A preparation and purification method of soybean oligopeptide and its application

Abstract

The present invention relates to a preparation and purification method of soybean oligopeptide and its application, and belongs to the technical field of oligopeptide preparation. The preparation and purification method of soybean oligopeptide of the present invention comprises soybean seed coat and seed embryo, wherein the seed coat is first fermented and then enzymatically hydrolyzed to obtain a peptide-containing solution, and the seed embryo is enzymatically hydrolyzed step by step to obtain a peptide-containing solution, after the peptide-containing solutions prepared by the seed coat and the seed embryo are combined, an adsorbent is used to remove impurities, and finally a soybean oligopeptide with high purity is obtained. The soybean oligopeptide obtained by the present invention has good moisturizing performance, can protect the skin, and resist light damage to the skin.

Description

Technical Field

[0001] The invention belongs to the technical field of oligopeptide preparation, and relates to a preparation and purification method of soybean oligopeptide and application thereof. Background Art

[0002] Soybean, soybean or soya bean is a leguminous plant native to East Asia, widely cultivated for its edible beans and used in a variety of applications. Traditional unfermented food uses of soybeans include soy milk, which is made from soy milk and tofu skin. Fermented soy products include soy sauce, bean paste, natto and tempeh. Defatted (skimmed) soybean meal is an important and inexpensive source of protein for animal feed and many packaged meals. Soybeans are rich in nutrients such as protein, fat, carbohydrates and minerals such as calcium, phosphorus and iron, and are the plant with the highest protein content in nature.

[0003] Soybean peptides are protein hydrolysates obtained by enzymatic hydrolysis of soybean protein. The biologically active ingredients contained in soybean peptides have multiple functions such as anti-cancer, hypoglycemic, lipid-lowering, and anti-oxidation (anti-aging). It is a highly potential functional base material with broad market application prospects. Summary of the invention

[0004] The main purpose of the present invention is to provide a method for preparing and purifying soybean oligopeptides. The soybean oligopeptides obtained by the present invention have good moisturizing and antioxidant properties, have a high protective effect on the skin, and can avoid skin damage caused by light.

[0005] The present invention adopts the following technical solutions to achieve the above purpose:

[0006] A method for preparing and purifying soybean oligopeptides, comprising the following steps:

[0007] Step 1, soaking the dried soybeans in an aqueous solution with a pH value of 7.5-8.0, separating the seed coat and the embryo, and washing them with deionized water until they are neutral for later use;

[0008] Step 2, adding 3-5 times the mass of water to the separated seed coat and grinding it into a homogenate, adding sucrose to the homogenate, inoculating Aspergillus niger spore suspension after mixing, adjusting the pH to 6.0-6.5, fermenting at 25-30° C. for 2-3 hours, heating and boiling, cooling to room temperature, inoculating Lactobacillus acidophilus liquid, fermenting anaerobically at 25-30° C. for 3-5 hours, centrifuging, and taking precipitate I; washing precipitate I to neutrality, adding an aqueous solution containing Bacillus subtilisin, enzymolyzing at 40-45° C. for 4-6 hours, centrifuging, and setting supernatant I and precipitate II for later use;

[0009] Step 3, adding 3-5 times the mass of the separated embryo to water and grinding to make a homogenate, adjusting the pH to 5.0-6.0, adding papain, enzymolysis at 40-45° C. for 1-2 hours, centrifuging, and setting the supernatant II and precipitate III aside; adding an aqueous solution containing bromelain to the precipitate III, adjusting the pH to 7.5-8.0, enzymolysis at 40-45° C. for 1-2 hours, centrifuging, and setting the supernatant III and precipitate IV aside;

[0010] Step 4, combining supernatant I, supernatant II, and supernatant III to obtain a mixed solution; allowing the mixed solution to stand for 1.5-2 hours, adding an adsorbent, and centrifuging to obtain supernatant IV; concentrating and drying supernatant IV to obtain soybean oligopeptide.

[0011] As a preferred embodiment, the amount of sucrose added in the above step 2 is 10-15 g / L.

[0012] As a preferred embodiment, the inoculation amount of the Aspergillus niger spore suspension in the above step 2 is 10-12 ml / L.

[0013] As a preferred embodiment, the spore concentration of the Aspergillus niger spore suspension in step 2 is 2-2.5×10 5 cfu / mL.

[0014] As a preferred embodiment, the inoculation amount of the Lactobacillus acidophilus liquid in the above step 2 is 10-15 ml / L.

[0015] As a preferred method, the effective viable count of the Lactobacillus acidophilus solution in step 2 is 4-5×10 5 cfu / mL.

[0016] As a preferred embodiment, the liquid ratio of the precipitate I to the aqueous solution containing subtilisin in the above step 2 is 1:(5-8) in kg / L.

[0017] As a preferred embodiment, the content of subtilisin in the aqueous solution containing subtilisin in step 2 is 5-8 g / L.

[0018] As a preferred embodiment, the amount of papain added in the above step 3 is 5-6 g / L.

[0019] As a preferred embodiment, the solid-liquid ratio of the precipitate III to the aqueous solution containing bromelain in the above step 3 is 1:(5-8) in kg / L.

[0020] As a preferred embodiment, the content of bromelain in the bromelain-containing aqueous solution in the above step 3 is 3-6 g / L.

[0021] As a preferred embodiment, the amount of adsorbent added in the above step 4 is 15-20 g / L.

[0022] As a preferred embodiment, the method for preparing the adsorbent in step 4 above comprises the following steps:

[0023] The precipitate II obtained in the above step 2 and the precipitate IV obtained in step 3 are mixed to obtain a mixture; the mixture is placed in an oxygen-free environment at 600-800° C. and calcined for 40-60 minutes. After the calcination is completed, molten polyethylene glycol is added, mixed, cooled to room temperature, and crushed to obtain an adsorbent.

[0024] As a preferred embodiment, in the above-mentioned adsorbent preparation method, the amount of polyethylene glycol added is 0.1-0.15 times the mass of the mixture.

[0025] As a preferred embodiment, in the above-mentioned adsorbent preparation method, the molecular weight of polyethylene glycol is 1000-1500.

[0026] The present invention provides a specific use of the soybean oligopeptide, that is, the soybean oligopeptide can be applied to cosmetics. The cosmetics prepared from the soybean oligopeptide of the present invention have good moisturizing, anti-oxidation and skin protection effects, and can resist light damage to the skin.

[0027] The present invention has the following beneficial effects:

[0028] 1. In the preparation process of soybean oligopeptides of the present invention, the peptides are extracted by using soybean seed coat and seed embryo respectively. In addition to protein components, soybean seed coat also contains cellulose, alkaloids, flavonoids, fat and other components. The presence of such components will interfere with the extraction of peptides, resulting in more impurities and lower purity in the prepared peptides. Therefore, in the preparation process, the seed coat is fermented by Aspergillus niger to decompose the cellulose therein. The addition of sucrose can induce Aspergillus niger to secrete cellulase, destroy the tissue structure, and fully release the alkaloids and other components therein. At the same time, the protein can be appropriately decomposed. After the fermentation of Aspergillus niger is completed, in an anaerobic environment, the specific acid production of the decomposition of sugar components by Lactobacillus acidophilus is utilized to fully decompose the decomposed sugar components. The organic acids such as lactic acid and acetic acid produced by the fermentation can reduce the pH of the fermentation liquid. The protein is precipitated and precipitated under acidic conditions. The solid components after fermentation are mainly composed of protein and peptides. The protein is decomposed by enzymatic hydrolysis to obtain a crude soybean oligopeptide. The main component of soybean embryo is protein, and different proteases are used to decompose it under different conditions. By utilizing the different protease decomposition sites, crude soybean oligopeptides with different molecular weights are obtained through enzymatic hydrolysis. In the present invention, the residue after peptide extraction and polyethylene glycol are used to prepare modified biochar, and the adsorption properties of biochar are used to adsorb impurity components such as polysaccharides in the crude soybean oligopeptide. The adsorbent has a small adsorption effect on proteins and peptides, and does not affect the yield of peptides while achieving purification.

[0029] 2. In vitro experiments and mouse experiments have confirmed that the soybean oligopeptide of the present invention has good moisturizing and antioxidant properties, can reduce the damage caused by UVB irradiation to mouse skin, reduce inflammatory reactions, and improve antioxidant activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 : Hygroscopic properties of different soybean peptides in performance test (I) (RH=40%);

[0031] Figure 2 : Hygroscopic properties of different soybean peptides in performance test (I) (RH=80%);

[0032] Figure 3 : Moisture residual rate of different soybean peptides in performance test (I);

[0033] Figure 4 : DPPH scavenging rate of different soybean peptides in performance test (I), * indicates P < 0.05 compared with the group in Example 1;

[0034] Figure 5 : ABTS clearance rate of different soybean peptides in performance test (I); * indicates P < 0.05 compared with the group in Example 1;

[0035] Figure 6 : Skin condition of mice in performance test (II), where A is the blank group mice, B is the model group mice, C is the test group 1 mice, and D is the test group 8 mice. DETAILED DESCRIPTION

[0036] The present invention is further illustrated below in conjunction with specific examples, and it should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. After reading the present invention, modifications of various equivalent forms of the present invention by those skilled in the art all fall within the scope protected by the claims of the present application. Aspergillus niger and Lactobacillus acidophilus used in the following embodiments are existing preserved strains purchased from the General Microbiological Center of the China Microbiological Culture Collection Administration Committee, and are used after expanded culture in our company's laboratory; all types of hydrolases used are existing commercially available products, which can be purchased through online or offline channels.

[0037] Example 1 Preparation of soybean oligopeptides

[0038] Step 1, soaking the dried soybeans in an aqueous solution with a pH value of 8.0, separating the seed coat and the embryo, and washing them with deionized water until they are neutral for later use;

[0039] Step 2: add 5 times the mass of water to the separated seed coat and grind it into a homogenate, add 15 g / L sucrose to the homogenate, mix well and inoculate 12 ml / L of Aspergillus niger spore suspension (activate and expand the Aspergillus niger strain numbered CGMCC 3.11598 according to the instructions to prepare a spore concentration of 2.5×10 5 cfu / mL spore suspension), adjust pH to 6.5, ferment at 30℃ for 2h, heat to boil, cool to room temperature, and inoculate Lactobacillus acidophilus liquid at an inoculum volume of 15ml / L (Lactobacillus acidophilus strain numbered CGMCC 1.1854 was activated and expanded according to the instructions to prepare an effective viable count of 5×10 5 cfu / mL of bacterial liquid), fermented anaerobically at 30℃ for 3h, centrifuged, and took precipitate I; washed precipitate I until neutral, added aqueous solution containing 8g / L subtilisin (CAS No. 9014-01-1, enzyme activity of 150u / mg) at a solid-liquid ratio of 1:8kg / L, enzymolyzed at 45℃ for 4h, centrifuged, and supernatant I and precipitate II were set aside;

[0040] Step 3, the separated embryos are added with water 5 times their mass, ground into a homogenate, the pH is adjusted to 6.0, 5 g / L of papain (CAS No. 9001-73-4, enzyme activity is 800 u / mg) is added, enzymolysis is carried out at 45° C. for 1 hour, centrifugation is performed, and supernatant II and precipitate III are set aside; precipitate III is taken, and an aqueous solution containing 3 g / L of bromelain (CAS No. 37189-34-7, enzyme activity is 300 u / mg) is added at a solid-liquid ratio of 1:8 kg / L, the pH is adjusted to 7.5, enzymolysis is carried out at 45° C. for 1 hour, centrifugation is performed, and supernatant III and precipitate IV are set aside;

[0041] Step 4, combining supernatant I, supernatant II, and supernatant III to obtain a mixed solution, and allowing the mixed solution to stand for 2 hours;

[0042] Step 5, during the standing period, prepare an adsorbent according to the following steps: mix the precipitate II obtained in the above step 2 and the precipitate IV obtained in step 3 to obtain a mixture; heat 0.15 times the mass of polyethylene glycol 1500 of the mixture to a molten state; calcine the mixture in an oxygen-free environment at 800° C. for 40 minutes, add the molten polyethylene glycol 1500 after the calcination is completed, mix well, cool to room temperature, and crush to obtain an adsorbent;

[0043] Step 6, adding 20 g / L of the prepared adsorbent to the standing mixed solution, centrifuging to obtain supernatant IV, concentrating and drying the supernatant IV to obtain soybean oligopeptide.

[0044] Example 2 Preparation of soybean oligopeptides

[0045] Step 1, soaking the dried soybeans in an aqueous solution with a pH value of 7.5, separating the seed coat and the embryo, and washing them with deionized water until they are neutral for later use;

[0046] Step 2: add 3 times the mass of water to the separated seed coat and grind it into a homogenate, add 10 g / L sucrose to the homogenate, mix well and inoculate the Aspergillus niger spore suspension at an inoculum volume of 10 ml / L (activate and expand the Aspergillus niger strain numbered CGMCC 3.17612 according to the instructions to prepare a spore concentration of 2×10 5 cfu / mL spore suspension), adjust pH to 6.0, ferment at 25℃ for 3h, heat to boil, cool to room temperature, and inoculate Lactobacillus acidophilus liquid at an inoculum volume of 10ml / L (Lactobacillus acidophilus strain numbered CGMCC 1.12735 was activated and expanded according to the instructions to prepare an effective viable count of 4×10 5 cfu / mL of bacterial liquid), fermented anaerobically at 25°C for 5h, centrifuged, and collected precipitate I; washed precipitate I until neutral, added with an aqueous solution containing 5g / L subtilisin (CAS No. 9014-01-1, enzyme activity of 150u / mg) at a solid-liquid ratio of 1:5kg / L, enzymatically hydrolyzed at 40°C for 6h, centrifuged, and supernatant I and precipitate II were set aside;

[0047] Step 3, adding 3 times the mass of water to the separated embryo to grind into a homogenate, adjusting the pH to 5.0, adding 6 g / L papain (CAS No. 9001-73-4, enzyme activity of 800 u / mg), enzymolysis at 40 ° C for 2 hours, centrifuging, and setting the supernatant II and precipitate III aside; taking precipitate III, adding 6 g / L of bromelain (CAS No. 37189-34-7, enzyme activity of 300 u / mg) at a solid-liquid ratio of 1:5 kg / L, adjusting the pH to 8.0, enzymolysis at 40 ° C for 2 hours, centrifuging, and setting the supernatant III and precipitate IV aside;

[0048] Step 4, combining supernatant I, supernatant II, and supernatant III to obtain a mixed solution, and allowing the mixed solution to stand for 1.5 hours;

[0049] Step 5, during the standing period, prepare an adsorbent according to the following steps: mix the precipitate II obtained in the above step 2 and the precipitate IV obtained in step 3 to obtain a mixture; heat 0.1 times the mass of polyethylene glycol 1000 of the mixture to a molten state; calcine the mixture in an oxygen-free environment at 600° C. for 60 minutes, add the molten polyethylene glycol 1000 after the calcination, mix well, cool to room temperature, and crush to obtain an adsorbent;

[0050] Step 6, adding 15 g / L of the prepared adsorbent to the standing mixed solution, centrifuging to obtain supernatant IV, concentrating and drying the supernatant IV to obtain soybean oligopeptide.

[0051] Example 3 Preparation of soybean oligopeptides

[0052] Step 1, soaking the dried soybeans in an aqueous solution with a pH value of 8.0, separating the seed coat and the embryo, and washing them with deionized water until they are neutral for later use;

[0053] Step 2: add 4 times the mass of water to the separated seed coat and grind it into a homogenate, add 13 g / L sucrose to the homogenate, mix well and inoculate the Aspergillus niger spore suspension at an inoculum volume of 11 ml / L (activate and expand the Aspergillus niger strain numbered CGMCC 3.11598 according to the instructions to prepare a spore concentration of 2.3×10 5 cfu / mL spore suspension), adjust pH to 6.5, ferment at 30℃ for 2h, heat to boil, cool to room temperature, and inoculate Lactobacillus acidophilus liquid at an inoculum volume of 13ml / L (Lactobacillus acidophilus strain numbered CGMCC 1.1854 was activated and expanded according to the instructions to prepare an effective viable count of 4.5×10 5 cfu / mL of bacterial liquid), fermented anaerobically at 30℃ for 2h, centrifuged, and took precipitate I; washed precipitate I until neutral, added aqueous solution containing 6g / L subtilisin (CAS No. 9014-01-1, enzyme activity of 150u / mg) at a solid-liquid ratio of 1:6kg / L, enzymolyzed at 40℃ for 5h, centrifuged, and supernatant I and precipitate II were set aside;

[0054] Step 3, the separated embryos are added with water 4 times their mass, and ground into a homogenate, the pH is adjusted to 6.0, papain (CAS No. 9001-73-4, enzyme activity is 800u / mg) is added at 6g / L, enzymolysis is carried out at 40°C for 1.5h, centrifugation is performed, and supernatant II and precipitate III are set aside; precipitate III is taken, and an aqueous solution containing 5g / L of bromelain (CAS No. 37189-34-7, enzyme activity is 300u / mg) is added at a solid-liquid ratio of 1:7kg / L, the pH is adjusted to 8.0, enzymolysis is carried out at 40°C for 1.5h, centrifugation is performed, and supernatant III and precipitate IV are set aside;

[0055] Step 4, combining supernatant I, supernatant II, and supernatant III to obtain a mixed solution, and allowing the mixed solution to stand for 2 hours;

[0056] Step 5, during the standing period, prepare an adsorbent according to the following steps: mix the precipitate II obtained in the above step 2 and the precipitate IV obtained in step 3 to obtain a mixture; heat 0.12 times the mass of polyethylene glycol 1200 of the mixture to a molten state; calcine the mixture in an oxygen-free environment at 700° C. for 50 minutes, add the molten polyethylene glycol 1200 after the calcination, mix well, cool to room temperature, and crush to obtain an adsorbent;

[0057] Step 6, adding 18 g / L of the prepared adsorbent to the standing mixed solution, centrifuging to obtain supernatant IV, concentrating and drying the supernatant IV to obtain soybean oligopeptide.

[0058] Example 4 Preparation of soybean oligopeptides

[0059] Step 1, soaking the dried soybeans in an aqueous solution with a pH value of 7.5, separating the seed coat and the embryo, and washing them with deionized water until they are neutral for later use;

[0060] Step 2: add 4 times the mass of water to the separated seed coat and grind it into a homogenate, add 12 g / L sucrose to the homogenate, mix well and inoculate 12 ml / L of Aspergillus niger spore suspension (activate and expand the Aspergillus niger strain numbered CGMCC 3.17612 according to the instructions to prepare a spore concentration of 2.5×10 5 cfu / mL spore suspension), adjust pH to 6.0, ferment at 25℃ for 3h, heat to boil, cool to room temperature, and inoculate Lactobacillus acidophilus liquid at an inoculum volume of 12ml / L (Lactobacillus acidophilus strain numbered CGMCC 1.12735 was activated and expanded according to the instructions to prepare an effective viable count of 4.3×10 5 cfu / mL of bacterial liquid), fermented anaerobically at 30℃ for 3h, centrifuged, and took precipitate I; washed precipitate I until neutral, added aqueous solution containing 8g / L subtilisin (CAS No. 9014-01-1, enzyme activity of 150u / mg) at a solid-liquid ratio of 1:6kg / L, enzymolyzed at 45℃ for 4h, centrifuged, and supernatant I and precipitate II were set aside;

[0061] Step 3, the separated embryos are added with water 4 times their mass, and ground into a homogenate, the pH is adjusted to 6.0, 5.5 g / L of papain (CAS No. 9001-73-4, enzyme activity is 800 u / mg) is added, enzymolysis is carried out at 45° C. for 1 hour, centrifugation is performed, and supernatant II and precipitate III are set aside; take precipitate III, add an aqueous solution containing 4 g / L of bromelain (CAS No. 37189-34-7, enzyme activity is 300 u / mg) at a solid-liquid ratio of 1:5 kg / L, adjust the pH to 8.0, enzymolysis is carried out at 40° C. for 2 hours, centrifugation is performed, and supernatant III and precipitate IV are set aside;

[0062] Step 4, combining supernatant I, supernatant II, and supernatant III to obtain a mixed solution, and allowing the mixed solution to stand for 1.5 hours;

[0063] Step 5, during the standing period, prepare an adsorbent according to the following steps: mix the precipitate II obtained in the above step 2 and the precipitate IV obtained in step 3 to obtain a mixture; heat 0.1 times the mass of polyethylene glycol 1000 of the mixture to a molten state; calcine the mixture in an oxygen-free environment at 800° C. for 40 minutes, add the molten polyethylene glycol 1000 after the calcination, mix well, cool to room temperature, and crush to obtain an adsorbent;

[0064] Step 6, adding 20 g / L of the prepared adsorbent to the standing mixed solution, centrifuging to obtain supernatant IV, concentrating and drying the supernatant IV to obtain soybean oligopeptide.

[0065] Comparative Example 1 Preparation of soybean peptide

[0066] Step 1: add 4 times the mass of dried soybeans to water with a pH value of 8.0, grind into a homogenate, add 13 g / L sucrose to the homogenate, mix well, and inoculate 11 ml / L of Aspergillus niger spore suspension (activate and expand the Aspergillus niger strain numbered CGMCC 3.11598 according to the instructions to prepare a spore concentration of 2.3 × 10 5 cfu / mL spore suspension), adjust pH to 6.5, ferment at 30℃ for 2h, heat to boil, cool to room temperature, and inoculate Lactobacillus acidophilus liquid at an inoculum volume of 13ml / L (Lactobacillus acidophilus strain numbered CGMCC 1.1854 was activated and expanded according to the instructions to prepare an effective viable count of 4.5×10 5 cfu / mL of bacterial liquid), fermented anaerobically at 30℃ for 2h, centrifuged, and took precipitate I; washed precipitate I until neutral, added aqueous solution containing 6g / L subtilisin (CAS No. 9014-01-1, enzyme activity of 150u / mg) at a solid-liquid ratio of 1:6kg / L, enzymolyzed at 40℃ for 5h, centrifuged, and supernatant I and precipitate II were set aside;

[0067] Step 2, allowing the supernatant I to stand for 2 h;

[0068] Step 3, during the standing period, prepare the adsorbent according to the following steps: take 0.12 times the mass of precipitate II of polyethylene glycol 1200 and heat it to a molten state; place precipitate II in an oxygen-free environment at 700° C. and calcine for 50 minutes, add molten polyethylene glycol 1200 after calcination, mix well, cool to room temperature, and crush to obtain the adsorbent;

[0069] Step 4, adding 18 g / L of the prepared adsorbent to the supernatant I after standing, centrifuging to obtain supernatant II, concentrating and drying the supernatant II to obtain soybean peptide.

[0070] Comparative Example 2 Preparation of soybean peptide

[0071] Step 1, soaking the dried soybeans in an aqueous solution with a pH value of 8.0, separating the seed coat and the embryo, and washing them with deionized water until they are neutral for later use;

[0072] Step 2, adding an aqueous solution containing 6 g / L of subtilisin (CAS No. 9014-01-1, enzyme activity of 150 u / mg) to the separated seed coat at a solid-liquid ratio of 1:6 kg / L, performing enzymolysis at 40° C. for 5 h, centrifuging, and keeping the supernatant I and precipitate I for later use;

[0073] Step 3, adding 4 times the mass of water to the separated embryo to grind into a homogenate, adjusting the pH to 6.0, adding 6 g / L of papain (CAS No. 9001-73-4, enzyme activity of 800 u / mg), enzymolysis at 40°C for 1.5 hours, centrifuging, and setting the supernatant II and precipitate II aside; adding 5 g / L of bromelain (CAS No. 37189-34-7, enzyme activity of 300 u / mg) to the precipitate II at a solid-liquid ratio of 1:7 kg / L, adjusting the pH to 8.0, enzymolysis at 40°C for 1.5 hours, centrifuging, and setting the supernatant III and precipitate III aside;

[0074] Step 4, combining supernatant I, supernatant II, and supernatant III to obtain a mixed solution, and allowing the mixed solution to stand for 2 hours;

[0075] Step 5, during the standing period, prepare an adsorbent according to the following steps: take the precipitate I obtained in the above step 2 and the precipitate III obtained in step 3 and mix them to obtain a mixture; place the mixture in an oxygen-free environment at 700° C. and calcine it for 50 minutes, cool it to room temperature, and crush it to obtain an adsorbent;

[0076] Step 6, adding 18 g / L of the prepared adsorbent to the standing mixed solution, centrifuging to obtain supernatant IV, concentrating and drying the supernatant IV to obtain soybean peptide.

[0077] Comparative Example 3 Preparation of soybean peptide

[0078] Step 1, adding 4 times the mass of dried soybeans to water with a pH value of 8.0, grinding to prepare a homogenate, adjusting the pH to 6.0, adding 6 g / L of papain (CAS No. 9001-73-4, enzyme activity of 800 u / mg), enzymolysis at 40°C for 1.5 hours, centrifuging, and setting the supernatant I and precipitate I aside; taking precipitate I, adding an aqueous solution containing 5 g / L of bromelain (CAS No. 37189-34-7, enzyme activity of 300 u / mg) at a solid-liquid ratio of 1:7 kg / L, adjusting the pH to 8.0, enzymolysis at 40°C for 1.5 hours, centrifuging, and setting the supernatant II and precipitate II aside;

[0079] Step 2, combining supernatant I and supernatant II to obtain a mixed solution, and allowing the mixed solution to stand for 2 hours;

[0080] Step 3, during the standing period, prepare the adsorbent according to the following steps: take 0.12 times the mass of precipitate II of polyethylene glycol 1200 and heat it to a molten state; place precipitate II in an oxygen-free environment at 700° C. and calcine for 50 minutes, add molten polyethylene glycol 1200 after calcination, mix well, cool to room temperature, and crush to obtain the adsorbent;

[0081] Step 4, adding 18 g / L of the prepared adsorbent to the standing mixed solution, centrifuging to obtain supernatant III, concentrating and drying the supernatant III to obtain soybean peptide.

[0082] Comparative Example 4 Preparation of soybean peptide

[0083] Step 1, soaking the dried soybeans in an aqueous solution with a pH value of 8.0, separating the seed coat and the embryo, and washing them with deionized water until they are neutral for later use;

[0084] Step 2: add 4 times the mass of water to the separated seed coat and grind it into a homogenate, add 13 g / L sucrose to the homogenate, mix well and inoculate the Aspergillus niger spore suspension at an inoculum volume of 11 ml / L (activate and expand the Aspergillus niger strain numbered CGMCC 3.11598 according to the instructions to prepare a spore concentration of 2.3×10 5 cfu / mL spore suspension), adjust pH to 6.5, ferment at 30℃ for 2h, heat to boil, cool to room temperature, and inoculate Lactobacillus acidophilus liquid at an inoculum volume of 13ml / L (Lactobacillus acidophilus strain numbered CGMCC 1.1854 was activated and expanded according to the instructions to prepare an effective viable count of 4.5×10 5cfu / mL of bacterial liquid), fermented anaerobically at 30℃ for 2h, centrifuged, and collected precipitate I; washed precipitate I until neutral, added with an aqueous solution containing 6g / L subtilisin (CAS No. 9014-01-1, enzyme activity of 150u / mg) at a solid-liquid ratio of 1:6kg / L, enzymolyzed at 40℃ for 5h, centrifuged, and collected supernatant I;

[0085] Step 3, adding water 4 times its mass to the separated embryo to grind into a homogenate, adjusting the pH to 7.0, adding papain (CAS No. 9001-73-4, enzyme activity 800u / mg) at 6g / L, performing enzymolysis at 40°C for 2h, centrifuging, and keeping the supernatant II for later use;

[0086] Step 4, combining supernatant I and supernatant II to obtain a mixed solution; slowly adding 3.0% ammonium sulfate solution by mass to the mixed solution, centrifuging to obtain a precipitate, and drying to obtain soybean peptide.

[0087] Performance Testing

[0088] 1. Determination of hygroscopicity, moisturizing and antioxidant functions of soybean oligopeptides

[0089] With reference to existing literature, the hygroscopicity, moisturizing and antioxidant properties of soybean oligopeptides were determined

[0090] 1. Moisture absorption and moisturizing performance test

[0091] At room temperature, 1.0 g of soybean oligopeptides of Example 1 to Example 4 and soybean peptides of Comparative Example 1 to Comparative Example 4 were weighed and placed in a drying bottle, and then placed in an environment with a humidity of 40% and 80%. They were weighed after 6 h, 12 h, 24 h, 36 h, 48 h, and 72 h, respectively, and the moisture absorption rate was calculated and the moisture absorption performance was compared. The moisture absorption of the peptides was then continuously observed. The results are shown in Table 1. Figure 1 , Figure 2 .

[0092]

[0093] Wherein, M0 is the mass before moisture absorption, g; Mn is the mass after moisture absorption, g.

[0094] At room temperature, the soybean oligopeptides of Example 1 to Example 4 and the soybean peptides of Comparative Example 1 to Comparative Example 4 with a water content of 20% were placed in a drying bottle, and then placed as a whole in a desiccator containing dry silica gel. They were weighed after being placed for 12h, 24h, 36h, 48h, and 72h, and the moisture residual rate was calculated to compare the moisturizing properties. The results are shown in FIG. Figure 3 .

[0095]

[0096] Where Hn is the mass after placement, g; H0 is the mass before placement, g.

[0097] 2. Antioxidant performance test

[0098] Take the soybean oligopeptides of Example 1 to Example 4 and the soybean peptides of Comparative Example 1 to Comparative Example 4 respectively, add deionized water to prepare a 5 mg / mL peptide solution, take 2 mL of the peptide solution and 2 mL of DPPH-ethanol solution (0.15 mmol / L, dissolved in 95% ethanol) and mix them evenly, react in a dark environment for 30 minutes, measure the absorbance value (A1) of the mixed solution, measure the absorbance value (A2) of 2 mL of the peptide solution + 2 mL of 98% ethanol mixed solution as a control, measure the absorbance value (A0) of 2 mL of DPPH-ethanol solution + 2 mL of deionized water mixed solution as a blank, and calculate the DPPH clearance rate; V C As a positive control. Figure 4 .

[0099]

[0100] 7mmol / L ABTS and 140mmol / L potassium persulfate were allowed to stand overnight at room temperature in a dark environment to obtain a base solution, and the base solution was diluted with phosphate buffer until the absorbance at 734nm was 0.70±0.02 to obtain a diluted solution; respectively, soybean oligopeptides of Example 1-Example 4 and soybean peptides of Comparative Example 1-Comparative Example 4 were taken, deionized water was added, and a 5mg / mL peptide solution was prepared, 1mL of the peptide solution was mixed with 4mL of the diluted solution, and the reaction was carried out for 6min, and the absorbance value (A1) of the mixed solution was measured, and the absorbance value (A2) of the mixed solution of 1mL peptide solution + 4mL deionized water was measured as a control, and the absorbance value (A0) of the mixed solution of 4mL of the diluted solution + 1mL of deionized water was measured as a blank, and the ABTS clearance rate was calculated, and VC was used as a positive control. Results are shown in Figure 5 .

[0101]

[0102] 3. Results and Analysis

[0103] 3.1 Moisture absorption and moisturizing properties

[0104] since Figure 1 It can be seen from the results that the hygroscopic curves of the peptides of Examples 1 to 4 and Comparative Examples 1 to 4 first increase and then tend to be flat. As time goes by, the groups that adsorb water molecules in the peptides tend to be saturated, and the hygroscopic rate stabilizes at the maximum value. In comparison, the oligopeptides obtained in Examples 1 to 4 of the present invention and the soybean peptides obtained in Comparative Example 4 have better hygroscopic properties and are finally completely dissolved. The peptides obtained in Comparative Examples 1 to 3 reach the maximum hygroscopic value in 72 hours, and the maximum hygroscopic capacity is less than that of the oligopeptides of the present invention.

[0105] since Figure 2 It can be seen from the results that as time goes by, the residual water content of the peptides of Examples 1 to 4 and Comparative Examples 1 to 4 gradually decreases, and the difference becomes more and more obvious. The oligopeptides of Examples 1 to 4 have a better moisturizing effect. The oligopeptides of the present invention contain more hydrophilic groups, which can effectively reduce the evaporation of water after combining with water molecules, and the effect is better than the soybean peptides obtained in Comparative Examples 1 to 4.

[0106] 3.2 Antioxidant properties

[0107] since Figure 4 , Figure 5 It can be seen from the results that the soybean oligopeptides obtained in Examples 1 to 4 of the present invention have strong scavenging capabilities for DPPH and ABTS, have good antioxidant capacity, and are significantly better than the soybean peptides obtained in Comparative Examples 1 to 4.

[0108] (II) Protective effect of soybean oligopeptides on skin

[0109] Refer to existing literature to model and explore the protective effect of soybean oligopeptides on the skin

[0110] 1. Experimental Materials and Procedures

[0111] 1.1 Test materials

[0112] SPF female Kunming mice (license number SCXK (Lu) 2022-0006), weighing 20 ± 2g, 3-4 weeks old, were adaptively fed for 7 days before the test. The mice were randomly divided into 10 groups, 10 in each group, namely, blank group, model group, test group 1 (administered with oligopeptide of Example 1), test group 2 (administered with oligopeptide of Example 2), test group 3 (administered with oligopeptide of Example 3), test group 4 (administered with oligopeptide of Example 4), test group 5 (administered with soybean peptide of Comparative Example 1), test group 6 (administered with soybean peptide of Comparative Example 2), test group 7 (administered with soybean peptide of Comparative Example 3), and test group 8 (administered with soybean peptide of Comparative Example 4).

[0113] The oligopeptides obtained in Examples 1 to 4 and the soybean peptides obtained in Comparative Examples 1 to 4 were dissolved in distilled water to prepare a 10 IU / mL peptide solution, which was prepared before use.

[0114] 1.2 Experimental process

[0115] The mice in each group were depilated 1 hour before the experiment, with an area of ​​about 2.0 cm × 3.0 cm on the back. Except for the blank group, the mice were placed about 15 cm away from the UVB irradiator and irradiated with UVB in the following manner: the minimum erythema dose was about 120 mJ / cm2, and the irradiation dose gradually increased over time. The irradiation lasted for 14 days, with an irradiation time of 1 hour on the 1st to 3rd day, 1.5 hours on the 4th to 8th day, and 2 hours on the last 6 days. After each irradiation, the mice in each group were returned to the breeding room, and the depilatory area of ​​the mice in each experimental group was smeared with 1 mL of the corresponding peptide solution, and the model group was smeared with 1 mL of distilled water. After the smearing was completed, the drug administration area was covered with plastic wrap for 1 hour.

[0116] 1.3 Detection indicators

[0117] After the experiment, blood was collected from the mouse eyeballs, and the levels of interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor α (TNF-α) in serum were determined by ELISA. Back skin tissue was obtained, subcutaneous fat and connective tissue were removed, and physiological saline was added to make a homogenate. The levels of malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) in skin tissue were determined according to the instructions of the kit.

[0118] 2. Results and Analysis

[0119] 2.1 Changes in mouse skin appearance

[0120] The back skin of mice in the blank group was intact; after 14 days of irradiation, mice in the model group developed a large amount of pigmentation, erythema and wrinkles on their backs, and local ulceration and scabs appeared; the backs of mice in test groups 1-4 showed varying degrees of redness, but no ulceration and scabs; the backs of mice in test groups 5-8 were obviously red, and some ulceration and scabs appeared. Figure 6 .

[0121] 2.2 Changes of serum inflammatory factors in mice

[0122] As shown in Table 1, the levels of IL-1, IL-6 and TNF-α in the serum of mice in groups 1 to 4 were significantly reduced, indicating that the oligopeptide of the present invention can effectively reduce the release of inflammatory factors in photodamaged skin and alleviate inflammation.

[0123] Table 1 Contents of inflammatory factors in mouse serum

[0124]

[0125] Note: Compared with Example 1, * P<0.05

[0126] 2.3 Changes in MDA content and antioxidant enzyme activity in mouse skin tissue

[0127] From the results in Table 2, it can be seen that the MDA content of mice in test groups 1-4 is significantly low, and the MDA content of mice in test groups 5-8 is high. MDA is a traditional Chinese medicine marker for tissue damage. The reduction in its content indicates that the soybean oligopeptide of the present invention can inhibit lipid peroxidation in mice.

[0128] From the results in Table 2, it can be seen that the SOD, CAT, and GSH-Px levels of mice in test groups 1-4 were significantly higher, and the SOD, CAT, and GSH-Px levels of mice in test groups 5-8 were significantly lower. SOD, CAT, and GSH-Px are antioxidant enzymes in the body, which can form an antioxidant defense system with other antioxidants. The increase in their content indicates that the body has a strong ability to scavenge oxygen free radicals.

[0129] Table 2 Changes of MDA content and antioxidant enzyme activity in mouse skin tissue

[0130]

[0131] Note: * Compared with the group in Example 1, P < 0.05

[0132] From the above results, it can be seen that the soybean oligopeptides prepared by the present invention can reduce skin inflammatory response, increase the activity of antioxidant enzymes, thereby resisting light damage and protecting the skin.

Claims

1. A method for preparing and purifying soybean oligopeptides, characterized in that: The following steps are involved: Step 1, soaking the dried soybeans in an aqueous solution with a pH value of 7.5-8.0, separating the seed coat and the embryo, and washing them with deionized water until they are neutral for later use; Step 2, adding 3-5 times the mass of the seed coat to the separated seed coat and grinding it into a homogenate, adding sucrose to the homogenate, inoculating Aspergillus niger spore suspension after mixing, adjusting the pH to 6.0-6.5, fermenting at 25-30° C. for 2-3 hours, heating and boiling, cooling to room temperature, inoculating Lactobacillus acidophilus liquid, fermenting anaerobically at 25-30° C. for 3-5 hours, centrifuging, and taking precipitate I; washing precipitate I to neutrality, adding an aqueous solution containing subtilisin, enzymolyzing at 40-45° C. for 4-6 hours, centrifuging, and reserving supernatant I and precipitate II for later use; wherein the Aspergillus niger is Aspergillus niger with a strain number of CGMCC 3.11598, and the Lactobacillus acidophilus is Lactobacillus acidophilus with a strain number of CGMCC 1.1854; Step 3, adding 3-5 times the mass of the separated embryo to water and grinding to make a homogenate, adjusting the pH to 5.0-6.0, adding papain, enzymolysis at 40-45° C. for 1-2 hours, centrifuging, and setting the supernatant II and precipitate III aside; adding an aqueous solution containing bromelain to the precipitate III, adjusting the pH to 7.5-8.0, enzymolysis at 40-45° C. for 1-2 hours, centrifuging, and setting the supernatant III and precipitate IV aside; Step 4, combining supernatant I, supernatant II, and supernatant III to obtain a mixed solution; allowing the mixed solution to stand for 1.5-2 hours, adding an adsorbent, centrifuging to obtain supernatant IV, concentrating and drying supernatant IV to obtain soybean oligopeptide; the preparation method of the adsorbent is: taking the precipitate II obtained in step 2 and the precipitate IV obtained in step 3 and mixing them to obtain a mixture; placing the mixture in an oxygen-free environment at 600-800°C and calcining it for 40-60 minutes, adding molten polyethylene glycol after the calcination is completed, mixing, cooling to room temperature, and crushing to obtain the adsorbent.

2. The method according to claim 1, characterized in that The amount of sucrose added in step 2 is 10-15 g / L.

3. The method according to claim 1, characterized in that In the step 2, the inoculation amount of the Aspergillus niger spore suspension is 10-12 ml / L, and the inoculation amount of the Lactobacillus acidophilus bacterial solution is 10-15 ml / L.

4. The method according to claim 1, characterized in that: In the step 2, the liquid ratio of precipitate I to the aqueous solution containing subtilisin is 1:(5-8) in kg / L; the content of subtilisin in the aqueous solution containing subtilisin is 5-8 g / L.

5. The method according to claim 1, characterized in that The amount of papain added in step 3 is 5-6 g / L.

6. The method according to claim 1, characterized in that In the step 3, the solid-liquid ratio of the precipitate III to the aqueous solution containing bromelain is 1:(5-8) in kg / L; the content of bromelain in the aqueous solution containing bromelain is 3-6 g / L.

7. The method according to claim 1, characterized in that The amount of adsorbent added in step 4 is 15-20 g / L.

8. The method according to claim 1, characterized in that In the adsorbent preparation method of step 4, the amount of polyethylene glycol added is 0.1-0.15 times the mass of the mixture; the molecular weight of the polyethylene glycol is 1000-1500.

9. Use of the soybean oligopeptide prepared according to the method according to any one of claims 1 to 8 in anti-inflammatory, antioxidant and anti-UVB light damage cosmetics.

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