Preparation method and application of cross-linked agarose embedded silica gel powder beads

Abstract

The invention belongs to the technical field of separation and purification of lecithin, and discloses a preparation method and application of cross-linked agarose embedded silica gel powder beads, comprising preparation of bead-shaped agarose gel liquid and preparation of cross-linked agarose embedded silica gel powder. According to the method, the cross-linked agarose embedded silica gel powder is used as a filler in the chromatography, so that the problems of unsmooth speed and even blockage caused by using silica gel micro powder as the filler can be solved, the adsorption capacity of silica gel is not influenced by the addition of agarose, and the extraction efficiency of lecithin in earthworms is improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of separating and purifying lecithin, in particular, relates to a preparation method and application of cross-linked agarose embedded silica gel powder beads. BACKGROUND

[0002] Lecithin, also known as egg yolk, is a phospholipid substance widely present in animal and plant tissues and egg yolk, which is yellow-brown in appearance and has oiliness. Lecithin is an important component of cell membranes and organelle membranes, and is crucial for maintaining the structure and function of cells. Lecithin has a history of more than a hundred years and is widely used. Today, lecithin has been positioned as the third nutrient along with protein and vitamins. Liposomes are a double-layer membrane structure composed of phospholipids (such as lecithin) and cholesterol, etc., which have properties similar to cell membranes. In the targeted drug delivery system, liposomes can encapsulate drugs inside or adsorb them on the surface, and then enter the human body through intravenous injection, oral administration or other routes. The role of lecithin in liposomes is crucial. First, it is one of the main components of the liposome membrane, which gives the liposome good biocompatibility and stability. Second, the structural characteristics of lecithin enable it to interact with cell membranes, promoting the fusion of liposomes with target cells and achieving targeted release of drugs.

[0003] Therefore, lecithin is widely used in food, health care products and pharmaceutical fields.

[0004] Lecithin is widely present in animal and plant tissues, especially in raw materials such as soybeans, eggs, milk, and earthworms. Therefore, lecithin can be obtained from these natural raw materials through specific extraction processes. In the prior art, silica gel column chromatography separation method is usually used for lecithin extraction. The separation principle of silica gel chromatography is that substances with higher polarity are easily adsorbed by silica gel, and substances with weaker polarity flow out of the silica gel chromatography column. The whole chromatography process is a cycle of adsorption, desorption, re-adsorption, and re-desorption, which can avoid chemical pollution caused by the use of organic solvents and other chemicals in separation and purification.

[0005] To improve the adsorption capacity of silica gel and increase its specific surface area, fine silica gel powder is usually used to improve the purification rate of lecithin. However, when the silica gel particles are finer, the specific surface area increases, but the flow rate in the adsorption process decreases, which also affects the production efficiency and quality of lecithin.

[0006] Agarose can be used as a separation medium in chromatographic separation methods and has good flowability. However, compared with silica gel, agarose has poor mechanical strength and is easily damaged under high pressure. Therefore, appropriate operating conditions are required when agarose is used as packing material in chromatographic separation, which is complicated to use. Therefore, the development of cross-linked agarose embedded silica gel powder as packing material in chromatographic separation can overcome the problem of slow speed and even blockage caused by fine silica gel powder as packing material.

[0007] Lecithin is a sub-project in the earthworm extraction industry chain. If calculated based on the dry weight of earthworms, the lecithin yield is 2%-3%. Earthworm lecithin can regulate blood lipids and has unique antioxidant properties. In addition, earthworm lecithin is a component of cell membranes and plays an important role in maintaining the permeability and fluidity of cell membranes, thereby participating in physiological processes such as cell signal transfer and material transport. Therefore, extracting lecithin from earthworm fat is not only a scientific attempt to tap new biological resources, but also an industrial innovation to address the challenges of sustainable development. Its unique biological activity, environmental value, and commercial potential make it a promising candidate for a place in the high-end health industry.

[0008] A Chinese patent with application number CN2021116619810 provides a method for extracting soy lecithin, which relates to the technical field of phospholipid preparation. The invention uses supercritical CO2 ethanol mixed solvent as the extractant for supercritical CO2 extraction of soy powder, and uses water-organic solvent as the mobile phase for high-performance liquid chromatography separation of the obtained soy lecithin crude extract, further improving the purity of soy lecithin. In the mobile phase, the water volume fraction is 0-20%, and the organic solvent includes one or several of chlorinated hydrocarbon solvents, lower alcohols, and tetrahydrofuran. The supercritical CO2 extraction of the invention obtains soy lecithin crude extract with high content and high purity, simple operation, no solvent residue, and green environmental protection. The invention further improves the purity of soy lecithin by high-performance liquid chromatography separation. The supercritical CO2 extraction and high-performance liquid chromatography separation method used in the invention obtains soy lecithin with a purity of more than 99%; However, the filler of the chromatographic column in the invention includes silica gel, which is a fine powder that affects flow rate in industrial production and is not conducive to quantitative production. The mobile phase system is a mixture of lower alcohols, chlorinated hydrocarbon solvents, tetrahydrofuran, lower alcohol-water mixed solvents, water-lower alcohol-chlorinated hydrocarbon solvent mixed solvents, or tetrahydrofuran-lower alcohol-chlorinated hydrocarbon solvents, which may cause chemical pollution. SUMMARY

[0009] The main technical problem to be solved by the invention is to provide a preparation method and application of cross-linked agarose-embedded silica gel powder beads for extracting lecithin from earthworms. The cross-linked agarose-embedded silica gel powder beads are used as the filler in the layer adsorption method, which can solve the problem of slow speed or even blockage caused by silica gel fine powder as the filler, and does not affect the adsorption capacity of silica gel due to the addition of agarose, thereby improving the extraction efficiency of lecithin from earthworms.

[0010] To solve the above technical problems, the invention provides the following technical solutions: A preparation method of cross-linked agarose-embedded silica gel powder beads, comprising the following steps: S1, 5 grams of water-soluble carboxymethyl cellulose sodium is added to 500 ml of water to completely dissolve to prepare a first mixed solution; S2, 20 g of agarose is added to the first mixed solution, and 200 grams of silica gel powder with a particle size of 70 μm is added, and stirring is performed to prepare a stirring slurry; S3, the stirring slurry is placed in a pressure cooker, and the valve is heated at high pressure for 20 minutes. After the valve is lowered, the cover is slowly stirred to be uniform, and the temperature is lowered to 65-55°C in the room temperature, to prepare an agarose silica gel glue liquid; S4, the agarose silica gel glue liquid is quickly transferred to the spray gun (1) for spraying to form beads. The mist droplets sprayed from the outlet of the spray gun (1) complete the cooling and solidification in the natural falling process to form agarose silica gel beads, which are placed in a conical flask.

[0011] S5, using a separatory funnel, a saturated aqueous solution of epoxy chloropropane, sodium hydroxide and sodium borohydride are added to the conical flask containing the agarose silica gel beads, and the device is placed in a water bath and stirred at 65°C. After stirring for 70 minutes, the stirring is stopped, the conical flask is sealed with a sealing film, and the temperature is maintained for 50 minutes. After 50 minutes, the conical flask is removed and cooled to room temperature. S6, the reaction in the conical flask cooled to room temperature in S5 is filtered with a Buchner funnel, and washed with anhydrous ethanol three times to remove residual water and small molecule reagents in the structure, thereby obtaining cross-linked agarose embedded silica gel powder beads. The cross-linked agarose embedded silica gel powder beads are stored in a silica gel dryer for storage.

[0012] The particle size of the cross-linked agarose embedded silica gel powder beads is 90 μm.

[0013] The following is a further optimization of the technical solution of the present application: The amount of the saturated aqueous solution of epoxy chloropropane added in S5 is 8 times the total volume of the beads, the amount of sodium hydroxide is 6 grams, and the amount of sodium borohydride is 1500 mg.

[0014] Further optimization: the upper end of the spray gun is provided with an air inlet, and the two ends of the spray gun are provided with glue liquid inlets which penetrate the gun body of the spray gun, and the air inlet is in communication with the glue liquid inlets.

[0015] Further optimization: the upper end of the spray gun is provided with an air inlet, and the two ends of the spray gun are provided with glue liquid inlets which penetrate the gun body of the spray gun, and the air inlet is in communication with the glue liquid inlets.

[0016] Further optimization: the spray gun is further provided with a mist droplet outlet which is in communication with the glue liquid inlets.

[0017] The application of the cross-linked agarose-embedded silica gel beads prepared in this invention in the extraction of lecithin involves using the cross-linked agarose-embedded silica gel beads as an adsorbent to adsorb and extract lecithin from earthworm fat.

[0018] The present invention, by adopting the above technical solution, has the following beneficial effects: Compared with other adsorption fillers with the same function, the cross-linked agarose-embedded silica powder beads prepared by this invention have better flowability due to their bead shape. In industrial production applications, the flow rate is smooth and less prone to clogging. At the same time, the cross-linked agarose-embedded silica powder beads have a large specific surface area, ensuring a certain amount of lecithin adsorption. Therefore, they are suitable for the extraction and production of lecithin from earthworm fat, providing a beneficial technical foundation for the next step of industrial production of earthworm lecithin.

[0019] The present invention will be further described below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0020] Figure 1 This is a schematic diagram illustrating the working principle of the spray gun in Embodiment 1 of the present invention; Figure 2 This is a schematic diagram of the bead-like structure of cross-linked agarose-embedded silica powder beads in Example 1 of the present invention; Figure 3 This is a schematic diagram of the silica gel particle structure in Embodiment 1 of the present invention; Figure 4 This is a schematic diagram of the silica powder structure in Embodiment 1 of the present invention; Figure 5 This is an elution diagram of earthworm lecithin separated by a cross-linked agarose-embedded silica gel bead chromatography column in Example 2 of the present invention; Figure 6 This is a schematic diagram of the thin-layer chromatography of the reference sample and the lecithin sample extracted from cross-linked agarose-embedded silica beads in Example 2 of the present invention; Figure 7 This is a schematic diagram of thin-layer chromatography (TLC) spectra of lecithin samples extracted with different types of adsorbents in Example 2 of the present invention. Figure 8 The thin-layer chromatography comparison experiment of adsorbing lecithin using cross-linked agarose-encapsulated silica beads in Example 2 of this invention is illustrated by the linear regression equation used to represent the experimental data processing.

[0021] In the diagram: 1. Spray gun; 11. Air inlet; 12. Adhesive inlet; 13. Hot water inlet; 14. Hot water outlet; 15. Droplet outlet. Detailed Implementation

[0022] Example 1: As Figures 1-2 As shown: A method for preparing cross-linked agarose-embedded silica powder beads includes the following steps: S1, 5 grams of water-soluble carboxymethyl cellulose sodium is added to 500 ml of water to completely dissolve to prepare a first mixed solution; S2, 20 g of agarose is added to the first mixed solution, and 150 grams of silica gel powder with a particle size of 70 μm is added, and stirring is performed to prepare a stirring slurry; S3, the stirring slurry is placed in a sterilization pot, the valve is maintained at steam for 20 minutes, after the valve is opened, the cover is slowly stirred to be uniform, and the stirring is prevented from generating bubbles, and the temperature is reduced to 65-55°C in the room temperature to prepare an agarose silica gel glue solution; S4, the agarose silica gel glue solution is quickly transferred to the spray gun 1 to spray the beads, the mist droplets sprayed from the outlet of the spray gun 1 complete the cooling and solidification in the natural falling process to prepare the agarose silica gel beads, which are loaded into a conical flask; S5, using a separatory funnel, a saturated aqueous solution of epoxy chloropropane, sodium hydroxide and sodium borohydride are added to the conical flask containing the agarose silica gel beads, and the device is placed in a water bath and stirred at 65°C under constant temperature conditions, and the stirring is prevented from generating bubbles, and the hydroxyl group of the agarose in the agarose silica gel beads reacts with the epoxy group of the epoxy chloropropane under alkaline conditions to form a covalent cross-linking reaction between adjacent agarose chains, and the originally linear agarose molecular structure is converted into a three-dimensional network structure, and the silica gel powder is firmly wrapped therein. Further improve the filtration area, increase the stability and structural strength of the carrier, thereby enhancing the fluidity. After stirring for 70 minutes, stop stirring, seal the conical flask with a sealing film, continue to keep warm for 50 min, and then take out the conical flask and cool to room temperature. In this embodiment 1, the amount of the added saturated aqueous solution of epoxy chloropropane is 8 times the total volume of the beads, the amount of sodium hydroxide is 6 grams, and the amount of sodium borohydride is 1500 mg; S6, the reaction in the conical flask cooled to room temperature in S5 is filtered with a Buchner funnel, and washed with anhydrous ethanol three times to remove residual water and small molecule reagents in the structure, thereby obtaining cross-linked agarose embedded silica gel powder beads, which are stored in a silica gel dryer for storage.

[0023] In S4, the spray gun 1 is a tool for atomizing and spraying liquid by using compressed air or other gas, and the specific principle is known and will not be described here.

[0024] In this embodiment 1, the upper end of the spray gun 1 is provided with an air inlet 11, and the two ends of the spray gun 1 are provided with glue liquid inlets 12, which penetrate the gun body of the spray gun 1, and the air inlet 11 is in communication with the glue liquid inlet 12; The upper end of the gun body of the spray gun 1 is also provided with a hot water inlet 13 and a hot water outlet 14 on both sides of the air inlet 11, and hot water can enter from the hot water inlet 13 to increase the temperature in the gun body, realize the heat preservation of the glue liquid, and then flow out from the hot water outlet 14 for recycling.

[0025] The spray gun 1 is also provided with a mist outlet 15, which is in communication with the glue liquid inlet 12.

[0026] In use, the agarose glue liquid in s4 is simultaneously input from the two glue liquid inlets 12 of the spray gun 1, so as to improve the efficiency of bead making. During this period, hot water at a temperature of 60 DEG C is flowed from the hot water inlet 13, the agarose glue liquid is kept at 60 DEG C, the glue liquid is placed to be solidified, high-pressure air is input from the air inlet 11 to act on the agarose glue liquid, and finally the mist drops are output from the mist outlet 15 to complete the cooling and solidification in the process of natural falling body, so as to make agarose silica gel beads.

[0027] As shown in Figures 2-4 The cross-linked agarose embedded silica gel powder bead has a particle size of 90 mu m, a large specific surface area, and good fluidity. The silica gel particles and silica gel powder have poor fluidity, and when produced industrially as a filler, are prone to blockage.

[0028] Example 2: Application of cross-linked agarose embedded silica gel powder beads in extraction of lecithin from earthworm fat, column chromatography is used to extract lecithin from earthworm fat, cross-linked agarose embedded silica gel powder beads are used as adsorbent, and the effect of cross-linked agarose embedded silica gel powder beads as adsorbent is compared through experimental data.

[0029] The column chromatography experiment steps of using cross-linked agarose embedded silica gel powder beads to extract lecithin from earthworm fat are as follows: b1, take 20g of earthworm fat, take 100ml of chloroform:methanol=1:1.5 mixed solvent; b2, add 20g of earthworm fat to 100ml of mixed solvent to dissolve, so as to become earthworm fat dissolved liquid, add the earthworm fat dissolved liquid into a centrifugal tube, centrifuge at 3000r / min for 15min, collect the supernatant, then perform vacuum rotary evaporation on the supernatant, and then send the remaining concentrated liquid into a vacuum drying box for vacuum drying, and the obtained solid is crude phospholipid; b3, take 5g of crude phospholipid, mix and dissolve with 100ml of anhydrous ethanol, centrifuge at 3000r / min for 15min, collect the supernatant, then perform vacuum rotary evaporation on the supernatant, and the remaining concentrated liquid 10ml is used as the column loading liquid; b4, take the cross-linked agarose embedded silica gel powder beads stored in the silica gel dryer in example 1 S6, first use microwave low temperature to activate for 60s, then put into 85% alcohol to swell, stir to form a suspension, then fill the suspension into a chromatography column with an inner diameter of 1.5cm and a length of 40cm as a chromatography medium; b5, the upper column liquid is added to the chromatography column to interact with the chromatography medium, and then elution is performed using an eluent to separate and collect, and the collected eluent is scanned using a UV-visible spectrophotometer at a wavelength of 209 nm to detect the characteristic absorption peak of lecithin, and finally the purified lecithin is obtained by acetone precipitation.

[0030] By Figure 5 It can be seen that the purification degree of lecithin is relatively high.

[0031] The steps of the comparative experiment of thin layer chromatography using cross-linked agarose embedded silica gel powder beads to adsorb lecithin are as follows: 1. Reference lecithin standard preparation: c1. A certain amount of lecithin standard purchased from a special reagent department is weighed and prepared into 20 mg / ml, 40 mg / ml, 60 mg / ml, 80 mg / ml and 100 mg / ml ethanol solutions; c2. Take 10 uL of lecithin ethanol solution of different concentrations in c1 and spot them on the thin layer chromatography plate, with the corresponding numbers a, b, c, d, e from high concentration to low concentration; c3. Then place the thin layer chromatography plate in the chromatography cylinder for development, and add the developing agent to the chromatography cylinder, and the composition of the developing agent is chloroform / methanol / acetone / acetic acid / water=35 / 25 / 14 / 4 / 2 (volume ratio); c4. After development, place the thin layer chromatography plate in a sealed container containing crystalline iodine for color development. The color development results are shown in Figure 6 .

[0032] The experimental process of lecithin standard is prior art, and the specific process will not be described here.

[0033] As Figure 6 shown, where a, b, c, d, e show the spectra of the five lecithin standard samples.

[0034] 2. Experimental steps of lecithin obtained by cross-linked agarose embedded silica gel powder beads adsorbing earthworm fat and lecithin obtained by ordinary silica gel powder adsorbing earthworm fat: d1. Prepare two portions of lecithin mother liquor with a lecithin content of 50 mg / ml; d2. One portion of the mother liquor uses cross-linked agarose embedded silica gel powder beads as the adsorbent to perform the experiment according to the experimental steps b1-b5, and the residual liquid and the mother liquor after adsorption are analyzed by thin layer chromatography; d3. The other portion of the mother liquor uses silica gel powder (70 μm) as the adsorbent to perform the experiment according to the experimental steps b1-b5, and the residual liquid and the mother liquor after adsorption are analyzed by thin layer chromatography.

[0035] As Figure 6As shown, the spectra labeled 1 and 3 are the lecithin content in the two mother liquors; labeled 2 is the spectra of the residual liquid after adsorption using cross-linked agarose-encapsulated silica beads as adsorbent; labeled 4 is the spectra of the residual liquid after adsorption using silica micropowder as adsorbent.

[0036] The spectral data were measured and analyzed, and the experimental data were processed using a linear regression equation, as shown in the table below. Figure 8 As shown: The experimental results are shown in the table below: Sample No. Original lecithin content (50 mg / ml) Cross-linked agarose-embedded silica gel powder beads 1 stock solution 38 mg / ml Cross-linked agarose-embedded silica gel powder beads 2 residual solution 0.11 mg / ml Silica gel powder 3 stock solution 37 mg / ml Silica gel powder 4 residual solution 0.077 mg / ml Experimental results show that: 1) The low concentration of lecithin in the residual liquid indicates good adsorption. 2) The test results of the two groups of samples were not significantly different, indicating that the cross-linked agarose-embedded silica powder beads do not affect the adsorption capacity, and the cross-linked agarose-embedded silica powder column has a smooth flow rate and is not easily blocked; 3) When using cross-linked agarose-encapsulated silica gel beads as an adsorbent for industrial-scale batch adsorption and extraction of lecithin from earthworm fat, work efficiency can be improved.

[0037] Example 3: Comparative experiment on the effects of different types of adsorbents: In Example 3, lecithin standard, solvent (ethanol, ether, chloroform, etc.) extraction product, cross-linked agarose-embedded silica gel beads adsorption product, granular silica gel column adsorption product, and micro-powdered silica gel adsorption product were selected and subjected to C1-C4 chemical experiments to obtain thin-layer chromatograms.

[0038] like Figure 7 As shown, F is the chromatogram of lecithin standard, G is the chromatogram of solvent extraction product, H is the chromatogram of cross-linked agarose-embedded silica gel beads adsorption product, I is the chromatogram of granular silica gel column adsorption product, and J is the chromatogram of micronized silica gel adsorption product.

[0039] Figure 7 Experiments show that using cross-linked agarose-encapsulated silica gel beads as an adsorbent results in a large specific surface area, increasing the loading capacity. Furthermore, it is non-toxic, produces high-purity lecithin, and ensures uniform flow without clogging, thus improving lecithin extraction efficiency.

[0040] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A preparation method of cross-linked agarose embedded silica gel powder beads, comprising the following steps: S1, 5 grams of water-soluble sodium carboxymethyl cellulose is completely dissolved in 500 ml of water to prepare a first mixed solution; S2, 20 g of agarose is added to the first mixed solution, and then 200 grams of silica gel powder with a particle size of 70 μm is added, and stirring is performed to prepare a stirring slurry; S3, the stirring slurry is placed in a pressure cooker, and high-pressure heating is performed for 20 minutes, after the valve is lowered, the cover is opened, and slow stirring is performed until uniform, and the temperature is reduced to 65-55°C in the room temperature, to prepare an agarose silica gel glue solution; S4, the agarose silica gel glue solution is quickly transferred to a spray gun (1) for spraying to form beads, the droplets sprayed from the outlet of the spray gun (1) complete cooling and solidification in the natural falling process to form agarose silica gel beads, which are placed in a conical flask; S5, a separatory funnel is used to add an epoxy chloropropane saturated aqueous solution, sodium hydroxide and sodium borohydride into the conical flask containing the agarose silica gel beads, and the device is placed in a water bath, and constant temperature heating is performed at 65°C, and stirring is performed for 70 minutes, then the stirring is stopped, the conical flask is sealed with a sealing film, and continues to be kept warm for 50 minutes, after 50 minutes, the conical flask is taken out and cooled to room temperature; S6, the reaction in the conical flask cooled to room temperature in S5 is filtered with a Buchner funnel, and washed with anhydrous ethanol three times to remove residual water and small molecule reagents in the structure, thereby obtaining cross-linked agarose embedded silica gel powder beads, which are stored in a silica gel dryer for storage; The cross-linked agarose embedded silica gel powder beads have a particle size of 90 μm.

2. The method for preparing cross-linked agarose-embedded silica gel powder beads according to claim 1, characterized in that: The amount of the epoxy chloropropane saturated aqueous solution added in S5 is 8 times the total volume of the beads, the amount of sodium hydroxide is 6 grams, and the amount of sodium borohydride is 1500 mg.

3. The method for preparing cross-linked agarose-embedded silica powder beads according to claim 2, characterized in that: An air inlet (11) is arranged at the upper end of the spray gun (1), and glue liquid inlets (12) are arranged at both ends of the spray gun (1), the glue liquid inlets (12) penetrate the gun body of the spray gun (1), and the air inlet (11) is in communication with the glue liquid inlets (12).

4. The method for preparing cross-linked agarose-embedded silica powder beads according to claim 3, characterized in that: Hot water inlets (13) and hot water outlets (14) are further arranged at both ends of the air inlet (11) at the upper end of the gun body of the spray gun (1), the hot water can enter from the hot water inlets (13) to increase the temperature in the gun body, thereby achieving heat preservation of the glue liquid, and then the hot water flows out from the hot water outlets (14) for recycling.

5. The method for preparing cross-linked agarose-embedded silica powder beads according to claim 4, characterized in that: A droplet outlet (15) is further arranged on the spray gun (1), and the droplet outlet (15) is in communication with the glue liquid inlets (12).

6. Use of cross-linked agarose-embedded silica gel powder beads according to claim 5, characterized in that: The cross-linked agarose embedded silica gel powder beads are used as an adsorbent to adsorb and extract lecithin in earthworm fat.

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