阅读说明：本技术 青稞蛋白质的分离纯化方法 (Separation and purification method of highland barley protein ) 是由 张春颖 于 2019-10-12 设计创作，主要内容包括：本发明公开了一种青稞蛋白质的分离纯化方法,其主要包括以下步骤：从青稞米中提取蛋白提取液；葡聚糖醛基化；醛基化的葡聚糖与聚烯丙胺反应制备微凝胶溶液；以异丙醇作为芯液,将制得的微凝胶溶液与芯液通过喷丝头共同挤出,浸入异丙醇凝固浴中进行非溶剂相转化取出干燥,获得中空纤维膜,将一束中空纤维膜制成中空纤维膜组件,利用中空纤维膜组件过滤,过滤完毕后用蒸馏水反向洗涤中空纤维膜组件,洗涤液离心,取沉淀真空冷冻干燥,得到纯化的青稞蛋白质。本发明所述青稞蛋白质的分离纯化方法,方便实用,适用性广,利用微凝胶溶液制成中空纤维膜,再加工成膜组件,对青稞蛋白质进行分离纯化,最终获得的蛋白质纯度高且提取率高。(The invention discloses a method for separating and purifying highland barley protein, which mainly comprises the following steps: extracting protein extract from highland barley; d, glucuronylation; reacting the aldehyde dextran with polyallylamine to prepare microgel solution; using isopropanol as core liquid, extruding the prepared microgel solution and the core liquid together through a spinning nozzle, immersing the extruded microgel solution and the core liquid into isopropanol coagulation bath for non-solvent phase conversion, taking out and drying to obtain a hollow fiber membrane, preparing a bundle of hollow fiber membranes into a hollow fiber membrane assembly, filtering by using the hollow fiber membrane assembly, reversely washing the hollow fiber membrane assembly by using distilled water after filtering, centrifuging washing liquid, taking precipitate, and freeze-drying in vacuum to obtain purified highland barley protein. The separation and purification method of the highland barley protein is convenient and practical, has wide applicability, utilizes the microgel solution to prepare the hollow fiber membrane, processes the hollow fiber membrane into the membrane forming assembly, and separates and purifies the highland barley protein, so that the finally obtained protein has high purity and high extraction rate.)

1. The method for separating and purifying the highland barley protein is characterized by mainly comprising the following steps:

step one, grinding cleaned and soaked highland barley, putting the highland barley into a high-pressure tank, pressurizing to 1.0-2.0 MPa, maintaining for 5-10 min after pressurization, instantly relieving pressure, filtering the mixture in the high-pressure tank, and taking filtrate to obtain protein extract;

Step two, taking glucan and KIO₄dissolving in water, mixing, and mixing the dextran and KIO₄The concentration of the solution is 0.3-0.5 mol/L, the solution is stirred for 10-12 h in a dark place at the temperature of 25 ℃, and anhydrous BaCl is added into the system₂Filtering, adding Na into the filtrate₂SO₄Filtering to obtain aldehyde-modified dextran solution, wherein the dextran, KIO₄，BaCl₂And Na₂SO₄The molar ratio of (a) to (b) is 10-12: 1:1: 1;

dissolving polyallylamine in water, adjusting the pH value to 9.5-10, slowly dripping the aldehyde dextran solution prepared in the third step while stirring, stirring for 2 hours, adding NaBH into the system₄reacting for 8-10 h to obtain a microgel solution, wherein the polyallylamine and the NaBH are₄the molar ratio of the glucan to the glucan in the step two is 1.5:1.5: 1;

Step four, taking isopropanol as core liquid, extruding the microgel solution prepared in the step three and the core liquid together through a spinning nozzle, immersing the extruded core liquid into isopropanol coagulation bath for non-solvent phase conversion for 2-4 h, taking out and drying to obtain a hollow fiber membrane, wherein the inner diameter of the hollow fiber membrane is 0.5-0.8 mm, and the outer diameter of the hollow fiber membrane is 1-1.5 mm;

Step five, putting a bundle of hollow fiber membrane into a cylindrical shell made of ABS material, sealing one end of the fiber bundle, bonding the other end of the fiber bundle together by epoxy resin to prepare a hollow fiber membrane component, filtering by using the hollow fiber membrane component, mixing the protein extract obtained in the step one with water, permeating the hollow fiber membrane under pressure, allowing the permeated liquid to flow out of a membrane core, reversely washing the hollow fiber membrane component by using distilled water after filtering, centrifuging the washing liquid, taking the precipitate, and freeze-drying in vacuum to obtain the purified protein.

2. The method for separating and purifying highland barley protein as claimed in claim 1, wherein the aperture of the outer surface of the hollow fiber membrane prepared in the fourth step is 0.1-0.5 μm.

3. The method for separating and purifying highland barley proteins as claimed in claim 1, wherein in the first step, the pressure in the high pressure tank is increased to 1.5MPa and the pressure is maintained for 10 min.

4. The method for separating and purifying highland barley protein as claimed in claim 1, wherein the glucan and KIO mixed in the second step₄The concentration of the solution is 0.5mol/L, the stirring time is 12h, and the glucan and the KIO in the system₄，BaCl₂And Na₂SO₄In a molar ratio of 12:1:1: 1.

5. The method for separating and purifying highland barley protein as claimed in claim 1, wherein the concentration of polyallylamine solution in the third step is 0.2mol/L, pH is 9.8, and NaBH is added into the system₄The post-reaction time was 10 h.

6. The method for separating and purifying highland barley protein as claimed in claim 1, wherein the time of phase inversion of the non-solvent in the fourth step is 2 hours, and the inner diameter of the hollow fiber membrane is 0.8mm and the outer diameter is 1.5 mm.

7. The method for separating and purifying highland barley protein as claimed in claim 1, wherein in the fourth step, the extruding speed of the microgel solution and the core liquid through the spinneret is 0.05 m/s.

8. The method for separating and purifying highland barley protein as claimed in claim 1, wherein the length of the fiber bundle in the fifth step is 30 cm.

9. The method for separating and purifying highland barley proteins as claimed in claim 1, wherein in the fifth step, the pressure is increased to 0.5MPa when the filtration is carried out by using a hollow fiber membrane module.

Technical Field

The present invention relates to a method for separating and purifying proteins. More specifically, the invention relates to a method for separating and purifying highland barley protein.

Background

The highland barley is used as the first grain crop in Tibetan regions in China, has high yield, rich and complete nutrition and has the characteristics of high protein, high fiber, high vitamin, low fat, low sugar and the like, the protein is separated and extracted from the highland barley, the protein source can be widened, the problem of residual local highland barley can be solved, and the advantage of local resources is converted into economic advantage.

at present, there are many methods for extracting plant proteins at home and abroad, and the methods mainly include a solution extraction method, a physical extraction method, an ion exchange method, an ultrafiltration method, a biological enzyme method and the like. In the field of vegetable protein research, research documents on functional properties of soybean protein isolate, rapeseed protein isolate, peanut protein isolate and the like are relatively extensive, while highland barley is used as an excellent vegetable protein resource, and domestic research on highland barley protein extraction processes mainly focuses on alkaline extraction and enzymatic extraction due to the limitation of planting areas. However, the traditional highland barley protein extraction method has the advantages of small treatment capacity, high energy consumption and low protein extraction rate, and strong alkali or excessive enzymolysis easily causes the weakening and even complete loss of certain functional characteristics of the protein, so that the technical level of the process still needs to be further improved in the highland barley protein extraction process.

Disclosure of Invention

an object of the present invention is to solve at least the above problems and to provide at least the advantages described later.

the invention also aims to provide a method for separating and purifying the highland barley protein, which has large treatment capacity and low energy consumption, and the hollow fiber membrane prepared from the microgel solution is used for filtering the protein extract and then reversely washing to obtain the purified highland barley protein, so that the protein keeps the original functional activity, and the finally obtained protein has high purity and high extraction rate.

In order to achieve these objects and other advantages in accordance with the present invention, there is provided a method for separating and purifying highland barley protein, which comprises the following steps:

step one, grinding cleaned and soaked highland barley, putting the highland barley into a high-pressure tank, pressurizing to 1.0-2.0 MPa, maintaining for 5-10 min after pressurization, instantly relieving pressure, filtering the mixture in the high-pressure tank, and taking filtrate to obtain protein extract;

Step two, taking glucan and KIO₄dissolving in water, mixing, and mixing the dextran and KIO₄the concentration of the solution is 0.3-0.5 mol/L, the solution is stirred for 10-12 h in a dark place at the temperature of 25 ℃, and anhydrous BaCl is added into the system₂Filtering, adding Na into the filtrate₂SO₄Filtering to obtain aldehyde-modified dextran solution, wherein the dextran, KIO₄，BaCl₂And Na₂SO₄the molar ratio of (a) to (b) is 10-12: 1:1: 1;

dissolving polyallylamine in water, adjusting the pH value to 9.5-10, slowly dripping the aldehyde dextran solution prepared in the third step while stirring, stirring for 2 hours, adding NaBH into the system₄Reacting for 8-10 h to obtain a microgel solution, wherein the polyallylamine and the NaBH are₄The molar ratio of the glucan to the glucan in the step two is 1.5:1.5: 1;

step four, taking isopropanol as core liquid, extruding the microgel solution prepared in the step three and the core liquid together through a spinning nozzle, immersing the extruded core liquid into isopropanol coagulation bath for non-solvent phase conversion for 2-4 h, taking out and drying to obtain a hollow fiber membrane, wherein the inner diameter of the hollow fiber membrane is 0.5-0.8 mm, and the outer diameter of the hollow fiber membrane is 1-1.5 mm;

step five, putting a bundle of hollow fiber membrane into a cylindrical shell made of ABS material, sealing one end of the fiber bundle, bonding the other end of the fiber bundle together by epoxy resin to prepare a hollow fiber membrane component, filtering by using the hollow fiber membrane component, mixing the protein extract obtained in the step one with water, permeating the hollow fiber membrane under pressure, allowing the permeated liquid to flow out of a membrane core, reversely washing the hollow fiber membrane component by using distilled water after filtering, centrifuging the washing liquid, taking the precipitate, and freeze-drying in vacuum to obtain the purified protein.

preferably, the pore diameter of the outer surface of the hollow fiber membrane prepared in the fourth step is 0.1-0.5 μm.

Preferably, in the first step, the pressure in the high-pressure tank is increased to 1.5MPa, and the pressure is maintained for 10 min.

Preferably, the glucan and the KIO mixed in the second step₄The concentration of the solution is 0.5mol/L, the stirring time is 12h, and the glucan and the KIO in the system₄，BaCl₂and Na₂SO₄in a molar ratio of 12:1:1: 1.

Preferably, the concentration of the polyallylamine solution in the step three is 0.2mol/L, the pH value is 9.8, and NaBH is added into the system₄The post-reaction time was 10 h.

Preferably, the phase inversion time of the non-solvent in the fourth step is 2h, and the inner diameter of the hollow fiber membrane is 0.8mm and the outer diameter is 1.5 mm.

Preferably, in the fourth step, the extruding speed of the microgel solution and the core liquid through the spinneret is 0.05 m/s.

Preferably, the length of the fiber bundle in the fifth step is 30 cm.

Preferably, in the fifth step, the pressure is increased to 0.5MPa during filtration using the hollow fiber membrane module.

The invention at least comprises the following beneficial effects:

Firstly, the instant blasting technology is utilized, the cleaned highland barley is placed in high pressure, and the pressure is instantaneously relieved, so that the cell breakage rate is improved, and the processing time is short;

Secondly, oxidizing glucan hydroxyl into aldehyde group, then utilizing the reaction of the amine group and the aldehyde group of polyallylamine to crosslink the glucan and the aldehyde group, finally adding sodium borohydride for reduction to obtain stable microgel, processing the prepared microgel into a hollow fiber membrane, and then preparing a membrane forming component; the membrane prepared from the microgel has a uniform three-dimensional network-shaped framework, a uniform membrane structure, and a pore diameter of the outer surface of the membrane controlled to be 0.1-0.5 mu m, can well retain highland barley protein, and finally obtains high-purity protein and high-extraction rate.

Thirdly, compared with the traditional highland barley protein extraction method, the method has the advantages of large treatment capacity and low energy consumption, the protein is purified by utilizing the hollow fiber membrane, and the original functional activity of the protein is kept because no enzyme or strong alkali is added in the separation process.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.

Detailed Description

The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.

it will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.

It is to be noted that the experimental methods described in the following embodiments are all conventional methods unless otherwise specified, and the reagents and materials are commercially available unless otherwise specified.

< example 1>

Step one, grinding cleaned and soaked highland barley, putting the highland barley into a high-pressure tank, pressurizing to 1.5MPa, maintaining for 10min after pressurization, instantly relieving pressure, filtering the mixture in the high-pressure tank, and taking filtrate to obtain protein extract;

Step two, taking glucan and KIO₄Dissolving in water, mixing, and mixing the dextran and KIO₄The concentration of the solution is 0.5mol/L, the solution is stirred for 12 hours in the dark at the temperature of 25 ℃, and anhydrous BaCl is added into the system₂Filtering, adding Na into the filtrate₂SO₄Filtering to obtain aldehyde-modified dextran solution, wherein the dextran, KIO₄，BaCl₂And Na₂SO₄In a molar ratio of 12:1:1: 1;

Dissolving polyallylamine in water, adjusting the pH value to 9.8, slowly dripping the aldehyde dextran solution prepared in the step three while stirring, stirring for 2 hours, adding NaBH into the system₄Reacting for 10h to obtain a microgel solution, wherein the polyallylamine and the NaBH are reacted₄The molar ratio of the glucan to the glucan in the step two is 1.5:1.5: 1;

step four, using isopropanol as core liquid, extruding the microgel solution prepared in the step three and the core liquid together through a spinning nozzle at the extrusion speed of 0.05m/s, immersing the microgel solution and the core liquid into isopropanol coagulation bath for non-solvent phase conversion for 2 hours, taking out and drying to obtain a hollow fiber membrane, wherein the inner diameter of the hollow fiber membrane is 0.8mm, the outer diameter of the hollow fiber membrane is 1.5mm, and the outer surface aperture of the hollow fiber membrane is 0.2 mu m;

step five, putting a bundle of hollow fiber membrane with the length of 30cm into a cylindrical shell made of ABS material, sealing one end of the fiber bundle, bonding the other end of the fiber bundle together by epoxy resin to prepare a hollow fiber membrane component, filtering by using the hollow fiber membrane component, mixing the protein extracting solution obtained in the step one with water, enabling the mixed solution of the protein extracting solution to permeate the hollow fiber membrane under the pressure of 0.5MPa, enabling the permeate to flow out of the membrane core, reversely washing the hollow fiber membrane component by using distilled water after the filtration is finished, centrifuging the washing solution, taking the precipitate, and freeze-drying in vacuum to obtain the purified protein.

< example 2>

Step one, grinding cleaned and soaked highland barley, putting the highland barley into a high-pressure tank, pressurizing to 2MPa, maintaining for 5min after pressurization, instantly relieving pressure, filtering the mixture in the high-pressure tank, and taking filtrate to obtain protein extract;

step two, taking glucan and KIO₄Dissolving in water, mixing, and mixing the dextran and KIO₄the concentration of the solution is 0.3mol/L, the solution is stirred for 10 hours in the dark at the temperature of 25 ℃, and anhydrous BaCl is added into the system₂Filtering, adding Na into the filtrate₂SO₄Filtering to obtain aldehyde-modified dextran solution, wherein the dextran, KIO₄，BaCl₂And Na₂SO₄In a molar ratio of 10:1:1: 1;

Dissolving polyallylamine in water, adjusting the pH value to 9.8, slowly dripping the aldehyde dextran solution prepared in the step three while stirring, stirring for 2 hours, adding NaBH into the system₄Reacting for 8h to obtain a microgel solution, wherein the polyallylamine and the NaBH are reacted₄the molar ratio of the glucan to the glucan in the step two is 1.5:1.5: 1;

step four, using isopropanol as core liquid, extruding the microgel solution prepared in the step three and the core liquid together through a spinning nozzle at the extrusion speed of 0.05m/s, immersing the microgel solution and the core liquid into isopropanol coagulation bath for non-solvent phase conversion for 4 hours, taking out and drying to obtain a hollow fiber membrane, wherein the inner diameter of the hollow fiber membrane is 0.5mm, the outer diameter of the hollow fiber membrane is 1.0mm, and the outer surface aperture of the hollow fiber membrane is 0.4 mu m;

step five, putting a bundle of hollow fiber membrane with the length of 30cm into a cylindrical shell made of ABS material, sealing one end of the fiber bundle, bonding the other end of the fiber bundle together by epoxy resin to prepare a hollow fiber membrane component, filtering by using the hollow fiber membrane component, mixing the protein extracting solution obtained in the step one with water, enabling the mixed solution of the protein extracting solution to permeate the hollow fiber membrane under the pressure of 0.5MPa, enabling the permeate to flow out of the membrane core, reversely washing the hollow fiber membrane component by using distilled water after the filtration is finished, centrifuging the washing solution, taking the precipitate, and freeze-drying in vacuum to obtain the purified protein.

< example 3>

Step one, grinding cleaned and soaked highland barley, putting the highland barley into a high-pressure tank, pressurizing to 1.0MPa, maintaining for 10min after pressurization, instantly relieving pressure, filtering the mixture in the high-pressure tank, and taking filtrate to obtain protein extract;

Step two, taking glucan and KIO₄Dissolving in water, mixing, and mixing the dextran and KIO₄The concentration of the solution is 0.4mol/L, the solution is stirred for 11 hours in the dark at the temperature of 25 ℃, and anhydrous BaCl is added into the system₂Filtering, adding Na into the filtrate₂SO₄Filtering to obtain aldehyde-modified dextran solution, wherein the dextran, KIO₄，BaCl₂And Na₂SO₄In a molar ratio of 11:1:1: 1;

Dissolving polyallylamine in water, adjusting the pH value to 9.8, slowly dripping the aldehyde dextran solution prepared in the step three while stirring, stirring for 2 hours, adding NaBH into the system₄reacting for 9h to obtain a microgel solution, wherein the polyallylamine and the NaBH are reacted₄The molar ratio of the glucan to the glucan in the step two is 1.5:1.5: 1;

Step four, using isopropanol as core liquid, extruding the microgel solution prepared in the step three and the core liquid together through a spinning nozzle at the extrusion speed of 0.05m/s, immersing the microgel solution and the core liquid into isopropanol coagulation bath for non-solvent phase conversion for 3h, taking out and drying to obtain a hollow fiber membrane, wherein the inner diameter of the hollow fiber membrane is 0.6mm, the outer diameter of the hollow fiber membrane is 1.3mm, and the outer surface aperture of the hollow fiber membrane is 0.1 mu m;

Step five, putting a bundle of hollow fiber membrane with the length of 30cm into a cylindrical shell made of ABS material, sealing one end of the fiber bundle, bonding the other end of the fiber bundle together by epoxy resin to prepare a hollow fiber membrane component, filtering by using the hollow fiber membrane component, mixing the protein extracting solution obtained in the step one with water, enabling the mixed solution of the protein extracting solution to permeate the hollow fiber membrane under the pressure of 0.5MPa, enabling the permeate to flow out of the membrane core, reversely washing the hollow fiber membrane component by using distilled water after the filtration is finished, centrifuging the washing solution, taking the precipitate, and freeze-drying in vacuum to obtain the purified protein.

< comparative example 1>

Step one, grinding cleaned and soaked highland barley, putting the highland barley into a high-pressure tank, pressurizing to 1.5MPa, maintaining for 10min after pressurization, instantly relieving pressure, filtering the mixture in the high-pressure tank, and taking filtrate to obtain protein extract;

Step two, taking glucan and KIO₄Dissolving in water, mixing, and mixing the dextran and KIO₄the concentration of the solution is 0.5mol/L, the solution is stirred for 12 hours in the dark at the temperature of 25 ℃, and anhydrous BaCl is added into the system₂Filtering, adding Na into the filtrate₂SO₄filtering to obtain aldehyde-modified dextran solution, wherein the dextran, KIO₄，BaCl₂And Na₂SO₄in a molar ratio of 12:1:1: 1;

Dissolving polyallylamine in water, adjusting the pH value to 9.8, slowly dripping the aldehyde dextran solution prepared in the step three while stirring, stirring for 2 hours, adding NaBH into the system₄Reacting for 14h to obtain a microgel solution, wherein the polyallylamine and the NaBH are reacted₄The molar ratio of the glucan to the glucan in the step two is 1.5:1.5: 1;

Step four, using isopropanol as core liquid, extruding the microgel solution prepared in the step three and the core liquid together through a spinning nozzle at the extrusion speed of 0.1m/s, immersing the microgel solution and the core liquid into isopropanol coagulation bath for non-solvent phase conversion for 5 hours, taking out and drying to obtain a hollow fiber membrane, wherein the inner diameter of the hollow fiber membrane is 0.8mm, the outer diameter of the hollow fiber membrane is 1mm, and the outer surface aperture of the hollow fiber membrane is 1 mu m;

Step five, putting a bundle of hollow fiber membrane with the length of 30cm into a cylindrical shell made of ABS material, sealing one end of the fiber bundle, bonding the other end of the fiber bundle together by epoxy resin to prepare a hollow fiber membrane component, filtering by using the hollow fiber membrane component, mixing the protein extracting solution obtained in the step one with water, enabling the mixed solution of the protein extracting solution to permeate the hollow fiber membrane under the pressure of 0.5MPa, enabling the permeate to flow out of the membrane core, reversely washing the hollow fiber membrane component by using distilled water after the filtration is finished, centrifuging the washing solution, taking the precipitate, and freeze-drying in vacuum to obtain the purified protein.

< comparative example 2>

Step one, grinding cleaned and soaked highland barley, putting the highland barley into a high-pressure tank, pressurizing to 1.5MPa, maintaining for 10min after pressurization, instantly relieving pressure, filtering the mixture in the high-pressure tank, and taking filtrate to obtain protein extract;

Step two, taking glucan and KIO₄Dissolving in water, mixing, and mixing the dextran and KIO₄The concentration of the solution is 0.5mol/L, the solution is stirred for 12 hours in the dark at the temperature of 25 ℃, and anhydrous BaCl is added into the system₂filtering, adding Na into the filtrate₂SO₄Filtering to obtain aldehyde-modified dextran solution, wherein the dextran, KIO₄，BaCl₂And Na₂SO₄In a molar ratio of 12:1:1: 1;

Dissolving polyallylamine in water, adjusting the pH value to 9.8, slowly dripping the aldehyde dextran solution prepared in the step three while stirring, stirring for 2 hours, adding NaBH into the system₄reacting for 10h to obtain a microgel solution, wherein the polyallylamine and the NaBH are reacted₄The molar ratio of the glucan to the glucan in the step two is 1.5:1.5: 1;

Fourthly, placing the microgel solution prepared in the third step on non-woven fabric, blade-coating the non-woven fabric into a flat membrane, then placing the non-woven fabric with the flat membrane in an isopropanol coagulating bath for non-solvent phase conversion for 4 hours, taking out and drying to obtain a composite flat membrane, wherein the thickness of the composite flat membrane is 0.5mm, and the average pore diameter is 0.3 micron;

And step five, filtering the protein extracting solution by using the composite flat membrane prepared in the step four under 0.5MPa, washing the surface of the composite flat membrane after filtering is finished, collecting washing liquid, centrifuging, taking the precipitate, and drying in vacuum to obtain the purified protein.

< comparative example 3>

The traditional enzymolysis method is adopted to extract and purify the highland barley protein, and the specific steps are as follows:

Soaking semen Avenae Nudae in distilled water, grinding, homogenizing, adding cellulase at an amount of 15EGU/g, performing enzymolysis for 4 hr at 45 deg.C and pH of 7.0, and purifying.

< extraction yield and purity test >

Taking the same amount of minced highland barley as a sample, extracting and purifying the sample by the methods of the examples and the comparative examples respectively to prepare corresponding products, and then calculating the extraction rate and the purity of the protein according to the following formula, wherein the quality of the protein is determined by adopting a micro Kjeldahl method.

Protein extraction (%) × 100% (mass of protein in product/mass of protein in sample);

Protein purity (%) × 100% (mass of protein in product/mass of product).

The specific extraction rate and purity test results are shown in table 1, and the test results show that the average extraction rate of the highland barley protein product prepared by the method is 78.38%, the average purity of the product reaches 92.55%, and compared with comparative examples 1-3, the extraction rate and purity of the protein are obviously improved.

TABLE 1

< film porosity test >

The porosity of each of the hollow fiber membranes and the composite flat sheet membranes obtained in examples 1, 2 and 3 and comparative examples 1 and 2 was measured by mercury intrusion method, and the measurement results are shown in table 2:

TABLE 2

< Membrane flux test >

Pure water flux was measured at a pressure of 0.5MPa for each of the hollow fiber membranes and the composite flat sheet membranes obtained in the above examples 1, 2, 3 and comparative examples 1, 2, and the test results are shown in table 3:

TABLE 3

The experimental results show that in examples 1, 2 and 3, the microgel is dissolved to prepare the hollow fiber membrane, and the method is used for separating and purifying the protein, the hollow fiber membrane is prepared by using the microgel, the porosity of the membrane is high, the flux is large, the pore diameter of the outer surface of the membrane is controlled to be 0.1-0.5 mu m, the protein in the highland barley can be well intercepted, the finally obtained protein is high in purity and extraction rate, in addition, the hollow fiber membrane is used for purifying the protein, strong acid and strong base are not added in the separation process, so that the protein keeps the original functional activity, the treatment capacity is large, the energy consumption is low, and the processing process is energy-saving and environment-friendly. In comparative example 1, the microgel solution is placed for too long time, the extrusion speed of the hollow fiber membrane is high, so that the inner diameter of the prepared hollow fiber membrane is small, the pore diameter of the surface of the membrane is increased, the membrane flux is maximum, but the porosity of the membrane is low, so that the retention rate of protein is reduced, and the final extraction rate is low. Comparative example 2 compared with example 1, the final extraction rate and purity were lower than those of examples 1, 2 and 3 when the hollow fiber membrane was replaced with the composite flat membrane and the protein extract was separated, mainly because the final purity of the protein was lower due to the limited contact area between the flat membrane and the protein extract during the separation process, the lower separation efficiency compared with the hollow fiber membrane, the lower flux of the membrane, the easy blockage of the membrane during the treatment process, and the influence on the retention of the protein. Comparative example 3 the extraction by the traditional enzymolysis method, the extraction rate of the final protein is greatly influenced by the enzymolysis temperature and pH, and the extraction rate is lower than that of the separation and purification method of the invention.

The number of apparatuses and the scale of the process described herein are intended to simplify the description of the present invention. Applications, modifications and variations of the present invention will be apparent to those skilled in the art.

while embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable to various fields of endeavor for which the invention may be embodied with additional modifications as would be readily apparent to those skilled in the art, and the invention is therefore not limited to the details given herein and to the examples shown and described without departing from the generic concept as defined by the claims and their equivalents.