A preparation method of a pH-responsive nanofiltration membrane for purifying small molecule peptides

Abstract

The patent relates to a preparation method of a nanofiltration membrane for purifying small molecule peptides. The pH-responsive nanofiltration membrane comprises a base membrane and a separation surface layer. The base membrane is an asymmetric commercial polymer membrane, and the separation surface layer is formed by interfacial polymerization of a material with pH-sensitive groups and a polybasic acid chloride. The advantage of the present application is that the pH-responsive material is added to the aqueous monomer. Compared with the traditional method, the introduction of the pH-sensitive polymer material not only responds to different feed pH during the filtration process, but also can control the polymer chain state by changing the pH of the feed liquid, thereby adjusting the membrane pore size and precisely separating small molecule peptides. In addition, the pH-sensitive group has a charge, which enhances the surface charge density of the membrane. The charge effect and the screening effect work together to facilitate the separation of small molecule peptides from inorganic salts and other amino acids, while maintaining the biological activity of small molecules. The preparation method of the present application is simple, and the obtained pH-responsive nanofiltration membrane has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The application relates to a preparation method of a pH-responsive nanofiltration membrane, and belongs to the technical field of membrane preparation. BACKGROUND

[0002] Peptides are organic compounds, which are obtained by dehydration of amino acids and contain carboxyl and amino groups, and are amphoteric compounds. Peptides with a molecular weight of 50-5000 are called peptides. Peptides with a molecular weight of 5000-10000 are called large peptides. Peptides with a molecular weight of 50-2000 are called small peptides, oligopeptides, and low-molecular-weight peptides, and are also called small-molecule active polypeptides. Biologists call peptides "amino acid chains", and call small-molecule active polypeptides "bioactive peptides". Generally, peptides with ten or fewer amino acids are more medically and commercially practical. Small peptides act as carriers and transport tools in organisms, and deliver nutrients to various parts of the human body to fully exert their functions. Small peptides act as neurotransmitters in organisms, and transmit information. Small peptides have strong activity and diversity, and can comprehensively regulate physiological functions of the human body and enhance physiological activity of the human body.

[0003] Soybean peptides are small-molecule fragments composed of 2-10 amino acids, which are obtained by enzymatic decomposition of large-molecule soybean proteins by biological technology. Soybean peptides are rich in 22 kinds of amino acids, including 9 kinds of essential amino acids that cannot be synthesized by the human body. Soybean peptides are small-molecule products of hydrolysis of soybean proteins, and have multiple health benefits. In addition to improving immunity, soybean peptides can affect the electron transfer capacity of amino acid residues and up-regulate the activity of cellular antioxidant enzymes, regulate intestinal flora, and reduce cholesterol.

[0004] At present, there are three methods for preparing soybean peptides: enzyme hydrolysis, chemical hydrolysis and microbial fermentation. The commonly used methods in production are enzyme hydrolysis and microbial fermentation. The soybean peptides produced by enzyme hydrolysis are easy to separate and purify, and the amino acids are not easily destroyed, which has obvious advantages for industrial production. At present, the commonly used enzyme preparations for hydrolyzing soybean protein mainly include papain, alkaline protease, neutral protease and complex protease. The optimum pH of these enzymes is about 7. In industrial production, after the enzyme hydrolysis reaction is completed, 10% trichloroacetic acid is used to adjust the pH value to the isoelectric point of soybean protein (pH value is 4.3). On the one hand, it is to remove the unhydrolyzed protein, and on the other hand, the pH of the enzyme hydrolysate is adjusted to the isoelectric point. At this time, the net charge of protein at the isoelectric point is zero, which is easy to precipitate. At this time, the product obtained by centrifugation has low purity and may lose protein. At the same time, it is necessary to add ethanol for washing to remove residual trichloroacetic acid, and the operation is relatively complex. In the subsequent purification and refining experiment, due to the very small content of the target active peptide in the production reaction system, traditional separation techniques are often helpless, and modern separation techniques such as adsorption separation, chromatography separation, ultrafiltration membrane separation and reverse osmosis must be used. In addition, techniques such as decolorization, deodorization and debittering are also required. However, these methods consume a lot of energy and have low yield. For example, the chromatography method is highly selective, but it consumes a lot of toxic and expensive solvents, and it provides low mass transfer and low output.

[0005] Studies have shown that the yield, purity and product quality of membrane technology separation are comparable to those of chromatography, but the production rate is higher and the cost is lower. Membrane filtration can be carried out at low temperature, which consumes less energy and does not require additives or solvents, and can be easily scaled up to high flow rate, which can improve batch processing capacity compared with chromatography. Chinese invention patent application 201610536913.4, a new soybean peptide separation and purification method, is characterized by two-stage separation. The molecular weight of the filtrate after the first filtration is 300 Da to 3000 Da, and the average molecular weight after the second filtration is less than 1000 Da. Although the final soybean peptide has a concentrated molecular weight distribution and an average of less than 1000 Da, it does not completely achieve the fractional separation of soybean peptides with different molecular weights. The present application uses pH-responsive nanofiltration membranes, which change the pore size of the membranes by adjusting the pH of the feed liquid, achieving the purpose of separating soybean peptides with different molecular weights by membrane separation. The equipment is simpler and has more industrial application prospects.

[0006] In recent years, the use of stimuli-responsive materials has been a growing topic due to their potential applications in many fields. For nanofiltration membranes, it can overcome the shortcomings of traditional nanofiltration membranes that cannot adapt to environmental changes. One potential advantage of using pH-sensitive polymer materials as membranes is that they not only respond differently to different feed pH during filtration, but also can control the state of polymer chains during membrane formation by changing the pH of the feed solution, thereby adjusting the pore size of the membrane. Therefore, we can precisely control the pore size of the membrane by changing the pH of the feed solution, and more efficiently separate the feed solution.

[0007] Since the final pH of the enzymatic experiment is around 7, and the enzymatic solution mainly contains soybean peptides with a molecular weight of 150-1000 Da, and other small molecule amino acids. If the precipitation method is used to separate soybean peptides, there will be the disadvantages of low yield, low purity and high energy consumption. The present application uses a pH-responsive nanofiltration membrane to purify soybean peptides. Specifically, by adjusting the pH of the enzymatic solution, the pore size of the membrane will change with the change of the pH, thereby achieving the separation of soybean peptides with different molecular weights using one nanofiltration membrane. SUMMARY

[0008] The technical problem to be solved by the present application is to provide a preparation method of a pH-responsive nanofiltration membrane for purifying small molecule peptides. By adjusting the pH of the feed solution, the molecular weight cut-off of the prepared nanofiltration membrane can be increased from 400 Da to 600 Da, thereby obtaining soybean peptides with different molecular weights, so that in the production process of small molecule peptides, efficient separation is achieved while ensuring their biological activity.

[0009] To achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0010] (1) Preparation of water phase monomer mixed solution

[0011] The substances involved in the preparation process of the water phase monomer mixed solution are composed of the following mass percentages: surfactant 0.5-1.5%, chitosan (different molecular weights) 0.05-0.2%, diamine organic matter 0.5-2.5%, and the remaining amount is ultrapure water. Prepare a certain concentration of acetic acid aqueous solution, weigh a certain amount of chitosan powder, slowly add it to the acetic acid aqueous solution, then magnetically stir for 1-2 h. After the chitosan is completely dissolved, add the diamine organic matter, wait for it to completely dissolve, then add the surfactant dropwise. Stir the mixed solution with a magnet for 10-15 min to obtain the water phase monomer mixed solution;

[0012] Prepare 2 mol / L sodium hydroxide solution, immerse the commercial membrane in the prepared sodium hydroxide solution, and place it in a constant temperature water bath at 60 DEG C for 2 h, then take out the membrane and wash it with pure water for 3 times, and then immerse it in pure water for 24 h for standby;

[0013] (2) Preparation of pH-responsive nanofiltration membrane

[0014] The water phase solution prepared in (1) is stirred uniformly and then is left to stand for 5 min for standby, the commercial membrane is taken out from pure water, and the excess water is wiped off by using industrial wiping paper; 1,3,5-benzene tricarbonyl chloride is dissolved in an organic solvent to prepare an oil phase solution, and the mass fraction of 1,3,5-benzene tricarbonyl chloride in the oil phase solution is 0.15-0.3%. 25 mL of the water phase solution is poured on the surface of the base membrane, and after being kept for 1-3 min, the excess water phase solution is wiped off by using industrial wiping paper, and then 25 mL of the oil phase solution is poured to cause an interfacial polymerization reaction, and after being reacted for 1-2 min, the excess oil phase solution is poured out, and after being washed twice with an organic solvent, the prepared nanofiltration membrane is immersed in solutions with different pH values for 30 min, and the membrane is freeze-dried and stored for use.

[0015] Preferably, the surfactant in step (1) is one of trimethylamine, triethylamine, dodecyl dimethyl benzyl ammonium bromide.

[0016] Preferably, the chitosan with a molecular weight of 50000 Da, 100000 Da or 200000 Da in step (1) is one of them.

[0017] Preferably, the diamine organic matter in step (1) is one of o-phenylenediamine, m-phenylenediamine or p-phenylenediamine.

[0018] Preferably, the commercial membrane in step (1) is one of polyacrylonitrile, polyvinylidene fluoride, polyether sulfone or polysulfone.

[0019] Preferably, the organic solvent in step (2) is one of n-hexane, n-heptane, dodecane or tetradecane.

[0020] By selecting chitosan as a pH-responsive material, the chitosan is mixed with o-phenylenediamine at a certain proportion as a water phase monomer and an acyl chloride monomer to perform an interfacial polymerization reaction, in a low-pH buffer solution, most of the functional amine groups of chitosan are protonated. The electrostatic repulsion between the charged chitosan chains causes the polymer network to swell, and the buffer solution diffuses and swells inside the gel. When the pH value of the solution increases, the ionization degree of chitosan decreases, forming a gel with a more compact structure and a lower equilibrium swelling ratio (Seq). By adjusting the pH value of the feed solution, the swelling and shrinking of the cross-linked network of the polyamide layer are affected, so that the pore size of the membrane can be accurately controlled. In addition, compared with other pH-responsive materials, chitosan has better anti-pollution performance and degradability.

[0021] Advantages:

[0022] Since the pH-responsive material is introduced by mixing with traditional water-phase monomers, the cross-linking degree of the polyamide layer is more intensive. The fermentation liquor of soybean peptides is acidic, and by slightly adjusting the pH of the fermentation liquor, soybean peptide solutions with different molecular weights can be obtained. The membrane preparation method of the present application can be used for the purification of various small molecule peptides, and has wide applicability.

[0023] The chitosan used in the present application is a linear polymer, and the membrane surface formed by it has a large number of functional amine groups. As the pH of the solution decreases, the degree of protonation of these functional amine groups increases, and in turn the degree of ionization increases. The increase in the degree of ionization will cause the electrostatic repulsion between adjacent NH 3+ groups to increase, resulting in chain expansion, which in turn affects the relaxation of the macromolecular chain, and the membrane pore size becomes smaller. Conversely, as the pH value increases, the degree of ionization decreases, the molecular chain contracts, and the membrane pore size becomes larger. By changing the pH of the feed liquid to control the membrane pore size, soybean peptides with different molecular weights can be obtained, and the presence of amine groups can increase the charge density on the membrane surface and enhance the Donnan effect. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Surface scanning electron micrograph of the nanofiltration membrane of Example 1

[0025] Figure 2 Surface scanning electron micrograph of the nanofiltration membrane of Example 3 after soaking in a solution with pH = 3.4 for half an hour and drying

[0026] Figure 3 Surface scanning electron micrograph of the nanofiltration membrane of Example 3 after soaking in a solution with pH = 9.4 for half an hour and drying

[0027] Figure 4 Cross-sectional scanning electron micrograph of the nanofiltration membrane of Example 5 DETAILED DESCRIPTION

[0028] The present application will be further described in detail by specific embodiments. However, those skilled in the art will understand that the following examples are only used to illustrate the present application and should not be regarded as limiting the scope of the present application.

[0029] The pH-responsive nanofiltration membrane prepared by the present application can be used for the purification of small molecule peptides, so the rejection rate of small molecule peptides, the permeation rate of inorganic salts, the water flux and the anti-fouling performance are four important parameters for evaluating the nanofiltration membrane.

[0030] The test conditions for water flux, small molecule peptide rejection rate and inorganic salt permeation rate are: dead-end filtration device, soybean peptide concentration of 2000 mg / L, inorganic salt is NaCl commonly found in soybean peptide fermentation broth, concentration of 500 mg / L, test temperature is 25°C, pH = 1-12, test pressure is 0.6 MPa.

[0031] The soybean peptide rejection rate is defined as:

[0032] In the formula, R1 represents the soybean peptide rejection rate, C p1 and C f1 respectively represent the soybean peptide concentrations in the permeate and the raw material liquid.

[0033] The NaCl permeation rate is defined as:

[0034] In the formula, R2 represents the soybean peptide rejection rate, C p2 and C f2 respectively represent the NaCl concentrations in the permeate and the raw material liquid.

[0035] The water flux is defined as: the water volume that passes through the effective area of the membrane per unit time under unit pressure.

[0036] Example 1:

[0037] (1) Under room temperature conditions, 2 mL of acetic acid was weighed into 100 mL of ultrapure water, and magnetic stirring was performed for 2 min to mix it uniformly. Then, 0.05 g of chitosan (MW = 200000) was slowly added to the acetic acid aqueous solution, and continuous stirring was performed for 1-2 h to fully dissolve it. Then, 0.5 g of p-phenylenediamine was added, and stirring was continued until complete dissolution. Finally, 0.5 mL of triethylamine was added, and the mixed solution was magnetically stirred for 10 min to obtain the water phase monomer mixed solution. A 2 mol / L sodium hydroxide solution was prepared, and the PAN commercial membrane was immersed in the prepared sodium hydroxide solution. The membrane was immersed in a constant temperature water bath at 60°C for 2 h, taken out, washed with pure water for 3 times, and then immersed in pure water for 24 h for standby;

[0038] (2) The water phase solution prepared in (1) is stirred uniformly and then left to stand for 5 min for standby, a commercial membrane is taken out from pure water, and the excess water is wiped off using an industrial wiping paper; 1,3,5-benzene tricarbonyl chloride is dissolved in n-heptane to prepare an oil phase solution with a concentration of 0.15 %w / v. 25 mL of the water phase mixed solution is poured on the surface of the base membrane, and after being kept for 2 min, the excess water phase solution is wiped off using an industrial wiping paper, and then 25 mL of the oil phase solution is poured to cause an interfacial polymerization reaction, after 1 min of reaction, the excess oil phase solution is poured out, and after being washed twice with n-heptane solution, different pH solutions are prepared respectively using sodium hydroxide and hydrochloric acid, and the prepared nanofiltration membrane is soaked in the solutions with different pH values for 30 min, and the prepared membrane is freeze-dried for storage and use.

[0039] The performance of the nanofiltration membrane prepared in Example 1 is tested, and the water flux of the nanofiltration membrane after being soaked in a solution with pH = 4.3 for half an hour is 39.8 L·m -2 ·h -1 ·bar -1 , the soybean peptide retention rate is 87.5%, and the NaCl permeation rate is 93.8%.

[0040] Example 2:

[0041] (1) At room temperature, 2 mL of acetic acid is weighed into 100 mL of ultrapure water, and is stirred magnetically for 2 min to mix uniformly, then 0.07 g of chitosan (MW = 200000) is slowly added to the acetic acid aqueous solution, and is continuously stirred for 1-2 h to dissolve completely, then 0.6 g of p-phenylenediamine is added, and is continuously stirred to dissolve completely, finally 0.6 mL of triethylamine is added, and the mixed solution is magnetically stirred for 10 min to obtain a water phase monomer mixed solution; a 2 mol / L sodium hydroxide solution is prepared, a PAN commercial membrane is soaked in the prepared sodium hydroxide solution, and is soaked in a constant temperature water bath at 60 ℃ for 2 h, then the membrane is taken out and washed with pure water for 3 times, and then is soaked in pure water for 24 h for standby;

[0042] (2) The water phase solution prepared in (1) is stirred uniformly and then left to stand for 5 min for standby, a commercial membrane is taken out from pure water, and the excess water is wiped off using an industrial wiping paper; 1,3,5-benzene tricarbonyl chloride is dissolved in n-heptane to prepare an oil phase solution with a concentration of 0.15 %w / v. 25 mL of the water phase mixed solution is poured on the surface of the base membrane, and after being kept for 2 min, the excess water phase solution is wiped off using an industrial wiping paper, and then 15 mL of the oil phase solution is poured to cause an interfacial polymerization reaction, after 1 min of reaction, the excess oil phase solution is poured out, and after being washed twice with n-heptane solution, different pH solutions are prepared respectively using sodium hydroxide and hydrochloric acid, and the prepared nanofiltration membrane is soaked in the solutions with different pH values for 30 min, and the prepared membrane is freeze-dried for storage and use.

[0043] The performance test was carried out on the nanofiltration membrane prepared in Example 2. The water flux of the nanofiltration membrane after soaking in a solution with pH = 4.1 for half an hour was 37.5 L·m -2 ·h -1 ·bar -1 The soy peptide retention rate was 94.3%, and the NaCl permeation rate was 91.8%.

[0044] Example 3:

[0045] (1) At room temperature, 2 mL of acetic acid was weighed into 100 mL of ultrapure water, and magnetic stirring was carried out for 2 min to mix it evenly. Then 0.09 g of chitosan (MW = 200000) was slowly added to the acetic acid aqueous solution, and continuous stirring was carried out for 1-2 h to fully dissolve. Then 0.7 g of p-phenylenediamine was added, and stirring was continued until complete dissolution. Finally, 0.7 mL of triethylamine was added, and the mixed solution was magnetically stirred for 10 min to obtain the water phase monomer mixed solution. A 2 mol / L sodium hydroxide solution was prepared, and the PAN commercial membrane was soaked in the prepared sodium hydroxide solution. The membrane was washed with pure water for 3 times, and then soaked in pure water for 24 h for standby;

[0046] (2) The water phase solution prepared in (1) was stirred evenly and stood for 5 min for standby. The commercial membrane was taken out from pure water, and the excess water was wiped off with industrial wiping paper. 1,3,5-benzene tricarbonyl chloride was dissolved in n-heptane to prepare an oil phase solution with a concentration of 0.15 %w / v. 25 mL of the water phase mixed solution was poured onto the surface of the base membrane, and after 2 min, the excess water phase solution was wiped off with industrial wiping paper, and then 25 mL of the oil phase solution was poured to occur interfacial polymerization. After 1 min of reaction, the excess oil phase solution was poured out, and the prepared nanofiltration membrane was soaked in different pH solutions prepared by sodium hydroxide and hydrochloric acid for 30 min. The prepared membrane was freeze-dried and stored for use.

[0047] The performance test was carried out on the nanofiltration membrane prepared in Example 3. The water flux of the nanofiltration membrane after soaking in a solution with pH = 4.3 for half an hour was 34.8 L·m -2 ·h -1 ·bar -1 The soy peptide retention rate was 96.5%, and the NaCl permeation rate was 92.8%.

[0048] Example 4:

[0049] (1) Under the room temperature, 2 mL acetic acid was added into 100 mL ultrapure water, and stirred magnetically for 2 min to mix evenly. Then 1.1 g chitosan (MW=150000) was slowly added into the acetic acid aqueous solution, and continuously stirred for 1-2 h to fully dissolve. Then 0.8 g p-phenylenediamine was added, and continuously stirred until completely dissolved. Finally, 0.8 mL triethylamine was added, and the mixed solution was stirred magnetically for 10-15 min to obtain the aqueous monomer mixed solution. A 2 mol / L sodium hydroxide solution was prepared, and the PAN commercial film was immersed in the prepared sodium hydroxide solution. The film was taken out and washed with pure water for 3 times, and then immersed in pure water for 24 h for standby;

[0050] (2) The aqueous solution prepared in (1) was stirred evenly and stood for 5 min for standby. The commercial film was taken out from the pure water, and the excess water was wiped off with industrial wiping paper. 1,3,5-benzene tricarbonyl chloride was dissolved in n-heptane to prepare an oil phase solution with a concentration of 0.15 %w / v. 25 mL of the aqueous mixed solution was poured onto the surface of the base film, and after 2 min, the excess aqueous solution was wiped off with industrial wiping paper. Then 25 mL of the oil phase solution was poured to occur interfacial polymerization reaction. After 2 min, the excess oil phase solution was poured out, and the prepared nanofiltration membrane was immersed in different pH solutions prepared by sodium hydroxide and hydrochloric acid for 30 min. The prepared membrane was freeze-dried for storage and use.

[0051] The performance of the nanofiltration membrane prepared in Example 4 was tested. The water flux of the nanofiltration membrane after immersion in a solution with pH=4.3 for half an hour was 32.4 L·m -2 ·h -1 ·bar -1 , the soybean peptide retention rate was 92.2%, and the NaCl permeation rate was 91.8%.

[0052] Example 5:

[0053] (1) Under the room temperature, 2 mL acetic acid was added into 100 mL ultrapure water, and stirred magnetically for 2 min to mix evenly. Then 1.1 g chitosan (MW=150000) was slowly added into the acetic acid aqueous solution, and continuously stirred for 1-2 h to fully dissolve. Then 0.8 g p-phenylenediamine was added, and continuously stirred until completely dissolved. Finally, 0.8 mL triethylamine was added, and the mixed solution was stirred magnetically for 10-15 min to obtain the aqueous monomer mixed solution. A 2 mol / L sodium hydroxide solution was prepared, and the PAN commercial film was immersed in the prepared sodium hydroxide solution. The film was taken out and washed with pure water for 3 times, and then immersed in pure water for 24 h for standby;

[0054] (2) The water phase solution prepared in (1) is stirred uniformly and then left to stand for 5 min for standby, a commercial membrane is taken out from pure water, and the excess water is wiped off using an industrial wiping paper; 1,3,5-benzene tricarbonyl chloride is dissolved in n-heptane to prepare an oil phase solution with a concentration of 0.15 %w / v. 25 mL of the water phase mixed solution is poured on the surface of the base membrane, and after being kept for 2 min, the excess water phase solution is wiped off using an industrial wiping paper, and then 25 mL of the oil phase solution is poured to cause an interfacial polymerization reaction. After 1 min of reaction, the excess oil phase solution is poured out, and the prepared nanofiltration membrane is soaked in a solution with different pH values prepared by sodium hydroxide and hydrochloric acid respectively for 30 min. The prepared membrane is freeze-dried for storage and use.

[0055] The performance of the nanofiltration membrane prepared in Example 5 is tested. The water flux of the nanofiltration membrane after being soaked in a solution with pH = 4.3 for half an hour is 36.8 L·m -2 ·h -1 ·bar -1 , the soybean peptide retention rate is 92.4%, and the NaCl permeation rate is 90.2%.

[0056] Example 6:

[0057] (1) At room temperature, 2 mL of acetic acid is weighed into 100 mL of ultrapure water, and is stirred magnetically for 2 min to mix uniformly. Then 0.5 g of chitosan (MW = 100000) is slowly added into the acetic acid aqueous solution, and is continuously stirred for 1-2 h to dissolve fully. Then 1 g of p-phenylenediamine is added, and is continuously stirred to dissolve completely. Finally, 1 mL of triethylamine is added, and the mixed solution is stirred magnetically for 15 min to obtain a water phase monomer mixed solution. A 2 mol / L sodium hydroxide solution is prepared, and a PAN commercial membrane is soaked in the prepared sodium hydroxide solution. The membrane is washed with pure water for 3 times in a constant temperature water bath at 60 ℃ for 2 h, and is then soaked in pure water for 24 h for standby;

[0058] (2) The water phase solution prepared in (1) is stirred uniformly and then left to stand for 5 min for standby, a commercial membrane is taken out from pure water, and the excess water is wiped off using an industrial wiping paper; 1,3,5-benzene tricarbonyl chloride is dissolved in n-heptane to prepare an oil phase solution with a concentration of 0.15 %w / v. 25 mL of the water phase mixed solution is poured on the surface of the base membrane, and after being kept for 2 min, the excess water phase solution is wiped off using an industrial wiping paper, and then 25 mL of the oil phase solution is poured to cause an interfacial polymerization reaction. After 1 min of reaction, the excess oil phase solution is poured out, and the prepared nanofiltration membrane is soaked in a solution with different pH values prepared by sodium hydroxide and hydrochloric acid respectively for 30 min. The prepared membrane is freeze-dried for storage and use.

[0059] The performance test was carried out on the nanofiltration membrane prepared in Example 6. The water flux of the nanofiltration membrane after soaking in a solution with pH = 4.3 for half an hour was 47.6 L·m -2 ·h -1 ·bar -1 The soy peptide retention rate was 97.1%, and the NaCl permeation rate was 98.7%.

[0060] Example 7:

[0061] (1) Under room temperature conditions, 2 mL of acetic acid was weighed into 100 mL of ultrapure water, and magnetic stirring was performed for 2 min to mix it uniformly. Then, 0.7 g of chitosan (MW = 100000) was slowly added to the acetic acid aqueous solution, and continuous stirring was performed for 1-2 h to fully dissolve it. Then, 1.1 g of p-phenylenediamine was added, and stirring was continued until complete dissolution. Finally, 1.2 mL of triethylamine was added, and the mixed solution was magnetically stirred for 10-15 min to obtain an aqueous monomer mixed solution. A 2 mol / L sodium hydroxide solution was prepared, and the PAN commercial membrane was soaked in the prepared sodium hydroxide solution. The membrane was washed with pure water for 3 times, and then soaked in pure water for 24 h for standby;

[0062] (2) The aqueous solution prepared in (1) was stirred uniformly and stood for 5 min for standby. The commercial membrane was taken out from pure water, and the excess water was wiped off using industrial wiping paper. 1,3,5-benzene tricarbonyl chloride was dissolved in n-heptane to prepare an oil phase solution with a concentration of 0.2 %w / v. 25 mL of the aqueous mixed solution was poured onto the surface of the base membrane, and after 2 min, the excess aqueous solution was wiped off using industrial wiping paper. Then, 25 mL of the oil phase solution was poured to occur an interfacial polymerization reaction. After 2 min of reaction, the excess oil phase solution was poured out, and the prepared nanofiltration membrane was soaked in different pH solutions prepared by sodium hydroxide and hydrochloric acid for 30 min. The prepared membrane was freeze-dried for storage and use.

[0063] The performance test was carried out on the nanofiltration membrane prepared in Example 7. The water flux of the nanofiltration membrane after soaking in a solution with pH = 4.6 for half an hour was 42.3 L·m -2 ·h -1 ·bar -1 The soy peptide retention rate was 95.6%, and the NaCl permeation rate was 94.5%.

[0064] Example 8:

[0065] (1) At room temperature, 2 mL of acetic acid was weighed into 100 mL of ultrapure water, and magnetic stirring was performed for 2 min to mix it evenly. Then 2 g of chitosan (MW=50000) was slowly added to the acetic acid aqueous solution, and continuous stirring was performed for 1-2 h to fully dissolve. Then 1.2 g of p-phenylenediamine was added, and stirring was continued until complete dissolution. Finally, 1.3 mL of triethylamine was added, and the mixed solution was magnetically stirred for 15 min to obtain the aqueous monomer mixed solution. A 2 mol / L sodium hydroxide solution was prepared, and the PAN commercial film was immersed in the prepared sodium hydroxide solution. The film was immersed in a constant temperature water bath at 60°C for 2 h, taken out, washed with pure water for 3 times, and then immersed in pure water for 24 h for standby;

[0066] (2) The aqueous solution prepared in (1) was stirred evenly and stood for 5 min for standby. The commercial film was taken out from pure water, and the excess water was wiped off with industrial wiping paper. 1,3,5-benzene tricarbonyl chloride was dissolved in n-heptane to prepare an oil phase solution with a concentration of 0.25% w / v. 25 mL of the aqueous mixed solution was poured onto the surface of the base film, and after 2 min, the excess aqueous solution was wiped off with industrial wiping paper, and then 25 mL of the oil phase solution was poured to occur interfacial polymerization. After 2 min of reaction, the excess oil phase solution was poured out, washed twice with n-heptane solution, and then immersed in different pH solutions prepared by sodium hydroxide and hydrochloric acid respectively for 30 min. The prepared nanofiltration membrane was freeze-dried and stored for use.

[0067] The performance of the nanofiltration membrane prepared in Example 8 was tested. The water flux of the nanofiltration membrane after soaking in a solution with pH=3.3 for half an hour was 46.7 L·m -2 ·h -1 ·bar -1 , the soybean peptide retention rate was 92.5%, and the NaCl permeation rate was 91.8%.

Claims

1. A method for preparing a pH-responsive nanofiltration membrane for purifying small molecule peptides, the specific steps of which are as follows: (1) Preparation of aqueous monomer mixed solution The substances involved in the preparation of the aqueous monomer mixed solution are composed of the following mass percentages: surfactant 0.5-1.5%, chitosan 0.05-0.2%, diamine organic compound 0.5-2.5%, and the remainder is an aqueous acetic acid solution. Prepare an aqueous acetic acid solution of a certain concentration, weigh a certain amount of chitosan powder, and slowly add it to the aqueous acetic acid solution, followed by magnetic stirring for 1-2 hours. After the chitosan is completely dissolved, add the diamine organic compound, wait for it to completely dissolve, and then add the surfactant dropwise. Stir the mixed solution magnetically for 10-15 minutes to obtain the aqueous monomer mixed solution. Prepare a 2 mol / L sodium hydroxide solution, immerse the base membrane in the prepared sodium hydroxide solution, and keep it in a constant temperature water bath at 60 ℃ for 2 h. Remove the base membrane, wash it 3 times with pure water, and then soak it in pure water for 24 h for later use. (2) Preparation of pH-responsive nanofiltration membranes After stirring the aqueous monomer mixture prepared in (1) evenly, let it stand for 5 minutes for later use. Take out the base membrane from pure water and wipe off the excess water with industrial wiping paper. Dissolve 1,3,5-benzenetricarboxyl chloride in an organic solvent to prepare an oil phase solution with a mass fraction of 0.15-0.3% of 1,3,5-benzenetricarboxyl chloride in the oil phase solution. Pour 25 mL of the aqueous phase solution onto the surface of the base membrane and keep it for 1-3 minutes. Wipe off the excess aqueous phase solution with industrial wiping paper. Then pour in 25 mL of the oil phase solution to carry out the interfacial polymerization reaction. After reacting for 1-2 minutes, pour out the excess oil phase solution. After washing twice with organic solvent, immerse the prepared nanofiltration membrane in solutions with different pH values ​​for 30 minutes. Freeze-dry the membrane and store it for use.

2. The preparation method according to claim 1, characterized in that... In step (1), the surfactant is one of trimethylamine, triethylamine, or dodecyl dimethyl benzyl ammonium bromide.

3. The preparation method according to claim 1, characterized in that... In step (1), the molecular weight of chitosan is one of 50,000 Da, 100,000 Da, or 200,000 Da.

4. The preparation method according to claim 1, characterized in that... In step (1), the diamine organic compound is one of o-phenylenediamine, m-phenylenediamine, or p-phenylenediamine.

5. The preparation method according to claim 1, characterized in that... In step (1), the base film is one of polyacrylonitrile, polyvinylidene fluoride, polyethersulfone, or polysulfone.

6. The preparation method according to claim 1, characterized in that... The organic solvent mentioned in step (2) is one of n-hexane, n-heptane, dodecane, and tetradecane.

7. A pH-responsive nanofiltration membrane prepared by the method for preparing a pH-responsive nanofiltration membrane for purifying small molecule peptides according to any one of claims 1-6, characterized in that: The pore size of the nanofiltration membrane changes under different pH conditions. After treatment with a solution at pH 3.5 for 30 minutes, the pure water flux of the nanofiltration membrane is ≥30 L·m. -2 ·h -1 ·bar -1 In the separation test of soybean peptides and NaCl, the soybean peptide retention rate was ≥95%, the NaCl permeation rate was ≥90%, and the membrane flux recovery rate was ≥95%, showing a significant anti-biofouling effect.

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