Method for purifying pea albumin PA1a by using negatively charged polysaccharide
阅读说明：本技术 一种利用负电荷多糖纯化豌豆清蛋白PA1a的方法 (Method for purifying pea albumin PA1a by using negatively charged polysaccharide ) 是由 李兴飞 华欲飞 杨淑暖 陈业明 孔祥珍 张彩猛 于 2020-08-04 设计创作，主要内容包括：一种利用负电荷多糖纯化豌豆清蛋白PA1a的方法,属于蛋白质提取技术领域。本发明使用了天然可食性的负电荷多糖作为回收材料,利用负电荷多糖可以优先结合非目标蛋白沉淀,并与目标蛋白发生相分离的技术原理,实现豌豆清蛋白PA1a的分离纯化,制备单一的PA1a。从资源回收利用的角度来看,本发明可以实现高附加值利用豌豆分离蛋白副产物,并从中制备高纯度的高含硫活性蛋白组分,为后续的研究提供技术支持。本发明可以提高豌豆乳清的综合利用,PA1a蛋白回收率高,纯度高,且本发明对设备要求低,操作简单快速,无环境污染问题。(A method for purifying pea albumin PA1a by using negatively charged polysaccharide belongs to the technical field of protein extraction. The invention uses natural edible negative-charge polysaccharide as a recovery material, and utilizes the technical principle that the negative-charge polysaccharide can be preferentially combined with non-target protein precipitation and is separated from target protein, so as to realize the separation and purification of pea albumin PA1a and prepare single PA1 a. From the perspective of resource recycling, the method can realize the utilization of the pea protein isolate byproduct with high added value, and prepare high-purity high-sulfur active protein components from the pea protein isolate byproduct, thereby providing technical support for subsequent research. The invention can improve the comprehensive utilization of pea whey, has high PA1a protein recovery rate and high purity, and has low requirement on equipment, simple and quick operation and no environmental pollution.)
1. A method for purifying pea albumin PA1a by using negatively charged polysaccharide is characterized in that: the pea whey liquid is taken as a raw material, and natural edible negative charge polysaccharide is taken as a recovery material to be compounded; by utilizing the principle that negatively charged polysaccharide is preferentially combined with non-target protein precipitate and is separated from target protein, the non-target protein in pea whey is precipitated by adjusting complex coacervation conditions, so that the target protein is separated and purified, and the single pea albumin PA1a is obtained.
2. The method for purifying pea albumin PA1a by using negatively charged polysaccharide according to claim 1, wherein the steps are as follows:
(1) pretreatment of pea whey liquid: adjusting pH of pea whey to 4.0-5.0 with hydrochloric acid, and standing at 4-10 deg.C for 24-48 hr; centrifuging to remove precipitate, adding alkali liquor to adjust the pH of the supernatant to 7.0-8.0 to obtain pretreated pea whey, and measuring the protein content;
(2) PA2 and phytohemagglutinin in the negatively charged polysaccharide complex coacervation precipitated pea whey protein: taking solid polysaccharide sample powder, and preparing a polysaccharide solution with the mass concentration of 0.1-0.6%; adding polysaccharide solution with corresponding volume into the pea whey prepared in the step (1) according to the mass ratio of protein to polysaccharide of 10-5:1, adjusting the pH to 3.5-4.6 by using hydrochloric acid, stirring at room temperature for 15-30min, centrifuging at 4000-6000rpm for 10-20 min, obtaining a precipitate, namely a complex coacervation precipitate of polysaccharide-PA 2/phytohemagglutinin, and collecting supernatant to obtain crude purified protein liquid enriched with PA1 a;
(3) purifying PA1a pea albumin: regulating the pH value of the crude and pure PA1 a-enriched protein liquid obtained in the step (2) to 7.0-8.0, carrying out ultrafiltration by using ultrafiltration equipment with the molecular weight cutoff of 10-100 kDa to remove polysaccharide, centrifuging at 3500-;
(4) freeze-drying PA1a protein: and (3) freezing the PA1a protein liquid obtained in the step (3) at-30 ℃ for 24-48h, and carrying out vacuum freeze drying on the frozen sample at-40 ℃ to-60 ℃ under 5-10 Pa for 48-72 h to obtain PA1a pea albumin.
3. The method for purifying pea albumin PA1a by using negatively charged polysaccharide according to claim 2, wherein the step of purifying comprises the following steps: the pea whey liquid in the step (1) is derived from a by-product of pea protein isolate processing and supernatant liquid of pea vermicelli wastewater after pea protein isolate precipitation, and the protein content of the pea whey liquid is 0.1-0.6%.
4. The method for purifying pea albumin PA1a by using negatively charged polysaccharide according to claim 2, wherein the step of purifying comprises the following steps: the centrifugation conditions in step (1) are as follows: 4000-8000 rpm, 15-30 min.
5. The method for purifying pea albumin PA1a by using negatively charged polysaccharide according to claim 2, wherein the step of purifying comprises the following steps: the negative charge polysaccharide in the step (2) is specifically sulfuric acid group polysaccharide and carboxyl polysaccharide; the relative molecular weight range is greater than 100 kDa.
6. The method for purifying pea albumin PA1a by using negatively charged polysaccharide according to claim 5, wherein the step of purifying comprises the following steps: the sulfate group polysaccharide is dextran sulfate, carrageenan (kappa-, iota-, lambda), chondroitin sulfate or fucoidin; the carboxyl polysaccharide is carboxymethyl cellulose, sodium alginate, xanthan gum or pectin.
7. The method for purifying pea albumin PA1a by using negatively charged polysaccharide according to claim 2, wherein the step of purifying comprises the following steps: the mass concentration of the negatively charged polysaccharide in the step (2) is consistent with the mass concentration of the protein in the used pea whey liquid.
8. The method for purifying pea albumin PA1a by using negatively charged polysaccharide according to claim 2, wherein the step of purifying comprises the following steps: performing ultrafiltration by using ultrafiltration equipment with molecular weight cut-off of 10-100 kDa to remove polysaccharide in the step (3), and preferentially selecting an ultrafiltration membrane with high molecular weight cut-off; ultrafiltering and concentrating with ultrafiltration equipment with cut-off molecular weight of 1000-.
Technical Field
The invention relates to a method for purifying pea albumin PA1a by using negatively charged polysaccharide, belonging to the technical field of protein extraction.
Background
In recent years, plant proteins have attracted more and more interest, and the dietary structure of people is gradually shifted to sustainable plant components. Among them, peas are a good source of not only starch products but also protein products as sustainable crops. Pea protein is used as hypoallergenic protein, has no transgenic risk and has high nutritional value. Pea protein milk drinks, fermented milk and the like are receiving much attention in European and American countries and are becoming important substitutes for milk protein drinks. In China, the pea protein is low in utilization rate, and the pea protein is mostly utilized in pea protein isolate, so that pea whey protein accounting for 20% -30% of the content of the pea protein is wasted. The sulfur content of the pea whey protein is high, the sulfur content can account for more than two thirds of the sulfur-containing protein of the total peas, the waste of the pea whey protein not only causes the loss of a large amount of sulfur-containing protein, but also causes environmental problems due to the large amount of emission.
Pea whey is rich in a plurality of 2S and 7S whey protein components, mainly comprising PA2, PA1 and phytohemagglutinin, and has different physiological activities. PA1 is one of the major pea 2S whey proteins, has sulfur-containing amino acids accounting for about 50% of the total sulfur content in the seeds, and is composed of PA1a and PA1b, wherein PA1a has a molecular weight of about 6 kDa. PA1 has been shown to be an insect toxin bioactive peptide that has a toxic effect on a variety of insects. At present, PA1a is purified mainly by the method provided by John. A. GATEHOUSE and is obtained by eluting with Sephadex G-75 and DEAE-cellulose chromatographic columns. Although the traditional chromatographic column separation has high selectivity, the high price and the low separation speed thereof cause the high cost of the separation and purification of PA1 a. The polysaccharide/protein electrostatic action driven complex coacervation method can not only rapidly and efficiently prepare the PA1a, but also can obtain a large amount of purified PA1a at low cost.
Disclosure of Invention
The invention aims to overcome the defects and provides a method for purifying pea albumin PA1a by using negatively charged polysaccharide, which is rapid and efficient and has low cost.
According to the technical scheme, the method for purifying pea albumin PA1a by using negatively charged polysaccharide provided by the invention is characterized in that pea protein and bean vermicelli wastewater processing by-products pea whey are taken as raw materials, different types of negatively charged polysaccharide solutions with certain concentrations are selected as recovery materials, and the negatively charged polysaccharide solutions are added into the pea whey according to a certain mass ratio for selective complex coagulation; by utilizing the principle that negatively charged polysaccharide is preferentially combined with non-target protein precipitate and is separated from target protein, PA2 and phytohemagglutinin in pea whey protein are removed by precipitation by controlling the use amount of the polysaccharide and the pH of the solution, and supernatant containing PA1a is obtained; the single PA1a protein component is prepared by adjusting the pH value, ultrafiltering and centrifuging to remove residual polysaccharide and small molecular substances, and then the single pea albumin PA1a product with higher purity is obtained by freeze drying.
The flow chart of the invention is shown in figure 1, and the specific steps are as follows:
(1) pretreatment of pea whey liquid: adjusting pH of pea whey to 4.0-5.0 with hydrochloric acid, standing at 4-10 deg.C for 24-48h, centrifuging to remove precipitate, adding alkali solution to adjust pH of supernatant to 7.0-8.0 to obtain pretreated pea whey, and measuring protein content.
(2) PA2 and phytohemagglutinin in the negatively charged polysaccharide complex coacervation precipitated pea whey protein: taking solid polysaccharide sample powder, and preparing a polysaccharide solution with the mass concentration of 0.1-0.6%; adding polysaccharide solution with corresponding volume into the pea whey prepared in the step (1) according to the mass ratio of protein to polysaccharide of 10-5:1, adjusting the pH to 3.5-4.6 by using hydrochloric acid, stirring at room temperature for 15-30min, centrifuging at 4000-6000rpm for 10-20 min, and removing precipitates, wherein the precipitates are complex coacervation precipitates of polysaccharide-
(3) Purifying PA1a pea albumin: adjusting the pH of the crude and pure PA1 a-enriched protein liquid obtained in the step (2) to 7.0-8.0, carrying out ultrafiltration by an ultrafiltration device with the molecular weight cutoff of 10-100 kDa to remove polysaccharide, centrifuging at 3500-.
(4) Freeze-drying PA1a protein: and (3) freezing the PA1a protein liquid obtained in the step (3) at-30 ℃ for 24-48h, and carrying out vacuum freeze drying on the frozen sample at-40 ℃ to-60 ℃ under 5-10 Pa for 48-72 h to obtain PA1a pea albumin.
Furthermore, the pea milk clear liquid in the step (1) is derived from a by-product of pea protein isolate processing and a supernatant of pea vermicelli wastewater after pea protein isolate precipitation, and the protein content of the pea milk clear liquid is 0.1-0.6%.
Further, the centrifugation conditions in step (1) are: 4000-8000 rpm, 15-30 min.
Further, the negatively charged polysaccharides in the step (2) are sulfate group polysaccharides and carboxyl polysaccharides: sulfate group polysaccharide: including but not limited to dextran sulfate, carrageenan (kappa-, iota-, lambda), chondroitin sulfate, fucoidan; carboxyl polysaccharide: including but not limited to carboxymethyl cellulose, sodium alginate, xanthan gum, pectin; the relative molecular weight range is greater than 100 kDa.
Further, the mass concentration of the negatively charged polysaccharide in the step (2) is consistent with the mass concentration of the protein in the used pea whey; if the mass concentration of the polysaccharide changes, the volume of the polysaccharide solution needs to be adjusted to ensure that the mass ratio of the protein to the polysaccharide in the final mixed solution is kept unchanged.
Further, performing ultrafiltration to remove polysaccharide by using ultrafiltration equipment with molecular weight cut-off of 10-100 kDa in the step (3), and preferentially selecting an ultrafiltration membrane with high molecular weight cut-off; ultrafiltering and concentrating with ultrafiltration equipment with cut-off molecular weight of 1000-.
Further, the PA1a pea albumin prepared in the step (4) is subjected to polyacrylamide gel electrophoresis SDS-PAGE and gel filtration-high performance liquid chromatography SEC-HPLC to detect the purity of a sample, the protein content is determined by a BCA method, the total sugar content is determined by a phenol-sulfuric acid method GB/T15672-.
In the invention, because the isoelectric point of the pea whey protein is concentrated at about pH 4.5-4.8, when the pH of the mixed solution of the pea whey protein and the polysaccharide is adjusted to be below the isoelectric point of the protein, the negatively charged polysaccharide and the positively charged pea whey protein are interacted by electrostatic attraction to form insoluble complex coacervate, and the insoluble complex coacervate can be separated by centrifugation. Therefore, the invention controls the complex coacervation condition of the pea whey protein and the negatively charged polysaccharide, so that all the pea whey protein PA2 and the phytohemagglutinin and the negatively charged polysaccharide form insoluble complex coacervate, and the insoluble complex coacervate is precipitated by centrifugation, and only the target pea albumin PA1a is left in the supernatant.
The invention uses natural edible polysaccharide with negative charge as raw material for recovering and purifying protein, and utilizes the principle that non-target protein (
The invention has the beneficial effects that: the method has the advantages of simple operation flow, low equipment requirement and easy scale production; the polysaccharide dosage is less, and the polysaccharide can be recycled through membrane separation; the purity of the purified and recovered PA1a protein is over 90 percent, the recovery rate is over 80 percent, and the natural activity is still maintained.
Drawings
FIG. 1 is a flow chart of a separation and purification process of PA1 a.
FIG. 2 SEC-HPLC detection profile of PA1a protein purity.
FIG. 3 is an SDS-PAGE profile of the purity of PA1a protein.
Description of reference numerals:
Detailed Description