An extraction process of marine biological protein for sensitive skin whitening

Abstract

This invention relates to the field of biological protein extraction technology, specifically disclosing an extraction process for marine biological proteins used for skin whitening in sensitive skin, comprising the following steps: S1, pretreatment; S2, acidic extraction; S3, enzymatic hydrolysis; S4, ultrafiltration and centrifugation. In the acidic extraction process, tannic acid, phytic acid, sulfonated soybean lecithin, and pepsin are added; in the enzymatic hydrolysis process, anthocyanins and magnesium gluconate are added. Through the synergistic effect of these processes, the purity and extraction rate of the marine biological protein are improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological protein extraction, and more particularly to an extraction process of marine biological protein for sensitive skin whitening. BACKGROUND

[0002] Squid collagen is a protein extracted from squid skin, cartilage and other by-products. It is emerging in the fields of food, cosmetics and biomedicine due to its unique advantages such as low denaturation temperature, high solubility and low immunogenicity.

[0003] In the prior art, the extraction of squid collagen is mainly based on acid method and enzyme method. When the acid method is used to extract squid collagen, the natural triple helix structure can be well preserved, and the operation is simple, but the extraction yield is low. When the enzyme method is used to extract squid collagen, the extraction conditions are mild, but the squid skin collagen is tightly wrapped by the dense structure of the epidermis layer and melanin, and a single protease (such as trypsin, papain, etc.) is difficult to open the outer barrier and hydrolyze the internal collagen at the same time, resulting in low purity and extraction rate of the extracted squid collagen.

[0004] Therefore, it is particularly important to provide an extraction process of marine biological protein for sensitive skin whitening. SUMMARY

[0005] In order to solve the problems mentioned in the background, the present application provides an extraction process of marine biological protein for sensitive skin whitening.

[0006] The present application provides an extraction process of marine biological protein for sensitive skin whitening, which adopts the following technical scheme:

[0007] An extraction process of marine biological protein for sensitive skin whitening, comprising the following steps:

[0008] S1, pretreatment: thaw and clean the squid skin, soak it in alkali solution for 10-14h, wash it, then soak it in acid solution for 8-10h, wash it, drain it, cut it, dry it, sieve it, and obtain pretreated squid skin;

[0009] S2. Acidic Extraction: Add pretreated squid skin to an acetic acid aqueous solution, add pepsin, heat to 36-40℃, stir for 1.2-1.6h, then add tannic acid, phytic acid, and sulfonated soybean lecithin, continue stirring for 0.2-0.4h, centrifuge, collect the supernatant, adjust the pH of the supernatant to 2.8-3.4, centrifuge, collect the supernatant, combine the two supernatants, add sodium chloride aqueous solution, collect the precipitate, wash, and dry to obtain crude squid protein extract. The mass ratio of pretreated squid skin, acetic acid aqueous solution, pepsin, tannic acid, phytic acid, sulfonated soybean lecithin, and sodium chloride aqueous solution is 1:12-16:0.001-0.003:0.004-0.006:0.01-0.03:0.03-0.05:8-12.

[0010] S3. Enzymatic hydrolysis: Add crude squid protein extract, anthocyanins, and magnesium gluconate to deionized water, adjust the pH to 6.4-6.8, then add papain, perform a first enzymatic hydrolysis, centrifuge, and collect the supernatant to obtain the first enzymatic hydrolysate; adjust the pH of the first enzymatic hydrolysate to 7.8-8.6, then add trypsin and alkaline protease, perform a second enzymatic hydrolysis, centrifuge, and collect the supernatant to obtain the second enzymatic hydrolysate. The mass ratio of crude squid protein extract, anthocyanins, magnesium gluconate, deionized water, and papain is 2-4:0.03-0.05:0.006-0.008:90-110:0.008-0.014; the mass ratio of the first enzymatic hydrolysate, trypsin, and alkaline protease is 40-60:0.006-0.01:0.002-0.005.

[0011] S4. Ultrafiltration and centrifugation: Centrifuge, ultrafilter, and freeze-dry the secondary enzymatic hydrolysate to obtain marine biological protein for skin whitening of sensitive skin.

[0012] Further, in S1, the alkaline solution is an aqueous solution of sodium hydroxide with a mass fraction of 0.4-0.6%, and the acid solution is an aqueous solution of acetic acid with a mass fraction of 0.1-0.3%.

[0013] Furthermore, in S2, the mass fraction of the acetic acid aqueous solution is 0.46-0.54%.

[0014] Furthermore, in S2, the mass fraction of the sodium chloride aqueous solution is 20%.

[0015] Furthermore, in S3, the enzymatic hydrolysis temperature for the first enzymatic hydrolysis is 42-46℃, and the enzymatic hydrolysis time is 2.6-3h; the enzymatic hydrolysis temperature for the second enzymatic hydrolysis is 46-50℃, and the enzymatic hydrolysis time is 2.2-2.6h.

[0016] Furthermore, the sulfonated soybean lecithin in S2 is specifically obtained by the following steps:

[0017] A1. Add soybean lecithin and fumaric acid to an ethanol aqueous solution, stir evenly, heat to reflux temperature, stir for 1.4-1.8 h, filter, wash, and dry to obtain carboxylated soybean lecithin. The mass ratio of soybean lecithin, fumaric acid and ethanol aqueous solution is 1:0.05-0.07:50-60. In the above process, using ethanol aqueous solution as solvent, the active hydroxyl groups on soybean lecithin undergo esterification reaction with fumaric acid to obtain carboxylated soybean lecithin.

[0018] A2. Add carboxylated soybean lecithin and sodium metabisulfite to deionized water, adjust the pH to 6.4-6.8, heat to 76-80℃, stir for 2.4-2.8 hours, filter, wash, and dry to obtain sulfonated soybean lecithin. The mass ratio of carboxylated soybean lecithin, sodium metabisulfite, and deionized water is 1:3.6-4.2:60-70. In the above process, using deionized water as a solvent, carboxylated soybean lecithin can undergo a Michael addition reaction with sodium metabisulfite to obtain sulfonated soybean lecithin.

[0019] Furthermore, in A1, the mass fraction of the aqueous ethanol solution is 40-50%.

[0020] In summary, the present invention has the following beneficial effects:

[0021] (1) In the technical solution of the present invention, tannic acid, phytic acid, sulfonated soybean lecithin and pepsin are added during the acid extraction process. Tannic acid can not only create and maintain an acidic environment, but also form multiple hydrogen bonds with amino and carboxyl groups in the protein, thereby competitively destroying the original hydrogen bonds inside the squid protein, making the three-dimensional structure of the protein "relaxed" and exposing more enzyme cleavage sites, which is convenient for enzymatic hydrolysis in subsequent steps. Phytic acid can not only chelate the metal ions such as Ca²⁺, Mg²⁺ and Cu²⁺ contained in the pretreated squid skin, avoiding their influence on the activity of pepsin, but also work synergistically with sulfonated soybean lecithin to promote protein dispersion and dissolution. Sulfonated soybean lecithin can not only promote the emulsification and dispersion of marine biological proteins in acidic aqueous solution, but also reduce the interfacial tension, helping water and pepsin to better contact and wet the protein substrate. Pepsin can efficiently hydrolyze specific amino acid sites in the peptide chain, decomposing large molecular proteins into small molecular peptides and amino acids. Through the synergistic effect of the above substances, the purity and extraction rate of marine biological collagen are improved.

[0022] (2) In the technical solution of the present invention, anthocyanins and magnesium gluconate are added during the enzymatic hydrolysis process. Anthocyanins can not only reduce enzyme inactivation caused by lipid peroxidation and maintain the activity of papain, trypsin and alkaline protease, but also bind to the enzyme surface through hydrogen bonding and hydrophobic interaction to stabilize the tertiary structure of the enzyme. The presence of magnesium gluconate can not only directly bind to the active site of the enzyme to stabilize its catalytic conformation and improve catalytic efficiency, but also neutralize the negative charge on the surface of squid protein, reduce the electrostatic repulsion between the protease and the protein substrate, promote their binding, and further improve the purity and extraction rate of marine biological collagen. Detailed Implementation

[0023] The present invention will be further described in detail below with reference to the embodiments.

[0024] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0025] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0026] The squid skin was purchased from a squid processing plant in Zhoushan; the disodium hydrogen phosphate-potassium dihydrogen phosphate buffer was BB028-disodium hydrogen phosphate-potassium dihydrogen phosphate buffer with a pH of 6.98, which was commercially available from Ise-Kyu (Lianyungang, Jiangsu) Biotechnology Co., Ltd.

[0027] The citrate-sodium citrate buffer solution was commercially available from Guangzhou Hewei Pharmaceutical Technology Co., Ltd. with a pH of 6.6; pepsin was purchased from Shandong Fangchang Biotechnology Co., Ltd., CAS No. 9001-75-6; papain was purchased from Sichuan Huatang Jurui Biotechnology Co., Ltd., CAS No. 9001-73-4; trypsin was purchased from Thermo Fisher Scientific (China) Co., Ltd., CAS No. 9002-07-7; and alkaline protease was purchased from Chengdu Wanxiang Hongrun Biotechnology Co., Ltd., CAS No. 9014-01-1.

[0028] Example 1

[0029] An extraction process for marine biological proteins used for whitening sensitive skin includes the following steps:

[0030] S1. Pretreatment: Thaw and clean the squid skin, soak it in a 0.4% sodium hydroxide aqueous solution for 10 hours, wash it, soak it in a 0.1% acetic acid aqueous solution for 8 hours, wash it, drain it, chop it, control the vacuum degree to -0.1MPa, dry it at 30℃ to constant weight, and pass it through a 200-mesh sieve to obtain pretreated squid skin.

[0031] S2. Acidic Extraction: Pretreated squid skin was added to a 0.46% acetic acid aqueous solution, along with pepsin. The mixture was heated to 40°C and stirred at 800 rpm for 1.2 hours. Tannic acid, phytic acid, and sulfonated soybean lecithin were then added, and stirring continued for another 0.2 hours while maintaining the same stirring speed. The mixture was then centrifuged at 8000 rpm for 16 minutes. The supernatant was collected, and the pH of the supernatant was adjusted to 2.8 with a 1% citric acid aqueous solution. The mixture was then centrifuged at 8800 rpm for 10 minutes. The supernatant was collected. The supernatants were combined and a 20% sodium chloride aqueous solution was added. The precipitate was collected and washed three times each with anhydrous ethanol and deionized water (each time the mass of anhydrous ethanol and deionized water was 25% of the mass of the acetic acid aqueous solution). The precipitate was then freeze-dried at -40℃, under a vacuum of 11 Pa, for 22 hours to obtain crude squid protein extract. The mass ratio of pretreated squid skin, acetic acid aqueous solution, pepsin, tannic acid, phytic acid, sulfonated soybean lecithin, and sodium chloride aqueous solution was 1:12:0.004:0.001:0.01:0.03:8.

[0032] S3. Enzymatic hydrolysis: Squid protein crude extract, anthocyanins, and magnesium gluconate were added to deionized water. The pH was adjusted to 6.4 using disodium hydrogen phosphate-potassium dihydrogen phosphate buffer. Papain was then added for a single enzymatic hydrolysis, controlled at 42℃ for 3 hours. The mixture was centrifuged at 9000 rpm for 14 minutes. The supernatant was collected as the first hydrolysate. The pH of the first hydrolysate was then adjusted to 7.8 using 0.8% Tris-HCl buffer. Pancreatic enzyme was then added... The protease and alkaline protease were subjected to a second enzymatic hydrolysis, with the hydrolysis temperature controlled at 46℃ and the hydrolysis time at 2.6h. After centrifugation, the centrifugation speed was controlled at 8200rpm and the centrifugation time was 16min. The supernatant was collected to obtain the second enzymatic hydrolysate. The mass ratio of squid protein crude extract, anthocyanins, magnesium gluconate, deionized water, and papain was 2:0.03:0.006:90:0.008, and the mass ratio of the first enzymatic hydrolysate, trypsin, and alkaline protease was 40:0.006:0.002.

[0033] S4. Ultrafiltration and centrifugation: Centrifuge the secondary enzymatic hydrolysate at 8000g and 4℃ for 30min. Transfer the supernatant obtained after centrifugation to a 3kDa ultrafiltration tube and centrifuge at 3000g and 4℃ for 50min to complete ultrafiltration. Freeze-dry at -40℃ and 11Pa for 34h. After freeze-drying, marine biological protein for sensitive skin whitening is obtained.

[0034] The sulfonated soybean lecithin is prepared by the following steps:

[0035] A1. Add soybean lecithin and fumaric acid to a 40% (w / w) aqueous ethanol solution, control the stirring speed at 600 rpm, stir for 22 min until homogeneous, raise the temperature to reflux temperature, maintain the stirring speed at a constant speed, and continue stirring for 1.4 h. Filter, wash three times with deionized water (each time the mass of deionized water is 50% of the mass of the aqueous ethanol solution), and dry at 55℃ to constant weight to obtain carboxylated soybean lecithin, wherein the mass ratio of soybean lecithin, fumaric acid and aqueous ethanol solution is 1:0.05:50;

[0036] A2. Carboxylated soybean lecithin and sodium metabisulfite were added to deionized water, and the pH was adjusted to 6.4 with citrate-sodium citrate buffer. The temperature was raised to 76°C, and the stirring speed was controlled at 600 rpm for 2.8 h. After the reaction was completed, the mixture was filtered and washed three times each with 70% ethanol aqueous solution and deionized water (each ethanol aqueous solution and deionized water were 20% of the mass of citrate-sodium citrate buffer). The mixture was dried at 62°C to constant weight to obtain sulfonated soybean lecithin. The mass ratio of carboxylated soybean lecithin, sodium metabisulfite and deionized water was 1:3.6:60.

[0037] Example 2

[0038] An extraction process for marine biological proteins used for whitening sensitive skin includes the following steps:

[0039] S1. Pretreatment: Thaw and clean the squid skin, soak it in a 0.5% sodium hydroxide aqueous solution for 12 hours, wash it, soak it in a 0.2% acetic acid aqueous solution for 9 hours, wash it, drain it, chop it, control the vacuum degree to -0.09MPa, dry it at 32℃ to constant weight, and pass it through a 240-mesh sieve to obtain pretreated squid skin.

[0040] S2. Acidic Extraction: Pretreated squid skin was added to a 0.5% acetic acid aqueous solution, along with pepsin. The mixture was heated to 38°C and stirred at 850 rpm for 1.4 hours. Tannic acid, phytic acid, and sulfonated soybean lecithin were then added, and stirring continued for 0.3 hours while maintaining the same stirring speed. The mixture was then centrifuged at 8200 rpm for 14 minutes. The supernatant was collected, and the pH of the supernatant was adjusted to 3.1 with a 3% citric acid aqueous solution. The mixture was then centrifuged at 8600 rpm for 12 minutes. The supernatant was collected and combined... The supernatant was added twice, and a 20% sodium chloride aqueous solution was added. The precipitate was collected and washed four times each with anhydrous ethanol and deionized water (each time the mass of anhydrous ethanol and deionized water was 25% of the mass of acetic acid aqueous solution). The precipitate was freeze-dried at -38℃, under a vacuum of 12 Pa, for 24 hours to obtain crude squid protein extract. The mass ratio of pretreated squid skin, acetic acid aqueous solution, pepsin, tannic acid, phytic acid, sulfonated soybean lecithin, pepsin, and sodium chloride aqueous solution was 1:14:0.002:0.005:0.02:0.04:10.

[0041] S3. Enzymatic hydrolysis: Squid protein crude extract, anthocyanins, and magnesium gluconate were added to deionized water. The pH was adjusted to 6.6 using disodium hydrogen phosphate-potassium dihydrogen phosphate buffer. Papain was then added for a single enzymatic hydrolysis, controlled at 44℃ for 2.8 hours. The mixture was centrifuged at 9200 rpm for 12 minutes. The supernatant was collected as the first hydrolysate. The pH of the first hydrolysate was then adjusted to 8.2 using 1% Tris-HCl buffer. Trypsin was then added... The squid protein extract and alkaline protease were subjected to a second enzymatic hydrolysis, with the hydrolysis temperature controlled at 48℃ and the hydrolysis time at 2.4 h. After centrifugation, the centrifugation speed was controlled at 8400 rpm and the centrifugation time was 14 min. The supernatant was collected to obtain the second enzymatic hydrolysate. The mass ratio of squid protein crude extract, anthocyanins, magnesium gluconate, deionized water, and papain was 3:0.04:0.007:100:0.011; the mass ratio of the first enzymatic hydrolysate, trypsin, and alkaline protease was 50:0.008:0.0035.

[0042] S4. Ultrafiltration and centrifugation: Centrifuge the secondary enzymatic hydrolysate at 8500g and 4℃ for 25min. Transfer the supernatant obtained after centrifugation to a 3kDa ultrafiltration tube and centrifuge at 3500g and 4℃ for 45min to complete ultrafiltration. Freeze-dry at -35℃, vacuum degree of 13Pa, and drying time of 36h. After freeze-drying, marine biological protein for sensitive skin whitening is obtained.

[0043] The sulfonated soybean lecithin is prepared by the following steps:

[0044] A1. Add soybean lecithin and fumaric acid to a 45% (w / w) aqueous ethanol solution, control the stirring speed at 650 rpm, stir for 20 min until homogeneous, raise the temperature to reflux temperature, maintain the stirring speed, and continue stirring for 1.6 h. Filter, wash 4 times with deionized water (each time the mass of deionized water is 50% of the mass of the aqueous ethanol solution), and dry at 60℃ to constant weight to obtain carboxylated soybean lecithin, wherein the mass ratio of soybean lecithin, fumaric acid and aqueous ethanol solution is 1:0.06:55;

[0045] A2. Carboxylated soybean lecithin and sodium metabisulfite were added to deionized water, and the pH was adjusted to 6.6 with citrate-sodium citrate buffer. The temperature was raised to 78°C, and the stirring speed was controlled at 550 rpm for 2.6 h. After the reaction was completed, the mixture was filtered and washed four times each with 75% ethanol aqueous solution and deionized water (each time the mass of ethanol aqueous solution and deionized water was 50% of the mass of citrate-sodium citrate buffer). The mixture was dried at 68°C to constant weight to obtain sulfonated soybean lecithin. The mass ratio of carboxylated soybean lecithin, sodium metabisulfite and deionized water was 1:3.9:65.

[0046] Example 3

[0047] An extraction process for marine biological proteins used for whitening sensitive skin includes the following steps:

[0048] S1. Pretreatment: Thaw and clean the squid skin, soak it in a 0.6% sodium hydroxide aqueous solution for 14 hours, wash it, soak it in a 0.3% acetic acid aqueous solution for 10 hours, wash it, drain it, chop it, control the vacuum degree to -0.08MPa, dry it at 34℃ to constant weight, and pass it through a 280-mesh sieve to obtain pretreated squid skin;

[0049] S2. Acidic Extraction: Pretreated squid skin was added to a 0.54% acetic acid aqueous solution, along with pepsin. The mixture was heated to 36°C and stirred at 900 rpm for 1.6 hours. Tannic acid, phytic acid, and sulfonated soybean lecithin were then added, and stirring continued for 0.4 hours while maintaining the same stirring speed. The mixture was then centrifuged at 8400 rpm for 12 minutes. The supernatant was collected, and the pH of the supernatant was adjusted to 3.4 with a 5% citric acid aqueous solution. The mixture was then centrifuged at 8400 rpm for 14 minutes. The supernatant was collected. The supernatants from the two treatments were combined, and a 20% sodium chloride aqueous solution was added. The precipitate was collected and washed five times each with anhydrous ethanol and deionized water (each time the mass of anhydrous ethanol and deionized water was 25% of the mass of the acetic acid aqueous solution). The precipitate was then freeze-dried at -35°C, under a vacuum of 13 Pa, for 26 hours to obtain crude squid protein extract. The mass ratio of pretreated squid skin, acetic acid aqueous solution, pepsin, tannic acid, phytic acid, sulfonated soybean lecithin, and sodium chloride aqueous solution was 1:16:0.003:0.006:0.03:0.05:12.

[0050] S3. Enzymatic hydrolysis: Squid protein crude extract, anthocyanins, and magnesium gluconate were added to deionized water. The pH was adjusted to 6.8 using disodium hydrogen phosphate-potassium dihydrogen phosphate buffer. Papain was then added for a single enzymatic hydrolysis, controlled at 46℃ for 2.6 hours. The mixture was centrifuged at 9400 rpm for 10 minutes. The supernatant was collected as the first hydrolysate. The pH of the first hydrolysate was then adjusted to 8.6 using 1.2% Tris-HCl buffer. Papain was then added... Trypsin and alkaline protease were subjected to a second enzymatic hydrolysis, with the hydrolysis temperature controlled at 50℃ and the hydrolysis time at 2.2 h. After centrifugation, the centrifugation speed was controlled at 8600 rpm and the centrifugation time was 16 min. The supernatant was collected to obtain the second enzymatic hydrolysate. The mass ratio of squid protein crude extract, anthocyanins, magnesium gluconate, deionized water, and papain was 4:0.05:0.008:110:0.014; the mass ratio of the first enzymatic hydrolysate, trypsin, and alkaline protease was 60:0.01:0.005.

[0051] S4. Ultrafiltration and centrifugation: Centrifuge the secondary enzymatic hydrolysate at 9000g and 4℃ for 20min. Transfer the supernatant obtained after centrifugation to a 3kDa ultrafiltration tube and centrifuge at 4000g and 4℃ for 40min to complete ultrafiltration. Freeze-dry at -30℃ and 15Pa for 38h. After freeze-drying, marine biological protein for sensitive skin whitening is obtained.

[0052] The sulfonated soybean lecithin is prepared by the following steps:

[0053] A1. Add soybean lecithin and fumaric acid to a 50% (w / w) aqueous ethanol solution, control the stirring speed at 700 rpm, stir for 18 min until homogeneous, raise the temperature to reflux temperature, maintain the stirring speed, and continue stirring for 1.8 h. Filter, wash 5 times with deionized water (each time the mass of deionized water is 50% of the mass of the aqueous ethanol solution), and dry at 65℃ to constant weight to obtain carboxylated soybean lecithin, wherein the mass ratio of soybean lecithin, fumaric acid and aqueous ethanol solution is 1:0.07:60;

[0054] A2. Carboxylated soybean lecithin and sodium metabisulfite were added to deionized water, and the pH was adjusted to 6.8 with citrate-sodium citrate buffer. The temperature was raised to 80°C, and the stirring speed was controlled at 500 rpm for 2.8 h. After the reaction was completed, the mixture was filtered and washed 5 times each with 80% ethanol aqueous solution and deionized water (each ethanol aqueous solution and deionized water were 50% of the mass of citrate-sodium citrate buffer). The mixture was dried at 74°C to constant weight to obtain sulfonated soybean lecithin. The mass ratio of carboxylated soybean lecithin, sodium metabisulfite and deionized water was 1:4.2:70.

[0055] Comparative Example 1

[0056] Comparative Example 1 is the same as Example 1, except that the sulfonated soybean lecithin in Example 1 is replaced with soybean lecithin, while the other steps and raw materials are the same as in Example 1.

[0057] Comparative Example 2

[0058] Comparative Example 2 is the same as Example 1, except that the tannic acid in Example 1 is replaced with phytic acid in equal mass, while the other steps and raw materials are the same as in Example 1.

[0059] Comparative Example 3

[0060] Comparative Example 3 is the same as Example 1, except that the phytic acid in Example 1 is replaced with tannic acid, while the other steps and raw materials are the same as in Example 1.

[0061] Comparative Example 4

[0062] Comparative Example 4 is the same as Example 1, except that the anthocyanins in Example 1 are replaced with magnesium gluconate, while the other steps and raw materials are the same as in Example 1.

[0063] Comparative Example 5

[0064] Comparative Example 4 is the same as Example 1, except that the magnesium gluconate in Example 1 is replaced with anthocyanins, while the other steps and raw materials are the same as in Example 1.

[0065] Performance testing

[0066] 1. Determination of the yield and extraction rate of marine biological proteins used for skin whitening in sensitive skin.

[0067] 1.1 Purity determination: The purity was determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

[0068] 1.2 Extraction Rate Determination: The purity and extraction rate of the marine biological proteins prepared in Examples 1-3 and Comparative Examples 1-5 were determined according to GB / T 9695.23-2008 "Determination of Hydroxyproline Content in Meat and Meat Products". The extraction rate of marine biological proteins is the ratio of the total mass of extracted collagen to the total mass of collagen in the squid skin raw material. The specific conversion formula is as follows:

[0069]

[0070] B = A × 7.64

[0071] In the formula: A is the percentage content of hydroxyproline; M1 is the mass of hydroxyproline, µg; M2 is the total mass of collagen in the pretreated squid skin raw material, mg; B is the collagen extraction rate.

[0072] The specific test results are shown in Table 1.

[0073] Table 1. Results of purity and extraction rate determination of marine biological proteins prepared in Examples 1-3 and Comparative Examples 1-5.

[0074]

[0075] As can be seen from Table 1, the marine biological proteins prepared by the methods provided in Examples 1-3 have higher purity and extraction rate compared to Comparative Examples 1-5.

[0076] 2. Irritating

[0077] Marine biological proteins for sensitive skin whitening prepared in Examples 1-3 and Comparative Examples 1-5 of this invention were used as test samples. The irritant properties of the above-mentioned marine biological proteins for sensitive skin whitening were verified by patch test. The specific method was as follows: 56 volunteers aged 18-50 years were selected, and the wells of the patch tester were numbered. The test sample was placed into the corresponding numbered well of the patch tester, with a dosage of 0.02 mL per well. The patch tester containing the test sample was applied to the skin of the test volunteer's arm with hypoallergenic adhesive tape. The patch was then gently pressed with the palm of the hand to make it evenly applied to the skin. The patch tester was removed after 24 hours, and the skin condition at the wound site was observed. The identification criteria are shown in Table 2.

[0078] Table 2. Irritation assay of marine biological proteins prepared in Examples 1-3 and Comparative Examples 1-5

[0079]

[0080] The results of the patch test were all negative, indicating that the marine biological protein for sensitive skin whitening prepared using this invention meets the requirement of being non-irritating.

[0081] 3. Antioxidant activity assay

[0082] Accurately weigh 3.94 mg of DPPH and prepare a 0.1 mmol / L DPPH solution. Take 2 mL of the marine biological protein aqueous solution for sensitive skin whitening prepared in Examples 1-3 and Comparative Examples 1-5 with a mass concentration of 0.2 mg / mL and mix it with 2 mL of DPPH solution. React at room temperature in the dark for 30 min. Measure the absorbance at a wavelength of 517 nm. Record the absorbance value as A. i Anhydrous ethanol and water were used to replace DPPH solution and peptide solution, respectively, as the control group and blank group. The absorbance was measured and recorded as A. j And A0; using VC as a control, its DPPH free radical scavenging ability was evaluated. The antioxidant capacity calculation formula is as follows:

[0083]

[0084] The specific results are shown in Table 3.

[0085] Table 3. Results of antioxidant activity assays for marine biological proteins prepared in Examples 1-3 and Comparative Examples 1-5.

[0086]

[0087] As shown in Table 3, the antioxidant properties of the marine biological proteins prepared in Examples 1-3 of this invention are significantly better than those in Comparative Examples 1-5. This indicates that the marine biological proteins prepared by this invention can reduce free radical damage and indirectly improve skin color problems caused by oxidative stress, such as dullness and yellowing. They have a good whitening effect and have broad application prospects in the cosmetics field.

[0088] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. An extraction process for marine biological proteins used for whitening sensitive skin, characterized in that, Includes the following steps: S1. Pretreatment: Thaw and clean the squid skin, soak it in alkaline solution for 10-14 hours, wash it, soak it in acid solution for 8-10 hours, wash it, drain it, chop it, dry it, and sieve it to obtain pretreated squid skin. S2. Acidic extraction: Add pretreated squid skin to an acetic acid aqueous solution, add pepsin, heat to 36-40℃, stir for 1.2-1.6h, then add tannic acid, phytic acid and sulfonated soybean lecithin, continue stirring for 0.2-0.4h, centrifuge, collect the supernatant, adjust the pH of the supernatant to 2.8-3.4, centrifuge, collect the supernatant, combine the two supernatants, add sodium chloride aqueous solution, collect the precipitate, wash, dry, and obtain crude squid protein extract; S3. Enzymatic hydrolysis: Add squid protein crude extract, anthocyanins and magnesium gluconate to deionized water, adjust the pH to 6.4-6.8, then add papain, perform a first enzymatic hydrolysis, centrifuge, and collect the supernatant to obtain the first enzymatic hydrolysate; adjust the pH of the first enzymatic hydrolysate to 7.8-8.6, then add trypsin and alkaline protease, perform a second enzymatic hydrolysis, centrifuge, and collect the supernatant to obtain the second enzymatic hydrolysate; S4. Ultrafiltration and centrifugation: Centrifuge, ultrafilter, and freeze-dry the secondary enzymatic hydrolysate to obtain marine biological protein for skin whitening of sensitive skin.

2. The extraction process of marine biological proteins for sensitive skin whitening according to claim 1, characterized in that, In S1, the alkaline solution is a sodium hydroxide aqueous solution with a mass fraction of 0.4-0.6%, and the acid solution is an acetic acid aqueous solution with a mass fraction of 0.1-0.3%.

3. The extraction process of marine biological proteins for sensitive skin whitening according to claim 1, characterized in that, In S2, the mass fraction of the acetic acid aqueous solution is 0.46-0.54%, and the mass fraction of the sodium chloride aqueous solution is 20%.

4. The extraction process of marine biological proteins for sensitive skin whitening according to claim 1, characterized in that, In step S2, the mass ratio of pretreated squid skin, acetic acid aqueous solution, pepsin, tannic acid, phytic acid, sulfonated soybean lecithin, and sodium chloride aqueous solution is 1:12-16:0.001-0.003:0.004-0.006:0.01-0.03:0.03-0.05:8-12.

5. The extraction process of marine biological proteins for sensitive skin whitening according to claim 1, characterized in that, In S3, the mass ratio of squid protein crude extract, anthocyanins, magnesium gluconate, deionized water, and papain is 2-4:0.03-0.05:0.006-0.008:90-110:0.008-0.014; the mass ratio of primary enzymatic hydrolysate, trypsin, and alkaline protease is 40-60:0.006-0.01:0.002-0.

005.

6. The extraction process of marine biological proteins for sensitive skin whitening according to claim 1, characterized in that, In S3, the enzymatic hydrolysis temperature for the first enzymatic hydrolysis is 42-46℃ and the enzymatic hydrolysis time is 2.6-3h; the enzymatic hydrolysis temperature for the second enzymatic hydrolysis is 46-50℃ and the enzymatic hydrolysis time is 2.2-2.6h.

7. The extraction process of marine biological proteins for sensitive skin whitening according to claim 1, characterized in that, The sulfonated soybean lecithin in S2 is specifically prepared by the following steps: A1. Add soybean lecithin and fumaric acid to an ethanol aqueous solution, stir evenly, heat to reflux temperature, stir for 1.4-1.8 h, filter, wash, and dry to obtain carboxylated soybean lecithin. A2. Add carboxylated soybean lecithin and sodium metabisulfite to deionized water, adjust the pH to 6.4-6.8, heat to 76-80℃, stir for 2.4-2.8 hours, filter, wash, and dry to obtain sulfonated soybean lecithin.

8. The extraction process of marine biological proteins for sensitive skin whitening according to claim 7, characterized in that, In A1, the mass ratio of soybean lecithin, fumaric acid, and ethanol aqueous solution is 1:0.05-0.07:50-60.

9. The extraction process of marine biological proteins for sensitive skin whitening according to claim 7, characterized in that, In A2, the mass ratio of carboxylated soybean lecithin, sodium metabisulfite, and deionized water is 1:3.6-4.2:60-70.

10. The extraction process of marine biological protein for sensitive skin whitening according to claim 7, characterized in that, In A1, the mass fraction of the aqueous ethanol solution is 40-50%.

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