Ganoderma lucidum spore oil enriched with ergosterol and preparation method of ganoderma lucidum spore oil

Abstract

The invention belongs to the technical field of ganoderma lucidum spore oil extraction, and relates to ergosterol-enriched ganoderma lucidum spore oil and a preparation method thereof. According to the method, supercritical CO2 is adopted for extracting ganoderma lucidum spore oil, a dielectrophoresis technology is adopted for removing free fatty acid, the acid value and the peroxide value of the spore oil are reduced, rancidity of the spore oil in the storage process is prevented, a large number of fat-soluble impurities in ergosterol crystals are removed and separated out through recrystallization, and then the ergosterol crystals are enriched into the spore oil through secondary dissolution of supercritical CO2. And finally, removing the plasticizer in the spore oil by adopting a molecularly imprinted polymer, and carrying out reduced pressure distillation to prepare the ganoderma lucidum spore oil with the ergosterol content of 210 mg / 100g or above and the total triterpenoids content of 40 g / 100g or above.

Description

Technical Field

[0001] The invention relates to the technical field of ganoderma lucidum spore oil extraction, in particular to ergosterol-enriched ganoderma lucidum spore oil and a preparation method thereof. Background Art

[0002] Ganoderma lucidum spore oil is an oily lipid extracted from broken Ganoderma lucidum spores through a specific technology. It integrates multiple active ingredients of Ganoderma lucidum spores and has the effects of improving immunity, protecting cardiovascular and cerebrovascular systems, calming the nerves and promoting sleep, protecting the liver and detoxifying.

[0003] There are various methods for preparing Ganoderma lucidum spore oil, including ultrasound, enzymatic hydrolysis, supercritical extraction, and microbial fermentation. Supercritical CO2 extraction is widely used due to its efficiency and high purity. Traditional supercritical CO2-extracted Ganoderma lucidum spore oil contains very low levels of ergosterol and also contains a large amount of plasticizers with similar molecular weight and polarity to ergosterol. The additional steps required to remove the plasticizers can lead to ergosterol loss. Furthermore, some free fatty acids in the spore oil can accelerate its rancidity, making it unsuitable for long-term storage and consumption during processing into health supplements.

[0004] A Chinese patent with the authorization number CN 108485794 B discloses a method for preparing Ganoderma lucidum spore oil. The technical solution uses unbroken Ganoderma lucidum spore powder as raw material, and obtains Ganoderma lucidum spore oil through germination, enzymatic hydrolysis, and ultrasonic freeze-thaw extraction. The spore powder is prepared by non-critical extraction, with a spore powder wall-breaking rate of 98.1% and a spore oil yield of 32.4%.

[0005] Chinese patent application publication number CN 112442419 A discloses a method for preparing Ganoderma lucidum spore oil. The technical solution uses multiple quick freezing and thawing, a roller press to break the wall of Ganoderma lucidum spore powder, and a supercritical CO2 extraction process to prepare Ganoderma lucidum spore oil.

[0006] Based on the above two technical solutions, Ganoderma lucidum spore oil can be prepared. However, both technical solutions aim to provide methods for preparing Ganoderma lucidum spore oil. However, the purification of Ganoderma lucidum spore oil and the enrichment of the effective substance ergosterol have not been mentioned. Therefore, the effective substance ergosterol in the spore oil may be lost, thereby affecting the efficacy of the spore oil, as well as the subsequent processing and storage stability. Therefore, deep processing of the spore oil, gentle impurity removal, and enrichment of more nutrients are the key to preparing high-quality Ganoderma lucidum spore oil. Summary of the Invention

[0007] The present invention provides ergosterol-enriched Ganoderma lucidum spore oil and a preparation method thereof. The Ganoderma lucidum spore oil prepared by this method has an ergosterol content of at least 210 mg / 100 g and a total triterpenoid content of at least 40 g / 100 g. This method utilizes supercritical CO2 extraction to obtain the Ganoderma lucidum spore oil, and then utilizes low-temperature crystallization, dielectrophoresis, and molecularly imprinted polymer-specific adsorption to remove impurities. This method overcomes the problems encountered in conventional techniques, such as residual organic solvents and plasticizers, loss of triterpenoid active ingredients, accumulation of oxidation products, and turbidity caused by wax esters.

[0008] The present invention provides a method for preparing ergosterol-enriched Ganoderma lucidum spore oil, comprising:

[0009] Step A1: adding the broken wall Ganoderma lucidum spore powder into a supercritical CO2 extraction kettle, extracting with supercritical CO2, and collecting the extract and crystals.

[0010] Step A2, diluting the extract with a second solvent, treating it with dielectrophoresis to obtain a crude product of Ganoderma lucidum spore oil with a low ergosterol content, and adding vitamin E to obtain a crude product of Ganoderma lucidum spore oil;

[0011] Step A3: Recrystallize the precipitated crystals in an ethanol-n-hexane mixed solution to obtain ergosterol, mix the ergosterol with the crude product of Ganoderma lucidum spore oil, put the mixture into a reactor, and dissolve the mixture twice using supercritical CO2 to obtain a crude product of Ganoderma lucidum spore oil enriched with ergosterol.

[0012] Step A4: subjecting the crude ergosterol-enriched Ganoderma lucidum spore oil to column chromatography on a molecularly imprinted polymer column and distillation to obtain ergosterol-enriched Ganoderma lucidum spore oil.

[0013] Preferably, in step A1, the supercritical CO2 extraction process parameters are: CO2 flow rate is 300-500 L / h, low pressure section pressure is 8-12 MPa, temperature is 30-40°C, extraction time is 1-2 h, high pressure section pressure is 20-30 MPa, temperature is 40-45°C, and extraction time is 2-3 h.

[0014] Preferably, in step A2, the second solvent is any one or more of n-hexane and isopropanol; and the mass ratio of the extract, the second solvent and vitamin E is 1:(0.5-1.5):(0.0015-0.003).

[0015] Preferably, in step A2, the dielectrophoresis process parameters are: flow rate of 0.5-1 μL / min, temperature of 25-30° C., low frequency band voltage of 5-10 V, frequency of 50-80 kHz, and electrophoresis time of 1-10 min. Subsequently, the parameters are adjusted to: low frequency band voltage of 10-25 V, frequency of 400-800 kHz, and electrophoresis time of 5-10 min. The first stage of electrophoresis removes free fatty acids from the spore oil, and the second stage removes unsaturated fatty acids from the spore oil, thereby reducing the acid value and peroxide value index of the spore oil and reducing the risk of rancidity of the spore oil.

[0016] Preferably, in step A3, the mass ratio of ethanol to n-hexane in the ethanol-n-hexane mixed solution is 1:(2-3).

[0017] Preferably, in step A3, the recrystallization temperature is 50-60° C. and the recrystallization time is 15-30 min. Recrystallization removes impurities co-crystallized with ergosterol, such as high-content wax esters, by utilizing the different solubility of impurities and ergosterol.

[0018] Preferably, in step A3, the parameters of the supercritical CO2 secondary dissolution are: CO2 flow rate of 1 to 3 L / min, pressure of 20 to 35 MPa, temperature of 40 to 60°C, and dissolution time of 60 to 150 min. Ergosterol is dissolved in spore oil. Since the solubility of ergosterol in spore oil is low, by increasing the pressure and adjusting the temperature, the solubility of CO2 for ergosterol is reduced, while the solubility of spore oil for ergosterol is enhanced. At this time, ergosterol is released from the CO2 fluid and redissolved in the spore oil, achieving directional dissolution.

[0019] Preferably, in step A4, the mobile phase used in the column chromatography is any one or more of methanol-acetonitrile (volume ratio of 9:1) and methanol-acetic acid (volume ratio of 9:1).

[0020] Preferably, in step A4, the distillation temperature is 40-60° C. and the vacuum degree is 0.1-1 Pa.

[0021] The present invention provides a method for preparing the molecularly imprinted polymer in the molecularly imprinted polymer column in step A4, comprising:

[0022] Step S1, dispersing the template molecule, di-n-butyl phthalate, di(2-ethyl)hexyl phthalate, and diisononyl phthalate in an acetonitrile-dimethyl sulfoxide mixed solution, stirring, adding ethylene glycol dimethacrylate and azobisisobutyronitrile, and reacting under nitrogen protection to obtain a molecularly imprinted polymer-plasticizer;

[0023] Step S2: washing the molecularly imprinted polymer-plasticizer with a methanol-dichloromethane mixed solution, drying, soaking in a methanol-acetic acid mixed solution, and washing to obtain a molecularly imprinted polymer.

[0024] Preferably, in step S1, the mass ratio of acetonitrile to dimethyl sulfoxide in the acetonitrile-dimethyl sulfoxide mixed solution is (10-20):1.

[0025] Preferably, in step S1, the mass ratio of the template molecule, di-n-butyl phthalate, di(2-ethyl)hexyl phthalate, diisononyl phthalate, acetonitrile-dimethyl sulfoxide mixed solution, ethylene glycol dimethacrylate, and azobisisobutyronitrile is 1: (0.023-0.03): (0.035-0.045): (0.036-0.045): (10-15): (0.05-0.1): (0.1-0.4).

[0026] Preferably, in step S1, the stirring time is 1 to 3 hours.

[0027] Preferably, in step S1, the reaction temperature is 60-70° C., and the reaction time is 20-24 h.

[0028] Preferably, in step S2, the mass ratio of methanol to dichloromethane in the methanol-dichloromethane mixed solution is (2-4):1.

[0029] Preferably, in step S2, the drying temperature is 40-50° C., and the drying time is 10-12 hours.

[0030] Preferably, in step S2, the mass ratio of methanol to acetic acid in the methanol-acetic acid mixed solution is (8-9):1.

[0031] Preferably, in step S2, the soaking time is 40 to 48 hours.

[0032] The present application provides a method for preparing the molecular template in step S1, comprising:

[0033] Step B1, dispersing γ-Fe2O3 magnetic nanoparticles in an ethanol aqueous solution, adjusting the pH to 4-5 with acetic acid, adding 3-(triethoxysilyl)-N-vinylpropylamine, and reacting under inert gas protection to obtain organosilicon-modified γ-Fe2O3;

[0034] Step B2, dispersing the organosilicon-modified γ-Fe2O3 in toluene, adding 4-dimethylaminopyridine and methacrylic anhydride, and reacting under nitrogen protection to obtain an intermediate;

[0035] Step B3: dissolving the intermediate in a first solvent, adding methyl methacrylate and azobisisobutyronitrile, and reacting under inert gas protection to obtain a template molecule, the structural formula of which is as follows:

[0036]

[0037] Preferably, in step B1, the mass ratio of ethanol to water in the ethanol aqueous solution is 1:(1-1.5).

[0038] Preferably, in step B1, the mass ratio of the γ-Fe2O3 magnetic nanoparticles, the ethanol aqueous solution, and 3-(triethoxysilyl)-N-vinylpropylamine is 1:(10-15):(0.15-0.35).

[0039] Preferably, in step B1, the reaction temperature is 50-60° C., and the reaction time is 8-10 h.

[0040] Preferably, in step B2, the mass ratio of the organosilicon-modified γ-Fe2O3, toluene, 4-dimethylaminopyridine and methacrylic anhydride is 1:(10-20):(0.01-0.05):(1-1.5).

[0041] Preferably, in step B2, the reaction temperature is 80-90° C., and the reaction time is 8-12 h.

[0042] Preferably, in step B3, the first solvent is any one or more of toluene and dimethylformamide.

[0043] Preferably, in step B3, the mass ratio of the intermediate, the first solvent, methyl methacrylate, and azobisisobutyronitrile is 1:(10-20):(2-3):(0.05-0.1).

[0044] Preferably, in step B3, the reaction temperature is 70-80° C., and the reaction time is 8-12 h.

[0045] The invention provides ergosterol-enriched Ganoderma lucidum spore oil, which is prepared by adopting the above-mentioned method for enriching Ganoderma lucidum spore oil. In the Ganoderma lucidum spore oil, the ergosterol content is above 210 mg / 100 g, and the total content of triterpenoid compounds is above 40 g / 100 g.

[0046] Compared with the prior art, the beneficial effects of the present invention are embodied in:

[0047] (1) The present application uses supercritical CO2 extraction to prepare Ganoderma lucidum spore oil, and adopts dielectrophoresis treatment to remove free fatty acids in the spore oil, which can effectively reduce the acid value and peroxide value of the spore oil and inhibit the rancidity of the spore oil. Polar molecules such as free fatty acids in the spore oil are affected by the positive dielectrophoretic force in the electric field, migrate to the high electric field area and enrich. Other non-polar substances such as neutral triglycerides are less affected by the dielectrophoretic force and remain in the oil phase. This method can accurately remove impurities. Compared with traditional alkali refining, it does not require chemical reagents, avoids saponification loss, and retains more active ingredients (such as triterpenes and sterols). Since a high-temperature system is not used, the oxidation of spore oil caused by high temperature can be avoided. Experimental data show that dielectrophoresis treatment can reduce the acid value and peroxide value of spore oil. The acid value is 0.63-0.65 mg / g (in terms of kOH) and the peroxide value is 0.18-0.25 g / 100 g.

[0048] (2) This application synthesizes a molecularly imprinted polymer with γ-Fe2O3 magnetic nanoparticles as the core, which can specifically adsorb common plasticizers in spore oil, thereby improving the safety and stability of spore oil. The molecularly imprinted polymer uses γ-Fe2O3 magnetic nanoparticles as the core, and paramagnetic nanoparticles are dispersed with the assistance of a magnetic field, which can reduce the agglomeration of molecularly imprinted polymer particles, allowing the target to diffuse into the imprinted cavity more quickly, thereby achieving precise and rapid adsorption. At the same time, the magnetic nanoparticles, as a skeleton material, can enhance the physical stability of the molecularly imprinted polymer and reduce the particle breakage caused by friction in column chromatography of traditional molecularly imprinted polymers. After recovery through the magnetic field, it can be reused with a simple cleaning. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 The present invention is a flow chart for the preparation of ergosterol-enriched Ganoderma lucidum spore oil.

[0050] Figure 2 The synthetic route of the template molecule.

[0051] Figure 3 Schematic diagram of the synthetic route of molecularly imprinted polymers.

[0052] Figure 4 This is a product picture of Ganoderma lucidum spore oil enriched with ergosterol. DETAILED DESCRIPTION

[0053] The following examples are only used to more clearly illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention.

[0054] Unless otherwise specified, the reagents and equipment involved in the following examples were purchased from commercial channels.

[0055] Example 1

[0056] like Figure 1 As shown, a Ganoderma lucidum spore oil enriched with ergosterol, the preparation method of which comprises:

[0057] Step S1: Disperse 10 g of γ-Fe2O3 magnetic nanoparticles in 100 g of ethanol aqueous solution (50 g of ethanol and 50 g of water), adjust the pH to 4-5 with acetic acid, add 1.5 g of 3-(triethoxysilyl)-N-vinylpropylamine, and react at 50°C for 10 h under inert gas protection to obtain organosilicon-modified γ-Fe2O3.

[0058] Step S2: Disperse 10 g of organosilicon-modified γ-Fe2O3 in 100 g of toluene, add 0.1 g of 4-dimethylaminopyridine and 10 g of methacrylic anhydride, and react at 80° C. for 12 h under nitrogen protection to obtain an intermediate.

[0059] Step S3: Dissolve 10 g of the intermediate in 100 g of toluene, add 20 g of methyl methacrylate and 0.5 g of azobisisobutyronitrile, and react at 70° C. for 12 h under inert gas protection to obtain a template molecule, such as Figure 2 shown.

[0060] Step S4: 10 g of the template molecule, 0.23 g of di-n-butyl phthalate, 0.35 g of di(2-ethyl)hexyl phthalate, and 0.36 g of diisononyl phthalate were dispersed in 100 g of an acetonitrile-dimethyl sulfoxide mixed solution (91 g of acetonitrile and 9 g of dimethyl sulfoxide), stirred for 1 h, 0.5 g of ethylene glycol dimethacrylate and 1 g of azobisisobutyronitrile were added, and the mixture was reacted at 60° C. for 24 h under nitrogen protection to obtain a molecularly imprinted polymer-plasticizer.

[0061] Step S5: 10 g of molecularly imprinted polymer-plasticizer was washed with 100 g of methanol-dichloromethane mixed solution (66 g of methanol and 34 g of dichloromethane), dried at 40° C. for 12 h, and soaked with 100 g of methanol-acetic acid mixed solution (89 g of methanol and 11 g of acetic acid) for 48 h, and washed to obtain a molecularly imprinted polymer, such as Figure 3 shown.

[0062] Step A1, 10kg of broken wall Ganoderma lucidum spore powder was put into a supercritical CO2 extraction kettle, and supercritical CO2 extraction was performed with a CO2 flow rate of 300L / h, a low pressure section pressure of 8MPa, a temperature of 30°C, an extraction time of 2h, a high pressure section pressure of 20MPa, a temperature of 40°C, and an extraction time of 3h, collecting 1820g of extract and 652g of precipitated crystals.

[0063] Step A2, add 900g of n-hexane to dilute the extract, inject at a flow rate of 0.5μL / min, set the system temperature to 25°C, and electrophoresis for 10min at a low frequency band voltage of 5V and a frequency of 50kHz. Then adjust the low frequency band voltage to 10V and electrophoresis for 5min at a frequency of 400kHz to obtain a low-ergosterol Ganoderma lucidum spore oil crude product, add 2.73g of vitamin E to obtain a Ganoderma lucidum spore oil crude product.

[0064] Step A3, 652g of crystals were dissolved in a 1000g ethanol-n-hexane mixed solution (333g of ethanol and 667g of n-hexane), and recrystallized at 50°C for 30min to obtain ergosterol, which was mixed with the crude product of Ganoderma lucidum spore oil and put into a reactor. The mixture was dissolved in supercritical CO2 for a second time at a CO2 flow rate of 1L / min, a pressure of 20MPa, and a temperature of 40°C for 150min to obtain a crude product of Ganoderma lucidum spore oil enriched with ergosterol.

[0065] Step A4: The crude product of ergosterol-enriched Ganoderma lucidum spore oil was placed in a column containing a molecularly imprinted polymer, and column chromatography was performed using a methanol-acetonitrile mixed solution (methanol and acetonitrile volume ratio of 9:1) as the mobile phase. The collected liquid was distilled at 0.1 Pa and 40° C. to obtain ergosterol-enriched Ganoderma lucidum spore oil, such as Figure 4 shown.

[0066] Example 2

[0067] like Figure 1 As shown, a Ganoderma lucidum spore oil enriched with ergosterol, the preparation method of which comprises:

[0068] Step S1: Disperse 10 g of γ-Fe2O3 magnetic nanoparticles in 120 g of ethanol aqueous solution (54 g of ethanol and 66 g of water), adjust the pH to 4-5 with acetic acid, add 2.5 g of 3-(triethoxysilyl)-N-vinylpropylamine, and react at 55°C for 9 h under inert gas protection to obtain organosilicon-modified γ-Fe2O3.

[0069] Step S2: Disperse 10 g of organosilicon-modified γ-Fe2O3 in 150 g of toluene, add 0.3 g of 4-dimethylaminopyridine and 13 g of methacrylic anhydride, and react at 85° C. for 10 h under nitrogen protection to obtain an intermediate.

[0070] Step S3: Dissolve 10 g of the intermediate in 150 g of dimethylformamide, add 25 g of methyl methacrylate and 0.75 g of azobisisobutyronitrile, and react at 75° C. for 10 h under inert gas protection to obtain a template molecule, such as Figure 2 shown.

[0071] Step S4: 10 g of the template molecule, 0.27 g of di-n-butyl phthalate, 0.4 g of di(2-ethyl)hexyl phthalate, and 0.4 g of diisononyl phthalate were dispersed in 120 g of an acetonitrile-dimethyl sulfoxide mixed solution (112.5 g of acetonitrile and 7.5 g of dimethyl sulfoxide), stirred for 2 h, 0.75 g of ethylene glycol dimethacrylate and 2.5 g of azobisisobutyronitrile were added, and the mixture was reacted at 65° C. for 22 h under nitrogen protection to obtain a molecularly imprinted polymer-plasticizer.

[0072] Step S5: 10 g of molecularly imprinted polymer-plasticizer was washed with 100 g of methanol-dichloromethane mixed solution (75 g of methanol and 25 g of dichloromethane), dried at 45° C. for 10 h, soaked with 100 g of methanol-acetic acid mixed solution (90 g of methanol and 10 g of acetic acid) for 44 h, and washed to obtain a molecularly imprinted polymer, such as Figure 3 shown.

[0073] Step A1, put 10kg of broken wall Ganoderma lucidum spore powder into a supercritical CO2 extraction kettle, use supercritical CO2 extraction, CO2 flow rate is 400L / h, low pressure section pressure is 10MPa, temperature is 35°C, extraction time is 2h, high pressure section pressure is 25MPa, temperature is 40°C, extraction time is 2h, collect 2100g of extract and 853g of precipitated crystals.

[0074] Step A2, add 2000g of isopropanol to dilute the extract, inject at a flow rate of 1 μL / min, set the system temperature to 30°C, and perform electrophoresis for 5 minutes at a low frequency band voltage of 10V and a frequency of 65kHz. Then adjust the low frequency band voltage to 20V and the frequency to 600kHz for an electrophoresis time of 7 minutes to obtain a crude product of Ganoderma lucidum spore oil with a low content of ergosterol. Add 6.3g of vitamin E to obtain a crude product of Ganoderma lucidum spore oil.

[0075] Step A3, 853g of crystals were dissolved in 1000g of ethanol-n-hexane mixed solution (250g of ethanol and 750g of n-hexane), and recrystallized at 55°C for 20min to obtain ergosterol, which was mixed with the crude product of Ganoderma lucidum spore oil and put into a reactor. The mixture was dissolved in supercritical CO2 for a second time at a CO2 flow rate of 2L / min, a pressure of 25MPa, and a temperature of 50°C for 100min to obtain a crude product of Ganoderma lucidum spore oil enriched with ergosterol.

[0076] Step A4: The crude product of ergosterol-enriched Ganoderma lucidum spore oil was placed in a column containing a molecularly imprinted polymer, and column chromatography was performed using a methanol-acetic acid mixed solution (methanol and acetic acid volume ratio of 9:1) as the mobile phase. The collected liquid was distilled at 0.5 Pa and 50° C. to obtain ergosterol-enriched Ganoderma lucidum spore oil, such as Figure 4 shown.

[0077] Example 3

[0078] like Figure 1 As shown, a Ganoderma lucidum spore oil enriched with ergosterol, the preparation method of which comprises:

[0079] Step S1: Disperse 10 g of γ-Fe2O3 magnetic nanoparticles in 150 g of ethanol aqueous solution (60 g of ethanol and 90 g of water), adjust the pH to 4-5 with acetic acid, add 3.5 g of 3-(triethoxysilyl)-N-vinylpropylamine, and react at 60°C for 8 h under inert gas protection to obtain organosilicon-modified γ-Fe2O3 magnetic nanoparticles.

[0080] Step S2: Disperse 10 g of organosilicon-modified γ-Fe2O3 magnetic nanoparticles in 200 g of toluene, add 0.5 g of 4-dimethylaminopyridine and 15 g of methacrylic anhydride, and react at 90° C. for 8 h under nitrogen protection to obtain an intermediate.

[0081] Step S3: Dissolve 10 g of the intermediate in 200 g of toluene, add 30 g of methyl methacrylate and 1 g of azobisisobutyronitrile, and react at 80° C. for 8 h under inert gas protection to obtain a template molecule, such as Figure 2 shown.

[0082] Step S4: 10 g of the template molecule, 0.3 g of di-n-butyl phthalate, 0.45 g of di(2-ethyl)hexyl phthalate, and 0.45 g of diisononyl phthalate were dispersed in 150 g of an acetonitrile-dimethyl sulfoxide mixed solution (143 g of acetonitrile and 7 g of dimethyl sulfoxide), stirred for 3 h, 1 g of ethylene glycol dimethacrylate and 4 g of azobisisobutyronitrile were added, and the mixture was reacted at 70° C. for 20 h under nitrogen protection to obtain a molecularly imprinted polymer-plasticizer.

[0083] Step S5: 10 g of molecularly imprinted polymer-plasticizer was washed with 100 g of methanol-dichloromethane mixed solution (80 g of methanol and 20 g of dichloromethane), dried at 50° C. for 10 h, soaked with 100 g of methanol-acetic acid mixed solution (90 g of methanol and 10 g of acetic acid) for 40 h, and washed to obtain a molecularly imprinted polymer, such as Figure 3 shown.

[0084] Step A1, put 10kg of broken wall Ganoderma lucidum spore powder into a supercritical CO2 extraction kettle, use supercritical CO2 extraction, CO2 flow rate is 500L / h, low pressure section pressure is 12MPa, temperature is 40°C, extraction time is 1h, high pressure section pressure is 30MPa, temperature is 45°C, extraction time is 2h, collect 1950g of extract and 804g of precipitated crystals.

[0085] Step A2, add 3000g of n-hexane to dilute the extract, inject at a flow rate of 0.5μL / min, set the system temperature to 30°C, and perform electrophoresis at a low frequency band voltage of 10V and a frequency of 80kHz for 1min, then adjust the low frequency band voltage to 25V and the frequency to 800kHz for 5min to obtain a crude product of Ganoderma lucidum spore oil with a low content of ergosterol, add 3.9g of vitamin E to obtain a crude product of Ganoderma lucidum spore oil.

[0086] Step A3, 804g of crystals were dissolved in 1000g of ethanol-n-hexane mixed solution (250g of ethanol and 750g of n-hexane), and recrystallized at 60°C for 15min to obtain ergosterol, which was mixed with the crude product of Ganoderma lucidum spore oil and put into a reactor. The mixture was dissolved in supercritical CO2 for a second time at a CO2 flow rate of 3L / min, a pressure of 35MPa, and a temperature of 60°C for 60min to obtain a crude product of Ganoderma lucidum spore oil enriched with ergosterol.

[0087] Step A4: The crude product of ergosterol-enriched Ganoderma lucidum spore oil was placed in a column containing a molecularly imprinted polymer, and column chromatography was performed using a methanol-acetonitrile mixed solution (methanol and acetonitrile volume ratio of 9:1) as the mobile phase. The collected liquid was distilled at 1 Pa and 60° C. to obtain ergosterol-enriched Ganoderma lucidum spore oil, such as Figure 4 shown.

[0088] Comparative Example 1

[0089] A preparation method of ergosterol-enriched Ganoderma lucidum spore oil is different from that of Example 2 in that in step S1, γ-Fe2O3 magnetic nanoparticles are replaced with titanium dioxide nanoparticles (non-magnetic).

[0090] Comparative Example 2

[0091] A preparation method of ergosterol-enriched Ganoderma lucidum spore oil is different from that of Example 2 in that a single functional monomer, di-n-butyl phthalate, is used in step S4.

[0092] Comparative Example 3

[0093] A preparation method of ergosterol-enriched Ganoderma lucidum spore oil is different from that of Example 2 in that step A2 does not include dielectrophoresis treatment.

[0094] Comparative Example 4

[0095] A Ganoderma lucidum spore oil enriched with ergosterol, the preparation method of which is different from that of Example 2 in that the crystallization in step A3 is not subjected to recrystallization.

[0096] Table 1 Test items

[0097]

[0098]

[0099] Table 2 Acid value and peroxide value index determination data of Ganoderma lucidum spore oil

[0100] Sample number Acid value (in kOH, mg / g) Peroxide value (g / 100g) Example 1 0.65 0.23 Example 2 0.63 0.18 Example 3 0.63 0.25 Comparative Example 1 0.96 1.053 Comparative Example 2 1.05 0.95 Comparative Example 3 3.26 1.255 Comparative Example 4 1.35 1.320

[0101] Acid value is a parameter that reflects the free fatty acid content in oils and fats. It is the number of milligrams of 0.1 mol / L KOH standard solution consumed per gram of oil and fat. It is an important indicator for measuring whether the oil has deteriorated. For used oils, a higher acid value means the quality of the oil is worse. As shown in Table 2, the acid value of the Ganoderma lucidum spore oils prepared using the methods of Examples 1 to 3 is all below 1 mg / g. The acid value of the spore oil prepared in Comparative Example 1 is 0.96 mg / g. The acid value of the spore oils prepared in Comparative Examples 2 to 4 is all above 1 mg / g. The acid value of the spore oil prepared in Comparative Example 3 exceeds 2 mg / g. Experiments have shown that the Ganoderma lucidum spore oils prepared using Examples 1 to 3 contain fewer free fatty acids and are of better quality.

[0102] Peroxide value is an early indicator of oil rancidity, primarily reflecting the degree of oxidation. It can be used to determine whether a food has oxidized and deteriorated. Products with high peroxide values often have low-quality raw materials or improper processing. As shown in Table 1, the peroxide values of the Ganoderma lucidum spore oils prepared in Examples 1 to 3 were all below 0.25, while the peroxide values of the Ganoderma lucidum spore oils prepared in Comparative Examples 1, 3, and 4 were above 1. The peroxide value of the Ganoderma lucidum spore oil prepared in Comparative Example 2 was 0.95. Experiments have shown that the Ganoderma lucidum spore oils prepared using the methods of Examples 1 to 3 contain fewer impurities that cause oil rancidity and have better oil quality.

[0103] Among them, the acid value and peroxide value of the spore oil prepared in Comparative Example 3 were higher than those of the other groups. In Comparative Example 3, the crude spore oil product was not treated with dielectrophoresis. Therefore, the content of substances such as free fatty acids in the oil was high, resulting in a high acid value and peroxide value of the spore oil. The spore oil prepared in Comparative Example 4 was not treated with recrystallization when the crystals precipitated in step A1 were treated. Therefore, the fat-soluble impurities in the crystals, such as wax esters, were high, resulting in a high acid value and peroxide value of the spore oil, as well as a darker color and a turbid appearance. White floccules would appear when the spore oil was left standing at normal temperature and pressure.

[0104] Table 3 Determination data of lead, total arsenic and total mercury content in Ganoderma lucidum spore oil

[0105]

[0106] As shown in Table 3, no heavy metals such as lead, total arsenic, mercury, cadmium, nickel, and chromium were detected in the Ganoderma lucidum spore oils prepared by the methods of Examples 1 to 3 and Comparative Examples 1 to 4.

[0107] Table 4 Determination data of plasticizer content in Ganoderma lucidum spore oil

[0108]

[0109] As shown in the data in Table 4, no plasticizer was detected in the Ganoderma lucidum spore oils prepared in Examples 1 to 3 and Comparative Examples 3 to 4. Trace amounts of plasticizer residues were detected in the spore oils prepared in Comparative Examples 1 and 2. In Comparative Example 1, γ-Fe2O3 magnetic nanoparticles were not used, but non-magnetic titanium dioxide nanoparticles were used. Its separation efficiency and adaptability to complex systems were significantly lower than those of the molecularly imprinted polymer using γ-Fe2O3 magnetic nanoparticles. The molecularly imprinted polymer using γ-Fe2O3 magnetic nanoparticles has good dispersibility, high mass transfer efficiency, and short adsorption equilibrium time, and can achieve "instant absorption and separation" under magnetic field drive. In Comparative Example 2, only a single functional monomer was used, with fewer action sites, and usually only a single target was targeted. However, the use of multiple functional monomers can simultaneously imprint multiple types of targets.

[0110] Table 5 Determination data of moisture, volatile matter content and solvent residue in Ganoderma lucidum spore oil

[0111]

[0112] As shown in the data in Table 5, the contents of water and volatile matter in the spore oils prepared in Examples 1 to 3 and Comparative Examples 1 to 4 are all lower than 0.1%, which are all within the specified ranges of the corresponding standard documents. The low water content makes the Ganoderma lucidum spore oil more transparent and has a low volatile matter content, which can improve the olfactory comfort of the product. In addition, no chemical reagent residues were detected in the Ganoderma lucidum spore oils prepared by the above method. Experiments have proved that the above preparation method is rigorous, green and safe.

[0113] Table 6 Determination data of ergosterol and total triterpenoid contents in Ganoderma lucidum spore oil

[0114] Sample number Ergosterol content (mg / 100g) Total triterpenoid content (g / 100g) Example 1 325 47.8 Example 2 350 49.5 Example 3 327 47.3 Comparative Example 1 274 40.11 Comparative Example 2 215 41.28 Comparative Example 3 232 40.17 Comparative Example 4 238 41.05

[0115] As shown in Table 6, the ergosterol content enriched in the Ganoderma lucidum spore oil prepared in Examples 1 to 3 is above 210 mg / 100 g, while the ergosterol content in the spore oil prepared in Comparative Examples 1 to 4 is below 210 mg / 100 g. Experiments have shown that the methods of Examples 1 to 3 can be used to prepare Ganoderma lucidum spore oil with a high ergosterol content; in Comparative Examples 1 to 4, the residual impurities that were not removed interfere with the identification of ergosterol during detection, resulting in quantitative errors, or causing oxidation, resulting in a decrease in the actual content of ergosterol. The total triterpene content in the spore oil prepared in Examples 1 to 3 and Comparative Examples 1 to 4 is above 40 g / 100 g, and the total triterpene content in the spore oil prepared in Examples 1 to 3 is higher than that in the spore oil prepared in Comparative Examples 1 to 4.

[0116] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing ergosterol-enriched Ganoderma lucidum spore oil, characterized in that: include: Step A1: adding the broken spore powder of Ganoderma lucidum into a supercritical CO2 extraction kettle, extracting with supercritical CO2, collecting the extract and precipitating crystals; Step A2, diluting the extract with a second solvent, treating it with dielectrophoresis to obtain a crude product of Ganoderma lucidum spore oil with a low ergosterol content, and adding vitamin E to obtain a crude product of Ganoderma lucidum spore oil; Step A3, recrystallizing the precipitated crystals in an ethanol-n-hexane mixed solution to obtain ergosterol, mixing the ergosterol with the crude product of Ganoderma lucidum spore oil, adding the mixture to a reactor, and performing secondary dissolution using supercritical CO2 to obtain a crude product of Ganoderma lucidum spore oil enriched with ergosterol; Step A4: subjecting the crude ergosterol-enriched Ganoderma lucidum spore oil to column chromatography on a molecularly imprinted polymer column and distillation to obtain ergosterol-enriched Ganoderma lucidum spore oil.

2. The method for preparing ergosterol-enriched Ganoderma lucidum spore oil according to claim 1, wherein: In the step A1, the supercritical CO2 extraction process parameters are: CO2 flow rate of 300-500 L / H, low pressure section pressure of 8-12 MPa, temperature of 30-40°C, extraction time of 1-2 h, high pressure section pressure of 20-30 MPa, temperature of 40-45°C, extraction time of 2-3 h; in the step A2, the second solvent is any one or more of n-hexane and isopropanol; the extract, the second solvent and the vitamin The mass ratio of element E is 1: (0.5-1.5): (0.0015-0.003); the dielectrophoresis process parameters are: flow rate of 0.5-1 μL / min, temperature of 25-30°C, low-frequency band voltage of 5-10 V, frequency of 50-80 kHz, and electrophoresis time of 1-10 min, and then the parameters are adjusted to: low-frequency band voltage of 10-25 V, frequency of 400-800 kHz, and electrophoresis time of 5-10 min.

3. The method for preparing ergosterol-enriched Ganoderma lucidum spore oil according to claim 1, wherein: In the step A3, the mass ratio of ethanol to n-hexane in the ethanol-n-hexane mixed solution is 1:(2-3); the recrystallization temperature is 50-60°C, and the recrystallization time is 15-30 min; the parameters of the supercritical CO2 secondary dissolution are: CO2 flow rate of 1-3 L / min, pressure of 20-35 MPa, temperature of 40-60°C, and dissolution time of 60-150 min; in the step A4, the mobile phase used in the column chromatography is any one or more of methanol-acetonitrile and methanol-acetic acid, the volume ratio of methanol to acetonitrile is 9:1, and the volume ratio of methanol to acetic acid is 9:1; the distillation temperature is 40-60°C, and the vacuum degree is 0.1-1 Pa.

4. The method for preparing ergosterol-enriched Ganoderma lucidum spore oil according to claim 1, wherein: In step A4, the method for preparing the molecularly imprinted polymer in the molecularly imprinted polymer column comprises: Step S1, dispersing the template molecule, di-n-butyl phthalate, di(2-ethyl)hexyl phthalate, and diisononyl phthalate in an acetonitrile-dimethyl sulfoxide mixed solution, stirring, adding ethylene glycol dimethacrylate and azobisisobutyronitrile, and reacting under nitrogen protection to obtain a molecularly imprinted polymer-plasticizer; Step S2: washing the molecularly imprinted polymer-plasticizer with a methanol-dichloromethane mixed solution, drying, soaking in a methanol-acetic acid mixed solution, and washing to obtain a molecularly imprinted polymer.

5. The method for preparing ergosterol-enriched Ganoderma lucidum spore oil according to claim 4, wherein: In step S1, the mass ratio of acetonitrile and dimethyl sulfoxide in the acetonitrile-dimethyl sulfoxide mixed solution is (10-20):1; the mass ratio of the template molecule, di-n-butyl phthalate, di(2-ethyl)hexyl phthalate, diisononyl phthalate, acetonitrile-dimethyl sulfoxide mixed solution, ethylene glycol dimethacrylate, and azobisisobutyronitrile is 1:(0.023-0.03):(0.035-0.045):(0.036-0.045):(10-15):(0.05-0.1):(0.1-0.4); the stirring time is 1-3 hours; the reaction temperature is 60-70°C, and the reaction time is 20-24 hours.

6. The method for preparing ergosterol-enriched Ganoderma lucidum spore oil according to claim 4, wherein: In step S2, the mass ratio of methanol to dichloromethane in the methanol-dichloromethane mixed solution is (2-4):1; the drying temperature is 40-50°C, and the drying time is 10-12 hours; the mass ratio of methanol to acetic acid in the methanol-acetic acid mixed solution is (8-9):1; and the soaking time is 40-48 hours.

7. The method for preparing ergosterol-enriched Ganoderma lucidum spore oil according to claim 4, wherein: In step S1, the method for preparing the template molecule includes: Step B1, dispersing γ-Fe2O3 magnetic nanoparticles in an ethanol aqueous solution, adjusting the pH to 4-5 with acetic acid, adding 3-(triethoxysilyl)-N-vinylpropylamine, and reacting under inert gas protection to obtain organosilicon-modified γ-Fe2O3; Step B2, dispersing the organosilicon-modified γ-Fe2O3 in toluene, adding 4-dimethylaminopyridine and methacrylic anhydride, and reacting under nitrogen protection to obtain an intermediate; Step B3: dissolving the intermediate in a first solvent, adding methyl methacrylate and azobisisobutyronitrile, and reacting under inert gas protection to obtain a template molecule, the structural formula of which is as follows:

8. The method for preparing ergosterol-enriched Ganoderma lucidum spore oil according to claim 7, wherein: In the step B1, the mass ratio of ethanol to water in the ethanol-water solution is 1:(1-1.5); the mass ratio of the γ-Fe2O3 magnetic nanoparticles, the ethanol-water solution, and 3-(triethoxysilyl)-N-vinylpropylamine is 1:(10-15):(0.15-0.35); the reaction temperature is 50-60°C, and the reaction time is 8-10 hours; in the step B2, the mass ratio of the organosilicon-modified γ-Fe2O3, toluene, 4-dimethylaminopyridine, and methacrylic anhydride is 1:(10-20):(0.01-0.05):(1-1.5); the reaction temperature is 80-90°C, and the reaction time is 8-12 hours.

9. The method for preparing ergosterol-enriched Ganoderma lucidum spore oil according to claim 7, wherein: In step B3, the first solvent is any one or more of toluene and dimethylformamide; the mass ratio of the intermediate, the first solvent, methyl methacrylate, and azobisisobutyronitrile is 1:(10-20):(2-3):(0.05-0.1); the reaction temperature is 70-80°C, and the reaction time is 8-12 hours.

10. A Ganoderma lucidum spore oil enriched in ergosterol, prepared by the method for preparing a Ganoderma lucidum spore oil enriched in ergosterol according to any one of claims 1 to 9, characterized in that: In the Ganoderma lucidum spore oil, the content of ergosterol is above 210 mg / 100 g, and the content of total triterpenes is above 40 g / 100 g.

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