Method for efficiently recovering active glycoprotein Patatin from potato starch processing waste juice

Abstract

The invention discloses a method for recovering active glycoprotein Patatin from potato starch processing waste juice, and belongs to the technical field of protein extraction. According to the method, ammonium sulfate fractional precipitation, anion exchange chromatography, Con A affinity chromatography, gel chromatography and other means are combined to achieve efficient separation of the active Patatin protein, the obtained Patatin protein retains the original protein structure and biological function activity, and compared with an existing method, the separation purity is better, the efficiency is higher, and the method is suitable for industrial production. The obtained Patatin protein has a complete protein advanced structure and functional activity, and has a better application scene.

Description

Technical Field

[0001] The invention relates to a method for recovering active glycoprotein Patatin from potato starch processing waste juice, belonging to the technical field of protein extraction. Background Art

[0002] Potato wastewater is a byproduct of potato starch processing, primarily composed of potato protein. Potato protein has a balanced amino acid profile, with essential amino acids accounting for nearly half of the total amino acid content, significantly higher than the 36.0% recommended by the FAO / WHO standard for protein and close to the 49.7% amino acid content of egg protein, resulting in extremely high nutritional value. Furthermore, potato protein contains a high absorbable content and is non-allergenic, making it safer and easier for the human body to absorb and utilize.

[0003] At present, in industrial production, potato starch processing waste juice is often discharged directly, which not only causes certain pollution to the ecological environment, but also wastes high-quality protein resources. Therefore, recovering the protein components in potato waste juice has very important economic significance.

[0004] Potato protein primarily consists of high-molecular-weight proteins, the glycoprotein patatin, and low-molecular-weight protease inhibitors. Patatin, the glycoprotein, is the primary component of potato protein, accounting for approximately 40% of soluble protein. Its molecular weight varies between 40 and 45 kDa, depending on potato variety. Patatin is a high-quality plant glycoprotein with diverse physiological functions. Its essential amino acid index (EAAI) is as high as 76%, significantly exceeding the nutritional value of soy protein. It also has numerous positive effects on human health, including antioxidant activity, cholesterol and blood pressure regulation, and acyl hydrolase activity. Furthermore, compared to other potato protein components, patatin possesses excellent properties for food processing applications, such as gelling, emulsification, and foaming properties. It can be added to foods as a food ingredient or supplement. Furthermore, patatin can be used as a red wine clarifier or as a flavor enhancer in cheese, releasing free fatty acids to increase the content of flavor compounds.

[0005] Currently, potato protein recovery primarily relies on methods such as acid-base precipitation, expanded bed adsorption, and enzymatic methods. However, these methods exhibit certain limitations in practical applications, such as insufficient separation of different protein components (particularly difficult to separate patatin and low-molecular-weight protease inhibitors), high energy consumption, cumbersome operations, and the potential risk of protein denaturation. In industrial production, most companies use acid-thermal flocculation to recover potato protein. While this method can reduce production costs and simplify equipment requirements, it often results in a near-complete loss of the protein's functional properties and activity, significantly limiting its application in the food and pharmaceutical industries. Summary of the Invention

[0006] In view of the shortcomings and deficiencies of the existing technology, the main purpose of the present invention is to provide a method for efficiently recovering the active glycoprotein Patatin from potato starch processing waste juice, aiming to solve the problem of protein inactivation during the separation and purification of potato glycoprotein Patatin, and to reduce the pollution problem caused by the direct discharge of potato processing waste liquid to the environment, thereby increasing the added value of potato product processing.

[0007] In order to achieve the above objectives, the technical solutions provided are as follows: The present invention provides a method for recovering active glycoprotein Patatin from potato starch processing waste juice, the method comprising the following steps: (1) The pretreated potato starch processing waste juice was subjected to ammonium sulfate graded precipitation, and the protein precipitates of each grade were collected and placed in buffer A for dialysis. The dialyzed protein solution was centrifuged and the supernatant was collected; (2) Separating the supernatant obtained in step (1) through an anion exchange chromatography column and a PreCap Con A affinity chromatography column to obtain a target protein eluate, and then dialyzing in ultrapure water to obtain a Patatin protein solution; (3) The patatin protein solution obtained in step (2) is ultrafiltered and concentrated, the concentrate is passed through a Sephadex G-75 gel chromatography column, and the target protein is eluted with ultrapure water. The eluate is collected and freeze-dried to obtain the product.

[0008] In one embodiment, the pretreatment in step (1) is to filter the potato starch processing waste juice and then centrifuge it to remove impurities; the filter mesh size is 200-300 mesh; the centrifugation parameters are: 10000-12000 r / min, time is 10-20 min, and temperature is 4-8°C.

[0009] In one embodiment, the ammonium sulfate graded precipitation in step (1) is specifically performed by adding ammonium sulfate to the potato starch processing waste juice to a saturation of 1, then precipitating at 0-4°C for 30-60 min, centrifuging, and collecting the protein precipitate; continuing to add ammonium sulfate to the supernatant to a saturation of 2, then precipitating at 0-4°C for 30-60 min, centrifuging, and collecting the protein precipitate; and repeating the ammonium sulfate graded precipitation.

[0010] In one embodiment, during the ammonium sulfate graded precipitation process, the ammonium sulfate saturation levels are 20%, 30%, 40%, 50%, 60%, and 70%, respectively; wherein saturation 1 is 20%, saturation 2 is 30%, and the saturation levels increase sequentially.

[0011] In one embodiment, the centrifugation parameters are: 10,000-12,000 r / min, time 15-20 min, and temperature 4-8°C.

[0012] In one embodiment, the buffer A in step (1) is 20±5 mmol / L NaH2PO4-Na2HPO4, pH=7.2.

[0013] In one embodiment, the dialysis in step (1) is specifically to dissolve the protein precipitates of each level in buffer A, and then load them into a dialysis bag with a molecular weight cutoff of 1000-15000 Da, and place them in buffer A, and dialyze them at 4-8°C for 6-14 hours; wherein the buffer A added to dissolve the protein precipitate is 1 g of wet protein plus 10-20 mL of buffer A; the dialysis bag is preferably a dialysis bag with a molecular weight cutoff of 3500 Da; and the volume ratio of protein solution to dialysate is preferably 1:50.

[0014] In one embodiment, the centrifugation parameters in step (1) are: 10,000-12,000 r / min, centrifugation at 4-8°C for 10-15 min.

[0015] In one embodiment, the anion exchange chromatography separation column in step (2) is an IexCap Smac Q chromatography column.

[0016] In one embodiment, the separation by anion exchange chromatography column in step (2) is specifically as follows: the supernatant obtained in step (1) is loaded onto an anion exchange chromatography IexCap Smac Q column, impurities are first washed with buffer A, and then the target protein is eluted with buffer B using a linear elution program, the eluates are collected in separate tubes, and SDS-PAGE analysis is performed on them, and the eluates containing the target protein are pooled according to the analysis results.

[0017] In one embodiment, the flushing volume of buffer A is 3 to 5 column volumes.

[0018] In one embodiment, the buffer B is 20±5 mmol / L NaH2PO4-Na2HPO4, 1 mol / LNaCl, pH=7.2.

[0019] In one embodiment, the linear elution program is specifically: 0-20 min, 0-60% buffer B, flow rate 2 mL / min; 20-25 min, 60-100% buffer B, flow rate 2 mL / min.

[0020] In one embodiment, the volume collected by the sub-tubes is 1-4 mL / tube, preferably 2 mL / tube.

[0021] In one embodiment, the pooled eluates containing the target protein specifically collect the target protein eluates eluted when the NaCl concentration in the eluate is 50-150 mmol / L.

[0022] In one embodiment, the separation by PreCap Con A affinity chromatography column in step (2) is specifically as follows: the target protein eluate collected by separation by IexCap Smac Q chromatography column is dialyzed and then loaded onto a PreCap Con A affinity chromatography column, impurity proteins are first washed with buffer C, and then the target protein is eluted with buffer D using a linear elution program, the eluates are collected in separate tubes, and SDS-PAGE analysis is performed on them, and the eluates of the target protein are pooled according to the analysis results.

[0023] In one embodiment, the target protein eluate separated and collected by the IexCap Smac Q chromatographic column is dialyzed, specifically placing the collected target protein eluate in buffer C and dialyzing for 6 to 14 hours at 4 to 8°C; wherein buffer C is 20±5 mmol / L Tris-HCl, 0.5 mol / L NaCl, 1 mmol / L CaCl2, 1 mmol / L MnCl2, pH 7.4; the molecular weight range of the dialysis bag is 1000-15000 Da.

[0024] In one embodiment, the buffer C wash volume is 3 to 5 column volumes.

[0025] In one embodiment, the buffer D is 20±5 mmol / L Tris-HCl, 0.5 mol / L NaCl, 1 mmol / L CaCl2, 1 mmol / L MnCl2, 0.2 mol / L α-D-methyl mannoside, pH 7.4.

[0026] In one embodiment, the linear elution program is specifically: 0-20 min, 0-100% buffer D, flow rate 2 mL / min.

[0027] In one embodiment, the volume collected by the sub-tubes is 1-4 mL / tube, preferably 2 mL / tube.

[0028] In one embodiment, the pooled eluates containing the target protein specifically collect the target protein eluates eluted when the concentration of α-D-methyl mannoside in buffer D is 10-20 mmol / L.

[0029] In one embodiment, the molecular weight cut-off of the ultrafiltration tube for ultrafiltration concentration in step (3) is 10,000 to 12,000 Da, and the concentration factor is 10 to 15 times.

[0030] In one embodiment, the concentrated liquid in step (3) needs to be passed through a 0.45 μm water filter before passing through the column.

[0031] In one embodiment, the ultrapure water elution rate in step (3) is 1-1.5 mL / min.

[0032] In one embodiment, the step (3) of collecting the eluate specifically comprises: collecting the eluate in separate tubes, performing SDS-PAGE analysis on the eluate, and pooling all the eluates containing the target protein based on the analysis results.

[0033] In one embodiment, the collection standard of step (3) is 1-2 mL / tube; preferably 1 mL / tube.

[0034] The present invention also provides application of the above method in the technical field of deep processing of potatoes.

[0035] The present invention also provides a method for increasing the activity of potato glycoprotein Patatin, which comprises the following steps: (1) The pretreated potato starch processing waste juice was subjected to ammonium sulfate graded precipitation, and the protein precipitates of each grade were collected and placed in buffer A for dialysis. The dialyzed protein solution was centrifuged and the supernatant was collected; (2) Separating the supernatant obtained in step (1) through an anion exchange chromatography column and a PreCap Con A affinity chromatography column to obtain a target protein eluate, and then dialyzing in ultrapure water to obtain a Patatin protein solution; (3) The patatin protein solution obtained in step (2) is ultrafiltered and concentrated, and the concentrate is passed through a Sephadex G-75 gel chromatography column, and the target protein is eluted with ultrapure water. The eluates are collected in separate tubes and subjected to SDS-PAGE analysis. Based on the analysis results, all eluates containing the target protein are pooled and freeze-dried to obtain the product.

[0036] Beneficial effects The method provided by the present invention for recovering active Patatin protein from potato waste juice based on the combination of ammonium sulfate precipitation, anion exchange chromatography, affinity chromatography, and gel chromatography has the following advantages over the prior art: (1) The efficient adsorption of Patatin protein by anion exchange chromatography IexCap Smac Q column and affinity chromatography effectively removes non-protein impurities and some large molecular weight miscellaneous proteins, thereby obtaining a high-purity Patatin protein. Gel chromatography is further used to remove small molecular proteins such as protease inhibitors to obtain a high-purity Patatin protein. This method is simple in process, has a high recovery rate, and the recovered protein has a high purity. (2) The present invention does not use organic solvents, strong acids, strong bases and other reagents, and the recovery process does not require high-temperature heating and denaturation. The recovery method conditions are mild, and the function of potato patatin protein is effectively retained during the extraction process. The isolated patatin protein has a normal protein structure and excellent antioxidant activity and lipase activity; (3) The reagents used in the present invention are all buffer solutions with water and salt as the main components. The consumables used can also be recycled and reused. The preparation method is sustainable and helps promote the application of potato protein in the fields of food, cosmetics, medicine, etc., and reduces resource waste and pollution in the potato processing process. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Schematic diagram of the separation and purification steps of Patatin protein in potato starch processing waste juice of the present invention; Figure 2 This is an SDS-PAGE image of potato protein precipitated with different ammonium sulfate saturations in Example 1; Figure 3 This is an SDS-PAGE image of the anion exchange chromatography column separating the Patatin protein in Example 1; Figure 4 This is an SDS-PAGE image of the Patatin protein separated by Con A affinity chromatography column in Example 1; Figure 5 This is an SDS-PAGE image of the Sephadex G-75 gel chromatography separation of the Patatin protein in Example 1; Figure 6 This is a diagram of the circular dichroism spectrum analysis data of the Patatin protein obtained in Example 1; Figure 7 This is a graph showing the fluorescence spectrum analysis data of the Patatin protein obtained in Example 1; Figure 8 This is a graph showing the antioxidant activity analysis data of the Patatin protein obtained in Example 1; Figure 9 This is a graph showing the lipase activity analysis data of the Patatin protein obtained in Example 1; Figure 10 This is an SDS-PAGE image of the Patatin protein separated by anion exchange chromatography IexCap Smac DEAE in Comparative Example 2; Figure 11 This is the SDS-PAGE image of the isolated and purified Patatin protein in Comparative Example 3; Figure 12 This is the SDS-PAGE image of Patatin protein separated by Sephadex G-100 gel chromatography column in Comparative Example 4. DETAILED DESCRIPTION

[0038] The technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention. The following specific embodiments further describe the present invention.

[0039] The test method involved in the present invention is: 1. Protein content detection method Protein concentration was determined according to the BCA protein assay kit instructions. A standard curve was constructed using bovine serum albumin (BSA) as the standard protein.

[0040] To determine the protein concentration of a sample, prepare an appropriate amount of BCA working solution at a volume ratio of Solution A:Solution B = 50:1 and mix thoroughly. Take 100 μL of the dialyzed protein sample and use 100 μL of dialysis buffer as a blank control. Add 2 mL of BCA working solution to each sample and control. Incubate at 37°C for 30 minutes, then cool to room temperature and measure the absorbance at 562 nm. Substitute the measured absorbance value (y) into the BSA protein standard curve equation y = 0.0011x. The resulting x is the protein sample concentration.

[0041] 2. Protein purity detection method Protein samples were collected at different stages and prepared for electrophoresis at a sample volume to loading buffer ratio of 5:1. SDS-PAGE analysis was performed on a 12% acrylamide gel. Electrophoresis conditions were: 60 V for 20–30 min, followed by 120 V for 60–80 min, until the bromophenol blue indicator reached the gel substrate. Protein expression yield was determined using Coomassie Brilliant Blue staining and a BCA assay.

[0042] Sources of raw materials involved in the present invention: The waste juice from potato starch processing came from Inner Mongolia Huaou Starch Factory; Buffer A is 20 mmol / L NaH2PO4-Na2HPO4, pH = 7.2; Buffer B is 20 mmol / L NaH2PO4-Na2HPO4, 1 M NaCl, pH = 7.2; Buffer C is 20 mmol / L Tris-HCl, 0.5 mol / L NaCl, 1 mmol / L CaCl2, 1 mmol / L MnCl2, pH 7.4; Buffer D is 20 mmol / L Tris-HCl, 0.5 mol / L NaCl, 1 mmol / L CaCl2, 1 mmol / L MnCl2, 0.2 mol / L α-D-methyl mannoside, pH 7.4.

[0043] Example 1 A method for efficiently recovering active glycoprotein Patatin from potato starch processing waste juice comprises the following steps: (1) Fresh potato starch processing waste juice was filtered through gauze (300 mesh) to remove the residue, and then centrifuged at 12,000 r / min for 10 min to remove the precipitate to obtain potato waste juice; accurately measure 500 mL of potato waste juice into a beaker, slowly add 53 g of ammonium sulfate powder and stir to dissolve it until its saturation reaches 20%, and precipitate protein at 4°C for 60 min; then centrifuge at 10,000 r / min and 4°C for 10 min to collect the protein precipitate (named 20% protein precipitate); Pour the supernatant back into the beaker and add 29 g of ammonium sulfate to the beaker to reach 30% saturation. Precipitate the protein at 4°C for 60 min. Centrifuge at 10,000 rpm and 4°C for 10 min to collect the protein precipitate (named 30% protein precipitate). The supernatant was poured back into the beaker again, and 31 g, 32.5 g, 35 g, and 37.5 g of ammonium sulfate were added to the beaker repeatedly to make the saturation reach 40%, 50%, 60%, and 70%, respectively. Then, the mixture was centrifuged at 10,000 r / min and 4°C for 10 min to collect the protein precipitates. The collected protein precipitates were named 40%, 50%, 60%, and 70% protein precipitates, respectively. (2) Add 50 mL of buffer A (20 mmol / L NaH2PO4-Na2HPO4, pH = 7.2) to the proteins precipitated with ammonium sulfate at different saturations to resuspend and dissolve the proteins. Centrifuge at 10,000 r / min, 4°C for 15 min to remove the insoluble protein precipitate. Analyze the supernatant after centrifugation by SDS-PAGE. The results are as follows: Figure 2 As shown in the figure, at 20-50% ammonium sulfate saturation, most of the Patatin protein can be precipitated, while some miscellaneous proteins and other non-protein impurities are removed. (3) Collect the potato protein precipitated with ammonium sulfate in the 20-50% saturation range and resuspend and dissolve it in 100 mL of buffer A. Then, dialyze the protein solution in 5 L of buffer A at 4°C for 12 h. Centrifuge the dialyzed protein solution at 10,000 r / min and 4°C for 10 min, and collect the protein supernatant for later use. (4) Load the IexCap Smac Q column into the column valve of the AKTA rapid protein purifier, first wash the system and column with ultrapure water, and then balance with buffer A; use the sample pump to draw the protein supernatant obtained in step (3) for sample loading, rinse with 20 mL of buffer A to remove unbound impurities, and then use buffer B (20 mmol / L NaH2PO4-Na2HPO4, 1 mol / L NaCl, pH=7.2) to elute the target protein with a linear elution program (0-30 min, 0-100% buffer B, flow rate 2 mL / min), monitor the protein at 280 nm, and collect the eluted protein at 2 mL / tube; perform SDS-PAGE analysis on the protein supernatant, flow-through, and eluate; the results are shown in the figure below. Figure 3 As shown, Patatin protein can be eluted when the NaCl concentration in the eluent is 50-150 mmol / L, while other impurities exist in the elution range of different salt concentrations; all Patatin proteins were pooled and then dialyzed in 2 L of buffer C (20 mmol / L Tris-HCl, 0.5 mol / L NaCl, 1 mmol / L CaCl2, 1 mmol / L MnCl2, pH 7.4) at 4°C for 12 h; (5) Load the PreCap Con A affinity chromatography column into the column valve of the AKTA rapid protein purifier, first clean the system and column with ultrapure water, and then balance with buffer C; load the protein solution after dialyzed in step (4) into the PreCapCon A affinity chromatography column, rinse the impurities with buffer C for 3 column volumes (15 mL), and then elute the target protein with buffer D (20 mmol / L Tris-HCl, 0.5 mol / L NaCl, 1 mmol / L CaCl2, 1 mmol / L MnCl2, 0.2 mol / L α-D-methyl mannoside, pH 7.4) using a linear elution program (0-25 min, 0-100% buffer D, flow rate 2 mL / min), monitor the protein at 280 nm, and collect the eluted protein at 2 mL / tube; perform SDS-PAGE analysis on the protein supernatant, flow-through, and eluate. The results are as follows: Figure 4 As shown, high molecular weight impurities in the sample were removed in the form of flow-through, while a small amount of low molecular weight protease inhibitors co-eluted with Patatin protein when the α-D-methyl mannoside concentration in buffer D was 10-20 mmol / L. All Patatin proteins in the collection tube were pooled and then dialyzed into 2 L of ultrapure water at 4°C for 12 h. (6) The Patatin protein solution after dialyzed in step (5) was concentrated 10 times using an ultrafiltration tube with a molecular weight cutoff of 10,000 Da to a protein concentration of 10 mg / mL, and then filtered through a 0.45 μm filter for later use; the Sephadex G-75 gel chromatography column was loaded into the column valve of the AKTA rapid protein purifier, and the system and the chromatography column were cleaned and balanced with ultrapure water. The above concentrated protein solution was loaded into the Sephadex G-75 gel chromatography column, and the chromatography column was rinsed with ultrapure water at a flow rate of 1.5 mL / min. According to the chromatogram monitored by the UV detector at 280 nm, the eluted protein was collected at 1 mL / tube, and the eluted target protein was analyzed by SDS-PAGE. The results are as follows: Figure 5 As shown; the Patatin protein after purification by Con A affinity chromatography contains some low molecular weight protease inhibitors, but after Sephadex G-75 gel chromatography separation, the protease inhibitors can be effectively removed to obtain high-purity Patatin protein; the elution fraction containing Patatin protein is collected to obtain.

[0044] Structure and activity identification 1. Analysis of the secondary structure of Patatin protein using far-UV CD spectrometer After freeze-drying the eluted fraction of the Patatin protein obtained in Example 1, the protein powder was dissolved in ultrapure water to a final concentration of 0.02 mg / mL, and then 200 μL was taken and placed in a cuvette for CD analysis; the CD test conditions were preferably: scanning wavelength range 190-260 nm, bandwidth 1 nm, scanning step length 1 nm, slit width 0.02 nm, path 1 mm, and scanning speed 50 nm / min; ultrapure water was used as a blank control, and the Patatin protein sample spectrum was baseline corrected by subtracting the blank control spectrum.

[0045] The results are as follows Figure 6 As shown, the isolated and extracted Patatin protein has a typical α-helix characteristic spectrum, forming obvious negative peaks at 208 and 220 nm, which is completely consistent with other naturally extracted and recombinantly expressed Patatin proteins, indicating that the correct secondary structure has been formed.

[0046] 2. Analyze the tertiary structure of Patatin protein using intrinsic fluorescence spectrometer After freeze-drying the eluted fraction of the Patatin protein obtained in Example 1, the protein powder was dissolved in ultrapure water to a final concentration of 0.4 mg / mL. Then, 2-4 mL was taken and placed in a quartz cuvette for fluorescence spectral analysis at an emission wavelength of 300-500 nm.

[0047] The results are as follows Figure 7 As shown, Patatin protein has the highest absorption at a wavelength of 330 nm, which is basically consistent with the prior art reports.

[0048] 3. Analyze the antioxidant activity of Patatin protein using DPPH antioxidant kit After freeze-drying the patatin protein elution fraction obtained in Example 1, the protein powder was dissolved in buffer A and then subjected to a DPPH antioxidant activity assay. Specifically, 400 μL of the patatin protein solution was mixed with 600 μL of the DPPH working solution. The mixture was then incubated in the dark at room temperature for 30 minutes. After centrifugation at 4000 rpm for 5 minutes, the absorbance was measured at 517 nm. Vitamin E was used as a positive control, and the antioxidant activity of the patatin protein was ultimately converted into vitamin E equivalents.

[0049] The results are as follows Figure 8As shown, compared with the potato protein Patatin-B2 disclosed in the prior art (DOI: 10.1016 / j.synbio.2025.01.003; 2025.02), the natural Patatin protein extracted in Example 1 has excellent antioxidant activity, and the DPPH scavenging rate can reach 50% at a concentration of 2 mg / mL. The unit mass of Patatin protein is equivalent to the antioxidant activity produced by 13.5 mg of vitamin E, and has higher antioxidant activity than Patatin-B2.

[0050] 4. The lipase activity of Patatin protein was analyzed using p-nitrophenyl acetate (p-NPC2), butyrate (p-NPC4), hexanoate (p-NPC6), octanoate (p-NPC8) and laurate (p-NPC12) with different carbon chain lengths.

[0051] After freeze-drying the eluted fraction of the Patatin protein obtained in Example 1, the protein powder was dissolved in buffer A to form a Patatin protein solution with a concentration of 1 mg / mL; and then the lipase activity was determined; Specifically, buffer A was used to dissolve a series of p Prepare a 10 mmol / L substrate stock solution of p-NPC2-12. Add 20 μL of this stock solution to 160 μL of buffer A. Then quickly add 20 μL of a 1 mg / mL patatin solution. Incubate at 37°C for 5 minutes, and measure absorbance at 405 nm. Buffer A was added to the substrate instead of the patatin solution as a blank control. One unit (U) of enzyme activity is defined as the amount of enzyme required to decompose p-NPC2-12 per minute to release 1 μmol / L of p-nitrophenol. The absorbance at 405 nm for 1 μmol / L of p-nitrophenol is 0.01. Patatin protein activity is expressed as U / mg.

[0052] The results are as follows Figure 9 As shown, Patatin protein is resistant to p-nitrophenyl acetate p -NP2 showed the best hydrolysis effect, and as the carbon chain lengthened, the lipase activity gradually decreased. This result was consistent with the substrate specificity of Patatin protein reported previously, indicating that the Patatin protein extracted in Example 1 had good lipase activity.

[0053] 5. The performance of the Patatin protein obtained in each step of Example 1 was analyzed. The results are shown in Table 1: Table 1. Potato glycoproteins obtained in various steps

[0054] As shown in Table 1, the purity of the potato glycoprotein Patatin obtained in Example 1 gradually increased from the original 23.4% to 95.5%, and the lipase hydrolysis activity per unit mass of the obtained protein also increased from 20.2 U / mg in the original potato waste juice to 71.8 U / mg.

[0055] Comparative Example 1 The difference from Example 1 is that step (4) uses a cation exchange chromatography column IexCap Smac S to separate the Patatin protein, but the Patatin protein cannot be bound to the IexCap Smac S column within the pH range of 6.0-9.0, and almost all potato proteins flow out in the form of flow-through. Only when the pH is adjusted to below 4.0 can the Patatin protein bind to the filler in the IexCap Smac S column. However, after high salt is used to elute the protein at this time, the activity of the obtained Patatin protein is severely lost and it cannot catalyze the hydrolysis of fatty acyl groups.

[0056] Comparative Example 2 The difference from Example 1 is that in step (4), the anion chromatography column IexCap Smac Q is replaced by the anion chromatography column IexCap Smac DEAE, and the other parameters and conditions are the same as those in step (4); the results are as follows Figure 10 As shown in the figure, compared with the separation effect of anion exchange chromatography IexCap Smac Q, after separation by IexCap Smac DEAE, although some low molecular weight impurities can be removed, a large amount of impurities still exist in the target protein fraction, and the separation effect is poor.

[0057] Comparative Example 3 The difference from Example 1 is that step (5) uses Sephadex G-75 gel chromatography column for separation, and step (6) uses PreCap Con A affinity chromatography column for separation. Other parameters and conditions are the same as those in Example 1.

[0058] The results are as follows Figure 11 As shown, Figure 5 Compared with the high-purity Patatin protein after separation, if Sephadex G-75 gel chromatography column is first used for separation and then PreCap Con A affinity chromatography column is used for separation, the obtained Patatin protein still contains more high molecular weight impurities and low molecular weight protease inhibitors and other impurities, resulting in low product purity.

[0059] Comparative Example 4 The difference from Example 1 is that in step (6), a Sephadex G-100 gel chromatography column is used instead of a Sephadex G-75 gel chromatography column, and other parameters and conditions are the same as those in Example 1.

[0060] The results are as follows Figure 12 As shown in the figure, when Sephadex G-100 column was used, low molecular weight protease inhibitors still existed in the obtained Patatin protein, indicating that Sephadex G-100 column could not effectively separate Patatin protein and low molecular weight protease inhibitors.

[0061] Comparative Example 5 The difference in effect between the technical solution of Example 1 and the existing method is shown in Table 2: Table 2. Recovery effects of different methods on potato protein Patatin

[0062] [1] Gao Jie. Research on the recovery and properties of protein from potato starch wastewater[D]. Shaanxi University of Science and Technology, 2012. [2]Waglay A, Karboune S, Alli I. Potato protein isolates: Recovery and characterization of their properties[J]. Food Chemistry, 2014, 142: 373-382.. [3] Liu Weicong. Research on protein recovery method and process design in potato starch production wastewater[D]. Heilongjiang Oriental University, 2022. As shown in Table 2, the existing technologies use flocculation, acid-base precipitation, acid-heat method, and ammonium sulfate precipitation method to separate crude protein or partial glycoprotein and protease inhibitors from potato processing wastewater. However, the purity, yield, and efficiency of the resulting Patatin protein are not ideal. Moreover, the biological activity of the potato protein obtained by the above methods is basically lost, making it difficult to achieve large-scale application. In addition, a large amount of waste pollutants are generated during the separation and purification process of the above methods.

[0063] The present invention uses ammonium sulfate fractionated precipitation, anion exchange chromatography, Con A affinity chromatography and gel chromatography for the first time to achieve efficient separation of active Patatin protein. The obtained Patatin protein retains the original protein structure and biological functional activity. Compared with existing methods, the separation purity is better and the efficiency is higher. The obtained Patatin protein has a complete protein high-level structure and functional activity, and has better application scenarios.

[0064] The embodiments provided above are not intended to limit the scope of the present invention, nor are the steps described to limit their execution order. Any obvious improvements to the present invention made by those skilled in the art in combination with existing common knowledge shall fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for recovering active glycoprotein Patatin from potato starch processing waste juice, characterized in that: The method comprises the following steps: (1) The pretreated potato starch processing waste juice was subjected to ammonium sulfate graded precipitation, and the protein precipitates of each grade were collected and placed in buffer A for dialysis. The dialyzed protein solution was centrifuged and the supernatant was collected; (2) Separating the supernatant obtained in step (1) through an anion exchange chromatography column and a PreCap Con A affinity chromatography column to obtain a target protein eluate, and then dialyzing in ultrapure water to obtain a Patatin protein solution; (3) The patatin protein solution obtained in step (2) is ultrafiltered and concentrated, the concentrate is passed through a Sephadex G-75 gel chromatography column, and the target protein is eluted with ultrapure water. The eluate is collected and freeze-dried to obtain the product.

2. The method according to claim 1, wherein The ammonium sulfate graded precipitation in step (1) specifically comprises adding ammonium sulfate to the potato starch processing waste juice to a saturation of 1, then precipitating at 0-4°C for 30-60 min, centrifuging, and collecting the protein precipitate; continuing to add ammonium sulfate to the supernatant to a saturation of 2, then precipitating at 0-4°C for 30-60 min, centrifuging, and collecting the protein precipitate; and repeating the ammonium sulfate graded precipitation.

3. The method according to claim 1, characterized in that During the ammonium sulfate graded precipitation process, the ammonium sulfate saturation levels are 20%, 30%, 40%, 50%, 60%, and 70%, respectively.

4. The method according to claim 1, wherein The anion exchange chromatography column in step (2) is an IexCapSmac Q chromatography column.

5. The method according to claim 4, wherein The separation by anion exchange chromatography column in step (2) is specifically as follows: the supernatant obtained in step (1) is loaded onto an IexCap Smac Q chromatography column, impurity proteins are first washed with buffer A, and then the target protein is eluted with buffer B using a linear elution program, the eluates are collected in separate tubes, and SDS-PAGE analysis is performed on them, and the eluates containing the target protein are pooled according to the analysis results; wherein buffer A is 20±5 mmol / L NaH2PO4-Na2HPO4, pH=7.2; buffer B is 20±5 mmol / L NaH2PO4-Na2HPO4, 1 mol / L NaCl, pH=7.

2.

6. The method according to claim 5, wherein The pooled eluates containing the target protein specifically collect the target protein eluates eluted when the NaCl concentration in the eluate is 50-150 mmol / L.

7. The method according to claim 1, wherein The separation by PreCap Con A affinity chromatography column in step (2) is specifically as follows: the target protein eluate collected by separation by IexCap Smac Q chromatography column is dialyzed and then loaded onto PreCap Con A affinity chromatography column, the impurity protein is first washed with buffer C, and then the target protein is eluted with buffer D using a linear elution program, the eluate is collected in separate tubes, and SDS-PAGE analysis is performed on it, and the eluate of the target protein is pooled according to the analysis results; wherein buffer C is 20±5 mmol / L Tris-HCl, 0.5 mol / L NaCl, 1 mmol / L CaCl2, 1 mmol / L MnCl2, pH 7.4; buffer D is 20±5 mmol / L Tris-HCl, 0.5 mol / L NaCl, 1 mmol / L CaCl2, 1 mmol / L MnCl2, 0.2 mol / L α-D-methyl mannoside, pH 7.

4.

8. The method according to claim 7, characterized in that The pooled eluate containing the target protein specifically collects the target protein eluate eluted when the α-D-methyl mannoside concentration in the eluate is 10-20 mmol / L.

9. Use of the method according to any one of claims 1 to 8 in the field of potato deep processing technology.

10. A method for increasing the activity of potato glycoprotein Patatin, characterized in that: The method comprises the following steps: (1) The pretreated potato starch processing waste juice was subjected to ammonium sulfate graded precipitation, and the protein precipitates of each grade were collected and placed in buffer A for dialysis. The dialyzed protein solution was centrifuged and the supernatant was collected; (2) Separating the supernatant obtained in step (1) through an anion exchange chromatography column and a PreCap Con A affinity chromatography column to obtain a target protein eluate, and then dialyzing in ultrapure water to obtain a Patatin protein solution; (3) The patatin protein solution obtained in step (2) is ultrafiltered and concentrated, the concentrate is passed through a Sephadex G-75 gel chromatography column, and the target protein is eluted with ultrapure water. The eluate is collected and freeze-dried to obtain the product.

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