Black chenopodium quinoa willd active peptide with hair follicle cell growth promoting, hair growing and hair care activity, and preparation method and application of black chenopodium quinoa willd active peptide

Abstract

The invention relates to the technical field of deep processing of black chenopodium quinoa, in particular to a black chenopodium quinoa active peptide capable of promoting hair follicle cell growth and hair growth and care activity and a preparation method and application of the black chenopodium quinoa active peptide. The black chenopodium quinoa active peptide is prepared by the following steps: crushing, pretreating with amylase, performing alkali extraction and acid precipitation, performing double-enzyme step-by-step hydrolysis with alkaline protease and pepsase, and performing ultrafiltration to cut off a fragment with the molecular weight of less than 5000Da, thereby obtaining the black chenopodium quinoa active peptide. In addition, seven brand-new non-toxic characteristic peptide fragments are further screened out, the 5 alpha-reductase and a GSK-3beta receptor can be efficiently combined, and hair follicle cell proliferation is promoted and oxidative damage is relieved by inhibiting the 5 alpha-reductase and activating Wnt / beta-catenin and Nrf2 / HO-1 signal channels. The peptide is natural and safe, the process is simple, the black quinoa peptide is expanded to the field of hair growth and hair care for the first time, the market gap related to grain peptide is filled, and high-value utilization of black quinoa is achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of deep processing of black quinoa, and particularly relates to a black quinoa active peptide with the activities of promoting hair follicle cell growth and hair growth, and a preparation method and application thereof. BACKGROUND

[0002] As a cereal with high nutritional value and wide application potential, black quinoa is not only balanced in protein and amino acid content, but also rich in essential fatty acids, minerals, vitamins, dietary fiber and carbohydrates, and is an ideal source of healthy food. Among them, the high-quality protein contained in black quinoa can provide a variety of essential amino acids, which belongs to high-quality plant protein. Based on the excellent functional properties and rich nutritional content of the protein, it has great development potential and broad application prospects in the field of healthy food.

[0003] At present, the research on black quinoa protein is relatively mature, but the functional mechanism analysis of black quinoa peptide still has a technical bottleneck to be broken through. Black quinoa peptide is a small molecule active peptide segment prepared by modern biological enzymatic hydrolysis technology with black quinoa as raw material. In recent years, it has attracted widespread attention due to its unique nutritional properties and multifunctional biological activity. In the prior art, the yield of active peptide can be improved by genetic engineering technology to modify protease or combined enzyme system, and the key active short peptide sequence can be determined by mass spectrometry and molecular docking technology, and the binding mechanism of the target is explored, and then the core path of the activity of black quinoa peptide is preliminarily revealed. In the aspect of functional properties, black quinoa peptide has been confirmed to have multiple biological activities such as antioxidant, antihypertensive, immune regulation, and maintenance of intestinal health.

[0004] In recent years, affected by factors such as accelerated pace of life and increased environmental pressure, hair loss and damaged hair quality have become increasingly common, and consumers have an increasingly urgent demand for natural and safe hair growth products, and there is a clear supply gap in the related market. However, in the prior art, the application of natural active ingredients in the field of hair growth and hair care still has a major gap, and the application scenarios of cereal peptides do not cover the field of hair growth and hair care, which cannot meet the above market demand, and the potential value of black quinoa can still be fully tapped. SUMMARY

[0005] In order to further expand the application scenarios of natural active ingredients in the field of hair growth and hair care and break through the application limitations of cereal peptides, the present application provides a preparation method of a black quinoa active peptide with the activities of promoting hair follicle cell growth and hair growth, comprising the following steps: S1, grinding black quinoa in a grinder, and passing through a 20-40 mesh sieve to obtain black quinoa powder; S2, once pretreatment: the black chenopodium album powder is mixed with water in a mass ratio of 1: (8-12), the temperature of the system is adjusted to 45-55 DEG C, amylase is added, and enzymolysis is carried out for 0.5-1.5 h, and the first pretreatment mixed liquor is obtained after enzyme inactivation; S3, secondary pretreatment: the temperature of the first pretreatment mixed liquor is adjusted to 50-55 DEG C, the pH is adjusted to 9.5-10.5, and extraction is carried out for 0.5-1.5 h, the supernatant is obtained by centrifugation, the pH is adjusted to 3.8-4.2, and the black chenopodium album protein precipitate is obtained by precipitation at 4 DEG C for 0.5-1.5 h and centrifugation again; S4, once hydrolysis: the black chenopodium album protein precipitate is mixed with pure water in a mass ratio of 1: (2-3), the temperature of the system is adjusted to 50-55 DEG C, alkaline protease is added, and enzymolysis is carried out for 1-3 h, and the first hydrolysis liquor is obtained after enzyme inactivation; S5, secondary hydrolysis: the temperature of the first hydrolysis liquor is adjusted to (37+ / -2) DEG C, pepsin is added, and enzymolysis is carried out for 1-3 h, the pH is adjusted to 3.8-4.2, and the second hydrolysis liquor is obtained after enzyme inactivation; S6, the second hydrolysis liquor is filtered and ultrafiltered to obtain the black chenopodium album active peptide filtrate, wherein the ultrafiltration molecular weight cut-off is <5000 Da.

[0006] In some embodiments, the black chenopodium album active peptide filtrate is subjected to spray drying to obtain black chenopodium album active peptide powder.

[0007] In some embodiments, in step S2, the addition amount of amylase is 7.0%-9.0% of the mass of black chenopodium album powder, and the pH of the enzymolysis system is 7.0; in step S4, the addition amount of alkaline protease is 0.1%-0.2% of the mass of black chenopodium album powder, and the pH of the enzymolysis system is 8.0-9.0; in step S5, the addition amount of pepsin is 0.2%-0.3% of the mass of black chenopodium album powder, and the pH of the enzymolysis system is 2.0-3.0.

[0008] In some embodiments, the enzyme inactivation treatment temperature is 85-90 DEG C, and the treatment time is 15-20 min.

[0009] In some embodiments, the centrifugation is carried out at 4000-6000 rpm for 5-10 min.

[0010] The application also provides a black chenopodium album active peptide prepared according to the preparation method of the black chenopodium album active peptide.

[0011] In some embodiments, the black quinoa active peptides include any one or more amino acid sequences selected from Val-Val-Val-Pro-Gln (VVVPQ), Leu-Leu-Pro-Ser (LLPS), Leu-Leu-Pro-His (LLPH), Thr-Tyr-Leu-Val (TYLV), Leu-Leu-Leu (LLL), Leu-Val-Leu (LVL), and Ile-Phe (IF).

[0012] The present invention also provides a functional product whose components include the black quinoa active peptides as described above.

[0013] In some embodiments, the functional product includes at least one of the following functions: (1) Inhibits 5α-reductase; (2) Promotes hair follicle cell growth; (3) Antioxidant; (4) Promote hair growth and protect hair.

[0014] This invention also provides the application of the black quinoa active peptide as described above in the preparation of products for inhibiting 5α-reductase activity, inhibiting GSK-3β receptor activity, activating the Wnt / β-catenin signaling pathway, upregulating CTNNB1 gene expression, upregulating β-catenin gene expression, upregulating Wnt5b gene expression, upregulating IGF-I gene expression, activating the Nrf2 / HO-1 antioxidant signaling pathway, upregulating Nrf2 gene expression, and upregulating HO-1 gene expression.

[0015] Compared with the prior art, the present invention has the following beneficial effects: This invention employs a pretreatment process involving pulverization, enzymatic hydrolysis, and alkaline extraction followed by acid precipitation. The product is then obtained through stepwise hydrolysis with a dual enzyme of Bacillus licheniformis alkaline protease and pepsin, followed by solid-liquid separation, ultrafiltration to remove active fragments with a molecular weight cutoff of less than 5000 Da, and drying. This preparation process is simple to operate, requires minimal production equipment, is easily implemented industrially, and yields products with excellent activity. In Example 1, the product exhibits a 5α-reductase inhibition rate of 62.82%, significantly superior to existing methods using alternative enzymes or single-enzyme hydrolysis.

[0016] Based on the optimized preparation method described above, this invention successfully screened seven novel, non-toxic, and highly active characteristic peptides using a combination of separation, purification, and bioinformatics techniques. These peptides are Val-Val-Val-Pro-Gln, Leu-Leu-Pro-Ser, Leu-Leu-Pro-His, Thr-Tyr-Leu-Val, Leu-Leu-Leu, Leu-Val-Leu, and Ile-Phe. These characteristic peptides can bind efficiently to 5α-reductase and GSK-3β receptors, while exhibiting no obvious bitterness or astringency, possessing a pleasant taste and flavor. This effectively fills the functional gap in the field of black quinoa peptides for hair growth and care, and demonstrates stronger targeting.

[0017] Meanwhile, the hair growth and hair care mechanism of the active peptides of this invention is clearly defined, exerting their effects through multiple synergistic pathways: First, they can directly inhibit 5α-reductase activity, reduce dihydrotestosterone production, and block the miniaturization process of hair follicles from the source; Second, in human dermal papilla cells (hDPCs) experiments, it has been confirmed that they can significantly upregulate the expression of CTNNB1, β-catenin, Wnt5b, and IGF-I genes, effectively activating the Wnt / β-catenin signaling pathway, thereby promoting the survival and proliferation of dermal papilla cells; Third, they can upregulate the expression of Nrf2 and HO-1 genes, activate antioxidant signaling pathways, enhance the cell's own antioxidant defense capabilities, reduce the damage of oxidative stress to hair follicle cells, create a favorable microenvironment for hair growth, and ensure the significant effect of hair growth and hair care.

[0018] In summary, this invention expands the application of black quinoa peptides to the field of hair growth and care, filling the supply gap of grain peptides in this niche market. Furthermore, since the raw material is derived from natural black quinoa, it is highly safe and meets the current consumer demand for natural care products. This not only realizes the high-value utilization of black quinoa, but also provides a new approach for the in-depth application of natural active ingredients in the field of hair care. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a diagram showing the expression of the CTNNB1 gene in hDPCs cells during Experiment 2 of this invention. Figure 2 This is a diagram showing the expression of the β-catenin gene in hDPCs cells during Experiment 2 of this invention. Figure 3This is a diagram showing the expression of the Wnt5b gene in hDPCs cells during Experiment 2 of this invention. Figure 4 This is a diagram showing the expression of the IGF-I gene in hDPCs cells during Experiment 2 of this invention. Figure 5 This is a diagram showing the expression of the Nrf2 gene in hDPCs cells during Experiment 2 of this invention. Figure 6 This is a diagram showing the expression of the HO-1 gene in hDPCs cells during Experiment 2 of this invention. Figure 7 This is a diagram showing the docking conformation of VVVPQ with 5α-reductase and GSK-3β molecules in Experiment 3 of this invention; Figure 8 This is a diagram showing the docking conformation of LLL with 5α-reductase and GSK-3β molecules in Experiment 3 of this invention; Figure 9 This is a diagram showing the docking conformation of LVL with 5α-reductase and GSK-3β molecules in Experiment 3 of this invention; Figure 10 This is a diagram showing the docking conformation of LLPH with 5α-reductase and GSK-3β molecules in Experiment 3 of this invention; Figure 11 This is a diagram showing the docking conformation of LLPS with 5α-reductase and GSK-3β molecules in Experiment 3 of this invention. Figure 12 This is a diagram showing the docking conformation of TYLV with 5α-reductase and GSK-3β molecules in Experiment 3 of this invention; Figure 13 This is a conformation diagram of the docking of IF with 5α-reductase and GSK-3β molecules in Experiment 3 of this invention. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, 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, not all embodiments, and the technical features designed in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other. 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.

[0022] This invention provides a black quinoa active peptide with the activity of promoting hair follicle cell growth and hair growth and conditioning, which is prepared by the following steps: S1. Add black quinoa to a grinder and grind it, then pass it through a 20-40 mesh sieve to obtain black quinoa flour; S2. First pretreatment: Mix black quinoa flour and water at a mass ratio of 1:(8~12) evenly, adjust the system temperature to 45~55℃ and pH to 7.0, add 7.0%~9.0% of the mass of black quinoa flour amylase, enzymatically hydrolyze for 0.5~1.5 h, and then inactivate the enzyme at 85~90℃ for 15~20 min to obtain the first pretreatment mixture; S3. Secondary pretreatment: Adjust the temperature of the pretreatment mixture to 50-55℃, adjust the pH to 9.5-10.5 with NaOH solution, extract for 0.5-1.5h, centrifuge at 4000-6000rpm for 5-10min, take the supernatant, adjust the pH to 3.8-4.2 with HCl solution, precipitate at 4℃ for 0.5-1.5h, centrifuge again to obtain black quinoa protein precipitate; S4. First hydrolysis: Mix black quinoa protein precipitate with pure water at a mass ratio of 1:(2~3), adjust the system temperature to 50~55℃ and pH to 8.0~9.0, add 0.1%~0.2% of black quinoa flour mass of alkaline protease, hydrolyze for 1~3 hours, and obtain the first hydrolysate after enzyme inactivation. S5. Secondary hydrolysis: Adjust the temperature of the first hydrolysate to (37±2)℃ and the pH to 2.0~3.0, add 0.2%~0.3% of pepsin by weight of black quinoa flour, hydrolyze for 1~3 hours, adjust the pH to 3.8~4.2, and obtain the second hydrolysate after enzyme inactivation; S6. Filter and ultrafilter the second hydrolysate to obtain black quinoa active peptide filtrate, wherein the molecular weight cutoff for ultrafiltration is <5000 Da; S7. Spray dry the black quinoa active peptide filtrate to obtain black quinoa active peptide powder.

[0023] The alkaline protease is Bacillus licheniformis alkaline protease with an enzyme activity of 400,000 U / g; the pepsin has an enzyme activity of 80,000 U / g.

[0024] This invention provides the following Examples 1-3 for preparing a black quinoa active peptide with the activities of promoting hair follicle cell growth and hair growth and conditioning: Example 1 S1. Add black quinoa to a grinder and grind it. Then pass it through a 30-mesh sieve to obtain black quinoa powder. S2. First pretreatment: Mix 300g of black quinoa flour with 3000g of water evenly, adjust the system temperature to 50℃, adjust the pH to 7.0, add 24g of amylase, enzymatically hydrolyze for 1h, and then inactivate the enzyme at 85℃ for 20min to obtain the first pretreatment mixture. S3. Secondary pretreatment: The temperature of the mixture from the first pretreatment was lowered to 50℃, the pH was adjusted to 10.0 with NaOH solution, and the mixture was extracted for 1 hour. After centrifugation at 5000 rpm for 10 minutes, the supernatant was collected, the pH was adjusted to 4.0 with HCl solution, and the mixture was placed at 4℃ for 1 hour to precipitate. After centrifugation at 5000 rpm for 10 minutes, black quinoa protein precipitate was obtained. S4. First hydrolysis: Mix black quinoa protein precipitate with pure water at a mass ratio of 1:2, adjust the system temperature to 55℃, adjust the pH to 8.5, add 0.45g alkaline protease, hydrolyze for 2h, and then inactivate the enzyme at 85℃ for 20min to obtain the first hydrolysate. S5. Secondary hydrolysis: The temperature of the first hydrolysate was lowered to 37℃, the pH was adjusted to 2.5, 0.75g of pepsin was added, and the hydrolysis was carried out for 2 hours. Then, the pH was adjusted to 4.0 with HCl solution, and the enzyme was inactivated at 85℃ for 20 minutes to obtain the second hydrolysate. S6. Filter and ultrafilter the second hydrolysate to obtain black quinoa active peptide filtrate, wherein the molecular weight cutoff for ultrafiltration is <5000 Da; S7. Spray dry the black quinoa active peptide filtrate to obtain black quinoa active peptide powder.

[0025] Example 2 S1. Add black quinoa to a grinder and grind it. Then pass it through a 30-mesh sieve to obtain black quinoa powder. S2. First pretreatment: Mix 300g of black quinoa flour with 2400g of water evenly, adjust the system temperature to 45℃, adjust the pH to 7.0, add 21g of amylase, enzymatically hydrolyze for 1.5h, and then inactivate the enzyme at 85℃ for 20min to obtain the first pretreatment mixture. S3. Secondary pretreatment: The temperature of the mixture from the first pretreatment was lowered to 50℃, the pH was adjusted to 9.5 with NaOH solution, and the mixture was extracted for 1.5h. After centrifugation at 5000rpm for 10min, the supernatant was collected, the pH was adjusted to 3.8 with HCl solution, and the mixture was placed at 4℃ for 0.5h to precipitate. After centrifugation at 4000rpm for 10min, black quinoa protein precipitate was obtained. S4. First hydrolysis: Mix black quinoa protein precipitate with pure water at a mass ratio of 1:2, adjust the system temperature to 50℃, adjust the pH to 9.0, add 0.30g alkaline protease, hydrolyze for 3h, and then inactivate the enzyme at 85℃ for 15min to obtain the first hydrolysate. S5. Secondary hydrolysis: The temperature of the first hydrolysate was lowered to 37℃, the pH was adjusted to 2.0, 0.60g of pepsin was added, and the hydrolysate was carried out for 3 hours. Then the pH was adjusted to 4.0 with HCl solution, and the enzyme was inactivated at 85℃ for 15 minutes to obtain the second hydrolysate. S6. Filter and ultrafilter the second hydrolysate to obtain black quinoa active peptide filtrate, wherein the molecular weight cutoff for ultrafiltration is <5000 Da; S7. Spray dry the black quinoa active peptide filtrate to obtain black quinoa active peptide powder.

[0026] Example 3 S1. Add black quinoa to a grinder and grind it. Then pass it through a 30-mesh sieve to obtain black quinoa powder. S2. First pretreatment: Mix 300g of black quinoa flour with 3600g of water evenly, adjust the system temperature to 55℃, adjust the pH to 7.0, add 27g of amylase, enzymatically hydrolyze for 0.5h, and then inactivate the enzyme at 90℃ for 15min to obtain the first pretreatment mixture. S3. Secondary pretreatment: The temperature of the mixture from the first pretreatment was lowered to 50℃, the pH was adjusted to 10.5 with NaOH solution, and the mixture was extracted for 0.5h. After centrifugation at 5000rpm for 10min, the supernatant was collected, the pH was adjusted to 4.2 with HCl solution, and the mixture was placed at 4℃ for 1.5h to precipitate. After centrifugation at 6000rpm for 5min, black quinoa protein precipitate was obtained. S4. First hydrolysis: Mix black quinoa protein precipitate with pure water at a mass ratio of 1:2, adjust the system temperature to 55℃, adjust the pH to 8.0, add 0.60g alkaline protease, hydrolyze for 1h, and then inactivate the enzyme at 85℃ for 20min to obtain the first hydrolysate. S5. Secondary hydrolysis: The temperature of the first hydrolysate was lowered to 37℃, the pH was adjusted to 3.0, 0.90g of pepsin was added, and the hydrolysate was carried out for 1 hour. Then the pH was adjusted to 4.2 with HCl solution, and the enzyme was inactivated at 85℃ for 20 minutes to obtain the second hydrolysate. S6. Filter and ultrafilter the second hydrolysate to obtain black quinoa active peptide filtrate, wherein the molecular weight cutoff for ultrafiltration is <5000 Da; S7. Spray dry the black quinoa active peptide filtrate to obtain black quinoa active peptide powder.

[0027] The present invention also provides the following comparative examples 1 to 9: Comparative Example 1 This comparative example prepared a black quinoa active peptide, which differs from Example 1 of the present invention in that: in step S5, 0.75g of pepsin was replaced with 0.60g of fig protease, the pH was adjusted to 5.7, and the enzymatic hydrolysis temperature was 65℃. All other components, ratios, operating steps, and process parameters remained consistent with Example 1.

[0028] It should be noted that, based on the protein content of black quinoa, the total amount of Bacillus licheniformis alkaline protease and fig protease used in this comparative example is equal to the total amount of Bacillus licheniformis alkaline protease and pepsin used in Example 1. The specific calculation process is as follows: The amount of black quinoa flour added is 300g, and the protein content is 10%, which means that 300g of black quinoa flour contains 30g of black quinoa protein.

[0029] In Example 1, the amount of Bacillus licheniformis alkaline protease added was 0.45g, with an enzyme activity of 400,000 U / g. Converted to per unit weight of black quinoa protein, this corresponds to an enzyme activity of 400,000 U / g × 0.45g / 30g = 6000 U / g. Similarly, the amount of pepsin added was 0.75g, with an enzyme activity of 80,000 U / g. Converted to per unit weight of black quinoa protein, this corresponds to an enzyme activity of 80,000 U / g × 0.75g / 30g = 2000 U / g. Therefore, in Example 1, based on the black quinoa protein content, the total amount of Bacillus licheniformis alkaline protease and pepsin used was 8000 U / g.

[0030] In Comparative Example 1, the amount of Bacillus licheniformis alkaline protease added was 0.45g. Based on the protein content of black quinoa, the amount of Bacillus licheniformis alkaline protease was 400,000 U / g × 0.45g / 30g = 6000 U / g. The amount of fig protease added was 0.60g, with an enzyme activity of 100,000 U / g. Based on the protein content of black quinoa, the amount of fig protease was 100,000 U / g × 0.60g / 30g = 2000 U / g. Therefore, in Comparative Example 1, based on the protein content of black quinoa, the total amount of Bacillus licheniformis alkaline protease and fig protease was 8000 U / g, which is the same as the total amount of Bacillus licheniformis alkaline protease and pepsin used in Example 1.

[0031] Comparative Example 2 This comparative example prepared a black quinoa active peptide, which differs from Example 1 of the present invention in that: in step S5, 0.75g of pepsin was replaced with 0.24g of trypsin, the pH was adjusted to 7.5, and the enzymatic hydrolysis temperature was 50℃. All other components, ratios, operating steps, and process parameters remained consistent with Example 1.

[0032] It should be noted that, based on the protein content of black quinoa, the total amount of Bacillus licheniformis alkaline protease and trypsin used in this comparative example is equal to the total amount of Bacillus licheniformis alkaline protease and pepsin used in Example 1. The specific calculation process is as follows: The amount of black quinoa flour added is 300g, and the protein content is 10%, which means that 300g of black quinoa flour contains 30g of black quinoa protein.

[0033] In Example 1, based on the protein content of black quinoa, the total amount of Bacillus licheniformis alkaline protease and pepsin used was 8000 U / g.

[0034] In Comparative Example 2, the amount of Bacillus licheniformis alkaline protease added was 0.45g. Based on the protein content of black quinoa, the amount of Bacillus licheniformis alkaline protease was 400,000 U / g × 0.45g / 30g = 6000 U / g. The amount of trypsin added was 0.24g, with an enzyme activity of 250,000 U / g. Based on the protein content of black quinoa, the amount of trypsin was 250,000 U / g × 0.24g / 30g = 2000 U / g. Therefore, in Comparative Example 2, based on the protein content of black quinoa, the total amount of Bacillus licheniformis alkaline protease and trypsin was 8000 U / g, which is the same as the total amount of Bacillus licheniformis alkaline protease and pepsin used in Example 1.

[0035] Comparative Example 3 This comparative example prepared a black quinoa active peptide, which differs from Example 1 of the present invention in that: in step S4, the amount of Bacillus licheniformis alkaline protease added was changed from 0.45g to 0.375g; in step S5, 0.75g of pepsin was replaced with 0.60g of proline endopeptidase, the pH was adjusted to 5.0, and the enzymatic hydrolysis temperature was 60℃. All other components, ratios, operating steps, and process parameters remained consistent with Example 1.

[0036] It should be noted that, based on the protein content of black quinoa, the total amount of Bacillus licheniformis alkaline protease and proline endopeptidase used in this comparative example is equal to the total amount of Bacillus licheniformis alkaline protease and pepsin used in Example 1. The specific calculation process is as follows: The amount of black quinoa flour added is 300g, and the protein content is 10%, which means that 300g of black quinoa flour contains 30g of black quinoa protein.

[0037] In Example 1, based on the protein content of black quinoa, the total amount of Bacillus licheniformis alkaline protease and pepsin used was 8000 U / g.

[0038] In Comparative Example 3, the amount of Bacillus licheniformis alkaline protease added was 0.375g. Based on the protein content of black quinoa, the amount of Bacillus licheniformis alkaline protease was 400,000 U / g × 0.375g / 30g = 5000 U / g. The amount of proline endonuclease added was 0.60g, with an enzyme activity of 150,000 U / g. Based on the protein content of black quinoa, the amount of proline endonuclease was 150,000 U / g × 0.60g / 30g = 3000 U / g. Therefore, in Comparative Example 3, based on the protein content of black quinoa, the total amount of Bacillus licheniformis alkaline protease and proline endonuclease was 8000 U / g, which is the same as the total amount of Bacillus licheniformis alkaline protease and pepsin used in Example 1.

[0039] Comparative Example 4 This comparative example prepared a black quinoa active peptide, which differs from Example 1 of the present invention in that: in step S4, the amount of Bacillus licheniformis alkaline protease added was changed from 0.45g to 0.15g; in step S5, 0.75g of pepsin was replaced with 0.30g of acidic protease, the pH was adjusted to 2.5, and the enzymatic hydrolysis temperature was 37℃. All other components, ratios, operating steps, and process parameters remained consistent with Example 1.

[0040] It should be noted that, based on the protein content of black quinoa, the total amount of Bacillus licheniformis alkaline protease and acidic protease used in this comparative example is equal to the total amount of Bacillus licheniformis alkaline protease and pepsin used in Example 1. The specific calculation process is as follows: The amount of black quinoa flour added is 300g, and the protein content is 10%, which means that 300g of black quinoa flour contains 30g of black quinoa protein.

[0041] In Example 1, based on the protein content of black quinoa, the total amount of Bacillus licheniformis alkaline protease and pepsin used was 8000 U / g.

[0042] In Comparative Example 4, the amount of Bacillus licheniformis alkaline protease added was 0.15g. Based on the protein content of black quinoa, the amount of Bacillus licheniformis alkaline protease was 400,000 U / g × 0.15g / 30g = 2000 U / g. The amount of acidic protease added was 0.30g, with an enzyme activity of 600,000 U / g. Based on the protein content of black quinoa, the amount of acidic protease was 600,000 U / g × 0.30g / 30g = 6000 U / g. Therefore, in Comparative Example 4, based on the protein content of black quinoa, the total amount of Bacillus licheniformis alkaline protease and acidic protease was 8000 U / g, which is the same as the total amount of Bacillus licheniformis alkaline protease and pepsin used in Example 1.

[0043] Comparative Example 5 This comparative example prepared a black quinoa active peptide, which differs from Example 1 of the present invention in that: in step S5, 0.75g of pepsin was replaced with 0.30g of papain, the pH was adjusted to 6.5, and the enzymatic hydrolysis temperature was 55℃. All other components, ratios, operating steps, and process parameters remained consistent with Example 1.

[0044] It should be noted that, based on the protein content of black quinoa, the total amount of Bacillus licheniformis alkaline protease and papain used in this comparative example is equal to the total amount of Bacillus licheniformis alkaline protease and pepsin used in Example 1. The specific calculation process is as follows: The amount of black quinoa flour added is 300g, and the protein content is 10%, which means that 300g of black quinoa flour contains 30g of black quinoa protein.

[0045] In Example 1, based on the protein content of black quinoa, the total amount of Bacillus licheniformis alkaline protease and pepsin used was 8000 U / g.

[0046] In Comparative Example 5, the amount of Bacillus licheniformis alkaline protease added was 0.45g. Based on the protein content of black quinoa, the amount of Bacillus licheniformis alkaline protease was 400,000 U / g × 0.45g / 30g = 6000 U / g. The amount of papain added was 0.30g, with an enzyme activity of 200,000 U / g. Based on the protein content of black quinoa, the amount of papain was 200,000 U / g × 0.30g / 30g = 2000 U / g. Therefore, in Comparative Example 5, based on the protein content of black quinoa, the total amount of Bacillus licheniformis alkaline protease and papain was 8000 U / g, which is the same as the total amount of Bacillus licheniformis alkaline protease and pepsin used in Example 1.

[0047] Comparative Example 6 This comparative example prepared a black quinoa bioactive peptide, which differs from Example 1 of the present invention in that: in step S4, the amount of Bacillus licheniformis alkaline protease added was changed from 0.45g to 0.4125g; in step S5, 0.75g of pepsin was replaced with 0.15g of neutral protease, the pH was adjusted to 6.5, and the enzymatic hydrolysis temperature was 55℃. All other components, ratios, operating steps, and process parameters remained consistent with Example 1.

[0048] It should be noted that, based on the protein content of black quinoa, the total amount of Bacillus licheniformis alkaline protease and neutral protease used in this comparative example is equal to the total amount of Bacillus licheniformis alkaline protease and pepsin used in Example 1. The specific calculation process is as follows: The amount of black quinoa flour added is 300g, and the protein content is 10%, which means that 300g of black quinoa flour contains 30g of black quinoa protein.

[0049] In Example 1, based on the protein content of black quinoa, the total amount of Bacillus licheniformis alkaline protease and pepsin used was 8000 U / g.

[0050] In Comparative Example 6, the amount of Bacillus licheniformis alkaline protease added was 0.4125g. Based on the protein content of black quinoa, the amount of Bacillus licheniformis alkaline protease was 400,000 U / g × 0.4125g / 30g = 5500 U / g. The amount of neutral protease added was 0.15g, with an enzyme activity of 500,000 U / g. Based on the protein content of black quinoa, the amount of neutral protease was 500,000 U / g × 0.15g / 30g = 2500 U / g. Therefore, in Comparative Example 6, based on the protein content of black quinoa, the total amount of Bacillus licheniformis alkaline protease and neutral protease was 8000 U / g, which is the same as the total amount of Bacillus licheniformis alkaline protease and pepsin used in Example 1.

[0051] Comparative Example 7 This comparative example prepared a black quinoa active peptide, which differs from Example 1 of the present invention in that: in step S4, the amount of Bacillus licheniformis alkaline protease added was changed from 0.45g to 0.60g; and step S5 was omitted. All other components, ratios, operating steps, and process parameters remained the same as in Example 1.

[0052] It should be noted that, based on the protein content of black quinoa, the amount of Bacillus licheniformis alkaline protease used in this comparative example is equal to the total amount of Bacillus licheniformis alkaline protease and pepsin used in Example 1. The specific calculation process is as follows: The amount of black quinoa flour added is 300g, and the protein content is 10%, which means that 300g of black quinoa flour contains 30g of black quinoa protein.

[0053] In Example 1, based on the protein content of black quinoa, the total amount of Bacillus licheniformis alkaline protease and pepsin used was 8000 U / g.

[0054] In Comparative Example 7, the amount of Bacillus licheniformis alkaline protease added was 0.60g. Based on the protein content of black quinoa, the amount of Bacillus licheniformis alkaline protease used was 400,000 U / g × 0.60g / 30g = 8,000 U / g, which is the same as the total amount of Bacillus licheniformis alkaline protease and pepsin used in Example 1.

[0055] Comparative Example 8 This comparative example prepared a black quinoa active peptide, which differs from Example 1 of the present invention in that: in step S5, 0.75g of pepsin was replaced with 0.30g of bromelain, the pH was adjusted to 6.8, and the enzymatic hydrolysis temperature was 45℃. All other components, ratios, operating steps, and process parameters remained consistent with Example 1.

[0056] It should be noted that, based on the protein content of black quinoa, the total amount of Bacillus licheniformis alkaline protease and bromelain used in this comparative example is equal to the total amount of Bacillus licheniformis alkaline protease and pepsin used in Example 1. The specific calculation process is as follows: The amount of black quinoa flour added is 300g, and the protein content is 10%, which means that 300g of black quinoa flour contains 30g of black quinoa protein.

[0057] In Example 1, based on the protein content of black quinoa, the total amount of Bacillus licheniformis alkaline protease and pepsin used was 8000 U / g.

[0058] In Comparative Example 8, the amount of Bacillus licheniformis alkaline protease added was 0.45g. Based on the protein content of black quinoa, the amount of Bacillus licheniformis alkaline protease was 400,000 U / g × 0.45g / 30g = 6000 U / g. The amount of bromelain added was 0.30g, with an enzyme activity of 200,000 U / g. Based on the protein content of black quinoa, the amount of bromelain was 200,000 U / g × 0.30g / 30g = 2000 U / g. Therefore, in Comparative Example 8, based on the protein content of black quinoa, the total amount of Bacillus licheniformis alkaline protease and bromelain was 8000 U / g, which is the same as the total amount of Bacillus licheniformis alkaline protease and pepsin used in Example 1.

[0059] Comparative Example 9 This comparative example prepared a black quinoa active peptide, which differs from Example 1 of the present invention in that: in step S5, 0.75g of pepsin was replaced with 0.60g of compound enzyme preparation, the pH was adjusted to 7.0, and the enzymatic hydrolysis temperature was 55℃. All other components, ratios, operating steps, and process parameters remained consistent with Example 1.

[0060] It should be noted that, based on the protein content of black quinoa, the total amount of Bacillus licheniformis alkaline protease and compound enzyme preparation used in this comparative example is equal to the total amount of Bacillus licheniformis alkaline protease and pepsin used in Example 1. The specific calculation process is as follows: The amount of black quinoa flour added is 300g, and the protein content is 10%, which means that 300g of black quinoa flour contains 30g of black quinoa protein.

[0061] In Example 1, based on the protein content of black quinoa, the total amount of Bacillus licheniformis alkaline protease and pepsin used was 8000 U / g.

[0062] In Comparative Example 9, the amount of Bacillus licheniformis alkaline protease added was 0.45g. Based on the protein content of black quinoa, the amount of Bacillus licheniformis alkaline protease was 400,000 U / g × 0.45g / 30g = 6000 U / g. The amount of the compound enzyme preparation added was 0.60g, with an enzyme activity of 100,000 U / g. Based on the protein content of black quinoa, the amount of the compound enzyme preparation was 100,000 U / g × 0.60g / 30g = 2000 U / g. Therefore, in Comparative Example 9, based on the protein content of black quinoa, the total amount of Bacillus licheniformis alkaline protease and the compound enzyme preparation was 8000 U / g, which is the same as the total amount of Bacillus licheniformis alkaline protease and pepsin used in Example 1.

[0063] Based on the above embodiments and comparative examples, the present invention also conducted the following experiments: Experiment 1: Determination of 5α-reductase inhibition rate Androgenetic alopecia (AGA), also known as seborrheic alopecia, is one of the most common types of hair loss in clinical practice. Modern medicine believes that high levels of dihydrotestosterone (DHT), which binds to androgen receptors and affects cellular processes, are a key factor leading to androgenetic alopecia. 5α-reductase is a crucial enzyme in the conversion of testosterone to DHT; therefore, inhibiting 5α-reductase activity is one of the important means of preventing androgenetic alopecia.

[0064] The black quinoa active peptides obtained in Examples 1-3 and Comparative Examples 1-9 were prepared into 25 mg / mL peptide solutions for 5α-reductase inhibition rate determination. The 5α-reductase inhibition rate was determined using the type II (SRD5A2) 5α-reductase inhibition rate assessment kit. The determination data are summarized in Table 1. Table 1. Data on the inhibition rate of 5α-reductase by active peptides from black quinoa.

[0065] According to the data in Table 1, the black quinoa active peptides prepared in Examples 1-3 and Comparative Examples 1-9 all have a certain 5α-reductase inhibitory ability; and the 5α-reductase inhibitory ability of Examples 1-3 is significantly higher than that of Comparative Examples 1-9. Among them, the black quinoa active peptide prepared in Example 1 has the highest 5α-reductase inhibition rate, which is 62.82%.

[0066] Experiment 2: Test on the hair growth and hair care capabilities of black quinoa active peptides 2.1 Cytotoxicity assay Cell viability was determined using the CCK8 assay (Cell Counting Kit-8). Experimental groups were as follows: (1) Experimental group: Complete culture medium containing black quinoa active peptide solutions of 0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL and 2.0 mg / mL respectively; (2) Positive control group: complete culture medium containing minoxidil at concentrations of 0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL, and 2.0 mg / mL, respectively; (3) Control group: Complete culture medium with fresh culture medium.

[0067] The results, through significance analysis, selected the highest non-toxic concentration of 2 mg / mL as the cell drug concentration.

[0068] 2.2 Determination of expression levels of key genes in human dermal papilla cells The CTNNB1 gene encodes β-catenin, a core transcriptional regulator in the Wnt signaling pathway, which plays a crucial role in hair follicle development, hair life cycle regulation, and stem cell activity. Recent studies have shown that abnormal expression and function of the CTNNB1 gene are directly associated with hair follicle degeneration and hair loss; therefore, activation of this gene-mediated pathway is considered an important potential target for promoting hair growth.

[0069] The core characteristic of the classic Wnt signaling pathway is Wnt protein-induced β-catenin-dependent transcriptional activation, and the regulatory role of this mechanism has been verified in relevant animal experiments. Xie et al. demonstrated that the Wnt signaling pathway can participate in regulating feather follicle development and feather growth during chick embryonic development by injecting different doses of the Wnt signaling pathway inhibitor DKK1 into fertilized eggs. In addition, the Wnt5b gene also has specific germinal regulation functions, mainly by regulating hair follicle cell migration, differentiation, and hair follicle morphogenesis, activating hair follicle stem cell activity, and inducing hair follicles to smoothly enter the growth phase from the resting phase.

[0070] In addition to the Wnt signaling pathway, a core regulatory network, insulin-like growth factor-I (IGF-I) is also a key regulator of the hair follicle anagen phase. As one of the core factors maintaining the hair follicle anagen phase, IGF-I can regulate hair follicle cell activity and promote local angiogenesis through multiple synergistic pathways, and form an interactive regulatory network with other signaling pathways to jointly ensure the normal growth and metabolism of hair follicles.

[0071] Based on the aforementioned Wnt / β-catenin signaling pathway and the core mechanism of IGF-I in regulating hair follicle growth, to further investigate whether the black quinoa active peptides provided by this invention can exert a hair growth-promoting effect by regulating the expression of these key genes, this invention selected Example 1, which showed a better inhibition rate of 5α-reductase, and Comparative Examples 2, 3, 4, and 9 for the following cell experiments: Cell seeding: Healthy human dermal papilla cells (hDPCs) were seeded into 12-well plates at a density of 50,000 cells / well and cultured in a 37°C, 5% CO2 incubator for 24 hours.

[0072] Drug incubation: Before adding the drugs, prepare complete culture media containing the optimal concentration of black quinoa active peptides prepared in Example 1 and Comparative Examples 2, 3, 4 and 9 respectively, shake well and set aside; discard the original culture media in the well plate, and add complete culture media containing the corresponding samples to the experimental groups for 24 hours.

[0073] Key gene detection: After drug incubation, RNA was extracted, reverse transcribed, and Real-time qPCR was performed to detect key genes for hair growth. Primers were designed and synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the primer sequences are listed in Table 2 below; the GAPDH gene was used as an internal reference gene, with 3 replicates in each group; the relative expression level of each gene was calculated using 2-ΔΔCt.

[0074] Table 2 Primer sequence listing

[0075] Figure 1 To determine the expression of the CTNNB1 gene in hDPCs cells, based on Figure 1 It can be seen that the expression level of CTNNB1 gene in the experimental group was mostly higher than that in the NC group, indicating that the presence of black quinoa active peptides can upregulate the expression of CTNNB1 gene. Among them, the upregulation in Example 1 was the most obvious, with the relative expression level of CTNNB1 gene exceeding 5.0. Therefore, it can be inferred that the black quinoa active peptides prepared in Example 1 of this invention can promote the nuclear accumulation of β-catenin and enhance its transcriptional activity by activating the CTNNB1 signaling pathway in human dermal papilla cells.

[0076] Figure 2 To determine the expression of the β-catenin gene in hDPCs cells, based on Figure 2 It can be seen that Example 1 significantly upregulated the expression level of the β-catenin gene, to approximately 4.7, while the upregulation of the β-catenin gene expression level in Comparative Examples 2, 3, and 9 was relatively small. Furthermore, the expression level of the β-catenin gene under Comparative Example 4 was not upregulated compared to the NC group. Therefore, it can be inferred that the black quinoa active peptide prepared in Example 1 of this invention can promote hair follicle growth by regulating the expression level of upstream genes, accumulating β-catenin in the cell nucleus, enhancing its interaction with transcription factors, initiating the transcription of downstream target genes, and thus promoting hair follicle growth.

[0077] Figure 3 To determine the expression status of the Wnt5b gene in hDPCs cells, based on Figure 3 It can be seen that Example 1 significantly upregulated the expression level of the Wnt5b gene and activated the Wnt / β-catenin signaling pathway to promote hair follicle growth, which is consistent with the effect of black quinoa active peptide on β-catenin gene expression.

[0078] Figure 4 To determine the expression of the IGF-I gene in hDPCs cells, based on Figure 4 It can be seen that Example 1 significantly upregulated the expression level of hair follicle growth factor IGF-I, promoting the survival and proliferation of human hair papilla cells.

[0079] In summary, the black quinoa active peptides provided by the technical solution of this invention can upregulate the expression levels of CTNNB1, β-catenin, Wnt5b and IGF-I genes, activate the Wnt / β-catenin signaling pathway, promote hair follicle structure repair, thereby promoting the growth and proliferation of hair follicle cells, and achieving the effect of hair growth and hair care.

[0080] 2.3 Determination of antioxidant gene expression levels Nuclear factor erythroid 2-associated factor 2 (Nrf2) is an important transcription factor, a basic leucine zipper protein, widely present in various cells and tissues. It is an important factor in the endogenous cellular defense mechanism in response to oxidative damage, playing a key role in regulating cellular oxidative stress, lipid peroxidation, and ferroptosis, and participating in the restoration of intracellular homeostasis.

[0081] Heme oxygenase-1 (HO-1) is an important downstream protein of Nrf2 and a crucial metabolic enzyme that plays a key role in regulating cellular antioxidant stress responses. Upon oxidative stress, Nrf2 migrates from the cytoplasm into the nucleus and binds to antioxidant response elements (ARES) in DNA, with heme oxygenase-1 being one of its target genes. Through binding to ARES, Nrf2 regulates the transcription and expression of HO-1, thereby enhancing the cell's antioxidant defense capabilities.

[0082] To clarify the regulatory role of black quinoa bioactive peptides on the expression levels of Nrf2 and HO-1, two key antioxidant genes, in human dermal papilla cells, and to verify whether they exert their antioxidant effect by activating the Nrf2 / HO-1 pathway, this invention conducted gene expression level detection through in vitro cell experiments. The specific steps are as follows: Cell seeding: Healthy human dermal papilla cells (hDPCs) were seeded into 12-well plates at a density of 50,000 cells / well and cultured in a 37°C, 5% CO2 incubator for 24 hours.

[0083] Drug incubation: Before adding the drugs, prepare complete culture media containing the optimal concentration of black quinoa active polypeptides prepared in Example 1 and Comparative Examples 2, 3, 4 and 9 respectively, shake well and set aside; discard the original culture media in the well plate, and add complete culture media containing the corresponding samples to the experimental groups for 24 hours.

[0084] Key gene detection: After drug incubation, RNA was extracted, reverse transcribed, and Real-time qPCR was used to detect the expression of antioxidant genes. Primers were designed and synthesized by Sangon Biotech (Shanghai) Co., Ltd.; the GAPDH gene was used as an internal reference gene, with three replicates in each group; the relative expression level of each gene was calculated using 2-ΔΔCt.

[0085] Table 3 Primer sequence listing

[0086] Figure 5 This indicates the expression status of the Nrf2 gene in hDPCs cells. Figure 6 The expression status of the HO-1 gene in hDPCs cells.

[0087] according to Figure 5 It can be seen that the relative expression level of the Nrf2 gene under the treatment with the black quinoa active peptides prepared in Example 1 was significantly upregulated to about 7.0, showing a significant difference compared with the NC group and each control group; and according to Figure 6 It can be seen that the relative expression level of the HO-1 gene under the action of the black quinoa active peptide prepared in Example 1 increased significantly to about 5.5. Among the comparative examples, only the relative expression level of the HO-1 gene in Comparative Example 4 was slightly increased to about 1.5, but it was much lower than that in Example 1. The relative expression level of the HO-1 gene in Comparative Examples 2 and 3 was not significantly different from that in the NC group, and the expression level in Comparative Example 9 was further reduced.

[0088] In summary, it can be inferred that black quinoa bioactive peptides can not only significantly upregulate the expression of the upstream transcription factor Nrf2, but also simultaneously increase the expression level of its downstream target protein HO-1. This means that black quinoa bioactive peptides can effectively activate the Nrf2 antioxidant signaling pathway, thereby enhancing the antioxidant defense capacity of human dermal papilla cells, reducing the damage to hair follicle cells caused by oxidative stress (such as lipid peroxidation, ferroptosis, etc.), and thus maintaining the normal function of dermal papilla cells, providing a favorable cellular microenvironment for hair growth, and ultimately playing a role in promoting hair growth.

[0089] Experiment 3 Screening of bioactive peptide fragments from black quinoa In this experiment, the active peptides of black quinoa prepared in Example 1 were screened and analyzed.

[0090] 3.1 Mass Spectrometry Experimental Methods (1) Preprocessing In this experiment, the active peptides of black quinoa were pretreated by reductive alkylation. The specific steps are as follows: Weigh 3 mg of the black quinoa active peptide powder prepared in Example 1, add it to a centrifuge tube, and then use a pipette to transfer 300 µL of 50 mmol / L NH4HCO3 solution of black quinoa active peptide powder, and shake to mix thoroughly. Accurately transfer 200µL of sample into a 10kDa ultrafiltration tube using a pipette, and centrifuge at 4℃ and 12000rpm for 10min. Add 200 μL of water, centrifuge at 4℃ and 12000 rpm for 10 min, repeat the washing 3 times, take the portion less than 10 kDa, and retain the portion greater than 10 kDa; Use a pipette to transfer 100 μg of a solution less than 10 kDa into a 1.5 mL centrifuge tube, and use a pipette to add 50 mmol / L NH4HCO3 to make up the volume to 100 μL for subsequent experiments; Use a pipette to add 1 μL of 1 mmol / L DTT solution to the sample to make the final DTT concentration 10 mmol / L, and reduce it in a water bath at 56 °C for 1 h; Use a pipette to transfer 2 μL of 1 mmol / L IAM solution into the sample to make the final IAM concentration 20 mmol / L, and react in the dark at room temperature for 40 min. Use a pipette to add 1 μL of 1 mmol / L DTT solution to the sample to bring the final DTT concentration to 10 mmol / L, in order to neutralize unreacted IAM.

[0091] (2) C18 desalination (Stage-Tip) Desalting was performed using C18 stage-tip, followed by vacuum drying at 45°C.

[0092] 3.2 Computer Usage Requirements The processed black quinoa active peptides were detected using liquid chromatography-mass spectrometry.

[0093] (1) Liquid chromatography conditions 1) Pre-column: 150 μm d. × 50 mm, packing: Reprosil-Pur120C18-AQ 3 μm; Analytical column: 150 μm d. × 170 mm, packing: Reprosil-Pur120C18-AQ 1.9 μm; 2) Mobile phase A: 0.1% FA; 3) Mobile phase B: 0.1% FA, 80% ACN; 4) Flow rate: 600 nL / min; 5) Analysis time for each component: 66 min; 6) Specific chromatographic conditions are listed in Table 4: Table 4 Specific chromatographic conditions for liquid chromatography

[0094] (2) Mass spectrometry conditions Level 1 mass spectrometry parameters: 1) Resolution: 70,000; 2) AGCtarget: 3e6; 3) MaximumIT: 100 ms; 4) Scanrange: 100to1500 m / z.

[0095] Secondary mass spectrometry parameters: 1) Resolution: 17,500; 2) AGCtarget: 1e5; 3) MaximumIT: 50 ms; 4) TopN: 20; 5) NCE / steppedNCE: 28.

[0096] Raw data was obtained through mass spectrometry.

[0097] 3.3 Search Conditions The raw mass spectrometry files were searched using software to retrieve the target protein database. The search parameters were as follows: 1) Fixed modifications: Carbamidomethyl(C); 2) Variable modifications: Oxidation (M), Acetyl (Peptide N-term); 3) Enzyme: Nonspecific; 4) Database: uniprotkb_Acipenser(genus)_2024_11_13; uniprotkb_Oreochromis(genus)_2024_12_05; 5) Peptide Mass Tolerance: 20 ppm; 6) Secondary mass spectrometry bias (FragmentMassTolerance): 0.02 Da.

[0098] 3.4 Peptide screening and molecular docking Raw files acquired by mass spectrometry were searched for peptides with an abundance greater than 2,700,000,000. After molecular docking, 15 peptides were obtained. A search of the BIOPEP peptide database revealed that all 15 peptides were novel and previously unreported, and all were predicted to be non-toxic. Peptide-related information is listed in Table 5. Simultaneously, molecular docking software was used to perform molecular docking of these 15 peptides with 5α-reductase (PDB: 7BW1) and GSK-3β (PDB: 1R0E) receptors. The results are summarized in Table 5.

[0099] Table 5. Peptide information and receptor binding energy

[0100] 5α-reductase is a key membrane-bound enzyme that plays a crucial role in steroid hormone metabolism. Its primary function is to catalyze the conversion of testosterone to dihydrotestosterone (DHT), which causes hair follicle miniaturization and atrophy. Therefore, 5α-reductase is an important target for hair care, and inhibiting 5α-reductase is a vital pathway for preventing hair loss.

[0101] Glycogen synthase kinase-3β (GSK-3β) is key to regulating hair follicle cell activity and signaling pathways. This pathway is the "start switch" for hair follicles to enter the growth phase from the resting phase. In the absence of Wnt signal, GSK-3β phosphorylates β-catenin, leading to its degradation and affecting hair follicle growth. Therefore, GSK-3β is a key regulatory target for the treatment of hair loss.

[0102] Peptides 1-5 and 7-15 bind well to the 5α-reductase receptor, indicating that, combined with previous experimental results, these peptides can inhibit the action of 5α-reductase after binding, thereby slowing down hair loss. All 15 peptides can bind to the GSK-3β receptor, indicating that, combined with previous experimental results, these peptides activate related signaling pathways after binding to GSK-3β, thus promoting hair follicle regeneration.

[0103] Based on binding energy and peptide abundance, seven sequences, LLPH, VVVPQ, IF, LLPS, LLL, TYLV, and LVL, were selected as characteristic peptide sequences in black quinoa active peptides that promote hair follicle cell growth and hair growth and protection.

[0104] More specifically, such as Figure 7 As shown, VVVPQ interacts with 5α-reductase via hydrophobic interactions and covalent hydrogen bonds, and with GSK-3β via hydrophobic interactions, covalent hydrogen bonds, and salt bridges; Figure 8 As shown, LLL binds to 5α-reductase via hydrophobic interactions, covalent hydrogen bonds, and salt bridges, and binds to GSK-3β via hydrophobic interactions, covalent hydrogen bonds, and salt bridges; as Figure 9 As shown, LVL binds to 5α-reductase via hydrophobic interactions, covalent hydrogen bonds, and salt bridges, and to GSK-3β via hydrophobic interactions and salt bridges; Figure 10 As shown, LLPH binds to 5α-reductase via hydrophobic interactions and covalent hydrogen bonds, and also binds to GSK-3β via hydrophobic interactions and covalent hydrogen bonds; Figure 11As shown, LLPS binds to 5α-reductase via hydrophobic interactions and covalent hydrogen bonds, and to GSK-3β via hydrophobic interactions, covalent hydrogen bonds, and salt bridges; Figure 12 As shown, TYLV binds to 5α-reductase via hydrophobic interactions and covalent hydrogen bonds, and binds to GSK-3β via hydrophobic interactions and covalent hydrogen bonds; as Figure 13 As shown, IF binds to 5α-reductase via hydrophobic interactions, covalent hydrogen bonds, and salt bridges, and also binds to GSK-3β via hydrophobic interactions, covalent hydrogen bonds, and salt bridges.

[0105] It should also be noted that the terms used in this invention, such as ultrafiltration, centrifugation, spray drying, mass spectrometry, molecular docking, and RNA extraction, are names of processing steps commonly used by those skilled in the art, and their names can accurately describe their processing procedures, so they will not be repeated in this invention.

[0106] The raw materials used in this invention, such as alkaline protease (400,000 U / g), pepsin (80,000 U / g), fig protease (100,000 U / g), trypsin (250,000 U / g), proline endonuclease (150,000 U / g), acidic protease (150,000 U / g), papain (400,000 U / g), neutral protease (80,000 U / g), bromelain (200,000 U / g), and compound enzyme preparations (100,000 U / g), are all commercially available enzymes that can be purchased and obtained by those skilled in the art. In addition, unless otherwise specified, other raw materials used in this invention can also be conventional commercial products in the field or prepared by conventional methods in the field. That is, reagents, instruments, and consumables used in this invention, unless otherwise specified, can also be purchased from the market.

[0107] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing black quinoa bioactive peptides, characterized in that, Includes the following steps: S1. Add black quinoa to a grinder and grind it, then pass it through a 20-40 mesh sieve to obtain black quinoa flour; S2. First pretreatment: Mix black quinoa flour and water at a mass ratio of 1:(8~12) evenly, adjust the system temperature to 45~55℃, add amylase, enzymatically hydrolyze for 0.5~1.5h, and obtain the first pretreatment mixture after enzyme inactivation. S3. Secondary pretreatment: Adjust the temperature of the pretreatment mixture to 50~55℃ and the pH to 9.5~10.5, extract for 0.5~1.5h, centrifuge to collect the supernatant, adjust the pH to 3.8~4.2, precipitate at 4℃ for 0.5~1.5h, centrifuge again to obtain black quinoa protein precipitate. S4. First hydrolysis: Mix black quinoa protein precipitate with pure water at a mass ratio of 1:(2~3), adjust the system temperature to 50~55℃, add alkaline protease, hydrolyze for 1~3 hours, and obtain the first hydrolysate after enzyme inactivation. S5. Secondary hydrolysis: Adjust the temperature of the first hydrolysate to (37±2)℃, add pepsin, hydrolyze for 1~3 h, adjust the pH to 3.8~4.2, and obtain the second hydrolysate after enzyme inactivation; S6. Filter and ultrafilter the second hydrolysate to obtain black quinoa active peptide filtrate, wherein the molecular weight cutoff for ultrafiltration is <5000 Da.

2. The method for preparing black quinoa active peptides according to claim 1, characterized in that: The black quinoa active peptide filtrate was spray-dried to obtain black quinoa active peptide powder.

3. The method for preparing black quinoa active peptides according to claim 1, characterized in that: In step S2, the amount of amylase added is 7.0% to 9.0% of the mass of black quinoa flour, and the pH of the enzymatic hydrolysis system is 7.

0. In step S4, the amount of alkaline protease added is 0.1% to 0.2% of the mass of black quinoa flour, and the pH of the enzymatic hydrolysis system is 8.0 to 9.0; In step S5, the amount of pepsin added is 0.2% to 0.3% of the mass of black quinoa flour, and the pH of the enzymatic hydrolysis system is 2.0 to 3.

0.

4. The method for preparing black quinoa active peptides according to claim 1, characterized in that: The enzyme inactivation treatment temperature is 85~90℃, and the treatment time is 15~20min.

5. The method for preparing black quinoa active peptides according to claim 1, characterized in that: The centrifugation is performed at 4000~6000 rpm for 5~10 minutes.

6. A black quinoa bioactive peptide, characterized in that: The black quinoa active peptide was prepared according to the preparation method described in any one of claims 1 to 5.

7. The black quinoa active peptide according to claim 6, characterized in that: Including any one or more amino acid sequences from Val-Val-Val-Pro-Gln (VVVPQ), Leu-Leu-Pro-Ser (LLPS), Leu-Leu-Pro-His (LLPH), Thr-Tyr-Leu-Val (TYLV), Leu-Leu-Leu (LLL), Leu-Val-Leu (LVL), and Ile-Phe (IF).

8. A functional product, characterized in that: Its components include the black quinoa active peptide as described in claim 7.

9. The functional product according to claim 8, characterized in that, Includes at least one of the following functions: (1) Inhibits 5α-reductase; (2) Promotes hair follicle cell growth; (3) Antioxidant; (4) Promote hair growth and protect hair.

10. The use of the black quinoa active peptide as described in claim 7 in the preparation of a product for inhibiting 5α-reductase activity, inhibiting GSK-3β receptor activity, activating the Wnt / β-catenin signaling pathway, upregulating CTNNB1 gene expression, upregulating β-catenin gene expression, upregulating Wnt5b gene expression, upregulating IGF-I gene expression, activating the Nrf2 / HO-1 antioxidant signaling pathway, upregulating Nrf2 gene expression, and upregulating HO-1 gene expression.

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