WO2022028221A1 - 一种利用负电荷多糖纯化豌豆清蛋白PA1a的方法 - Google Patents

一种利用负电荷多糖纯化豌豆清蛋白PA1a的方法 Download PDF

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WO2022028221A1
WO2022028221A1 PCT/CN2021/106242 CN2021106242W WO2022028221A1 WO 2022028221 A1 WO2022028221 A1 WO 2022028221A1 CN 2021106242 W CN2021106242 W CN 2021106242W WO 2022028221 A1 WO2022028221 A1 WO 2022028221A1
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pa1a
pea
protein
negatively charged
polysaccharide
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李兴飞
华欲飞
龙杰
张彩猛
杨淑暖
陈业明
孔祥珍
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Jiangnan University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/30—Extraction; Separation; Purification by precipitation

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  • the invention relates to a method for purifying pea albumin PA1a by utilizing negatively charged polysaccharide, and belongs to the technical field of protein extraction.
  • peas are not only a good source of starch products, but also a good source of protein products.
  • pea protein has no risk of genetic modification and has high nutritional value.
  • pea protein milk drinks and fermented milk have attracted much attention, and have increasingly become important substitutes for milk protein drinks.
  • the utilization rate of pea protein is not only low, but the utilization of pea protein is mostly concentrated in pea protein isolate, resulting in waste of pea whey protein, which accounts for 20%-30% of pea protein content.
  • the sulfur content of pea whey protein is high, and the sulfur content can account for more than two-thirds of the total pea sulfur-containing protein.
  • the waste of pea whey protein not only causes a large loss of sulfur-containing protein, but also causes environmental problems.
  • Pea whey is rich in a variety of 2S and 7S whey protein components, mainly including PA2, PA1 and lectins, all of which have different physiological activities.
  • PA1 is one of the main pea 2S whey proteins, and its sulfur-containing amino acids account for about 50% of the total sulfur content in seeds. It is composed of PA1a and PA1b, and PA1a has a molecular weight of about 6kDa.
  • PA1 has been confirmed to be an insect toxin bioactive peptide with toxic effects on a variety of insects.
  • the purification of PA1a is mainly provided by John.A.GATEHOUSE, and it is obtained by Sephadex G-75 and DEAE-cellulose chromatographic column.
  • the purpose of the present invention is to overcome the above deficiencies, and to provide a method for purifying pea albumin PA1a using negatively charged polysaccharides, which is fast, efficient, and low-cost.
  • the technical solution of the present invention provides a method for purifying pea albumin PA1a by using negatively charged polysaccharides, using pea protein and pea whey, a by-product of vermicelli wastewater processing, as raw materials, and selecting different types of negatively charged polysaccharide solutions of a certain concentration.
  • a recycled material As a recycled material, it is added to pea whey according to a certain amount of material for selective complex coagulation; using the principle that negatively charged polysaccharides preferentially bind to non-target proteins to precipitate and separate from the target proteins, by controlling the amount of polysaccharides and the pH of the solution , to remove PA2 and lectin from pea whey protein by precipitation to obtain a supernatant containing PA1a; by adjusting pH, ultrafiltration and centrifugation to remove residual polysaccharides and small molecular substances, a single PA1a protein fraction was prepared, and then frozen The single pea albumin PA1a product with higher purity is obtained by drying.
  • Pretreatment of pea whey liquid adjust the pH of pea whey liquid to 4.0-5.0 with hydrochloric acid, let stand at 4-10°C for 24-48h, remove the precipitate by centrifugation, and add lye to adjust the pH of the supernatant to 4.0-5.0. 7.0-8.0, obtain the pretreated pea whey liquid, and measure the protein content.
  • Negatively charged polysaccharide complex coagulation to precipitate PA2 and lectin in pea whey protein take the solid polysaccharide sample powder, prepare a polysaccharide solution with a mass concentration of 0.1%-0.6%; according to the protein:polysaccharide mass ratio of 10-5:1 A corresponding volume of polysaccharide solution was added to the pea whey liquid prepared in step (1), and the pH was adjusted to 3.5-4.6 with hydrochloric acid, stirred at room temperature for 15-30min, centrifuged at 4000-6000rpm for 10-20min, and removed the precipitation, the precipitation was the polysaccharide- Complex precipitates of PA2/phytohemagglutinin. The remaining supernatant was collected to obtain the crude purified protein solution enriched in PA1a.
  • PA1a pea albumin the crude purified protein solution enriched with PA1a obtained in step (2) was adjusted to pH 7.0-8.0, and the polysaccharide was removed by ultrafiltration with an ultrafiltration device with a molecular weight cut-off of 10-100 kDa, 3500 Centrifuge at -5000rpm for 15-30min, repeat the ultrafiltration 1-2 times, collect the permeate, and then use an ultrafiltration device with a molecular weight cut-off of 1000-3500Da to carry out ultrafiltration and concentration to remove small molecular substances, and collect the retentate to obtain purified PA1a protein solution.
  • the pea whey liquid described in step (1) is derived from the by-product of pea protein isolate processing and the supernatant after pea vermicelli wastewater is precipitated by pea protein isolate, and its protein content is 0.1%-0.6%.
  • centrifugation conditions in step (1) are: 4000-8000rpm, 15-30min.
  • the negatively charged polysaccharides described in step (2) are sulfated polysaccharides and carboxyl polysaccharides: sulfated polysaccharides: including but not limited to dextran sulfate, carrageenan ( ⁇ -, ⁇ -, ⁇ ), sulfated cartilage Fucoidan, fucoidan; carboxypolysaccharide: including but not limited to carboxymethyl cellulose, sodium alginate, xanthan gum, pectin; its relative molecular weight range is greater than 100kDa.
  • the mass concentration of the negatively charged polysaccharide described in step (2) is consistent with the protein mass concentration in the pea whey liquid used; if the polysaccharide mass concentration changes, the volume of the polysaccharide solution needs to be adjusted to ensure that the protein in the final mixed solution is The polysaccharide mass ratio remained unchanged.
  • the polysaccharide is removed by ultrafiltration with an ultrafiltration device with a molecular weight cut-off of 10-100kDa, preferably an ultrafiltration membrane with a high molecular weight cut-off; ultrafiltration equipment with a molecular weight cut-off of 1000-3500Da is used for ultrafiltration. Filtration and concentration to remove small molecular substances, preferably ultrafiltration membranes with low molecular weight cut-off.
  • the prepared PA1a pea albumin described in step (4) is subjected to polyacrylamide gel electrophoresis SDS-PAGE and gel filtration-high performance liquid chromatography SEC-HPLC to detect the purity of the sample, and the protein content is determined by the BCA method, The total sugar content was determined by the phenol-sulfuric acid method GB/T 15672-2009, the moisture content was determined by the drying method GB/T 5497-1985, and the ash content was determined by the burning gravimetric method.
  • the isoelectric point of pea whey protein is concentrated at about pH 4.5-4.8, when the pH of the mixed solution of pea whey protein and polysaccharide is adjusted to be below the isoelectric point of protein, the negatively charged polysaccharide will interact with the charged polysaccharide. Positively charged pea whey proteins interact via electrostatic attraction to form insoluble complex aggregates that can be separated by centrifugation. Therefore, in the present invention, by controlling the complex coagulation conditions of pea whey protein and negatively charged polysaccharides, all the pea whey protein PA2 and lectin and negatively charged polysaccharides form insoluble complex aggregates, which are precipitated by centrifugation to promote the supernatant. Only the target pea albumin PA1a remained.
  • the present invention uses natural edible negatively charged polysaccharides as raw materials for recovering and purifying proteins, utilizes non-target proteins (PA2 and lectin) and polysaccharides to produce complex coagulation precipitation under the condition of carrying opposite charges, and the target protein PA1a does not participate in the formation of complex coagulation According to the principle of the compound, a single purified PA1a protein can be obtained. In addition, the prepared complex aggregate can be directly utilized as a food material. From the perspective of resource recovery and utilization, the present invention can not only prepare high-purity pea albumin component-PA1a with low loss and high selectivity, but also recover other protein components in pea whey, providing technology for subsequent development and research. support.
  • the beneficial effects of the invention are as follows: the operation process of the invention is simple, the equipment requirements are low, and the scale is easy; the amount of polysaccharide is small, and it can be recycled through membrane separation; retains its natural activity.
  • FIG. 1 Process flow diagram of separation and purification of PA1a.
  • lane 1 represents marker
  • lane 2 represents purified PA1a
  • lane 3 represents pea whey protein fraction.
  • Pretreatment of pea whey adjust the pH to 4.5 with 2mol/L hydrochloric acid pea whey, stand at 4°C for 48h, centrifuge at 8000rpm for 20min, remove the precipitation, add 1mol/L NaOH solution to adjust the supernatant The pH of the liquid was adjusted to 7.0 to obtain a pretreated pea whey liquid.
  • Negatively charged polysaccharide complex coagulation and precipitation of PA2 and lectin in pea whey protein Weigh 400 mg of carboxymethyl cellulose powder (molecular weight 250 kDa), dissolve in 100 mL of deionized water, and magnetically stir for 2 hours to completely dissolve. Add 20% (v/v) carboxymethyl cellulose solution to the pea whey, mix well, adjust the pH to 4.12, stir magnetically for 15 min, centrifuge (4000 rpm, 20 min), remove the complex aggregates, and collect The supernatant is the crude protein extract of PA1a and carboxymethyl cellulose.
  • PA1a pea albumin adjust the pH of the above solution to 7.0, ultrafiltration (100kDa) centrifugation for 30min to remove polysaccharides, repeat ultrafiltration 1 time, combine the permeate, use ultrafiltration with a molecular weight cut-off of 3500Da to centrifuge for 30min to remove small molecules Substances were collected, and the retentate was collected to obtain the purified PA1a protein solution.
  • the protein content of the prepared PA1a was determined, the protein purity was detected by SEC-HPLC, and analyzed by SDS-PAGE electrophoresis.
  • the SEC-HPLC detection chromatogram of the protein purity of PA1a is shown in Figure 2. As can be seen from the figure, the protein purity of PA1a is 93.82%.
  • the SDS-PAGE detection map of PA1a protein purity is shown in Figure 3. It can be seen from the figure that the protein purity of PA1a is about 97.25%.
  • Pretreatment of pea whey Adjust the pH of the pea whey to 4.6, stand at 4°C for 24h, centrifuge at 4000rpm for 30min, remove the precipitation, add 1mol/L NaOH solution to adjust the pH of the supernatant to 7.5, A pretreated pea whey was obtained.
  • Negatively charged polysaccharide complex coagulation and precipitation of PA2 and lectins in pea whey protein Weigh 450 mg of dextran sulfate powder (molecular weight 500 kDa), dissolve in 100 mL of deionized water, and magnetically stir for 1 h to completely dissolve. Add 10% (v/v) dextran sulfate solution to the pea whey, mix well, adjust the pH to 4.60, stir magnetically for 25 min, centrifuge (6000 rpm, 15 min), remove the complex aggregate precipitate, collect the clear to obtain the crude protein extract of PA1a and dextran sulfate.
  • PA1a pea albumin adjust the pH of the above solution to 7.5, ultrafiltration (50kDa) centrifugation for 20min to remove polysaccharides, repeat ultrafiltration 2 times, combine the permeate, use ultrafiltration with a molecular weight cut-off of 1000Da to centrifuge for 25min to remove small molecules Substances were collected, and the retentate was collected to obtain the purified PA1a protein solution.
  • the protein content of the prepared PA1a was determined, the protein purity was detected by SEC-HPLC, and analyzed by SDS-PAGE electrophoresis.
  • Pretreatment of pea whey Adjust the pH of the pea whey to 4.8, stand at 4°C for 48h, centrifuge at 6000rpm for 15min, remove the precipitation, add 1mol/L NaOH solution to adjust the pH of the supernatant to 7.0, A pretreated pea whey was obtained.
  • Negatively charged polysaccharide complex coagulation and precipitation of PA2 and lectin in pea whey protein Weigh 500 mg of sodium alginate powder (molecular weight 390 kDa), dissolve it in 100 mL of deionized water, and magnetically stir for 2 hours to completely dissolve it. Add 20% (v/v) sodium alginate solution to the pea whey, mix well, adjust the pH to 3.9, stir magnetically for 30 min, centrifuge (4000 rpm, 20 min), remove the complex aggregate precipitate, and collect the supernatant , that is, the crude protein extract of PA1a and sodium alginate is obtained.
  • PA1a pea albumin adjust the pH of the above solution to 8.0, ultrafiltration (100kDa) centrifugation for 25min to remove polysaccharides, repeat ultrafiltration 1 time, combine the permeate, and use ultrafiltration with a molecular weight cut-off of 3500Da to centrifuge for 20min to remove small molecules Substances were collected, and the retentate was collected to obtain the purified PA1a protein solution.
  • the protein content of the prepared PA1a was determined, the protein purity was detected by SEC-HPLC, and analyzed by SDS-PAGE electrophoresis.
  • the PA1a obtained in the above example has a protein content of about 91.75% on a dry basis, a total sugar of 3.65%, and an ash content of 4.32%.

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Abstract

提供一种利用负电荷多糖纯化豌豆清蛋白PA1a的方法，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，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。可以提高豌豆乳清的综合利用，PA1a蛋白回收率高，纯度高，且对设备要求低，操作简单快速，无环境污染问题。

Description

一种利用负电荷多糖纯化豌豆清蛋白PA1a的方法 技术领域
本发明涉及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。
背景技术
近年来，植物蛋白引起越来越多人的兴趣，人们的饮食结构也逐渐向可持续的植物成分转变。其中，豌豆作为可持续作物，不仅是淀粉产品的良好来源，更是蛋白产品的良好来源。豌豆蛋白作为低致敏性蛋白，没有转基因风险且具有较高的营养价值。在欧美国家，豌豆蛋白乳饮料和发酵乳等备受关注，越来越成为牛乳蛋白饮品的重要替代品。在我国，豌豆蛋白不仅利用率低，且对豌豆蛋白的利用多集中在豌豆分离蛋白，造成占豌豆蛋白含量20％-30％的豌豆乳清蛋白浪费。豌豆乳清蛋白中硫含量高，硫含量可占总豌豆含硫蛋白的三分之二以上，豌豆乳清蛋白的浪费不仅使得含硫蛋白的大量损失，且大量排放也致使环境问题。
豌豆乳清中富含多种2S和7S乳清蛋白组分，主要包括PA2，PA1和植物凝集素，均具有不同的生理活性。PA1是主要的豌豆2S乳清蛋白之一，其含硫氨基酸约占种子中总含硫量的50％，是由PA1a和PA1b组成，其中PA1a分子量约6kDa。PA1已经被证实是对多种昆虫具有毒害作用的昆虫毒素生物活性肽。目前对PA1a的纯化是以John.A.GATEHOUSE提供的方式为主，采用Sephadex G-75和DEAE-纤维素色谱柱洗脱得到。传统的色谱柱分离虽具有较高的选择性，但其昂贵的价格和低的分离速度使得PA1a的分离纯化具有较高的成本。而多糖/蛋白静电作用驱动的复凝聚法制备不仅能快速、高效，而且能以低成本大量得到纯化的PA1a。
发明内容
本发明的目的是克服上述不足之处，提供一种利用负电荷多糖纯化豌豆清蛋白PA1a的方法，该方法快速高效，成本低廉。
本发明的技术方案，本发明提供的一种利用负电荷多糖纯化豌豆清蛋白PA1a的方法，以豌豆蛋白及粉丝废水加工副产物豌豆乳清为原料，选取一定浓度的不同类型的负电荷多糖溶液作为回收材料，按照一定的物质量比例加入到豌豆乳清中进行选择性复凝聚；利用负电 荷多糖优先结合非目标蛋白沉淀，并与目标蛋白发生相分离的原理，通过控制多糖用量和溶液pH，沉淀去除豌豆乳清蛋白中PA2和植物凝集素，获得含有PA1a的上清液；通过调节pH，超滤离心除残留多糖及除小分子物质，制备得到单一的PA1a蛋白组分，再经冷冻干燥得到纯度较高的单一豌豆清蛋白PA1a产品。
本发明流程图如图1所示，具体步骤如下：
(1)豌豆乳清液的预处理：用盐酸将豌豆乳清液调节pH至4.0-5.0，在4-10℃下静置24-48h，离心除沉淀，加入碱液调节上清液pH至7.0-8.0，得到预处理的豌豆乳清液，测定蛋白质含量。
(2)负电荷多糖复凝聚沉淀豌豆乳清蛋白中PA2和植物凝集素：取固体多糖样品粉末，配制质量浓度为0.1％-0.6％的多糖溶液；按照蛋白质:多糖质量比10-5:1向步骤(1)制备的豌豆乳清液中添加相应体积的多糖溶液，并用盐酸调节pH至3.5-4.6，室温搅拌15-30min，4000-6000rpm离心10-20min，去除沉淀，沉淀即为多糖-PA2/植物凝集素的复凝聚沉淀物。收集剩余上清液，即得富集PA1a的粗提纯蛋白液。
(3)纯化PA1a豌豆清蛋白：将步骤(2)得到的富集PA1a的粗提纯蛋白液，调节pH至7.0-8.0，用截留分子量为10-100kDa的超滤设备进行超滤除去多糖，3500-5000rpm离心15-30min，重复超滤1-2次，收集透过液，再用截留分子量为1000-3500Da的超滤设备进行超滤浓缩，除去小分子物质，收集截留液，即得纯化的PA1a蛋白液。
(4)PA1a蛋白冷冻干燥：将步骤(3)所得PA1a蛋白液，于-30℃冷冻24-48h，冷冻样品置于-40～-60℃、5～10Pa下进行真空冷冻干燥48-72h，即制备得到PA1a豌豆清蛋白。
进一步地，步骤(1)中所述的豌豆乳清液来源于豌豆分离蛋白加工的副产物及豌豆粉丝废水经沉淀豌豆分离蛋白后的上清液，其蛋白质含量为0.1％-0.6％。
进一步地，步骤(1)中的离心条件为：4000-8000rpm，15-30min。
进一步地，步骤(2)所述的负电荷多糖为硫酸基团多糖和羧基多糖：硫酸基团多糖：包括但不限于硫酸葡聚糖，卡拉胶(κ-，ι-，λ)，硫酸软骨素，岩藻多糖；羧基多糖：包括但不限于羧甲基纤维素，海藻酸钠，黄原胶，果胶；其相对分子量范围大于100kDa。
进一步地，步骤(2)所述的负电荷多糖的质量浓度与所使用的豌豆乳清液中蛋白质量浓度保持一致；如果多糖质量浓度发生变化，需调整多糖溶液体积，保证最终混合溶液中蛋白质多糖质量比保持不变。
进一步地，步骤(3)所述的用截留分子量为10-100kDa的超滤设备进行超滤除去多糖，优先选择截留分子量高的超滤膜；用截留分子量为1000-3500Da的超滤设备进行超滤浓缩，除去小分子物质，优先选择截留分子量低的超滤膜。
进一步地，步骤(4)所述的制备得到的PA1a豌豆清蛋白经过聚丙烯酰胺凝胶电泳SDS-PAGE及凝胶过滤-高效液相色谱SEC-HPLC检测样品纯度，经BCA法测定蛋白含量，经过苯酚硫酸法GB/T 15672-2009测定总糖含量，经过干燥法GB/T 5497-1985测定水分，经过灼烧重量法测定灰分。
本发明中，由于豌豆乳清蛋白的等电点集中于pH 4.5-4.8左右，当调节豌豆乳清蛋白与多糖的混合溶液的pH至蛋白质等电点以下后，带负电荷的多糖会与带正电荷的豌豆乳清蛋白通过静电吸引发生相互作用，形成不溶性的复凝聚物，并可以通过离心进行分离。因此，本发明通过控制豌豆乳清蛋白与负电荷多糖的复凝聚条件，将全部的豌豆乳清蛋白PA2和植物凝集素与负电荷多糖形成不溶性复凝聚物，并通过离心沉淀下来，促使上清中仅剩余目标豌豆清蛋白PA1a。
本发明使用了天然可食性的负电荷多糖作为回收纯化蛋白的原料，利用非目标蛋白质(PA2和植物凝集素)与多糖在携带相反电荷条件下产生复凝聚沉淀，目标蛋白PA1a不参与形成复凝聚物的原理，得到单一的纯化的PA1a蛋白，此外，制备的复凝聚物可以作为食品材料直接利用。从资源回收利用的角度来看，本发明不仅可以低损耗、高选择性地制备高纯度豌豆清蛋白组分—PA1a，还可以回收豌豆乳清中其它蛋白组分，为后续的开发研究提供技术支持。
本发明的有益效果：本发明操作流程简单，设备要求低，易于规模化；多糖用量少，且可通过膜分离回收利用；纯化回收的PA1a蛋白纯度超过90％，回收率超过80％，仍然保留其天然活性。
附图说明
图1.PA1a的分离纯化工艺流程图。
图2.PA1a蛋白纯度的SEC-HPLC检测图谱。
图3.PA1a蛋白纯度的SDS-PAGE检测图谱。
附图标记说明：泳道1代表marker；泳道2代表纯化的PA1a；泳道3代表豌豆乳清蛋白组分。
具体实施方式
实施例1
(1)豌豆乳清液的预处理：将用2mol/L盐酸豌豆乳清液调节pH至4.5，在4℃下静置48h，8000rpm离心20min，除沉淀，加入1mol/L NaOH溶液调节上清液pH至7.0，得到预处理的豌豆乳清液。
(2)负电荷多糖复凝聚沉淀豌豆乳清蛋白中PA2和植物凝集素：称取400mg的羧甲 基纤维素粉末(分子量250kDa)，溶解于100mL去离子水中，磁力搅拌2h使其完全溶解。向豌豆乳清液中加入20％(v/v)的羧甲基纤维素溶液，混合均匀，将pH调至4.12，磁力搅拌15min，离心(4000rpm，20min)，除去复凝聚物沉淀物，收集上清，即得PA1a与羧甲基纤维素的粗蛋白提取液。
(3)纯化PA1a豌豆清蛋白：调节上述溶液pH至7.0，超滤(100kDa)离心30min除去多糖，重复超滤1次，合并透过液，用截留分子量为3500Da的超滤离心30min除去小分子物质，收集截留液，即得纯化的PA1a蛋白液。
(4)PA1a蛋白冷冻干燥：将以上蛋白纯化液于-30℃冷冻48h，置于-60℃、5Pa条件下冷冻干燥48h，即制备得到PA1a豌豆清蛋白。
对制备的PA1a测定蛋白含量，SEC-HPLC检测蛋白纯度，并做SDS-PAGE电泳分析。
PA1a蛋白纯度的SEC-HPLC检测图谱具体如图2所示，由图可知，PA1a的蛋白纯度为93.82％。
PA1a蛋白纯度的SDS-PAGE检测图谱如图3所示，由图可知，PA1a的蛋白纯度约为97.25％.
经测定，以上实例所得PA1a，蛋白含量约占干基的92.68％，总糖2.3％，灰分4.85％。
实施例2
(1)豌豆乳清液的预处理：将豌豆乳清液调节pH至4.6，在4℃下静置24h，4000rpm离心30min，除沉淀，加入1mol/L NaOH溶液调节上清液pH至7.5，得到预处理的豌豆乳清液。
(2)负电荷多糖复凝聚沉淀豌豆乳清蛋白中PA2和植物凝集素：称取450mg的硫酸葡聚糖粉末(分子量500kDa)，溶解于100mL去离子水中，磁力搅拌1h使其完全溶解。向豌豆乳清液中加入10％(v/v)的硫酸葡聚糖溶液，混合均匀，将pH调至4.60，磁力搅拌25min，离心(6000rpm，15min)，除去复凝聚物沉淀物，收集上清，即得PA1a与硫酸葡聚糖的粗蛋白提取液。
(3)纯化PA1a豌豆清蛋白：调节上述溶液pH至7.5，超滤(50kDa)离心20min除去多糖，重复超滤2次，合并透过液，用截留分子量为1000Da的超滤离心25min除去小分子物质，收集截留液，即得纯化的PA1a蛋白液。
(4)PA1a蛋白冷冻干燥：将以上蛋白纯化液于-30℃冷冻24h，置于-50℃、10Pa条件下冷冻干燥48h，即制备得到PA1a豌豆清蛋白。
对制备的PA1a测定蛋白含量，SEC-HPLC检测蛋白纯度，并做SDS-PAGE电泳分析。
经测定，以上实例所得PA1a，蛋白含量约占干基的90.81％，总糖4.22％，灰分4.57％。
实施例3
(1)豌豆乳清液的预处理：将豌豆乳清液调节pH至4.8，在4℃下静置48h，6000rpm离心15min，除沉淀，加入1mol/L NaOH溶液调节上清液pH至7.0，得到预处理的豌豆乳清液。
(2)负电荷多糖复凝聚沉淀豌豆乳清蛋白中PA2和植物凝集素：称取500mg的海藻酸钠粉末(分子量390kDa)，溶解于100mL去离子水中，磁力搅拌2h使其完全溶解。向豌豆乳清液中加入20％(v/v)的海藻酸钠溶液，混合均匀，将pH调至3.9，磁力搅拌30min，离心(4000rpm，20min)，除去复凝聚物沉淀物，收集上清，即得PA1a与海藻酸钠的粗蛋白提取液。
(3)纯化PA1a豌豆清蛋白：调节上述溶液pH至8.0，超滤(100kDa)离心25min除去多糖，重复超滤1次，合并透过液，用截留分子量为3500Da的超滤离心20min除去小分子物质，收集截留液，即得纯化的PA1a蛋白液。
(4)PA1a蛋白冷冻干燥：将以上蛋白纯化液于-30℃冷冻36h，置于-60℃、10Pa条件下冷冻干燥60h，即制备得到PA1a豌豆清蛋白。
对制备的PA1a测定蛋白含量，SEC-HPLC检测蛋白纯度，并做SDS-PAGE电泳分析。
经测定，以上实例所得PA1a，蛋白含量约占干基的91.75％，总糖3.65％，灰分4.32％。
由于实例中所使用的三种负电荷多糖的分子量大小以及均一性都不同，因此造成所得的PA1a中残留的总糖和蛋白质含量存在一定的差异性。

Claims (8)

  1. 一种利用负电荷多糖纯化豌豆清蛋白PA1a的方法，其特征在于：以豌豆乳清液为原料，使用天然可食性的负电荷多糖作为回收材料，进行复配；利用负电荷多糖优先结合非目标蛋白沉淀，并与目标蛋白发生相分离的原理，通过调节复凝聚条件，沉淀豌豆乳清中的非目标蛋白，实现目标蛋白的分离纯化，得到单一的豌豆清蛋白PA1a。
  2. 根据权利要求1所述利用负电荷多糖纯化豌豆清蛋白PA1a的方法，其特征在于步骤如下：
    (1)豌豆乳清液的预处理：用盐酸将豌豆乳清液调节pH至4.0-5.0，在4-10℃下静置24-48h；离心除沉淀，加入碱液调节上清液pH至7.0-8.0，得到预处理的豌豆乳清液，测定蛋白质含量；
    (2)负电荷多糖复凝聚沉淀豌豆乳清蛋白中PA2和植物凝集素：取固体多糖样品粉末，配制质量浓度为0.1％-0.6％的多糖溶液；按照蛋白质:多糖质量比10-5:1向步骤(1)制备的豌豆乳清液中添加相应体积的多糖溶液，并用盐酸调节pH至3.5-4.6，室温搅拌15-30min，4000-6000rpm离心10-20min，沉淀即为多糖-PA2/植物凝集素的复凝聚沉淀物，收集上清液，即得富集PA1a的粗提纯蛋白液；
    (3)纯化PA1a豌豆清蛋白：将步骤(2)得到的富集PA1a的粗提纯蛋白液，调节pH至7.0-8.0，用截留分子量为10-100kDa的超滤设备进行超滤除去多糖，3500-5000rpm离心15-30min，重复超滤1-2次，收集透过液，再用截留分子量为1000-3500Da的超滤设备进行超滤浓缩，除去小分子物质，收集截留液，即得纯化的PA1a蛋白液；
    (4)PA1a蛋白冷冻干燥：将步骤(3)所得PA1a蛋白液，于-30℃冷冻24-48h，冷冻样品置于-40～-60℃、5～10Pa下进行真空冷冻干燥48-72h，即制备得到PA1a豌豆清蛋白。
  3. 根据权利要求2所述利用负电荷多糖纯化豌豆清蛋白PA1a的方法，其特征在于：步骤(1)所述的豌豆乳清液来源于豌豆分离蛋白加工的副产物及豌豆粉丝废水经沉淀豌豆分离蛋白后的上清液，其蛋白质含量为0.1％-0.6％。
  4. 根据权利要求2所述利用负电荷多糖纯化豌豆清蛋白PA1a的方法，其特征在于：步骤(1)中的离心条件为：4000-8000rpm，15-30min。
  5. 根据权利要求2所述利用负电荷多糖纯化豌豆清蛋白PA1a的方法，其特征在于：步骤(2)所述的负电荷多糖具体为硫酸基团多糖和羧基多糖；其相对分子量范围大于100kDa。
  6. 根据权利要求5所述利用负电荷多糖纯化豌豆清蛋白PA1a的方法，其特征在于：所述硫酸基团多糖为硫酸葡聚糖，卡拉胶(κ-，ι-，λ)，硫酸软骨素或岩藻多糖；所述羧基多糖为羧甲基纤维素，海藻酸钠，黄原胶或果胶。
  7. 根据权利要求2所述利用负电荷多糖纯化豌豆清蛋白PA1a的方法，其特征在于：步骤(2)所述的负电荷多糖的质量浓度与所使用的豌豆乳清液中蛋白质量浓度保持一致。
  8. 根据权利要求2所述利用负电荷多糖纯化豌豆清蛋白PA1a的方法，其特征在于：步骤(3)所述的用截留分子量为10-100kDa的超滤设备进行超滤除去多糖，优先选择截留分子量高的超滤膜；用截留分子量为1000-3500Da的超滤设备进行超滤浓缩，除去小分子物质，优先选择截留分子量低的超滤膜。
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