Preparation method of high-antioxidant activity bird's nest peptide and tablet

Abstract

The present application relates to the technical field of protein and peptide, and particularly relates to a kind of high anti-oxygen activity bird's nest peptide and preparation method of its tablet.The present application includes the following steps: preparation of modified bird's nest product;Multiple enzymolysis of modified bird's nest product;Strengthening of bird's nest peptide anti-oxygen stability.The present application can not only enhance the antioxidant capacity of bird's nest by the free radical reaction of resveratrol grafted on the surface of bird's nest protein, but also improve the water solubility of bird's nest, which is helpful for the subsequent enzyme and bird's nest to better contact, improve the enzymolysis efficiency, avoid the reduction of bird's nest peptide anti-oxygen activity caused by external oxidation factors and too long enzymolysis time, then the bird's nest is glycosylated by chitosan, which can not only improve the dispersion emulsification stability of modified bird's nest product, enhance the contact area of subsequent enzyme and modified bird's nest product, further enhance the antioxidant capacity and the efficiency of subsequent enzymolysis.

Description

Technical Field

[0001] This invention relates to the field of protein and peptide technology, and in particular to a method for preparing a bird's nest peptide with high antioxidant activity and its tablets. Background Technology

[0002] Bird's nest has been hailed as a precious tonic and health-preserving product since ancient times. It is rich in nutrients, especially proteins, polysaccharides, and amino acids, which have certain health benefits. In recent years, with in-depth research, the active ingredients of bird's nest, such as bird's nest peptides, have gradually become a research hotspot in the fields of biomedicine and functional foods due to their various biological activities such as anti-oxidation, anti-aging, and immune regulation.

[0003] The active ingredients in bird's nest are mainly composed of proteins and carbohydrates. Due to the complex structure and poor solubility of the raw protein, it is difficult to directly utilize its bioactive components. To address this, scientists use enzymatic hydrolysis technology to convert the macromolecules in bird's nest into small peptides, thereby improving its bioavailability. However, existing technologies often suffer from insufficient enzymatic hydrolysis in bird's nest. This leads to the frequent addition of chemical reagents such as surfactants or the use of physical methods such as ultrasound and microwave to improve the efficiency of enzymatic hydrolysis. However, regardless of whether chemical reagents are added or physical methods are used, the activity of bird's nest peptides after enzymatic hydrolysis is affected, resulting in low product quality. Summary of the Invention

[0004] To address the aforementioned technical deficiencies, this invention presents a method for preparing bird's nest peptides with high antioxidant activity that can effectively improve the enzymatic hydrolysis efficiency of bird's nest, ensure the stability of the enzymatic hydrolysis system, and prevent interference from external oxidative factors. The prepared bird's nest peptides exhibit high antioxidant activity and yield.

[0005] Furthermore, this application provides a method for preparing bird's nest peptides with high antioxidant activity, comprising the following steps:

[0006] S1: Preparation of modified bird's nest products

[0007] Soak the bird's nest in clean water until softened, filter, and then shear it in a colloid mill to obtain bird's nest paste. Mix ascorbic acid, deionized water, and hydrogen peroxide solution evenly and add it to the bird's nest paste. After thorough shaking and stirring, add resveratrol, stir at low temperature, and dialyze to obtain resveratrol-grafted bird's nest paste. Mix chitosan and resveratrol-grafted bird's nest paste evenly in PBS solution, freeze-dry, add saturated KCl solution, seal, let stand, and filter to obtain modified bird's nest product.

[0008] S2: Multiple enzymatic hydrolysis of modified bird's nest products

[0009] After mixing and stirring the modified bird's nest product with purified water, the pH was adjusted, the temperature was raised and chitosanase was added for enzymatic hydrolysis, and then papain was added for enzymatic hydrolysis to obtain a preliminary enzymatic hydrolyzed bird's nest solution. The preliminary enzymatic hydrolyzed bird's nest solution was then enzymatically hydrolyzed using flavor protease under high hydrostatic pressure, and the solution was filtered and centrifuged to obtain a bird's nest peptide solution.

[0010] S3: Enhanced antioxidant stability of bird's nest peptides

[0011] Bird's nest peptide solution, yeast extract and trehalose were mixed and subjected to ultrasonic water bath treatment to obtain bird's nest peptide antioxidant solution. Bird's nest peptide antioxidant solution and β-cyclodextrin were mixed, homogenized and freeze-dried, and then ground into powder to obtain bird's nest peptide with high antioxidant activity.

[0012] Further, step S1, preparing the modified bird's nest product, includes the following steps:

[0013] S1.1: Place 2-3 parts by weight of bird's nest in a container, add 80-100 parts by weight of water, and soak in an environment of 12-15℃ for 3-5 hours. Then filter it using a filter screen with a pore size of 0.2-0.5mm to obtain soaked bird's nest. Place the soaked bird's nest in a colloid mill, adjust the gap between the grinding teeth of the colloid mill to 150-200μm, and shear for 30-35 minutes to obtain bird's nest paste.

[0014] S1.2: Place 0.2-0.3 parts by weight of ascorbic acid and 60-80 parts by weight of deionized water in a container, stir at 150-200 rpm for 8-10 minutes, then add 0.8-1 parts by weight of hydrogen peroxide solution, continue stirring at the same speed for 8-10 minutes, then add 10-12 parts by weight of bird's nest paste, and thoroughly shake and stir to obtain a bird's nest paste pretreatment solution. Add resveratrol obtained through grape extraction to the bird's nest paste pretreatment solution, allowing the resveratrol to... The final concentration of the bird's nest paste pretreatment solution is 0.4-0.5 mmol / L. Then, the bird's nest paste pretreatment solution with added resveratrol is placed in an environment of 3-4℃ and stirred at a stirring speed of 80-120 rpm for 20-24 hours. Then, it is put into a dialysis bag with a molecular weight cutoff of 1000-1500 Da for water dialysis for 25-30 hours to obtain resveratrol-grafted bird's nest paste. The resveratrol-grafted bird's nest paste is stored in an anaerobic environment at 2-4℃ for later use.

[0015] S1.3: Chitosan and resveratrol-grafted bird's nest paste were placed in a container at a mass ratio of 1:(1.5-2), and 100-120 times the volume of PBS solution was added and stirred evenly. Then, the mixture was placed in a freeze dryer for freeze drying to obtain modified bird's nest freeze-dried product. The modified bird's nest freeze-dried product was soaked in saturated KCl solution for sealed storage, and then allowed to stand at 50-55℃ for 20-24 hours. The modified bird's nest product was obtained by filtration.

[0016] Furthermore, the multiple enzymatic hydrolysis of the modified bird's nest product in step S2 includes the following steps:

[0017] S2.1: Mix 1-2 parts by weight of modified bird's nest product and 60-80 parts by weight of purified water in a container. While stirring, add 0.1-0.2 mol / L acetic acid aqueous solution to adjust the pH to 5.5-6. After heating to 42-45℃, add chitosanase at an enzyme ratio of 2000-2500 U / mg. Maintain the temperature and stir magnetically for 3-4 minutes. Then, heat to 55-60℃ and add papain at an enzyme ratio of 4000-4500 U / mg. Maintain the temperature and stir magnetically for 6-8 minutes. Then, heat to 95-100℃ and keep warm for 10-15 minutes to inactivate the enzyme, obtaining a preliminary enzymatically hydrolyzed bird's nest solution.

[0018] S2.2: Add the preliminary enzymatically hydrolyzed bird's nest solution to PBS solution at a volume ratio of 1:(1-2), with a PBS concentration of 40-50 mmol / L and a pH of 7.4. Then add flavor protease at an enzyme ratio of 6000-7500 U / mg. After mixing evenly, vacuum pack the mixture in a polyethylene bag and place it in a high hydrostatic pressure treatment device. Adjust the temperature to 50-55℃ and the hydrostatic pressure to 100-150 MPa for 3-5 hours to obtain the enzymatically hydrolyzed bird's nest solution. Treat the enzymatically hydrolyzed bird's nest solution at 95-100℃ for 15-20 minutes. After cooling to 25-30℃, filter it through a 1-2 mm filter membrane and centrifuge it at 10000-12000 rpm for 15-20 minutes. Retain the supernatant to obtain the bird's nest peptide solution.

[0019] Furthermore, the enhancement of the antioxidant stability of bird's nest peptides in step S3 includes the following steps:

[0020] S3.1: Mix 10-15 parts by weight of bird's nest peptide solution, 0.4-0.6 parts by weight of yeast extract and 0.3-0.4 parts by weight of trehalose in a sealed container, and then place it in an ultrasonic water bath. Adjust the power of the ultrasonic water bath to 350-400W and the temperature to 50-52℃. After ultrasonic treatment for 6-8 minutes, stop the ultrasonic treatment and maintain the temperature for 20-25 minutes in the water bath to obtain bird's nest peptide antioxidant solution.

[0021] S3.2: Mix the bird's nest peptide antioxidant solution and β-cyclodextrin in a mass ratio of 1:(3-4) and place them in a homogenizer. Adjust the homogenization power to 6-7.5kw and homogenize for 10-20 minutes. Then place them in a vacuum dryer and freeze-dry at -70℃ to -40℃. Finally, grind them into powder to obtain bird's nest peptide with high antioxidant activity.

[0022] Furthermore, in step S1.2, the concentration of ascorbic acid and hydrogen peroxide solution in deionized water is 4-4.5 mol / L.

[0023] Furthermore, the PBS solution in step S1.3 is prepared by mixing KH2PO4, Na2HPO4 and NaCl with deionized water in a mass ratio of 1:6:33, with a concentration of 20-25 mmol / L and a pH of 7.2-7.4.

[0024] Furthermore, the enzyme-to-dry weight ratio in step S2 refers to the ratio of enzyme activity to the dry weight of the modified bird's nest product.

[0025] Furthermore, the glutathione content of the yeast extract in step S3.1 is 30%.

[0026] Furthermore, this application provides a bird's nest peptide with high antioxidant activity, which is prepared by the above-mentioned method for preparing a bird's nest peptide with high antioxidant activity.

[0027] Furthermore, this application provides a method for preparing a high antioxidant activity bird's nest peptide tablet, comprising the following steps: mixing the high antioxidant activity bird's nest peptide prepared by the above-mentioned method, starch, and microcrystalline cellulose in a mass ratio of 1:(1-1.5):(0.5-1) until homogeneous, then adding 25-30wt% ethanol and continuing to stir to obtain a soft mass, which is then passed through a 20-25 mesh sieve to obtain soft mass particles, and then mixing the soft mass particles, carboxymethyl starch, and magnesium stearate in a mass ratio of 1:(0.2-0.25):(0.015-0.02) until homogeneous, and then placing the mixture in a tableting machine for tableting to obtain a high antioxidant activity bird's nest peptide tablet.

[0028] The beneficial effects are: 1. This invention prepares bird's nest paste by foaming the bird's nest and then shearing it into nanoparticles in a colloid mill. The bird's nest paste is then placed in a mixed solution of ascorbic acid, deionized water, and hydrogen peroxide solution, and resveratrol is added with continuous stirring. During this process, ascorbic acid, through its reducing properties, promotes the generation of highly reactive hydroxyl radicals from hydrogen peroxide. These highly reactive hydroxyl radicals act on the proteins in the bird's nest, initiating a free radical reaction. At this time, resveratrol also participates in the free radical reaction through its phenolic hydroxyl groups, thereby grafting onto the protein surface. This not only enhances the antioxidant capacity of the bird's nest but also... Furthermore, it can improve the water solubility of bird's nest, which helps subsequent enzymes to better contact the bird's nest, improves enzymatic hydrolysis efficiency, and avoids the reduction of antioxidant activity of bird's nest peptides caused by external oxidative factors and excessively long enzymatic hydrolysis time. Then, by glycosylation modification of bird's nest with chitosan, it can not only improve the dispersion and emulsification stability of modified bird's nest products and increase the contact area between subsequent enzymes and modified bird's nest products, thus improving enzymatic hydrolysis efficiency, but also further enhance antioxidant capacity. In summary, it improves the enzymatic hydrolysis efficiency and antioxidant capacity of bird's nest, thereby ensuring the high antioxidant activity of bird's nest peptides obtained during and after enzymatic hydrolysis.

[0029] This invention involves uniformly dispersing modified bird's nest products in purified water, then adjusting the pH and temperature to perform enzymatic hydrolysis of chitosan. This process breaks down partially glycosylated chitosan into smaller monosaccharides, reducing the shielding effect of chitosan on bird's nest proteins. This makes it easier for subsequent proteases to bind to the proteins and catalyze hydrolysis. Simultaneously, by controlling the hydrolysis time and maintaining an appropriate glycosylation state, the solution system is stabilized, promoting the pre-hydrolysis of papain and the complete hydrolysis of flavor proteases, thereby improving the activity, nutritional value, and flavor of the bird's nest peptide products.

[0030] This invention involves ultrasonically mixing bird's nest peptide solution, yeast extract, and trehalose in a water bath to obtain an antioxidant solution of bird's nest peptide. Then, the antioxidant solution of bird's nest peptide is homogenized with β-cyclodextrin and freeze-dried. The bird's nest peptide, yeast extract, and trehalose are coated together by β-cyclodextrin, which not only further enhances the antioxidant capacity of bird's nest peptide, but also avoids the influence of external environment and tableting on the activity of bird's nest peptide. Attached Figure Description

[0031] Figure 1 This is a flowchart illustrating the preparation method of the highly antioxidant bird's nest peptide used in the embodiments of the present invention. Detailed Implementation

[0032] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] Example 1: A method for preparing bird's nest peptides with high antioxidant activity, such as... Figure 1 As shown, it includes the following steps:

[0034] S1: Preparation of modified bird's nest products

[0035] S1.1: Place 2 parts by weight of bird's nest in a container, add 80 parts by weight of water, and soak in an environment of 12°C for 3 hours. Then filter it using a filter screen with a pore size of 0.2 mm to obtain soaked bird's nest. Place the soaked bird's nest in a colloid mill, adjust the gap between the grinding teeth of the colloid mill to 150 μm, and shear for 30 minutes to obtain bird's nest paste.

[0036] S1.2: Place 0.2 parts by weight of ascorbic acid and 60 parts by weight of deionized water in a container, stir at 150 rpm for 8 minutes, then add 0.8 parts by weight of 4 mol / L hydrogen peroxide solution, continue stirring at the same speed for 8 minutes, then add 10 parts by weight of bird's nest paste, and shake and stir thoroughly to obtain bird's nest paste pretreatment solution. Add resveratrol obtained by grape extraction to bird's nest paste pretreatment solution to make the final concentration of resveratrol in bird's nest paste pretreatment solution 0.4 mmol / L. Then place the bird's nest paste pretreatment solution with resveratrol in an environment of 3°C and stir at 80 rpm for 20 hours. Then put it into a dialysis bag with a molecular weight cutoff of 1000 Da for water dialyzing for 25 hours to obtain resveratrol-grafted bird's nest paste. Store the resveratrol-grafted bird's nest paste in an anaerobic environment at 2°C for later use.

[0037] S1.3: Chitosan and resveratrol-grafted bird's nest paste were placed in a container at a mass ratio of 1:1.5, and 100 times the volume of PBS solution was added and stirred evenly. The PBS solution was prepared by mixing KH2PO4, Na2HPO4 and NaCl with deionized water at a mass ratio of 1:6:33, with a concentration of 20 mmol / L and a pH of 7.2. Then, the mixture was placed in a freeze dryer for freeze drying to obtain modified bird's nest freeze-dried product. The modified bird's nest freeze-dried product was soaked in a saturated KCl solution for sealed storage, and then allowed to stand at 50°C for 20 hours. After filtration, the modified bird's nest product was obtained.

[0038] S2: Multiple enzymatic hydrolysis of modified bird's nest products

[0039] S2.1: Mix 1 part by weight of modified bird's nest product and 60 parts by weight of purified water in a container. While stirring, add 0.1 mol / L acetic acid aqueous solution to adjust the pH to 5.5. After heating to 42℃, add chitosanase at an enzyme ratio of 2000 U / mg. Maintain the temperature and stir magnetically for 3 minutes. Then, heat to 55℃ and add papain at an enzyme ratio of 4000 U / mg. Maintain the temperature and stir magnetically for 6 minutes. Then, heat to 95℃ and keep warm for 10 minutes to inactivate the enzyme and obtain a preliminary enzymatic hydrolysed bird's nest solution.

[0040] S2.2: The preliminary enzymatically hydrolyzed bird's nest solution was added to PBS solution at a volume ratio of 1:1. The PBS solution concentration was 40 mmol / L and the pH was 7.4. Then, flavor protease was added at an enzyme ratio of 6000 U / mg. After mixing evenly, the mixture was vacuum-packed in a polyethylene bag and placed in a high hydrostatic pressure treatment device. The temperature was adjusted to 50℃ and the hydrostatic pressure to 100 MPa. The treatment lasted for 3 hours to obtain the enzymatically hydrolyzed bird's nest solution. The enzymatically hydrolyzed bird's nest solution was treated at 95℃ for 15 minutes. After cooling to 25℃, it was filtered through a 1 mm filter membrane and then centrifuged at 10000 rpm for 15 minutes. The supernatant was retained to obtain the bird's nest peptide solution.

[0041] S3: Enhanced antioxidant stability of bird's nest peptides

[0042] S3.1: Mix 10 parts by weight of bird's nest peptide solution, 0.4 parts by weight of yeast extract with 30% glutathione content and 0.3 parts by weight of trehalose in a sealed container, and then place it in an ultrasonic water bath. Adjust the power of the ultrasonic water bath to 350W and the temperature to 50℃. After ultrasonic treatment for 6 minutes, stop the ultrasonic treatment and maintain the temperature for 20 minutes in the water bath to obtain bird's nest peptide antioxidant solution.

[0043] S3.2: Mix the bird's nest peptide antioxidant solution and β-cyclodextrin in a mass ratio of 1:3 and place them in a homogenizer. Adjust the homogenization power to 6kw and homogenize for 10 minutes. Then place them in a vacuum dryer and freeze-dry at -70℃. Finally, grind them into powder to obtain bird's nest peptide with high antioxidant activity.

[0044] A method for preparing a high antioxidant activity bird's nest peptide tablet includes the following steps: The high antioxidant activity bird's nest peptide prepared by the above-mentioned method, starch, and microcrystalline cellulose are mixed evenly at a mass ratio of 1:1:0.5. Then, 25wt% ethanol is added and the mixture is stirred further to obtain a soft mass. After passing through a 20-mesh sieve, soft mass particles are obtained. The soft mass particles, carboxymethyl starch, and magnesium stearate are mixed evenly at a mass ratio of 1:0.2:0.015 and then placed in a tableting machine for tableting to obtain a high antioxidant activity bird's nest peptide tablet.

[0045] Example 2: A method for preparing bird's nest peptides with high antioxidant activity, such as... Figure 1 As shown, it includes the following steps:

[0046] S1: Preparation of modified bird's nest products

[0047] S1.1: Place 3 parts by weight of bird's nest in a container, add 100 parts by weight of water, and soak in an environment of 12°C for 3 hours. Then filter it using a filter sieve with a pore size of 0.2 mm to obtain soaked bird's nest. Place the soaked bird's nest in a colloid mill, adjust the gap between the grinding teeth of the colloid mill to 150 μm, and shear for 30 minutes to obtain bird's nest paste.

[0048] S1.2: Place 0.3 parts by weight of ascorbic acid and 80 parts by weight of deionized water in a container, stir at 150 rpm for 8 minutes, then add 1 part by weight of 4 mol / L hydrogen peroxide solution, continue stirring at the same speed for 8 minutes, then add 12 parts by weight of bird's nest paste, and after thorough shaking and stirring, obtain bird's nest paste pretreatment solution. Add resveratrol obtained by grape extraction to the bird's nest paste pretreatment solution to make the final concentration of resveratrol in the bird's nest paste pretreatment solution 0.4 mmol / L. Then place the bird's nest paste pretreatment solution with resveratrol in an environment of 3°C and stir at 80 rpm for 20 hours. Then put it into a dialysis bag with a molecular weight cutoff of 1000 Da for water dialyzing for 25 hours to obtain resveratrol-grafted bird's nest paste. Store the resveratrol-grafted bird's nest paste in an anaerobic environment at 2°C for later use.

[0049] S1.3: Chitosan and resveratrol-grafted bird's nest paste were placed in a container at a mass ratio of 1:2. 100 times the volume of PBS solution was added and stirred evenly. The PBS solution was prepared by mixing KH2PO4, Na2HPO4 and NaCl with deionized water at a mass ratio of 1:6:33, with a concentration of 20 mmol / L and a pH of 7.2. The mixture was then placed in a freeze dryer for freeze drying to obtain modified bird's nest freeze-dried product. The modified bird's nest freeze-dried product was soaked in a saturated KCl solution for sealed storage and then allowed to stand at 50°C for 20 hours. The product was then filtered to obtain the modified bird's nest product.

[0050] S2: Multiple enzymatic hydrolysis of modified bird's nest products

[0051] S2.1: Mix 2 parts by weight of modified bird's nest product and 80 parts by weight of purified water in a container. While stirring, add 0.1 mol / L acetic acid aqueous solution to adjust the pH to 5.5. After heating to 42℃, add chitosanase at an enzyme ratio of 2000 U / mg. Maintain the temperature and stir magnetically for 4 minutes. Then, heat to 55℃ and add papain at an enzyme ratio of 4000 U / mg. Maintain the temperature and stir magnetically for 6 minutes. Then, heat to 95℃ and keep warm for 10 minutes to inactivate the enzyme and obtain a preliminary enzymatic hydrolysed bird's nest solution.

[0052] S2.2: The preliminary enzymatically hydrolyzed bird's nest solution was added to PBS solution at a volume ratio of 1:2. The PBS solution concentration was 40 mmol / L and the pH was 7.4. Then, flavor protease was added at an enzyme ratio of 6000 U / mg. After mixing evenly, the mixture was vacuum-packed in a polyethylene bag and placed in a high hydrostatic pressure treatment device. The temperature was adjusted to 50℃ and the hydrostatic pressure to 100 MPa. The treatment lasted for 8 hours to obtain the enzymatically hydrolyzed bird's nest solution. The enzymatically hydrolyzed bird's nest solution was treated at 95℃ for 15 minutes. After cooling to 25℃, it was filtered through a 1 mm filter membrane and then centrifuged at 10000 rpm for 15 minutes. The supernatant was retained to obtain the bird's nest peptide solution.

[0053] S3: Enhanced antioxidant stability of bird's nest peptides

[0054] S3.1: Mix 15 parts by weight of bird's nest peptide solution, 0.6 parts by weight of yeast extract with 30% glutathione content and 0.4 parts by weight of trehalose in a sealed container, and then place it in an ultrasonic water bath. Adjust the power of the ultrasonic water bath to 350W and the temperature to 50℃. After ultrasonic treatment for 6 minutes, stop the ultrasonic treatment and maintain the temperature for 20 minutes in the water bath to obtain bird's nest peptide antioxidant solution.

[0055] S3.2: Mix the bird's nest peptide antioxidant solution and β-cyclodextrin in a mass ratio of 1:4 and place them in a homogenizer. Adjust the homogenization power to 6kw and homogenize for 10 minutes. Then place them in a vacuum dryer and freeze-dry at -70℃. Finally, grind them into powder to obtain bird's nest peptide with high antioxidant activity.

[0056] A method for preparing a high antioxidant activity bird's nest peptide tablet includes the following steps: The high antioxidant activity bird's nest peptide prepared by the above-mentioned method, starch, and microcrystalline cellulose are mixed evenly at a mass ratio of 1:1.5:1. Then, 30wt% ethanol is added and stirring is continued to obtain a soft mass. After passing through a 20-mesh sieve, soft mass particles are obtained. The soft mass particles, carboxymethyl starch, and magnesium stearate are mixed evenly at a mass ratio of 1:0.25:0.02 and then placed in a tableting machine for tableting to obtain a high antioxidant activity bird's nest peptide tablet.

[0057] Example 3: A method for preparing bird's nest peptides with high antioxidant activity, such as... Figure 1 As shown, it includes the following steps:

[0058] S1: Preparation of modified bird's nest products

[0059] S1.1: Place 2 parts by weight of bird's nest in a container, add 80 parts by weight of water, and soak in an environment of 15°C for 5 hours. Then filter it using a filter screen with a pore size of 0.5 mm to obtain soaked bird's nest. Place the soaked bird's nest in a colloid mill, adjust the gap between the grinding teeth of the colloid mill to 200 μm, and shear for 35 minutes to obtain bird's nest paste.

[0060] S1.2: Place 0.2 parts by weight of ascorbic acid and 60 parts by weight of deionized water in a container, stir at 200 rpm for 10 minutes, then add 0.8 parts by weight of 4.5 mol / L hydrogen peroxide solution, continue stirring at the same speed for 10 minutes, then add 10 parts by weight of bird's nest paste, and shake thoroughly to obtain bird's nest paste pretreatment solution. Add resveratrol obtained by grape extraction to the bird's nest paste pretreatment solution to make the final concentration of resveratrol in the bird's nest paste pretreatment solution 0.5 mmol / L. Then place the bird's nest paste pretreatment solution with resveratrol in an environment of 4°C and stir at 120 rpm for 24 hours. Then put it into a dialysis bag with a molecular weight cutoff of 1500 Da for water dialyzing for 30 hours to obtain resveratrol-grafted bird's nest paste. Store the resveratrol-grafted bird's nest paste in an anaerobic environment at 4°C for later use.

[0061] S1.3: Chitosan and resveratrol-grafted bird's nest paste were placed in a container at a mass ratio of 1:2. 120 times the volume of PBS solution was added and stirred until homogeneous. The PBS solution was prepared by mixing KH2PO4, Na2HPO4 and NaCl with deionized water at a mass ratio of 1:6:33, with a concentration of 25 mmol / L and a pH of 7.4. The mixture was then freeze-dried in a freeze dryer to obtain the modified bird's nest freeze-dried product. The modified bird's nest freeze-dried product was then soaked in a saturated KCl solution for sealed storage. After standing at 55°C for 24 hours, the mixture was filtered to obtain the modified bird's nest product.

[0062] S2: Multiple enzymatic hydrolysis of modified bird's nest products

[0063] S2.1: Mix 1 part by weight of modified bird's nest product and 60 parts by weight of purified water in a container. While stirring, add 0.2 mol / L acetic acid aqueous solution to adjust the pH to 6. After heating to 45°C, add chitosanase at an enzyme ratio of 2500 U / mg. Maintain the temperature and stir magnetically for 4 minutes. Then, heat to 60°C and add papain at an enzyme ratio of 4500 U / mg. Maintain the temperature and stir magnetically for 8 minutes. Then, heat to 100°C and keep warm for 15 minutes to inactivate the enzyme, thus obtaining a preliminary enzymatically hydrolyzed bird's nest solution.

[0064] S2.2: The preliminary enzymatically hydrolyzed bird's nest solution was added to PBS solution at a volume ratio of 1:2. The PBS solution concentration was 50 mmol / L and the pH was 7.4. Then, flavor protease was added at an enzyme ratio of 7500 U / mg. After mixing evenly, the mixture was vacuum-packed in a polyethylene bag and placed in a high hydrostatic pressure treatment device. The temperature was adjusted to 55℃ and the hydrostatic pressure to 150 MPa. The treatment was carried out for 5 hours to obtain the enzymatically hydrolyzed bird's nest solution. The enzymatically hydrolyzed bird's nest solution was treated at 100℃ for 20 minutes. After cooling to 30℃, it was filtered through a 2 mm filter membrane and then centrifuged at 12000 rpm for 20 minutes. The supernatant was retained to obtain the bird's nest peptide solution.

[0065] S3: Enhanced antioxidant stability of bird's nest peptides

[0066] S3.1: Mix 10 parts by weight of bird's nest peptide solution, 0.4 parts by weight of yeast extract with 30% glutathione content and 0.3 parts by weight of trehalose in a sealed container, and then place it in an ultrasonic water bath. Adjust the power of the ultrasonic water bath to 400W and the temperature to 52℃. After ultrasonic treatment for 8 minutes, stop the ultrasonic treatment and maintain the temperature for 25 minutes in the water bath to obtain bird's nest peptide antioxidant solution.

[0067] S3.2: Mix the bird's nest peptide antioxidant solution and β-cyclodextrin in a mass ratio of 1:3 and place them in a homogenizer. Adjust the homogenization power to 7.5kw and homogenize for 20 minutes. Then place them in a vacuum dryer and freeze-dry at -40℃. Finally, grind them into powder to obtain bird's nest peptide with high antioxidant activity.

[0068] A method for preparing a high antioxidant activity bird's nest peptide tablet includes the following steps: The high antioxidant activity bird's nest peptide prepared by the above-mentioned method, starch, and microcrystalline cellulose are mixed evenly at a mass ratio of 1:1:0.5. Then, 25wt% ethanol is added and the mixture is stirred to obtain a soft mass. After passing through a 25-mesh sieve, soft mass particles are obtained. The soft mass particles, carboxymethyl starch, and magnesium stearate are mixed evenly at a mass ratio of 1:0.2:0.015 and then placed in a tableting machine for tableting to obtain a high antioxidant activity bird's nest peptide tablet.

[0069] Comparative Example 1: The difference between Comparative Example 1 and Example 1 is that step S1.2 was removed in Comparative Example 1, and the resveratrol-grafted bird's nest paste in step S1.3 was replaced with an equal mass of bird's nest paste. The remaining steps were the same as in Example 1, and a bird's nest peptide solution was prepared. This was denoted as Comparative Example 1.

[0070] Comparative Example 2: The difference between Comparative Example 2 and Example 1 is that chitosan was not added in step S1.3 to obtain the modified bird's nest product. The remaining steps were the same as in Example 1 to obtain the bird's nest peptide solution, which is referred to as Comparative Example 2.

[0071] Comparative Example 3: The difference between Comparative Example 3 and Example 1 is that chitosanase was not added for enzymatic hydrolysis in step S2.1. After adjusting the pH, the temperature was directly raised to 55°C, and papain was added at an enzyme-to-base ratio of 4000 U / mg. The temperature was maintained and the mixture was magnetically stirred for 6 minutes. Then the temperature was raised to 95°C and kept warm for 10 minutes to inactivate the enzyme, thus obtaining a preliminary enzymatic hydrolyzed bird's nest solution. The remaining steps were the same as in Example 1, and a bird's nest peptide solution was obtained, which was designated as Comparative Example 3.

[0072] Comparative Example 4: The difference between Comparative Example 4 and Example 1 is that step S3.1 was removed, and the bird's nest peptide antioxidant solution in step S3.2 was replaced with an equal mass of bird's nest peptide solution to obtain bird's nest peptide. The bird's nest peptide with high antioxidant activity was replaced with bird's nest peptide. In the process of preparing high antioxidant activity bird's nest peptide tablets, 0.4 parts by weight of yeast extract and 0.3 parts by weight of trehalose were added. The remaining steps were the same as in Example 1 to obtain high antioxidant activity bird's nest peptide tablets, which were recorded as Comparative Example 4.

[0073] Experiment 1: Take the bird's nest peptide solution prepared in the examples and Comparative Examples 1-3, and measure their absorbance at A516nm using an ELISA reader according to the instructions of the DPPH free radical scavenging kit. Record this absorbance as bird's nest peptide absorbance. Use 80% methanol as a blank control to measure the absorbance at A516nm and record this absorbance as blank absorbance. Calculate the DPPH free radical scavenging rate: DPPH free radical scavenging rate = 1 - bird's nest peptide absorbance / blank absorbance × 100%. Record this as the experimental group. Repeat the experiment once in parallel and record this as the parallel group. Record the data and make a table as shown in Table 1.

[0074] Table 1: DPPH free radical scavenging rate of bird's nest peptides

[0075]

[0076] Experiment 2: The bird's nest peptide solution prepared in the examples and Comparative Examples 1-3 were taken, and the content of bird's nest peptide in the solution was determined using a UV-Vis spectrophotometer. The yield of bird's nest peptide was calculated. The yield of bird's nest peptide was calculated as (mass of bird's nest peptide / mass of bird's nest) × 100%. The data are shown in Table 2.

[0077] Table 2: Yield of Bird's Nest Peptides

[0078]

[0079] As can be seen from the data of Examples and Comparative Example 1 in Tables 1 and 2, the DPPH free radical scavenging rate of the bird's nest peptides obtained without resveratrol treatment was reduced, and the yield of bird's nest peptides was also reduced. This proves that resveratrol treatment of bird's nest not only enhances the antioxidant capacity of bird's nest, but also improves the enzymatic hydrolysis efficiency.

[0080] As can be seen from the data in Examples and Comparative Example 2 in Tables 1 and 2, the DPPH free radical scavenging rate of the bird's nest peptides obtained without chitosan-grafted resveratrol glycosylation modification was lower, and the yield of bird's nest peptides was also lower. This proves that chitosan glycosylation modification of bird's nest can not only improve the dispersion and emulsification stability of the modified bird's nest product, enhance the contact area between the subsequent enzyme and the modified bird's nest product, and improve the enzymatic hydrolysis efficiency, but also further enhance the antioxidant capacity.

[0081] As can be seen from the data in Examples and Comparative Example 3 in Tables 1 and 2, the DPPH free radical scavenging rate of the bird's nest peptides obtained without enzymatic hydrolysis of the modified bird's nest products by chitosanase decreased, and the yield of bird's nest peptides also decreased. This proves that chitosanase can improve the activity of bird's nest peptide products and increase the yield of bird's nest peptides.

[0082] Experiment 3: Take the high antioxidant active bird's nest peptide tablets prepared in the example and Comparative Example 4, and mix them with DPPH standard solution at a mass ratio of 1:3 at room temperature. Let them stand for 35 minutes, and read the absorbance at 516 nm using a UV spectrophotometer. Record this as the absorbance of the experimental group. Mix deionized water with DPPH standard solution at a mass ratio of 1:3, let them stand for 35 minutes, and read the absorbance at 516 nm using a UV spectrophotometer. Record this as the absorbance of the blank group. Calculate the DPPH free radical scavenging rate: DPPH free radical scavenging rate = 1 - (absorbance of experimental group / absorbance of blank group) × 100%. Repeat the experiment three times in parallel and take the average value. Record the data and make a table as shown in Table 3.

[0083] Table 3: DPPH free radical scavenging rate of high antioxidant activity bird's nest peptide tablets

[0084]

[0085] As can be seen from the data in Table 3 of the examples and Comparative Example 4, the antioxidant properties of the high-antioxidant-activity bird's nest peptide tablets were reduced when β-cyclodextrin was not used to encapsulate the bird's nest peptide, yeast extract, and trehalose. Furthermore, as can be seen from the DPPH free radical scavenging rates of the examples in Table 1, the antioxidant capacity of the high-antioxidant-activity bird's nest peptide tablets was greater than that of the bird's nest peptide itself. This demonstrates that encapsulating the bird's nest peptide, yeast extract, and trehalose together with β-cyclodextrin can not only further enhance the antioxidant capacity of the bird's nest peptide but also prevent the influence of the external environment and tableting process on the activity of the bird's nest peptide.

[0086] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A method for preparing high-antioxidant activity of bird's nest peptides, characterized in that, Comprising the following steps: S1: preparing a modified bird's nest product The bird's nest is soaked in clean water, filtered, and then placed in a colloid mill for shearing to obtain bird's nest slurry. Ascorbic acid, deionized water, and hydrogen peroxide solution are stirred and mixed uniformly, then added to the bird's nest slurry. After sufficient oscillation and stirring, resveratrol is added. After stirring at low temperature, dialysis is performed to obtain resveratrol grafted bird's nest slurry. Chitosan and resveratrol grafted bird's nest slurry are mixed uniformly in a PBS solution. After freeze-drying, saturated KCl solution is added for sealing. After standing and filtering, a modified bird's nest product is obtained; S2: multiple enzymatic hydrolysis of the modified bird's nest product The modified bird's nest product and purified water are mixed and stirred, and the pH is adjusted. Chitosanase is added for enzymatic hydrolysis, followed by the addition of papain for enzymatic hydrolysis after warming. A preliminary enzymatic hydrolysis bird's nest solution is obtained. Flavor protease is used for enzymatic hydrolysis treatment of the preliminary enzymatic hydrolysis bird's nest solution under high hydrostatic pressure. Filtration and centrifugation yield a bird's nest peptide solution; S3: strengthening of the antioxidant stability of the bird's nest peptide 10-15 parts by weight of the bird's nest peptide solution, 0.4-0.6 parts by weight of yeast extract, and 0.3-0.4 parts by weight of trehalose are mixed and subjected to ultrasonic water bath treatment to obtain a bird's nest peptide antioxidant solution. The bird's nest peptide antioxidant solution is mixed with β-cyclodextrin, homogenized, freeze-dried, and ground into powder to obtain a bird's nest peptide with high antioxidant activity. The glutathione content of the yeast extract is 30%; Step S1 includes the following steps: S1.1: 2-3 parts by weight of bird's nest are placed in a container, 80-100 parts by weight of clean water are added, and the container is soaked in an environment at 12-15°C for 3-5 hours. Then, a filter screen with a pore size of 0.2-0.5 mm is used for filtration to obtain soaked bird's nest. The soaked bird's nest is placed in a colloid mill, the gap between the colloid mill teeth is adjusted to 150-200 μm, and shearing is performed for 30-35 minutes to obtain bird's nest slurry; S1.2: 0.2-0.3 parts by weight of ascorbic acid and 60-80 parts by weight of deionized water are placed in a container. Stirring is performed at a stirring speed of 150-200 rpm for 8-10 minutes, then 0.8-1 part by weight of hydrogen peroxide solution is added. Stirring is continued at the same stirring speed for 8-10 minutes, then 10-12 parts by weight of bird's nest slurry is added. After sufficient oscillation and stirring, a bird's nest slurry pretreatment solution is obtained. Resveratrol extracted from grapes is added to the bird's nest slurry pretreatment solution, and the final concentration of resveratrol in the bird's nest slurry pretreatment solution is 0.4-0.5 mmol / L. Then, the bird's nest slurry pretreatment solution containing resveratrol is placed in an environment at 3-4°C and stirred at a stirring speed of 80-120 rpm for 20-24 hours. Then, the dialysis bag with a molecular weight cutoff of 1000-1500 Da is loaded for water dialysis, which is continued for 25-30 hours to obtain resveratrol grafted bird's nest slurry. The resveratrol grafted bird's nest slurry is stored in an anaerobic environment at 2-4°C for standby use; S1.3: The grafting of chitosan and white veratral on the bird's nest pulp is placed in a container with a mass ratio of 1: (1.5-2), 100-120 times the volume of PBS solution is added and stirred uniformly, then placed in a freeze dryer for freeze-drying to obtain modified bird's nest freeze-dried material, the modified bird's nest freeze-dried material is soaked in saturated KCl solution for sealed storage, then placed at 50-55℃ for 20-24 hours, and filtered to obtain the modified bird's nest product.

2. The method for preparing a bird's nest peptide with high antioxidant activity according to claim 1, characterized in that, Step S2 of the multiple enzymatic hydrolysis of the modified bird's nest product includes the following steps: S2.1: 1-2 parts by weight of the modified bird's nest product and 60-80 parts by weight of purified water are mixed and placed in a container, 0.1-0.2 mol / L acetic acid aqueous solution is added while stirring to adjust the pH to 5.5-6, then heated to 42-45℃, and chitosanase is added at an enzyme-to-substrate ratio of 2000-2500 U / mg, the temperature is maintained for 3-4 minutes of magnetic stirring, then heated to 55-60℃, and papain is added at an enzyme-to-substrate ratio of 4000-4500 U / mg, the temperature is maintained for 6-8 minutes of magnetic stirring, then heated to 95-100℃ and incubated for 10-15 minutes to inactivate the enzyme, obtaining a preliminary enzymatic hydrolysis bird's nest solution; S2.2: The preliminary enzymatic hydrolysis bird's nest solution is added to PBS solution at a volume ratio of 1: (1-2), the PBS solution has a concentration of 40-50 mmol / L and a pH of 7.4, flavor protease is then added at an enzyme-to-substrate ratio of 6000-7500 U / mg, the mixture is uniformly mixed and then vacuum packaged using a polyethylene bag, placed in a high hydrostatic pressure treatment device, the temperature is adjusted to 50-55℃, the hydrostatic pressure is adjusted to 100-150 Mpa, and the treatment is performed for 3-5 hours to obtain an enzymatic hydrolysis bird's nest solution, the enzymatic hydrolysis bird's nest solution is treated at 95-100℃ for 15-20 minutes, cooled to 25-30℃, filtered using a filter membrane with a thickness of 1-2 mm, then placed in a centrifuge and centrifuged at a speed of 10000-12000 rpm for 15-20 minutes, the supernatant is retained, and a bird's nest peptide solution is obtained.

3. The method for preparing a bird's nest peptide with high antioxidant activity according to claim 2, characterized in that, Step S3 of the strengthening of the antioxidant stability of the bird's nest peptide includes the following steps: S3.1: 10-15 parts by weight of the bird's nest peptide solution, 0.4-0.6 parts by weight of yeast extract, and 0.3-0.4 parts by weight of trehalose are mixed and placed in a sealed container, then placed in an ultrasonic water bath, the power of the ultrasonic water bath is adjusted to 350-400 W and the temperature is adjusted to 50-52℃, ultrasonic treatment is performed for 6-8 minutes, then the ultrasonic treatment is stopped, the temperature is maintained for 20-25 minutes of water bath, and a bird's nest peptide antioxidant solution is obtained; S3.2: The bird's nest peptide antioxidant solution and β-cyclodextrin are mixed at a mass ratio of 1: (3-4) and placed in a homogenizer, the homogenization power is adjusted to 6-7.5 kw for 10-20 minutes of homogenization, then placed in a vacuum dryer, freeze-dried at minus 70℃ to minus 40℃, then ground into powder, and a bird's nest peptide with high antioxidant activity is obtained.

4. The method for preparing a bird's nest peptide with high antioxidant activity according to claim 1, characterized in that, In step S1.2, the concentration of ascorbic acid and hydrogen peroxide solution in deionized water is 4-4.5 mol / L.

5. The method for preparing a bird's nest peptide with high antioxidant activity according to claim 1, characterized in that, The PBS solution in step S1.3 is prepared by KH2PO4, Na2HPO4 and NaCl with a mass ratio of 1:6:33 and deionized water, with a concentration of 20-25 mmol / L and a pH of 7.2-7.

4.

6. The method for preparing a bird's nest peptide with high antioxidant activity according to claim 2, characterized in that, The enzyme activity to dry weight ratio of the modified bird's nest product in step S2 refers to the ratio of enzyme activity to dry weight of the modified bird's nest product.

7. A method for preparing a high-antioxidant activity bird's nest peptide tablet, characterized in that, The method comprises the following steps: The high-antioxidant bird's nest peptide prepared by the method of any one of claims 1-6 is mixed with starch and microcrystalline cellulose at a mass ratio of 1:(1-1.5):(0.5-1) to prepare a soft material, and then 25-30 wt% of ethanol is added for further stirring to obtain a soft material granule. The soft material granule, carboxymethyl starch, and magnesium stearate are mixed at a mass ratio of 1:(0.2-0.25):(0.015-0.02) to obtain a high-antioxidant bird's nest peptide tablet.

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