtain a secondary crystallization sample;

[0089] (6) The secondary crystallized sample was placed in a 43°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 As shown, the total yield is 61.4% and the product purity is 99.5%.

[0090] Example 4

[0091] This embodiment provides a method for separating and purifying 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 95% ethanol aqueous solution, and other conditions remain unchanged. The total yield is 50.7% and the product purity is 99.4%.

[0092] Example 5

[0093] This embodiment provides a method for separating and purifying 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 total crystallization yield is 44.5% and the product purity is 99.2%.

[0094] Example 6

[0095] This embodiment provides a method for separating and purifying cannabinoid phenol (CBG) from microbial fermentation cells. The specific steps are different from those of Example 1 only in that a mixed solution of n-heptane:ethyl acetate = 98:2 (V / V) is used for elution in step (4), and other conditions remain unchanged. The total yield is 7.6% and the product purity is 99.5%.

[0096] Example 7

[0097] This embodiment provides a method for separating and purifying cannabinoid phenol (CBG) from microbial fermentation cells. The specific steps are different from those of Example 1 only in that in step (5), the equal volume of n-heptane is replaced by a mixed solution of n-heptane:ethyl acetate = 4:1 (V / V), and other conditions remain unchanged. The total yield is 21.8% and the product purity is 99.0%.

[0098] Example 8

[0099] This embodiment provides a method for separating and purifying cannabinoid phenol (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 solution is placed in a -10°C refrigerator for 24 hours for crystallization, filtered at 4°C, and rinsed once with n-heptane to obtain a crystalline sample. Other conditions remain unchanged. The total yield was 70.3% and the product purity was 96.5%.

[0100] Example 9

[0101] This embodiment provides a method for separating and purifying 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 solution is placed in a 0°C refrigerator for 12 hours, filtered at 4°C, and rinsed with n-heptane to obtain a primary crystallization sample; the primary crystallization sample is heated and re-dissolved with n-heptane (1:15 g / mL), and then placed at 25°C room temperature for 12 hours, filtered at 4°C, and rinsed with n-heptane to obtain a secondary crystallization sample. Other conditions remain unchanged. The total yield was 53.8% and the product purity was 99.4%.

[0102] Comparative Example 1

[0103] This comparative example provides a method for separating and purifying cannabinoid phenol (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.

[0104] Comparative Example 2

[0105] This comparative example provides a method for separating and purifying cannabinoid phenol (CBG) from microbial fermentation cells. The specific steps thereof are different from those of Example 1 only in step (3) and step (4). Steps (1), (2) and (5) are the same as those of Example 1, and are specifically as follows:

[0106] (3) concentrating the decolorized liquid to obtain CBG extract;

[0107] (4) Weigh 4 times the weight of the CBG extract and load it into a 100-200 mesh silica gel column. Load the CBG extract onto the top of the silica gel column and elute 7 BV with a mixed solution of n-heptane:ethyl acetate = 4:1 (V / V). Collect the eluate and concentrate it to obtain the CBG extract after chromatography. The total yield was 11.7% and the product purity was 99.5%.

[0108] Comparative Example 3

[0109] This comparative example provides a method for separating and purifying cannabinoids (CBG) from microbial fermentation cells, which comprises the following steps:

[0110] (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;

[0111] (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%.

[0112] The yield and purity results of the cannabinoid products obtained in Examples 1-9 are summarized in Table 1:

[0113] Table 1

[0114] Group Yield (%) purity(%) Example 1 63.9 99.5 Example 2 59.6 99.6 Example 3 61.4 99.5 Example 4 50.7 99.4 Example 5 44.5 99.2 Example 6 7.6 99.5 Example 7 21.8 99.0 Example 8 70.3 96.5 Example 9 53.8 99.4

[0115] From the data results in Table 1, it can be seen that the separation and purification method involved in the present invention uses microbial fermentation cells as raw materials, and sequentially undergoes organic solvent extraction, decolorization, solid phase dispersion of samples with a small amount of silica gel, and then silica gel column elution and crystallization to obtain a high-yield and high-purity cannabinoid product. The separation and purification method is simple and quick to operate, does not require special production equipment, has low production costs, and is green and environmentally friendly.

[0116] 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.

[0117] 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.

[0118] 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 separating and purifying 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 from the liquid to obtain a decolorized solution; (3) mixing the decolorizing solution with silica gel for solid phase dispersion, or concentrating and / or drying the decolorizing solution and re-dissolving it with an alcohol aqueous solution and then mixing it with silica gel for solid phase dispersion, removing the solvent, and obtaining a solid phase dispersed sample; (4) loading the solid phase dispersed sample onto the top of a silica gel column, eluting with a solvent, collecting the eluate, concentrating and / or drying it to obtain a crude product; (5) The crude product is redissolved in a solvent and subjected to crystallization to obtain cannabinoids.

2. The method according to claim 1, characterized in that The powdering in step (1) is performed until the bacterial cell particle size is 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 in step (1) comprises methanol aqueous solution and / or ethanol 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; Preferably, after the solid-liquid separation in step (2), the separated solid is rinsed.

5. The method according to any one of claims 1 to 4, characterized in that The silica gel in step (3) is 100-300 mesh silica gel; Preferably, the volume fraction of alcohol in the alcohol aqueous solution in step (3) is 95%-100%; Preferably, the alcohol aqueous solution in step (3) comprises methanol aqueous solution and / or ethanol aqueous solution; Preferably, the method of removing the solvent in step (3) includes natural evaporation.

6. The method according to any one of claims 1 to 5, characterized in that The silica gel column in step (4) contains 100-300 mesh silica gel; Preferably, the total amount of silica gel in the silica gel column in step (4) is 0.5-6 times that of the solid phase dispersed sample; Preferably, the solvent in step (4) is a mixture of n-heptane and ethyl acetate; Preferably, the volume ratio of n-heptane to ethyl acetate is (4-9):1; Preferably, the solvent elution in step (4) is performed for a total of 5-10 column volumes.

7. The method according to any one of claims 1 to 6, characterized in that The solvent used for the re-dissolution in step (5) is a C5-C8 alkane, more preferably n-heptane; Preferably, the ratio of the amount of the solvent used in step (5) to the mass of the crude product is 1:(8-15) g / mL.

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 the re-dissolution solvent 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.

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