Method for separating and purifying hydroxytyrosol and preparing inclusion compound

Abstract

The invention discloses a method for separating and purifying hydroxytyrosol and preparing an inclusion compound, and belongs to the field of biochemical engineering separation and purification. The fermentation liquor is concentrated through membrane separation, an adsorption material is added into the concentrated liquor, vacuum concentration and evaporation drying are conducted, and a mixture is obtained; performing supercritical extraction on the mixture in a supercritical fluid extraction tank; three times of molecular distillation are adopted, and the high-purity hydroxytyrosol is finally obtained by controlling the conditions of each time of molecular distillation; and dissolving hydroxypropyl-beta-cyclodextrin in water, adding the obtained hydroxytyrosol into the hydroxypropyl-beta-cyclodextrin, carrying out nitrogen protection, heating and stirring for reaction, cooling the reactant, and freeze-drying to obtain the hydroxytyrosol inclusion compound. According to the method, no organic solvent is used, the purification cost is reduced, hydroxytyrosol is prepared into the inclusion compound, the light stability and the heat stability of hydroxytyrosol are remarkably improved on the premise of ensuring the activity, and the method is beneficial to expanding application of hydroxytyrosol.

Description

Technical Field

[0001] The invention belongs to the field of biochemical separation and purification, and specifically relates to a method for separating and purifying hydroxytyrosol and preparing an inclusion compound. Background Art

[0002] Hydroxytyrosol, also known as 3,4-dihydroxyphenylethanol, is a natural polyphenol compound. Hydroxytyrosol has been widely used in cosmetics, health products and food additives for its strong antioxidant and anti-inflammatory activities. It has the effects of whitening, anti-aging, cardiovascular protection, and improving cognitive ability.

[0003] Currently, commercial hydroxytyrosol is mainly extracted from natural plants or obtained by chemical synthesis. Plant extraction requires the consumption of a large amount of raw materials, is severely affected by the planting area and growth cycle, and has a low yield. The chemical synthesis method is also limited by the high cost of substrates, complex processes, environmental pollution and other issues. In recent years, it has become a reality to efficiently produce hydroxytyrosol using cheap raw materials through biosynthesis technology. The hydroxytyrosol obtained by biological fermentation has low content and is difficult to purify. It often requires the combined use of membrane separation, column chromatography, molecular distillation and other technologies to obtain high-purity hydroxytyrosol. In particular, the use of column chromatography requires the consumption of a large amount of organic solvents, which greatly increases its production cost.

[0004] SFE (supercritical fluid extraction) has been used to extract hydroxytyrosol from olive oil due to its advantages of being green, environmentally friendly and low cost. However, the processing objects of SFE technology are limited to solid, oily products or low-water-content matrices. The hydroxytyrosol obtained by biological fermentation is an aqueous solution system with a high water content and cannot be directly purified using SFE technology. In addition, hydroxytyrosol is greatly affected by air, light and heat. If the separation method is not selected properly, not only will high-purity hydroxytyrosol fail to be obtained, but additional impurities will be introduced due to oxidation, degradation and other factors, greatly affecting its downstream applications. Therefore, the purification of hydroxytyrosol fermentation broth in aqueous solution systems still faces great challenges.

[0005] Studies have shown that hydroxytyrosol can improve the antioxidant level of liver cells, reduce liver cell damage caused by ethanol, thereby increasing liver cell survival rate, reducing the levels of aspartate aminotransferase and alanine aminotransferase, and effectively protecting liver cells. High-purity hydroxytyrosol is unstable to light and heat, and is easily oxidized and discolored during use, limiting its application in food, health products, skin care products, etc. Summary of the invention

[0006] In order to solve the problems in the prior art, the present invention proposes a method for separating, purifying and preparing inclusion compounds of hydroxytyrosol. The method solves the difficulty of extracting hydroxytyrosol from hydroxytyrosol fermentation broth. First, the fermentation broth is subjected to membrane separation to remove macromolecular impurities and achieve preliminary concentration of hydroxytyrosol. Then, an adsorbent material with a suitable mesh size and amount is added and combined with vacuum concentration and evaporation technology to obtain a mixture suitable for SFE purification treatment. Then, SFE is used for purification. SFE not only avoids the use of organic solvents and reduces production costs, but also hydroxytyrosol is purified in CO. 2 The present invention further adopts three-step molecular distillation to obtain high-purity hydroxytyrosol. At the same time, it is prepared into a hydroxytyrosol inclusion compound, which significantly improves the stability and retains good antioxidant and potential liver protection properties.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is:

[0008] The present invention provides a method for separating and purifying hydroxytyrosol and preparing an inclusion compound, comprising the following steps:

[0009] S1: using membrane separation to concentrate the hydroxytyrosol fermentation liquid obtained by the biological fermentation method to obtain a concentrated liquid; adding an adsorption material to the concentrated liquid, and vacuum concentrating and evaporating to obtain a mixture;

[0010] S2: The mixture obtained in S1 is placed in a supercritical fluid extraction tank with supercritical CO 2 Supercritical extraction is carried out with 2% NH4O2 as the extractant and water as the entrainer;

[0011] S3: subjecting the extract obtained in S2 to the first molecular distillation, with feed insulation temperature of 70-90 °C, heavy phase insulation temperature of 70-90 °C, evaporation temperature of 90-120 °C, condensation temperature of 5-20 °C, vacuum degree of 4000-5000 Pa, and retaining the heavy phase component;

[0012] The heavy phase component is subjected to a second molecular distillation, with a feed insulation temperature of 70-90°C, a heavy phase insulation temperature of 70-100°C, an evaporation temperature of 110-160°C, a condensation temperature of 40-70°C, a vacuum degree of 5-100 Pa, and the heavy phase component is retained;

[0013] The heavy phase component obtained by the second molecular distillation is subjected to a third molecular distillation, with a feed insulation temperature of 90-110°C, a heavy phase insulation temperature of 100-120°C, an evaporation temperature of 120-150°C, a condensation temperature of 40-70°C, a vacuum degree of 0.5-5 Pa, and the light phase is retained as high-purity hydroxytyrosol;

[0014] S4: dissolving hydroxypropyl-β-cyclodextrin in water, adding the hydroxytyrosol obtained in S3 to the hydroxypropyl-β-cyclodextrin, heating and stirring the reaction under nitrogen protection, and after cooling the reactants, freeze-drying them into hydroxytyrosol inclusion complexes.

[0015] It should be noted that the production of hydroxytyrosol by biological fermentation has been reported. In order to increase the yield of hydroxytyrosol, researchers have tried to construct some high-yield hydroxytyrosol engineering bacteria, which can obtain fermentation broth containing hydroxytyrosol under certain fermentation conditions. The raw material of the method of the present invention can be any hydroxytyrosol fermentation broth obtained by biological fermentation, typically but not limited to, for example, the fermentation broth obtained by the strain and method disclosed in CN202210930591.7 "Construction method and application of recombinant Escherichia coli for fermentation production of hydroxytyrosol".

[0016] As a preferred embodiment of the present invention, membrane separation uses at least two of a ceramic membrane, an ultrafiltration membrane, a nanofiltration membrane, and a reverse osmosis membrane, and the membrane separation removes macromolecular impurities such as mycelium and protein, while achieving preliminary concentration of hydroxytyrosol; the concentration of hydroxytyrosol in the final concentrated solution obtained by membrane separation is 5 to 40 g / L, and preferably the concentrated solution with a hydroxytyrosol concentration of 15 to 40 g / L obtained by conventional membrane separation is obtained.

[0017] As a preferred embodiment of the present invention, the adsorption material is one or more of silica gel (100-400 mesh), adsorption resin (20-60 mesh), activated carbon (16-400 mesh), preferably 300-400 mesh silica gel or 45-60 mesh adsorption resin; the amount of adsorption material added is 0.5-2 times the mass of hydroxytyrosol in the concentrated solution, preferably 1-2 times; vacuum concentration and evaporation are carried out at 40-100°C, preferably at 60-85°C, and the vacuum degree is controlled at -0.09MPa or below. Among them, the present invention does not specifically limit the type of adsorption resin, and typically, but not limiting, styrene-divinylbenzene copolymer, acrylic resin, macroporous resin XDA-200B, etc. can be selected.

[0018] Based on the characteristics of the selected adsorption material and the mixture obtained by the vacuum concentration and evaporation method, the present invention further designs a preferred scheme for supercritical extraction, wherein the extraction pressure of the supercritical extraction is 20-40 MPa, the extraction temperature is 35-55°C; the entrainer is water, and the volume percentage of the entrainer is 0.1-5%; the extraction time is 1-4 h, and CO 2 The flow rate is 10~40 L / h, and the separation pressure is 10~20 MPa. Further preferably, the extraction pressure of supercritical extraction is 25~35 MPa, the extraction temperature is 35~45°C; the entrainer volume percentage is 2~3%; the extraction time is 1.5~2.5 h, CO 2The flow rate is 20~35 L / h and the separation pressure is 10~15 MPa.

[0019] As a preferred embodiment of the present invention, the scraper speed used in the three molecular distillations is 350-450 rpm / min.

[0020] According to an embodiment of the present invention, the conditions for the first molecular distillation are preferably: feed insulation temperature of 75~85°C, heavy phase insulation temperature of 75~85°C, evaporation temperature of 100~110°C, condensation temperature of 5~10°C, vacuum degree of 4000~4500 Pa, and heavy phase components are retained.

[0021] The conditions for the second molecular distillation are preferably: feed insulation temperature of 75~85°C, heavy phase insulation temperature of 85~95°C, evaporation temperature of 130~150°C, condensation temperature of 45~65°C, vacuum degree of 5~20 Pa, and heavy phase components are retained.

[0022] The conditions for the third molecular distillation are preferably: feed insulation temperature of 95-110°C, heavy phase insulation temperature of 100-110°C, evaporation temperature of 125-140°C, condensation temperature of 45-65°C, vacuum degree of 0.5-2 Pa, and light phase. The light phase contains high-purity hydroxytyrosol, and the purity detected by HPLC is ≥99%.

[0023] As a preferred embodiment of the present invention, hydroxypropyl-β-cyclodextrin is dissolved in water and heated in a water bath to 40-70° C., preferably 45-60° C. The mass of hydroxytyrosol added is 1 / 10-1 / 5 of that of hydroxypropyl-β-cyclodextrin, and the heating and stirring reaction time is 4-5 h.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] Hydroxytyrosol biological fermentation liquid has low content, and the common problems in the extraction process are high purification cost, large amount of organic solvent, and great difficulty in purification. The method of the present invention adopts vacuum concentration and drying to convert the hydroxytyrosol fermentation liquid from liquid to solid, which solves the problem that SFE cannot extract hydroxytyrosol from aqueous solution, and uses an adsorption material of suitable size and amount during vacuum concentration and drying. At the same time, the adsorption material can also adsorb pigments and impurities to improve the purification efficiency. In the whole purification process, no organic solvent is used, which greatly reduces the purification cost. The overall method of the present invention realizes the application of the SFE method to the separation process of hydroxytyrosol fermentation products, solves the problem that the fermentation liquid is easy to form a viscous substance during the drying process and cannot be effectively dispersed, resulting in low SFE extraction efficiency. 2Under the extraction system, the risk of hydroxytyrosol being oxidized during the purification process is reduced. The present invention further optimizes the process parameters of SFE and molecular distillation processes to obtain high-purity hydroxytyrosol. The present invention is suitable for the separation and purification of hydroxytyrosol fermentation broth obtained by biological fermentation method, does not need to use organic solvents, and well protects hydroxytyrosol during the separation process to avoid its degradation or oxidation, and the obtained hydroxytyrosol has high yield and high purity.

[0026] In addition, in the process of preparing hydroxytyrosol into an inclusion compound, unlike the prior art, the present invention does not use protective agents such as vitamin C and vitamin C ethyl ether, but adopts nitrogen protection, thereby reducing the influence of the addition of other reagents on the quality and activity of the inclusion compound. Under the premise of ensuring the activity, the inclusion compound can significantly improve its light and heat stability, which is conducive to the expansion of the application of hydroxytyrosol. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is a preparation flow chart of the method of the present invention;

[0028] Figure 2 This is a HPLC analysis chart of hydroxytyrosol obtained after three molecular distillations of the present invention;

[0029] Figure 3 is the DPPH clearance rate; Figure 3 The middle vertical axis is the DPPH radical scavenging rate (%);

[0030] Figure 4 It is a test for liver cell viability; Figure 4 The vertical axis is cell viability (%). DETAILED DESCRIPTION

[0031] The present invention is further described and illustrated below in conjunction with specific embodiments. The embodiments are merely exemplary of the present disclosure and do not define the scope of limitation. The technical features of each embodiment of the present invention may be combined accordingly without conflicting with each other.

[0032] The fermentation broth used in the subsequent embodiments of the present invention is obtained by the method and strain disclosed in the invention patent "Construction method and application of recombinant Escherichia coli for fermentation production of hydroxytyrosol" with application number CN202210930591.7. The general fermentation method is to culture the recombinant Escherichia coli BLAH constructed in the document in LB medium, inoculate it into TB culture medium at an inoculum of 0.5-2% for expansion culture, then add 0.1-0.3 mM inducer for induction culture, continue to culture at 30 ° C and 220r / min for product accumulation, and obtain a fermentation broth containing a certain concentration of hydroxytyrosol.

[0033] It should be noted that the fermentation broth obtained by the above method and the separation and purification in the embodiment of the present invention is only illustrative and does not constitute a limitation on the fermentation broth raw material used in the method of the present invention. The raw material of the method of the present invention can be any hydroxytyrosol fermentation broth obtained by a biological fermentation method.

[0034] Example 1

[0035] In this example, 65 L of fermentation liquid with a hydroxytyrosol concentration of 7 g / L was used, and concentrated through a 50 nm ceramic membrane, an ultrafiltration membrane with a molecular weight cutoff of 10KD, and a nanofiltration membrane with a molecular weight cutoff of 100 D to obtain a 30 g / L concentrate, and 200-300 mesh silica gel with twice the mass of hydroxytyrosol was added, stirred, and placed in a rotary evaporator at 85°C and a vacuum degree of -0.09 MPa to evaporate to dryness. The mixture of hydroxytyrosol and silica gel was placed in an SFE extraction tank, and the extraction conditions were: extraction pressure: 30 MPa, extraction temperature: 35°C; extraction time: 2.0 h, CO 2 Flow rate: 30 L / h, entrainer: 2% water (in the present invention, the entrainer content is volume percentage, the same below), separation pressure: 10 MPa. The SFE extract was added to the molecular distillation apparatus for three separations under the following conditions: First separation: feed insulation temperature 80 ° C, heavy phase insulation temperature: 80 ° C, evaporation temperature 110 ° C, condensation temperature 10 ° C, scraper speed: 410 rpm / min, vacuum: 4400 Pa. The heavy phase component was retained. The heavy phase component was used for the second separation: feed insulation temperature 80 ° C, heavy phase insulation temperature: 90 ° C, evaporation temperature 145 ° C, condensation temperature 55 ° C, scraper speed: 415 rpm / min, vacuum: 10 Pa. The heavy phase component was retained. The obtained heavy phase component was separated for the third time: feed insulation temperature 100°C, heavy phase insulation temperature: 110°C, evaporation temperature 135°C, condensation temperature 55°C, scraper speed: 415 rpm / min, vacuum degree: 1 Pa. The light phase was retained to obtain 232 g of high-purity hydroxytyrosol, with a purity of 99.7% detected by HPLC and a total yield of 51%. Figure 2 The HPLC analysis chart of hydroxytyrosol is shown in Figure 2. 2 g of hydroxytyrosol was added to an aqueous solution containing 12 g of hydroxypropyl-β-cyclodextrin, heated to 50°C under nitrogen protection, stirred for 5 h, slowly cooled, and placed in a freeze dryer to obtain a hydroxytyrosol inclusion complex.

[0036] Example 2

[0037] In this example, 58 L of fermentation broth with a hydroxytyrosol concentration of 7.7 g / L was used, and concentrated through a 50 nm ceramic membrane, an ultrafiltration membrane with a molecular weight cutoff of 10KD and 3KD, and a nanofiltration membrane with a molecular weight cutoff of 100 D to obtain a 35 g / L concentrate, and 1 times the mass of hydroxytyrosol with 200-300 mesh silica gel and 1 times the amount of 40-60 mesh XDA-200B adsorption resin were added, stirred, and placed in a rotary evaporator at 80°C and a vacuum degree of -0.09 MPa to evaporate to dryness. The mixture of hydroxytyrosol and silica gel was placed in an SFE extraction tank, and the extraction conditions were: extraction pressure: 32 MPa, extraction temperature: 40°C; extraction time: 2.0 h, CO 2 Flow rate: 35 L / h, entrainer: 3% water, separation pressure: 15 MPa. The SFE extract was added to the molecular distillation instrument for three separations under the following conditions: First separation: feed insulation temperature 80 ℃, heavy phase insulation temperature: 80 ℃, evaporation temperature 110 ℃, condensation temperature 5 ℃, scraper speed: 450 rpm / min, vacuum degree: 4000 Pa. The heavy phase component was retained. The heavy phase component was used for the second separation: feed insulation temperature 80 ℃, heavy phase insulation temperature: 90 ℃, evaporation temperature 150 ℃, condensation temperature 60 ℃, scraper speed: 420rpm / min, vacuum degree: 5Pa. The heavy phase component was retained. The obtained heavy phase component was separated for the third time: feed insulation temperature 110 ℃, heavy phase insulation temperature: 110 ℃, evaporation temperature 125 ℃, condensation temperature 50 ℃, scraper speed: 430 rpm / min, vacuum degree: 1.5 Pa. The light phase was retained to obtain 214 g of high-purity hydroxytyrosol, with a purity of 99.3% as determined by HPLC and a total yield of 47.9%. 2 g of hydroxytyrosol was added to an aqueous solution containing 14 g of hydroxypropyl-β-cyclodextrin, heated to 60°C under nitrogen protection, stirred for 4 h, slowly cooled, and placed in a freeze dryer to obtain a hydroxytyrosol inclusion complex.

[0038] Example 3

[0039] In this example, 100 L of fermentation broth with a hydroxytyrosol concentration of 7.4 g / L was used, and concentrated through a 50 nm ceramic membrane, an ultrafiltration membrane with a molecular weight cutoff of 10KD, and a nanofiltration membrane with a molecular weight cutoff of 100 D to obtain a 40 g / L concentrate, and 1.5 times the mass of hydroxytyrosol, 40-60 mesh XDA-200B adsorption resin and 3% (g / mL) Zhuxi 313 type activated carbon were added, stirred, and placed in a rotary evaporator at 80°C and vacuum degree -0.09MPa to evaporate to dryness. The mixture of hydroxytyrosol and silica gel was placed in an SFE extraction tank, and the extraction conditions were: extraction pressure: 30 MPa, extraction temperature: 45°C; extraction time: 2.0 h, CO 2Flow rate: 30 L / h, entrainer: 3% water, separation pressure: 12 MPa. The SFE extract was added to the molecular distillation instrument for three separations under the following conditions: First separation: feed insulation temperature 80 ℃, heavy phase insulation temperature: 80 ℃, evaporation temperature 110 ℃, condensation temperature 10 ℃, scraper speed: 430 rpm / min, vacuum degree: 4300 Pa. The heavy phase component was retained. The heavy phase component was used for the second separation: feed insulation temperature 80 ℃, heavy phase insulation temperature: 90 ℃, evaporation temperature 145 ℃, condensation temperature 60 ℃, scraper speed: 420 rpm / min, vacuum degree: 10Pa. The heavy phase component was retained. The obtained heavy phase component was separated for the third time: feed insulation temperature 110 ℃, heavy phase insulation temperature: 110 ℃, evaporation temperature 135 ℃, condensation temperature 50 ℃, scraper speed: 430 rpm / min, vacuum degree: 1.0 Pa. The light phase was retained to obtain 355 g of high-purity hydroxytyrosol, with a purity of 99.4% as determined by HPLC and a total yield of 48%. 10 g of hydroxytyrosol was added to an aqueous solution containing 60 g of hydroxypropyl-β-cyclodextrin, heated to 60°C under nitrogen protection, stirred for 4 h, slowly cooled, and placed in a freeze dryer to obtain a hydroxytyrosol inclusion complex.

[0040] Comparative Example 1

[0041] In this comparative example, 10 L of fermentation liquid with a hydroxytyrosol concentration of 5.9 g / L was concentrated by a 50 nm ceramic membrane, an ultrafiltration membrane with a molecular weight cutoff of 10KD, and a nanofiltration membrane with a molecular weight cutoff of 100 D to obtain a 35 g / L concentrate. The concentrate was directly placed in an SFE extraction tank, and the extraction conditions were: extraction pressure: 30 MPa, extraction temperature: 35°C; extraction time: 2.0 h, CO 2 Flow rate: 30L / h, entrainer: 2% water, separation pressure: 10 MPa. The difference from Example 1 is that the hydroxytyrosol concentrate is not concentrated into a solid by vacuum drying, but directly subjected to SFE extraction. Almost no extract is obtained after SFE extraction. This may be because the solubility of hydroxytyrosol in water is much greater than that in a supercritical fluid system. Therefore, converting the hydroxytyrosol concentrate from a liquid into a solid is a key point of the present invention.

[0042] Comparative Example 2

[0043] In this comparative example, 60 L of fermentation liquid with a hydroxytyrosol concentration of 7.1 g / L was concentrated by a 50 nm ceramic membrane, an ultrafiltration membrane with a molecular weight cutoff of 10KD, and a nanofiltration membrane with a molecular weight cutoff of 100 D to obtain a 32 g / L concentrate. The concentrate was placed in a rotary evaporator at 85°C and a vacuum degree of -0.09 MPa to dryness to obtain a hydroxytyrosol concentrate. The hydroxytyrosol concentrate was placed in an SFE extraction tank, and the extraction conditions were: extraction pressure: 30 MPa, extraction temperature: 35°C; extraction time: 2.0 h, CO 2 Flow rate: 30 L / h, entrainer: 2% water, separation pressure: 10 MPa. The difference from Example 1 is that no adsorbent is added during the evaporation of the hydroxytyrosol nanofiltration membrane concentrate. After evaporation, the hydroxytyrosol concentrate is a viscous colloid that is not easy to disperse. After SFE extraction, only 8 g of extract is obtained with a purity of 65%. Subsequent inclusion compounds are not prepared. Comparative Example 2 does not add adsorbent during evaporation. After evaporation, the sample becomes a "maltose"-like viscous substance with poor dispersibility, which greatly limits the contact area between the supercritical fluid and the hydroxytyrosol viscous substance, thereby greatly reducing the extraction rate of SFE.

[0044] Comparative Example 3

[0045] In this comparative example, 55 L of fermentation liquid with a hydroxytyrosol concentration of 7.6 g / L was concentrated through a 50 nm ceramic membrane, an ultrafiltration membrane with a molecular weight cutoff of 10KD, and a nanofiltration membrane with a molecular weight cutoff of 100 D to obtain a 36 g / L concentrate, and 200-300 mesh silica gel with twice the mass of hydroxytyrosol was added, stirred, and placed in a rotary evaporator at 85°C and a vacuum degree of -0.09 MPa for evaporation. The hydroxytyrosol and silica gel concentrate were placed in an SFE extraction tank, and the extraction conditions were: extraction pressure: 30 MPa, extraction temperature: 35°C; extraction time: 2.0 h, CO 2Flow rate: 30 L / h, entrainer: 10% water, separation pressure: 10 MPa. The SFE extract was added to the molecular distillation instrument for three separations under the following conditions: First separation: feed insulation temperature 80 ℃, heavy phase insulation temperature: 80 ℃, evaporation temperature 110 ℃, condensation temperature 10 ℃, scraper speed: 410 rpm / min, vacuum degree: 4400 Pa. The heavy phase component was retained. The heavy phase component was used for the second separation: feed insulation temperature 80 ℃, heavy phase insulation temperature: 90 ℃, evaporation temperature 145 ℃, condensation temperature 55 ℃, scraper speed: 415 rpm / min, vacuum degree: 10 Pa. The heavy phase component was retained. The obtained heavy phase component was separated for the third time: feed insulation temperature 100 ℃, heavy phase insulation temperature: 110 ℃, evaporation temperature 135 ℃, condensation temperature 55 ℃, scraper speed: 415 rpm / min, vacuum degree: 1 Pa. The light phase was retained to obtain 250 g of light red hydroxytyrosol, with a purity of 95.2% as determined by HPLC and a total yield of 59.8%. The colorless hydroxytyrosol obtained in the example is different in appearance and purity. The difference between this comparative example and Example 1 is that the proportion of entrainer during the SFE extraction process reaches 10%. In the supercritical fluid state, CO 2 And 5% or less water miscible.When the ratio of entrainer water exceeds 5%, it is now in a sub-supercritical fluid state, and the property is closer to liquid.In this comparative example, the entrainer ratio has reached 10%, which is closer to the characteristics of liquid water, and the water-soluble pigment and impurity extracted are more, thereby reducing the purity and properties of subsequent hydroxytyrosol.This comparative example does not carry out the preparation of inclusion compound to sample.

[0046] Comparative Example 4

[0047] In this comparative example, 50 L of fermentation liquid with a hydroxytyrosol concentration of 6.5 g / L was concentrated through a 50 nm ceramic membrane, an ultrafiltration membrane with a molecular weight cutoff of 10KD, and a nanofiltration membrane with a molecular weight cutoff of 100 D to obtain a 32 g / L concentrate, and 200-300 mesh silica gel with twice the mass of hydroxytyrosol was added, stirred, and evaporated to dryness in a rotary evaporator at 85°C and a vacuum degree of -0.09 MPa. The mixture of hydroxytyrosol and silica gel was placed in an SFE extraction tank, and the extraction conditions were: extraction pressure: 30 MPa, extraction temperature: 35°C; extraction time: 2.0 h, CO 2Flow rate: 30 L / h, entrainer: 2% water, separation pressure: 10 MPa. The SFE extract was added to the molecular distillation instrument for three separations under the following conditions: First separation: feed insulation temperature 80 ℃, heavy phase insulation temperature: 80 ℃, evaporation temperature 110 ℃, condensation temperature 10 ℃, scraper speed: 410 rpm / min, vacuum degree: 4400 Pa. The heavy phase component was retained. The heavy phase component was used for the second separation: feed insulation temperature 80 ℃, heavy phase insulation temperature: 90 ℃, evaporation temperature 145 ℃, condensation temperature 55 ℃, scraper speed: 415 rpm / min, vacuum degree: 10 Pa. The heavy phase component was retained. The obtained heavy phase component was separated for the third time: feed insulation temperature 100 ℃, heavy phase insulation temperature: 110 ℃, evaporation temperature 135 ℃, condensation temperature 55 ℃, scraper speed: 415 rpm / min, vacuum degree: 1 Pa. The light phase was retained to obtain 159 g of high-purity hydroxytyrosol, with a purity of 99.2% detected by HPLC and a total yield of 48.9%. 2 g of hydroxytyrosol was added to an aqueous solution containing 12 g of hydroxypropyl-β-cyclodextrin, heated to 50 ° C, stirred for 5 h, slowly cooled, and placed in a freeze dryer to obtain a hydroxytyrosol inclusion compound. The difference from Example 1 is that there is no nitrogen protection during the preparation of the inclusion compound, and the color of the solution gradually turns red during the heating process until the solution becomes wine red. Obviously, hydroxytyrosol is oxidized in this process, which illustrates the necessity of adding nitrogen protection in the present invention.

[0048] Performance Testing

[0049] Test Example 1

[0050] Stability test:

[0051] Preparation of reference solution: Hydroxytyrosol reference substance (Aladdin Chemical Reagent Co., Ltd., purity 99.5%) was prepared into a 1.0 mg / mL aqueous solution.

[0052] Preparation of hydroxytyrosol solution: The hydroxytyrosol sample prepared in Example 1 was prepared into a 1.0 mg / mL aqueous solution.

[0053] Preparation of hydroxytyrosol inclusion complex solution: A sample of hydroxytyrosol inclusion complex (Example 1) was prepared into a 1.0 mg / mL aqueous solution.

[0054] The content of hydroxytyrosol in the sample = (As×mr×Pr) / (Ar×ms)×100%

[0055] Where As is the peak area of ​​hydroxytyrosol in the sample solution chromatogram, Ar is the peak area of ​​hydroxytyrosol in the reference solution chromatogram, ms is the sample weight of hydroxytyrosol sample (g), mr is the sample weight of hydroxytyrosol reference (g), and Pr is the content of hydroxytyrosol reference. The arithmetic mean of the results of two parallel determinations is used as the reported value, and the absolute difference between the two parallel determination results is not greater than 2% of the arithmetic mean.

[0056] The purity of hydroxytyrosol in the sample was calculated by the peak area percentage using the area normalization method. The arithmetic mean of the results of two parallel determinations was used as the reported value, and the absolute difference between the two parallel determinations was no greater than 2% of the arithmetic mean.

[0057] The hydroxytyrosol and hydroxytyrosol inclusion complex solutions were irradiated with 4500 Lx intensity light, analyzed by high performance liquid chromatography (HPLC), and the content was calculated. The hydroxytyrosol content in the samples at 0 d, 7 d, 14 d and 28 d was calculated, and the results are shown in Table 1. The HPLC method is as follows: chromatographic column: Hypersil ODS-2 (250*4.6 mm, 5 μm); column temperature: 35 ℃; mobile phase A: 0.1% formic acid aqueous solution; mobile phase B: methanol; elution: 0-18 min, 20% B; flow rate: 1.0 mL / min; detection wavelength: 210 nm.

[0058] Table 1 - Light test of hydroxytyrosol and its inclusion complex

[0059]

[0060] The results showed that under strong light irradiation, hydroxytyrosol and its inclusion complex would degrade to varying degrees. At 28 days, the content of hydroxytyrosol had dropped to 53.5% of the original content, while that of the inclusion complex dropped to 79.2%, showing better stability.

[0061] The two groups of experimental samples were placed in an oven at 40 ℃ and 60 ℃ to determine their thermal stability. The results are listed in Table 2. Hydroxytyrosol is sensitive to heat, and the higher the temperature, the faster it degrades. The stability of hydroxytyrosol inclusion complex at 40 ℃ and 60 ℃ is better than that of hydroxytyrosol.

[0062] Table 2 - Thermal stability of hydroxytyrosol and its inclusion complexes

[0063]

[0064] Test Example 2

[0065] Antioxidant activity

[0066] Determination of DPPH radical scavenging rate: Hydroxytyrosol and its inclusion complexes (Examples 1-3) were prepared into 1 mg / mL by adding water, 0.2 mL of the solution was taken, 0.2 mL of 0.2 mmol / L DPPH anhydrous ethanol solution was added, and after mixing evenly, the mixture was reacted at room temperature in the dark for 30 min, and then the sample absorbance was measured at 517 nm using an enzyme marker. A mixed solution of water and DPPH anhydrous ethanol was used as a blank group. A mixed solution of the sample solution and anhydrous ethanol was used as a control group. Vitamin C was used as a positive control group. The DPPH radical scavenging rate (R1, %) was calculated according to the formula, and the results are shown in Figure 3 .

[0067] R1 / %=[1-(A 样 -A 对 ) / A 空 ]*100%

[0068] Where: A 样 is the absorbance of the sample group; A 对 is the absorbance of the control group; A 空 is the absorbance of the blank group.

[0069] The DPPH radical scavenging rates of hydroxytyrosol and Examples 1 to 3 were significantly higher than that of vitamin C, and the two were comparable, which means that the inclusion process did not affect their antioxidant activity.

[0070] Test Example 3

[0071] Hepatocyte viability assay

[0072] Referring to the patent "Active peptides with antioxidant and alcoholic liver damage protection effects and their applications" (patent application number: 202311804900.7), an ethanol-damaged HepG2 cell model was constructed: HepG2 cells were inoculated in a 96-well plate and cultured for 24 hours before the culture medium was replaced. The control group only added a culture medium containing 40 mg / mL ethanol; the experimental group added a culture medium containing 40 mg / mL ethanol and 0.05 mg / mL hydroxytyrosol or hydroxytyrosol inclusion complex; the blank group added fresh culture medium and continued to culture for 24 hours. The cell survival rate was determined by the MTT method, see for details. Figure 4 . Compared with the blank group, the cell viability of the control group was 47.5%, which was able to significantly inhibit cell viability (P<0.05), indicating that the cell injury model was successfully constructed. When hydroxytyrosol and hydroxytyrosol inclusion complex were added, the cell viability was 75.3% and 73.9%, respectively, which were higher than that of the control group, indicating that hydroxytyrosol and its inclusion complex can reduce the damaging effect of ethanol on cells, and the effects of the two are comparable. The present invention shows the potential ability of hydroxytyrosol inclusion complex in the pre-protection of alcoholic liver injury.

[0073] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. For ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for separating, purifying and preparing an inclusion compound of hydroxytyrosol, characterized in that: The steps include: S1: using membrane separation to concentrate the hydroxytyrosol fermentation liquid obtained by the biological fermentation method to obtain a concentrated liquid; adding an adsorption material to the concentrated liquid, and vacuum concentrating and evaporating to obtain a mixture; S2: placing the mixture obtained in S1 in a supercritical fluid extraction tank, and performing supercritical extraction using supercritical CO2 as an extractant and water as an entrainer; S3: subjecting the extract obtained in S2 to the first molecular distillation, with feed insulation temperature of 70-90 °C, heavy phase insulation temperature of 70-90 °C, evaporation temperature of 90-120 °C, condensation temperature of 5-20 °C, vacuum degree of 4000-5000 Pa, and retaining the heavy phase component; The heavy phase component is subjected to a second molecular distillation, with a feed insulation temperature of 70-90 °C, a heavy phase insulation temperature of 70-100 °C, an evaporation temperature of 110-160 °C, a condensation temperature of 40-70 °C, a vacuum degree of 5-100 Pa, and the heavy phase component is retained; The heavy phase component obtained by the second molecular distillation is subjected to a third molecular distillation, with a feed insulation temperature of 90-110°C, a heavy phase insulation temperature of 100-120°C, an evaporation temperature of 120-150°C, a condensation temperature of 40-70°C, a vacuum degree of 0.5-5 Pa, and the light phase is retained as high-purity hydroxytyrosol; S4: dissolving hydroxypropyl-β-cyclodextrin in water, adding the hydroxytyrosol obtained in S3 to the hydroxypropyl-β-cyclodextrin, heating and stirring the reaction under nitrogen protection, and after cooling the reactants, freeze-drying them into hydroxytyrosol inclusion complexes.

2. The method for separating, purifying and preparing an inclusion compound of hydroxytyrosol according to claim 1, wherein: In S1, membrane separation uses at least two of a ceramic membrane, an ultrafiltration membrane, a nanofiltration membrane, and a reverse osmosis membrane, and the concentration of hydroxytyrosol in the final concentrated solution obtained by membrane separation is 5-40 g / L.

3. The method for separating, purifying and preparing an inclusion compound of hydroxytyrosol according to claim 1, characterized in that: In S1, the adsorption material is one or more of silica gel, adsorption resin, and activated carbon. The amount of adsorption material added is 0.5 to 2 times the mass of hydroxytyrosol in the concentrated solution. The vacuum concentration and evaporation are carried out at 40 to 100° C., and the vacuum degree is controlled at -0.09 MPa or below.

4. The method for separating, purifying and preparing an inclusion compound of hydroxytyrosol according to claim 3, characterized in that: The silica gel shall be 100-400 mesh silica gel, the adsorption resin shall be 20-60 mesh adsorption resin, and the activated carbon shall be 16-400 mesh activated carbon.

5. The method for separating, purifying and preparing inclusion compound of hydroxytyrosol according to claim 1, characterized in that, In S2, the extraction pressure of supercritical extraction is 20~40 MPa, the extraction temperature is 35~55℃; the entrainer is water, the volume percentage is 0.1~5%; the extraction time is 1~4 h, the CO2 flow rate is 10~40 L / h, and the separation pressure is 10~20 MPa.

6. The method for separating, purifying and preparing an inclusion compound of hydroxytyrosol according to claim 5, characterized in that: In S2, the extraction pressure of supercritical extraction is 25~35 MPa, the extraction temperature is 35~45℃; the volume percentage of the entrainer is 2~3%; the extraction time is 1.5~2.5 h, the CO2 flow rate is 20~35 L / h, and the separation pressure is 10~15 MPa.

7. The method for separating, purifying and preparing inclusion compound of hydroxytyrosol according to claim 1, characterized in that: The conditions for the first molecular distillation are: feed insulation temperature is 75~85 ℃, heavy phase insulation temperature is 75~85 ℃, evaporation temperature is 100~110 ℃, condensation temperature is 5~10 ℃, vacuum degree is 4000~4500 Pa, and heavy phase components are retained.

8. The method for separating, purifying and preparing inclusion compound of hydroxytyrosol according to claim 1, characterized in that: The conditions for the second molecular distillation are: feed insulation temperature is 75~85 ℃, heavy phase insulation temperature is 85~95 ℃, evaporation temperature is 130~150 ℃, condensation temperature is 45~65 ℃, vacuum degree is 5~20 Pa, and heavy phase components are retained.

9. The method for separating, purifying and preparing an inclusion compound of hydroxytyrosol according to claim 1, characterized in that: The conditions for the third molecular distillation are: feed insulation temperature is 95~110℃, heavy phase insulation temperature is 100~110℃, evaporation temperature is 125~140℃, condensation temperature is 45~65℃, vacuum degree is 0.5~2 Pa, and the light phase is retained.

10. The method for separating, purifying and preparing inclusion compound of hydroxytyrosol according to claim 1, characterized in that: In S4, the mass of hydroxytyrosol added is 1 / 10~1 / 5 of hydroxypropyl-β-cyclodextrin, and the heating and stirring reaction time is 4~5 h.

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