Method for extracting and purifying a polygalacturonase-inhibiting protein from a pyrus communis

Abstract

The application discloses a method for extracting and purifying a pectinase inhibitor protein of fragrant pear, which comprises the following steps: firstly, crushing fragrant pear to obtain a crushed product of fragrant pear; secondly, precipitating the crushed product after filtration by using an ammonium sulfate solution; thirdly, dissolving the precipitate by using a buffer solution, performing dialysis preliminary purification, and obtaining a crude extract; and fourthly, purifying the crude extract by using a gel filtration chromatography column, obtaining a purified extract, and drying the purified extract to obtain the product. The application uses natural fragrant pear as a raw material, does not depend on chemical synthesis or genetic engineering technology, and conforms to the green, environment-friendly and sustainable development concept. The extraction and purification process of the application comprises multiple purification processes such as filtration, precipitation, dissolution, dialysis and filter membrane filtration, and uses conventional laboratory technologies such as filter paper filtration, centrifugation and dialysis, so that the whole process is easy to implement and repeat, the feasibility and reliability of the experiment are improved, and the purity, biological activity and extraction rate of the extract are significantly improved.

Description

Technical Field

[0001] This invention relates to the field of protein extraction technology. More specifically, this invention relates to a method for extracting and purifying polygalacturonase-inhibited protein from pear. Background Technology

[0002] Korla fragrant pears are a specialty fruit of Xinjiang, mainly distributed in the Korla region. They are highly favored by consumers both domestically and internationally for their thin skin, delicate flesh, crisp and juicy texture, sweet and sour taste, and rich nutritional value. They are also a key export product for Xinjiang. In recent years, the planting area and yield of Korla fragrant pears have increased year by year, becoming a major source of income for local farmers. However, fragrant pears are susceptible to infection by various pathogens during post-harvest storage, transportation, and sales, posing a serious threat to the development of the fragrant pear industry and export trade. The main pathogenic fungi include Alternaria, Aspergillus niger, Botrytis cinerea, and Penicillium expansum. Currently, chemical preservatives are mainly used for treatment, which, while effective, affects the quality of the fruit, thus restricting the export of fragrant pears. Furthermore, Xinjiang is also my country's largest grape production base. The main pathogen causing post-harvest diseases in grapes is Botrytis cinerea. Currently, SO2-based chemical preservatives are used for grapes, which damage the grape fruit and pollute the environment. Therefore, there is a need to develop highly efficient and low-toxicity biological preservatives for Xinjiang's specialty fruits and vegetables.

[0003] The cell wall is the first line of defense for plant cells against pathogen infection. Inhibiting pathogen degradation of the cell wall is an important pathway for plants to resist infection. Polygalacturonase-inhibiting protein (PGIP) can inhibit polygalacturonase (PG), activating the plant's defense system to protect the integrity of the cell wall. Studies have shown that PGIP has an inhibitory effect on pathogenic fungi's PG, and specifically inhibits only the PG secreted by pathogenic fungi, but not the PG produced by plants, bacteria, or other microorganisms. PGIP is concentrated in the plant cell wall, but its distribution varies among different varieties, developmental stages, and organs. Since plants have low protein content, it is necessary to separate and purify extracts containing PGIP.

[0004] Currently, many scholars have conducted in-depth research on the isolation, purification, structure, and function of PGIP. Commonly used separation methods include acetone, ammonium sulfate, and polyethylene glycol precipitation; commonly used purification methods include chromatography, electrophoresis, and ultrafiltration. However, existing PGIP extraction and purification technologies still suffer from problems such as low extraction rates and poor bioactivity due to protein hydrolysis and incomplete removal of impurities during the extraction and purification process. Furthermore, there are currently no reports on PGIP from Korla fragrant pear. Therefore, it is essential to develop a rapid and efficient method for the isolation and purification of Korla fragrant pear PGIP to lay a foundation for its development and utilization. Summary of the Invention

[0005] One object of the present invention is to solve at least the above-mentioned problems and to provide at least the advantages that will be described later.

[0006] Another objective of this invention is to provide a method for extracting and purifying polygalacturonase inhibitory protein from pear, which at least solves the aforementioned technical problems.

[0007] To achieve these objectives and other advantages according to the present invention, a method for extracting and purifying polygalacturonase inhibitory protein from pear is provided, comprising the following steps:

[0008] Step 1: Remove the core from the pear and crush it using an ultra-fine grinder to obtain pear powder.

[0009] Step 2: Filter the crushed pears with filter paper, collect the first supernatant, precipitate the first supernatant with ammonium sulfate solution, centrifuge to remove the solvent, and collect the precipitate;

[0010] Step 3: Dissolve the precipitate with a buffer solution, centrifuge to collect the third supernatant, dialyze the third supernatant with a buffer solution, and then filter it through a 0.45 μm filter membrane to obtain the crude extract;

[0011] Step 4: Purify the crude extract using a gel filtration chromatography column to obtain a purified extract, which is then freeze-dried to obtain pear polygalacturonase inhibitor protein.

[0012] In step one, during the crushing process using an ultra-fine pulverizer, a stabilizer is added to the ultra-fine pulverizer and crushed together. The weight ratio of the stabilizer to the cored pear fruit is 0.01~0.03:1. The stabilizer includes 0.005~0.01 parts of dithiothreitol, 0.15~0.25 parts of polyvinylpyrrolidone, and 0.3~0.5 parts of protease inhibitor.

[0013] Preferably, in the method for extracting and purifying pear polygalacturonase inhibitory protein, step two involves precipitating the first supernatant with ammonium sulfate solution, centrifuging to remove the solvent, and collecting the precipitate. Specifically, this includes:

[0014] Step 201: Add ammonium sulfate to the first supernatant until the saturation is 35%, let it stand at 4°C for 1-2 hours, and then centrifuge at 2000-3000 r / min to obtain the first precipitate and the second supernatant A;

[0015] Step 202: Add ammonium sulfate to the second supernatant A until the saturation is 55%, let it stand at 4°C for 1-2 hours, and then centrifuge at 4000-5500 r / min to obtain the second precipitate and the second supernatant B.

[0016] Step 203: Add ammonium sulfate to the second supernatant B until the saturation is 75%, let it stand at 4°C for 1-2 hours, and then centrifuge at 5000-6500 r / min to obtain the third precipitate.

[0017] Preferably, in the method for extracting and purifying polygalacturonase inhibitory protein from pear, 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.

[0018] Preferably, in the method for extracting and purifying the polygalacturonase inhibitory protein from pear, step three specifically comprises:

[0019] Step 301: Dissolve the first precipitate with the first buffer solution and centrifuge to collect the third supernatant A. Dialyze the third supernatant A with the first buffer solution using a dialysis bag with a molecular weight cutoff of 10 kDa. Dialyze at 4°C for 24 h, changing the dialysis solution twice during the process to obtain dialysate A. The first buffer solution is a 0.1–0.2 mol / L phosphate buffer solution containing 0.01–0.02 mol / L sodium chloride and a pH of 6.8–7.5.

[0020] Step 302: Dissolve the second precipitate with the second buffer solution and centrifuge to collect the third supernatant B. Dialyze the third supernatant A with the second buffer solution using a dialysis bag with a molecular weight cutoff of 10 kDa. Dialyze at 4°C for 24 h, changing the dialysis solution twice during the process to obtain dialysate B. The second buffer solution is a 0.02–0.03 mol / L phosphate buffer solution containing 0.01–0.02 mol / L sodium chloride and with a pH of 6.8–7.5.

[0021] Step 303: Dissolve the third precipitate with the third buffer solution and collect the third supernatant C by centrifugation. Dialyze the third supernatant C with the third buffer solution using a dialysis bag with a molecular weight cutoff of 10 kDa. Dialyze at 4°C for 24 h, changing the dialysis solution twice during the process to obtain dialysate C. The third buffer solution is a 0.008–0.012 mol / L phosphate buffer solution containing 0.01–0.02 mol / L sodium chloride and a pH of 6.8–7.5.

[0022] Step 304: Mix dialysate A, dialysate B, and dialysate C, and transfer them to distilled water. Dialyze at 4°C for 24-48 hours to obtain dialysate E. Use a dialysis bag with a molecular weight cutoff of 10 kDa as the dialysis membrane. Replace the dialysate every 12 hours. Filter dialysate E through a 0.45 μm filter membrane to obtain the crude extract.

[0023] Preferably, in the method for extracting and purifying polygalacturonase inhibitory protein from pear, in step two, before filtering the pear pulverized material with filter paper, a pretreatment is performed: the pear pulverized material is placed in an ice bath at 0°C, acetone with 5 times its weight of the pear pulverized material is added, stirred evenly, filtered, and washed with trimethylsilyl ether to obtain the pretreated pear pulverized material, which is then filtered with filter paper.

[0024] Preferably, in the method for extracting and purifying the polygalacturonase inhibitor protein from pear, the protease inhibitor is diisopropylfluorophosphate or iodoacetic acid.

[0025] Preferably, in the method for extracting and purifying the polygalacturonase inhibitory protein from pear, step four specifically comprises:

[0026] Step 401: The crude extract is pre-treated with pH 5.5 containing 5 × 10⁻⁶ mg / L. -5 A CM-Sephadex C-50 column was equilibrated with 25 mmol / L sodium acetate buffer containing mol / L EDTA-Na2, followed by a pH 5.5 solution containing 5 × 10 mol / L EDTA-Na2. -5 Wash with 25 mmol / L sodium acetate buffer containing mol / L EDTA-Na2 until A 280 If the concentration is less than 0.02, then perform gradient elution with sodium acetate buffer containing 0.1-1 mol / L NaCl and collect the preliminarily purified active component.

[0027] Step 402: Load the pre-purified active component onto a solution pre-treated at pH 5.5 containing 5 × 10⁻⁶ mg / L. -5A Sephadex G-100 column (1 cm × 70 cm) equilibrated with 25 mmol / L sodium acetate buffer containing 5 × 10 mol / L EDTA-Na2 was used. -5 The active fraction was eluted with 25 mmol / L sodium acetate buffer containing 1 mol / L LEDTA-Na2, and the purified extract was collected.

[0028] Preferably, in the method for extracting and purifying the polygalacturonase inhibitory protein from pear, both the CM-Sephadex C-50 gel and the Sephadex G-100 gel are pretreated before being loaded onto the column, specifically as follows:

[0029] Swelling treatment: Immerse the gel in an ethanol solution with a volume fraction of 50-60%. After removing the ethanol solution, immerse it in sterile water and stir it magnetically at room temperature for at least 24 hours. Then remove the gel and rinse it with sterile water until the washing water is colorless. Wrap the gel in three layers of sterile filter cloth and let it stand for 30 minutes. Replace the filter cloth every 10 minutes during this period.

[0030] Acid-base treatment: After swelling, the gel was immersed in an equal volume of 1.0 mol / L NaOH solution with stirring at 50 rpm for 1 hour. After immersion, the gel was removed and washed with sterile water until the washing solution was neutral. Then, the gel was immersed in an equal volume of 0.1 mol / L HCl solution with stirring at 50 rpm for 1 hour. After immersion, the gel was removed and washed with sterile water until the washing solution was neutral. The gel was then wrapped in three layers of sterile filter cloth and allowed to stand for 2 hours, with the filter cloth being replaced every 30 minutes.

[0031] Degassing treatment: The acid-base treated gel is placed in an ultrasonic device and ultrasonically treated at a frequency of 20~40kHz for 20 minutes. Then the gel is placed in a vacuum filtration flask, the mouth of the flask is plugged with a rubber bulb, and the pressure is reduced for 30 minutes to remove the tiny air bubbles in the gel.

[0032] The present invention has at least the following beneficial effects:

[0033] 1. This invention uses natural fragrant pear fruit as raw material, without relying on chemical synthesis or genetic engineering technology, which is in line with the green, environmentally friendly and sustainable development concept. The extraction and purification process of this invention includes a multi-step purification process such as filtration, precipitation, dissolution, dialysis and membrane filtration. It uses conventional laboratory techniques such as filter paper filtration, centrifugation and dialysis, which makes the whole process easy to implement and repeat, improves the feasibility and reliability of the experiment, and significantly improves the purity, bioactivity and extraction rate of the extract, providing high-quality raw materials for subsequent research and application. Furthermore, by optimizing the extraction and purification steps, cumbersome operations and reagent consumption are reduced, thereby reducing production costs.

[0034] 2. This invention utilizes an ultrafine pulverizer to crush pear fruit and adds stabilizers, effectively improving the uniformity and efficiency of cell crushing. This allows for the more complete release of polygalacturonase inhibitory proteins within the cells, while preventing protein degradation during the pulverization process, thereby increasing the extraction rate of polygalacturonase inhibitory proteins. The stabilizers include dithiothreitol, polyvinylpyrrolidone, and protease inhibitors. These stabilizers work together to provide a stable microenvironment for the proteins, helping to maintain their structural and functional integrity. Adding stabilizers during the ultrafine pulverization process not only effectively prevents protein degradation and inactivation during pear pulverization, ensuring the bioactivity of the final product, but also helps improve the physicochemical properties of the solution, such as pH, ionic strength, and viscosity. This ensures that the proteins maintain their native conformation and activity during dissolution, while also significantly affecting protein solubility and stability. This facilitates the separation of proteins from cell fragments during subsequent extraction, thereby improving protein extraction efficiency and bioactivity.

[0035] 3. Dithiothreitol (DTT): DTT is a commonly used reducing agent that prevents protein degradation due to oxidation during extraction. It reacts with disulfide bonds in proteins, reducing them to thiol groups, thereby protecting the protein's conformation and activity. Polyvinylpyrrolidone (PVP): PVP has good water solubility and can form a protective film covering the protein surface, preventing contact with oxygen, enzymes, or other harmful substances in the air, thus reducing protein degradation and denaturation. Since cells or tissues release proteases during lysis, these enzymes may react with the proteins to be extracted, leading to protein decomposition. Therefore, protease inhibitors are added to the stabilizer to inhibit protease activity, prevent protein hydrolysis during extraction, maintain protein integrity, and thus improve extraction efficiency, purity, and the bioactivity of the extracted product.

[0036] 4. This invention uses ammonium sulfate concentration gradient precipitation to precipitate the first supernatant. The pretreated sample is added to the ammonium sulfate gradient solution in sequence. Under centrifugation, the protein will precipitate in different ammonium sulfate solutions according to the concentration gradient. This can separate a variety of proteins with different molecular weights, enhance the biological activity of the final extract, and produce highly purified protein.

[0037] 5. Sodium L-ascorbate is the sodium salt of vitamin C and has antioxidant properties. Adding sodium L-ascorbate during ammonium sulfate precipitation of proteins prevents oxidative denaturation. Disodium ethylenediaminetetraacetate (EDTA) is a commonly used metal ion chelating agent that effectively removes metal ions such as iron and copper ions from solution. Adding EDTA during ammonium sulfate precipitation helps remove harmful metal ions, thereby improving protein purity. Further purification of the precipitate using multi-stage dialysis, and targeted dialysis of the precipitates obtained from each stage, significantly improves protein bioactivity, extraction rate, and extraction purity.

[0038] 6. Treating the pulverized pear with acetone can disrupt cell membranes and break the binding between proteins and lipids. After acetone treatment, trimethylsilyl ether is used for further treatment. The ether can passivate some of the exposed active groups of the protein, reducing damage to the active groups during the extraction process. The stabilizer in the pretreatment process plays a protective role for the protein, improves the protein's biological activity, and can also improve the purity and extraction rate of the extracted protein.

[0039] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Detailed Implementation

[0040] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0041] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0042] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0043] <Example 1>

[0044] This invention provides a method for extracting and purifying polygalacturonase inhibitory protein from pear, comprising the following steps:

[0045] Step 1: Raw material crushing

[0046] Core the fragrant pears, add a stabilizer, and crush them together in an ultrafine pulverizer. The weight ratio of the stabilizer to the cored fragrant pears is 0.01:1. The stabilizer includes 0.005 parts of dithiothreitol, 0.15 parts of polyvinylpyrrolidone, and 0.3 parts of a protease inhibitor. The mixture is crushed in an ultrafine pulverizer to obtain fragrant pear powder. The protease inhibitor is diisopropylfluorophosphate or iodoacetic acid.

[0047] Step 2: Precipitation Extraction

[0048] Place the crushed pear powder in an ice bath at 0°C, add acetone at 5 times its weight, stir well, filter, and wash with trimethylsilyl ether to obtain the pretreated pear powder. Filter the pretreated pear powder through filter paper and collect the first supernatant. Precipitate the first supernatant with ammonium sulfate solution, centrifuge to remove the solvent, and collect the precipitate. Specifically:

[0049] Step 201: Add ammonium sulfate to the first supernatant until the saturation is 35%, let it stand at 4°C for 1 hour, and then centrifuge at 2000 r / min to obtain the first precipitate and the second supernatant A;

[0050] Step 202: Add ammonium sulfate to the second supernatant A until the saturation is 55%, let it stand at 4°C for 1 hour, and then centrifuge at 4000 r / min to obtain the second precipitate and the second supernatant B;

[0051] Step 203: Add ammonium sulfate to the second supernatant B until the saturation is 75%, let it stand at 4°C for 1 hour, and then centrifuge at 5000 r / min to obtain the third precipitate;

[0052] In steps 201, 202, and 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 / second supernatant A / second supernatant B.

[0053] Step 3: Dialysis purification

[0054] The precipitate was dissolved in a buffer solution, specifically as follows:

[0055] Step 301: Dissolve the first precipitate with the first buffer solution and centrifuge to collect the third supernatant A. Dialyze the third supernatant A with the first buffer solution using a dialysis bag with a molecular weight cutoff of 10 kDa. Dialyze at 4°C for 24 h, changing the dialysis solution twice during the process to obtain 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.

[0056] Step 302: Dissolve the second precipitate with the second buffer solution and centrifuge to collect the third supernatant B. Dialyze the third supernatant A with the second buffer solution using a dialysis bag with a molecular weight cutoff of 10 kDa. Dialyze at 4°C for 24 hours, changing the dialysis solution twice during the process to obtain 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.

[0057] Step 303: Dissolve the third precipitate with the third buffer solution and centrifuge to collect the third supernatant C. Dialyze the third supernatant C with the third buffer solution using a dialysis bag with a molecular weight cutoff of 10 kDa. Dialyze at 4°C for 24 h, changing the dialysis solution twice during the process to obtain 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.

[0058] Step 304: Mix dialysate A, dialysate B, and dialysate C, and transfer them to distilled water. Dialyze at 4°C for 24 hours to obtain dialysate E. Use a dialysis bag with a molecular weight cutoff of 10 kDa as the dialysis membrane. Replace the dialysate every 12 hours during the dialysis process. Filter dialysate E through a 0.45 μm filter membrane to obtain the crude extract.

[0059] Step 4: Column chromatography purification

[0060] The crude extract was purified using gel filtration chromatography to obtain a purified extract, which was then freeze-dried to obtain pear polygalacturonase inhibitor protein. The purification process was as follows:

[0061] Step 401: The crude extract is pre-treated with pH 5.5 containing 5 × 10⁻⁶ mg / L. -5 A CM-Sephadex C-50 column was equilibrated with 25 mmol / L sodium acetate buffer containing mol / L EDTA-Na2, followed by a pH 5.5 solution containing 5 × 10 mol / L EDTA-Na2. -5 Wash with 25 mmol / L sodium acetate buffer containing mol / L EDTA-Na2 until A 280 If the concentration is less than 0.02, then perform gradient elution with sodium acetate buffer containing 0.1 mol / L NaCl and collect the preliminarily purified active component.

[0062] Step 402: Load the pre-purified active component onto a solution pre-treated at pH 5.5 containing 5 × 10⁻⁶ mg / L. -5 A Sephadex G-100 column (1 cm × 70 cm) equilibrated with 25 mmol / L sodium acetate buffer containing 5 × 10 mol / L EDTA-Na2 was used. -5The active fraction was eluted with 25 mmol / L sodium acetate buffer containing mol / L LEDTA-Na2, and the purified extract was collected.

[0063] Both CM-Sephadex C-50 gel and Sephadex G-100 gel underwent pretreatment before being loaded onto the column, specifically as follows:

[0064] Swelling treatment: Immerse the gel in a 50% ethanol solution by volume. After removing the ethanol solution, immerse it in sterile water and stir magnetically at room temperature for at least 24 hours. Then remove the gel and rinse it with sterile water until the washing water is colorless. Wrap the gel in three layers of sterile filter cloth and let it stand for 30 minutes, changing the filter cloth every 10 minutes during this period.

[0065] Acid-base treatment: After swelling, the gel was immersed in an equal volume of 1.0 mol / L NaOH solution with stirring at 50 rpm for 1 hour. After immersion, the gel was removed and washed with sterile water until the washing solution was neutral. Then, the gel was immersed in an equal volume of 0.1 mol / L HCl solution with stirring at 50 rpm for 1 hour. After immersion, the gel was removed and washed with sterile water until the washing solution was neutral. The gel was then wrapped in three layers of sterile filter cloth and allowed to stand for 2 hours, with the filter cloth being replaced every 30 minutes.

[0066] Degassing treatment: The acid-base treated gel is placed in an ultrasonic device and ultrasonically treated at a frequency of 20~40kHz for 20 minutes. Then the gel is placed in a vacuum filtration flask, the mouth of the flask is plugged with a rubber bulb, and the pressure is reduced for 30 minutes to remove the tiny air bubbles in the gel.

[0067] <Example 2>

[0068] This invention provides a method for extracting and purifying polygalacturonase inhibitory protein from pear, comprising the following steps:

[0069] Step 1: Raw material crushing

[0070] Core the fragrant pears, add a stabilizer, and crush them together in an ultrafine pulverizer. The weight ratio of the stabilizer to the cored fragrant pears is 0.03:1. The stabilizer includes 0.01 parts of dithiothreitol, 0.25 parts of polyvinylpyrrolidone, and 0.5 parts of a protease inhibitor. The mixture is crushed using an ultrafine pulverizer to obtain fragrant pear powder. The protease inhibitor is diisopropylfluorophosphate or iodoacetic acid.

[0071] Step 2: Precipitation Extraction

[0072] Place the crushed pear powder in an ice bath at 0°C, add acetone at 5 times its weight, stir well, filter, and wash with trimethylsilyl ether to obtain the pretreated pear powder. Filter the pretreated pear powder through filter paper and collect the first supernatant. Precipitate the first supernatant with ammonium sulfate solution, centrifuge to remove the solvent, and collect the precipitate. Specifically:

[0073] Step 201: Add ammonium sulfate to the first supernatant until the saturation is 35%, let it stand at 4°C for 2 hours, and then centrifuge at 3000 r / min to obtain the first precipitate and the second supernatant A;

[0074] Step 202: Add ammonium sulfate to the second supernatant A until the saturation is 55%, let it stand at 4°C for 2 hours, and then centrifuge at 5500 r / min to obtain the second precipitate and the second supernatant B;

[0075] Step 203: Add ammonium sulfate to the second supernatant B until the saturation is 75%, let it stand at 4°C for 2 hours, and then centrifuge at 6500 r / min to obtain the third precipitate and the second supernatant C;

[0076] In steps 201, 202, and 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 / second supernatant A / second supernatant B.

[0077] Step 3: Dialysis purification

[0078] The precipitate was dissolved in a buffer solution, specifically as follows:

[0079] Step 301: Dissolve the first precipitate with the first buffer solution and centrifuge to collect the third supernatant A. Dialyze the third supernatant A with the first buffer solution using a dialysis bag with a molecular weight cutoff of 10 kDa. Dialyze at 4°C for 24 h, changing the dialysis solution twice during the process to obtain 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.

[0080] Step 302: Dissolve the second precipitate with the second buffer solution and centrifuge to collect the third supernatant B. Dialyze the third supernatant A with the second buffer solution using a dialysis bag with a molecular weight cutoff of 10 kDa. Dialyze at 4°C for 24 hours, changing the dialysis solution twice during the process to obtain 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.

[0081] Step 303: Dissolve the third precipitate with the third buffer solution and centrifuge to collect the third supernatant C. Dialyze the third supernatant C with the third buffer solution using a dialysis bag with a molecular weight cutoff of 10 kDa. Dialyze at 4°C for 24 h, changing the dialysis solution twice during the process to obtain 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.

[0082] Step 304: Mix dialysate A, dialysate B, and dialysate C, and transfer them to distilled water. Dialyze at 4°C for 48 hours to obtain dialysate E. Use a dialysis bag with a molecular weight cutoff of 10 kDa as the dialysis membrane. Replace the dialysate every 12 hours during the dialysis process. Filter dialysate E through a 0.45 μm filter membrane to obtain the crude extract.

[0083] Step 4: Column chromatography purification

[0084] The crude extract was purified using gel filtration chromatography to obtain a purified extract, which was then freeze-dried to obtain pear polygalacturonase inhibitor protein. The purification process was as follows:

[0085] Step 401: The crude extract is pre-treated with pH 5.5 containing 5 × 10⁻⁶ mg / L. -5 A CM-Sephadex C-50 column was equilibrated with 25 mmol / L sodium acetate buffer containing mol / L EDTA-Na2, followed by a pH 5.5 solution containing 5 × 10 mol / L EDTA-Na2. -5 Wash with 25 mmol / L sodium acetate buffer containing mol / L EDTA-Na2 until A 280 If the concentration is less than 0.02, then perform gradient elution with sodium acetate buffer containing 1 mol / L NaCl and collect the preliminarily purified active component.

[0086] Step 402: Load the pre-purified active component onto a solution pre-treated at pH 5.5 containing 5 × 10⁻⁶ mg / L. -5 A Sephadex G-100 column (1 cm × 70 cm) equilibrated with 25 mmol / L sodium acetate buffer containing 5 × 10 mol / L EDTA-Na2 was used. -5 The active fraction was eluted with 25 mmol / L sodium acetate buffer containing mol / L LEDTA-Na2, and the purified extract was collected.

[0087] Both CM-Sephadex C-50 gel and Sephadex G-100 gel underwent pretreatment before being loaded onto the column, specifically as follows:

[0088] Swelling treatment: Immerse the gel in a 60% ethanol solution by volume. After removing the ethanol solution, immerse it in sterile water and stir magnetically at room temperature for at least 24 hours. Then remove the gel and rinse it with sterile water until the washing water is colorless. Wrap the gel in three layers of sterile filter cloth and let it stand for 30 minutes, changing the filter cloth every 10 minutes during this period.

[0089] Acid-base treatment: After swelling, the gel was immersed in an equal volume of 1.0 mol / L NaOH solution with stirring at 50 rpm for 1 hour. After immersion, the gel was removed and washed with sterile water until the washing solution was neutral. Then, the gel was immersed in an equal volume of 0.1 mol / L HCl solution with stirring at 50 rpm for 1 hour. After immersion, the gel was removed and washed with sterile water until the washing solution was neutral. The gel was then wrapped in three layers of sterile filter cloth and allowed to stand for 2 hours, with the filter cloth being replaced every 30 minutes.

[0090] Degassing treatment: The acid-base treated gel is placed in an ultrasonic device and ultrasonically treated at a frequency of 20~40kHz for 20 minutes. Then the gel is placed in a vacuum filtration flask, the mouth of the flask is plugged with a rubber bulb, and the pressure is reduced for 30 minutes to remove the tiny air bubbles in the gel.

[0091] <Example 3>

[0092] This invention provides a method for extracting and purifying polygalacturonase inhibitory protein from pear, comprising the following steps:

[0093] Step 1: Raw material crushing

[0094] Core the fragrant pears, add a stabilizer, and crush them together in an ultrafine pulverizer. The weight ratio of the stabilizer to the cored fragrant pears is 0.02:1. The stabilizer includes 0.008 parts of dithiothreitol, 0.2 parts of polyvinylpyrrolidone, and 0.4 parts of a protease inhibitor. The mixture is crushed in an ultrafine pulverizer to obtain fragrant pear powder. The protease inhibitor is diisopropylfluorophosphate or iodoacetic acid.

[0095] Step 2: Precipitation Extraction

[0096] Place the crushed pear powder in an ice bath at 0°C, add acetone at 5 times its weight, stir well, filter, and wash with trimethylsilyl ether to obtain the pretreated pear powder. Filter the pretreated pear powder through filter paper and collect the first supernatant. Precipitate the first supernatant with ammonium sulfate solution, centrifuge to remove the solvent, and collect the precipitate. Specifically:

[0097] Step 201: Add ammonium sulfate to the first supernatant until the saturation is 35%, let it stand at 4°C for 1.5 h, and then centrifuge at 2500 r / min to obtain the first precipitate and the second supernatant A;

[0098] Step 202: Add ammonium sulfate to the second supernatant A until the saturation is 55%, let it stand at 4°C for 1.5 h, and then centrifuge at 5000 r / min to obtain the second precipitate and the second supernatant B;

[0099] Step 203: Add ammonium sulfate to the second supernatant B until the saturation is 75%, let it stand at 4°C for 1.5 h, and then centrifuge at 6000 r / min to obtain the third precipitate and the second supernatant C;

[0100] In steps 201, 202 and 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 / second supernatant A / second supernatant B.

[0101] Step 3: Dialysis purification

[0102] The precipitate was dissolved in a buffer solution, specifically as follows:

[0103] Step 301: Dissolve the first precipitate with the first buffer solution and centrifuge to collect the third supernatant A. Dialyze the third supernatant A with the first buffer solution using a dialysis bag with a molecular weight cutoff of 10 kDa. Dialyze at 4°C for 24 h, changing the dialysis solution twice during the process to obtain 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.

[0104] Step 302: Dissolve the second precipitate with the second buffer solution and centrifuge to collect the third supernatant B. Dialyze the third supernatant A with the second buffer solution using a dialysis bag with a molecular weight cutoff of 10 kDa. Dialyze at 4°C for 24 hours, changing the dialysis solution twice during the process to obtain 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.

[0105] Step 303: Dissolve the third precipitate with the third buffer solution and centrifuge to collect the third supernatant C. Dialyze the third supernatant C with the third buffer solution using a dialysis bag with a molecular weight cutoff of 10 kDa. Dialyze at 4°C for 24 h, changing the dialysis solution twice during the process to obtain 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.

[0106] Step 304: Mix dialysate A, dialysate B, and dialysate C, and transfer them to distilled water. Dialyze at 4°C for 24-48 hours to obtain dialysate E. Use a dialysis bag with a molecular weight cutoff of 10 kDa as the dialysis membrane. Replace the dialysate every 12 hours during the dialysis process. Filter dialysate E through a 0.45 μm filter membrane to obtain the crude extract.

[0107] Step 4: Column chromatography purification

[0108] The crude extract was purified using gel filtration chromatography to obtain a purified extract, which was then freeze-dried to obtain pear polygalacturonase inhibitor protein. The purification process was as follows:

[0109] Step 401: The crude extract is pre-treated with pH 5.5 containing 5 × 10⁻⁶ mg / L. -5 A CM-Sephadex C-50 column was equilibrated with 25 mmol / L sodium acetate buffer containing mol / L EDTA-Na2, followed by a pH 5.5 solution containing 5 × 10 mol / L EDTA-Na2. -5 Wash with 25 mmol / L sodium acetate buffer containing mol / L EDTA-Na2 until A 280 If the concentration is less than 0.02, then perform gradient elution with sodium acetate buffer containing 0.5 mol / L NaCl and collect the preliminarily purified active component.

[0110] Step 402: Load the pre-purified active component onto a solution pre-treated at pH 5.5 containing 5 × 10⁻⁶ mg / L. -5 A Sephadex G-100 column (1 cm × 70 cm) equilibrated with 25 mmol / L sodium acetate buffer containing 5 × 10 mol / L EDTA-Na2 was used. -5 The active fraction was eluted with 25 mmol / L sodium acetate buffer containing mol / L LEDTA-Na2, and the purified extract was collected.

[0111] Both CM-Sephadex C-50 gel and Sephadex G-100 gel underwent pretreatment before being loaded onto the column, specifically as follows:

[0112] Swelling treatment: Immerse the gel in a 55% ethanol solution by volume. After removing the ethanol solution, immerse it in sterile water and stir it magnetically at room temperature for at least 24 hours. Then remove the gel and rinse it with sterile water until the washing water is colorless. Wrap the gel in three layers of sterile filter cloth and let it stand for 30 minutes, changing the filter cloth every 10 minutes during this period.

[0113] Acid-base treatment: After swelling, the gel was immersed in an equal volume of 1.0 mol / L NaOH solution with stirring at 50 rpm for 1 hour. After immersion, the gel was removed and washed with sterile water until the washing solution was neutral. Then, the gel was immersed in an equal volume of 0.1 mol / L HCl solution with stirring at 50 rpm for 1 hour. After immersion, the gel was removed and washed with sterile water until the washing solution was neutral. The gel was then wrapped in three layers of sterile filter cloth and allowed to stand for 2 hours, with the filter cloth being replaced every 30 minutes.

[0114] Degassing treatment: The acid-base treated gel is placed in an ultrasonic device and ultrasonically treated at a frequency of 20~40kHz for 20 minutes. Then the gel is placed in a vacuum filtration flask, the mouth of the flask is plugged with a rubber bulb, and the pressure is reduced for 30 minutes to remove the tiny air bubbles in the gel.

[0115] <Comparative Example 1>

[0116] The difference from Example 3 is that no stabilizer is added during the ultrafine pulverizer crushing process; the other conditions and parameters are the same as in Example 3.

[0117] <Comparative Example 2>

[0118] The difference from Example 3 is that in step two, only step 201 is used for precipitation, and step 301 in step three is used for precipitation extraction and dialysis purification. The other conditions and parameters are the same as in Example 3.

[0119] <Comparative Example 3>

[0120] The difference from Example 3 is that in step two, only step 202 is used for precipitation, and step 302 in step three is used for precipitation extraction and dialysis purification. The other conditions and parameters are the same as in Example 3.

[0121] <Comparative Example 4>

[0122] The difference from Example 3 is that in step two, only step 203 is used for precipitation, and step 303 in step three is used for precipitation extraction and dialysis purification. The other conditions and parameters are the same as in Example 3.

[0123] <Comparative Example 5>

[0124] The difference from Example 3 is that, in steps 201, 202 and 203, before adding ammonium sulfate, 0.02~0.05 mol / L of L-ascorbic acid sodium salt and 0.03~0.05 mol / L of ethylenediaminetetraacetic acid disodium salt are not added to the first supernatant / second supernatant A / second supernatant B; the other conditions and parameters are the same as in Example 3.

[0125] <Comparative Example 6>

[0126] The difference from Example 3 is that no pretreatment was performed before filtering the pulverized pear with filter paper; the other conditions and parameters are the same as in Example 3.

[0127] <Comparative Example 7>

[0128] Step 1: Remove the core from the pear and crush it using an ultra-fine grinder to obtain pear powder.

[0129] Step 2: Filter the pulverized pear mixture using filter paper, collect the first supernatant, precipitate the first supernatant with ammonium sulfate solution, centrifuge to remove the solvent, and collect the precipitate; the ammonium sulfate solution should be saturated at 55%.

[0130] Step 3: The crude extract was purified by Sephadex G-100 molecular sieve gel filtration chromatography to obtain a purified extract, which was then freeze-dried to obtain pear polygalacturonase inhibitor protein. Each of the above steps was carried out using existing technical means.

[0131] <Experimental Example 1>

[0132] The extraction rate and purity of polygalacturonase inhibitory protein in Examples 1-3 and Comparative Examples 1-7 of the present invention were calculated. The extraction rate and purity were calculated as follows: a standard solution of the protein (PGIP) at the inhibition concentration was prepared, and a standard curve between protein content and absorbance was plotted by ultraviolet spectrophotometry; the absorbance value of the first supernatant before extraction was tested, and the protein content A1 mg in the first supernatant was obtained using the standard curve; in the same way, the PGIP content A2 mg in the final freeze-dried extract (the weight of the final freeze-dried extract is A mg) was obtained. The extraction rate = A2 / A1%; purity = A2 / A%; the results are shown in Table 1.

[0133] <Experimental Example 2>

[0134] PGIP can specifically bind to PG, thereby reducing PG activity. The activity of PGIP can be indirectly assessed by measuring the degree of PG degradation of pectinic acid under conditions of PG presence and absence of PGIP. Generally, the content of the degradation product galacturonic acid is directly proportional to the activity of PG; therefore, the amount of galacturonic acid produced can be used to reflect the activity of PG and further evaluate the inhibitory effect of PGIP (a higher inhibition rate indicates higher activity).

[0135] Pear PGIP extract: Extracted from pears and used as the sample to be tested.

[0136] PG solution: a polygalacturonase solution with known activity, used as a reaction substrate.

[0137] Pectic acid solution: used as a substrate for PG to generate galacturonic acid.

[0138] DNS reagent (3,5-dinitrosalicylic acid reagent): used to react with galacturonic acid to produce a reddish-brown substance, which is convenient for determination.

[0139] Buffer solution: Used to adjust the pH and ionic strength of the reaction system.

[0140] Spectrophotometer: Used to measure absorbance, thereby calculating the content of galacturonic acid.

[0141] Control group: Take a certain amount of pectic acid solution, add an appropriate amount of buffer solution (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.

[0142] Experimental group: Based on the control group, an appropriate amount of pear PGIP extract (purified extract dissolved in buffer) was added, mixed well, and then placed in a constant temperature water bath for incubation.

[0143] After the reaction is complete, remove the reaction system and cool it to room temperature.

[0144] The absorbance of the reaction system at a specific wavelength (e.g., 540 nm) is determined using a spectrophotometer.

[0145] The content of galacturonic acid is calculated based on the absorbance value.

[0146] The galacturonic acid content of the control group and the experimental group was compared to evaluate the inhibitory effect of PGIP on PG activity.

[0147] The inhibition rate can be calculated as (galacturonic acid content in the control group - galacturonic acid content in the experimental group) / galacturonic acid content in the control group × 100%.

[0148] The inhibition rate of polygalacturonase inhibitor protein against PG in Examples 1-3 and Comparative Examples 1-7 of the present invention was tested according to the above test method. The results are shown in Table 1.

[0149] Table 1 Extraction rate, purity, and inhibition rate

[0150]

[0151] As shown in Table 1, the extraction and purification methods for pear polygalacturonase inhibitory protein provided in Examples 1-3 of this invention significantly improved the protein extraction rate and protein purity compared to Comparative Examples 1-7, while exhibiting higher inhibitory activity. This demonstrates that by using pear as raw material and combining it with reasonable, simple, and easy-to-operate process steps, this invention can obtain pear polygalacturonase inhibitory protein with high extraction rate, high purity, and good biological activity, providing high-quality raw materials for subsequent research and application.

[0152] The number of devices and processing scale described herein are for the purpose of simplifying the description of the invention. Applications, modifications, and variations of the invention will be readily apparent to those skilled in the art.

[0153] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details.

Claims

1. A method for extracting and purifying polygalacturonase inhibitory protein from pear, characterized in that, Includes the following steps: Step 1: Remove the core from the pear and crush it using an ultra-fine grinder to obtain pear powder. Step 2: Filter the crushed pears with filter paper, collect the first supernatant, precipitate the first supernatant with ammonium sulfate solution, centrifuge to remove the solvent, and collect the precipitate; Step 3: Dissolve the precipitate with a buffer solution, centrifuge to collect the third supernatant, dialyze the third supernatant with a buffer solution, and then filter it through a 0.45 μm filter membrane to obtain the crude extract; Step 4: Purify the crude extract using a gel filtration chromatography column to obtain a purified extract, which is then freeze-dried to obtain pear polygalacturonase inhibitor protein. In step one, during the crushing process using an ultrafine pulverizer, a stabilizer is added to the ultrafine pulverizer and crushed simultaneously. The weight ratio of the stabilizer to the cored pear fruit is 0.01–0.03:

1. The stabilizer includes 0.005–0.01 parts of dithiothreitol, 0.15–0.25 parts of polyvinylpyrrolidone, and 0.3–0.5 parts of a protease inhibitor. The protease inhibitor is diisopropylfluorophosphate or iodoacetic acid. In step two, the first supernatant is precipitated with ammonium sulfate solution, the solvent is removed by centrifugation, and the precipitate is collected. Specifically, this includes: Step 201: Add ammonium sulfate to the first supernatant until the saturation is 35%, let it stand at 4°C for 1-2 hours, and then centrifuge at 2000-3000 r / min to obtain the first precipitate and the second supernatant A; Step 202: Add ammonium sulfate to the second supernatant A until the saturation is 55%, let it stand at 4°C for 1-2 hours, and then centrifuge at 4000-5500 r / min to obtain the second precipitate and the second supernatant B; Step 203: Add ammonium sulfate to the second supernatant B until the saturation is 75%, let it stand at 4°C for 1-2 hours, and then centrifuge at 5000-6500 r / min to obtain the third precipitate; In steps 201, 202, and 203, before adding ammonium sulfate, 0.02–0.05 mol / L of L-ascorbic acid sodium salt and 0.03–0.05 mol / L of ethylenediaminetetraacetic acid disodium salt are added to the first supernatant / second supernatant A / second supernatant B. In step two, before filtering the pear powder with filter paper, it was pretreated by placing the pear powder in a 0°C ice bath, adding acetone at 5 times its weight, stirring evenly, filtering, and rinsing with trimethylsilyl ether to obtain the pretreated pear powder, which was then filtered with filter paper.

2. The method for extracting and purifying pear polygalacturonase inhibitory protein as described in claim 1, characterized in that, Step three specifically involves: Step 301: Dissolve the first precipitate with the first buffer solution and centrifuge to collect the third supernatant A. Dialyze the third supernatant A with the first buffer solution using a dialysis bag with a molecular weight cutoff of 10 kDa. Dialyze at 4°C for 24 h, changing the dialysis solution twice during the process to obtain dialysate A. The first buffer solution is a 0.1–0.2 mol / L phosphate buffer solution containing 0.01–0.02 mol / L sodium chloride and a pH of 6.8–7.

5. Step 302: Dissolve the second precipitate with the second buffer solution and centrifuge to collect the third supernatant B. Dialyze the third supernatant A with the second buffer solution using a dialysis bag with a molecular weight cutoff of 10 kDa. Dialyze at 4°C for 24 h, changing the dialysis solution twice during the process to obtain dialysate B. The second buffer solution is a 0.02–0.03 mol / L phosphate buffer solution containing 0.01–0.02 mol / L sodium chloride and with a pH of 6.8–7.

5. Step 303: Dissolve the third precipitate with the third buffer solution and collect the third supernatant C by centrifugation. Dialyze the third supernatant C with the third buffer solution using a dialysis bag with a molecular weight cutoff of 10 kDa. Dialyze at 4°C for 24 h, changing the dialysis solution twice during the process to obtain dialysate C. The third buffer solution is a 0.008–0.012 mol / L phosphate buffer solution containing 0.01–0.02 mol / L sodium chloride and a pH of 6.8–7.

5. Step 304: Mix dialysate A, dialysate B, and dialysate C, and transfer them to distilled water. Dialyze at 4°C for 24-48 hours to obtain dialysate E. Use a dialysis bag with a molecular weight cutoff of 10 kDa as the dialysis membrane. Replace the dialysis solution every 12 hours during the process. Filter dialysate E through a 0.45 μm filter membrane to obtain the crude extract.

3. The method for extracting and purifying pear polygalacturonase inhibitory protein as described in claim 1, characterized in that, Step four is as follows: Step 401: The crude extract is pre-treated with pH 5.5 containing 5 × 10⁻⁶ mg / L. -5 A CM-Sephadex C-50 column was equilibrated with 25 mmol / L sodium acetate buffer containing mol / L EDTA-Na2, followed by pH 5.5, containing 5 × 10 mol / L EDTA-Na2. -5 Wash with 25 mmol / L sodium acetate buffer containing mol / L EDTA-Na2 until A 280 If the concentration is less than 0.02, then perform gradient elution with sodium acetate buffer containing 0.1–1 mol / L NaCl and collect the preliminarily purified active component. Step 402: Load the pre-purified active component onto a solution pre-treated at pH 5.5 containing 5 × 10⁻⁶ mg / L. -5 A Sephadex G-100 column, 1 cm × 70 cm in size, equilibrated with 25 mmol / L sodium acetate buffer containing 5 × 10 mol / L EDTA-Na2, was used. -5 The active fraction was eluted with 25 mmol / L sodium acetate buffer containing 1 mol / L EDTA-Na2 and collected to obtain the purified extract.

4. The method for extracting and purifying pear polygalacturonase inhibitory protein as described in claim 3, characterized in that, Both CM-Sephadex C-50 gel and Sephadex G-100 gel underwent pretreatment before being loaded onto the column, specifically as follows: Swelling treatment: Immerse the gel in an ethanol solution with a volume fraction of 50-60%. After removing the ethanol solution, immerse it in sterile water and stir it magnetically at room temperature for at least 24 hours. Then remove the gel and rinse it with sterile water until the washing water is colorless. Wrap the gel in three layers of sterile filter cloth and let it stand for 30 minutes, changing the filter cloth every 10 minutes during this period. Acid-base treatment: After swelling, the gel was immersed in an equal volume of 1.0 mol / L NaOH solution and stirred at 50 rpm for 1 hour. After immersion, the gel was removed and washed with sterile water until the washing solution was neutral. Then, the gel was immersed in an equal volume of 0.1 mol / L HCl solution and stirred at 50 rpm for 1 hour. After immersion, the gel was removed and washed with sterile water until the washing solution was neutral. The gel was then wrapped in three layers of sterile filter cloth and allowed to stand for 2 hours, with the filter cloth being replaced every 30 minutes. Degassing treatment: The acid-base treated gel is placed in an ultrasonic device and ultrasonically treated at a frequency of 20-40 kHz for 20 min. Then the gel is placed in a vacuum filtration flask, the mouth of the flask is plugged with a rubber bulb, and the pressure is reduced for 30 min to remove the tiny air bubbles in the gel.

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