Antioxidant nano collagen peptide powder and preparation method thereof

Abstract

The invention relates to the technical field of food processing, in particular to antioxidant nano collagen peptide powder and a preparation method thereof.The antioxidant nano collagen peptide powder is prepared by forming antioxidant micelles wrapping collagen peptide through collagen peptide liquid and a complex antioxidant, loading the antioxidant micelles on a porous carrier and then conducting cross-linking through transglutaminase; tea polyphenol and whey protein are covalently combined to prepare a composite antioxidant, rapid free radical neutralization, lactoferrin inhibition of an oxidation source and synergistic effect of collagen peptide self-antioxidant groups are provided, multistage barrier design is combined, oxygen diffusion is isolated through a micelle hydrophobic core, and the contact area of micelles and the outside is reduced through physical adsorption of a porous carrier, so that the anti-oxidation effect of the collagen peptide is improved. Finally, the micelles are fixed in the carrier by utilizing an enzyme cross-linked network, so that the antioxidant nano collagen peptide powder has efficient and long-acting antioxidant capacity.

Description

Technical Field

[0001] The present invention relates to the technical field of food processing, and more specifically, to an antioxidant nano - collagen peptide powder and a preparation method thereof. Background Art

[0002] Collagen peptide is a small - molecule active peptide substance generated by the enzymatic hydrolysis of collagen. Its molecular weight is between amino acids and proteins, and it has the characteristics of low molecular weight, high skin - affinity, and easy absorption. With the growth of consumers' demand for health and beauty, collagen peptide has rapidly emerged in the large - health field with its unique nutritional value and biological activity, and the market scale has continued to expand.

[0003] However, traditional collagen peptide products are vulnerable to free - radical attacks during storage or digestion, resulting in peptide - chain breakage and loss of activity, which affects their bioavailability and antioxidant activity. In view of this, we propose an antioxidant nano - collagen peptide powder and a preparation method thereof. Summary of the Invention

[0004] The purpose of the present invention is to provide an antioxidant nano - collagen peptide powder and a preparation method thereof to solve the problems raised in the above - mentioned background art.

[0005] To achieve the above purpose, the present invention provides an antioxidant nano - collagen peptide powder, which at least includes antioxidant micelles encapsulating collagen peptides, a porous carrier, and transglutaminase; Among them, the antioxidant micelles encapsulating collagen peptides are prepared by dissolving a composite antioxidant, collagen peptide solution, pea protein isolate, lecithin, and short - chain inulin in phosphate buffer c.

[0006] Preferably, the preparation method of the composite antioxidant is as follows: Dissolve tea polyphenols and whey protein in phosphate buffer a with a pH of 7.0 - 7.2, add polyphenol oxidase, react at 37 °C for 1 - 1.5 hours, heat at 80 °C for 10 minutes to inactivate the enzyme, and use an ultrafiltration membrane with a molecular weight cut - off of 10 kDa to remove unbound small molecules to obtain the composite antioxidant.

[0007] Among them, the tea polyphenols are 0.1 - 0.5 parts by weight, the whey protein is 1 - 3 parts by weight, the polyphenol oxidase is 0.08 - 0.19 parts by weight, and the phosphate buffer a is 95 - 100 parts by weight.

[0008] Preferably, the composite antioxidant is 0.9 - 1.3 parts by weight, the collagen peptide solution is 4 - 6 parts by weight, the pea protein isolate is 2 - 5 parts by weight, the lecithin is 1 - 2 parts by weight, the short - chain inulin is 0.7 - 0.9 parts by weight, and the phosphate buffer c is 75 - 84 parts by weight.

[0009] Preferably, the preparation method of the porous carrier is as follows: Mix high amylose corn starch with acetic acid buffer solution with a pH of 5.0 - 6.0 to prepare a starch suspension of 5 - 10% w / v. Add α - amylase and place it in a constant temperature water bath at 55 ± 0.5°C for a reaction time of 12 - 15 h. Heat the temperature to 80 - 85°C and maintain it for 10 - 15 minutes, then centrifuge and wash. Dissolve lactoferrin in acetic acid buffer solution with a pH of 5.0 - 6.0 to prepare a lactoferrin solution of 0.5 - 1% w / v, and mix the lactoferrin solution and the porous carrier at a mass ratio of 5 - 5.5:1, stir at 37°C for 2 hours, centrifuge to remove the unadsorbed protein, and freeze - dry to obtain the porous carrier.

[0010] Preferably, the amylose content of the high amylose corn starch is 70 - 75%.

[0011] Preferably, the concentration of the α - amylase is 0.5 - 1.0 U / g of high amylose corn starch.

[0012] Through the catalysis of polyphenol oxidase, tea polyphenols are covalently bonded with whey protein to form a stable composite antioxidant, providing antioxidant effects; pea protein isolate and lecithin, as amphiphilic substances, promote the formation of micelles by ultrasonic treatment to encapsulate collagen peptides. Short - chain inulin (degree of polymerization 2 - 10) is adsorbed on the surface of micelles through hydrogen bonds to promote the stability of micelles. High amylose corn starch is partially hydrolyzed by α - amylase to form a porous structure with a high specific surface area, increasing the adsorption sites. Lactoferrin is deposited on the inner wall of the pores to provide active sites, and peptide segments are adsorbed under vacuum. Transglutaminase catalyzes the cross - linking reaction between micelles and carriers to form a three - dimensional network structure, constructing a "micelle - carrier" covalent connection, further locking collagen peptides and antioxidants to prevent shedding; through enzymatic modification, micelle encapsulation, porous loading, and cross - linking linkage, the risk of collagen exposure is reduced, and the efficient protection of active ingredients is achieved.

[0013] On the other hand, the present invention provides a preparation method of an antioxidant nano - collagen peptide powder for an antioxidant nano - collagen peptide powder described in any one of the above, comprising the following steps: S1.1. Dissolve the substrate in phosphate buffer b at 35 - 37°C and pH 6.5 - 7.5, add collagenase, add 0.5 - 1 mM calcium chloride, enzymatically hydrolyze at 100 - 200 rpm for 4 - 6 h, then add a vitamin C - citric acid complex with a mass fraction of 1 - 2%, adjust the pH to 4 - 4.5, and collect and concentrate the active peptide segments with a 3 - 10 kDa ultrafiltration membrane to obtain a collagen peptide solution; S1.2. Dissolve the compound antioxidant, collagen peptide solution, pea protein isolate, lecithin, and short-chain inulin in phosphate buffer c with a pH of 7.2 - 7.4, and perform ultrasonic treatment at a power of 200 - 300 W for 3 - 5 minutes at 37°C. Then stir for 1 - 1.5 hours, and centrifuge to remove unbound components to obtain antioxidant micelles encapsulating collagen peptides. S1.3. Mix the porous carrier with the antioxidant micelles, and treat the mixture under vacuum conditions at a pH of 7.4 for 25 - 30 min, wash and dry to obtain a negative carrier. Immerse the negative carrier in a transglutaminase solution containing 0.1 - 0.3% CaCl₂ and an enzyme activity of 0.5 - 2 U / g of the negative carrier, and impregnate at a pH of 7.0 - 7.5 and 50°C for 2 - 3 h to obtain antioxidant nano - collagen peptide powder.

[0014] Preferably, in S1.1, the substrate is a mixture of yak bone peptide and fish collagen peptide in a mass ratio of 1:1 - 2, and the mass ratio of the substrate to phosphate buffer b is 1:10 - 15.

[0015] Preferably, in S1.1, the mass ratio of collagenase to the substrate is 1:40 - 50.

[0016] Preferably, in S1.3, the porous carrier and the antioxidant micelles are mixed in a mass ratio of 1:3 - 5.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: In this antioxidant nano - collagen peptide powder and its preparation method, the compound antioxidant combines the strong antioxidant property of tea polyphenols and the stability of whey protein to achieve the slow release of the antioxidant and continuously neutralize free radicals. The pea protein isolate, lecithin, and short - chain inulin form antioxidant micelles encapsulating collagen peptides through electrostatic interaction, hydrophobic interaction, and hydrogen bond force. The double - layer structure of the porous carrier and the micelles isolates oxygen and free radicals from contacting the collagen peptides, delaying oxidative degradation. Moreover, the synergistic effect of loading on the porous carrier and enzyme cross - linking further locks the active components while reducing oxidative degradation caused by environmental oxygen penetration, making the collagen peptide firmly bound and its activity stable. Specific Embodiments

[0018] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0019] An antioxidant nano - collagen peptide powder of the present invention comprises at least antioxidant micelles encapsulating collagen peptides, a porous carrier, and transglutaminase. Among them, the antioxidant micelles encapsulating collagen peptides are prepared by dissolving a composite antioxidant, collagen peptide solution, pea protein isolate, lecithin, and short-chain inulin in phosphate buffer c; The vitamin C-citric acid complex is a product obtained by mixing vitamin C and citric acid in a mass ratio of 1:2.

[0020] The following examples of the present invention are used: Tea polyphenols are purchased from Wuhan Yuancheng Technology Development Co., Ltd.

[0021] Whey protein is purchased from Guangdong Daxiao Chemical Industry Co., Ltd.

[0022] Polyphenol oxidase is purchased from Shanghai Lianshuo Biotechnology Co., Ltd.

[0023] Phosphate buffer is purchased from Shanghai Bangjing Industrial Co., Ltd.

[0024] High amylose corn starch is purchased from Wuhan Kangcan Biotechnology Co., Ltd.

[0025] α-Amylase is purchased from Shanghai Jianglai Biotechnology Co., Ltd.

[0026] Acetate buffer is purchased from North China Weiye Metrology Group Co., Ltd.

[0027] Transglutaminase uses the transglutaminase of Pangbo Biological.

[0028] Polyphenol oxidase is purchased from Shanghai Lianshuo Biotechnology Co., Ltd.

[0029] Whey protein is purchased from Guangdong Daxiao Chemical Industry Co., Ltd.

[0030] Polyphenol oxidase is purchased from Shanghai Lianshuo Biotechnology Co., Ltd.

[0031] Pea protein isolate is purchased from Foodchem.

[0032] Lecithin is purchased from Tesco Chemical Industry (Hubei) Co., Ltd.

[0033] Short-chain inulin is purchased from Hebei Hongtao Bioengineering Co., Ltd.

[0034] Example 1: An antioxidant nano-collagen peptide powder and its preparation method, comprising the following steps: Prepare components: 0.1 part by weight of tea polyphenols, 3 parts by weight of whey protein, 0.19 part by weight of polyphenol oxidase, 100 parts by weight of phosphate buffer a; the amylose content of high amylose corn starch is 70%; the concentration of α-amylase is 1.0 U / g of high amylose corn starch; Dissolve tea polyphenols and whey protein in phosphate buffer a with a pH of 7.0, add polyphenol oxidase, react at 37°C for 1 hour, heat at 80°C for 10 minutes to inactivate the enzyme, and use a ultrafiltration membrane with a molecular weight cut-off of 10 kDa to remove unbound small molecules to obtain a composite antioxidant; Mix high amylose corn starch with acetic acid buffer with a pH of 6.0 to prepare a 5% w / v starch suspension, add α-amylase and place in a constant temperature water bath at 55°C for 12 h, raise the temperature to 80°C and maintain for 10 minutes, centrifuge and wash. Dissolve lactoferrin in acetic acid buffer with a pH of 6.0 to prepare a 1% w / v lactoferrin solution, and mix the lactoferrin solution and the porous carrier at a mass ratio of 5:1, stir at 37°C for 2 hours, centrifuge to remove unadsorbed protein, and freeze-dry to obtain a porous carrier; S1.1. Mix yak bone peptide and fish collagen peptide as a substrate in a mass ratio of 1:2 and dissolve in phosphate buffer b at 37°C and pH 7.0, add collagenase, add 1 mM calcium chloride, where the mass ratio of the substrate to phosphate buffer b is 1:10, and the mass ratio of collagenase to the substrate is 1:50. Enzymatically hydrolyze at 200 rpm for 6 h, then add a 1% mass fraction of vitamin C-citric acid complex, adjust the pH to 4.5, and use a 3-10 kDa ultrafiltration membrane to collect and concentrate the active peptide segments to obtain a collagen peptide solution; S1.2. Dissolve 1.3 parts by weight of the composite antioxidant, 6 parts by weight of the collagen peptide solution, 5 parts by weight of pea protein isolate, 2 parts by weight of lecithin, and 0.9 parts by weight of short-chain inulin in 84 parts by weight of phosphate buffer c with a pH of 7.4, ultrasonically treat at a power of 200 W at 37°C for 5 minutes, then stir for 1 hour, centrifuge to remove unbound components to obtain antioxidant micelles encapsulating collagen peptides; S1.3. Mix the porous carrier and the antioxidant micelles at a mass ratio of 1:3, treat the mixture under a vacuum condition of 50 mbar and pH of 7.4 for 30 min, wash and dry to obtain a negative carrier. Immerse the negative carrier in a transglutaminase solution containing 0.1% CaCl2 and an enzyme activity of 2 U / g of the negative carrier, and impregnate at pH 7.5 and 50°C for 3 h to obtain antioxidant nano-collagen peptide powder.

[0035] Example 2: An antioxidant nano-collagen peptide powder and its preparation method, comprising the following steps: Prepare components: 0.3 parts by weight of tea polyphenols, 3 parts by weight of whey protein, 0.19 parts by weight of polyphenol oxidase, 100 parts by weight of phosphate buffer a; the amylose content of high amylose corn starch is 70%; the concentration of α-amylase is 1.0 U / g of high amylose corn starch; Dissolve tea polyphenols and whey protein in phosphate buffer a with a pH of 7.0, add polyphenol oxidase, react at 37 °C for 1 hour, heat at 80 °C for 10 minutes to inactivate the enzyme, and use an ultrafiltration membrane with a molecular weight cut-off of 10 kDa to remove unbound small molecules to obtain a composite antioxidant; Mix high amylose corn starch with acetic acid buffer at a pH of 6.0 to prepare a 5% w / v starch suspension, add α-amylase and place it in a constant temperature water bath at 55 °C for 12 h, raise the temperature to 80 °C and maintain for 10 minutes, centrifuge and wash. Dissolve lactoferrin in acetic acid buffer at a pH of 6.0 to prepare a 1% w / v lactoferrin solution, and mix the lactoferrin solution with the porous carrier at a mass ratio of 5:1, stir at 37 °C for 2 hours, centrifuge to remove unadsorbed protein, and freeze-dry to obtain a porous carrier; S1.1. Mix yak bone peptide and fish collagen peptide as a substrate in a mass ratio of 1:2 and dissolve it in phosphate buffer b at 37 °C and pH 7.0, add collagenase, and add 1 mM calcium chloride. Among them, the mass ratio of the substrate to phosphate buffer b is 1:10, and the mass ratio of collagenase to the substrate is 1:50. Enzymatically hydrolyze at 200 rpm for 6 h, then add a 1% mass fraction of vitamin C-citric acid complex, adjust the pH to 4.5, and collect and concentrate the active peptide segments with a 3-10 kDa ultrafiltration membrane to obtain a collagen peptide solution; S1.2. Dissolve 1.3 parts by weight of the composite antioxidant, 6 parts by weight of the collagen peptide solution, 5 parts by weight of pea protein isolate, 2 parts by weight of lecithin, and 0.9 parts by weight of short-chain inulin in 84 parts by weight of phosphate buffer c with a pH of 7.4, and ultrasonically treat at a power of 200 W at 37 °C for 5 minutes, then stir for 1 hour, centrifuge to remove unbound components to obtain antioxidant micelles encapsulating collagen peptides; S1.3. Mix the porous carrier and the antioxidant micelles at a mass ratio of 1:3, treat the mixture under a vacuum condition of 50 mbar and at a pH of 7.4 for 30 min, wash and dry to obtain a negative carrier. Immerse the negative carrier in a transglutaminase solution containing 0.1% CaCl2 and an enzyme activity of 2 U / g of the negative carrier, and immerse it at a pH of 7.5 and 50 °C for 3 h to obtain antioxidant nano-collagen peptide powder.

[0036] Example 3: An antioxidant nano-collagen peptide powder and its preparation method, comprising the following steps: Prepare components: 0.5 part by weight of tea polyphenols, 3 parts by weight of whey protein, 0.19 part by weight of polyphenol oxidase, 100 parts by weight of phosphate buffer a; the amylose content of high amylose corn starch is 70%; the concentration of α-amylase is 1.0 U / g of high amylose corn starch; Dissolve tea polyphenols and whey protein in phosphate buffer a with a pH of 7.0, add polyphenol oxidase, react at 37 °C for 1 hour, heat at 80 °C for 10 minutes to inactivate the enzyme, and use a ultrafiltration membrane with a molecular weight cut-off of 10 kDa to remove unbound small molecules to obtain a composite antioxidant; Mix high amylose corn starch with acetic acid buffer with a pH of 6.0 to prepare a 5% w / v starch suspension, add α-amylase and place it in a constant temperature water bath at 55 °C for 12 h, raise the temperature to 80 °C and maintain for 10 minutes, centrifuge and wash. Dissolve lactoferrin in acetic acid buffer with a pH of 6.0 to prepare a 1% w / v lactoferrin solution, and mix the lactoferrin solution with the porous carrier at a mass ratio of 5:1, stir at 37 °C for 2 hours, centrifuge to remove unadsorbed protein, and freeze-dry to obtain the porous carrier; S1.1. Mix yak bone peptide and fish collagen peptide as a substrate in a mass ratio of 1:2 and dissolve it in phosphate buffer b at 37 °C and pH 7.0, add collagenase, add 1 mM calcium chloride, wherein the mass ratio of the substrate to phosphate buffer b is 1:10, and the mass ratio of collagenase to the substrate is 1:50, enzymatically hydrolyze at 200 rpm for 6 h, then add a 1% mass fraction of vitamin C-citric acid complex, adjust the pH to 4.5, and collect and concentrate the active peptide segments with a 3-10 kDa ultrafiltration membrane to obtain a collagen peptide solution; S1.2. Dissolve 1.3 parts by weight of the composite antioxidant, 6 parts by weight of the collagen peptide solution, 5 parts by weight of pea protein isolate, 2 parts by weight of lecithin, and 0.9 parts by weight of short-chain inulin in 84 parts by weight of phosphate buffer c with a pH of 7.4, perform ultrasonic treatment at 37 °C with a power of 200 W for 5 minutes, then stir for 1 hour, centrifuge to remove unbound components to obtain antioxidant micelles encapsulating collagen peptides; S1.3. Mix the porous carrier and the antioxidant micelles at a mass ratio of 1:3, treat the mixture under a vacuum condition of 50 mbar and pH 7.4 for 30 min, wash and dry to obtain a negative carrier, immerse the negative carrier in a transglutaminase solution containing 0.1% CaCl2 and an enzyme activity of 2 U / g of the negative carrier, and immerse at pH 7.5 and 50 °C for 3 h to obtain antioxidant nano-collagen peptide powder.

[0037] Example 4: An antioxidant nano-collagen peptide powder and its preparation method, comprising the following steps: Prepare components: 0.5 part by weight of tea polyphenols, 3 parts by weight of whey protein, 0.19 part by weight of polyphenol oxidase, 100 parts by weight of phosphate buffer a; the amylose content of high amylose corn starch is 70%; the concentration of α-amylase is 5.0 U / g of high amylose corn starch; Dissolve tea polyphenols and whey protein in phosphate buffer a with a pH of 7.0, add polyphenol oxidase, react at 37 °C for 1 hour, heat at 80 °C for 10 minutes to inactivate the enzyme, and use a ultrafiltration membrane with a molecular weight cut-off of 10 kDa to remove unbound small molecules to obtain a composite antioxidant; Mix high amylose corn starch with acetic acid buffer with a pH of 6.0 to prepare a 5% w / v starch suspension, add α-amylase and place in a constant temperature water bath at 55 °C for 12 h, raise the temperature to 80 °C and maintain for 10 minutes, centrifuge and wash. Dissolve lactoferrin in acetic acid buffer with a pH of 6.0 to prepare a 1% w / v lactoferrin solution, and mix the lactoferrin solution with the porous carrier at a mass ratio of 5:1, stir at 37 °C for 2 hours, centrifuge to remove unadsorbed protein, and freeze-dry to obtain the porous carrier; S1.1: Mix yak bone peptide and fish collagen peptide as a substrate in a mass ratio of 1:2 and dissolve in phosphate buffer b at 37 °C and pH 7.0, add collagenase, add 1 mM calcium chloride, where the mass ratio of the substrate to phosphate buffer b is 1:10, and the mass ratio of collagenase to the substrate is 1:50, enzymatically hydrolyze at 200 rpm for 6 h, then add a 1% mass fraction of vitamin C-citric acid complex, adjust the pH to 4.5, and collect and concentrate the active peptide segments with a 3-10 kDa ultrafiltration membrane to obtain a collagen peptide solution; S1.2: Dissolve 1.3 parts by weight of the composite antioxidant, 6 parts by weight of the collagen peptide solution, 5 parts by weight of pea protein isolate, 2 parts by weight of lecithin, and 0.9 parts by weight of short-chain inulin in 84 parts by weight of phosphate buffer c with a pH of 7.4, ultrasonically treat at a power of 200 W for 5 minutes at 37 °C, then stir for 1 hour, centrifuge to remove unbound components to obtain antioxidant micelles encapsulating collagen peptides; S1.3: Mix the porous carrier and the antioxidant micelles at a mass ratio of 1:3, treat the mixture under a vacuum condition of 50 mbar and pH 7.4 for 30 min, wash and dry to obtain a negative carrier. Immerse the negative carrier in a transglutaminase solution containing 0.1% CaCl2 and an enzyme activity of 2 U / g of the negative carrier, and immerse at pH 7.5 and 50 °C for 3 h to obtain antioxidant nano-collagen peptide powder.

[0038] Example 5: An antioxidant nano-collagen peptide powder and its preparation method, comprising the following steps: Prepare components: 0.5 parts by weight of tea polyphenols, 3 parts by weight of whey protein, 0.19 parts by weight of polyphenol oxidase, 100 parts by weight of phosphate buffer a; the amylose content of high amylose corn starch is 70%; the concentration of α-amylase is 1.0 U / g of high amylose corn starch; Dissolve tea polyphenols and whey protein in phosphate buffer a with a pH of 7.0, add polyphenol oxidase, react at 37°C for 1 hour, heat at 80°C for 10 minutes to inactivate the enzyme, and use an ultrafiltration membrane with a molecular weight cut-off of 10 kDa to remove unbound small molecules to obtain a composite antioxidant; Mix high amylose corn starch with acetic acid buffer with a pH of 6.0 to prepare a 5% w / v starch suspension, add α-amylase and react in a constant temperature water bath at 55°C for 12 h, raise the temperature to 80°C and maintain for 10 minutes, centrifuge and wash. Dissolve lactoferrin in acetic acid buffer with a pH of 6.0 to prepare a 1% w / v lactoferrin solution, and mix the lactoferrin solution with the porous carrier at a mass ratio of 5:1, stir at 37°C for 2 hours, centrifuge to remove unadsorbed protein, and freeze-dry to obtain the porous carrier; S1.1: Mix yak bone peptide and fish collagen peptide as a substrate in a mass ratio of 1:2 and dissolve it in phosphate buffer b at 37°C and pH 7.0, add collagenase, add 1 mM calcium chloride, where the mass ratio of the substrate to phosphate buffer b is 1:10, and the mass ratio of collagenase to the substrate is 1:50, enzymatically hydrolyze at 200 rpm for 6 h, then add a 1% mass fraction of vitamin C-citric acid complex, adjust the pH to 4.5, and collect and concentrate the active peptide segments with a 3-10 kDa ultrafiltration membrane to obtain a collagen peptide solution; S1.2: Dissolve 1.3 parts by weight of the composite antioxidant, 6 parts by weight of the collagen peptide solution, 5 parts by weight of pea protein isolate, 2 parts by weight of lecithin, and 0.9 parts by weight of short-chain inulin in 84 parts by weight of phosphate buffer c with a pH of 7.4, ultrasonically treat at 200 W power for 5 minutes at 37°C, then stir for 1 hour, centrifuge to remove unbound components to obtain antioxidant micelles encapsulating collagen peptides; S1.3: Mix the porous carrier and the antioxidant micelles at a mass ratio of 1:3, treat the mixed solution under a vacuum condition of 50 mbar and pH 7.4 for 30 min, wash and dry to obtain a negative carrier, immerse the negative carrier in a transglutaminase solution containing 0.1% CaCl2 and an enzyme activity of 2 U / g of the negative carrier, and immerse at pH 7.5 and 50°C for 5 h to obtain antioxidant nano-collagen peptide powder.

[0039] Comparative Example 1: The method of Example 3 was used without adding the composite antioxidant.

[0040] Comparative Example 2: The method of Example 3 was used, and the collagen peptide solution was directly loaded onto the porous carrier without preparing antioxidant micelles encapsulating collagen peptides.

[0041] Comparative Example 3: The method of Example 3 was used, and the antioxidant micelles encapsulating collagen peptides were directly immersed in the transglutaminase solution without loading the antioxidant micelles encapsulating collagen peptides onto the porous carrier.

[0042] Comparative Example 4: The method of Example 3 was adopted, and the carrier was not impregnated in the transglutaminase solution.

[0043] An antioxidant nano - collagen peptide powder prepared by using a composite antioxidant according to the present invention, wherein the inspection items and inspection standards for stability and antioxidant performance are as follows: Test for antioxidant activity (DPPH radical scavenging rate): DPPH radical is a stable nitrogen - centered radical, and its ethanol solution has a strong absorption peak at 517 nm. When an antioxidant reacts with DPPH, the radical is neutralized, the color of the solution changes from purple to yellow, and the absorbance decreases. The scavenging rate is proportional to the antioxidant ability. The specific test steps are as follows: Take 2 mL of the sample and add it to a test tube, then add 2 mL of a DPPH solution with a concentration of 0.1 mmol / L (prepared using an ethanol solution with a volume fraction of 95%) to the test tube. After mixing evenly, place it in the dark for 30 min, and measure the absorbance at a wavelength of 517 nm. Each group is tested with three parallel samples, and the average value is taken. Among them, the DPPH radical scavenging rate = (A0 - A) / A0×100%, where A0 is the absorbance of the DPPH solution after adding the sample, and A is the absorbance of the DPPH solution without adding the sample. Storage stability simulates long - term storage conditions through an accelerated experiment to evaluate the activity retention rate of collagen peptide under high - temperature and high - humidity environments for evaluating its stability. The accelerated experiment conditions are: 40°C, 75% RH, and the DPPH scavenging rate is tested at 3 and 6 months.

[0044] Through the above standards, the antioxidant nano - collagen peptide powders prepared in Examples 1 - 5 and Comparative Examples 1 - 4 were tested, and the obtained data are shown in Table 1: Table 1 Performance data of Examples 1 - 5 and Comparative Examples 1 - 4 The above data fully show that compared with Comparative Examples 1 - 4, Examples 1 - 5 can fully show the role of the composite antioxidant in terms of the stability and antioxidant activity of the antioxidant nano - collagen peptide powder.

[0045] Since the present invention uses a composite antioxidant to prepare the antioxidant nano - collagen peptide powder, the performance of the antioxidant nano - collagen peptide powder is effectively improved by the composite antioxidant, specifically as follows: It can be seen from Examples 1-3 that with the continuous increase in the content of the tea polyphenol component, the stability and antioxidant activity of the antioxidant nano-collagen peptide powder are significantly improved. The hydrophilic region of whey protein and the hydrophobic part of the tea polyphenol-quinone complex endow the composite antioxidant with amphiphilicity, enabling it to participate in micelle self-assembly together with pea protein isolate (hydrophilic) and lecithin (amphiphilic). The hydrophobic aromatic ring of tea polyphenols and the hydrophobic region of collagen peptides jointly form the micelle core. At the same time, the composite antioxidant itself is also encapsulated in the core as an antioxidant active ingredient. Therefore, when the content of tea polyphenols increases, more phenolic hydroxyl active sites are provided, which synergistically scavenge free radicals with collagen peptides, improving the stability and antioxidant activity of the antioxidant nano-collagen peptide powder.

[0046] It can be seen from Examples 3 and 4 that when the concentration of α-amylase is too high, the stability and antioxidant performance of the antioxidant nano-collagen peptide powder decrease. This is because when the concentration of α-amylase is too high, the excessive hydrolysis of high amylose corn starch leads to the loosening of the carrier skeleton, a decrease in mechanical strength, structural collapse, an inability to withstand the vacuum negative pressure, and easy leakage of micelles, thus reducing the stability and antioxidant ability of the antioxidant nano-collagen peptide powder.

[0047] It can be seen from Examples 3 and 5 that with the prolongation of the transglutaminase cross-linking time, the stability of the antioxidant nano-collagen peptide powder is effectively improved, but the antioxidant performance decreases. This is because the transglutaminase cross-linking network can effectively resist changes in the external environment, and the dense cross-linking network is also beneficial for reducing the contact between the active components and oxygen. However, when over-crosslinked, it will mask the carrier pores and active sites, preventing the timely release of collagen peptides and instead reducing their antioxidant activity.

[0048] According to the above test experiments, it is known that an antioxidant nano-collagen peptide powder prepared according to Example 3 has the optimal performance, so Example 3 is taken as the optimal example; It can be seen from the comparison between Example 3 and Comparative Examples 1-4 that: In Comparative Example 1, no composite antioxidant was added, and the stability and antioxidant effect of the antioxidant nano-collagen peptide powder were poorer. Lacking the exogenous antioxidant protection of tea polyphenols, the sulfhydryl (-SH) and proline hydroxyl (-OH) groups of collagen peptides were directly exposed to free radicals, resulting in peptide chain breakage and a significant increase in the oxidation degradation rate of collagen peptides. Therefore, the stability and antioxidant effect were poorer.

[0049] In Comparative Example 2, the collagen peptide solution was directly loaded onto the porous carrier without preparing antioxidant micelles encapsulating the collagen peptide. The stability and antioxidant performance of the antioxidant nano-collagen peptide powder were poorer. The collagen peptide that did not form micelles was directly adsorbed on the surface of the carrier, and the pore size of the porous carrier could not completely block small molecule free radicals, causing the collagen peptide solution to be directly exposed to the oxygen environment, thereby resulting in poorer stability and antioxidant performance of the antioxidant nano-collagen peptide powder.

[0050] In Comparative Example 3, the antioxidant micelles encapsulating the collagen peptide were directly immersed in the transglutaminase solution without loading the antioxidant micelles encapsulating the collagen peptide onto the porous carrier. The stability and antioxidant performance of the antioxidant nano-collagen peptide powder were poorer. Lacking the physical barrier of the porous carrier, the micelles were directly exposed to the external environment, and the micelle structure formed by lecithin and pea protein isolate was easily damaged by external forces, causing the collagen peptide to be released and oxidized in advance. Therefore, the stability and antioxidant performance of the antioxidant nano-collagen peptide powder were poorer.

[0051] In Comparative Example 4, the carrier was not immersed in the transglutaminase solution. For the antioxidant nano-collagen peptide powder, the stability and antioxidant performance of the antioxidant nano-collagen peptide powder were poorer. Lacking the ε-(γ-glutamyl) lysine isopeptide bond catalyzed by transglutaminase, the micelles and the carrier were only physically adsorbed and were easily dissociated in a solution with a higher ionic strength, thereby releasing the active components. Therefore, the stability and antioxidant performance of the antioxidant nano-collagen peptide powder were poor.

[0052] In summary, through a dynamic equilibrium antioxidant system, the covalent complex of tea polyphenols and whey protein provides rapid free radical neutralization, lactoferrin inhibits the source of oxidation, and the antioxidant groups of the collagen peptide act synergistically. Combined with a multi-level barrier design, the hydrophobic core of the micelles isolates oxygen diffusion, then the physical adsorption of the porous carrier reduces the contact area between the micelles and the outside world, and finally the enzyme cross-linking network fixes the micelles in the carrier, enabling the antioxidant nano-collagen peptide powder to have efficient and long-lasting antioxidant ability and extending the storage time.

[0053] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. The above embodiments and descriptions in the specification are only preferred examples of the present invention and are not used to limit the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed.

Claims

1. An antioxidant nano - collagen peptide powder, characterized in that: It includes at least antioxidant micelles encapsulating collagen peptides and a porous carrier; Among them, the antioxidant micelles encapsulating collagen peptides are prepared by dissolving a composite antioxidant, a collagen peptide solution, pea protein isolate, lecithin, and short-chain inulin in phosphate buffer c.

2. The antioxidant nano - collagen peptide powder according to claim 1, characterized in that: The preparation method of the composite antioxidant is as follows: Dissolve tea polyphenols and whey protein in phosphate buffer a with a pH of 7.0 - 7.2, add polyphenol oxidase, react at 37°C for 1 - 1.5 hours, heat at 80°C for 10 minutes to inactivate the enzyme, and remove unbound small molecules with a 10 kDa molecular weight cut-off ultrafiltration membrane to obtain the composite antioxidant; Among them, the tea polyphenols are 0.1 - 0.5 parts by weight, the whey protein is 1 - 3 parts by weight, the polyphenol oxidase is 0.08 - 0.19 parts by weight, and the phosphate buffer a is 95 - 100 parts by weight.

3. The antioxidant nano - collagen peptide powder according to claim 1, characterized in that: The composite antioxidant is 0.9 - 1.3 parts by weight, the collagen peptide solution is 4 - 6 parts by weight, the pea protein isolate is 2 - 5 parts by weight, the lecithin is 1 - 2 parts by weight, the short-chain inulin is 0.7 - 0.9 parts by weight, and the phosphate buffer c is 75 - 84 parts by weight.

4. The antioxidant nano - collagen peptide powder according to claim 1, wherein: The preparation method of the porous carrier is as follows: Mix high amylose corn starch with acetic acid buffer with a pH of 5.0 - 6.0 to prepare a 5 - 10% w / v starch suspension, add α-amylase, react in a constant temperature water bath at 55 ± 0.5°C for 12 - 15 h, raise the temperature to 80 - 85°C and maintain for 10 - 15 minutes, centrifuge and wash. Dissolve lactoferrin in acetic acid buffer with a pH of 5.0 - 6.0 to prepare a 0.5 - 1% w / v lactoferrin solution, and mix the lactoferrin solution with the porous carrier at a mass ratio of 5 - 5.5:1, stir at 37°C for 2 hours, centrifuge to remove unadsorbed protein, and freeze-dry to obtain the porous carrier.

5. The antioxidant nano - collagen peptide powder according to claim 4, wherein: The amylose content of the high amylose corn starch is 70 - 75%.

6. The antioxidant nano - collagen peptide powder according to claim 4, characterized in that: The concentration of the α-amylase is 0.5 - 1.0 U / g of high amylose corn starch.

7. A preparation method of an antioxidant nano collagen peptide powder, which is used to prepare the antioxidant nano collagen peptide powder described in any one of claims 1-6, and is characterized in that: It includes the following steps: S1.

1. Dissolve the substrate in phosphate buffer b at 35 - 37°C and pH 6.5 - 7.5, add collagenase, add 0.5 - 1 mM calcium chloride, enzymatically hydrolyze at 100 - 200 rpm for 4 - 6 h, then add a 1 - 2% mass fraction of vitamin C-citric acid complex, adjust the pH to 4 - 4.5, and collect and concentrate the active peptide segments with a 3 - 10 kDa ultrafiltration membrane to obtain the collagen peptide solution; S1.

2. Dissolve the composite antioxidant, the collagen peptide solution, the pea protein isolate, the lecithin, and the short-chain inulin in phosphate buffer c with a pH of 7.2 - 7.4, perform ultrasonic treatment at a power of 200 - 300 W at 37°C for 3 - 5 minutes, then stir for 1 - 1.5 hours, and centrifuge to remove unbound components to obtain the antioxidant micelles encapsulating collagen peptides; S1.

3. Mix the porous carrier with the antioxidant micelles, treat the mixture under vacuum at pH 7.4 for 25 - 30 min, wash and dry to obtain the negative carrier. Immerse the negative carrier in a transglutaminase solution containing 0.1 - 0.3% CaCl₂ and with an enzyme activity of 0.5 - 2 U / g of the negative carrier, and impregnate at 50 °C at pH 7.0 - 7.5 for 2 - 3 h to obtain the antioxidant nano - collagen peptide powder.

8. The preparation method of the antioxidant nano collagen peptide powder according to claim 7, characterized in that: In S1.1, the substrate is obtained by mixing yak bone peptide and fish collagen peptide in a mass ratio of 1:1 - 2, and the mass ratio of the substrate to phosphate buffer b is 1:10 - 15.

9. The preparation method of the antioxidant nano collagen peptide powder according to claim 7, characterized in that: In S1.1, the mass ratio of collagenase to the substrate is 1:40 - 50.

10. The preparation method of the antioxidant nano - collagen peptide powder according to claim 7, wherein: In S1.3, the porous carrier and the antioxidant micelles are mixed in a mass ratio of 1:3 - 5.

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