Pumpkin seed protein extraction process

Abstract

The invention relates to the field of protein extraction, in particular to a pumpkin seed protein extraction process which comprises the following steps: S1, crushing pumpkin seed meal, adding water and an antioxidant, extracting at 20-60 DEG C, and performing ultrafiltration to obtain an extracting solution; s2, adding hydrochloric acid into the extracting solution for acid precipitation, and filtering to obtain a protein solid; s3, washing and drying the protein solid to obtain the pumpkin seed protein. The antioxidant is added in the protein extraction process, so that the oxidation process of polyphenol can be effectively inhibited, and the generation of quinone substances in the extraction process of the pumpkin seed protein is reduced, so that the extraction rate of the pumpkin seed protein is effectively improved, and the oxidation rate of polyphenol is reduced.

Description

Technical Field

[0001] The present application relates to the field of protein extraction, and in particular to a pumpkin seed protein extraction process. Background Art

[0002] Pumpkin seed protein has a balanced amino acid composition and contains essential amino acids for the human body. It can make up for the deficiencies of cereal protein and is very suitable as a full-value plant protein supplement. In addition, pumpkin seed protein contains ingredients such as phytosterols, polyphenols, and squalene, which have antioxidant, anti-inflammatory, and lipid-regulating functions. Therefore, it also has potential value in the prevention of chronic diseases. Among them, the polyphenols in pumpkin seed protein are easily oxidized. The quinones generated after polyphenol oxidation are easily combined with the amino or sulfhydryl groups of proteins, causing protein cross-linking, denaturation or precipitation, which reduces the extraction rate and functional activity of the protein. At the same time, the browning reaction caused by polyphenol oxidation will change the color of the extract and reduce the sensory quality of the product. Therefore, there is an urgent need for a pumpkin seed protein extraction process that can reduce polyphenol oxidation. Summary of the Invention

[0003] In order to reduce the oxidation of polyphenols during the extraction process of pumpkin seed protein, the present application provides a pumpkin seed protein extraction process.

[0004] The present application provides a pumpkin seed protein extraction process, which adopts the following technical solution: A pumpkin seed protein extraction process comprises the following steps: S1: After the pumpkin seed meal is crushed, water and antioxidants are added to extract at 20-60°C, and then ultrafiltration is performed to obtain an extract; S2: adding hydrochloric acid to the extract for acid precipitation, and filtering to obtain protein solid; S3: After washing and drying the protein solid, pumpkin seed protein is obtained.

[0005] By adopting the above technical solution, the cell wall structure of the pumpkin seed meal is firstly destroyed by crushing so that the protein can be released into the solvent, and then the oxidation of polyphenols is inhibited by adding an antioxidant, thereby reducing the probability that quinone substances generated by the oxidation of polyphenols combine with proteins to affect the yield and quality of the protein, and the protein is extracted by water, and then the protein solution is purified by ultrafiltration, and finally pumpkin seed protein is obtained by acid precipitation, water washing and drying.

[0006] The present application can effectively inhibit the oxidation process of polyphenols by adding antioxidants during the protein extraction process, reduce the generation of quinone substances during the extraction process of pumpkin seed protein, thereby effectively improving the extraction rate of pumpkin seed protein and reducing the oxidation rate of polyphenols.

[0007] Preferably, the added amount of the antioxidant accounts for 0.3-1.5% of the mass of the pumpkin seed meal.

[0008] By adopting the above technical solution, when the added amount of antioxidant is too low, the antioxidant cannot completely chelate the copper ions in the active center of polyphenol oxidase, nor can it promptly remove oxidative intermediates such as hydroxyl radicals and quinones, causing polyphenols to be catalyzed and oxidized by polyphenol oxidase to generate dark quinone compounds, resulting in a browning reaction. At the same time, oxidation products such as quinones have strong reactivity and can covalently bind to the amino and thiol groups of proteins to form irreversible protein-polyphenol complexes, resulting in reduced purity of the extracted pumpkin seed protein.

[0009] When excessive antioxidants are added, high concentrations may alter the system's ionic strength or pH, leading to polyphenol precipitation due to changes in solvent polarity or chemical degradation at extreme pH. Furthermore, excessively high ionic strength in the system can disrupt the charge balance on the protein surface, triggering intermolecular hydrophobic aggregation, leading to protein precipitation or disruption of protein bioactivity, thus affecting the yield and quality of the extracted protein. Furthermore, excessive antioxidants may compete with polyphenols for binding sites, weakening the antioxidant's protective effect on polyphenols.

[0010] Preferably, the antioxidant is at least one of rosemary extract and sodium glycyrrhizate.

[0011] By adopting the above technical solution, rosemary extract contains a variety of phenolic substances, all of which have significant antioxidant capacity. Its molecular structure is rich in hydroxyl groups, which can act as hydrogen donors and combine with superoxide anions, hydroxyl radicals, and peroxyl radicals to interrupt the oxidation chain reaction, thereby preventing the oxidation of polyphenols. In addition, the activity of oxidases such as polyphenol oxidase and peroxidase depends on metal ions such as copper and iron. The phenolic components in rosemary can chelate metal ions with groups such as hydroxyl and carboxyl groups, thereby inhibiting the activity of enzymes and reducing the oxidation of polyphenols. At the same time, the antioxidant components in rosemary extract can reduce the quinones generated by the oxidation of polyphenols to polyphenols, preventing subsequent polymerization reactions. In addition, rosemary extract can bind to proteins through hydrogen bonds or hydrophobic interactions, changing their surface charge and reducing the aggregation tendency between protein molecules, thereby improving the solubility of proteins.

[0012] Sodium glycyrrhizate is the sodium salt form of glycyrrhizic acid, its core component being glycyrrhizic acid. It belongs to the triterpenoid saponin class of compounds, and its structure contains multiple hydroxyl, carboxyl, and glycosidic bonds. The hydroxyl groups act as hydrogen donors, directly neutralizing oxidative free radicals such as superoxide anions and hydroxyl radicals, thereby interrupting the chain reaction of polyphenol oxidation. The carboxyl and hydroxyl groups can also bind to the copper ions in the active center of polyphenol oxidase, chelating the metal ions and preventing the enzyme from participating in the catalytic reaction, thereby inhibiting its activity. Furthermore, as a saponin, sodium glycyrrhizate exhibits amphiphilic properties, dispersing hydrophobic components in the system and reducing the probability of direct contact between polyphenols and polyphenol oxidase, thereby reducing polyphenol oxidation. Furthermore, sodium glycyrrhizate can optimize the charge distribution of proteins by adjusting the ionic strength or pH of the solution, enhancing their hydrophilicity and thus promoting their solubility.

[0013] Preferably, the antioxidant is a mixture of rosemary extract and sodium glycyrrhizate.

[0014] By adopting the above technical solution, the antioxidant formed by compounding rosemary extract and sodium glycyrrhizate can inhibit the oxidation of polyphenols in the following ways: First, sodium glycyrrhizate can preferentially chelate free copper ions in the solution, reducing the supply of copper ions to the active center of polyphenol oxidase. The phenolic substances in the rosemary extract directly bind to the active center of polyphenol oxidase, blocking the binding of polyphenols to polyphenol oxidase. Second, sodium glycyrrhizate is highly water-soluble and can scavenge polar free radicals in an aqueous environment. The fat-soluble components in the rosemary extract can scavenge free radicals in an oil phase or cell membrane environment. Through the synergistic effect of the two, they can cover the water-fat two-phase system and expand the antioxidant range.

[0015] Sodium glycyrrhizate can also improve the dispersibility of rosemary extract in aqueous solution. The sodium glycyrrhizate molecule contains multiple polar groups and a hydrophobic steroid ring structure, which gives it surfactant properties. In aqueous solution, it can bind to the fat-soluble phenolic substances in rosemary extract through hydrophobic interactions, forming a micelle-like complex. The hydrophilic carboxyl and hydroxyl groups, facing outward, form hydrogen bonds with water molecules, "encapsulating" the fat-soluble phenolic substances in the aqueous phase, thereby improving the dispersibility of the rosemary extract in the aqueous phase.

[0016] In addition, the carboxyl group of sodium glycyrrhizate dissociates into negatively charged groups in water, making the surface of the formed complex negatively charged. The repulsion of like charges can prevent the mutual aggregation of phenolic particles. At the same time, the hydration of hydroxyl groups forms a hydration layer on the surface of the particles, which further stabilizes the dispersion system through "steric hindrance", reduces precipitation or stratification, and prolongs the uniform distribution time of the active ingredients in the solution.

[0017] Preferably, the mixing ratio of the rosemary extract to sodium glycyrrhizate is 1-3:1.

[0018] By adopting the above technical solution, when the proportion of rosemary extract is too large, the critical micelle concentration of sodium glycyrrhizate is insufficient, and sufficient micelles cannot be formed to encapsulate the fat-soluble components in rosemary, resulting in the precipitation, stratification or precipitation of the fat-soluble components. In addition, the negative charge provided by sodium glycyrrhizate is insufficient, which weakens the charge repulsion effect on the particle surface, increases the particle size of the dispersed phase, and significantly increases the turbidity of the system.

[0019] When the proportion of sodium glycyrrhizate is too large, the excess sodium glycyrrhizate will form high-density micelles, and the carboxyl groups in sodium glycyrrhizate will neutralize the surface charge of the protein, weaken the electrostatic repulsion, and promote the formation of irreversible aggregates between protein molecules through hydrophobic interaction, thereby reducing the solubility of the protein. At the same time, the excess sodium glycyrrhizate will competitively bind with the rosemary extract, reduce the antioxidant layer on the protein surface, and reduce the protein extraction rate.

[0020] Preferably, in S1, the mesh size of the crushed pumpkin seed meal is 40-80 mesh.

[0021] By adopting the above technical solution, when the mesh size is between 40 and 80, the gaps between pumpkin seed meal particles are moderate, and the antioxidant solvent can evenly penetrate into the interior of the pumpkin seed meal particles, thereby inhibiting the activity of polyphenol oxidase.

[0022] When the mesh size of pumpkin seed meal is too large, the high mechanical shear force and heat generated by excessive crushing may cause partial protein denaturation and reduce the functional properties of the protein. In addition, overly fine pumpkin seed meal particles are prone to agglomeration, hindering the penetration of antioxidant solvent into the interior of the pumpkin seed meal particles. At the same time, a large amount of free polyphenols are rapidly released, exceeding the inhibitory capacity of the antioxidant, resulting in the accumulation of polyphenol oxidation products, affecting the yield and quality of pumpkin seed protein.

[0023] When the mesh size of pumpkin seed meal is too small, the particles of pumpkin seed meal are coarse, and the unbroken cell structure prevents the release of internal protein, resulting in a lower extraction rate of pumpkin seed protein. In addition, the interior of the coarse particles is difficult to be covered by antioxidants, and the activity of polyphenol oxidase cannot be effectively inhibited, which intensifies the local oxidation reaction.

[0024] Preferably, in S1, the ultrafiltration pressure is 0.05-0.25 MPa, and the ultrafiltration temperature is 20-50°C.

[0025] By adopting the above technical solution, when the ultrafiltration pressure is too low, the driving force is insufficient, resulting in a significant reduction in the rate at which the solvent and small molecule impurities pass through the ultrafiltration membrane. In addition, the target protein's residence time on the ultrafiltration membrane surface is too long, making it easy for the impurities remaining in the membrane pores to undergo nonspecific adsorption or aggregation, resulting in some proteins being unable to be effectively retained, leading to a decrease in the extraction rate of pumpkin seed protein. When the ultrafiltration pressure is too high, the shear force generated by the high pressure may destroy the secondary structure of the protein, resulting in a decrease in the functional properties of the protein. In addition, some target proteins that exceed the membrane's molecular weight cut-off may be "forced to pass through" the membrane pores due to the high pressure. At the same time, the high pressure forces the membrane pores to expand, causing some of the proteins that should have been retained to pass through the ultrafiltration membrane, resulting in a decrease in the purity of the pumpkin seed protein finally obtained.

[0026] When the ultrafiltration temperature is too low, the viscosity of the solution will increase, causing the ultrafiltration flux to decrease. In addition, low temperatures may reduce the solubility of some proteins, leading to enhanced hydrophobic interactions between protein molecules, forming micron-sized aggregates, clogging the membrane pores and reducing the retention efficiency. At the same time, low temperatures will reduce the flexibility of the ultrafiltration membrane material and slightly shrink the pore size of the ultrafiltration membrane, resulting in the retention of small molecular impurities that should have passed through, affecting the purity of pumpkin seed protein. When the ultrafiltration temperature is too high, the structure of pumpkin seed protein will be destroyed, the hydrophobic groups will be exposed and irreversible aggregates will form, resulting in a decrease in the functional properties of the final product. In addition, high temperatures will accelerate the adsorption reaction between proteins and the ultrafiltration membrane surface, exacerbating membrane fouling.

[0027] Preferably, in S1, the crushed pumpkin seed meal is first soaked in an antioxidant solution for 20-30 minutes, and then water is added to extract the protein.

[0028] By adopting the above technical solution, the cell structure of pumpkin seed meal is destroyed after being crushed, and the activity of polyphenol oxidase is the highest at this time. After being activated, polyphenol oxidase will contact with the substrate to accelerate oxidation, thereby forming oxidation products. The present application soaks the crushed pumpkin seed meal in an antioxidant solution, allowing the antioxidant to fully contact the pumpkin seed meal through osmosis, and effectively reduces the oxidation of polyphenols by inhibiting the activity of polyphenol oxidase or interrupting the oxidation chain reaction.

[0029] In summary, this application includes at least one of the following beneficial technical effects: 1. The present invention effectively inhibits the oxidation process of polyphenols by adding antioxidants during protein extraction, reduces the generation of quinones during pumpkin seed protein extraction, thereby effectively improving the extraction rate of pumpkin seed protein and reducing the oxidation rate of polyphenols; 2. This application uses a mixture of sodium glycyrrhizate and rosemary extract as an antioxidant, which can effectively scavenge free radicals in the water-lipid two-phase system and inhibit the activity of polyphenol oxidase. In addition, sodium glycyrrhizate can promote the uniform dispersion of rosemary extract in the aqueous phase, further enhancing the antioxidant effect of rosemary extract. 3. The present application first performs antioxidant treatment on the crushed pumpkin seed meal and then extracts the protein, thereby inhibiting polyphenol oxidase when its activity is strongest and effectively reducing the polyphenol oxidation rate. DETAILED DESCRIPTION

[0030] The raw materials in this application include the following parts: Rosemary extract: Rosemary extract with purity ≥99% from Hubei Haijia Biotechnology Co., Ltd.; Rosemary extract with purity ≥99% from Sichuan Huanxu Biotechnology Co., Ltd. Sodium glycyrrhizate: a commercially available product with CAS number 71277-79-7.

[0031] The present application is further described in detail below with reference to the following examples and comparative examples.

[0032] Example 1 A pumpkin seed protein extraction process comprises the following steps: S1: After crushing 200 g of pumpkin seed meal into 60 mesh, add 2 L of water and 2.4 g of antioxidant and extract at 20-60°C and adjust the pH to 7-8.5. Then, ultrafiltration is performed at 0.15 MPa and 30°C to obtain an extract; S2: adding 1M hydrochloric acid to the extract to adjust the pH of the solution to 2-5, then performing acid precipitation and filtering to obtain protein solids; S3: The protein solid is washed with water, filtered, and then dried under conditions of vacuum degree > 0.07 MPa and temperature 40-80° C. to obtain pumpkin seed protein.

[0033] The antioxidant is a mixture of rosemary extract and sodium glycyrrhizate, and the mixing ratio of rosemary extract and sodium glycyrrhizate is 2:1.

[0034] Example 2-3 In Example 2-3, based on the preparation method of Example 1, the components of the antioxidant were adjusted. The specific adjustments are shown in Table 1.

[0035] Comparative Example 1 Comparative Example 1: Based on the preparation method of Example 1, the antioxidant in S1 was removed, and the other conditions remained unchanged.

[0036] Performance testing The pumpkin seed proteins of Examples 1-3 and Comparative Example 1 were analyzed, and the specific detection method was as follows: 1. Protein extraction rate The total protein mass of pumpkin seeds and the protein mass of pumpkin seed protein were determined by the Kjeldahl method to obtain the protein extraction rate. The protein extraction rate was calculated as follows: protein extraction rate = protein mass of pumpkin seed protein / total protein mass of pumpkin seeds × 100%.

[0037] 2. Polyphenol oxidation rate First, standard solutions at 0.1, 0.5, 1, 5, and 10 μg / mL were prepared. HPLC conditions were set as a C18 column, mobile phase A consisting of 0.1% formic acid in water, mobile phase B consisting of acetonitrile, a flow rate of 1.0 mL / min, and a column temperature of 30°C. Detection was performed at a wavelength of 280 nm. The standard solutions were injected and a standard curve was plotted. The polyphenol content of pumpkin seeds and pumpkin seed protein was calculated using the standard curve, thereby determining the polyphenol oxidation rate. The polyphenol oxidation rate was calculated as follows: Polyphenol oxidation rate = (total polyphenol content of pumpkin seeds - polyphenol content of pumpkin seed protein) / total polyphenol content of pumpkin seeds × 100%.

[0038] According to the above detection method, the performance test results of Examples 1-3 and Comparative Example 1 were obtained, and the test results are shown in Table 1.

[0039] Table 1 Antioxidant components and performance test table of Examples 1-3 and Comparative Example 1 Referring to Table 1, by comparing Examples 1-3 with Comparative Example 1, it can be seen that the protein extraction rate and polyphenol oxidation rate obtained in Example 1 are both higher than those in Example 2 and Example 3. This may be because the combination of rosemary extract and sodium glycyrrhizate can inhibit the activity of polyphenol oxidase and the scavenging of free radicals in the water-lipid phase. At the same time, sodium glycyrrhizate can also improve the dispersibility of rosemary extract in aqueous solution, further increasing the antioxidant range of rosemary extract, thereby reducing the oxidation of polyphenols and improving the protein extraction rate.

[0040] Examples 4-5 Example 4-5 Based on the preparation method of Example 1, the mixing ratio of rosemary extract and sodium glycyrrhizate was adjusted. The specific adjustment is shown in Table 2.

[0041] Comparative Examples 2-3 Comparative Example 2-3 Based on the preparation method of Example 1, the mixing ratio of rosemary extract and sodium glycyrrhizate was adjusted. The specific adjustment is shown in Table 2.

[0042] The pumpkin seed proteins of Examples 4-5 and Comparative Examples 2-3 were subjected to the above-mentioned performance tests, and the test results are shown in Table 2.

[0043] Table 2 Mixing ratio and performance test table of rosemary extract and sodium glycyrrhizate of Example 1, Examples 4-5 and Comparative Examples 2-3 Referring to Table 2, it can be seen from Comparative Example 1, Examples 4-5 and Comparative Examples 2-3 that when the mixing ratio of rosemary extract and sodium glycyrrhizate is 1-3:1, especially when the mixing ratio of rosemary extract and sodium glycyrrhizate is 2:1, the resulting protein extraction rate and polyphenol oxidation rate are ideal. This may be because when the proportion of rosemary extract is too large, the critical micelle concentration of sodium glycyrrhizate is insufficient, and enough micelles cannot be formed to encapsulate the fat-soluble components in rosemary, and the negative charge provided by sodium glycyrrhizate is insufficient, which weakens the charge repulsion reaction on the particle surface, making the antioxidant effect unsatisfactory; when the proportion of sodium glycyrrhizate is too large, excess sodium glycyrrhizate will form high-density micelles, and the carboxyl groups in sodium glycyrrhizate will neutralize the protein surface charge, weaken the electrostatic repulsion, prompt the protein molecules to form irreversible aggregates through hydrophobic interaction, reduce the solubility of the protein, and decrease the protein extraction rate.

[0044] Examples 6-7 In Example 6-7, based on the preparation method of Example 1, the amount of antioxidant added was adjusted. The specific adjustments are shown in Table 3.

[0045] Comparative Examples 4-5 Comparative Example 4-5 is based on the preparation method of Example 1, and the amount of antioxidant added is adjusted. The specific adjustment is shown in Table 3.

[0046] Performance testing The pumpkin seed proteins of Example 1, Examples 6-7 and Comparative Examples 4-5 were analyzed, and the specific detection method was as follows: protein purity The protein mass of pumpkin seed protein was tested using the Kjeldahl method to calculate the protein purity. The protein purity calculation formula is: protein purity = protein mass of pumpkin seed protein / pumpkin seed protein mass × 100%.

[0047] According to the above detection method, the detection results of Example 1, Examples 6-7 and Comparative Examples 4-5 were obtained, and the detection results are shown in Table 3.

[0048] Table 3 Antioxidant addition amount and performance test table of Example 1, Examples 6-7 and Comparative Examples 4-5 Referring to Table 3, it can be seen from the comparison of Example 1, Examples 6-7 and Comparative Examples 4-5 that when the added amount of the antioxidant is 0.3-1.5%, especially when the added amount of the antioxidant is 1.2%, the protein purity of the obtained pumpkin seed protein is higher, and the protein extraction rate and the polyphenol oxidation rate are both within the ideal range. This may be because when the added amount of the antioxidant is too low, the antioxidant cannot fully chelate the copper ions in the active center of polyphenol oxidase, nor can it promptly remove oxidation intermediates such as free radicals and quinones. At the same time, oxidation products such as quinones will covalently bind to the amino and sulfhydryl groups of the protein to form irreversible protein-polyphenol complexes, resulting in reduced protein purity; when the added amount of the antioxidant is too high, the charge balance on the protein surface is destroyed, triggering hydrophobic aggregation between molecules, leading to protein precipitation or destruction of the biological activity of the protein. At the same time, excessive antioxidants may compete with polyphenols for binding sites, weakening the protection of the antioxidants on polyphenols, thereby increasing the polyphenol oxidation rate.

[0049] Examples 8-9 In Example 8-9, based on the preparation method of Example 1, the mesh size of the crushed pumpkin seed meal was adjusted. The specific adjustments are shown in Table 4.

[0050] Comparative Examples 6-7 In Comparative Examples 6-7, based on the preparation method of Example 1, the mesh size of the crushed pumpkin seed meal was adjusted, and the specific adjustments are shown in Table 4.

[0051] The pumpkin seed proteins of Examples 8-9 and Comparative Examples 6-7 were subjected to the above-mentioned performance test, and the test results are shown in Table 4.

[0052] Table 4 Mesh size and performance test table of crushed pumpkin seed meal of Example 1, Examples 8-9 and Comparative Examples 6-7 Referring to Table 4, it can be seen from the comparison of Example 1, Examples 8-9 and Comparative Examples 6-7 that when the mesh number of the crushed pumpkin seed meal is 40-80 mesh, especially when the mesh number of the crushed pumpkin seed meal is 60 mesh, the protein extraction rate and the polyphenol oxidation rate are ideal. This may be because when the mesh number of the crushed pumpkin seeds is too large, the high mechanical shear force and heat generated by excessive crushing will cause protein deformation, and the overly fine pumpkin seed meal is prone to agglomeration, affecting the penetration of antioxidants into the pumpkin seed meal. At the same time, a large amount of free polyphenols are rapidly released, exceeding the inhibitory ability of the antioxidant, resulting in an increase in the polyphenol oxidation rate; when the mesh number of the crushed pumpkin seeds is too small, there is protein inside the pumpkin seed meal that is not released, resulting in a decrease in the protein extraction rate, and the coarse-grained pumpkin seed meal is difficult to be covered by the antioxidant, which intensifies the local oxidation reaction.

[0053] Examples 10-11 Examples 10-11 Based on the preparation method of Example 1, the pressure and temperature of ultrafiltration in S1 were adjusted. The specific adjustments are shown in Table 5.

[0054] Comparative Examples 8-9 Comparative Examples 8-9 Based on the preparation method of Example 1, the pressure and temperature of ultrafiltration in S1 were adjusted. The specific adjustments are shown in Table 5.

[0055] The pumpkin seed proteins of Examples 10-11 and Comparative Examples 8-9 were subjected to the above-mentioned performance tests, and the test results are shown in Table 5.

[0056] Table 5 Ultrafiltration pressure and temperature and performance test table of Example 1, Examples 10-11 and Comparative Examples 8-9 Referring to Table 5, it can be seen from the comparison of Example 1, Examples 10-11 and Comparative Examples 8-9 that when the ultrafiltration pressure is in the range of 0.05-0.25 MPa and the temperature is in the range of 20-50°C, especially when the ultrafiltration pressure is 0.15 MPa and the temperature is 30°C, the protein extraction rate and protein purity are the highest. This may be because when the ultrafiltration pressure is too low and the temperature is too low, the driving force is insufficient, resulting in a decrease in the rate at which the solvent and small molecule impurities pass through the ultrafiltration membrane, and the target protein retains for too long on the surface of the ultrafiltration membrane, which makes it easy for the impurities remaining in the membrane pores to undergo nonspecific adsorption or aggregation, resulting in some proteins being unable to be effectively retained, thereby reducing the protein extraction rate; when the ultrafiltration pressure and temperature are too high, the structure of the protein is destroyed, and some target proteins that exceed the membrane cut-off molecular weight will be "forced to pass through" the membrane pores, resulting in a decrease in protein purity.

[0057] Example 12 Example 12 Based on the preparation method of Example 1, the method of simultaneously extracting and antioxidant the crushed pumpkin seed meal in S1 is replaced by first soaking the crushed pumpkin seed meal in an antioxidant solvent for 20-30 minutes, then adding water to the system to a solid-liquid ratio of 1:10 and adjusting the pH to 7-8.5 for extraction, while the other conditions remain unchanged.

[0058] The pumpkin seed protein of Example 12 was subjected to the above-mentioned performance test, and the test results are shown in Table 6.

[0059] Table 6 Performance test table of Example 1 and Example 12 project Example 1 Example 12 Protein extraction rate / % 88.6 89.7 Polyphenol oxidation rate / % 11.5 10.8 Referring to Table 6, by comparing Example 1 and Example 12, it can be seen that subjecting the crushed pumpkin seed meal to antioxidant treatment before extraction can effectively improve the protein extraction rate and reduce the polyphenol oxidation rate. This may be because when the cell structure of the pumpkin seed meal is destroyed after crushing, the activity of polyphenol oxidase is highest. After being activated, polyphenol oxidase will contact with the substrate to accelerate oxidation. Soaking the crushed pumpkin seed meal in an antioxidant first can inhibit the activity of polyphenol oxidase as much as possible or interrupt the oxidation chain reaction, thereby reducing the oxidation of polyphenols.

[0060] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A pumpkin seed protein extraction process, characterized in that, The following steps are involved: S1: After the pumpkin seed meal is crushed, water and antioxidants are added to extract at 20-60°C, and then ultrafiltration is performed to obtain an extract; S2: adding hydrochloric acid to the extract for acid precipitation, and filtering to obtain protein solid; S3: After washing and drying the protein solid, pumpkin seed protein is obtained.

2. The extraction process of pumpkin seed protein according to claim 1, characterized in that: The added amount of the antioxidant accounts for 0.3-1.5% of the mass of the pumpkin seed meal.

3. The extraction process of pumpkin seed protein according to claim 2, characterized in that: The antioxidant is at least one of rosemary extract and sodium glycyrrhizate.

4. The extraction process of pumpkin seed protein according to claim 3, characterized in that: The antioxidant is a mixture of rosemary extract and sodium glycyrrhizate.

5. The extraction process of pumpkin seed protein according to claim 4, characterized in that: The mixing ratio of the rosemary extract and sodium glycyrrhizate is 1-3:

1.

6. The extraction process of pumpkin seed protein according to claim 1, characterized in that: In S1, the mesh size of the crushed pumpkin seed meal is 40-80 mesh.

7. The extraction process of pumpkin seed protein according to claim 1, characterized in that: In S1, the ultrafiltration pressure is 0.05-0.25 MPa, and the ultrafiltration temperature is 20-50°C.

8. The process for extracting pumpkin seed protein according to claim 1, wherein: In S1, the crushed pumpkin seed meal is first soaked in an antioxidant solution for 20-30 minutes, and then water is added to extract the protein.

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