Preparation process of nanoscale water-soluble squalene

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to a preparation process of nanoscale water-soluble squalene. Comprising the following steps: S1, treating corn flour, adding pure water, and performing enzymolysis by adopting compound enzyme to obtain corn saccharified liquid for later use; s2, inoculating saccharomyces cerevisiae GS-A3 into a culture medium containing the corn saccharification liquid, and fermenting; s3, centrifuging the fermentation liquor, and extracting by adopting supercritical CO2 to obtain a squalene crude product; and S4, carrying out nanocrystallization treatment after molecular distillation treatment. According to the method, corn is taken as a raw material, rich starch resources are utilized, the corn is converted into glucose through enzymolysis, squalene is generated in saccharomyces cerevisiae through an MVA way, high-yield and high-purity squalene is prepared, the stability of squalene is improved through a special nanocrystallization treatment mode, the storage life of squalene in the application process is prolonged, and the method is suitable for industrial production. And the bioavailability can be obviously improved. In addition, the technology used in the invention meets the requirements of green and environmental protection.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and particularly relates to a preparation process of nano-scale water-soluble squalene. Background Art

[0002] Squalene (C30H50) is a fully trans straight-chain unsaturated alkene formed by six isoprene units connected end to end. Since it was initially derived from the liver of sharks, it is called squalene, also known as shark liver oil, and is a triterpenoid substance with important medicinal and health care values. The molecular weight of squalene is 410.7 g / mol, the density is 0.8584 mg / mL, the melting point is -75 °C, the boiling point is 285 °C, and the refractive index is 1.49. Due to the presence of six double bonds in its structure, squalene is extremely unstable and prone to oxidation. Therefore, squalene has powerful physiological and pharmacological activities: such as antioxidant, enhancing the body's immunity, antibacterial and anti-inflammatory, anti-cancer, anti-fatigue, preventing cardiovascular diseases, cell recovery, etc. functions, and has been widely concerned due to its extremely high application value in the fields of cosmetics, nutritional supplements, functional foods, pharmaceutical products, etc. Previously, the development and research of squalene mainly used shark liver oil as the raw material. However, with the concept of species protection and ecological awareness deeply rooted in people's hearts, it has become increasingly important to find substances that can replace shark liver oil to protect shark species. There are many alternative squalene extraction substances available. Considering economic rationality and safety and health, extracting squalene from vegetable oils is the preferred method.

[0003] Traditional preparation methods mainly rely on extracting from deep-sea shark livers, mostly using large shark livers as raw materials, and there are problems such as resource shortage, high cost, and low product purity. Although chemical synthesis methods can be produced on a large scale, they have the defects of environmental pollution and insufficient product activity. In comparison, the biological synthesis of squalene has the advantages of short production cycle, environmental friendliness, simple extraction, and mild production conditions.

[0004] Squalene is an important intermediate in the ergosterol synthesis pathway in Saccharomyces cerevisiae. Squalene is produced through the mevalonate pathway (MVA pathway) in Saccharomyces cerevisiae, and its biological metabolic pathway is as Figure 1 shown. Currently, the synthesis of squalene using Saccharomyces cerevisiae has problems such as insufficient supply of precursor substances, poor product stability, and low product yield.

[0005] In addition, squalene, as a lipophilic compound, is soluble in acetone, ether, petroleum ether, and ethyl acetate, and insoluble in water. Due to its poor water solubility and easy oxidation, etc., it has faced severe challenges in the specific application process.

[0006] Based on this, we have proposed a nano-scale water-soluble squalene and its preparation process, hoping to solve the deficiencies in the existing technology. Summary of the Invention

[0007] The object of the present invention is to provide a preparation process of nano - scale water - soluble squalene for existing problems.

[0008] The present invention is achieved through the following technical solutions:

[0009] A preparation process of nano - scale water - soluble squalene includes the following steps:

[0010] S1. Add corn flour into 1 - butyl - 3 - methylimidazolium chloride ionic liquid. After heating to 50 - 60 °C, perform ultrasonic treatment and then centrifuge. Then place it in a vacuum drying oven and dry at 60 - 70 °C. Add pure water to a solid - liquid ratio of 1:3 - 4, and then perform enzymatic hydrolysis with a composite enzyme to obtain a corn saccharification liquid for standby.

[0011] S2. Inoculate Saccharomyces cerevisiae GS - A3 (CCTCC NO: M20211191) into a culture medium containing the above - mentioned corn saccharification liquid, and perform fermentation to obtain a fermentation liquid for standby.

[0012] S3. After centrifuging the fermentation liquid, obtain crude squalene by supercritical CO2 extraction.

[0013] S4. Subject the crude squalene to molecular distillation treatment and then perform nano - scale treatment.

[0014] Furthermore, the ultrasonic treatment time in step S1 is 30 - 40 min;

[0015] During centrifugation, the rotation speed of the centrifuge is 6000 - 8000 r / min, and the centrifugation time is 15 - 20 min;

[0016] The composite enzyme is α - amylase and glucoamylase, and their mass ratio is 1:1 - 2;

[0017] The enzymatic hydrolysis temperature is 50 - 60 °C, and the enzymatic hydrolysis time is 5 - 6 h.

[0018] Furthermore, the preparation of the culture medium containing the above - mentioned corn saccharification liquid in step S2 includes the following steps:

[0019] (1) Place wheat, rice bran, tomato, and Ligusticum chuanxiong in a freeze - drying oven for drying respectively. Weigh 20 - 30 parts of dried wheat, 10 - 20 parts of rice bran, 20 - 30 parts of tomato, and 8 - 13 parts of Ligusticum chuanxiong, and place them together in a cryogenic grinder for grinding. Use ethanol as the extractant, and utilize a Soxhlet extraction device to extract active ingredients under ultrasonic assistance. After extraction is completed, rotate and evaporate to remove the extractant, and then perform freeze - drying to obtain an auxiliary agent.

[0020] (2) Prepare the culture medium according to the following formula: corn saccharified liquid (glucose concentration 30 - 50 g / L), yeast extract 2 - 5 g / L, (NH4)2SO4 1 - 3 g / L, MgSO4·7H2O 0.5 - 1.0 g / L, auxiliary agent 4 - 5 g / L, and adjust the pH to 5.0 - 6.0.

[0021] Furthermore, during the cryogenic grinding in step (1), control the temperature at -80 to -60 °C and the rotation speed at 6000 - 8000 r / min.

[0022] Furthermore, the fermentation conditions in step S2 are: ferment at 28 - 32 °C and 180 - 200 r / min for 50 - 60 h to obtain the fermentation broth.

[0023] Furthermore, the conditions for supercritical CO2 extraction in step S3 are: 25 MPa, 50 - 60 °C;

[0024] The vacuum degree of the molecular distillation in step S4 is 1×10 -4 Pa, and the temperature is 160 - 180 °C.

[0025] Furthermore, step S4 is specifically as follows:

[0026] S401. Add the squalene after molecular distillation treatment to ethanol, then add α-tocopherol with a mass 0.1 - 0.3 times that of squalene and curcumin with a mass 0.1 - 0.2 times that of squalene. After heating to 40 - 50 °C and stirring evenly, add zein with a weight 1 - 2 times that of squalene, and continue to stir until the zein dissolves to obtain a mixed solution. Under the action of ultrasonic waves, drop the mixed solution into water at a speed of 2 - 3 mL / min. After the dropping is completed, stir at 100 - 200 r / min for 2 - 3 h, then centrifuge at 6000 - 8000 r / min and collect the precipitate. Wash the precipitate with deionized water by centrifugation 3 - 4 times and then place it in a vacuum drying oven, and dry it at 50 - 60 °C for 8 - 12 h to obtain squalene nanocrystal core powder;

[0027] S402. Add carboxymethyl-β-cyclodextrin to dimethyl sulfoxide to prepare a carboxymethyl-β-cyclodextrin solution with a concentration of 0.3 - 0.5 mol / L. In another reaction flask, add folic acid to dimethyl sulfoxide, stir and dissolve it, then add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, and stir at 100 - 200 r / min for 3 - 4 h under room temperature and dark conditions to obtain a folic acid solution;

[0028] The folic acid solution was added dropwise to the carboxymethyl-β-cyclodextrin solution at a rate of 2 - 3 mL / min. During the dropping process, it was stirred at a speed of 100 - 200 r / min. After the dropping was completed, stirring was continued for 12 - 16 h, and then it was transferred to a dialysis bag and dialyzed with deionized water for 2 - 3 d. After dialysis was completed, freeze-drying was carried out to obtain modified carboxymethyl-β-cyclodextrin;

[0029] S403: The squalene nanocrystal core powder obtained in step S401 was ultrasonically dispersed in deionized water to obtain a squalene nanocrystal core suspension. The modified carboxymethyl-β-cyclodextrin was ultrasonically dispersed in deionized water. After being evenly dispersed, under the rotational speed condition of 200 - 300 r / min, the squalene nanocrystal core suspension was added dropwise to it at a rate of 2 - 3 mL / min. After the dropping was completed, stirring was continued for 2 - 3 h, and then it was placed in a constant temperature environment of 30 - 40 °C and allowed to stand for 12 - 20 h. Then, it was centrifuged at 6000 - 8000 r / min for 10 - 20 min, and the precipitate was collected. After centrifugally washing 3 - 4 times with deionized water, it was placed in a vacuum drying oven and dried at 50 - 60 °C for 8 - 12 h to obtain the nanoparticles with a middle layer coating;

[0030] S404: The nanoparticles with a middle layer coating were ultrasonically dispersed in deionized water to form a suspension. Under the stirring condition of 200 - 300 r / min, the sodium alginate solution and the chitosan solution were simultaneously added dropwise to the suspension at a rate of 1.5 - 2.5 mL / min. After the dropping was completed, stirring was continued for 3 - 4 h, and then it was allowed to stand at 30 - 40 °C for 14 - 18 h. After centrifuging at 6000 - 8000 r / min for 15 - 20 min, the precipitate was collected. After centrifugally washing 3 - 4 times with deionized water, it was placed in a vacuum drying oven and dried at 50 - 60 °C for 8 - 12 h to obtain the product.

[0031] Furthermore, the mass-volume ratio of squalene to ethanol in step S401 is 1 g:10 - 30 mL.

[0032] Furthermore, the mass-volume ratio of folic acid to dimethyl sulfoxide in step S402 is 1 g:10 - 20 mL;

[0033] The molar ratio of folic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide is 1:1.2 - 1.5:1.2 - 1.5;

[0034] The molar ratio of folic acid to carboxymethyl-β-cyclodextrin is 1:0.5 - 1.

[0035] Further, the preparation method of the oxidized sodium alginate solution described in step S404 is as follows: Dissolve sodium alginate in deionized water, add nanocellulose accounting for 4-6% of the mass of sodium alginate, and then add a hydrogen peroxide solution with a mass fraction of 20-30%, and stir and mix evenly to obtain the oxidized sodium alginate solution;

[0036] The mass-volume ratio of sodium alginate to deionized water is 1 g: 20-40 mL;

[0037] The volume ratio of the hydrogen peroxide solution to deionized water is 1: 40-80;

[0038] The preparation method of the chitosan solution is as follows: Add chitosan to an acetic acid solution with a mass fraction of 1-2% and stir and mix evenly to obtain the chitosan solution. The mass-volume of chitosan to the acetic acid solution is 1 g: 40-50 mL.

[0039] The present invention has the following advantages compared with the prior art:

[0040] 1. The present invention uses corn as a raw material, utilizes the rich starch resources, enzymatically converts them into glucose, realizes low-cost and sustainable raw material supply, and effectively solves the problem of resource shortage relying on extracting squalene from deep-sea shark livers. Pretreat corn flour with ionic liquid, which can destroy the crystalline structure of starch granules and dissolve part of lignin at the same time. Then, combined with the enzymatic hydrolysis of composite enzymes and the action of ultrasonic waves, they cooperate with each other to improve the conversion rate of glucose, and further improve the fermentation efficiency. Moreover, the enzymatic hydrolysis technology is green and environmentally friendly, with low treatment temperature and low energy consumption.

[0041] 2. The present invention optimizes the culture medium, adds a special additive to the culture medium. The additive contains phenolic substances such as ferulic acid that can induce and stimulate metabolism, optimizes the fermentation conditions, shortens the fermentation cycle, and can significantly increase the yield of the product.

[0042] 3. After centrifuging the fermentation broth, the present invention uses supercritical CO2 and molecular distillation technologies in combination to further increase the yield of the product, improve the purity, and avoid the residue of organic solvents, meeting the requirements of green environmental protection.

[0043] 4. The present invention uses a special nanonization technology to nanonize squalene. First, squalene forms nanocrystalline nuclei together with α-tocopherol, curcumin, zein, etc., increasing the stability and specific surface area of squalene, which helps to improve its bioavailability. Then, modified carboxymethyl-β-cyclodextrin is wrapped on the surface of the nanocrystalline nuclei. Its cavity can capture squalene molecules, further improving the stability of squalene and making the entire nanostructure have good solubility and dispersibility in aqueous solution. In addition, the modified carboxymethyl-β-cyclodextrin of this application can also endow the entire nanostructure with the ability to target specific cells and tissues. Finally, a dynamic covalent cross-linking layer is formed outside the nanostructure through a Schiff base reaction, making squalene not easily damaged in different pH environments, increasing the storage period of squalene during application, and also enabling the slow release of squalene according to environmental changes, thereby further improving the bioavailability of squalene. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 It is a metabolic pathway diagram of squalene biosynthesis;

[0045] Figure 2 It is a comparison chart of squalene content in each example and comparative example;

[0046] Figure 3 It is a comparison chart of squalene solubility in each example and comparative example. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0047] In order to further explain the present invention, the following specific embodiments are described below.

[0048] Example 1

[0049] A preparation process for nano-scale water-soluble squalene, comprising the following steps:

[0050] S1. Add corn flour to 1-butyl-3-methylimidazolium chloride ionic liquid. After heating to 50 °C, ultrasonically treat for 30 min and then centrifuge at 6000 r / min for 15 min. Then place it in a vacuum drying oven and dry at 60 °C. Add pure water to a solid-liquid ratio of 1:3, and then use a composite enzyme (α-amylase and glucoamylase with a mass ratio of 1:1) for enzymatic hydrolysis. The enzymatic hydrolysis temperature is 50 °C and the enzymatic hydrolysis time is 5 h to obtain corn sugar solution for standby;

[0051] S2. Inoculate Saccharomyces cerevisiae GS-A3 into the culture medium containing the above-mentioned corn sugar solution, and ferment at 28 °C and 180 r / min for 50 h to obtain a fermentation broth for standby;

[0052] The preparation of the culture medium containing the above-mentioned corn sugar solution includes the following steps:

[0053] (1) After drying wheat, rice bran, tomatoes, and chuanxiong in a freeze-dryer, weigh 20 parts of dried wheat, 10 parts of rice bran, 20 parts of tomatoes, and 8 parts of chuanxiong, and place them together in a cryogenic grinder for grinding at -80°C and 6000 r / min. Using ethanol as the extractant, and with the assistance of a Soxhlet extraction device and ultrasonic waves, extract the active ingredients. After extraction is completed, rotate and evaporate to remove the extractant, and then perform freeze-drying to obtain the auxiliary agent;

[0054] (2) Prepare the culture medium according to the following formula: corn hydrolysate (glucose concentration 30 g / L), yeast extract 2 g / L, (NH4)2SO4 1 g / L, MgSO4·7H2O 0.5 g / L, auxiliary agent 4 - 5 g / L, and adjust the pH to 5.0.

[0055] S3. After centrifuging the fermentation broth, extract the crude squalene using supercritical CO2 (25 MPa, 50°C);

[0056] S4. Subject the crude squalene to molecular distillation treatment (vacuum degree 1×10 -4 Pa, temperature 160°C), and then perform nanometerization treatment. Specifically:

[0057] S401. Add the squalene after molecular distillation treatment to ethanol, with the mass-volume ratio of squalene to ethanol being 1 g:10 mL. Then add α-tocopherol at 0.1 times the mass of squalene and curcumin at 0.1 times the mass of squalene. Heat to 40°C, stir and mix evenly, add zein at 1 times the weight of squalene, and continue to stir until the zein dissolves to obtain a mixed solution. Under the action of ultrasonic waves, drop the mixed solution into water at a speed of 2 mL / min. After dropping is completed, stir at 100 r / min for 2 h, then centrifuge at 6000 r / min to collect the precipitate. Wash the precipitate 3 times by centrifugation with deionized water and then place it in a vacuum drying oven. Dry it at 50°C for 8 h to obtain the squalene nanocrystal core powder;

[0058] S402. Add carboxymethyl-β-cyclodextrin to dimethyl sulfoxide to prepare a carboxymethyl-β-cyclodextrin solution with a concentration of 0.3 mol / L. In another reaction flask, add folic acid to dimethyl sulfoxide, with the mass-volume ratio of folic acid to dimethyl sulfoxide being 1 g:10 mL. Stir and dissolve, then add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide. Under room temperature and dark conditions, stir at 100 r / min for 3 h to obtain the folic acid solution;

[0059] The molar ratio of folic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide is 1:1.2:1.2;

[0060] The folic acid solution was added dropwise to the carboxymethyl-β-cyclodextrin solution at a rate of 2 mL / min, and the molar ratio of folic acid to carboxymethyl-β-cyclodextrin was 1:0.5. During the dropping process, it was stirred at a speed of 100 r / min. After the dropping was completed, stirring was continued for 12 h, and then it was transferred to a dialysis bag and dialyzed with deionized water for 2 d. After dialysis was completed, freeze-drying was carried out to obtain modified carboxymethyl-β-cyclodextrin;

[0061] S403. The squalene nanocrystal core powder obtained in step S401 was ultrasonically dispersed in deionized water to obtain a squalene nanocrystal core suspension. The modified carboxymethyl-β-cyclodextrin was ultrasonically dispersed in deionized water. After being uniformly dispersed, at a rotation speed of 200 r / min, the squalene nanocrystal core suspension was added dropwise thereto at a rate of 2 mL / min. After the dropping was completed, stirring was continued for 2 h, and then it was placed in a constant temperature environment at 30 °C and allowed to stand for 12 h. Then, it was centrifuged at 6000 r / min for 10 min, and the precipitate was collected. After centrifugally washing 3 times with deionized water, it was placed in a vacuum drying oven and dried at 50 °C for 8 h to obtain the nanoparticles with a middle layer coating;

[0062] S404. The nanoparticles with a middle layer coating were ultrasonically dispersed in deionized water to form a suspension. Under the stirring condition of 200 r / min, the sodium alginate solution and the chitosan solution were simultaneously added dropwise to the suspension at a rate of 1.5 mL / min. After the dropping was completed, stirring was continued for 3 h, and then it was allowed to stand at 30 °C for 14 h. After centrifuging at 6000 r / min for 15 min, the precipitate was collected. After centrifugally washing 3 times with deionized water, it was placed in a vacuum drying oven and dried at 50 °C for 8 h to obtain the product;

[0063] The preparation method of the sodium alginate solution is as follows: Sodium alginate was dissolved in deionized water, nanofibrillated cellulose accounting for 4% of the mass of sodium alginate was added, and then a hydrogen peroxide solution with a mass fraction of 20% was added, and they were stirred and mixed evenly to obtain the sodium alginate solution;

[0064] The mass-volume ratio of sodium alginate to deionized water is 1 g:20 mL;

[0065] The volume ratio of the hydrogen peroxide solution to deionized water is 1:40;

[0066] The preparation method of the chitosan solution is as follows: Chitosan was added to an acetic acid solution with a mass fraction of 1% and stirred and mixed evenly to obtain the chitosan solution, and the mass-volume of chitosan to the acetic acid solution is 1 g:40 mL.

[0067] Example 2

[0068] A preparation process of nanoscale water-soluble squalene, including the following steps:

[0069] S1. Add corn flour to 1-butyl-3-methylimidazolium chloride ionic liquid. After heating to 55 °C, perform ultrasonic treatment for 35 min, then centrifuge at 7000 r / min for 18 min. Then place it in a vacuum drying oven and dry at 65 °C. Add pure water to a solid-liquid ratio of 1:3.5, and then perform enzymatic hydrolysis using a composite enzyme (α-amylase and glucoamylase with a mass ratio of 1:1.5). The enzymatic hydrolysis temperature is 55 °C and the enzymatic hydrolysis time is 5.5 h to obtain corn saccharification liquid for standby;

[0070] S2. Inoculate Saccharomyces cerevisiae GS-A3 into the medium containing the above-mentioned corn saccharification liquid, and ferment at 30 °C and 190 r / min for 55 h to obtain fermentation broth for standby;

[0071] The preparation of the medium containing the above-mentioned corn saccharification liquid includes the following steps:

[0072] (1) Place wheat, rice bran, tomato, and chuanxiong in a freeze-drying oven for drying respectively. Weigh 25 parts of dried wheat, 15 parts of rice bran, 25 parts of tomato, and 10 parts of chuanxiong, and place them together in a cryogenic grinder at -70 °C and 7000 r / min for grinding. Use ethanol as the extractant, and use a Soxhlet extraction device to extract active ingredients under ultrasonic assistance. After extraction, rotate and evaporate to remove the extractant, and then perform freeze-drying to obtain an auxiliary agent;

[0073] (2) Prepare the medium according to the following formula: corn saccharification liquid (glucose concentration 40 g / L), yeast extract 3 g / L, (NH4)2SO4 2 g / L, MgSO4·7H2O 0.75 g / L, auxiliary agent 4.5 g / L, and adjust the pH to 5.5.

[0074] S3. After centrifuging the fermentation broth, extract squalene crude product by supercritical CO2 (25 MPa, 55 °C);

[0075] S4. Subject the squalene crude product to molecular distillation treatment (vacuum degree of 1×10 -4 Pa, temperature of 170 °C), and then perform nanometerization treatment. Specifically:

[0076] S401. Add the squalene after molecular distillation treatment to ethanol, with the mass-volume ratio of squalene to ethanol being 1 g:20 mL. Then add α-tocopherol at 0.2 times the mass of squalene and curcumin at 0.15 times the mass of squalene. After heating to 45 °C and stirring evenly, add zein at 1.5 times the weight of squalene, and continue stirring to dissolve zein to obtain a mixed solution. Under the action of ultrasonic waves, drop the mixed solution into water at a speed of 2.5 mL / min. After the dropping is completed, stir at 150 r / min for 2.5 h, then centrifuge at 7000 r / min and collect the precipitate. After centrifugally washing with deionized water 3.5 times, place it in a vacuum drying oven and dry at 55 °C for 10 h to obtain squalene nanocrystal core powder;

[0077] S402. Add carboxymethyl-β-cyclodextrin to dimethyl sulfoxide to prepare a carboxymethyl-β-cyclodextrin solution with a concentration of 0.4 mol / L. In another reaction flask, add folic acid to dimethyl sulfoxide, with the mass-volume of folic acid to dimethyl sulfoxide being 1 g:15 mL. After stirring and dissolving, add 1-ethyl-3-(3-dimethylpropylamine) carbodiimide hydrochloride and N-hydroxysuccinimide, and stir at 150 r / min for 3.5 h under room temperature and dark conditions to obtain a folic acid solution;

[0078] The molar ratio of folic acid, 1-ethyl-3-(3-dimethylpropylamine) carbodiimide hydrochloride, and N-hydroxysuccinimide is 1:1.3:1.3;

[0079] Drop the folic acid solution into the carboxymethyl-β-cyclodextrin solution at a speed of 2.5 mL / min, with the molar ratio of folic acid to carboxymethyl-β-cyclodextrin being 1:0.7. Stir at a speed of 150 r / min during the dropping process. After the dropping is completed, continue stirring for 14 h, then transfer it to a dialysis bag and dialyze with deionized water for 2.5 d. After dialysis is completed, perform freeze-drying to obtain modified carboxymethyl-β-cyclodextrin;

[0080] S403. Ultrasonically disperse the squalene nanocrystal core powder obtained in step S401 in deionized water to obtain a squalene nanocrystal core suspension. Ultrasonically disperse the modified carboxymethyl-β-cyclodextrin in deionized water. After dispersing evenly, under the condition of a rotation speed of 250 r / min, drop the squalene nanocrystal core suspension into it at a speed of 2.5 mL / min. After the dropping is completed, continue stirring for 2.5 h, then place it in a constant temperature environment at 35 °C and let it stand for 16 h. Then centrifuge at 7000 r / min for 15 min and collect the precipitate. After centrifugally washing with deionized water 3 times, place it in a vacuum drying oven and dry at 55 °C for 10 h to obtain the middle-layer coated nanoparticles;

[0081] S404. Ultrasonically disperse the nanoparticles coated in the middle layer into deionized water to form a suspension. Under the stirring condition of 250 r / min, simultaneously drop the sodium alginate oxide solution and the chitosan solution into the suspension at a speed of 2 mL / min. After the dropping is completed, continue stirring for 3.5 h, then let it stand at 35 °C for 16 h, centrifuge at 7000 r / min for 18 min, collect the precipitate, wash it 3 times by centrifugation with deionized water, and then place it in a vacuum drying oven and dry it at 55 °C for 10 h to obtain the product;

[0082] The preparation method of the sodium alginate oxide solution is as follows: Dissolve sodium alginate in deionized water, add nanocellulose accounting for 5% of the mass of sodium alginate, and then add a hydrogen peroxide solution with a mass fraction of 25%, and stir and mix evenly to obtain the sodium alginate oxide solution;

[0083] The mass-volume ratio of sodium alginate to deionized water is 1 g:30 mL;

[0084] The volume ratio of the hydrogen peroxide solution to deionized water is 1:60;

[0085] The preparation method of the chitosan solution is as follows: Add chitosan to an acetic acid solution with a mass fraction of 1.5% and stir and mix evenly to obtain the chitosan solution. The mass-volume of chitosan to the acetic acid solution is 1 g:45 mL.

[0086] Example 3

[0087] A preparation process of nano-scale water-soluble squalene includes the following steps:

[0088] S1. Add corn flour to 1-butyl-3-methylimidazolium chloride ionic liquid. After heating to 60 °C, perform ultrasonic treatment for 40 min, then centrifuge at 8000 r / min for 20 min, and then place it in a vacuum drying oven and dry it at 70 °C. Then add pure water to a solid-liquid ratio of 1:4, and then use a composite enzyme (α-amylase and glucoamylase with a mass ratio of 1:2) for enzymatic hydrolysis. The enzymatic hydrolysis temperature is 60 °C and the enzymatic hydrolysis time is 6 h to obtain corn sugar solution for standby;

[0089] S2. Inoculate Saccharomyces cerevisiae GS-A3 into the medium containing the above-mentioned corn sugar solution, and ferment it at 32 °C and 200 r / min for 60 h to obtain the fermentation broth for standby;

[0090] The preparation of the medium containing the above-mentioned corn sugar solution includes the following steps:

[0091] (1) After drying wheat, rice bran, tomatoes, and chuanxiong in a freeze-dryer respectively, weigh 30 parts of dried wheat, 20 parts of rice bran, 30 parts of tomatoes, and 13 parts of chuanxiong, and place them together in a cryogenic grinder at -60°C and 8000 r / min for grinding. Using ethanol as the extraction agent, with the assistance of a Soxhlet extraction device, carry out the extraction of active ingredients under ultrasonic waves. After the extraction is completed, rotate and evaporate to remove the extraction agent, and then carry out freeze-drying to obtain the auxiliary agent;

[0092] (2) Prepare the culture medium according to the following formula: corn saccharified liquid (glucose concentration 50 g / L), yeast extract 5 g / L, (NH4)2SO4 3 g / L, MgSO4·7H2O 1.0 g / L, auxiliary agent 5 g / L, and adjust the pH to 6.0.

[0093] S3. After centrifuging the fermentation broth, extract the crude squalene by supercritical CO2 (25 MPa, 60°C);

[0094] S4. Subject the crude squalene to molecular distillation treatment (vacuum degree 1×10 -4 Pa, temperature 180°C), and then carry out nanometerization treatment. Specifically:

[0095] S401. Add the squalene after molecular distillation treatment to ethanol, with the mass-volume ratio of squalene to ethanol being 1 g:30 mL. Then add α-tocopherol with a mass 0.3 times that of squalene and curcumin with a mass 0.2 times that of squalene. Heat to 50°C, stir and mix evenly, add zein with a weight 2 times that of squalene, and continue to stir until the zein dissolves to obtain a mixed solution. Under ultrasonic waves, drip the mixed solution into water at a speed of 3 mL / min. After the dripping is completed, stir at 200 r / min for 3 h, then centrifuge at 8000 r / min to collect the precipitate. Wash the precipitate 4 times by centrifugation with deionized water and then place it in a vacuum drying oven, and dry it at 60°C for 12 h to obtain squalene nanocrystal core powder;

[0096] S402. Add carboxymethyl-β-cyclodextrin to dimethyl sulfoxide to prepare a carboxymethyl-β-cyclodextrin solution with a concentration of 0.5 mol / L. In another reaction flask, add folic acid to dimethyl sulfoxide, with the mass-volume ratio of folic acid to dimethyl sulfoxide being 1 g:20 mL. Stir and dissolve, then add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, and stir at 200 r / min for 4 h under room temperature and dark conditions to obtain a folic acid solution;

[0097] The molar ratio of folic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide is 1:1.5:1.5;

[0098] The folic acid solution was dropped into the carboxymethyl-β-cyclodextrin solution at a rate of 3 mL / min, and the molar ratio of folic acid to carboxymethyl-β-cyclodextrin was 1:1. During the dropping process, it was stirred at a speed of 200 r / min. After the dropping was completed, stirring was continued for 16 h, and then it was transferred into a dialysis bag and dialyzed with deionized water for 3 d. After dialysis, it was freeze-dried to obtain modified carboxymethyl-β-cyclodextrin;

[0099] S403. The squalene nanocrystal core powder obtained in step S401 was ultrasonically dispersed in deionized water to obtain a squalene nanocrystal core suspension. The modified carboxymethyl-β-cyclodextrin was ultrasonically dispersed in deionized water. After being dispersed evenly, at a rotation speed of 300 r / min, the squalene nanocrystal core suspension was dropped into it at a rate of 3 mL / min. After the dropping was completed, stirring was continued for 3 h, and then it was placed in a constant temperature environment at 40 °C and allowed to stand for 20 h. Then, it was centrifuged at 8000 r / min for 20 min, and the precipitate was collected. After being centrifuged and washed 4 times with deionized water, it was placed in a vacuum drying oven and dried at 60 °C for 12 h to obtain the nanoparticles with a middle layer coating;

[0100] S404. The nanoparticles with a middle layer coating were ultrasonically dispersed in deionized water to form a suspension. Under the stirring condition of 300 r / min, the sodium alginate solution and the chitosan solution were simultaneously dropped into the suspension at a rate of 2.5 mL / min. After the dropping was completed, stirring was continued for 4 h, and then it was allowed to stand at 40 °C for 18 h. After centrifuging at 8000 r / min for 20 min, the precipitate was collected. After being centrifuged and washed 4 times with deionized water, it was placed in a vacuum drying oven and dried at 60 °C for 12 h to obtain the product;

[0101] The preparation method of the sodium alginate solution is as follows: Sodium alginate was dissolved in deionized water, nano-cellulose accounting for 6% of the mass of sodium alginate was added, and then a hydrogen peroxide solution with a mass fraction of 30% was added, and it was stirred and mixed evenly to obtain the sodium alginate solution;

[0102] The mass-volume ratio of sodium alginate to deionized water is 1 g:40 mL;

[0103] The volume ratio of the hydrogen peroxide solution to deionized water is 1:80;

[0104] The preparation method of the chitosan solution is as follows: Chitosan was added to an acetic acid solution with a mass fraction of 2% and stirred and mixed evenly to obtain the chitosan solution. The mass-volume of chitosan and the acetic acid solution is 1 g:50 mL.

[0105] Comparative Example 1

[0106] On the basis of Example 2, in step S1, the corn flour was not treated with ionic liquid and ultrasound, and pure water was directly added to the corresponding solid-liquid ratio, and the rest of the technical solutions were the same as those in Example 2.

[0107] Comparative Example 2

[0108] Based on Example 2, no auxiliary agent is added to the culture medium in step S2, and the rest of the technical solutions are the same as those of Example 2.

[0109] Comparative Example 3

[0110] Based on Example 2, the modified carboxymethyl-β-cyclodextrin in step S403 is replaced with unmodified carboxymethyl-β-cyclodextrin, and the rest of the technical solutions are the same as those of Example 2.

[0111] Comparative Example 4

[0112] Based on Example 2, step S4 is replaced with the following steps, and the rest of the technical solutions are the same as those of Example 2.

[0113] S4. Mix squalene and β-cyclodextrin at a ratio of 1:3 and ultrasonically treat for 15 min.

[0114] To compare the technical effects of the present invention, squalene was prepared by the methods of Examples 1 to 3 and Comparative Examples 1 to 4 respectively, and then the concentration of squalene in each group of samples was determined by high performance liquid chromatography. The specific test results are as Figure 2 shown.

[0115] From Figure 2 it can be seen that the concentration of squalene prepared by the method of the present invention is the highest, up to 272.2 mg / L. Moreover, the present invention uses corn as a raw material and pretreats it, and then significantly improves the yield of squalene through the optimization of the culture medium.

[0116] From Figure 3 it can be seen that the present invention significantly improves the water solubility of squalene by nanosizing squalene, and the solubility will increase with the increase of temperature.

[0117] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, makes equivalent substitutions or changes, and should be covered by the protection scope of the present invention.

Claims

1. A process for preparing nano-scale water-soluble squalene, characterized in that: The steps include: S1, adding corn flour to 1-butyl-3-methylimidazolium chloride ionic liquid, heating to 50-60° C., ultrasonically treating and centrifuging, then drying in a vacuum drying oven at 60-70° C., adding pure water to a solid-liquid ratio of 1:3-4, and then performing enzymolysis with a composite enzyme to obtain corn saccharification liquid for use; S2, inoculating brewer's yeast GS-A3 into the culture medium containing the corn saccharification liquid, and fermenting to obtain a fermentation liquid for later use; S3, after the fermentation liquid is centrifuged, supercritical CO2 is used to extract the crude squalene; S4. The crude squalene is subjected to molecular distillation and then nano-processed.

2. The process for preparing nano-scale water-soluble squalene according to claim 1, characterized in that: The ultrasonic treatment time in step S1 is 30 to 40 minutes; During the centrifugation, the speed of the centrifuge is 6000-8000 r / min, and the centrifugal treatment time is 15-20 min; The complex enzyme is α-amylase and saccharifying enzyme, and the mass ratio thereof is 1:1-2; The enzymatic hydrolysis temperature is 50-60°C, and the enzymatic hydrolysis time is 5-6h.

3. The process for preparing nano-scale water-soluble squalene according to claim 1, characterized in that: The preparation of the culture medium containing the corn saccharification liquid described in step S2 comprises the following steps: (1) After wheat, rice bran, tomato and Chuanxiong are dried in a freeze drying oven, 20 to 30 parts of the dried wheat, 10 to 20 parts of rice bran, 20 to 30 parts of tomato and 8 to 13 parts of Chuanxiong are weighed and placed in a deep cold mill for pulverization, ethanol is used as an extractant, and a Soxhlet extraction device is used to extract the active ingredient under the assistance of ultrasound. After the extraction is completed, the extractant is removed by rotary evaporation, and then freeze-drying is performed to obtain an auxiliary agent; (2) Prepare the culture medium according to the following formula: corn saccharification solution (glucose concentration 30-50 g / L), yeast extract 2-5 g / L, (NH4)2SO4 1-3 g / L, MgSO4·7H2O 0.5-1.0 g / L, additive 4-5 g / L, and adjust the pH to 5.0-6.

0.

4. The process for preparing nano-scale water-soluble squalene according to claim 3, characterized in that: During the deep cold crushing in step (1), the temperature is controlled at -80 to -60°C and the rotation speed is 6000 to 8000 r / min.

5. The process for preparing nano-scale water-soluble squalene according to claim 1, characterized in that: The fermentation conditions described in step S2 are: fermentation treatment at 28-32° C. and 180-200 r / min for 50-60 hours to obtain a fermentation liquid.

6. The process for preparing nano-scale water-soluble squalene according to claim 1, characterized in that: The conditions of supercritical CO2 extraction in step S3 are: 25MPa, 50-60°C; The vacuum degree of the molecular distillation in step S4 is 1×10 -4 Pa, temperature is 160~180℃.

7. The process for preparing nano-scale water-soluble squalene according to claim 1, characterized in that: Step S4 is specifically as follows: S401, adding squalene treated by molecular distillation to ethanol, then adding α-tocopherol in an amount of 0.1 to 0.3 times the mass of squalene and curcumin in an amount of 0.1 to 0.2 times the mass of squalene, heating to 40 to 50° C. and stirring to mix, adding zein in an amount of 1 to 2 times the mass of squalene, continuing to stir to dissolve the zein to obtain a mixed solution, under the action of ultrasound, dropping the mixed solution into water at a speed of 2 to 3 mL / min, after the dropwise addition is completed, stirring at 100 to 200 r / min for 2 to 3 h, centrifuging at 6000 to 8000 r / min, collecting the precipitate, washing it by centrifugation with deionized water for 3 to 4 times, placing it in a vacuum drying oven, and drying it at 50 to 60° C. for 8 to 12 h to obtain squalene nanocrystalline core powder; S402, adding carboxymethyl-β-cyclodextrin to dimethyl sulfoxide to prepare a carboxymethyl-β-cyclodextrin solution with a concentration of 0.3-0.5 mol / L, in another reaction bottle, adding folic acid to dimethyl sulfoxide, stirring to dissolve, adding 1-ethyl-3-(3-dimethylpropylamine)carbodiimide hydrochloride and N-hydroxysuccinimide, stirring at room temperature and in the dark at 100-200 r / min for 3-4 hours to obtain a folic acid solution; The folic acid solution is added dropwise to the carboxymethyl-β-cyclodextrin solution at a speed of 2-3 mL / min, and stirred at a speed of 100-200 r / min during the dropping process. After the dropping is completed, stirring is continued for 12-16 hours, and then the solution is transferred to a dialysis bag and dialyzed with deionized water for 2-3 days. After the dialysis is completed, the solution is freeze-dried to obtain modified carboxymethyl-β-cyclodextrin; S403, ultrasonically dispersing the squalene nanocrystal core powder obtained in step S401 into deionized water to obtain a squalene nanocrystal core suspension, ultrasonically dispersing modified carboxymethyl-β-cyclodextrin into deionized water, and after uniform dispersion, dripping the squalene nanocrystal core suspension into the deionized water at a speed of 2-3 mL / min at a rotation speed of 200-300 r / min, continuing to stir for 2-3 h after the dripping is completed, and then placing it in a constant temperature environment of 30-40° C. for 12-20 h, and then centrifuging it at 6000-8000 r / min for 10-20 min, collecting the precipitate, washing it with deionized water for 3-4 times, and placing it in a vacuum drying oven, and drying it at 50-60° C. for 8-12 h to obtain nanoparticles coated with a middle layer; S404. Ultrasonic disperse the middle-layer coated nanoparticles into deionized water to form a suspension. Under stirring conditions of 200-300 r / min, simultaneously add the oxidized sodium alginate solution and the chitosan solution to the suspension at a rate of 1.5-2.5 mL / min. After the addition is completed, continue stirring for 3-4 hours, then let it stand at 30-40°C for 14-18 hours, centrifuge at 6000-8000 r / min for 15-20 minutes, collect the precipitate, wash it with deionized water by centrifugation for 3-4 times, place it in a vacuum drying oven, and dry it at 50-60°C for 8-12 hours.

8. The process for preparing nano-scale water-soluble squalene according to claim 7, characterized in that: The mass volume ratio of squalene to ethanol in step S401 is 1 g:10-30 mL.

9. The process for preparing nano-scale water-soluble squalene according to claim 7, characterized in that: The mass volume of folic acid and dimethyl sulfoxide in step S402 is 1 g: 10-20 mL; The molar ratio of folic acid, 1-ethyl-3-(3-dimethylpropylamine)carbodiimide hydrochloride, and N-hydroxysuccinimide is 1:1.2-1.5:1.2-1.5; The molar ratio of folic acid to carboxymethyl-β-cyclodextrin is 1:0.5-1.

10. The process for preparing nano-scale water-soluble squalene according to claim 7, characterized in that: The preparation method of the oxidized sodium alginate solution in step S404 is as follows: dissolving sodium alginate in deionized water, adding 4-6% nanocellulose by mass of the sodium alginate, and then adding 20-30% by mass of hydrogen peroxide solution, stirring and mixing to obtain the oxidized sodium alginate solution; The mass volume ratio of sodium alginate to deionized water is 1g:20-40mL; The volume ratio of hydrogen peroxide solution to deionized water is 1:40-80; The preparation method of the chitosan solution is as follows: chitosan is added into an acetic acid solution with a mass fraction of 1-2%, and the mixture is stirred and mixed to obtain the chitosan solution, wherein the mass volume of the chitosan and the acetic acid solution is 1g:40-50mL.

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