Preparation method of nanofiltration membrane with pH response for purifying small molecule peptide

Abstract

The invention relates to a preparation method of a nanofiltration membrane for purifying small molecule peptides, the nanofiltration membrane with pH response comprises a base membrane and a separation surface layer, the base membrane adopts a polymer asymmetric commercial membrane, and the separation surface layer is formed by a material with a pH sensitive group and multi-element acyl chloride through interfacial polymerization reaction. The method has the advantages that the pH response material is added into the water phase monomer, compared with a traditional method, the pH sensitive polymer material is introduced, not only is response to different feeding pH in the filtering process achieved, but also the polymer chain state can be controlled by changing the feeding liquid pH, and therefore the membrane pore diameter is adjusted, and small molecule peptide is accurately separated. In addition, the pH sensitive group carries charges, the charge density on the surface of the membrane is enhanced, the synergistic effect of the charge effect and the screening effect is beneficial to separation of small molecule peptide from inorganic salt and other amino acids, and meanwhile, the biological activity of small molecules is kept. The preparation method is simple, and the obtained nanofiltration membrane with pH response has a wide application prospect.

Description

Technical Field

[0001] The invention relates to a method for preparing a pH-responsive nanofiltration membrane, belonging to the technical field of membrane preparation. The nanofiltration membrane has the advantages of high pure water flux, high selectivity, good anti-fouling effect, etc., and is suitable for fields such as biomolecule purification. Background Art

[0002] Peptide is an organic compound formed by the dehydration of amino acids. It contains carboxyl and amino groups and is an amphoteric compound. Only those with a molecular weight between 50 and 5000 can be called peptides. Those with a molecular weight between 5000 and 10000 are called large peptides. Those with a molecular weight between 50 and 2000 are called small peptides, oligopeptides, and oligopeptides, also known as small molecule active polypeptides. Biologists call peptides "amino acid chains" and collectively refer to small molecule active polypeptides as "bioactive peptides." Usually, those with less than ten peptides are more medically and commercially practical. Small peptides act as carriers and transportation tools in organisms to transport the nutrients that people usually eat to various parts of the human body and give full play to their functions; small peptides act as neurotransmitters in organisms to transmit information; small peptides have extremely strong activity and diversity, and can comprehensively regulate human physiological functions and enhance human physiological activity.

[0003] Soy peptides are large-molecule soybean proteins that are broken down into small-molecule fragments consisting of 2 to 10 amino acids using biotechnology enzymes. Soy peptides are rich in 22 amino acids, including 9 essential amino acids that cannot be synthesized by the human body. Soy peptides are small-molecule products of soy protein hydrolysis and have a variety of health benefits. In addition to improving immunity; soy peptides can also affect the electron transfer capacity of amino acid residues and upregulate the activity of cellular antioxidant enzymes, regulate intestinal flora, and lower cholesterol.

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

[0005] Studies have shown that the yield, purity and product quality of separation using membrane technology are comparable to those of chromatography, but with higher productivity and lower cost. Membrane filtration can be performed at low temperatures, consumes less energy, does not require additives or solvents, and can be easily expanded to high flow rates, which can increase batch processing capacity compared to chromatography. Chinese invention patent application 201610536913.4, a new type of soybean peptide separation and purification method, is characterized by the use of two-stage separation, the molecular weight of the filtrate after the first stage of filtration is 300Da to 3000Da, and the average molecular weight after the second stage of filtration is less than 1000Da. Although the molecular weight distribution of the soybean peptides obtained in the end is concentrated, with an average of less than 1000Da, the graded separation of soybean peptides of different molecular weights is not fully achieved. The present invention uses a pH-responsive nanofiltration membrane, and by adjusting the pH of the feed liquid, the pore size of the membrane changes, thereby achieving the purpose of separating soybean peptides of different molecular weights with a membrane, and the equipment is simpler and has more prospects for industrial application.

[0006] In recent years, the use of stimuli-responsive materials has become an emerging topic due to their potential application prospects in many fields. For nanofiltration membranes, it can overcome the shortcomings of traditional nanofiltration membranes that cannot adapt to environmental changes. A potential benefit of using pH-sensitive polymer materials as membranes is that it will not only respond differently to different feed pHs during the filtration process, but also 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 achieve precise control of the membrane pore size by changing the pH value of the feed solution, and perform separation more efficiently.

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

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

[0009] In order to achieve the above object, the technical solution adopted by the present invention is: a method for preparing a pH-responsive nanofiltration membrane for purifying small molecule peptides, and the specific steps are as follows: (1) Preparation of aqueous monomer mixed solution The substances involved in the preparation process of the aqueous 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 remainder is ultrapure water. Prepare an acetic acid aqueous solution of a certain concentration, weigh a certain amount of chitosan powder, slowly add it to the acetic acid aqueous solution, and then stir magnetically for 1-2 hours. After the chitosan is completely dissolved, add the diamine organic matter, wait until it is completely dissolved, and then add the surfactant dropwise. Stir the mixed solution magnetically for 10-15 minutes to obtain an aqueous monomer mixed solution; Prepare 2 mol / L sodium hydroxide solution, soak the commercial membrane in the prepared sodium hydroxide solution, and keep it in a constant temperature water bath at 60 ℃ for 2 h. Take out the membrane and wash it with pure water for 3 times, and then soak it in pure water for 24 h for use. (2) Preparation of pH-responsive nanofiltration membrane The aqueous solution prepared in (1) was stirred evenly and then allowed to stand for 5 minutes for use. The commercial membrane was taken out from the pure water and excess water was wiped off with industrial wipe paper. 1,3,5-benzenetricarboxylic acid chloride was dissolved in an organic solvent to prepare an oil phase solution, and the mass fraction of 1,3,5-benzenetricarboxylic acid chloride in the oil phase solution was 0.15-0.3%. 25 mL of the aqueous solution was poured on the surface of the base membrane, and after keeping it for 1-3 minutes, the excess aqueous solution was wiped off with industrial wipe paper, and then 25 mL of the oil phase solution was poured in for interfacial polymerization reaction. After reacting for 1-2 minutes, the excess oil phase solution was poured out. After washing twice with organic solvent, the prepared nanofiltration membrane was immersed in solutions of different pH values ​​for 30 minutes, and the membrane was freeze-dried and stored for use.

[0010] Preferably, the surfactant in step (1) is one of trimethylamine, triethylamine and dodecyldimethylbenzyl ammonium bromide.

[0011] Preferably, the chitosan in step (1) is one of chitosans having molecular weights of 50,000 Da, 100,000 Da, and 200,000 Da.

[0012] Preferably, the diamine organic compound in step (1) is one of o-phenylenediamine, m-phenylenediamine and p-phenylenediamine.

[0013] Preferably, the commercial membrane in step (1) is one of polyacrylonitrile, polyvinylidene fluoride, polyether sulfone and polysulfone.

[0014] Preferably, the organic solvent in step (2) is one of n-hexane, n-heptane, dodecane and tetradecane.

[0015] The present invention selects chitosan as a pH-responsive material, mixes it with o-phenylenediamine in a certain proportion as an aqueous phase monomer and an acyl chloride monomer to carry out 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 diffuses and swells inside the gel. When the pH value of the solution increases, the degree of ionization of chitosan decreases, forming a gel with a denser structure and a lower equilibrium expansion ratio (Seq). By adjusting the pH value of the feed solution, the expansion and contraction of the cross-linked network of the polyamide layer is affected, thereby achieving precise control of the membrane pore size. In addition, compared with other pH-responsive materials, chitosan has better anti-pollution performance and degradability.

[0016] Beneficial effects: Since the pH responsive material is introduced by mixing with the traditional aqueous phase monomer, the cross-linking degree of the polyamide layer is more intensive. The fermentation broth of soybean peptide is acidic, and soybean peptide solutions of different molecular weights can be obtained by slightly adjusting the pH of the fermentation broth. The membrane preparation method of the present invention can be used for the purification of a variety of small molecule peptides and has a wide range of applicability.

[0017] The chitosan used in the present invention is a chain polymer. The membrane surface formed by it has a large number of functional amine groups. As the pH of the solution decreases, the protonation degree of these functional amine groups increases, and then the ionization degree increases. The increase in ionization degree will cause the adjacent NH 3+ The electrostatic repulsion between the groups increases, causing the chain to expand, which in turn affects the relaxation of the macromolecular chain and the membrane pore size becomes smaller. On the contrary, as the pH value increases, the degree of ionization decreases, the molecular chain shrinks, and the membrane pore size becomes larger. By changing the pH of the feed solution to control the pore size of the membrane, soybean peptides of different molecular weights can be obtained. At the same time, the presence of amine groups can increase the charge density on the membrane surface and enhance the Donan effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The surface scanning electron microscope image of the nanofiltration membrane of Example 1 is Figure 2 This is a scanning electron microscope image of the surface of the nanofiltration membrane of Example 3 after being immersed in a pH = 3.4 solution for half an hour and dried. Figure 3 This is a scanning electron microscope image of the surface of the nanofiltration membrane of Example 3 after being immersed in a pH = 9.4 solution for half an hour and dried. Figure 4 This is a cross-sectional scanning electron microscope image of the nanofiltration membrane of Example 5. DETAILED DESCRIPTION

[0019] The present invention is further described in detail below by specific embodiments. However, those skilled in the art will appreciate that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention.

[0020] The pH-responsive nanofiltration membrane prepared by the present invention can be used to purify small molecule peptides, so the retention rate of small molecule peptides, the permeability of inorganic salts, water flux and anti-fouling performance are four important parameters for evaluating the nanofiltration membrane.

[0021] The test conditions for water flux, small molecule peptide retention rate and inorganic salt permeability are as follows: dead-end filtration device, soybean peptide concentration of 2000 mg / L, inorganic salt selected as NaCl, which is more common in soybean peptide fermentation broth, with a concentration of 500 mg / L, test temperature of 25°C, pH=1-12, and test pressure of 0.6 MPa.

[0022] The soy peptide retention rate is defined as:

[0023] Where R1 represents the soybean peptide retention rate, C p1 and C f1 are the concentrations of soybean peptides in the permeate and raw material solution, respectively.

[0024] The NaCl permeability is defined as:

[0025] Where R2 represents the soybean peptide retention rate, C p2 and C f2 are the NaCl concentrations in the permeate and feed solution, respectively.

[0026] Water flux is defined as the volume of water that passes through the effective area of ​​the membrane per unit time under unit pressure.

[0027] Embodiment 1: (1) At room temperature, add 2 mL of acetic acid to 100 mL of ultrapure water and stir magnetically for 2 min to mix evenly. Then weigh 0.05 g of chitosan (MW=200000) and slowly add it to the aqueous solution of acetic acid. Stir continuously for 1-2 h until fully dissolved. Then add 0.5 g of p-phenylenediamine and continue stirring until completely dissolved. Finally, add 0.5 mL of triethylamine and stir the mixed solution magnetically for 10 min to obtain an aqueous monomer mixed solution. Prepare a 2 mol / L sodium hydroxide solution and soak the commercial PAN membrane in the prepared sodium hydroxide solution. Keep it in a constant temperature water bath at 60 °C for 2 h. Take out the membrane and wash it with pure water for 3 times. Then soak it in pure water for 24 h for use. (2) The aqueous solution prepared in (1) was stirred evenly and allowed to stand for 5 minutes for later use. The commercial membrane was taken out from the pure water and excess water was wiped off with industrial wipe paper. 1,3,5-Benzenetricarboxylic acid chloride was dissolved in n-heptane to prepare a 0.15% w / v oil phase solution. 25 mL of the aqueous mixed solution was poured on the surface of the base membrane. After keeping it for 2 minutes, the excess aqueous solution was wiped off with industrial wipe paper, and then 25 mL of the oil phase solution was poured in to cause interfacial polymerization reaction. After reacting for 1 minute, the excess oil phase solution was poured out. After washing twice with n-heptane solution, solutions of different pH values ​​were prepared with sodium hydroxide and hydrochloric acid, respectively. The prepared nanofiltration membrane was immersed in the solutions of different pH values ​​for 30 minutes. The prepared membrane was freeze-dried and stored for use.

[0028] The performance of the nanofiltration membrane prepared in Example 1 was tested. The water flux of the nanofiltration membrane after being immersed in a pH = 4.3 solution for half an hour was 39.8 L·m -2 ·h -1 bar -1 , the soybean peptide retention rate was 87.5% and the NaCl permeability was 93.8%.

[0029] Embodiment 2: (1) At room temperature, add 2 mL of acetic acid to 100 mL of ultrapure water and stir magnetically for 2 min to mix evenly. Then weigh 0.07 g of chitosan (MW=200000) and slowly add it to the aqueous solution of acetic acid. Stir continuously for 1-2 h until fully dissolved. Then add 0.6 g of p-phenylenediamine and continue stirring until completely dissolved. Finally, add 0.6 mL of triethylamine and stir the mixed solution magnetically for 10 min to obtain an aqueous monomer mixed solution. Prepare a 2 mol / L sodium hydroxide solution and soak the commercial PAN membrane in the prepared sodium hydroxide solution. Keep it in a constant temperature water bath at 60 °C for 2 h. Take out the membrane and wash it with pure water for 3 times. Then soak it in pure water for 24 h for use. (2) The aqueous solution prepared in (1) was stirred evenly and allowed to stand for 5 minutes. The commercial membrane was taken out from the pure water and excess water was wiped off with industrial wipe paper. 1,3,5-benzenetricarboxylic acid chloride was dissolved in n-heptane to prepare a 0.15% w / v oil phase solution. 25 mL of the aqueous mixed solution was poured on the surface of the base membrane. After keeping it for 2 minutes, the excess aqueous solution was wiped off with industrial wipe paper, and then 15 mL of the oil phase solution was poured in to cause interfacial polymerization reaction. After reacting for 1 minute, the excess oil phase solution was poured out. After washing twice with n-heptane solution, solutions of different pH values ​​were prepared with sodium hydroxide and hydrochloric acid, respectively. The prepared nanofiltration membrane was immersed in the solutions of different pH values ​​for 30 minutes. The prepared membrane was freeze-dried and stored for use.

[0030] The performance of the nanofiltration membrane prepared in Example 2 was tested. After being immersed in a pH = 4.1 solution for half an hour, the water flux of the nanofiltration membrane was 37.5 L·m -2 ·h -1 bar -1 The soybean peptide retention rate was 94.3% and the NaCl permeability was 91.8%.

[0031] Embodiment 3: (1) At room temperature, add 2 mL of acetic acid to 100 mL of ultrapure water and stir magnetically for 2 min to mix evenly. Then weigh 0.09 g of chitosan (MW=200000) and slowly add it to the aqueous solution of acetic acid. Stir continuously for 1-2 h until fully dissolved. Then add 0.7 g of p-phenylenediamine and continue stirring until completely dissolved. Finally, add 0.7 mL of triethylamine and stir the mixed solution magnetically for 10 min to obtain an aqueous monomer mixed solution. Prepare 2 mol / L sodium hydroxide solution, soak the commercial PAN membrane in the prepared sodium hydroxide solution, keep it in a constant temperature water bath at 60 °C for 2 h, take out the membrane, wash it with pure water three times, and then soak it in pure water for 24 h for use. (2) The aqueous solution prepared in (1) was stirred evenly and allowed to stand for 5 minutes for later use. The commercial membrane was taken out from the pure water and excess water was wiped off with industrial wipe paper. 1,3,5-Benzenetricarboxylic acid chloride was dissolved in n-heptane to prepare a 0.15% w / v oil phase solution. 25 mL of the aqueous mixed solution was poured on the surface of the base membrane. After keeping it for 2 minutes, the excess aqueous solution was wiped off with industrial wipe paper, and then 25 mL of the oil phase solution was poured in to cause interfacial polymerization reaction. After reacting for 1 minute, the excess oil phase solution was poured out. After washing twice with n-heptane solution, solutions of different pH values ​​were prepared with sodium hydroxide and hydrochloric acid, respectively. The prepared nanofiltration membrane was immersed in the solutions of different pH values ​​for 30 minutes. The prepared membrane was freeze-dried and stored for use.

[0032] The performance of the nanofiltration membrane prepared in Example 3 was tested. The water flux of the nanofiltration membrane after being immersed in a pH = 4.3 solution for half an hour was 34.8 L·m -2 ·h -1 bar -1 The soybean peptide retention rate was 96.5% and the NaCl permeability was 92.8%.

[0033] Embodiment 4: (1) At room temperature, add 2 mL of acetic acid to 100 mL of ultrapure water and stir magnetically for 2 min to mix evenly. Then weigh 1.1 g of chitosan (MW=150000) and slowly add it to the aqueous solution of acetic acid. Stir continuously for 1-2 h until fully dissolved. Then add 0.8 g of p-phenylenediamine and continue stirring until completely dissolved. Finally, add 0.8 mL of triethylamine and stir the mixed solution magnetically for 10-15 min to obtain an aqueous monomer mixed solution. Prepare 2 mol / L sodium hydroxide solution, soak the commercial PAN membrane in the prepared sodium hydroxide solution, keep it in a constant temperature water bath at 60 °C for 2 h, take out the membrane, wash it with pure water three times, and then soak it in pure water for 24 h for use. (2) The aqueous solution prepared in (1) was stirred evenly and allowed to stand for 5 minutes for later use. The commercial membrane was taken out from the pure water and excess water was wiped off with industrial wipe paper. 1,3,5-benzenetricarboxylic acid chloride was dissolved in n-heptane to prepare a 0.15% w / v oil phase solution. 25 mL of the aqueous mixed solution was poured on the surface of the base membrane. After keeping it for 2 minutes, the excess aqueous solution was wiped off with industrial wipe paper, and then 25 mL of the oil phase solution was poured in to cause interfacial polymerization reaction. After reacting for 2 minutes, the excess oil phase solution was poured out. After washing twice with n-heptane solution, solutions of different pH values ​​were prepared with sodium hydroxide and hydrochloric acid, respectively. The prepared nanofiltration membrane was immersed in the solutions of different pH values ​​for 30 minutes. The prepared membrane was freeze-dried and stored for use.

[0034] The performance of the nanofiltration membrane prepared in Example 4 was tested. The water flux of the nanofiltration membrane after being immersed in a pH = 4.3 solution 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 permeability was 91.8%.

[0035] Embodiment 5: (1) At room temperature, add 2 mL of acetic acid to 100 mL of ultrapure water and stir magnetically for 2 min to mix evenly. Then weigh 1.3 g of chitosan (MW=150000) and slowly add it to the aqueous solution of acetic acid. Stir continuously for 1-2 h until fully dissolved. Then add 0.9 g of p-phenylenediamine and continue stirring until completely dissolved. Finally, add 0.9 mL of triethylamine and stir the mixed solution magnetically for 15 min to obtain an aqueous monomer mixed solution. Prepare a 2 mol / L sodium hydroxide solution and soak the commercial PAN membrane in the prepared sodium hydroxide solution. Keep it in a constant temperature water bath at 60 °C for 2 h. Take out the membrane and wash it with pure water for 3 times. Then soak it in pure water for 24 h for use. (2) The aqueous solution prepared in (1) was stirred evenly and allowed to stand for 5 minutes for later use. The commercial membrane was taken out from the pure water and excess water was wiped off with industrial wipe paper. 1,3,5-Benzenetricarboxylic acid chloride was dissolved in n-heptane to prepare a 0.15% w / v oil phase solution. 25 mL of the aqueous mixed solution was poured on the surface of the base membrane. After keeping it for 2 minutes, the excess aqueous solution was wiped off with industrial wipe paper, and then 25 mL of the oil phase solution was poured in to cause interfacial polymerization reaction. After reacting for 1 minute, the excess oil phase solution was poured out. After washing twice with n-heptane solution, solutions of different pH values ​​were prepared with sodium hydroxide and hydrochloric acid, respectively. The prepared nanofiltration membrane was immersed in the solutions of different pH values ​​for 30 minutes. The prepared membrane was freeze-dried and stored for use.

[0036] The performance of the nanofiltration membrane prepared in Example 5 was tested. The water flux of the nanofiltration membrane after being immersed in a pH = 4.3 solution for half an hour was 36.8 L·m -2 ·h -1 bar -1 The soybean peptide retention rate was 92.4% and the NaCl permeability was 90.2%.

[0037] Embodiment 6: (1) At room temperature, add 2 mL of acetic acid to 100 mL of ultrapure water and stir magnetically for 2 min to mix evenly. Then weigh 0.5 g of chitosan (MW=100000) and slowly add it to the aqueous solution of acetic acid. Stir continuously for 1-2 h until fully dissolved. Then add 1 g of p-phenylenediamine and continue stirring until completely dissolved. Finally, add 1 mL of triethylamine and stir the mixed solution magnetically for 15 min to obtain an aqueous monomer mixed solution. Prepare a 2 mol / L sodium hydroxide solution and soak the commercial PAN membrane in the prepared sodium hydroxide solution. Keep it in a constant temperature water bath at 60 °C for 2 h. Take out the membrane and wash it with pure water for 3 times. Then soak it in pure water for 24 h for use. (2) The aqueous solution prepared in (1) was stirred evenly and allowed to stand for 5 minutes for later use. The commercial membrane was taken out from the pure water and excess water was wiped off with industrial wipe paper. 1,3,5-benzenetricarboxylic acid chloride was dissolved in n-heptane to prepare a 0.15% w / v oil phase solution. 25 mL of the aqueous mixed solution was poured on the surface of the base membrane. After keeping it for 2 minutes, the excess aqueous solution was wiped off with industrial wipe paper, and then 25 mL of the oil phase solution was poured in to cause interfacial polymerization reaction. After reacting for 2 minutes, the excess oil phase solution was poured out. After washing twice with n-heptane solution, solutions of different pH values ​​were prepared with sodium hydroxide and hydrochloric acid, respectively. The prepared nanofiltration membrane was immersed in the solutions of different pH values ​​for 30 minutes. The prepared membrane was freeze-dried and stored for use.

[0038] The performance of the nanofiltration membrane prepared in Example 6 was tested. The water flux of the nanofiltration membrane after being immersed in a pH = 4.3 solution for half an hour was 47.6 L·m -2 ·h -1 bar -1 The soybean peptide retention rate was 97.1% and the NaCl permeability was 98.7%.

[0039] Embodiment 7: (1) At room temperature, add 2 mL of acetic acid to 100 mL of ultrapure water and stir magnetically for 2 min to mix evenly. Then weigh 0.7 g of chitosan (MW=100000) and slowly add it to the aqueous solution of acetic acid. Stir continuously for 1-2 h until fully dissolved. Then add 1.1 g of p-phenylenediamine and continue stirring until completely dissolved. Finally, add 1.2 mL of triethylamine and stir the mixed solution magnetically for 10-15 min to obtain an aqueous monomer mixed solution. Prepare 2 mol / L sodium hydroxide solution, soak the commercial PAN membrane in the prepared sodium hydroxide solution, keep it in a constant temperature water bath at 60 °C for 2 h, take out the membrane, wash it with pure water for 3 times, and then soak it in pure water for 24 h for use. (2) The aqueous solution prepared in (1) was stirred evenly and allowed to stand for 5 minutes for later use. The commercial membrane was taken out from the pure water and excess water was wiped off with industrial wipe paper. 1,3,5-benzenetricarboxylic acid chloride was dissolved in n-heptane to prepare a 0.2% w / v oil phase solution. 25 mL of the aqueous mixed solution was poured on the surface of the base membrane. After keeping it for 2 minutes, the excess aqueous solution was wiped off with industrial wipe paper, and then 25 mL of the oil phase solution was poured in to cause interfacial polymerization reaction. After reacting for 2 minutes, the excess oil phase solution was poured out. After washing twice with n-heptane solution, solutions of different pH values ​​were prepared with sodium hydroxide and hydrochloric acid, respectively. The prepared nanofiltration membrane was immersed in the solutions of different pH values ​​for 30 minutes. The prepared membrane was freeze-dried and stored for use.

[0040] The performance of the nanofiltration membrane prepared in Example 7 was tested. The water flux of the nanofiltration membrane after being immersed in a pH = 4.6 solution for half an hour was 42.3 L·m -2 ·h -1 bar -1 The soybean peptide retention rate was 95.6% and the NaCl permeability was 94.5%.

[0041] Embodiment 8: (1) At room temperature, add 2 mL of acetic acid to 100 mL of ultrapure water and stir magnetically for 2 min to mix evenly. Then weigh 2 g of chitosan (MW=50000) and slowly add it to the aqueous solution of acetic acid. Stir continuously for 1-2 h until fully dissolved. Then add 1.2 g of p-phenylenediamine and continue stirring until completely dissolved. Finally, add 1.3 mL of triethylamine and stir the mixed solution magnetically for 15 min to obtain an aqueous monomer mixed solution. Prepare a 2 mol / L sodium hydroxide solution and soak the commercial PAN membrane in the prepared sodium hydroxide solution. Keep it in a constant temperature water bath at 60 °C for 2 h. Take out the membrane and wash it with pure water for 3 times. Then soak it in pure water for 24 h for use. (2) The aqueous solution prepared in (1) was stirred evenly and allowed to stand for 5 minutes for later use. The commercial membrane was taken out from the pure water and excess water was wiped off with industrial wipe paper. 1,3,5-benzenetricarboxylic acid chloride was dissolved in n-heptane to prepare a 0.25% w / v oil phase solution. 25 mL of the aqueous mixed solution was poured on the surface of the base membrane. After keeping it for 2 minutes, the excess aqueous solution was wiped off with industrial wipe paper, and then 25 mL of the oil phase solution was poured in to cause interfacial polymerization reaction. After reacting for 2 minutes, the excess oil phase solution was poured out. After washing twice with n-heptane solution, different pH solutions were prepared with sodium hydroxide and hydrochloric acid, respectively. The prepared nanofiltration membrane was immersed in the solutions with different pH values ​​for 30 minutes. The prepared membrane was freeze-dried and stored for use.

[0042] The performance of the nanofiltration membrane prepared in Example 8 was tested. The water flux of the nanofiltration membrane after being immersed in a pH = 3.3 solution 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 permeability 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 process of the aqueous monomer mixed solution are composed of the following mass percentages: 0.5-1.5% surfactant, 0.05-0.2% chitosan, 0.5-2.5% diamine organic matter, and the remainder is ultrapure water. Prepare an acetic acid aqueous solution of a certain concentration, weigh a certain amount of chitosan powder, slowly add it to the acetic acid aqueous solution, and then stir magnetically for 1-2 hours. After the chitosan is completely dissolved, add the diamine organic matter, wait until it is completely dissolved, and then add the surfactant dropwise. Stir the mixed solution magnetically for 10-15 minutes to obtain an aqueous monomer mixed solution; Prepare 2 mol / L sodium hydroxide solution, soak the commercial membrane in the prepared sodium hydroxide solution, and keep it in a constant temperature water bath at 60 ℃ for 2 h. Take out the membrane and wash it with pure water for 3 times, and then soak it in pure water for 24 h for use. (2) Preparation of pH-responsive nanofiltration membrane The aqueous solution prepared in (1) was stirred evenly and then allowed to stand for 5 minutes for use. The commercial membrane was taken out from the pure water and excess water was wiped off with industrial wipe paper. 1,3,5-benzenetricarboxylic acid chloride was dissolved in an organic solvent to prepare an oil phase solution, and the mass fraction of 1,3,5-benzenetricarboxylic acid chloride in the oil phase solution was 0.15-0.3%. 25 mL of the aqueous solution was poured on the surface of the base membrane, and after keeping it for 1-3 minutes, the excess aqueous solution was wiped off with industrial wipe paper, and then 25 mL of the oil phase solution was poured in to cause interfacial polymerization reaction. After reacting for 1-2 minutes, the excess oil phase solution was poured out. After washing twice with organic solvent, the prepared nanofiltration membrane was immersed in solutions of different pH values ​​for 30 minutes, and the membrane was freeze-dried and stored for use.

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

3. The preparation method according to claim 1, characterized in that The molecular weight of chitosan in step (1) is one of 50000 Da, 100000 Da, and 200000 Da.

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

5. The preparation method according to claim 1, characterized in that The commercial membrane in step (1) is one of polyacrylonitrile, polyvinylidene fluoride, polyether sulfone and polysulfone.

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

7. A method for preparing a pH-responsive nanofiltration membrane according to any one of claims 1 to 5, wherein the prepared pH-responsive nanofiltration membrane is characterized in that: Under different pH environments, the pore size of the nanofiltration membrane changes. After being treated with a solution at pH=3.5 for 30 min, the pure water flux of the nanofiltration membrane is ≥30 L·m -2 ·h -1 bar -1 In the separation test of soybean peptide and NaCl, the soybean peptide retention rate was ≥95%, the NaCl permeability was ≥90%, and the membrane flux recovery rate was ≥95%, which had obvious anti-biological pollution effect.

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