A method for preparing a hydrolyzed conchiolin and its use

Abstract

The present application relates to the biochemical field, provide a kind of preparation of hydrolyzed conchiolin method, comprising:1) pearl powder is added to weak acid aqueous solution, stir the suspension obtained 6 hours or more;2) after adjusting the pH of the suspension to 2.5-3.5, add acid protease treatment 2 hours or more;And 3) temperature inactivation of the acid protease, centrifugal, after adjusting the supernatant to neutral, over 20kD filter membrane, the filtrate obtained is hydrolyzed conchiolin solution.The present application also provides the use of the hydrolyzed conchiolin in the preparation of cosmetics.

Description

Technical Field

[0001] This invention relates to a method for preparing hydrolyzed conchiolin, and more particularly to a method for preparing hydrolyzed conchiolin by treating pearl powder with a weak acid and a protease. This invention also relates to the whitening applications of this hydrolyzed conchiolin. Background Technology

[0002] Conchiolin is a type of protein tightly bound to calcium salts, mainly found in shells and pearls, forming the lustrous structure of the nacreous layer. Some cosmetic manufacturers have begun using hydrolyzed (acid or alkali) or enzymatically hydrolyzed products of shells or pearls (called "hydrolyzed conchiolin") as nutritional additives in cosmetics, achieving effects such as moisturizing and whitening.

[0003] Chinese patent publication CN111053729A discloses a method for preparing hydrolyzed conchiolin, which includes soaking conchiolin powder in potassium hydroxide solution followed by treatment with protease. The hydrolyzed conchiolin prepared in this method is used in hair conditioner, but it does not mention whether it has endothelin antagonistic function.

[0004] Although hydrolyzed conchiolin has been used in a variety of cosmetic products, there is a lack of detailed descriptions of how to obtain it in existing literature. Summary of the Invention

[0005] On the one hand, this article provides a method for preparing hydrolyzed conchiolin, including:

[0006] 1) Add pearl powder to an aqueous solution containing acetic acid and oxalic acid, and stir to form a suspension for more than 6 hours;

[0007] 2) After adjusting the pH of the suspension to 2.5-3.5, add acidic protease and treat for at least 2 hours; and

[0008] 3) Inactivate the acidic protease by heating, centrifuge, adjust the resulting supernatant to neutral, and filter through a 20kD filter membrane. The resulting filtrate is the hydrolyzed shell scleroprotein solution.

[0009] In some embodiments, the aqueous solution containing acetic acid and oxalic acid has an acetic acid concentration of 1.5 M and an oxalic acid concentration of 1.0 M.

[0010] In some embodiments, the pearl powder is added to the aqueous solution containing acetic acid and oxalic acid in an amount of 10% to 35%.

[0011] In some embodiments, the amount of the acidic protease used is 0.5-1%.

[0012] In some embodiments, the method includes: pulverizing dried pearls and passing them through an 80-mesh sieve; adding them at a concentration of 25% to an aqueous solution containing 1.5M acetic acid and 1.0M oxalic acid; stirring the resulting suspension at room temperature for 8 hours; adjusting the pH of the suspension to 3.0 with sodium hydroxide powder; adding 0.5% acidic protease; stirring at 40°C for 3 hours; inactivating the enzyme by heating to 90°C for 20 minutes; centrifuging at 2000g for 10 minutes; adjusting the resulting supernatant to neutral with 0.5M sodium hydroxide; passing the supernatant through a 20kD ultrafiltration membrane; and collecting the filtrate.

[0013] In some embodiments, the method includes: pulverizing dried pearl powder and passing it through an 80-mesh sieve; adding it at a concentration of 30% to an aqueous solution containing 1.5M acetic acid and 1.0M oxalic acid; stirring the resulting mixture at room temperature for 6 hours; adjusting the pH of the suspension to 3.0 with sodium hydroxide powder; adding 1.0% acidic protease; stirring at 40°C for 2 hours; inactivating the enzyme by heating to 90°C for 20 minutes; centrifuging at 2000g for 10 minutes; adjusting the resulting supernatant to neutral with 0.5M sodium hydroxide; passing the supernatant through a 20kD ultrafiltration membrane; and collecting the filtrate.

[0014] In some embodiments, the method further includes concentrating the filtrate and then freeze-drying it to obtain hydrolyzed conchiolin powder.

[0015] On the other hand, the present invention provides hydrolyzed shell scleroprotein prepared by the above method.

[0016] On the other hand, the present invention provides the use of the above-mentioned hydrolyzed conchiolin in the preparation of whitening or anti-photoaging cosmetics.

[0017] The method for preparing hydrolyzed shell scleroprotein provided in this article is mild, simple to operate, and suitable for large-scale industrial production. Detailed Implementation

[0018] Unless otherwise stated, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art.

[0019] The term "includes" means "including but not limited to", but also refers to situations consisting only of the listed elements.

[0020] When referring to the pH value of a solution, the term "neutral" means a pH between 6 and 8, especially pH 7.0.

[0021] Unless otherwise stated, concentrations expressed as percentages in this document are mass concentrations (wt%).

[0022] The inventors have discovered a method for obtaining hydrolyzed conchiolin with endothelin antagonism and anti-photoaging effects, comprising first treating pearl powder with acetic acid and oxalic acid, and then enzymatically hydrolyzing it with acid protease.

[0023] The following detailed description is provided through examples.

[0024] Example 1: Dried pearl powder was pulverized and passed through an 80-mesh sieve. It was added at a concentration of 25% to an aqueous solution containing 1.5M acetic acid and 1.0M oxalic acid. The resulting mixture was stirred at room temperature for 8 hours. The pH was adjusted to approximately 3.0 with sodium hydroxide powder, and then 0.5% acidic protease (from Aspergillus niger, 30,000 U / g, Nanning Dongheng Huadao) was added. The mixture was stirred at 40°C for 3 hours. The enzyme was inactivated by heating to 90°C for 20 minutes. The mixture was centrifuged at 2000g for 10 minutes. The supernatant was adjusted to neutral with 0.5M sodium hydroxide, filtered through a 20kD ultrafiltration membrane, and the filtrate was collected as the hydrolyzed conchiolin solution. This hydrolyzed conchiolin solution can be stored at 4°C for a short period (no more than one week) or concentrated and then freeze-dried.

[0025] Example 2: Dried pearl powder was pulverized and passed through an 80-mesh sieve. It was added at a concentration of 30% to an aqueous solution containing 1.5M acetic acid and 1.0M oxalic acid. The resulting mixture was stirred at room temperature for 6 hours. The pH was adjusted to approximately 3.0 with sodium hydroxide powder, and 1.0% acidic protease was added. The mixture was stirred at 40°C for 2 hours. The enzyme was inactivated by heating to 90°C for 20 minutes. The mixture was centrifuged at 2000g for 10 minutes. The supernatant was adjusted to neutral with 0.5M sodium hydroxide, filtered through a 20kD ultrafiltration membrane, and the filtrate was collected as the hydrolyzed conchoidal scleroprotein solution.

[0026] Comparative Example 1

[0027] After pulverizing the dried pearl powder and passing it through an 80-mesh sieve, add it to a 1.5M acetic acid aqueous solution at a concentration of 25%. Stir the resulting mixture at room temperature for 8 hours. Adjust the pH to approximately 3.0 with sodium hydroxide powder, then add 0.5% acidic protease and stir at 40°C for 3 hours. Inactivate the enzyme by heating to 90°C for 20 minutes. Centrifuge at 2000g for 10 minutes, adjust the supernatant to neutral with 0.5M sodium hydroxide, filter through a 20kD ultrafiltration membrane, and collect the filtrate.

[0028] The difference from Example 1 is that oxalic acid is not used.

[0029] Comparative Example 2

[0030] After pulverizing the dried pearl powder and passing it through an 80-mesh sieve, add it to a 1.0M oxalic acid aqueous solution at a concentration of 25%. Stir the resulting mixture at room temperature for 8 hours. Adjust the pH to approximately 3.0 with sodium hydroxide powder, then add 0.5% acidic protease and stir at 40°C for 3 hours. Inactivate the enzyme by heating to 90°C for 20 minutes. Centrifuge at 2000g for 10 minutes, adjust the supernatant to neutral with 0.5M sodium hydroxide, filter through a 20kD ultrafiltration membrane, and collect the filtrate.

[0031] The difference from Example 1 is that acetic acid is not used.

[0032] Comparative Example 3

[0033] After drying, the pearl powder was crushed and passed through an 80-mesh sieve. It was then added at a concentration of 25% to an aqueous solution containing 1.5M acetic acid and 1.0M oxalic acid. The resulting mixture was stirred at room temperature for 8 hours. The pH was adjusted to approximately 3.0 with sodium hydroxide powder, and the mixture was centrifuged at 2000g for 10 minutes. The supernatant was adjusted to neutral with 0.5M sodium hydroxide, filtered through a 20kD ultrafiltration membrane, and the filtrate was collected.

[0034] The difference from Example 1 is that the acidic protease treatment step is not included.

[0035] Comparative Example 4

[0036] The dried pearl powder was pulverized and passed through an 80-mesh sieve. It was then added at a concentration of 25% to an aqueous solution containing 1.5M acetic acid and 1.0M oxalic acid. The resulting mixture was stirred at room temperature for 8 hours. The pH was adjusted to approximately 3.0 with sodium hydroxide powder, and then 1.0% acidic protease (Pepsin, extracted from pig stomach, 10,000 U / g, Shanghai Yuanye Biotechnology) was added. The mixture was stirred at 37℃ for 3 hours. The enzyme was then inactivated by heating to 90℃ for 20 minutes. The mixture was centrifuged at 2000g for 10 minutes. The supernatant was adjusted to neutral with 0.5M sodium hydroxide, filtered through a 20kD ultrafiltration membrane, and the filtrate was collected.

[0037] The difference from Example 1 is that pepsin is used instead of acidic protease for treatment.

[0038] Comparative Example 5

[0039] After drying, the pearl powder was crushed and passed through an 80-mesh sieve. It was then added to an aqueous solution containing 1.5M sulfuric acid at a concentration of 25%. The resulting mixture was stirred at room temperature for 8 hours. The pH was adjusted to approximately 3.0 with sodium hydroxide powder, and the mixture was centrifuged at 2000g for 10 minutes. The supernatant was adjusted to neutral with 0.5M sodium hydroxide, filtered through a 20kD ultrafiltration membrane, and the filtrate was collected.

[0040] Comparative Example 6

[0041] After pulverizing the dried pearl powder and passing it through an 80-mesh sieve, add it to a 1.5M sulfuric acid aqueous solution at a concentration of 25%. Stir the resulting mixture at room temperature for 8 hours. Adjust the pH to approximately 3.0 with sodium hydroxide powder, then add 0.5% acidic protease and stir at 40°C for 3 hours. Inactivate the enzyme by heating to 90°C for 20 minutes. Centrifuge at 2000g for 10 minutes, adjust the supernatant to neutral with 0.5M sodium hydroxide, filter through a 20kD ultrafiltration membrane, and collect the filtrate.

[0042] Comparative Example 7

[0043] This embodiment basically follows the method described in CN111053729A to prepare hydrolyzed shell scleroprotein.

[0044] After drying and crushing the seashells, pass them through an 80-mesh sieve. Add 2M potassium hydroxide aqueous solution equal to the weight of the seashell powder, stir, and soak for 5 hours to obtain the soaking material. Adjust the pH of the soaking material to 7.0 with concentrated hydrochloric acid, add 1% of the raw material mass of neutral proteinase (50,000 U / g, Hebei Zhongzhisheng Biotechnology Co., Ltd.) (since CN111053729A does not disclose the specific proteinase, neutral proteinase is used here instead), and incubate at 45℃ for 2 hours to obtain the enzymatic hydrolysate. Centrifuge 2000g of the enzymatic hydrolysate for 20 minutes to obtain the supernatant. Filter the supernatant through a 50kD ultrafiltration membrane to obtain the filtrate.

[0045] Example 3: This example tests the endothelin antagonistic function of the filtrates prepared in Examples 1 and 2 and Comparative Examples 1-7.

[0046] Experimental principle: Endothelin (such as that produced by keratinocytes under ultraviolet radiation) binds to melanocyte receptors, stimulating melanocyte proliferation and differentiation, activating tyrosinase activity, and ultimately increasing melanin synthesis. The endothelin antagonistic ability of the test substance can be reflected by detecting the melanin content of the cells using the sodium hydroxide lysis method.

[0047] Human A375 melanoma cells were diluted with DMEM medium (containing 10% FBS and 1% P / S) to a concentration of 1. 10 5Cell suspension of 100 μL / mL was seeded into 96-well plates. After 24 hours of adherent culture at 37°C and 5% CO2, the supernatant was discarded, and each group was cultured in fresh DMEM medium (containing 10% FBS and 1% P / S) supplemented with the test sample for another 72 hours. The cells and melanin granules were dissolved by rinsing twice with PBS buffer, and 100 μL of 1M NaOH solution containing 10% DSMO was added to each well. The plates were then incubated at 80°C with shaking for 10 min. The absorbance (OD value) was measured at 405 nm using a microplate reader. The blank wells (containing only medium) were zeroed. The relative melanin content of cells in each group was calculated using the OD value, with the blank control group recorded as 100%. Five replicates were set for each group, and the mean value was taken.

[0048] The experimental groups are as follows:

[0049] Blank control group: Fresh DMEM culture medium (without test substance);

[0050] Positive control group: fresh DMEM medium + 0.5 μg / mL endothelin;

[0051] Experimental group: fresh DMEM medium + 0.5 μg / mL endothelin + 5% (v / v) filtrate (previous experiments showed that adding up to 10% (v / v) of the above filtrate to the medium had no effect on cell growth).

[0052] The results are shown in Table 1.

[0053] Table 1. Results of endothelin antagonism in various filtrates

[0054]

[0055] p<0.05; p<0.01, vs positive control group

[0056] As shown in Table 1, only the filtrates from Examples 1 and 2 exhibited endothelin antagonistic activity. This indicates that treatment with acetic acid and oxalic acid, as well as acidic protease, is crucial for the presence of endothelin antagonistic activity in the resulting filtrates. The absence of either acetic acid or oxalic acid, or its substitution with other acids (such as sulfuric acid), or the absence of acidic protease treatment or its substitution with other proteases (such as pepsin), resulted in no tyrosinase antagonistic activity in the filtrates. Hydrolyzed conchiolin prepared by the method described in CN111053729A also did not exhibit endothelin antagonistic activity.

[0057] We hypothesize that an acidic environment facilitates the separation of conchoidal proteins from other components in pearl powder, and that oxalic acid reacts with dissociated calcium ions in the pearl powder to form insoluble precipitates, thus promoting this process. Furthermore, this weakly acidic environment (relative to sulfuric acid) may aid in the selective preliminary hydrolysis of conchoidal proteins, and further enzymatic hydrolysis by acidic proteases may yield peptides that inhibit tyrosinase. Further analysis is needed to determine which peptides exhibit endothelin antagonistic activity.

[0058] Example 4 We also found that the filtrates from Examples 1 and 2 have anti-photoaging effects.

[0059] Long-term exposure to ultraviolet (UV) radiation from the sun, especially UVB (290-320nm), can cause cell damage to the skin, resulting in obvious signs of skin aging, such as sagging skin, reduced moisture retention, wrinkles, and age spots.

[0060] HaCaT cells are immortalized human keratinocytes that are morphologically and biologically similar to normal keratinocytes. Their activity is easily affected by UVB irradiation, and therefore they are often used to evaluate the anti-photoaging effects of some test products. When cells are damaged, lactate dehydrogenase (LDH) is released extracellularly. The extent of cell damage can be determined by detecting the LDH content or activity in the cell culture supernatant.

[0061] HaCaT cells were harvested, and the cell suspension concentration was adjusted to 5%. 10 5 Cells / mL. 100 μL of cell suspension was seeded into each well of a 96-well cell culture plate and cultured at 37°C and 5% CO2 for 24 hours. The supernatant was then discarded. Each group was further cultured in fresh DMEM medium (containing 10% FBS and 1% P / S) supplemented with the test sample for 24 hours, followed by UVB treatment (50 mJ / cm²). 2 The culture was continued for 24 hours, and the LDH content in the culture supernatant was detected using an LDH kit (reflected by the absorbance value of formazan at a wavelength of 490 nm). The LDH content of the culture supernatant in the blank control group was recorded as 100%. Five replicates were set for each group, and the mean value was taken.

[0062] The experimental groups are as follows:

[0063] Blank control group: Fresh DMEM medium (without filtrate or UVB treatment);

[0064] Positive control group: Fresh DMEM medium + UVB treatment (without adding filtrate)

[0065] Experimental group: fresh DMEM medium + 5% (v / v) filtrate (previous experiments showed that adding up to 10% (v / v) of the above filtrate to the medium had no effect on cell growth) + UVB treatment.

[0066] The results are shown in Table 2.

[0067] Table 2. Results of photoaging resistance of various filtrates

[0068]

[0069] p<0.01; p<0.001, vs positive control group

[0070] As shown in Table 2, the filtrates from Examples 1 and 2 exhibit significant anti-UVB damage effects. Since the filtrates prepared in each comparative example did not demonstrate any anti-UVB damage effect, their specific data are not listed in Table 2.

Claims

1. A method for preparing hydrolyzed conchiolin, comprising: 1) Add pearl powder to an aqueous solution containing acetic acid and oxalic acid, and stir to form a suspension for more than 6 hours; 2) After adjusting the pH of the suspension to 2.5-3.5, add acidic protease and treat for more than 2 hours; 3) Inactivate the acidic protease by heating, centrifuge, adjust the supernatant to neutral, and filter through a 20kD filter membrane. The resulting filtrate is the hydrolyzed conchiolin protein solution. The aqueous solution containing acetic acid and oxalic acid has an acetic acid concentration of 1.5M and an oxalic acid concentration of 1.0M. The pearl powder is added to the aqueous solution containing acetic acid and oxalic acid at a concentration of 10% to 35%, and the amount of acidic protease is 0.5-1.0%. The acidic protease is derived from Aspergillus niger.

2. The method of claim 1, comprising: The dried pearl powder was crushed and passed through an 80-mesh sieve. It was added at a concentration of 25% to an aqueous solution containing 1.5M acetic acid and 1.0M oxalic acid. The resulting suspension was stirred at room temperature for 8 hours. The pH of the suspension was adjusted to 3.0 with sodium hydroxide powder, and 0.5% of the acidic protease was added. The mixture was stirred at 40°C for 3 hours. The enzyme was inactivated by heating to 90°C for 20 minutes. The mixture was centrifuged at 2000g for 10 minutes. The supernatant was adjusted to neutral with 0.5M sodium hydroxide and filtered through a 20kD ultrafiltration membrane. The filtrate was collected.

3. The method of claim 1, comprising: The dried pearl powder was pulverized and passed through an 80-mesh sieve. It was added at a concentration of 30% to an aqueous solution containing 1.5M acetic acid and 1.0M oxalic acid. The resulting mixture was stirred at room temperature for 6 hours. The pH of the suspension was adjusted to 3.0 with sodium hydroxide powder, and 1.0% of the acidic protease was added. The mixture was stirred at 40°C for 2 hours. The enzyme was inactivated by heating to 90°C for 20 minutes. The mixture was centrifuged at 2000g for 10 minutes. The supernatant was adjusted to neutral with 0.5M sodium hydroxide and filtered through a 20kD ultrafiltration membrane. The filtrate was collected.

4. The method according to any one of claims 1-3 further includes concentrating the filtrate and then freeze-drying it to obtain hydrolyzed conchiolin powder.

5. Hydrolyzed shellac protein prepared by the method according to any one of claims 1-4.

6. The use of the hydrolyzed conchiolin protein according to claim 5 in the preparation of whitening or anti-photoaging cosmetics.

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