Method for extracting and purifying bergamot pear polygalacturonase inhibitory protein

Abstract

The invention discloses a bergamot pear polygalacturonase inhibitory protein extraction and purification method, which comprises: 1, crushing bergamot pears to obtain crushed bergamot pears; 2, precipitating the filtered crushed material by using an ammonium sulfate solution; 3, dissolving the precipitate with a buffer solution, and performing dialysis and primary purification to obtain a crude extract; and 4, purifying the crude extract by using a gel filtration chromatography column to obtain a purified extract, and drying to obtain the traditional Chinese medicine composition. The natural bergamot pear fruits are used as raw materials, and the method does not depend on chemical synthesis or genetic engineering technology, and accords with green, environment-friendly and sustainable development concepts; the extraction and purification process comprises multi-step purification processes such as filtration, precipitation, dissolution, dialysis and filter membrane filtration, and conventional laboratory technologies such as filter paper filtration, centrifugation and dialysis are used, so that the whole process is easy to implement and repeat, the feasibility and the reliability of experiments are improved, and the cost is reduced. The purity, the biological activity and the extraction rate of the extract are remarkably improved.

Description

Technical Field

[0001] The present invention relates to the technical field of protein extraction, and more specifically to a method for extracting and purifying fragrant pear polygalacturonase inhibitory protein. Background Art

[0002] Korla pear is a specialty fruit of Xinjiang, mainly distributed in the Korla area. It is favored by domestic and foreign consumers for its thin skin, fine flesh, crisp and juicy, sweet and sour taste, and rich nutrition. It is also an advantageous fruit for Xinjiang to export and earn foreign exchange. In recent years, the planting area and output of Korla pear have increased year by year, becoming the main source of economic income for local farmers. However, pears are susceptible to infection by various pathogens in the storage, transportation, and sales links after harvest, posing a serious threat to the development of the pear industry and export trade. The main pathogenic fungi are Alternaria, Aspergillus niger, Botrytis cinerea, and Penicillium expansum. At present, chemical preservatives are mainly used for treatment. Although the effect is good, it affects the quality of the fruit, resulting in restrictions on the export of pears. In addition, Xinjiang is also the largest production base of grapes in my country. The pathogen that causes post-harvest diseases is mainly Botrytis cinerea. At present, grapes use SO 2 Chemical preservatives can harm grapes and pollute the environment. Therefore, it is necessary to develop efficient and low-toxic biological preservatives for Xinjiang's specialty fruits and vegetables.

[0003] The cell wall is the first barrier for plant cells to defend against pathogens. Inhibiting pathogens from degrading the cell wall is an important way for plants to resist infection. Polygalacturonase-inhibiting protein (PGIP) can inhibit polygalacturonase (PG) and activate the plant's defense system to protect the integrity of the cell wall. Studies have shown that PGIP has an inhibitory effect on pathogenic fungal PG, and only specifically inhibits the secretion of pathogenic fungi, but cannot inhibit the PG produced by plants, bacteria and other microorganisms. PGIP is concentrated in the cell wall of plants, but its distribution varies among different varieties, different developmental stages, and different organs. Plants have low protein content, so the extract containing PGIP needs to be separated and purified.

[0004] At present, many scholars have conducted in-depth research on the separation and purification, structure, function, etc. of PGIP. Common separation methods include acetone, ammonium sulfate and polyethylene glycol precipitation; Common purification methods include chromatography, electrophoresis, ultrafiltration, etc. The existing PGIP extraction and purification technology still has problems such as low PGIP extraction rate and poor biological activity of the extracted PGIP due to incomplete removal of impurities in the extracts due to protein hydrolysis during the extraction and purification process; and at present, there are no relevant reports on the research of Korla pear PGIP. Therefore, it is very necessary to construct a fast and efficient method for separating and purifying pear PGIP to lay a certain foundation for the development and utilization of PGIP. Summary of the invention

[0005] An object of the present invention is to solve at least the above problems and to provide at least the advantages which will be described hereinafter.

[0006] Another object of the present invention is to provide a method for extracting and purifying fragrant pear polygalacturonase inhibitory protein, which at least solves the above-mentioned technical problems.

[0007] In order to achieve these purposes and other advantages according to the present invention, a method for extracting and purifying fragrant pear polygalacturonase inhibitory protein is provided, which comprises the following steps: Step 1, remove the core of the fragrant pear fruit, and crush it with an ultra-fine grinder to obtain a fragrant pear crushed material; Step 2: Filter the crushed pear with filter paper to collect the first supernatant, precipitate the first supernatant with ammonium sulfate solution, remove the solvent by centrifugation, and collect the precipitate; Step 3, dissolving the precipitate with a buffer solution, collecting the third supernatant by centrifugation, dialyzing the third supernatant with a buffer solution, and then filtering with a 0.45 um filter membrane to obtain a crude extract; Step 4: Purify the crude extract using a gel filtration chromatography column to obtain a purified extract, and freeze-dry to obtain the fragrant pear polygalacturonase inhibitory protein; Among them, in step one, during the crushing process with an ultrafine grinder, a stabilizer is added to the ultrafine grinder and the crushing operation is performed together, and the weight ratio of the stabilizer to the pitted pear fruit is 0.01~0.03:1; the stabilizer includes 0.005~0.01 parts of dithiothreitol, 0.15~0.25 parts of polyvinyl pyrrolidone, and 0.3~0.5 parts of a protease inhibitor.

[0008] Preferably, the method for extracting and purifying the fragrant pear polygalacturonase inhibitory protein, in step 2, precipitating the first supernatant with an ammonium sulfate solution, removing the solvent by centrifugation, and collecting the precipitate, specifically comprises: Step 201, adding ammonium sulfate to the first supernatant to a saturation of 35%, standing at 4°C for 1-2 hours, and then centrifuging at 2000-3000 r / min to obtain a first precipitate and a second supernatant A; Step 202, adding ammonium sulfate to the second supernatant A until the saturation is 55%, standing at 4°C for 1-2 hours, and then centrifuging at 4000-5500 r / min to obtain a second precipitate and a second supernatant B; Step 203, adding ammonium sulfate to the second supernatant B to a saturation of 75%, standing at 4°C for 1-2 hours, and then centrifuging at 5000-6500 r / min to obtain a third precipitate.

[0009] Preferably, in the method for extracting and purifying the fragrant pear polygalacturonase inhibitory protein, in steps 201, 202 and 203, before adding ammonium sulfate, 0.02-0.05 mol / L of sodium L-ascorbate and 0.03-0.05 mol / L of disodium ethylenediaminetetraacetate are added to the first supernatant / second supernatant A / second supernatant B.

[0010] Preferably, in the method for extracting and purifying the fragrant pear polygalacturonase inhibitory protein, step three is specifically as follows: Step 301, dissolving the first precipitate with a first buffer solution, and collecting the third supernatant A by centrifugation, dialyzing the third supernatant A with a first buffer solution, using a dialysis bag with a molecular weight cutoff of 10 kDa as the dialysis membrane, dialyzing for 24 hours at 4°C, changing the dialysate twice during the period, and obtaining a dialysate A; the first buffer solution is a phosphate buffer solution containing 0.1-0.2 mol / L sodium chloride and a pH of 6.8-7.5; Step 302, dissolving the second precipitate with a second buffer solution, and collecting the third supernatant B by centrifugation, dialyzing the third supernatant A with a second buffer solution, using a dialysis bag with a molecular weight cutoff of 10 kDa as the dialysis membrane, dialyzing for 24 hours at 4°C, changing the dialysate twice during the period, and obtaining a dialysate B; the second buffer solution is a phosphate buffer solution containing 0.01-0.02 mol / L sodium chloride and a pH of 6.8-7.5 and a concentration of 0.02-0.03 mol / L; Step 303, dissolving the third precipitate with a third buffer solution, collecting a third supernatant C by centrifugation, dialyzing the third supernatant C with a third buffer solution, using a dialysis bag with a molecular weight cutoff of 10 kDa as the dialysis membrane, dialyzing for 24 hours at 4° C., replacing the dialysate twice during the period, and obtaining a dialysate C; the third buffer solution is a phosphate buffer solution containing 0.01 to 0.02 mol / L sodium chloride and a pH of 6.8 to 7.5 and 0.008 to 0.012 mol / L; Step 304: Mix dialysate A, dialysate B, and dialysate C, transfer them into distilled water, and dialyze them at 4°C for 24-48 hours to obtain dialysate E. A dialysis bag with a molecular weight cutoff of 10 kDa is selected as the dialysis membrane. The dialysate is replaced every 12 hours. The dialysate E is filtered with a 0.45 um filter membrane to obtain a crude extract.

[0011] Preferably, in the method for extracting and purifying the fragrant pear polygalacturonase inhibitory protein, in step 2, before filtering the fragrant pear crushed material with filter paper, it is also pretreated, the fragrant pear crushed material is placed in an ice bath at 0°C, acetone 5 times the weight of the fragrant pear crushed material is added, the fragrant pear crushed material is stirred evenly and then filtered, and the fragrant pear crushed material is rinsed with trimethylsilyl ether to obtain the pretreated fragrant pear crushed material, and the pretreated fragrant pear crushed material is filtered with filter paper.

[0012] Preferably, in the method for extracting and purifying the fragrant pear polygalacturonase inhibitory protein, the protease inhibitor is diisopropylfluorophosphate or iodoacetic acid.

[0013] Preferably, in the method for extracting and purifying the fragrant pear polygalacturonase inhibitory protein, step 4 is specifically as follows: Step 401: pre-treat the crude extract with a pH of 5.5 containing 5×10 -5 mol / L EDTA-Na 2 The CM-Sephadex C-50 column was equilibrated with 25 mmol / L sodium acetate buffer at pH 5.5 and then with 5×10 -5 mol / L EDTA-Na 2 Wash with 25mmol / L sodium acetate buffer until A 280 <0.02, and then gradient elution was performed with sodium acetate buffer containing 0.1~1 mol / L NaCl to collect the initially purified active components; Step 402: The preliminarily purified active component is preliminarily treated with a pH of 5.5 containing 5×10 -5 mol / L EDTA-Na 2 A Sephadex G-100 column (1 cm × 70 cm) was equilibrated with 25 mmol / L sodium acetate buffer and a column containing 5 × 10 -5 mol / LEDTA-Na 2 The extract was eluted with 25 mmol / L sodium acetate buffer and the active fraction was collected to obtain the purified extract.

[0014] Preferably, in the method for extracting and purifying the fragrant pear polygalacturonase inhibitory protein, the CM-Sephadex C-50 gel and the Sephadex G-100 gel are both pretreated before being loaded onto the column, specifically as follows: Swelling treatment: soak the gel in an ethanol solution with a volume fraction of 50-60%. After removing the ethanol solution, soak it in sterile water and soak it under magnetic stirring at room temperature for at least 24 hours. Then take out the gel and rinse it with sterile water until the washing water is colorless. Wrap the gel with three layers of sterile filter cloth and let it stand for 30 minutes. During this period, replace the filter cloth every 10 minutes. Acid-base treatment: soak the swollen gel in an equal volume of 1.0 mol / L NaOH solution, stir at 50 rpm / min for 1 h, take out the gel after soaking, and then wash the gel with sterile water until the washing solution is neutral; then soak the gel in an equal volume of 0.1 mol / L HCl solution, stir at 50 rpm / min for 1 h, take out the gel after soaking, and then wash the gel with sterile water until the washing solution is neutral; wrap the gel with three layers of sterile filter cloth and let it stand for 2 h, during which the filter cloth was replaced every 30 minutes; Degassing treatment: Place the gel after acid and alkali treatment in an ultrasonic device and perform ultrasonic treatment at a frequency of 20~40kHz for 20 minutes. Then place the gel in a suction filtration bottle, plug the bottle mouth with an ear bulb, and reduce the pressure and evacuate for 30 minutes to remove tiny bubbles in the gel.

[0015] The present invention has at least the following beneficial effects: 1. The present invention uses natural fragrant pear fruit as raw material, does not rely on chemical synthesis or genetic engineering technology, and conforms to the concept of green, environmental protection and sustainable development; the extraction and purification process of the present invention includes a multi-step purification process such as filtration, precipitation, dissolution, dialysis and membrane filtration, and uses conventional laboratory techniques such as filter paper filtration, centrifugation and dialysis, so that the whole process is easy to implement and repeat, improves the feasibility and reliability of the experiment, and significantly improves the purity, biological activity and extraction rate of the extract, providing high-quality raw materials for subsequent research and application, and further reduces cumbersome operations and reagent consumption by optimizing the extraction and purification steps, thereby reducing production costs; 2. The present invention adopts an ultrafine pulverizer to crush the fragrant pear fruit, and adds a stabilizer, thereby effectively improving the uniformity and efficiency of cell crushing, so that the polygalacturonase inhibiting protein in the cell can be released more fully, and at the same time, the degradation of the protein during the crushing process is avoided, thereby improving the extraction rate of the polygalacturonase inhibiting protein; the stabilizer includes dithiothreitol, polyvinyl pyrrolidone and protease inhibitors, and these stabilizers work together to provide a stable microenvironment for the protein, which helps to maintain the integrity of its structure and function. Adding the stabilizer during the crushing process of the ultrafine pulverizer not only effectively prevents the degradation and inactivation of the protein during the crushing process of the fragrant pear, ensures the biological activity of the final product, but also helps to improve the physicochemical properties of the solution, such as pH value, ionic strength and viscosity, thereby ensuring that the protein maintains its natural conformation and activity during the dissolution process, and at the same time has an important influence on the solubility and stability of the protein, which helps to make the protein easier to separate from the cell fragments in the subsequent extraction process, thereby achieving the effect of improving the extraction efficiency and biological activity of the protein; 3. Dithiothreitol (DTT): DTT is a commonly used reducing agent that can prevent protein from being degraded due to oxidation during the extraction process. It reacts with the disulfide bonds in the protein to reduce it to sulfhydryl groups, thereby protecting the conformation and activity of the protein; Polyvinylpyrrolidone (PVP): PVP has good water solubility and can form a protective film that covers the surface of the protein to prevent it from contacting with oxygen, enzymes or other harmful substances in the air, thereby reducing protein degradation and denaturation; Since cells or tissues release proteases when they are lysed, these enzymes may react with the protein to be extracted, causing protein decomposition. Therefore, protease inhibitors are added to the stabilizer to inhibit the activity of proteases, prevent protein hydrolysis during the extraction process, maintain protein integrity, thereby improving extraction efficiency and purity as well as improving the biological activity of the extracted product; 4. The present invention adopts an ammonium sulfate concentration gradient precipitation method to precipitate the first supernatant, and the pretreated samples are sequentially added to the ammonium sulfate gradient solution. Under the action of centrifugation, the protein will be precipitated in different ammonium sulfate solutions according to the concentration gradient, and a variety of proteins with different molecular weights can be separated, thereby enhancing the biological activity of the final extraction product and producing highly purified proteins; 5. Sodium L-ascorbate is the sodium salt form of vitamin C and has antioxidant properties. In the process of ammonium sulfate precipitation of protein, the addition of sodium L-ascorbate can prevent the protein from oxidative denaturation during the precipitation process. Disodium ethylenediaminetetraacetic acid (EDTA) is a commonly used metal ion chelating agent that can effectively remove metal ions in the solution, such as iron ions and copper ions. In the process of ammonium sulfate precipitation of protein, the addition of EDTA can help remove harmful metal ions, thereby improving the purity of the protein. The precipitate is further purified by a multi-stage dialysis method, and the precipitate obtained by the multi-stage precipitation treatment is dialyzed separately, which greatly improves the biological activity, extraction rate and extraction purity of the protein. 6. Treating the crushed pear with acetone can destroy the cell membrane and the combination of protein and lipid. After acetone treatment, it is treated with trimethylsilyl ether. Methyl ether can passivate some active groups exposed by the protein and reduce the damage of active groups during the extraction process. The stabilizer plays a protective role on the protein during the pretreatment process, improves the biological activity of the protein, and also improves the purity and extraction rate of the extracted protein.

[0016] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. DETAILED DESCRIPTION

[0017] The present invention is further described in detail below in conjunction with embodiments so that those skilled in the art can implement the invention with reference to the description.

[0018] It should be understood that the terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.

[0019] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0020] <Example 1> The present invention provides a method for extracting and purifying a fragrant pear polygalacturonase inhibitory protein, which comprises the following steps: Step 1: Crushing raw materials The pear fruit is pitted, a stabilizer is added, and the fruit is crushed in an ultrafine grinder, wherein the weight ratio of the stabilizer to the pitted pear fruit is 0.01:1; the stabilizer comprises 0.005 parts of dithiothreitol, 0.15 parts of polyvinyl pyrrolidone, and 0.3 parts of a protease inhibitor, and the fruit is crushed in an ultrafine grinder to obtain a pear crushed product; the protease inhibitor is diisopropyl fluorophosphoric acid or iodoacetic acid; Step 2: Precipitation Extraction The crushed pears are placed in an ice bath at 0°C, acetone is added in an amount of 5 times the weight of the crushed pears, the mixture is stirred evenly and then filtered, and the mixture is rinsed with trimethylsilyl ether to obtain the pretreated crushed pears. The pretreated crushed pears are filtered with filter paper to collect a first supernatant; the first supernatant is precipitated with an ammonium sulfate solution, the solvent is removed by centrifugation, and the precipitate is collected, specifically: Step 201, adding ammonium sulfate to the first supernatant to a saturation of 35%, standing at 4°C for 1 hour, and then centrifuging at 2000 r / min to obtain a first precipitate and a second supernatant A; Step 202, adding ammonium sulfate to the second supernatant A until the saturation is 55%, standing at 4°C for 1 hour, and then centrifuging at 4000 r / min to obtain a second precipitate and a second supernatant B; Step 203, adding ammonium sulfate to the second supernatant B until the saturation is 75%, standing at 4°C for 1 hour, and then centrifuging at 5000 r / min to obtain a third precipitate; Wherein, in step 201, step 202 and step 203, before adding ammonium sulfate, 0.02 mol / L of sodium L-ascorbate and 0.03 mol / L of disodium ethylenediaminetetraacetate are added to the first supernatant / the second supernatant A / the second supernatant B; Step 3: Dialysis purification Dissolve the precipitate with a buffer solution, specifically: Step 301, dissolving the first precipitate with a first buffer solution, and collecting the third supernatant A by centrifugation, dialyzing the third supernatant A with a first buffer solution, using a dialysis bag with a molecular weight cutoff of 10 kDa as the dialysis membrane, dialyzing for 24 hours at 4° C., changing the dialysate twice during the period, and obtaining a dialysate A; the first buffer solution is a 0.1 mol / L phosphate buffer solution containing 0.01 mol / L sodium chloride and a pH of 6.8; Step 302, dissolving the second precipitate with a second buffer solution, and collecting the third supernatant B by centrifugation, dialyzing the third supernatant A with a second buffer solution, using a dialysis bag with a molecular weight cutoff of 10 kDa as the dialysis membrane, dialyzing for 24 hours at 4° C., changing the dialysate twice during the period, and obtaining a dialysate B; the second buffer solution is a 0.02 mol / L phosphate buffer solution containing 0.01 mol / L sodium chloride and a pH of 6.8; Step 303, dissolving the third precipitate with a third buffer solution, collecting a third supernatant C by centrifugation, dialyzing the third supernatant C with a third buffer solution, using a dialysis bag with a molecular weight cutoff of 10 kDa as the dialysis membrane, dialyzing for 24 hours at 4° C., replacing the dialysate twice during the dialysate, and obtaining a dialysate C; the third buffer solution is a 0.008 mol / L phosphate buffer solution containing 0.01 mol / L sodium chloride and a pH of 6.8; Step 304: dialysate A, dialysate B, and dialysate C are mixed and transferred into distilled water, and dialyzed at 4° C. for 24 hours to obtain dialysate E. The dialysis membrane uses a dialysis bag with a molecular weight cutoff of 10 kDa. The dialysate is replaced every 12 hours. The dialysate E is filtered through a 0.45 um filter membrane to obtain a crude extract. Step 4: Column chromatography purification The crude extract was purified by gel filtration chromatography to obtain a purified extract, and then freeze-dried to obtain the fragrant pear polygalacturonase inhibitory protein; the purification process is specifically as follows: Step 401: pre-treat the crude extract with a pH of 5.5 containing 5×10 -5 mol / L EDTA-Na 2 The CM-Sephadex C-50 column was equilibrated with 25 mmol / L sodium acetate buffer at pH 5.5 and then with 5×10 -5 mol / L EDTA-Na 2 Wash with 25mmol / L sodium acetate buffer until A 280 <0.02, and then gradient elution was performed with sodium acetate buffer containing 0.1 mol / L NaCl to collect the initially purified active components; Step 402: The preliminarily purified active component is preliminarily treated with a pH of 5.5 containing 5×10 -5 mol / L EDTA-Na 2 A Sephadex G-100 column (1 cm × 70 cm) was equilibrated with 25 mmol / L sodium acetate buffer and a column containing 5 × 10 -5 mol / LEDTA-Na 2 25 mmol / L sodium acetate buffer was used for elution, and the active fraction was collected to obtain the purified extract; Among them, CM-Sephadex C-50 gel and Sephadex G-100 gel were pretreated before loading, specifically: Swelling treatment: soak the gel in a 50% ethanol solution by volume, remove the ethanol solution, and soak it in sterile water for at least 24 hours under magnetic stirring at room temperature. Then take out the gel and rinse it with sterile water until the washing water is colorless. Wrap the gel with three layers of sterile filter cloth and let it stand for 30 minutes, during which the filter cloth is replaced every 10 minutes. Acid-base treatment: soak the swollen gel in an equal volume of 1.0 mol / L NaOH solution, stir at 50 rpm / min for 1 h, take out the gel after soaking, and then wash the gel with sterile water until the washing solution is neutral; then soak the gel in an equal volume of 0.1 mol / L HCl solution, stir at 50 rpm / min for 1 h, take out the gel after soaking, and then wash the gel with sterile water until the washing solution is neutral; wrap the gel with three layers of sterile filter cloth and let it stand for 2 h, during which the filter cloth was replaced every 30 minutes; Degassing treatment: Place the gel after acid and alkali treatment in an ultrasonic device and perform ultrasonic treatment at a frequency of 20~40kHz for 20 minutes. Then place the gel in a suction filtration bottle, plug the bottle mouth with an ear bulb, and reduce the pressure and evacuate for 30 minutes to remove tiny bubbles in the gel.

[0021] <Example 2> The present invention provides a method for extracting and purifying a fragrant pear polygalacturonase inhibitory protein, which comprises the following steps: Step 1: Crushing raw materials The pear fruit is pitted, a stabilizer is added, and the fruit is crushed in an ultrafine grinder, wherein the weight ratio of the stabilizer to the pitted pear fruit is 0.03:1; the stabilizer comprises 0.01 parts of dithiothreitol, 0.25 parts of polyvinyl pyrrolidone, and 0.5 parts of a protease inhibitor, and the fruit is crushed in an ultrafine grinder to obtain a pear crushed product; the protease inhibitor is diisopropyl fluorophosphoric acid or iodoacetic acid; Step 2: Precipitation Extraction The crushed pears are placed in an ice bath at 0°C, acetone is added in an amount of 5 times the weight of the crushed pears, the mixture is stirred evenly and then filtered, and the mixture is rinsed with trimethylsilyl ether to obtain the pretreated crushed pears. The pretreated crushed pears are filtered with filter paper to collect a first supernatant; the first supernatant is precipitated with an ammonium sulfate solution, the solvent is removed by centrifugation, and the precipitate is collected, specifically: Step 201, adding ammonium sulfate to the first supernatant to a saturation of 35%, standing at 4°C for 2 hours, and then centrifuging at 3000 r / min to obtain a first precipitate and a second supernatant A; Step 202, adding ammonium sulfate to the second supernatant A until the saturation is 55%, standing at 4°C for 2 hours, and then centrifuging at 5500 r / min to obtain a second precipitate and a second supernatant B; Step 203, adding ammonium sulfate to the second supernatant B until the saturation is 75%, standing at 4°C for 2 hours, and then centrifuging at 6500 r / min to obtain a third precipitate and a second supernatant C; Wherein, in step 201, step 202 and step 203, before adding ammonium sulfate, 0.05 mol / L of sodium L-ascorbate and 0.05 mol / L of disodium ethylenediaminetetraacetate are added to the first supernatant / the second supernatant A / the second supernatant B; Step 3: Dialysis purification Dissolve the precipitate with a buffer solution, specifically: Step 301, dissolving the first precipitate with a first buffer solution, and collecting the third supernatant A by centrifugation, dialyzing the third supernatant A with a first buffer solution, using a dialysis bag with a molecular weight cutoff of 10 kDa as the dialysis membrane, dialyzing for 24 hours at 4° C., changing the dialysate twice during the period, and obtaining a dialysate A; the first buffer solution is a 0.2 mol / L phosphate buffer solution containing 0.02 mol / L sodium chloride and a pH of 7.5; Step 302, dissolving the second precipitate with a second buffer solution, and collecting the third supernatant B by centrifugation, dialyzing the third supernatant A with a second buffer solution, using a dialysis bag with a molecular weight cutoff of 10 kDa as the dialysis membrane, dialyzing for 24 hours at 4° C., changing the dialysate twice during the period, and obtaining a dialysate B; the second buffer solution is a 0.03 mol / L phosphate buffer solution containing 0.02 mol / L sodium chloride and a pH of 7.5; Step 303, dissolving the third precipitate with a third buffer solution, collecting a third supernatant C by centrifugation, dialyzing the third supernatant C with a third buffer solution, using a dialysis bag with a molecular weight cutoff of 10 kDa as the dialysis membrane, dialyzing for 24 hours at 4° C., replacing the dialysate twice during the period, and obtaining a dialysate C; the third buffer solution is a 0.012 mol / L phosphate buffer solution containing 0.02 mol / L sodium chloride and a pH of 7.5; Step 304: dialysate A, dialysate B, and dialysate C are mixed and transferred into distilled water, and dialyzed at 4° C. for 48 hours to obtain dialysate E. The dialysis membrane is a dialysis bag with a molecular weight cutoff of 10 kDa. The dialysate is replaced every 12 hours. The dialysate E is filtered through a 0.45 um filter membrane to obtain a crude extract. Step 4: Column chromatography purification The crude extract was purified by gel filtration chromatography to obtain a purified extract, and then freeze-dried to obtain the fragrant pear polygalacturonase inhibitory protein; the purification process is specifically as follows: Step 401: pre-treat the crude extract with a pH of 5.5 containing 5×10 -5 mol / L EDTA-Na 2 The CM-Sephadex C-50 column was equilibrated with 25 mmol / L sodium acetate buffer at pH 5.5 and then with 5×10 -5 mol / L EDTA-Na2 Wash with 25mmol / L sodium acetate buffer until A 280 <0.02, and then gradient elution was performed with sodium acetate buffer containing 1 mol / L NaCl to collect the initially purified active components; Step 402: The preliminarily purified active component is preliminarily treated with a pH of 5.5 containing 5×10 -5 mol / L EDTA-Na 2 A Sephadex G-100 column (1 cm × 70 cm) was equilibrated with 25 mmol / L sodium acetate buffer and a column containing 5 × 10 -5 mol / LEDTA-Na 2 25 mmol / L sodium acetate buffer was used for elution, and the active fraction was collected to obtain the purified extract; Among them, CM-Sephadex C-50 gel and Sephadex G-100 gel were pretreated before loading, specifically: Swelling treatment: soak the gel in a 60% ethanol solution by volume, remove the ethanol solution, soak it in sterile water, and soak it under magnetic stirring at room temperature for at least 24 hours. Then take out the gel and rinse it with sterile water until the washing water is colorless. Wrap the gel with three layers of sterile filter cloth and let it stand for 30 minutes. During this period, replace the filter cloth every 10 minutes. Acid-base treatment: soak the swollen gel in an equal volume of 1.0 mol / L NaOH solution, stir at 50 rpm / min for 1 h, take out the gel after soaking, and then wash the gel with sterile water until the washing solution is neutral; then soak the gel in an equal volume of 0.1 mol / L HCl solution, stir at 50 rpm / min for 1 h, take out the gel after soaking, and then wash the gel with sterile water until the washing solution is neutral; wrap the gel with three layers of sterile filter cloth and let it stand for 2 h, during which the filter cloth was replaced every 30 minutes; Degassing treatment: Place the gel after acid and alkali treatment in an ultrasonic device and perform ultrasonic treatment at a frequency of 20~40kHz for 20 minutes. Then place the gel in a suction filtration bottle, plug the bottle mouth with an ear bulb, and reduce the pressure and evacuate for 30 minutes to remove tiny bubbles in the gel.

[0022] <Example 3> The present invention provides a method for extracting and purifying a fragrant pear polygalacturonase inhibitory protein, which comprises the following steps: Step 1: Crushing raw materials The pear fruit is pitted, a stabilizer is added, and the fruit is crushed in an ultrafine grinder, wherein the weight ratio of the stabilizer to the pitted pear fruit is 0.02:1; the stabilizer comprises 0.008 parts of dithiothreitol, 0.2 parts of polyvinyl pyrrolidone, and 0.4 parts of a protease inhibitor, and the fruit is crushed in an ultrafine grinder to obtain a pear crushed product; the protease inhibitor is diisopropyl fluorophosphoric acid or iodoacetic acid; Step 2: Precipitation Extraction The crushed pears are placed in an ice bath at 0°C, acetone is added in an amount of 5 times the weight of the crushed pears, the mixture is stirred evenly and then filtered, and the mixture is rinsed with trimethylsilyl ether to obtain the pretreated crushed pears. The pretreated crushed pears are filtered with filter paper to collect a first supernatant; the first supernatant is precipitated with an ammonium sulfate solution, the solvent is removed by centrifugation, and the precipitate is collected, specifically: Step 201, adding ammonium sulfate to the first supernatant to a saturation of 35%, standing at 4°C for 1.5 hours, and then centrifuging at 2500 r / min to obtain a first precipitate and a second supernatant A; Step 202, adding ammonium sulfate to the second supernatant A until the saturation is 55%, standing at 4°C for 1.5 hours, and then centrifuging at 5000 r / min to obtain a second precipitate and a second supernatant B; Step 203, adding ammonium sulfate to the second supernatant B until the saturation is 75%, standing at 4°C for 1.5 hours, and then centrifuging at 6000 r / min to obtain a third precipitate and a second supernatant C; Wherein, in step 201, step 202 and step 203, before adding ammonium sulfate, 0.04 mol / L of sodium L-ascorbate and 0.04 mol / L of disodium ethylenediaminetetraacetate are added to the first supernatant / the second supernatant A / the second supernatant B; Step 3: Dialysis purification Dissolve the precipitate with a buffer solution, specifically: Step 301, dissolving the first precipitate with a first buffer solution, and collecting the third supernatant A by centrifugation, dialyzing the third supernatant A with a first buffer solution, using a dialysis bag with a molecular weight cutoff of 10 kDa as the dialysis membrane, dialyzing for 24 hours at 4° C., changing the dialysate twice during the period, and obtaining a dialysate A; the first buffer solution is a 0.15 mol / L phosphate buffer solution containing 0.015 mol / L sodium chloride and a pH of 7.2; Step 302, dissolving the second precipitate with a second buffer solution, and collecting the third supernatant B by centrifugation, dialyzing the third supernatant A with a second buffer solution, using a dialysis bag with a molecular weight cutoff of 10 kDa as the dialysis membrane, dialyzing for 24 hours at 4° C., changing the dialysate twice during the period, and obtaining a dialysate B; the second buffer solution is a 0.025 mol / L phosphate buffer solution containing 0.05 mol / L sodium chloride and a pH of 7.2; Step 303, dissolving the third precipitate with a third buffer solution, collecting a third supernatant C by centrifugation, dialyzing the third supernatant C with a third buffer solution, using a dialysis bag with a molecular weight cutoff of 10 kDa as the dialysis membrane, dialyzing for 24 hours at 4° C., replacing the dialysate twice during the dialysate, and obtaining a dialysate C; the third buffer solution is a 0.01 mol / L phosphate buffer solution containing 0.015 mol / L sodium chloride and a pH of 7.2; Step 304: dialysate A, dialysate B, and dialysate C are mixed and transferred into distilled water, and dialyzed at 4° C. for 24 to 48 hours to obtain dialysate E. The dialysis membrane is a dialysis bag with a molecular weight cutoff of 10 kDa, and the dialysate is replaced every 12 hours; the dialysate E is filtered through a 0.45 um filter membrane to obtain a crude extract; Step 4: Column chromatography purification The crude extract was purified by gel filtration chromatography to obtain a purified extract, and then freeze-dried to obtain the fragrant pear polygalacturonase inhibitory protein; the purification process is specifically as follows: Step 401: pre-treat the crude extract with a pH of 5.5 containing 5×10 -5 mol / L EDTA-Na 2 The CM-Sephadex C-50 column was equilibrated with 25 mmol / L sodium acetate buffer at pH 5.5 and then with 5×10 -5 mol / L EDTA-Na 2 Wash with 25mmol / L sodium acetate buffer until A 280 <0.02, and then gradient elution was performed with sodium acetate buffer containing 0.5 mol / L NaCl to collect the initially purified active components; Step 402: The preliminarily purified active component is preliminarily treated with a pH of 5.5 containing 5×10 -5 mol / L EDTA-Na 2 A Sephadex G-100 column (1 cm × 70 cm) was equilibrated with 25 mmol / L sodium acetate buffer and a column containing 5 × 10 -5 mol / LEDTA-Na 2 25 mmol / L sodium acetate buffer was used for elution, and the active fraction was collected to obtain the purified extract; Among them, CM-Sephadex C-50 gel and Sephadex G-100 gel were pretreated before loading, specifically: Swelling treatment: soak the gel in a 55% ethanol solution, remove the ethanol solution, and soak it in sterile water for at least 24 hours under magnetic stirring at room temperature. Then take out the gel and rinse it with sterile water until the washing water is colorless. Wrap the gel with three layers of sterile filter cloth and let it stand for 30 minutes, during which the filter cloth is replaced every 10 minutes. Acid-base treatment: soak the swollen gel in an equal volume of 1.0 mol / L NaOH solution, stir at 50 rpm / min for 1 h, take out the gel after soaking, and then wash the gel with sterile water until the washing solution is neutral; then soak the gel in an equal volume of 0.1 mol / L HCl solution, stir at 50 rpm / min for 1 h, take out the gel after soaking, and then wash the gel with sterile water until the washing solution is neutral; wrap the gel with three layers of sterile filter cloth and let it stand for 2 h, during which the filter cloth was replaced every 30 minutes; Degassing treatment: Place the gel after acid and alkali treatment in an ultrasonic device and perform ultrasonic treatment at a frequency of 20~40kHz for 20 minutes. Then place the gel in a suction filtration bottle, plug the bottle mouth with an ear bulb, and reduce the pressure and evacuate for 30 minutes to remove tiny bubbles in the gel.

[0023] <Comparative Example 1> The difference from Example 3 is that no stabilizer is added during the crushing process of the ultrafine grinder; the other conditions and parameters are the same as those of Example 3.

[0024] <Comparative Example 2> The difference from Example 3 is that in step 2, only step 201 is used for precipitation, and step 301 in step 3 is used for precipitation extraction and dialysis purification. The other conditions and parameters are the same as in Example 3.

[0025] <Comparative Example 3> The difference from Example 3 is that in step 2, only step 202 is used for precipitation, and step 302 in step 3 is used for precipitation extraction and dialysis purification. The other conditions and parameters are the same as in Example 3.

[0026] <Comparative Example 4> The difference from Example 3 is that in step 2, only step 203 is used for precipitation, and step 303 in step 3 is used for precipitation extraction and dialysis purification. The other conditions and parameters are the same as in Example 3.

[0027] <Comparative Example 5> The difference from Example 3 is that, in step 201, step 202 and step 203, before adding ammonium sulfate, 0.02-0.05 mol / L of sodium L-ascorbate and 0.03-0.05 mol / L of disodium ethylenediaminetetraacetate are not added to the first supernatant / the second supernatant A / the second supernatant B; the other conditions and parameters are the same as those in Example 3.

[0028] <Comparative Example 6> The difference from Example 3 is that the crushed fragrant pear is not pretreated before being filtered with filter paper; the other conditions and parameters are the same as those in Example 3.

[0029] <Comparative Example 7> Step 1, remove the core of the fragrant pear fruit, and crush it with an ultra-fine grinder to obtain a fragrant pear crushed material; Step 2: Filter the crushed pear with filter paper to collect the first supernatant, precipitate the first supernatant with ammonium sulfate solution, remove the solvent by centrifugation, and collect the precipitate; the saturation of the ammonium sulfate solution is 55%; Step 3: Purify the crude extract by Sephadex G-100 molecular sieve gel filtration chromatography to obtain a purified extract, and freeze-dry to obtain the fragrant pear polygalacturonase inhibitory protein. Each of the above steps is implemented by existing technical means.

[0030] <Test Example 1> The extraction rate and purity of the polygalacturonase inhibitory protein of Examples 1 to 3 and Comparative Examples 1 to 7 of the present invention were calculated, wherein the extraction rate and purity were calculated by preparing a standard solution of a protein with an inhibitory concentration (PGIP), and drawing a standard curve between protein content and absorbance by ultraviolet spectrophotometry; testing the absorbance value of the first supernatant before extraction, and using the standard curve to obtain the protein content A in the first supernatant. 1 mg; in the same way, the PGIP content A in the final freeze-dried extract was obtained 2 mg (the final weight of the freeze-dried extract is Amg), extraction rate = A 2 / A 1 %; Purity = A 2 / A%; the results are shown in Table 1.

[0031] <Test Example 2> PGIP can specifically bind to PG, thereby reducing the activity of PG. The activity of PGIP can be indirectly evaluated by measuring the degree of degradation of pectin acid by PG in the presence and absence of PGIP. Generally, the content of the degradation product galacturonic acid is proportional to the activity of PG, so the activity of PG can be reflected by measuring the amount of galacturonic acid produced, and the inhibitory effect of PGIP can be further evaluated (the higher the inhibition rate, the higher the activity).

[0032] Fragrant pear PGIP extract: extracted from fragrant pear, used as the sample to be tested.

[0033] PG solution: polygalacturonase solution with known activity, used as reaction substrate.

[0034] Pectic acid solution: as a substrate for PG, used to generate galacturonic acid.

[0035] DNS reagent (3,5-dinitrosalicylic acid reagent): used to react with galacturonic acid to produce a reddish-brown substance for easy determination.

[0036] Buffer: used to adjust the pH value and ionic strength of the reaction system.

[0037] Spectrophotometer: used to measure the absorbance and thus calculate the content of galacturonic acid.

[0038] Control group: Take a certain amount of pectin acid solution, add an appropriate amount of buffer (phosphate buffer solution) and DNS reagent, then add a certain amount of PG solution, mix well and place in a constant temperature water bath for a certain period of time.

[0039] Experimental group: On the basis of the control group, an appropriate amount of pear PGIP extract (purified extract dissolved in buffer) was added, mixed and placed in a constant temperature water bath for insulation.

[0040] After the reaction was completed, the reaction system was taken out and cooled to room temperature.

[0041] Use a spectrophotometer to measure the absorbance of the reaction system at a specific wavelength (such as 540 nm).

[0042] According to the absorbance value, the content of galacturonic acid was calculated.

[0043] The galacturonic acid content in the control group and the experimental group was compared to evaluate the inhibitory effect of PGIP on PG activity.

[0044] The inhibition rate can be calculated, that is, (galacturonic acid content in the control group - galacturonic acid content in the experimental group) / galacturonic acid content in the control group × 100%.

[0045] According to the above test method, the inhibition rate of the polygalacturonase inhibitory proteins of Examples 1 to 3 and Comparative Examples 1 to 7 of the present invention on PG was tested, and the results are shown in Table 1; Table 1 Extraction rate, purity and inhibition rate It can be seen from Table 1 that the extraction and purification methods of the fragrant pear polygalacturonase inhibitory protein provided in Examples 1-3 of the present invention significantly improve the protein extraction rate and protein purity compared with comparative examples 1 to 7, and at the same time have higher inhibitory activity. This shows that the present invention uses fragrant pear as a raw material, and cooperates with reasonable, simple and easy-to-operate process steps to obtain a fragrant pear polygalacturonase inhibitory protein with high extraction rate, high purity and good biological activity, which provides high-quality raw materials for subsequent research and application.

[0046] The number of devices and processing scales described here are used to simplify the description of the present invention. Applications, modifications and variations of the present invention will be obvious to those skilled in the art.

[0047] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.

Claims

1. A method for extracting and purifying fragrant pear polygalacturonase inhibitory protein, characterized in that: The following steps are involved: Step 1, remove the core of the fragrant pear fruit, and crush it with an ultra-fine grinder to obtain a fragrant pear crushed material; Step 2: Filter the crushed pear with filter paper to collect the first supernatant, precipitate the first supernatant with ammonium sulfate solution, remove the solvent by centrifugation, and collect the precipitate; Step 3, dissolving the precipitate with a buffer solution, collecting the third supernatant by centrifugation, dialyzing the third supernatant with a buffer solution, and then filtering with a 0.45 um filter membrane to obtain a crude extract; Step 4: Purify the crude extract using a gel filtration chromatography column to obtain a purified extract, and freeze-dry to obtain the fragrant pear polygalacturonase inhibitory protein; Among them, in step one, during the crushing process with an ultrafine grinder, a stabilizer is added to the ultrafine grinder and the crushing operation is performed together, and the weight ratio of the stabilizer to the pitted pear fruit is 0.01~0.03:1; the stabilizer includes 0.005~0.01 parts of dithiothreitol, 0.15~0.25 parts of polyvinyl pyrrolidone, and 0.3~0.5 parts of a protease inhibitor.

2. The method for extracting and purifying the fragrant pear polygalacturonase inhibitory protein according to claim 1, characterized in that: In step 2, the first supernatant is precipitated with an ammonium sulfate solution, the solvent is removed by centrifugation, and the precipitate is collected, which specifically includes: Step 201, adding ammonium sulfate to the first supernatant to a saturation of 35%, standing at 4°C for 1-2 hours, and then centrifuging at 2000-3000 r / min to obtain a first precipitate and a second supernatant A; Step 202, adding ammonium sulfate to the second supernatant A until the saturation is 55%, standing at 4°C for 1-2 hours, and then centrifuging at 4000-5500 r / min to obtain a second precipitate and a second supernatant B; Step 203, adding ammonium sulfate to the second supernatant B to a saturation of 75%, standing at 4°C for 1-2 hours, and then centrifuging at 5000-6500 r / min to obtain a third precipitate.

3. The method for extracting and purifying the fragrant pear polygalacturonase inhibitory protein according to claim 2, characterized in that: In step 201, step 202 and step 203, before adding ammonium sulfate, 0.02-0.05 mol / L of sodium L-ascorbate and 0.03-0.05 mol / L of disodium ethylenediaminetetraacetate are added to the first supernatant / second supernatant A / second supernatant B.

4. The method for extracting and purifying the fragrant pear polygalacturonase inhibitory protein according to claim 3, characterized in that: Step three is as follows: Step 301, dissolving the first precipitate with a first buffer solution, and collecting the third supernatant A by centrifugation, dialyzing the third supernatant A with a first buffer solution, using a dialysis bag with a molecular weight cutoff of 10 kDa as the dialysis membrane, dialyzing for 24 hours at 4° C., changing the dialysate twice during the period, and obtaining a dialysate A; the first buffer solution is a phosphate buffer solution containing 0.01 to 0.02 mol / L sodium chloride and a pH of 6.8 to 7.5 and a concentration of 0.1 to 0.2 mol / L; Step 302, dissolving the second precipitate with a second buffer solution, and collecting the third supernatant B by centrifugation, dialyzing the third supernatant A with a second buffer solution, using a dialysis bag with a molecular weight cutoff of 10 kDa as the dialysis membrane, dialyzing for 24 hours at 4° C., changing the dialysate twice during the period, and obtaining a dialysate B; the second buffer solution is a phosphate buffer solution containing 0.01 to 0.02 mol / L sodium chloride and a pH of 6.8 to 7.5 and a concentration of 0.02 to 0.03 mol / L; Step 303, dissolving the third precipitate with a third buffer solution, collecting a third supernatant C by centrifugation, dialyzing the third supernatant C with a third buffer solution, using a dialysis bag with a molecular weight cutoff of 10 kDa as the dialysis membrane, dialyzing for 24 hours at 4° C., replacing the dialysate twice during the period, and obtaining a dialysate C; the third buffer solution is a phosphate buffer solution containing 0.01 to 0.02 mol / L sodium chloride and a pH of 6.8 to 7.5 and 0.008 to 0.012 mol / L; Step 304: Mix dialysate A, dialysate B, and dialysate C, transfer the mixture into distilled water, and dialyze at 4° C. for 24 to 48 hours to obtain dialysate E. A dialysis bag with a molecular weight cutoff of 10 kDa is used as the dialysis membrane. The dialysate is replaced every 12 hours. Filter dialysate E with a 0.45 um filter membrane to obtain a crude extract.

5. The method for extracting and purifying the fragrant pear polygalacturonase inhibitory protein according to claim 1, characterized in that: In step 2, before filtering the fragrant pear crushed material with filter paper, it is also pretreated. The fragrant pear crushed material is placed in an ice bath at 0°C, acetone 5 times the weight of the fragrant pear crushed material is added, and after stirring evenly, it is filtered and rinsed with trimethylsilyl ether to obtain the pretreated fragrant pear crushed material, which is then filtered with filter paper.

6. The method for extracting and purifying the fragrant pear polygalacturonase inhibitory protein according to claim 2, characterized in that: Protease inhibitors were diisopropylfluorophosphate or iodoacetic acid.

7. The method for extracting and purifying the fragrant pear polygalacturonase inhibitory protein according to claim 1, characterized in that: Step 4 is as follows: Step 401: pre-treat the crude extract with a pH of 5.5 containing 5×10 -5 1 mol / L EDTA-Na2 in 25 mmol / L sodium acetate buffer, followed by a CM-Sephadex C-50 column at pH 5.5 containing 5×10 -5 25mmol / L sodium acetate buffer containing mol / L EDTA-Na2 was washed to A 280 <0.02, and then gradient elution was performed with sodium acetate buffer containing 0.1~1 mol / L NaCl to collect the initially purified active components; Step 402: The preliminarily purified active component is preliminarily treated with a pH of 5.5 containing 5×10 -5 A Sephadex G-100 column (1 cm × 70 cm) was equilibrated with 25 mmol / L sodium acetate buffer containing 5 × 10 -5 The extract was eluted with 25 mmol / L sodium acetate buffer containing mol / LEDTA-Na2 and the active fraction was collected to obtain the purified extract.

8. The method for extracting and purifying the fragrant pear polygalacturonase inhibitory protein according to claim 7, characterized in that: CM-Sephadex C-50 gel and Sephadex G-100 gel were pretreated before loading, specifically: Swelling treatment: soak the gel in an ethanol solution with a volume fraction of 50-60%. After removing the ethanol solution, soak it in sterile water and soak it under magnetic stirring at room temperature for at least 24 hours. Then take out the gel and rinse it with sterile water until the washing water is colorless. Wrap the gel with three layers of sterile filter cloth and let it stand for 30 minutes. During this period, replace the filter cloth every 10 minutes. Acid-base treatment: soak the swollen gel in an equal volume of 1.0 mol / L NaOH solution, stir at 50 rpm / min for 1 h, take out the gel after soaking, and then wash the gel with sterile water until the washing solution is neutral; then soak the gel in an equal volume of 0.1 mol / L HCl solution, stir at 50 rpm / min for 1 h, take out the gel after soaking, and then wash the gel with sterile water until the washing solution is neutral; wrap the gel with three layers of sterile filter cloth and let it stand for 2 h, during which the filter cloth was replaced every 30 minutes; Degassing treatment: Place the gel after acid and alkali treatment in an ultrasonic device and perform ultrasonic treatment at a frequency of 20-40kHz for 20 minutes. Then place the gel in a suction filtration bottle, plug the bottle mouth with an ear bulb, and reduce the pressure and evacuate for 30 minutes to remove tiny bubbles in the gel.

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