A Torreya grandis seed polypeptide with antioxidant and hyaluronidase inhibitory activities, its preparation and application

Abstract

This invention relates to a Torreya grandis seed polypeptide with antioxidant and hyaluronidase inhibitory activities, its preparation, and its application. The method includes the following steps: S1, mixing Torreya grandis seed powder and ethanol for defatting to obtain a solid phase; S2, preparing the solid phase into a solution, acidifying it, then enzymatically hydrolyzing it with pepsin, inactivating the enzyme, centrifuging to collect the liquid phase, and freeze-drying it to obtain Torreya grandis seed polypeptide powder. Compared with the prior art, this invention provides a simple, reasonable, and efficient preparation method for Torreya grandis seed polypeptide, exhibiting good activity. It also proposes the application of Torreya grandis seed polypeptide in antioxidant and hyaluronidase activity inhibition, expanding its application in the fields of cosmeceuticals and food.

Description

Technical Field

[0001] This invention belongs to the field of polypeptide preparation technology, and in particular relates to a Torreya grandis seed polypeptide with antioxidant and hyaluronidase inhibitory activities, its preparation and application. Background Technology

[0002] Chinese torreya (Torreya grandis) is a cultivated variety derived from wild torreya trees. It is primarily grown in the hilly regions of southern China, such as Jiangsu, Zhejiang, Hunan, Guizhou, Anhui, Fujian, and Jiangxi. With a cultivation history of over 1300 years, it is a rare and unique woody oilseed tree species native to my country. Studies have shown that Chinese torreya contains abundant active ingredients, such as fatty acids, amino acids, terpenoids, flavonoids, and phenolic acids. The seeds contain 10.3%–16.4% protein and 118.1 g / kg of amino acids. They also contain vitamins (niacin and folic acid), minerals (Mg, Ca, Fe, Zn, Se), tocopherols, sterols (β-sitosterol, brassosterol, stigmasterol), and polyphenols, exhibiting anti-inflammatory, antifungal, bactericidal, antiviral, and antitumor activities. It can be used to treat various diseases, including atherosclerosis, lowering blood lipids, and anthelmintics, demonstrating high medicinal value.

[0003] Research on Torreya grandis mainly focuses on its seed oil, inner seed coat, aril, and functional components such as flavonoids, polyphenols, and vitamins. Research and patents on Torreya grandis seed polypeptides are scarce. Currently, the preparation of Torreya grandis seed polypeptides primarily involves alkali extraction and acid precipitation of the seeds to extract proteins, followed by protease hydrolysis to obtain crude polypeptide extracts. This method is cumbersome, time-consuming, and has low extraction efficiency. Therefore, a simple, time-efficient, and high-yield method for preparing Torreya grandis seed polypeptides is needed.

[0004] Furthermore, existing technologies only study the antioxidant, anti-inflammatory, and angiotensin-converting enzyme-inhibiting activities of Torreya grandis seed polypeptides, but do not mention its hyaluronidase-inhibiting effect, which limits the application areas of Torreya grandis.

[0005] For example, CN115786430A discloses a blood pressure-lowering Torreya grandis polypeptide and its preparation method. The preparation method includes the following steps: S1, extracting Torreya grandis cake with water, separating the solid and liquid, and repeating the extraction of the residue 1-3 times, combining the extracts, and then concentrating to obtain a crude protein extract; S2, adjusting the pH of the above crude protein extract to 8-10, adding alkaline protease for enzymatic hydrolysis, then adjusting the pH to 6-7, adding papain for enzymatic hydrolysis, heating to 85-95℃ for 5-15 minutes after the enzymatic hydrolysis reaction, and cooling to below 30℃; S3, taking the crude polypeptide solution prepared in step S2, extracting polypeptides of 1000-5000 Da, and drying them to obtain the blood pressure-lowering Torreya grandis polypeptide. However, this enzymatic hydrolysis method has a low polypeptide yield and cannot be used for large-scale production. Summary of the Invention

[0006] The purpose of this invention is to overcome the defects of the prior art and provide a Torreya grandis seed polypeptide with antioxidant and hyaluronidase inhibitory activities, as well as its preparation and application. This invention prepares Torreya grandis seed polypeptide with good antioxidant activity and high hyaluronidase inhibition rate, expanding the application of Torreya grandis in the chemical industry, especially in the cosmetics industry.

[0007] The objective of this invention can be achieved through the following technical solutions:

[0008] This invention provides a method for preparing Torreya grandis seed polypeptides with antioxidant and hyaluronidase inhibitory activities, characterized by comprising the following steps:

[0009] S1, the solid phase obtained by defatting a mixture of Torreya grandis seed powder and ethanol;

[0010] S2, after the solid phase is prepared into a solution, it is acidified, then enzymatically hydrolyzed with pepsin, after enzyme inactivation, the liquid phase is collected by centrifugation and then freeze-dried to obtain Torreya seed polypeptide powder.

[0011] Furthermore, in step S1, the Torreya grandis seed powder is obtained by crushing Torreya grandis seed meal after oil extraction;

[0012] In step S1, the method of mixing Torreya grandis seed powder and ethanol includes: adding Torreya grandis seed powder to an ethanol solution;

[0013] In step S1, the ratio of the amount of Torreya grandis seed powder to the amount of ethanol in the mixture formed by Torreya grandis seed powder and ethanol is 1g:3-10ml.

[0014] In step S1, the ethanol concentration is 75%.

[0015] Furthermore, in step S1, the defatting specifically includes: mixing Torreya seed powder and ethanol, followed by ultrasonication and then allowing it to stand and soak.

[0016] In step S1, after the torreya seed powder and ethanol are mixed, the mixture is ultrasonicated for 20-60 minutes and then allowed to stand and soak for 2-6 hours to degrease.

[0017] In step S1, after defatting the Torreya seed powder, the supernatant is removed and dried to obtain a solid phase.

[0018] Furthermore, in step S2, preparing the solid into a solution includes: mixing the solid with water;

[0019] In step S2, the concentration of the solid phase in the solution is specifically 0.06–0.14 g / ml.

[0020] Furthermore, in step S2, the acidification specifically involves adjusting the pH of the solution prepared from the solid phase to 2-4.

[0021] In step S2, the pH of the solution prepared from the solid phase is adjusted to 2-4 using 1 ml / L hydrochloric acid.

[0022] Furthermore, in step S2, the enzymatic hydrolysis includes: adding pepsin to the acidified solid solution and then heating it in a water bath;

[0023] In step S2, the mass ratio of the pepsin to the solid phase is 1 to 5:100.

[0024] In step S2, the enzyme activity of the pepsin is 30-150 U / mg.

[0025] In step S2, the specific conditions for water bath heating during enzymatic hydrolysis are: heating and stirring at 30–50°C for 2–4 hours.

[0026] Furthermore, in step S2, the enzyme inactivation specifically involves heating the enzymatic hydrolysate in a water bath.

[0027] In step S2, the specific conditions for the enzyme-inactivating water bath heating are: heating at 90-100℃ for 10-30 minutes.

[0028] Furthermore, in step S2, the liquid phase is subjected to ultrafiltration before freeze drying;

[0029] This includes ultrafiltration of the liquid phase using 3kDa and 10kDa ultrafiltration membranes before freeze-drying;

[0030] The specific conditions for freeze drying are: vacuum degree of 26 Pa, temperature of -68℃, and time of 48 h.

[0031] The second aspect of the present invention provides a method for preparing Torreya grandis seed polypeptide with antioxidant and hyaluronidase inhibitory activities as described above, resulting in Torreya grandis seed polypeptide with antioxidant and hyaluronidase inhibitory activities.

[0032] The third aspect of the present invention provides the application of the Torreya grandis seed polypeptide with antioxidant and hyaluronidase inhibitory activities as described above in the preparation of antioxidant and anti-inflammatory products.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] 1. The Torreya grandis seed polypeptide provided by this invention has excellent antioxidant properties and hyaluronidase inhibitory effects, and has high efficacy when applied in the cosmetics field.

[0035] 2. The Torreya grandis seed polypeptide provided by this invention has excellent antioxidant properties and hyaluronidase inhibitory effects. When applied in the field of functional foods, it can be used as a raw material or excipient for health products, which has high benefits.

[0036] 3. This invention utilizes specific enzymatic cleavage of proteases to convert plant proteins into free amino acids and short peptide chains, exposing more active sites and preparing protein hydrolysates with enhanced biological activity. The antioxidant properties and hyaluronidase inhibitory effects of the Torreya grandis seed polypeptides obtained by this invention are significantly enhanced. Attached Figure Description

[0037] Figure 1 The graph shows the effect of substrate concentration on the yield of Torreya grandis seed polypeptide in Examples 1 to 5.

[0038] Figure 2 The graph shows the effect of enzyme addition on the yield of Torreya grandis seed polypeptide in Examples 1, 6 to 9.

[0039] Figure 3 The graph shows the effect of enzymatic hydrolysis time on the yield of Torreya grandis seed polypeptides in Examples 1, 10 to 13.

[0040] Figure 4 The figure shows the results of the ABTS+ free radical scavenging experiment for the T0, T1, T2, and T3 Torreya grandis seed polypeptide groups in Example 5.

[0041] Figure 5 The figure shows the DPPH free radical scavenging results for the T0, T1, T2, and T3 Torreya grandis seed polypeptide groups in Example 5.

[0042] Figure 6 The figure shows the hydroxyl radical scavenging results of the T0, T1, T2, and T3 Torreya grandis seed polypeptide groups in Example 5.

[0043] Figure 7 The figure shows the results of the hyaluronidase inhibition experiment for the T0, T1, T2, and T3 Torreya seed polypeptide groups in Example 6. Detailed Implementation

[0044] To facilitate understanding of this application, it will be described more fully below through embodiments. However, this application can be implemented in many different forms and is not limited to the embodiments or implementations described herein. Rather, these embodiments are provided to provide a more thorough and complete understanding of the disclosure of this application.

[0045] For simplicity, this application only explicitly discloses some numerical ranges. However, any lower limit can be combined with any upper limit to form a range not explicitly stated; and any lower limit can be combined with other lower limits to form a range not explicitly stated, just as any upper limit can be combined with any other upper limit to form a range not explicitly stated. Furthermore, although not explicitly stated, every point or individual value between the endpoints of the range is included within that range. Therefore, each point or individual value can be used as its own lower or upper limit and combined with any other point or individual value or with other lower or upper limits to form a range not explicitly stated.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. It should be noted that, unless otherwise stated, the term "and / or" as used herein includes any and all combinations of one or more of the associated listed items, "above," "below," includes the stated number, and "one or more" with "multiple" means two or more.

[0047] The raw materials involved in the following examples and implementation methods are shown below:

[0048] Torreya seed meal was purchased from Zhejiang Baifan Agricultural Development Co., Ltd.

[0049] Ethanol, analytical grade, purchased from Sinopharm Chemical Reagent Co., Ltd.

[0050] Hydrochloric acid, analytical grade, purchased from Sinopharm Chemical Reagent Co., Ltd.; pepsin, 3000 U mg -1 Purchased from Shanghai Bohr Chemical Reagent Co., Ltd.

[0051] Alkaline protease, 200,000 U g -1 Purchased from Jiangsu Yinong Biotechnology Co., Ltd.;

[0052] Papain, 800,000 U g -1 Purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0053] Trypsin, 500,000 U g -1 Purchased from Sinopharm Chemical Reagent Co., Ltd.

[0054] Neutral protease, 500,000 U g -1 Purchased from Jiangsu Yinong Biotechnology Co., Ltd.

[0055] Studies have found that the current preparation of Torreya grandis seed polypeptides mainly involves extracting proteins through alkaline extraction and acid precipitation of Torreya grandis seeds, followed by protease hydrolysis to obtain crude polypeptide extracts. This method is cumbersome, time-consuming, and has low extraction efficiency. Furthermore, the application of Torreya grandis seed polypeptides is mainly focused on its antioxidant, anti-inflammatory, and angiotensin-converting enzyme inhibitory activities.

[0056] Based on this, the first aspect of this application provides a method for preparing Torreya grandis seed polypeptides with antioxidant and hyaluronidase inhibitory activities, comprising the following steps:

[0057] S1, the solid phase obtained by defatting a mixture of Torreya grandis seed powder and ethanol;

[0058] S2, after the solid phase is prepared into a solution, it is acidified, then enzymatically hydrolyzed with pepsin, after enzyme inactivation, the liquid phase is collected by centrifugation and then freeze-dried to obtain Torreya seed polypeptide powder.

[0059] In some embodiments, in step S1, the Torreya seed powder is obtained by crushing Torreya seed meal after oil extraction. Here, crushing is carried out by a crusher, but ball milling or grinding can also be used.

[0060] In some embodiments, step S1, the method of mixing Torreya grandis seed powder and ethanol includes: adding Torreya grandis seed powder to an ethanol solution, wherein in the mixture formed by Torreya grandis seed powder and ethanol, the ratio of the amount of Torreya grandis seed powder to ethanol is 1g:3-10ml, preferably 1g:3-8ml, and even more preferably 1g:5ml, wherein the concentration of ethanol is preferably 75%.

[0061] In some embodiments, step S1, the defatting specifically includes: mixing Torreya grandis seed powder and ethanol, followed by sonication, and then allowing it to stand and soak. The mixture of Torreya grandis seed powder and ethanol is sonicated for 20–60 min, preferably 20–40 min, and more preferably 30 min, followed by standing and soaking for 2–6 h, preferably 2–4 h, and more preferably 3 h. Furthermore, after defatting the Torreya grandis seed powder, the supernatant is removed, and the solid phase is dried. The removal of the supernatant is performed according to conventional methods in the art, such as filtration or centrifugation, with centrifugation being preferred. The drying specifically includes: allowing it to stand naturally to dry or baking, with natural drying being preferred.

[0062] In some embodiments, step S2, preparing the solid into a solution, includes mixing the solid with water, specifically adding the solid to water, wherein the concentration of the solid in the solution is specifically 0.06 to 0.14 g / ml, preferably 0.06 to 0.14 g / ml, and even more preferably 0.10 g / ml.

[0063] In some embodiments, in step S2, the acidification specifically involves adjusting the pH of the solution prepared from the solid phase to 2-4, preferably 2-3, and even more preferably 2, wherein the pH of the solution prepared from the solid phase is adjusted to 2-4, preferably 2-3, and even more preferably 2, using 1 ml / L hydrochloric acid.

[0064] In some embodiments, step S2, the enzymatic hydrolysis includes: adding pepsin to the acidified solid solution and then heating in a water bath, wherein the mass ratio of pepsin to solid is 1-5:100, preferably 2-5, and even more preferably 4:100, the enzyme activity of the pepsin is 30-150 U / mg, and the specific conditions for water bath heating in the enzymatic hydrolysis are: heating and stirring at 30-50°C for 2-4 hours, preferably heating and stirring at 30-40°C for 2-3 hours, and even more preferably heating and stirring at 37°C for 2.5 hours.

[0065] In some embodiments, in step S2, the enzyme inactivation specifically involves heating the enzymatic hydrolysate in a water bath. The specific conditions for the water bath heating for enzyme inactivation are: heating at 90-100°C for 10-30 minutes, preferably heating at 95-100°C for 15-30 minutes, and even more preferably heating at 95°C for 15 minutes.

[0066] In some embodiments, step S2 further includes ultrafiltration of the liquid phase before freeze-drying, wherein the ultrafiltration uses 3kDa and 10kDa ultrafiltration membranes. In addition, the specific conditions for freeze-drying are: vacuum degree of 26Pa, temperature of -68℃, and time of 48h.

[0067] The method for preparing Torreya grandis seed polypeptides provided in this application can bring at least the following beneficial effects:

[0068] The embodiments of this application utilize specific enzymatic cleavage of proteases to convert plant proteins into free amino acids and short peptide chains, exposing more active sites and preparing protein hydrolysates with enhanced biological activity. The antioxidant properties and hyaluronidase inhibitory effects of the Torreya grandis seed polypeptides obtained in the embodiments of this application are significantly enhanced.

[0069] The second aspect of the embodiments of this application provides the above-mentioned Torreya grandis seed polypeptide with antioxidant and hyaluronidase inhibitory activities.

[0070] The Torreya seed polypeptide provided in this application can bring at least the following beneficial effects:

[0071] 1. The Torreya seed polypeptide provided in this application has excellent antioxidant properties and hyaluronidase inhibitory effects, and has high efficacy when applied in the cosmetics field.

[0072] 2. The Torreya seed polypeptide provided in this application has excellent antioxidant properties and hyaluronidase inhibitory effects. When applied in the field of functional foods, it can be used as a raw material or excipient for health products, which has high benefits.

[0073] The third aspect of this application provides the application of the above-mentioned Torreya grandis seed polypeptide with antioxidant and hyaluronidase inhibitory activities in the preparation of antioxidant and anti-inflammatory products.

[0074] The above implementation methods will be described in more detail below with reference to specific embodiments.

[0075] Preparation Example

[0076] Example 1

[0077] Torreya seed meal was pulverized in a pulverizer to obtain Torreya seed powder. Torreya seed powder was defatted with 75% ethanol at a ratio of 1g:5mL, ultrasonically treated for 30min, soaked at room temperature for 2h, centrifuged to remove the upper defatted ethanol solution, dried overnight to obtain defatted Torreya seeds.

[0078] Weigh out defatted Torreya grandis seeds and mix with water, specifically 10g of defatted Torreya grandis seeds and 100ml of water, stir well, then adjust the pH of the mixture to 2.0 with a 1mol / L HCl aqueous solution, add pepsin, the mass ratio of pepsin to defatted Torreya grandis seeds is 4%, so that the pepsin enzyme activity is 120U / mg, and stir in a water bath at 37℃ for 2.5h. After that, place the enzymatic hydrolysate in a water bath at 95℃ for 15 minutes to inactivate the enzyme, then centrifuge at 8000r / min for 20min, collect the supernatant, and use the resulting filtrate as the Torreya grandis seed polypeptide extract solution.

[0079] Example 2

[0080] Torreya seed meal was pulverized in a pulverizer to obtain Torreya seed powder. Torreya seed powder was defatted with 75% ethanol at a ratio of 1g:5mL, ultrasonically treated for 30min, soaked at room temperature for 2h, centrifuged to remove the upper defatted ethanol solution, dried overnight to obtain defatted Torreya seeds.

[0081] Weigh out defatted Torreya grandis seeds and mix with water, specifically 6g of defatted Torreya grandis seeds and 100ml of water, and stir well. Then adjust the pH of the mixture to 2.0 with a 1mol / L HCl aqueous solution. Add pepsin, with a pepsin-to-defatted Torreya grandis seed mass ratio of 4%, so that the pepsin enzyme activity is 120U / mg. Stir in a water bath at 37℃ for 2.5h. After that, place the enzymatic hydrolysate in a 95℃ water bath for 15 minutes to inactivate the enzyme, and then centrifuge at 8000r / min for 20min. Collect the supernatant, and use the resulting filtrate as the Torreya grandis seed polypeptide extract solution.

[0082] Example 3

[0083] Torreya seed meal was pulverized in a pulverizer to obtain Torreya seed powder. Torreya seed powder was defatted with 75% ethanol at a ratio of 1g:5mL, ultrasonically treated for 30min, soaked at room temperature for 2h, centrifuged to remove the upper defatted ethanol solution, dried overnight to obtain defatted Torreya seeds.

[0084] Weigh out defatted Torreya grandis seeds and mix with water, specifically 8g of defatted Torreya grandis seeds and 100ml of water, and stir well. Then adjust the pH of the mixture to 2.0 with a 1mol / L HCl aqueous solution. Add pepsin, with a pepsin-to-defatted Torreya grandis seed mass ratio of 4%, so that the pepsin enzyme activity is 120U / mg. Stir in a water bath at 37℃ for 2.5h. After that, place the enzymatic hydrolysate in a 95℃ water bath for 15 minutes to inactivate the enzyme, and then centrifuge at 8000r / min for 20min. Collect the supernatant, and use the resulting filtrate as the Torreya grandis seed polypeptide extract solution.

[0085] Example 4

[0086] Torreya seed meal was pulverized in a pulverizer to obtain Torreya seed powder. Torreya seed powder was defatted with 75% ethanol at a ratio of 1g:5mL, ultrasonically treated for 30min, soaked at room temperature for 2h, centrifuged to remove the upper defatted ethanol solution, dried overnight to obtain defatted Torreya seeds.

[0087] Weigh out defatted Torreya grandis seeds and mix with water, specifically 12g of defatted Torreya grandis seeds and 100ml of water, stir well, then adjust the pH of the mixture to 2.0 with a 1mol / L HCl aqueous solution, add pepsin, the mass ratio of pepsin to defatted Torreya grandis seeds is 4%, so that the pepsin enzyme activity is 120U / mg, and stir in a water bath at 37℃ for 2.5h. After that, place the enzymatic hydrolysate in a water bath at 95℃ for 15 minutes to inactivate the enzyme, then centrifuge at 8000r / min for 20min, collect the supernatant, and use the resulting filtrate as the Torreya grandis seed polypeptide extract solution.

[0088] Example 5

[0089] Torreya seed meal was pulverized in a pulverizer to obtain Torreya seed powder. Torreya seed powder was defatted with 75% ethanol at a ratio of 1g:5mL, ultrasonically treated for 30min, soaked at room temperature for 2h, centrifuged to remove the upper defatted ethanol solution, dried overnight to obtain defatted Torreya seeds.

[0090] Weigh out defatted Torreya grandis seeds and mix with water, specifically 14g of defatted Torreya grandis seeds and 100ml of water, and stir well. Then adjust the pH of the mixture to 2.0 with a 1mol / L HCl aqueous solution. Add pepsin, with a pepsin-to-defatted Torreya grandis seed mass ratio of 4%, so that the pepsin enzyme activity is 120U / mg. Stir in a water bath at 37℃ for 2.5h. After that, place the enzymatic hydrolysate in a 95℃ water bath for 15 minutes to inactivate the enzyme, and then centrifuge at 8000r / min for 20min. Collect the supernatant, and use the resulting filtrate as the Torreya grandis seed polypeptide extract solution.

[0091] Example 6

[0092] Torreya seed meal was pulverized in a pulverizer to obtain Torreya seed powder. Torreya seed powder was defatted with 75% ethanol at a ratio of 1g:5mL, ultrasonically treated for 30min, soaked at room temperature for 2h, centrifuged to remove the upper defatted ethanol solution, dried overnight to obtain defatted Torreya seeds.

[0093] Weigh out defatted Torreya grandis seeds and mix with water, specifically 10g of defatted Torreya grandis seeds and 100ml of water, stir well, then adjust the pH of the mixture to 2.0 with a 1mol / L HCl aqueous solution, add pepsin, the mass ratio of pepsin to defatted Torreya grandis seeds is 1%, so that the pepsin enzyme activity is 120U / mg, and stir in a water bath at 37℃ for 2.5h. After that, place the enzymatic hydrolysate in a water bath at 95℃ for 15 minutes to inactivate the enzyme, then centrifuge at 8000r / min for 20min, collect the supernatant, and use the resulting filtrate as the Torreya grandis seed polypeptide extract solution.

[0094] Example 7

[0095] Torreya seed meal was pulverized in a pulverizer to obtain Torreya seed powder. Torreya seed powder was defatted with 75% ethanol at a ratio of 1g:5mL, ultrasonically treated for 30min, soaked at room temperature for 2h, centrifuged to remove the upper defatted ethanol solution, dried overnight to obtain defatted Torreya seeds.

[0096] Weigh out defatted Torreya grandis seeds and mix with water, specifically 10g of defatted Torreya grandis seeds and 100ml of water, stir well, then adjust the pH of the mixture to 2.0 with a 1mol / L HCl aqueous solution, add pepsin, the mass ratio of pepsin to defatted Torreya grandis seeds is 2%, so that the pepsin enzyme activity is 120U / mg, and stir in a water bath at 37℃ for 2.5h. After that, place the enzymatic hydrolysate in a water bath at 95℃ for 15 minutes to inactivate the enzyme, then centrifuge at 8000r / min for 20min, collect the supernatant, and use the resulting filtrate as the Torreya grandis seed polypeptide extract solution.

[0097] Example 8

[0098] Torreya seed meal was pulverized in a pulverizer to obtain Torreya seed powder. Torreya seed powder was defatted with 75% ethanol at a ratio of 1g:5mL, ultrasonically treated for 30min, soaked at room temperature for 2h, centrifuged to remove the upper defatted ethanol solution, dried overnight to obtain defatted Torreya seeds.

[0099] Weigh out defatted Torreya grandis seeds and mix with water, specifically 10g of defatted Torreya grandis seeds and 100ml of water, and stir well. Then adjust the pH of the mixture to 2.0 with a 1mol / L HCl aqueous solution. Add pepsin, with a pepsin-to-defatted Torreya grandis seed mass ratio of 3%, so that the pepsin enzyme activity is 120U / mg. Stir in a water bath at 37℃ for 2.5h. After that, place the enzymatic hydrolysate in a 95℃ water bath for 15 minutes to inactivate the enzyme, and then centrifuge at 8000r / min for 20min. Collect the supernatant, and use the resulting filtrate as the Torreya grandis seed polypeptide extract solution.

[0100] Example 9

[0101] Torreya seed meal was pulverized in a pulverizer to obtain Torreya seed powder. Torreya seed powder was defatted with 75% ethanol at a ratio of 5g:5mL, ultrasonically treated for 30min, soaked at room temperature for 2h, centrifuged to remove the upper defatted ethanol solution, dried overnight to obtain defatted Torreya seeds.

[0102] Weigh out defatted Torreya grandis seeds and mix with water, specifically 10g of defatted Torreya grandis seeds and 100ml of water, and stir well. Then adjust the pH of the mixture to 2.0 with a 1mol / L HCl aqueous solution. Add pepsin, with a pepsin-to-defatted Torreya grandis seed mass ratio of 3%, so that the pepsin enzyme activity is 120U / mg. Stir in a water bath at 37℃ for 2.5h. After that, place the enzymatic hydrolysate in a 95℃ water bath for 15 minutes to inactivate the enzyme, and then centrifuge at 8000r / min for 20min. Collect the supernatant, and use the resulting filtrate as the Torreya grandis seed polypeptide extract solution.

[0103] Example 10

[0104] Torreya seed meal was pulverized in a pulverizer to obtain Torreya seed powder. Torreya seed powder was defatted with 75% ethanol at a ratio of 5g:5mL, ultrasonically treated for 30min, soaked at room temperature for 2h, centrifuged to remove the upper defatted ethanol solution, dried overnight to obtain defatted Torreya seeds.

[0105] Weigh out defatted Torreya grandis seeds and mix with water, specifically 10g of defatted Torreya grandis seeds and 100ml of water, stir well, then adjust the pH of the mixture to 2.0 with a 1mol / L HCl aqueous solution, add pepsin, the mass ratio of pepsin to defatted Torreya grandis seeds is 3%, so that the pepsin enzyme activity is 120U / mg, and stir in a water bath at 37℃ for 2h. After that, place the enzymatic hydrolysate in a water bath at 95℃ for 15 minutes to inactivate the enzyme, then centrifuge at 8000r / min for 20min, collect the supernatant, and use the resulting filtrate as the Torreya grandis seed polypeptide extract solution.

[0106] Example 11

[0107] Torreya seed meal was pulverized in a pulverizer to obtain Torreya seed powder. Torreya seed powder was defatted with 75% ethanol at a ratio of 5g:5mL, ultrasonically treated for 30min, soaked at room temperature for 2h, centrifuged to remove the upper defatted ethanol solution, dried overnight to obtain defatted Torreya seeds.

[0108] Weigh out defatted Torreya grandis seeds and mix with water, specifically 10g of defatted Torreya grandis seeds and 100ml of water, stir well, then adjust the pH of the mixture to 2.0 with a 1mol / L HCl aqueous solution, add pepsin, the mass ratio of pepsin to defatted Torreya grandis seeds is 3%, so that the pepsin enzyme activity is 120U / mg, and stir in a water bath at 37℃ for 3h. After that, place the enzymatic hydrolysate in a 95℃ water bath for 15 minutes to inactivate the enzyme, then centrifuge at 8000r / min for 20min, collect the supernatant, and use the resulting filtrate as the Torreya grandis seed polypeptide extract solution.

[0109] Example 12

[0110] Torreya seed meal was pulverized in a pulverizer to obtain Torreya seed powder. Torreya seed powder was defatted with 75% ethanol at a ratio of 5g:5mL, ultrasonically treated for 30min, soaked at room temperature for 2h, centrifuged to remove the upper defatted ethanol solution, dried overnight to obtain defatted Torreya seeds.

[0111] Weigh out defatted Torreya grandis seeds and mix with water, specifically 10g of defatted Torreya grandis seeds and 100ml of water, and stir well. Then adjust the pH of the mixture to 2.0 with a 1mol / L HCl aqueous solution. Add pepsin, with a pepsin-to-defatted Torreya grandis seed mass ratio of 3%, so that the pepsin enzyme activity is 120U / mg. Stir in a water bath at 37℃ for 3.5h. After that, place the enzymatic hydrolysate in a 95℃ water bath for 15 minutes to inactivate the enzyme, and then centrifuge at 8000r / min for 20min. Collect the supernatant, and use the resulting filtrate as the Torreya grandis seed polypeptide extract solution.

[0112] Example 13

[0113] Torreya seed meal was pulverized in a pulverizer to obtain Torreya seed powder. Torreya seed powder was defatted with 75% ethanol at a ratio of 5g:5mL, ultrasonically treated for 30min, soaked at room temperature for 2h, centrifuged to remove the upper defatted ethanol solution, dried overnight to obtain defatted Torreya seeds.

[0114] Weigh out defatted Torreya grandis seeds and mix with water, specifically 10g of defatted Torreya grandis seeds and 100ml of water, stir well, then adjust the pH of the mixture to 2.0 with a 1mol / L HCl aqueous solution, add pepsin, the mass ratio of pepsin to defatted Torreya grandis seeds is 3%, so that the pepsin enzyme activity is 120U / mg, and stir in a water bath at 37℃ for 4h. After that, place the enzymatic hydrolysate in a water bath at 95℃ for 15 minutes to inactivate the enzyme, then centrifuge at 8000r / min for 20min, collect the supernatant, and use the resulting filtrate as the Torreya grandis seed polypeptide extract solution.

[0115] Comparative Example 1

[0116] The only difference from Example 1 is that pepsin treatment was not added and the pH of the mixture was 5.6. All other operations and process conditions were the same as in Example 1.

[0117] Comparative Example 2

[0118] The only difference from Example 1 is that the added protease is an alkaline protease mixture with a pH of 8.0, and the water bath stirring temperature is 50°C. All other operations and process conditions are the same as in Example 1.

[0119] Comparative Example 3

[0120] The only difference from Example 1 is that the added protease is papain, the pH of the mixture is 6.2, and the water bath stirring temperature is 50°C. All other operations and process conditions are the same as in Example 1.

[0121] Comparative Example 4

[0122] The only difference from Example 1 is that the added protease is trypsin and the pH of the mixture is 8.0. All other operations and process conditions are the same as in Example 1.

[0123] Comparative Example 5

[0124] The only difference from Example 1 is that the added protease is a neutral protease, the pH of the mixture is 7.0, and the water bath stirring temperature is 50°C. All other operations and process conditions are the same as in Example 1.

[0125] Characterization example

[0126] Preparation of a standard curve for peptide content:

[0127] Using the absorbance of the Gly-Gly-Tyr-Arg tetrapeptide at 540 nm as the dependent variable and the Gly-Gly-Tyr-Arg tetrapeptide concentration as the independent variable, a linear regression equation was obtained, showing a good linear relationship.

[0128] The method for determining the polypeptide extraction rate is as follows:

[0129] Take 2.5 mL of sample solution, add 2.5 mL of 10% (w / v) trichloroacetic acid (TCA) aqueous solution, mix thoroughly on a vortex mixer, let stand for 10 min, then centrifuge at 6000 r / min for 15 min. Transfer the supernatant to a 50 mL volumetric flask and dilute to the mark with 5% TCA, then shake well. Next, take 6.0 mL of the above solution and place it in another test tube, add 4.0 mL of biuret reagent (sample solution:biuret reagent = 3:2, v / v), mix thoroughly on a vortex mixer, let stand for 10 min, centrifuge at 2000 r / min for 10 min, and measure the OD value of the supernatant at 540 nm. Determine the peptide concentration C (mg / mL) in the sample solution by referring to the standard curve, and then calculate the peptide content in the sample and the peptide yield.

[0130] The content of Torreya seed polypeptides in the Torreya seed polypeptide extract solution was determined according to the above method, and the content of Torreya seed polypeptides was calculated according to the Gly-Gly-Tyr-Arg tetrapeptide standard curve to obtain the polypeptide yield.

[0131] The Torreya grandis seed polypeptide extract obtained in the above embodiments was subjected to ultrafiltration treatment using 3kDa and 10kDa ultrafiltration membranes to obtain unseparated T0 Torreya grandis seed polypeptide group, T1 Torreya grandis seed polypeptide group with molecular weight >10kDa, T2 Torreya grandis seed polypeptide group with molecular weight 3-10kDa, and T3 Torreya grandis seed polypeptide group with molecular weight <3kDa; T0, T1, T2, and T3 Torreya grandis seed polypeptide groups were obtained by freeze drying and refrigerated for later use.

[0132] 1. Effect of substrate concentration on the yield of Torreya grandis seed polypeptides

[0133] It should be noted that the substrate here refers to the aforementioned solid phase.

[0134] In the experiment, 6g (Example 2), 8g (Example 3), 10g (Example 1), 12g (Example 4), and 14g (Example 5) of defatted Torreya grandis seeds were weighed sequentially and added to 100mL of deionized water. The extraction temperature was controlled at 50℃, and the pH was adjusted to 2.0 with 1mol / L HCl aqueous solution. Pepsin was added, and the mixture was stirred in a water bath for 3 hours. After the extraction, the enzyme hydrolysate was placed in a 95℃ water bath for 15 minutes to inactivate the enzyme, and then centrifuged at 8000r / min for 20 minutes. The supernatant was collected, and the polypeptide content was obtained by comparing with the polypeptide standard curve. The polypeptide yield was calculated. A bar chart was plotted to study the effect of different substrate concentrations on the polypeptide extraction yield of Torreya grandis seeds. Each experiment was repeated 3 times. The results are shown below. Figure 1 As shown.

[0135] Depend on Figure 1 It can be seen that as the substrate concentration increases, the peptide content increases, and the peptide yield shows a trend of first increasing and then decreasing. After the substrate concentration exceeds a certain amount, the enzyme reaction sites become saturated, resulting in a decrease in the overall yield. When the substrate concentration reaches 0.1 g / mL, the peptide yield of Torreya grandis seeds is the highest, at 1.061%.

[0136] 2. Effect of enzyme addition amount on the yield of Torreya grandis seed polypeptides

[0137] In this experiment, 10g of defatted Torreya grandis seeds were weighed and added to 100mL of deionized water. The pH was adjusted to 2.0 with 1mol / L HCl aqueous solution. The amount of pepsin added was set as follows: 0.1g (Example 6), 0.2g (Example 7), 0.3g (Example 8), 0.4g (Example 1), and 0.5g (Example 9). The mixture was stirred in a water bath for 3 hours. After the reaction, the enzyme was inactivated in a 95℃ water bath for 15 minutes, and then centrifuged at 8000r / min for 20 minutes. The supernatant was collected, and the peptide content was obtained by comparing with the peptide standard curve. The peptide yield was calculated. A bar chart was plotted to study the effect of different enzyme addition amounts on the peptide extraction yield of Torreya grandis seeds. Each experiment was repeated 3 times. The results are shown below. Figure 2 As shown.

[0138] from Figure 2 It can be seen that as enzyme activity increases, the peptide yield first increases and then decreases, with a significant increase in peptide yield. When the enzyme reaches a certain addition amount, further increasing the enzyme amount will lead to excessive hydrolysis of peptides into amino acids, resulting in a decrease in peptide yield. When the enzyme activity is 120,000 U / g, i.e., the addition amount is 0.4g, the peptide yield hydrolyzed by pepsin is the highest, at 1.490%.

[0139] 3. Effect of extraction time on the yield of Torreya grandis seed polypeptides

[0140] In this experiment, 10g of defatted Torreya grandis seeds were weighed and added to 100mL of deionized water (pH 8.0). Pepsin was added, and the water bath stirring time was set to 2h (Example 10), 2.5h (Example 1), 3h (Example 11), 3.5h (Example 12), and 4h (Example 13). After the water bath, the enzyme was inactivated in a 95℃ water bath for 15 minutes, followed by centrifugation at 8000r / min for 20min. The supernatant was collected, and the polypeptide content was determined by comparing with a polypeptide standard curve. The polypeptide yield was calculated. A bar chart was plotted to study the effect of different extraction times on the polypeptide extraction yield from Torreya grandis seeds. Each experiment was repeated three times. The results are shown below. Figure 3 As shown.

[0141] Depend on Figure 3 It can be seen that the yield of peptides hydrolyzed by the two proteases gradually increases with increasing extraction time. Figure 3 As the enzymatic hydrolysis time increases, specific sites on the protein molecules are gradually exposed, and the amount of peptides released in the hydrolysate gradually increases. The decrease in peptide yield may be because enzyme-specific binding sites still exist on the released peptide chains. As the enzymatic hydrolysis time is extended, the specific sites on the protein molecules are completely reacted, and the protease further binds to the peptides, causing the peptides to undergo excessive enzymatic cleavage, thus affecting the extraction of peptides. When the pepsin extraction time is 180 min, the peptide yield is the highest, which is 1.484%.

[0142] 4. Orthogonal experiment for optimizing the extraction process of polypeptides from Torreya grandis seeds

[0143] Orthogonal experimental analysis was conducted by combining the results of the single-factor experiments in the above-mentioned characterization examples 1 to 3.

[0144] 1) Selection of factor levels in orthogonal experiments

[0145] Using substrate concentration, enzyme activity, and extraction time as factors, the L9(3)2 enzyme was analyzed. 3 An orthogonal experiment was conducted (see Table 1). The optimal extraction process was determined based on the yield of defatted Torreya grandis seed polypeptides.

[0146] Table 1

[0147]

[0148] 2) Results of orthogonal experiments

[0149] Table 2 Results of orthogonal experiments for process optimization

[0150]

[0151]

[0152] From Table 2 and Figures 1-3The results showed that in the experiment of hydrolyzing Torreya grandis seeds with pepsin, enzyme activity had the most significant effect on polypeptide extraction, followed by time and substrate concentration. The results indicated that when the factor combination was A3B2C1, i.e., substrate concentration of 12%, enzyme activity of 120000 U / g, and time of 150 min, the yield of Torreya grandis seed polypeptides was the highest, at 0.79±0.0294%.

[0153] 5. Antioxidant property test

[0154] Preparation of Torreya grandis seed polypeptides:

[0155] Extraction conditions optimized by the above orthogonal experiments were used to prepare Torreya grandis seed polypeptides. Torreya grandis seed meal was pulverized in a pulverizer to obtain Torreya grandis seed powder. The Torreya grandis seed powder was defatted with 75% ethanol at a ratio of 1g:5mL, ultrasonicated for 30min, and soaked at room temperature for 2h. The upper defatted ethanol solution was removed, and the precipitate was dried overnight to obtain defatted Torreya grandis seeds. Weigh defatted Torreya grandis seeds and mix them with water at a ratio of 3g:25mL. Stir well and adjust the pH of the mixture to 2.0 with 1mol / L HCl aqueous solution. Add pepsin at a mass ratio of 4% to defatted Torreya grandis seeds to achieve an enzyme activity of 120U / mg. Stir in a water bath at 37℃ for 2.5h. After stirring, place the enzymatic hydrolysate in a water bath at 95℃ for 15 minutes to inactivate the enzyme, and then centrifuge at 8000r / min for 20min. Collect the supernatant and use the filtrate as the Torreya grandis seed polypeptide extract. The extract of Torreya grandis seeds was subjected to ultrafiltration treatment using 3kDa and 10kDa ultrafiltration membranes to obtain unseparated Torreya grandis seed polypeptide groups (T0), T1 Torreya grandis seed polypeptide groups with molecular weight >10kDa, T2 Torreya grandis seed polypeptide groups with molecular weight 3-10kDa, and T3 Torreya grandis seed polypeptide groups with molecular weight <3kDa. These were then freeze-dried under vacuum at 26Pa, -68℃, and for 48h to obtain T0, T1, T2, and T3 Torreya grandis seed polypeptide groups.

[0156] 1) Determination of ABTS+ free radical scavenging rate

[0157] 7 mmol / L ABTS + Mix the solution with an equal volume of 2.45 mmol / L potassium persulfate solution and let it stand in the dark at room temperature for 12–16 hours. Before use, dilute it with anhydrous ethanol to achieve an absorbance of 0.7 ± 0.02 at 734 nm. + Working solution. After the mixture was placed in the dark for 6 minutes, its absorbance was measured at 734 nm using a full-wavelength microplate reader.

[0158] Four groups of samples were prepared at concentration gradients of 0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 1.5 mg / mL, and 2.0 mg / mL, respectively, using the formulas T0, T1, T2, and T3.

[0159] The mixture was added according to groups A0, A1, and A2: A0 consisted of 600 μL of anhydrous ethanol plus 2.4 mL of ABTS. + Absorbance of the working solution; A1 is 600 μL of enzyme digest plus 2.4 mL of ABTS + The absorbance of the working solution; A2 is the absorbance of 600 μL of enzyme digest plus 2.4 mL of anhydrous ethanol. After incubation at room temperature in the dark for 20 min, the OD value is measured at 734 nm. The calculation formula is:

[0160] ABTS + Free radical scavenging rate (%) = (1 - (A1 - A2) / A0) × 100

[0161] The results are as follows Figure 4 ,from Figure 4 It can be seen that when the concentration of Torreya grandis seed polypeptide solution is 0.1–2.0 mg / mL, Torreya grandis seed polypeptide has an effect on ABTS. + The ability to scavenge free radicals is dose-dependent, IC50 50 The concentrations were 0.7058 mg / mL (T0), 0.2001 mg / mL (T1), 0.1229 mg / mL (T2), and 0.1221 mg / mL (T3), respectively. The results indicate that the ABTS peptides from Torreya grandis seeds possess strong ABTS activity. + Free radical scavenging ability, among which the T3 Torreya grandis seed polypeptide group with a molecular weight <3kDa has an IC50 value. 50 Minimum, ABTS + Free radicals have the strongest scavenging ability.

[0162] 2) DPPH free radical scavenging rate determination

[0163] Accurately weigh 0.004 g of DPPH and dilute to 100 mL with anhydrous ethanol. Let stand for no more than 4 hours to obtain the DPPH solution. Incubate the mixture in a 96-well plate in the dark for 30 min, and then measure its absorbance at 517 nm using a full-wavelength microplate reader.

[0164] Four groups of samples were prepared at concentration gradients of 0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 1.5 mg / mL, and 2.0 mg / mL, respectively, using the formulas T0, T1, T2, and T3.

[0165] The mixed solutions were added according to groups A0, A1, and A2: A0 was the absorbance of 50 μL anhydrous ethanol plus 100 μL LPPH working solution; A1 was the absorbance of 50 μL enzyme digest plus 100 μL LPPH working solution; and A2 was the absorbance of 50 μL enzyme digest plus 100 μL anhydrous ethanol. After incubating the 96-well plates in the dark for 30 min, the absorbance was measured at 517 nm using a full-wavelength microplate reader. The calculation formula is as follows:

[0166] DPPH radical scavenging rate (%) = (1 - (A1 - A2) / A0) × 100

[0167] The results are as follows Figure 5 ,from Figure 5 It can be seen that when the concentration of Torreya grandis seed polypeptide solution is 0.1–2.0 mg / mL, the unseparated T0 Torreya grandis seed polypeptide group and the T1 Torreya grandis seed polypeptide group with a molecular weight >10 kDa have significant DPPH free radical scavenging advantages, IC50 50 The concentrations were 0.8531 mg / mL (T0) and 0.9577 mg / mL (T1), respectively. The T2 Torreya grandis seed polypeptide group with a molecular weight of 3-10 kDa and the T3 Torreya grandis seed polypeptide group with a molecular weight of <3 kDa both had DPPH scavenging rates below 50% at concentrations of 0.01-2.0 mg / mL. Therefore, the unseparated T0 Torreya grandis seed polypeptide group had the strongest DPPH free radical scavenging rate.

[0168] 3) Hydroxyl radical scavenging rate determination

[0169] The salicylic acid-ferrous sulfate method was used. 9 mmol / L salicylic acid ethanol solution, 9 mmol / L FeSO4, sample solutions of different concentrations, and 1 mL each of 8.8 mmol / L H2O2 were added sequentially to the reaction system. After reacting in a constant temperature water bath at 37℃ for 15 min, the absorbance of the reaction solution was measured at 510 nm.

[0170] Four groups of samples were prepared at concentration gradients of 0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 1.5 mg / mL, and 2.0 mg / mL, respectively, using the formulas T0, T1, T2, and T3.

[0171] The reaction system was divided into groups A0, A1, and A2: A0 represents the absorbance of the reaction system where deionized water replaced the sample; A1 represents the absorbance of the reaction system where the sample was added; and A2 represents the absorbance of the reaction system where deionized water replaced the H2O2 solution. After reacting in a constant temperature water bath at 37℃ for 15 min, the absorbance of the reaction solution was measured at 510 nm. The calculation formula is as follows:

[0172] Hydroxyl radical scavenging rate (%) = (1 - (A1 - A2) / A0) × 100

[0173] The results are as follows Figure 6,from Figure 6 It can be seen that when the concentration of Torreya grandis seed polypeptide solution is 0.1–2.0 mg / mL, the scavenging ability of Torreya grandis seed polypeptide against hydroxyl radicals is dose-dependent, IC50... 50 The concentrations of the T1 Torreya grandis seed polypeptide group (with a molecular weight > 10 kDa) were 0.8873 mg / mL (T0), 1.5322 mg / mL (T2), and 0.8739 mg / mL (T3), respectively. At concentrations of 0.01–2.0 mg / mL, the hydroxyl radical scavenging rate did not exceed 50%. Therefore, the IC50 of the T3 Torreya grandis seed polypeptide group (with a molecular weight < 3 kDa) was significantly lower. 50 It has the lowest scavenging capacity for hydroxyl radicals and the strongest scavenging capacity.

[0174] 6. Hyaluronidase activity inhibition rate determination

[0175] Prepare the following solutions separately: an acetate buffer solution (0.2 mol / L acetic acid solution and 0.2 mol / L sodium acetate solution mixed in a certain ratio) at pH 5.6; a calcium chloride solution of 2.5 mmol / L; a hyaluronidase solution of 600 u / mL; a sodium hyaluronate solution of 0.5 mg / mL; a sodium hydroxide solution of 0.4 mol / L; an acetylacetone solution; and Ehrlich's reagent (0.8 g of p-dimethylaminobenzaldehyde dissolved in 15 mL of concentrated hydrochloric acid and 15 mL of anhydrous ethanol).

[0176] Add reagents according to Table 3, mix the system and develop color at room temperature for 20 min. After the process, measure the absorbance at 530 nm using an ELISA reader and calculate the hyaluronidase activity inhibition capacity of the Torreya grandis seed polypeptide solution. Each group has three parallel experiments.

[0177] Table 3 Hyaluronidase Activity Inhibition Experiment

[0178]

[0179]

[0180] The inhibition rate of hyaluronidase by Torreya grandis seed polypeptides reflects the strength of its anti-inflammatory activity; the higher the inhibition rate, the stronger the anti-inflammatory activity. The inhibition rate of hyaluronidase activity of Torreya grandis seed polypeptide solution is calculated according to the following formula:

[0181] Hyaluronidase inhibition rate (%) = (AB - C + D) / (AB) × 100

[0182] In the formula: C is the absorbance of the sample group, D is the absorbance of the sample control group, A is the absorbance of the negative control group, and B is the absorbance of the blank control group.

[0183] The results are as follows Figure 7 As shown, from Figure 7It can be seen that when the concentration of Torreya grandis seed polypeptide solution is 0.1–2.0 mg / mL, its inhibitory effect on hyaluronidase activity gradually increases with increasing concentration. Furthermore, the fraction separated by ultrafiltration membrane exhibits a significant inhibitory advantage on hyaluronidase activity compared to the unseparated fraction. Among them, the T3 Torreya grandis seed polypeptide group with a molecular weight <3 kDa showed the best scavenging activity; when the T3 concentration was 2.0 mg / mL, its hyaluronidase activity inhibition rate was 86.27%, indicating that the Torreya grandis seed polypeptide solution has a good inhibitory effect on hyaluronidase.

[0184] 7. Comparative Experiment

[0185] The polypeptide yield of the Torreya grandis seed polypeptide extract solutions prepared in Example 1 and Comparative Examples 1-5 was calculated using a standard curve. The test results are shown in Table 4.

[0186] Table 4

[0187] project Example 1 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Comparative Example 5 Peptide yield 0.79％ 0.21％ 0.67％ 0.41％ 0.52％ 0.39％

[0188] The test results in Table 4 show that:

[0189] (1) In Example 1, the yield of the Torreya grandis seed polypeptide extract liquid obtained by pepsin treatment was as high as 0.79%;

[0190] (2) In Comparative Example 1, the yield of peptides in the extract without protease treatment was only 0.21%;

[0191] (3) Comparative Examples 2, 3, 4 and 5 were treated with alkaline protease, papain, trypsin and neutral protease, respectively. The peptide yields of the Torreya grandis seed polypeptide extracts decreased by 0.12%, 0.38%, 0.27% and 0.40%, respectively. The reason may be that: Studies have shown that Torreya grandis seed protein has a high content of hydrophobic amino acids. Pepsin has specific cleavage of hydrophobic amino acids. After treatment with pepsin, more hydrophobic amino residue side chain groups are exposed, and more active peptides are released from the extract.

[0192] In summary, the type of protease used for hydrolysis is a key factor affecting the yield of peptides extracted from Torreya grandis seeds, and the extract solution obtained by treating with pepsin has the highest peptide yield.

[0193] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. The application of a Torreya grandis seed polypeptide with antioxidant and hyaluronidase inhibitory activities in the preparation of an anti-inflammatory product that inhibits hyaluronidase, characterized in that, The preparation method of the Torreya grandis seed polypeptide includes the following steps: S1, the solid phase obtained by defatting a mixture of Torreya grandis seed powder and ethanol; S2, after the solid phase is prepared into a solution, it is acidified, then enzymatically hydrolyzed with pepsin, after enzyme inactivation, the liquid phase is collected by centrifugation and then freeze-dried to obtain Torreya seed polypeptide powder.

2. The use of a torreya grandis seed polypeptide having antioxidant and hyaluronidase inhibitory activity according to claim 1 in the preparation of an anti-inflammatory product for inhibiting hyaluronidase, characterized in that, In step S1, the Torreya grandis seed powder is obtained by crushing Torreya grandis seed meal after oil extraction; And / or, in step S1, the method of mixing Torreya grandis seed powder and ethanol includes: adding Torreya grandis seed powder to an ethanol solution; And / or, in step S1, in the mixture of Torreya grandis seed powder and ethanol, the ratio of Torreya grandis seed powder to ethanol is 1g:3~10ml; And / or, in step S1, the ethanol concentration is 75%.

3. The application of the Torreya grandis seed polypeptide with antioxidant and hyaluronidase inhibitory activities according to claim 1 in the preparation of an anti-inflammatory product that inhibits hyaluronidase, characterized in that, In step S1, the defatting specifically includes: mixing Torreya grandis seed powder and ethanol, followed by ultrasonication and then allowing it to stand and soak. And / or, in step S1, after the torreya seed powder and ethanol are mixed, the mixture is ultrasonicated for 20-60 minutes and then allowed to stand and soak for 2-6 hours to degrease. And / or, in step S1, after defatting the Torreya grandis seed powder, the supernatant is removed and the product is dried to obtain a solid phase.

4. The application of the Torreya grandis seed polypeptide with antioxidant and hyaluronidase inhibitory activities according to claim 1 in the preparation of an anti-inflammatory product that inhibits hyaluronidase, characterized in that, In step S2, preparing the solid into a solution includes: mixing the solid with water; And / or, in step S2, the concentration of the solid phase in the solution is specifically 0.06~0.14 g / ml.

5. The application of the Torreya grandis seed polypeptide with antioxidant and hyaluronidase inhibitory activities according to claim 1 in the preparation of an anti-inflammatory product that inhibits hyaluronidase, characterized in that, In step S2, the acidification specifically involves adjusting the pH of the solution prepared from the solid phase to 2-4. And / or, in step S2, the pH of the solution prepared from the solid phase is adjusted to 2-4 using 1 ml / L hydrochloric acid.

6. The application of the Torreya grandis seed polypeptide with antioxidant and hyaluronidase inhibitory activities according to claim 1 in the preparation of an anti-inflammatory product that inhibits hyaluronidase, characterized in that, In step S2, the enzymatic hydrolysis includes: adding pepsin to the acidified solid solution and then heating it in a water bath; And / or, in step S2, the mass ratio of the pepsin to the solid is 1~5:100; And / or, in step S2, the enzyme activity of the pepsin is 30-150 U / mg; And / or, in step S2, the specific conditions for water bath heating in the enzymatic hydrolysis are: heating and stirring at 30~50℃ for 2~4 hours.

7. The application of the Torreya grandis seed polypeptide with antioxidant and hyaluronidase inhibitory activities according to claim 1 in the preparation of an anti-inflammatory product that inhibits hyaluronidase, characterized in that, In step S2, the enzyme inactivation specifically involves heating the enzymatic hydrolysate in a water bath. And / or, in step S2, the specific conditions for the enzyme-inactivating water bath heating are: heating at 90~100℃ for 10~30min.

8. The application of the Torreya grandis seed polypeptide with antioxidant and hyaluronidase inhibitory activities according to claim 1 in the preparation of an anti-inflammatory product that inhibits hyaluronidase, characterized in that, In step S2, the liquid phase is further subjected to ultrafiltration before freeze drying; And / or, prior to freeze-drying, the liquid phase may be ultrafiltered using 3 kDa and 10 kDa ultrafiltration membranes; And / or, the specific conditions for freeze drying are: a vacuum of 26 Pa, a temperature of -68 °C, and a time of 48 h.

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