Application of Xianglian protein peptide

Abstract

The present invention relates to the field of plant extraction technology and specifically provides the use of a Xianglian protein peptide. The present invention provides the use of a Xianglian protein peptide in preparing a product that promotes collagen secretion in cells. Compared with the prior art, the Xianglian protein peptide provided by the present invention can significantly promote collagen secretion in cells.

Description

Technical Field

[0001] The present invention relates to the technical field of plant extraction, and more particularly to the application of Xianglian protein peptide. Background Art

[0002] Xianglian, a specialty of Hunan Province, holds a National Geographical Indication in China. Lotus seeds are produced in Hunan, Hubei, Fujian, Jiangsu, Zhejiang, and Jiangxi. Xiangtan, located in the east-central part of Hunan Province, on the lower reaches of the Xiangjiang River, enjoys a humid climate with distinct four seasons, an average annual temperature of around 17°C, and abundant sunshine and rainfall, creating favorable climatic conditions for Xianglian production. Xianglian, known as "China's No. 1 Lotus Seed," is characterized by its high protein content, low fat content, and excellent taste. Xianglian varieties include Cunsanlian, as well as Furonglian and Taikonglian, which are bred from the Cunsanlian variety. Cunsanlian, a Xianglian variety passed down through generations in Xiangtan County, is named for its three connected seeds measuring one inch in length. These seeds are round and uniform, with a creamy white color and delicate texture. When cooked, they melt in the mouth, leaving a fragrant aroma. On May 24, 2010, the former General Administration of Quality Supervision, Inspection and Quarantine approved "Xianglian" as a protected Geographical Indication product.

[0003] Traditionally, lotus seeds have been used as both an edible vegetable and a medicinal herb. According to the Compendium of Materia Medica, lotus seeds have the functions of "tonifying the middle and nourishing the spirit, quenching thirst and removing heat, calming the mind and stopping diarrhea, harmonizing the heart and kidneys, consolidating essence and qi, strengthening tendons and bones, replenishing deficiency, benefiting the eyes and ears, and removing cold and dampness." Modern research has shown that lotus seeds are rich in protein, but current research on lotus seeds is limited to processing, breeding, and component analysis. Research on their functional components has primarily focused on starch polysaccharides and polyphenols, while research on the efficacy and activity of their proteins and hydrolyzed small-molecule peptides is even more limited.

[0004] Plant protein production is economical, requires no animal farming space restrictions, and produces no greenhouse gases. With the global population growing, arable land shrinking, and global warming emerging, the use of plant protein as an alternative to animal protein is gaining increasing attention. Common methods for extracting plant protein include alkaline dissolution and acid precipitation, enzyme extraction, organic solvent extraction, salt extraction, and reverse micelle extraction. Alkaline dissolution and acid precipitation is the most commonly used plant protein extraction method, leveraging the principle that plant proteins are readily soluble in alkaline environments and precipitate at their acidic isoelectric points. The advantages of alkaline dissolution and acid precipitation are ease of use and low cost. However, the disadvantage is that excessively high alkaline concentrations can cause the extracted protein to undergo the Maillard reaction, affecting its nutritional properties. Organic solvent extraction primarily targets proteins that are insoluble in water, acid, alkaline, or dilute salt solutions. These proteins bind strongly to lipids and are only soluble in highly lipophilic organic solvents such as ethanol and acetone. Furthermore, extraction must be performed at low temperatures to prevent protein denaturation.

[0005] Plant protein peptides are small molecule peptides formed by hydrolysis of plant proteins under specific conditions. They exhibit promising pharmacological activities, such as hypoglycemic and anti-cancer effects. Lotus seed protein peptides are a type of plant protein peptide, but there are currently no reports or trials on their efficacy in the cosmetics field. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide an application of Xianglian protein peptide. The Xianglian protein peptide provided by the present invention has a good effect on promoting cell secretion of collagen.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] Application of Xianglian protein peptide in the preparation of products that promote cell secretion of collagen.

[0009] In the present invention, the cells are skin cells, preferably fibroblasts, more preferably HFF-1 cells. The Xianglian protein peptide can act on the epidermis and dermis of skin cells.

[0010] In the present invention, the collagen is type I collagen.

[0011] In the present invention, the Xianglian protein peptide exists in the above-mentioned product in the form of a solution; in the solution of the Xianglian protein peptide, the concentration of the Xianglian protein peptide is 50 μg-200 μg / ml, preferably 200 μg / ml.

[0012] In some embodiments of the present invention, it has been demonstrated that 200 μg / ml of Xianglian protein peptide solution can significantly promote the secretion of type I collagen by HFF-1 cells.

[0013] In the present invention, the preparation method of the Xianglian protein peptide comprises:

[0014] (1) extracting the lotus seeds with an extraction solvent to obtain a lotus protein liquid;

[0015] (2) enzymatically hydrolyzing the Xianglian protein solution to obtain Xianglian protein peptides;

[0016] The extraction temperature is 30-50° C.; the extraction material-liquid ratio is 1:(10-50); and the extraction time is 20-140 minutes.

[0017] In the present invention, the Xiang lotus seeds are preferably Hunan Xiangtan lotus seeds; the extraction solvent is NaCl solution; in the NaCl solution, the concentration of NaCl is 0.05 to 0.25 mol / L, more preferably 0.25 mol / L.

[0018] In one embodiment of the present invention, the extraction temperature is 30° C.; the material-liquid ratio is 1:30; the extraction time is 80 min; and the concentration of NaCl in the NaCl solution is 0.15 mol / L.

[0019] In one embodiment of the present invention, the extraction temperature is 40° C.; the material-liquid ratio is 1:40; the extraction time is 110 min; and the concentration of NaCl in the NaCl solution is 0.15 mol / L.

[0020] In one embodiment of the present invention, the extraction temperature is 40° C.; the material-liquid ratio is 1:30; the extraction time is 140 min; and the concentration of NaCl in the NaCl solution is 0.2 mol / L.

[0021] In one embodiment of the present invention, the extraction temperature is 50° C.; the material-liquid ratio is 1:40; the extraction time is 80 min; and the concentration of NaCl in the NaCl solution is 0.2 mol / L.

[0022] In one embodiment of the present invention, the extraction temperature is 50° C.; the material-liquid ratio is 1:30; the extraction time is 110 min; and the concentration of NaCl in the NaCl solution is 0.25 mol / L.

[0023] In one embodiment of the present invention, the extraction temperature is 30° C.; the material-liquid ratio is 1:40; the extraction time is 140 min; and the concentration of NaCl in the NaCl solution is 0.25 mol / L.

[0024] In the present invention, the pH of the enzymatic hydrolysis is 8-10, the temperature of the enzymatic hydrolysis is 50-60°C, and the time of the enzymatic hydrolysis is 2-4 hours; the pH of the enzymatic hydrolysis is preferably 9, the temperature of the enzymatic hydrolysis is preferably 50°C, and the time of the enzymatic hydrolysis is preferably 3 hours.

[0025] In the present invention, the protease used for enzymatic hydrolysis is alkaline protease, and 6000-8000 U / g of alkaline protease is added to the Xianglian protein solution with a substrate concentration of 2%.

[0026] Before enzymatic hydrolysis of the Xianglian protein liquid, the present invention preferably removes impurities from the Xianglian protein liquid, then freeze-dries it, and then prepares it into a Xianglian protein liquid with a substrate concentration of 2-5%; the Xianglian protein liquid is preferably removed from the Xianglian protein liquid using an ultrafiltration membrane; the molecular weight of the ultrafiltration membrane is preferably 20kD.

[0027] The present invention also provides cosmetics, including the Xianglian protein peptide and cosmetically acceptable auxiliary materials.

[0028] In the present invention, the preparation method of the Xianglian protein peptide comprises:

[0029] (1) extracting the lotus seeds with an extraction solvent to obtain a lotus protein liquid;

[0030] (2) enzymatically hydrolyzing the Xianglian protein solution to obtain Xianglian protein peptides;

[0031] The extraction temperature is 30-50° C.; the extraction material-liquid ratio is 1:(10-50); and the extraction time is 20-140 minutes.

[0032] In the present invention, the extraction solvent is a NaCl solution; in the NaCl solution, the concentration of NaCl is 0.05 to 0.25 mol / L, more preferably 0.25 mol / L.

[0033] In the present invention, the pH of the enzymatic hydrolysis is 8-10, the temperature of the enzymatic hydrolysis is 50-60°C, and the time of the enzymatic hydrolysis is 2-4 hours; the pH of the enzymatic hydrolysis is preferably 9, the temperature of the enzymatic hydrolysis is preferably 50°C, and the time of the enzymatic hydrolysis is preferably 3 hours.

[0034] In the present invention, the protease used for enzymatic hydrolysis is alkaline protease, and 6000-8000 U / g of alkaline protease is added to the Xianglian protein solution with a substrate concentration of 2%.

[0035] Before enzymatic hydrolysis of the Xianglian protein liquid, the present invention preferably removes impurities from the Xianglian protein liquid, then freeze-dries it, and then prepares it into a Xianglian protein liquid with a substrate concentration of 2-5%; the Xianglian protein liquid is preferably removed from the Xianglian protein liquid using an ultrafiltration membrane; the molecular weight of the ultrafiltration membrane is preferably 20kD.

[0036] The cosmetics of the present invention are preferably toner, lotion, essence, facial mask, sunscreen, face cream, eye cream, hand cream or body cream; the facial mask is preferably a patch mask or a smear mask; the essence is preferably essence water or essence oil.

[0037] The excipients of the present invention are cosmetically acceptable excipients, preferably one or more of solvents, thickeners, humectants, film formers, liposomes, preservatives, colorants, pH regulators and flavors; the solvent is preferably water or an alcohol solvent.

[0038] Compared with large molecular proteins, the Xianglian protein peptide of the present invention can increase skin firmness, smooth static wrinkles, and improve skin appearance. It is a good helper for the human body to fight aging. Experiments of the present invention have confirmed that the Xianglian protein peptide has a good effect of promoting the synthesis of collagen secreted by fibroblasts.

[0039] This study uses Hunan lotus seeds as the research object and innovatively employs salt-dissolving extraction, membrane separation and impurity removal, and enzymatic hydrolysis to extract and prepare Hunan lotus protein peptides. The salt-dissolving extraction technique was optimized by analyzing the effects of lotus seeds from different origins, extraction solvents, salt concentrations, material-to-liquid ratios, extraction temperature, and time on Hunan lotus protein content. The protein solution was then concentrated and desalted using membrane separation technology. Finally, enzymatic hydrolysis was used to produce the Hunan lotus protein peptides.

[0040] The preparation process of the present invention is simple and fast, and can be easily scaled up for production. The salt dissolution and heating extraction technology, membrane separation technology, and enzymatic hydrolysis technology adopted in the present invention require relatively common instruments and equipment, the process operation flow is relatively simple, and the time cycle required for the process is short, which is conducive to the actual scale-up of the factory and energy saving and consumption reduction. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is a standard curve diagram of standard bovine serum albumin and absorbance;

[0042] Figure 2 This is a chart showing the protein content of different varieties of lotus seeds;

[0043] Figure 3 This is a graph showing the effect of different extraction solvents on the amount of Xianglian protein extracted;

[0044] Figure 4 This is the effect of different NaCl concentrations on the amount of Xianglian protein extracted;

[0045] Figure 5 This is the effect of different material-liquid ratios on the extraction of Xianglian protein;

[0046] Figure 6 This is the effect of different extraction temperatures on the extraction amount of Xianglian protein;

[0047] Figure 7 This is the effect of different extraction times on the amount of Xianglian protein extracted;

[0048] Figure 8 This is a graph showing the effects of Xianglian protein and Xianglian protein peptide on the collagen content secreted by HFF-1 cells. DETAILED DESCRIPTION

[0049] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0050] In order to further illustrate the present invention, the following examples are provided for detailed description. The raw materials used in the following examples of the present invention are all commercially available commodities.

[0051] 1.1 Drawing of bovine serum albumin standard curve

[0052] (1) Preparation of Coomassie Brilliant Blue G-250 staining working solution:

[0053] Dissolve 10 mg of Coomassie Brilliant Blue G-250 powder in 5 mL of 95% ethanol. Once completely dissolved, add the solution to 10 mL of 85% phosphoric acid while stirring. (Commercially available liquid phosphoric acid (H₃PO₄) should have a mass fraction of ≥85.0%, is a thick, transparent liquid, and is highly corrosive to the skin. Use a 5 mL pipette to slowly pipette.) Dilute to 100 mL with distilled water. Filter the solution through filter paper and store in a brown reagent bottle in a dark place. If the working solution is not used for an extended period, it can be stored in a refrigerator at 4°C for up to 1 year. Filter the solution again before use and warm it to room temperature before adding it to the reaction tube. Failure to do so will not affect the accuracy, but the measured OD value will be lower, placing higher demands on the instrument.

[0054] (2) Preparation of standard bovine serum albumin (BSA):

[0055] Accurately weigh 4 mg of BSA powder and dissolve it in 1 mL of 0.15 mol / L NaCl solution to prepare a 4 mg / mL standard protein solution. Store at -20°C.

[0056] (3) Determination of standard curve

[0057] Accurately measure 0.25 mL of 4 mg / mL standard protein solution and add 3.75 mL of water to make a solution containing 250 μg per 1 mL. Mix thoroughly to prepare the master solution for the serial dilutions shown in Table 1. Prepare the bovine serum albumin control solution series according to Table 1.

[0058] Table 1 Bovine serum albumin control solution series

[0059] Test tube number blank 1# 2# 3# 4# 5# Control solution 0mL 0.1mL 0.2mL 0.4mL 0.8mL 1.0mL water 1.0mL 0.9mL 0.8mL 0.6mL 0.2mL 0mL Coomassie Brilliant Blue Test Solution 4.0mL 4.0mL 4.0mL 4.0mL 4.0mL 4.0mL

[0060] After adding Coomassie Brilliant Blue solution, mix immediately. After 5 minutes, use a microplate reader to measure the absorbance at a wavelength of 595nm, and calculate the regression equation and correlation coefficient (Note: the concentration range of the standard curve can be adjusted according to the actual concentration, and r>0.980 is required). For specific results, see Figure 1 , Figure 1 The standard curve is shown in Figure 2. The linear regression equation is Y = 0.0038X + 0.04, R 2 =0.9955.

[0061] 1.2 Determination of lotus seed protein (soluble protein) content from different origins

[0062] Weigh 0.5g of lotus seed powder from four origins after defatting with petroleum ether and place it in a round-bottom flask. Add 10mL of pure water at a solid-liquid ratio of 1:20, and then place it in a 30℃ oil bath and stir for 20min. After the extraction is completed, place it in an ultracentrifuge (speed of 6000r / min) and centrifuge for 8min. The supernatant is diluted 50 times. Accurately aspirate 1mL of the diluted extract and place it in a 10mL stoppered test tube. Measure the absorbance value according to the steps in 1.1. The experimental results are as follows. Figure 2 shown.

[0063] Different geographical locations have different climates and soils, so the protein content in lotus seeds from different origins will also vary. Lotus seeds from four representative origins in China (Hunan Xiangtan lotus seeds, Hubei Honghu lotus seeds, Jiangxi Guangchang lotus seeds, Fujian Jianning lotus seeds) were selected for protein content determination and analysis. Figure 2 It can be seen that the protein content in Xianglian is much better than that in other production areas. This may be due to Xiangtan's unique geographical advantages: Xiangtan is located in the east-central part of Hunan Province, in the lower reaches of the Xiangjiang River. It has a humid climate, distinct four seasons, an average annual temperature of around 17°C, and plenty of sunshine and rainfall, which is more conducive to the enrichment of active substances.

[0064] 1.3 Effect of different extraction solvents on lotus seed protein extraction

[0065] Weigh 0.5 g of Xianglian lotus seed powder defatted with petroleum ether and place it in a round-bottom flask. Add 10 mL of pure water, PBS buffer, Tris-HCl buffer and NaCl salt solution (0.1 M) in a solid-liquid ratio of 1:20, and then place it in a 30°C oil bath and stir for 20 minutes. After the extraction is completed, place it in an ultracentrifuge (speed of 6000 r / min) and centrifuge for 8 minutes to obtain a supernatant diluted 50 times. Accurately aspirate 1 mL of the diluted extract and place it in a 10 mL stoppered test tube. Determine the absorbance value according to the steps in 1.1. The experimental results are as follows. Figure 3 shown.

[0066] Depend on Figure 3 It can be seen that NaCl solution has a better protein extraction effect than other solvents, and NaCl solution will be used in subsequent experiments.

[0067] 1.4 Optimization of Xianglian protein extraction process conditions

[0068] 1.4.1 Single-factor experiment

[0069] Protein from Xianglian was extracted by heating, and the effects of NaCl concentration, solid-liquid ratio, extraction temperature, and time on protein content were studied in detail. To obtain the optimal protein extraction process conditions, single-factor experiments were conducted on NaCl concentration, solid-liquid ratio, extraction temperature, and time, and the protein extraction process from Xianglian was further optimized.

[0070] (1) Effect of NaCl concentration

[0071] Weigh 0.5 g of Xianglian lotus seed powder defatted with petroleum ether and place it in a round-bottom flask. Add 0.05M, 0.1M, 0.15M, 0.2M, and 0.25M NaCl solutions at a solid-liquid ratio of 1:20, respectively. Then place it in a 30°C oil bath and stir for 20 minutes. After the extraction is completed, place it in an ultracentrifuge (speed of 6000r / min) and centrifuge for 8 minutes to obtain a supernatant diluted 50 times. Accurately aspirate 1 mL of the diluted extract and place it in a 10 mL stoppered test tube. Measure the absorbance value according to the steps in 1.1. The experimental results are as follows. Figure 4 shown.

[0072] Salt concentration is an important factor affecting the extraction effect. Figure 4 As can be seen from the figure, when the NaCl concentration gradually increases from 0.05M to 0.2M, the protein content also increases; when the concentration increases from 0.2M to 0.25M, the protein content decreases. The reason for this phenomenon is that when the salt concentration is low, as the concentration gradually increases, the salt dissolution phenomenon promotes the dissolution of protein; when the salt concentration is too high, the extraction rate decreases due to the salting-out phenomenon of protein.

[0073] (2) Influence of material-liquid ratio

[0074] Weigh 0.5 g of Xianglian lotus seed powder, defatted with petroleum ether, into a round-bottom flask. Add 0.1 M NaCl solution at solid-to-liquid ratios of 1:10, 1:20, 1:30, 1:40, and 1:50, respectively. Stir in a 30°C oil bath for 20 minutes. After extraction, centrifuge in an ultracentrifuge (6000 rpm) for 8 minutes. Dilute the supernatant 50-fold. Accurately pipette 1 mL of the diluted extract into a 10 mL stoppered test tube and measure the absorbance according to the procedure in 1.1.

[0075] Different material-liquid ratios also have a significant impact on the extraction efficiency. If the material-liquid ratio is too small, the lotus seed protein cannot be completely extracted. If the material-liquid ratio is too high, it will lead to higher process costs. Figure 5As shown in the figure, it can be seen that the protein content increases with the material-liquid ratio from 1:10 to 1:40, reaching the maximum at 1:40. It then shows a downward trend. This may be because the lotus seed protein extraction is in a saturated state. Further increasing the solvent amount will not only reduce the protein content per unit extract, but also cause waste of resources and increase costs.

[0076] (3) Effect of extraction temperature

[0077] Weigh 0.5 g of Xianglian lotus seed powder defatted with petroleum ether and place it in a round-bottom flask. Add 0.1 M NaCl salt solution at a solid-liquid ratio of 1:20, and then place it in an oil bath at 30°C, 40°C, 50°C, 60°C, and 70°C and stir for 20 minutes. After the extraction is completed, place it in an ultracentrifuge (speed of 6000r / min) and centrifuge for 8 minutes to obtain a supernatant diluted 50 times. Accurately aspirate 1 mL of the diluted extract and place it in a 10 mL stoppered test tube. Measure the absorbance value according to the steps in 1.1. The experimental results are as follows. Figure 6 shown.

[0078] Temperature also plays an important role in the extraction process. Too high or too low a temperature will have an adverse effect on the extraction effect. Figure 6 Results show that protein content increases with temperature, but decreases when the temperature exceeds 50°C. Appropriate temperature enhances molecular diffusion and promotes protein dissolution from plant cells. However, excessively high temperatures may cause protein denaturation, leading to a decrease in protein content.

[0079] (4) Effect of extraction time

[0080] Weigh 0.5 g of Xianglian lotus seed powder, defatted with petroleum ether, into a round-bottom flask. Add 0.1 M NaCl solution at a solid-to-liquid ratio of 1:20. Stir in a 30°C oil bath for 20, 50, 80, 110, and 140 minutes, respectively. After extraction, centrifuge in an ultracentrifuge (6000 rpm) for 8 minutes. Dilute the supernatant 50-fold. Accurately pipette 1 mL of the diluted extract into a 10 mL stoppered test tube and measure the absorbance according to the procedure in 1.1.

[0081] The present invention studies the effect of extraction time on protein extraction effect in the range of 20 to 140 minutes. Figure 7 As shown in the figure, the protein content gradually increases from 20 to 140 minutes, reaching its highest level at 110 minutes. As time continues to increase, the content decreases slightly. This may be because, within a certain range, the longer the extraction time, the more protein will be dissolved. However, too long an extraction time may damage the protein structure, thereby reducing the extraction efficiency.

[0082] 1.3.2 Example

[0083] Example 1

[0084] (1) Extraction: Weigh 0.5 g of Xianglian lotus seed powder defatted with petroleum ether and place it in a round-bottom flask. Add 10 mL of 0.15 M NaCl solution at a solid-liquid ratio of 1:30, and then place it in an oil bath at 30°C and stir for 80 min.

[0085] (2) Separation: After extraction, centrifuge in an ultracentrifuge (6000 rpm) for 8 min. Dilute the supernatant 50-fold. Accurately pipette 1 mL of the diluted extract into a 10 mL stoppered test tube. Measure the absorbance according to the steps in 1.1. The extraction yield of Xianglian protein was 7.29%.

[0086] Example 2

[0087] Referring to the steps in Example 1, the solid-liquid ratio was set to 1:40, the temperature of the oil bath was set to 40°C, the stirring extraction time was set to 110 minutes, and other conditions remained unchanged. The extraction rate of Xianglian protein was measured to be 7.77%.

[0088] Example 3

[0089] Referring to the steps in Example 1, the solid-liquid ratio was set to 1:50, the temperature of the oil bath was set to 50° C., the stirring extraction time was set to 140 min, and other conditions remained unchanged. The extraction rate of Xianglian protein was measured to be 9.38%.

[0090] Example 4

[0091] Referring to the steps in Example 1, the concentration of the NaCl solution was set to 0.2 M, the temperature of the oil bath was set to 40° C., the stirring extraction time was set to 140 min, and other conditions remained unchanged. The extraction rate of Xianglian protein was measured to be 9.87%.

[0092] Example 5

[0093] Referring to the steps in Example 4, the solid-liquid ratio was set to 1:40, the oil bath temperature was set to 50°C, the stirring extraction time was set to 80 minutes, and other conditions remained unchanged. The extraction rate of Xianglian protein was measured to be 8.42%.

[0094] Example 6

[0095] Referring to the steps in Example 4, the solid-liquid ratio was set to 1:50, the oil bath temperature was set to 30°C, the stirring extraction time was set to 110 minutes, and other conditions remained unchanged. The extraction rate of Xianglian protein was measured to be 9.49%.

[0096] Example 7

[0097] Referring to the steps in Example 1, the concentration of the NaCl solution was set to 0.25 M, the temperature of the oil bath was set to 50° C., the stirring extraction time was set to 110 min, and other conditions remained unchanged. The extraction rate of Xianglian protein was measured to be 10.83%.

[0098] Example 8

[0099] Referring to the steps in Example 7, the solid-liquid ratio was set to 1:40, the oil bath temperature was set to 30°C, the stirring extraction time was set to 140 minutes, and other conditions remained unchanged. The extraction rate of Xianglian protein was measured to be 11.98%.

[0100] Example 9

[0101] Referring to the steps in Example 7, the solid-liquid ratio was set to 1:50, the oil bath temperature was set to 40°C, the stirring extraction time was set to 80 minutes, and other conditions remained unchanged. The extraction rate of Xianglian protein was measured to be 9.07%.

[0102] From the extraction rate results of Examples 1 to 9, it can be seen that the optimal process conditions for extracting Xianglian protein are as in Example 8: NaCl concentration of 0.25M, solid-liquid ratio of 1:40g / mL, extraction temperature of 30°C, and extraction time of 140min. The results of the confirmatory experiment are shown in Table 2. The protein content (extraction rate) of three parallel experiments was 115.38mg / g (11.53%), 119.01mg / g (11.90%), and 116.33mg / g (11.63%), respectively. The average protein extraction amount (extraction rate) was 116.90mg / g (11.69%), and the RSD value was 1.65% (n=3). The calculation results show that under the optimal process conditions, the extraction amount (extraction rate%) of Xianglian protein is the highest, and the extraction process has good stability.

[0103] Table 2. Verification experiment results

[0104]

[0105] 1.4 Preparation of Xianglian protein

[0106] 1.4.1 Using membrane separation equipment to remove impurities from protein solution

[0107] 2-3 L of Xianglian protein extract was prepared according to the method in Example 8 and centrifuged at 8500 rpm for 15 min. The supernatant was removed and the protein and some small molecular impurities were removed using a membrane separation device (model: GY-UF-1812, ultrafiltration membrane molecular weight cut-off 20 kD). The conductivity of the solution was monitored in real time using a conductivity meter to maintain the conductivity of the protein solution between 1000 and 2000 μS / m.

[0108] 1.4.2 Preparation of Xianglian protein freeze-dried powder

[0109] The protein solution adsorbed after membrane separation and impurity removal in 1.4.1 was concentrated on a rotary evaporator (rotary evaporation temperature: 40-45°C), then freeze-dried and powdered, and refrigerated at 4°C for subsequent hydrolysis and efficacy experiments.

[0110] 1.5 Preparation of Xianglian protein peptide

[0111] Example 10

[0112] (1) Enzymatic hydrolysis: Prepare the freeze-dried powder obtained in 1.4.2 with a substrate concentration of 2% Xianglian protein solution. Adjust the pH to 9.0 according to the optimal hydrolysis pH of alkaline protease. After heating to 50°C, add 6000 U / g alkaline protease and hydrolyze at this constant temperature for 3 h (during which the pH is adjusted to maintain a constant pH value and the degree of hydrolysis (DH%) is calculated). After the hydrolysis time is reached, heat to 90°C and inactivate the enzyme for 10 min.

[0113] (2) Separation: The hydrolyzate was centrifuged at a speed of 4500 r / min for 15 min, the supernatant was concentrated using a rotary evaporator, and finally freeze-dried to obtain Xianglian protein peptide.

[0114] The pH-stat method is used to calculate the degree of hydrolysis:

[0115]

[0116] Where: A is the volume of the consumed NaOH standard solution, mL; Mb is the concentration of the NaOH standard solution, mol / L; α is the dissociation degree of the amino group, which is taken as 1 here; MP is the total amount of protein in the substrate, g; htot is the number of millimoles of protein bonds per gram of substrate, mmol / g, which is taken as 8.0 mmol / g.

[0117] 1.6 Experimental Example: Comparative Test of the Efficacy of Xianglian Protein VS Xianglian Protein Peptide Cosmetics

[0118] 1.6.1 Effect of active ingredients on the activation of type I collagen in HFF-1 (fibroblasts)

[0119] (1) Culture medium and solution preparation

[0120] DMEM high-glucose medium: Add one bag of DMEM high-glucose medium powder to 800 mL of purified water and stir to dissolve. Then add 3.7 g of NaHCO3 and continue stirring until the powder is completely dissolved. Adjust the pH to 7.1-7.2 and make up to 1 L. Filter and sterilize with a 0.22 μm filter, seal and store at 4°C.

[0121] DMEM high glucose complete medium: Add FBS to DMEM high glucose medium to make the content 10%

[0122] (2) Effects of test samples on collagen secretion by HFF-1 cells

[0123] 1. Cell seeding: 10,000 HFF-1 cells were seeded into a 96-well plate and cultured in a 37°C, 5% CO2 incubator for 24 hours.

[0124] 2. Experimental Grouping: A negative control group (NC) and a sample group were set up. Three concentration gradients were set in the sample group, and each group had 6 replicates. The Xianglian protein in the sample group was prepared according to Example 8, with concentrations of 50 μg / ml, 100 μg / ml, and 200 μg / ml, respectively; the Xianglian protein peptide was prepared according to step 1.5 above, with concentrations of 50 μg / ml, 100 μg / ml, and 200 μg / ml, respectively.

[0125] 3. Dilution and addition of test samples: Use complete culture medium as diluent and prepare sample working solutions of different concentrations according to the sample test concentration table, 100 μl / well. Add an equal amount of complete culture medium to the negative control group and culture in a 37°C, 5% CO2 incubator for 24 hours. Observe the cell morphology under a microscope.

[0126] 4. Take the supernatant from the well plate and detect the collagen content by ELISA. The operation steps are as follows:

[0127] A. Calculate the required enzyme label strips in advance, take out the kit 30 minutes before the experiment, and return it to room temperature.

[0128] B. Serial dilution of standard: dilute the standard to 200, 100, and 50 ng / mL using standard and sample diluent.

[0129] C. Collect the cell culture supernatant into a sterile centrifuge tube and centrifuge (4°C, 1000×g, 5 min). Take the supernatant and dilute it to an appropriate concentration as the test sample.

[0130] D. Add 100 μl of standard working solution and test sample (diluted 1-fold) to each reaction well. Set up 2 replicate wells for each group and incubate at 37°C for 90 min.

[0131] E. Discard the liquid, spin dry, add 100 μl of biotin-labeled antibody working solution to each reaction well, and incubate at 37°C for 60 min.

[0132] F. Discard the liquid, spin dry, add 300 μl of washing solution to each reaction well, soak for 1-2 minutes and spin dry. Repeat 4 times

[0133] Add 100 μl of HRP-labeled streptavidin working solution to each reaction well and incubate at 37°C for 30 min.

[0134] Add 300 μl of washing solution to each reaction well, wait 30 seconds, and spin dry the washing solution. Repeat 4 times

[0135] I. Add 90 μl of color developing reagent to each reaction well and develop the color in the dark at 37°C for about 15 minutes.

[0136] J Add 50 μl of stop solution to each reaction well and immediately measure the OD value at a wavelength of 450 nm using a microplate reader.

[0137] K is used to calculate the standard curve regression equation (R 2 >0.99), substitute the OD value of the sample well into the calculated concentration of the sample, and then multiply it by the dilution factor to get the actual concentration of the original sample.

[0138] 5. Statistical analysis: GraphPad Prism 8.0 software was used for data statistical analysis and graphing. Measurement data were expressed as x ± s. Differences between groups were analyzed using one-way ANOVA. P < 0.05 was considered statistically significant.

[0139] (3) Test results

[0140] 1. ELISA and sample test results

[0141] The collagen content of each group was detected by ELISA, and the standard curve y=0.0006x+0.0915, R 2 =0.9934 (abscissa x is the collagen content of the sample, ordinate y = absorbance).

[0142] Table 3

[0143]

[0144]

[0145] The collagen content was inferred from the absorbance of the standard curve, and its changing trend is shown in Table 3. Then the data were statistically analyzed to calculate the significant differences between the groups. The results are as follows Figure 8 As shown, Figure 8 In the table, * indicates significant difference compared with the negative control group (P<0.05), and Xianglian peptide is the Xianglian protein peptide in Table 3.

[0146] From Table 3 and Figure 8 The results show that compared to Xianglian protein, within the active ingredient concentration range of 50μg-200μg / ml, Xianglian protein peptide has a certain activation effect on HFF-1 type I collagen, especially at 200μg / ml, which has a significant difference in promoting collagen secretion compared to Xianglian protein peptide. Therefore, compared to protein, Xianglian protein peptide has the anti-aging effect of promoting type I collagen secretion.

[0147] The above description of the disclosed embodiments will enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is to be construed in the widest manner consistent with the principles and novel features disclosed herein.

Claims

1. Application of Xianglian protein peptide in the preparation of products that promote cell secretion of collagen; The cells are skin cells; The collagen is type I collagen; The preparation method of the Xianglian protein peptide comprises: (1) extracting lotus seeds with an extraction solvent to obtain a lotus protein solution; the extraction solvent is a NaCl solution; (2) The Xianglian protein liquid is enzymatically hydrolyzed to obtain Xianglian protein peptides; the pH of the enzymatic hydrolysis is 8-10, the enzymatic hydrolysis temperature is 50-60°C, and the enzymatic hydrolysis time is 2-4 hours; the protease used in the enzymatic hydrolysis is alkaline protease; the extraction temperature is 30-50°C; the extraction material-liquid ratio is 1:(10-50); and the extraction time is 20-140 minutes.

2. The use according to claim 1, characterized in that In the NaCl solution, the concentration of NaCl is 0.05-0.25 mol / L.

3. The use according to claim 1, characterized in that The extraction temperature was 30° C.; the solid-liquid ratio was 1:40; the extraction time was 140 min; and the concentration of NaCl in the NaCl solution was 0.25 mol / L.

4. The use according to claim 1, wherein the product is a cosmetic; the cosmetic comprises cosmetically acceptable excipients.

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