Chromatographic medium for polyphenol removal and method for preparing the same

Abstract

The present application relates to the technical field of organic polymer compounds, in particular to a chromatography medium for removing polyphenols and a preparation method thereof.The preparation method of the chromatography medium for removing polyphenols comprises the following steps: mixing a water-soluble inorganic salt, a dispersing agent and water to obtain an aqueous phase; mixing N-vinylpyrrolidone, a crosslinking agent one, a crosslinking agent two, a crosslinking agent three, an initiator and a pore-forming agent to obtain an oil phase; and adding the oil phase into the aqueous phase to perform suspension polymerization to obtain the chromatography medium for removing polyphenols.The chromatography medium has effective combination capacity for polyphenols in fermented beverages, the polyphenol adsorption rate is 40-46%, and after recovery, the chromatography medium can be repeatedly used for adsorbing polyphenols, the recovery loss rate is less than 1%, the recovery adsorption decrease rate is less than 2%, the circulation performance is good, the production cost is effectively reduced, the generation of solid waste is reduced, and the chromatography medium prepared by the present application has high mechanical performance.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of organic polymer compounds, in particular to a chromatography medium for removing polyphenols and a preparation method thereof. BACKGROUND

[0002] In fermented beverages such as beer, due to the precipitation and aggregation of polyphenols, precipitated substances affecting the quality of the beverage will be generated during storage. At present, silica hydrogel (SHG) and / or cross-linked polyvinylpyrrolidone particles (PVPP) are mainly added to unstable fermented beverages to remove polyphenols therein. However, although the above-mentioned methods can achieve the effect of removing polyphenols, they are often difficult to recover and have poor mechanical properties, and cannot be used to treat fermented beverages at a large flow rate. SUMMARY

[0003] Therefore, the technical problem to be solved by the present application is to overcome the defects of the prior art that the polyphenol removal medium is difficult to recover and has poor mechanical properties, thereby providing a chromatography medium for removing polyphenols and a preparation method thereof to solve the above-mentioned problems.

[0004] To achieve the above-mentioned purposes, the present application provides the following technical solutions:

[0005] A preparation method of a chromatography medium for removing polyphenols, comprising:

[0006] mixing a water-soluble inorganic salt, a dispersing agent and water to obtain an aqueous phase;

[0007] mixing N-vinylpyrrolidone, a cross-linking agent one, a cross-linking agent two, a cross-linking agent three, an initiator and a pore-forming agent to obtain an oil phase;

[0008] adding the oil phase to the aqueous phase to perform suspension polymerization to obtain the chromatography medium for removing polyphenols;

[0009] The cross-linking agent one comprises at least one of triallyl isocyanurate, N,N'-methylenebisacrylamide;

[0010] The cross-linking agent two comprises at least one of N,N'-divinylimidazolinone, N,N'-bisacryloyl ethylenediamine, N,N'-divinyl ethylene urea, N,N'-divinyl propylene urea;

[0011] The cross-linking agent three comprises at least one of allyl methacrylate, ethylene glycol diacrylate, ethylene glycol dimethacrylate, tetraethylene glycol acrylate.

[0012] Preferably, the water-soluble inorganic salt comprises at least one of sodium sulfate, sodium chloride, calcium chloride and potassium chloride;

[0013] And / or, the dispersing agent comprises at least one of gelatin, polyvinyl alcohol, carboxymethyl cellulose, hydroxypropyl methyl cellulose, polyvinyl pyrrolidone, starch.

[0014] Preferably, the initiator comprises at least one of benzoyl peroxide, azobisisobutyronitrile, azobisisoheptyl nitrile.

[0015] And / or, the porogen comprises at least one of toluene, dichloromethane, dichloroethane, butyl acetate, ethyl acetate.

[0016] Preferably, the mass ratio of the water-soluble inorganic salt, the dispersing agent and water is (25-50):(0.1-3):100.

[0017] Preferably, the mass ratio of the N-vinyl pyrrolidone, the crosslinking agent one, the crosslinking agent two, the crosslinking agent three, the initiator, the porogen is 100:(21-36):(11-26):(3-10):(0.7-1.8):(120-125).

[0018] Preferably, the volume ratio of the water phase and the oil phase is 1:(3-8).

[0019] Preferably, the temperature of the suspension polymerization is 50-90℃, and the time length of the suspension polymerization is 3-10h.

[0020] Preferably, the stirring treatment is performed after the liquid level stratification in the suspension polymerization.

[0021] And / or, the suspension polymerization is further extracted by an extractant, washed by water and screened.

[0022] Preferably, the stirring treatment is performed at a rotation speed of 100-500rpm / min.

[0023] And / or, the extractant comprises at least one of methanol, ethanol, acetone, methylal.

[0024] And / or, the particle size of the screening treatment is 100-3000μm.

[0025] The application further provides a chromatography medium for removing polyphenols, which is prepared by the above method.

[0026] The technical scheme of the application has the following advantages:

[0027] 1.A method for preparing a chromatographic medium for polyphenol removal, comprising: mixing a water-soluble inorganic salt, a dispersing agent and water to obtain an aqueous phase; mixing N-vinylpyrrolidone, a crosslinking agent one, a crosslinking agent two, a crosslinking agent three, an initiator and a porogen to obtain an oil phase; adding the oil phase to the aqueous phase to perform suspension polymerization to obtain the chromatographic medium for polyphenol removal; the crosslinking agent one comprises at least one of triallyl isocyanurate and N, N'-methylene bisacrylamide; the crosslinking agent two comprises at least one of N, N'-divinylimidazolinone, N, N'-bisacryloyl ethylenediamine, N, N'-divinyl ethylene urea and N, N'-divinyl propylene urea; and the crosslinking agent three comprises at least one of allyl methacrylate, ethylene glycol diacrylate, ethylene glycol dimethacrylate and tetraethylene glycol acrylate. In the prior art, crosslinked polyvinylpyrrolidone particles (PVPP) are generally produced by using one crosslinking agent, but the microspheres prepared by the method have rough surfaces, uneven pore distribution and poor strength, resulting in small pressure flow rate. However, the chromatographic medium for polyphenol removal prepared by the forward suspension polymerization method and using three crosslinking agents with different activities has smooth and uniform surfaces, uniform pore distribution and better strength than that of a single crosslinking agent (the pressure flow rate can reflect the spheroidal shape and strength, and the adsorption rate and regeneration amount can reflect the pore distribution), has effective binding capacity for polyphenols in fermented beverages, has a polyphenol adsorption rate of 40-45%, and can be repeatedly used for adsorbing polyphenols after recovery, with a recovery loss rate of <1%, a recovery adsorption decrease rate of <2% and good cycle performance, thereby effectively reducing production cost and reducing the generation of solid waste.

[0028] In addition, the chromatographic medium prepared by the method has high mechanical properties, and the linear flow rate of the chromatographic medium in an XK16 / 40 column can reach 1000 cm / h, and the pressure resistance is >1 MPa.

[0029] 2.In the method for preparing the chromatographic medium for polyphenol removal, the crosslinking agent does not contain benzene substances, and the use amount is greatly reduced, the microsphere body is polyvinylpyrrolidone, the separation efficiency of the microsphere for polyphenol removal is improved, and the environment is more friendly.

[0030] 3.In the method for preparing the chromatographic medium for polyphenol removal, a large amount of inorganic salt solution is added to the aqueous phase to reduce the solubility of N-vinylpyrrolidone in the aqueous phase, and the PVPP microspheres are stably prepared, the preparation problem of the N-vinylpyrrolidone microsphere body is solved, and the problem that the chromatographic medium cannot be repeatedly used for removing polyphenols in fermented beverages is solved. DETAILED DESCRIPTION

[0031] The following examples are provided to better enable those skilled in the art to better understand and practice the application, and are not intended to limit the scope of the application. They are provided solely for illustration of certain aspects of the application, and thus, any person skilled in the art will recognize various modifications and variations that can be made to the embodiments without departing from the scope and spirit of the application. Any such modifications to the embodiments of this application should be considered to fall within the scope of the application.

[0032] Unless otherwise indicated, conventional methods of molecular biology, microbiology, recombinant DNA techniques, and pharmacology, including siRNA, shRNA, and RNAi techniques, within the skill of the art were used for these experiments. Such techniques are explained fully in the literature. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rded., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); and Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Techniques, 4thed., John Wiley & Sons, Inc., Hoboken, N.J. (1999).

[0033] Example 1

[0034] The present example provides a method for preparing a chromatography medium for removing polyphenols, comprising the following steps:

[0035] 1) 100 g of sodium chloride, 12 g of polyvinylpyrrolidone K90 were added to 1200 g of water, and stirred and dissolved at 45°C to obtain an aqueous phase;

[0036] 2) 196 g of N-vinylpyrrolidone, 43.2 g of triallyl isocyanurate, 29.4 g of N,N'-divinylimidazolinone, 5.9 g of allyl methacrylate, 1.44 g of azobisisobutyronitrile, 120 g of butyl acetate, and 120 g of toluene were stirred and mixed to obtain an oil phase;

[0037] 3) The aqueous phase of step 1) and the oil phase of step 2) were weighed according to a volume ratio of 1:5, the oil phase was added to the aqueous phase, and after the liquid surface was layered, stirring was started to form an oil-in-water reverse suspension polymerization bead, the stirring speed was 200 rpm / min, then suspension polymerization was carried out at 80°C for 5 h, after the reaction was completed, the reaction product was soaked in ethanol, residual monomers and other solvents were removed, water was added to detect that no white turbidity flowed out of the ethanol, then the water was washed and sieved to obtain a spherical chromatography medium of 100-300 μm.

[0038] Example 2

[0039] The present example provides a method for preparing a chromatography medium for removing polyphenols, comprising the following steps:

[0040] 1) 150 g of sodium chloride, 14 g of polyvinylpyrrolidone K60 were added to 1200 g of water, and stirred and dissolved at 45°C to obtain an aqueous phase;

[0041] 2) Take 200g N-vinyl pyrrolidone, 52g N,N'-methylene bisacrylamide, 22g N,N'-bisacryloyl ethylene diamine, 20g ethylene glycol diacrylate, 2.88g azobis isopropyl cyanide, 240g ethyl acetate, mix by stirring to obtain the oil phase;

[0042] 3) According to the volume ratio of the water phase and the oil phase is 1:8, the water phase of step 1) and the oil phase of step 2) are weighed, the oil phase is added to the water phase, the stirring is started after the liquid surface is layered to form the oil-in-water reverse suspension polymerization beads, the stirring speed is 250 rpm / min, then the suspension polymerization is carried out at 80℃ for 5h, after the reaction is completed, the reaction product is poured into ethanol for soaking, the residual monomer and other solvents are removed, water is added to detect that there is no white turbidity flowing out of ethanol, then the spherical chromatography medium of 110-320μm is obtained after water washing and sieving.

[0043] Example 3

[0044] The embodiment provides a preparation method of a chromatography medium for removing polyphenols, comprising the following steps:

[0045] 1) 150g calcium chloride, 12g polyvinyl alcohol, 12g gelatin are added to 1200g water, and stirring and dissolving are carried out at 45℃ to obtain a water phase;

[0046] 2) 220g N-vinyl pyrrolidone, 66g triallyl isocyanurate, 35.2g N,N'-divinyl propylene urea, 8.8g tetraethylene glycol acrylate, 3.8g benzoyl peroxide, 120g dichloromethane, 150g toluene are mixed by stirring to obtain an oil phase;

[0047] 3) According to the volume ratio of the water phase and the oil phase is 1:3, the water phase of step 1) and the oil phase of step 2) are weighed, the oil phase is added to the water phase, the stirring is started after the liquid surface is layered to form the oil-in-water reverse suspension polymerization beads, the stirring speed is 200 rpm / min, then the suspension polymerization is carried out at 80℃ for 5h, after the reaction is completed, the reaction product is poured into ethanol for soaking, the residual monomer and other solvents are removed, water is added to detect that there is no white turbidity flowing out of ethanol, then the spherical chromatography medium of 200-500μm is obtained after water washing and sieving.

[0048] Example 4

[0049] The embodiment provides a preparation method of a chromatography medium for removing polyphenols, comprising the following steps:

[0050] 1) 300g sodium sulfate, 1.2g carboxymethyl cellulose are added to 1200g water, and stirring and dissolving are carried out at 45℃ to obtain a water phase;

[0051] 2) 200 g N-vinylpyrrolidone, 42 g triallyl isocyanurate, 52 g N,N'-divinylethylene urea, 6 g ethylene glycol dimethacrylate, 1.4 g azobisisobutyronitrile, 250 g dichloroethane, stirring and mixing to obtain an oil phase;

[0052] 3) The water phase of step 1) and the oil phase of step 2) are weighed according to a volume ratio of 1:5, the oil phase is added to the water phase, stirring is started after the liquid surface is layered to form an oil-in-water reverse suspension polymerization bead, the stirring speed is 100 rpm / min, then suspension polymerization is carried out at 50°C for 10 h, after the reaction is completed, the reaction product is soaked in acetone, residual monomers and other solvents are removed, water is added to detect that no white turbidity flows out of the acetone, then the water-washed product is sieved to obtain a spherical chromatographic medium of 100-300 μm.

[0053] Example 5

[0054] The present example provides a preparation method of a chromatographic medium for removal of polyphenols, comprising the following steps:

[0055] 1) 600 g potassium chloride, 36 g hydroxypropyl methyl cellulose are added to 1200 g water, stirring and dissolving at 45°C to obtain a water phase;

[0056] 2) 200 g N-vinylpyrrolidone, 72 g triallyl isocyanurate, 22 g N,N'-divinylimidazolidinone, 6 g allyl methacrylate, 3.6 g azobisisobutyronitrile, 240 g butyl acetate, stirring and mixing to obtain an oil phase;

[0057] 3) The water phase of step 1) and the oil phase of step 2) are weighed according to a volume ratio of 1:5, the oil phase is added to the water phase, stirring is started after the liquid surface is layered to form an oil-in-water reverse suspension polymerization bead, the stirring speed is 500 rpm / min, then suspension polymerization is carried out at 90°C for 3 h, after the reaction is completed, the reaction product is soaked in methylal, residual monomers and other solvents are removed, water is added to detect that no white turbidity flows out of the methylal, then the water-washed product is sieved to obtain a spherical chromatographic medium of 100-300 μm.

[0058] Comparative Example 1

[0059] The difference between the present comparative example and Example 1 is that only 78.5 g of triallyl isocyanurate is added as a crosslinking agent, and other conditions are the same as those of Example 1.

[0060] Comparative Example 2

[0061] The comparative example is different from example 1 in that 43.2 g of triallyl isocyanurate and 35.3 g of N,N'-divinylimidazolidinone are used as the crosslinking agent, and other conditions are the same as those in example 1.

[0062] Comparative example 3

[0063] The comparative example is different from example 1 in that 43.2 g of triallyl isocyanurate and 35.3 g of N,N'-divinylimidazolidinone are used as the crosslinking agent, and other conditions are the same as those in example 1.

[0064] Comparative example 4

[0065] The comparative example is different from example 1 in that 43.2 g of triallyl isocyanurate and 35.3 g of N,N'-divinylimidazolidinone are used as the crosslinking agent, and other conditions are the same as those in example 1.

[0066] Test example 1

[0067] The pressure flow rate and adsorption capacity of the chromatography medium prepared in examples 1-5 and comparative examples 1-4 are tested, wherein the pressure flow rate test is performed by using a GE AKTA chromatography system, an XK16 / 40 column, 0.10 mol / L NaCl, and an AKTA program, and the test results are shown in Table 1; the adsorption capacity test is performed by using a GE AKTA chromatography system, a 1 mL chromatography column, a detection wavelength of 280 nm, a mobile phase of 0.1% phosphoric acid aqueous solution: acetonitrile = 85:15, a flow rate of 1.000 mL / min, and a column temperature of 25°C, and the ultraviolet absorption maximum value is recorded to calculate the adsorption capacity, and the test results are shown in Table 2.

[0068] Table 1

[0069]

[0070] Table 2

[0071]

[0072]

[0073] According to the above test results, the adsorption rate of the chromatography medium prepared in the application for polyphenolic substances is 40-46%, and after recovery, it can be repeatedly used for adsorbing polyphenolic substances, and the loss rate after recovery is <1%, the adsorption decrease rate after recovery is <2%, and the cycle performance is good. In addition, the mechanical property of the chromatography medium prepared in the application is high, the linear flow rate of the chromatography medium in the XK16 / 40 column can reach 1000 cm / h, and the pressure resistance is >1 MPa.

[0074] Obviously, the above embodiments are merely example for clearly illustrating but not limitation to the embodiments. Based on the above description, other different forms of changes or variations can be made by those skilled in the art. Here, all the embodiments need not and can not be enumerated. The obvious changes or variations derived from the above description are still within the protection scope of the present application.

Claims

1. A method for preparing a chromatography medium for the removal of polyphenolic substances, characterized in that, include: Aqueous phase is obtained by mixing water-soluble inorganic salts, dispersants, and water; The oil phase is obtained by mixing N-vinylpyrrolidone, crosslinking agent one, crosslinking agent two, crosslinking agent three, initiator, and pore-forming agent. An oil phase was added to an aqueous phase for suspension polymerization to prepare a chromatography medium for the removal of polyphenols. The crosslinking agent is at least one of triallyl isocyanurate and N,N'-methylenebisacrylamide; The second crosslinking agent is at least one of N,N'-divinylimidazolinone, N,N'-bisacryloylethylenediamine, N,N'-divinylethylidene urea, and N,N'-divinylpropylidene urea. The crosslinking agent is at least one of allyl methacrylate, ethylene glycol diacrylate, ethylene glycol dimethacrylate, and tetraethylene glycol acrylate. The mass ratio of N-vinylpyrrolidone, crosslinking agent one, crosslinking agent two, crosslinking agent three, initiator, and porogen is 100:(21-36):(11-26):(3-10):(0.7-1.8):(120-125).

2. The preparation method according to claim 1, characterized in that, The water-soluble inorganic salt includes at least one of sodium sulfate, sodium chloride, calcium chloride, and potassium chloride; And / or, the dispersant includes at least one of gelatin, polyvinyl alcohol, carboxymethyl cellulose, hydroxypropyl methyl cellulose, polyvinylpyrrolidone, and starch.

3. The preparation method according to claim 1 or 2, characterized in that, The initiator includes at least one of benzoyl peroxide, azobisisobutyronitrile, and azobisisoheptanenitrile; And / or, the pore-forming agent includes at least one of toluene, dichloromethane, dichloroethane, butyl acetate, and ethyl acetate.

4. The preparation method according to claim 1 or 2, characterized in that, The mass ratio of the water-soluble inorganic salt, dispersant and water is (25-50):(0.1-3):

100.

5. The preparation method according to claim 1 or 2, characterized in that, The volume ratio of the aqueous phase to the oil phase is 1:(3-8).

6. The preparation method according to claim 1 or 2, characterized in that, The suspension polymerization temperature is 50-90℃, and the suspension polymerization time is 3-10 h.

7. The preparation method according to claim 1 or 2, characterized in that, In the suspension polymerization process, stirring is performed after the liquid surface has separated into layers. And / or, after the suspension polymerization, the process includes extraction with an extractant, washing with water, and sieving.

8. The preparation method according to claim 7, characterized in that, The stirring speed is 100-500 rpm; And / or, the extractant includes at least one of methanol, ethanol, acetone, and methylal; And / or, the particle size of the sieve is 100-3000 μm.

9. A chromatography medium for the removal of polyphenolic substances, characterized in that, It is prepared by the method for preparing a chromatographic medium for the removal of polyphenolic substances as described in any one of claims 1-8.

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