A small molecule active peptide from fish maw, an acne-removing composition, an acne-removing handmade soap, and a method for preparing the same.

Abstract

This invention relates to a small-molecule active peptide from fish maw, an acne-removing composition, an acne-removing handmade soap, and their preparation method, belonging to the field of biopharmaceutical manufacturing. The amino acid sequence of the small-molecule active peptide from fish maw is: Tyr-Gly-Arg-Gly-Pro. The acne-removing composition is made from the following components by weight percentage: 20-70% oil composition, 6-20% sodium hydroxide, 10-30% deionized water, 0.1-5% small-molecule active peptide from fish maw, 0.5-3% anti-inflammatory and soothing extract, and 0.05-5% essential oil; the sum of the weight percentages of all components in the acne-removing composition is 100%. This invention prepares highly active small-molecule active peptides from fish maw through targeted enzymatic hydrolysis with a compound enzyme, achieving multiple effects of anti-inflammatory acne removal and gentle cleansing. Specifically targeting facial acne-prone skin, it has significant anti-inflammatory effects and inhibits the activity of Propionibacterium acnes, making it more suitable for various skin types, especially acne-prone and sensitive skin.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical manufacturing technology, specifically relating to a small molecule active peptide from fish maw, an acne-removing composition, an acne-removing handmade soap, and their preparation methods. Background Technology

[0002] Facial acne is a common chronic inflammatory skin disease of the hair follicles and sebaceous glands. Its main causes include excessive sebum secretion, abnormal keratinization of cells around the hair follicles, and inflammation caused by Propionibacterium acnes. Currently, acne treatment products mainly work through the following mechanisms: 1. Inhibiting sebum secretion from the sebaceous glands, reducing oil production at the source, and thus effectively improving facial acne; 2. Promoting the renewal and shedding of keratinocytes, unclogging pores, and achieving acne-reducing effects; 3. Inhibiting the proliferation of microorganisms such as Propionibacterium acnes, thereby reducing inflammation; 4. Inhibiting the release of inflammatory factors, reducing acne redness and swelling, and promoting recovery.

[0003] Currently, most acne-removing handmade soaps on the market use ingredients such as salicylic acid, retinoic acid, and sulfur to achieve acne-removing effects. Although these ingredients are effective, they are too irritating and are mainly chemically synthesized, which can easily lead to dry skin, peeling, redness, and even damage to the skin barrier, causing skin sensitivity. They are not suitable for sensitive skin.

[0004] Fish maw (swim bladder) is rich in collagen, and its hydrolysate, fish maw peptides, has good skin compatibility. Traditional fish maw peptide extraction often uses single proteases (such as trypsin and papain), which has problems such as low enzymatic hydrolysis efficiency, large peptide molecular weight, and poor skin permeability. In addition, the content of active peptides in fish maw is low, making it difficult to exert effective acne-removing effects in handmade soaps.

[0005] Therefore, it is of great significance to develop a new type of handmade acne-removing soap that has both good acne-removing effects and gentle cleansing properties. Summary of the Invention

[0006] The purpose of this invention is to provide a small molecule active peptide from fish maw, an acne-removing composition, an acne-removing handmade soap, and a method for preparing the same, in order to solve the problems of low enzymatic hydrolysis efficiency, large peptide molecular weight, and poor skin permeability in existing fish maw peptide extraction methods, as well as the problems of strong irritation, easy to cause dry skin, peeling, redness, and sensitivity in existing acne-removing handmade soaps, which are not suitable for use on sensitive skin.

[0007] The technical solution adopted by this invention to solve the technical problem is as follows: The present invention provides a small molecule active peptide for fish maw, the amino acid sequence of which is: Tyr-Gly-Arg-Gly-Pro.

[0008] This invention provides an acne-removing composition containing the aforementioned small-molecule active peptides from fish maw, which is made of the following components by weight percentage: 20-70% oil composition, 6-20% sodium hydroxide, 10-30% deionized water, 0.1-5% small-molecule active peptides from fish maw, 0.5-3% anti-inflammatory and soothing extract, and 0.05-5% essential oil; the sum of the weight percentages of all components in the acne-removing composition is 100%.

[0009] Preferably, the oil composition comprises a hard oil, a soft oil, and a superfatting oil; the hard oil is selected from one or more of coconut oil, palm oil, and shea butter; the soft oil is selected from one or more of olive oil, grapeseed oil, castor oil, jojoba oil, and evening primrose oil; and the superfatting oil is selected from one or more of unrefined avocado oil, rice bran oil, and walnut oil.

[0010] Preferably, the anti-inflammatory and soothing extract is selected from one or more of the following: honeysuckle extract, centella asiatica extract, forsythia extract, aloe vera leaf extract, lithospermum erythrorhizon extract, verbena extract, menthol extract, and salvia miltiorrhiza extract.

[0011] Preferably, the essential oil is selected from one or more of tea tree oil, lavender oil, and peppermint oil.

[0012] Preferably, the preparation method of the small molecule active peptides of fish maw is as follows: (1) After crushing the raw fish maw, it is enzymatically hydrolyzed stepwise using a compound enzyme. The enzymatic hydrolysis pH is 7.5-9.0, the temperature is 45-55℃, and the time is 3-6 hours. After enzymatic hydrolysis, the enzyme is inactivated. After centrifugation, the hydrolysate is separated using membrane separation technology. The membrane solution is spray-dried to obtain fish maw oligopeptide powder. (2) Dissolve the fish maw oligopeptide powder in water to prepare a solution with a concentration of 10-100 mg / mL. Separate and purify the solution using Sephadex LH-20 column chromatography with a dextran gel. Measure the absorbance at 280 nm and collect the desired peak based on the absorbance value. Further purify the solution using high performance liquid chromatography with a detection wavelength of 280 nm. Collect the chromatographic peak with a retention time of 26 minutes. After concentration and freeze-drying, obtain the small molecule active peptides of fish maw.

[0013] Preferably, the complex enzyme is composed of alkaline protease, cellulase and trypsin in a mass ratio of (4-6):(3-5):(1-3).

[0014] Preferably, the mobile phase of the dextran gel Sephadex LH-20 column chromatography is 40% methanol, and the flow rate is 0.4-0.6 mL / min; the chromatographic conditions of the high-performance liquid chromatography are: C18 column, mobile phase A is water containing 0.06-0.08% trifluoroacetic acid by volume, mobile phase B is acetonitrile, and the gradient elution conditions are: 0-15 min, 3% B; 15-20 min, 3% B-10% B; 20-30 min, 10% B-20% B; 30-40 min, 20% B-35% B; and the flow rate is 0.8-1.0 mL / min.

[0015] Preferably, the proportion of peptides with a molecular weight of less than 1000 Da in the fish maw oligopeptide powder is greater than 90%.

[0016] This invention provides a method for preparing acne-removing handmade soap using the aforementioned acne-removing composition, comprising the following steps: (1) Preparation of alkaline solution: Add sodium hydroxide to deionized water, dissolve it completely and let it stand at room temperature; (2) Oil and fat classification: The oil and fat composition is classified into hard oil, soft oil and superfatting oil; (3) The soft oil and alkaline solution are mixed and stirred until a slight trace state is reached to obtain the first mixture; (4) Add hard oil to the first mixture and continue stirring until the medium trace state is reached to obtain the second mixture; (5) Add small molecule active peptides of fish maw, anti-inflammatory and soothing extract, essential oil and superfatting oil to the second mixture, stir thoroughly to obtain soap solution; (6) Molding and curing: Pour the soap liquid into the mold, keep it warm for 24-48 hours, then unmold it and let it air dry for 4-6 weeks under ventilation conditions to obtain acne-removing handmade soap.

[0017] The beneficial effects of this invention are: This invention uses small-molecule active peptides from fish maw as the core active ingredient, specifically targeting facial acne-prone skin for cleansing. It exhibits significant anti-inflammatory effects and inhibits the activity of Propionibacterium acnes, offering the following technical advantages compared to existing technologies: (1) In preparing small molecule active peptides of fish maw, the present invention uses a novel composite enzyme system composed of alkaline protease, cellulase and trypsin in a mass ratio of (4-6):(3-5):(1-3) to achieve directional enzymatic hydrolysis of fish maw peptides. The enzymatic hydrolysis efficiency is high, and the content of active peptide segments of fish maw in the obtained product is high and the proportion of peptides with a molecular weight of less than 1000 Da is greater than 90%.

[0018] (2) In preparing small molecule active peptides of fish maw, the present invention uses Sephadex LH-20 column chromatography and high performance liquid chromatography, which have the advantages of simple separation and purification steps and easy acquisition of small molecule active peptides of fish maw, thereby improving the skin penetration rate of small molecule active peptides of fish maw.

[0019] (3) The small molecule active peptides of fish maw prepared in this invention have the amino acid sequence Tyr-Gly-Arg-Gly-Pro. They can interact with the cell membrane of Propionibacterium acnes to destroy its integrity, leading to leakage of cell contents and cell death. At the same time, combined with essential oils and anti-inflammatory and soothing extracts, as well as the oil-removing and cleansing effects of handmade soap itself, they can achieve a synergistic effect of antibacterial, anti-inflammatory and oil-removing, reducing the formation and recurrence of acne.

[0020] (4) The present invention adopts the oil separation method of saponification, which can ensure that the oil is completely saponified, reduce the occurrence of rancidity, and avoid the influence of microorganisms on active ingredients. At the same time, through scientific oil ratio and superfatting process, the product ensures sufficient cleaning power while avoiding excessive degreasing. After washing, the skin feels refreshed and not tight, and will not cause dryness, peeling, redness, sensitivity and other phenomena. It is more friendly to various skin types, especially acne-prone skin and sensitive skin.

[0021] (5) The acne-removing handmade soap prepared by the present invention uses natural ingredients as the active ingredients for acne removal. No additional ingredients are added during the preparation process. It is mild and non-irritating, which is in line with the trend of green development. Detailed Implementation

[0022] In a first aspect, the present invention provides a small molecule active peptide from fish maw.

[0023] The present invention provides a small molecule active peptide for fish maw, the amino acid sequence of which is: Tyr-Gly-Arg-Gly-Pro.

[0024] Secondly, the present invention provides an acne-removing composition containing the above-mentioned small molecule active peptides from fish maw, wherein the acne-removing composition is made of the following components by weight percentage: 20-70% oil composition, 6-20% sodium hydroxide, 10-30% deionized water, 0.1-5% small molecule active peptides from fish maw, 0.5-3% anti-inflammatory and soothing extract, and 0.05-5% essential oil; the sum of the weight percentages of each component of the acne-removing composition is 100%.

[0025] According to a preferred embodiment of the present invention, the oil composition comprises hard oil, soft oil and superfatting oil.

[0026] According to a more preferred embodiment of the present invention, the hard oil is selected from one or more of coconut oil, palm oil and shea butter; the soft oil is selected from one or more of olive oil, grapeseed oil, castor oil, jojoba oil and evening primrose oil; and the superfatting oil is selected from one or more of unrefined avocado oil, rice bran oil and walnut oil.

[0027] According to a preferred embodiment of the present invention, the anti-inflammatory and soothing extract is selected from one or more of honeysuckle extract, centella asiatica extract, forsythia extract, aloe vera leaf extract, lithospermum erythrorhizon extract, verbena extract, menthol extract, and salvia miltiorrhiza extract.

[0028] According to a preferred embodiment of the present invention, the essential oil is selected from one or more of tea tree oil, lavender oil, and peppermint oil.

[0029] According to a preferred embodiment of the present invention, the specific preparation method of small molecule active peptides from fish maw is as follows: (1) Preparation of fish maw oligopeptide powder; After cleaning and crushing, the fish maw raw material is subjected to stepwise enzymatic hydrolysis using a complex enzyme composed of alkaline protease, cellulase, and trypsin in a mass ratio of (4-6):(3-5):(1-3). The hydrolysis pH is 7.5-9.0, the temperature is 45-55℃, and the time is 3-6 hours. After hydrolysis, the enzyme is inactivated, and the hydrolysate is centrifuged at 8000-10000 rpm for 10-15 minutes to remove particulate matter. Then, membrane separation technology is used to separate the oligopeptides, retaining those with a molecular weight cutoff of 3000-5000 Da. The membrane solution is then spray-dried to obtain fish maw oligopeptide powder. The proportion of peptides with a molecular weight less than 1000 Da in the obtained fish maw oligopeptide powder is greater than 90%.

[0030] According to a preferred embodiment of the present invention, the raw material for fish maw is selected from one or more of yellow croaker, cod, grouper, sea bass and sturgeon.

[0031] (2) Preparation of small molecule active peptides from fish maw; Small molecule active peptides from fish maw were obtained by separating and purifying fish maw oligopeptide powder. The fish maw oligopeptide powder was dissolved in water to prepare a solution with a concentration of 10-100 mg / mL. Separation and purification were performed using Sephadex LH-20 column chromatography with a mobile phase of 40% methanol and a flow rate of 0.4-0.6 mL / min. Absorbance was measured at 280 nm, and the desired peak was collected based on the absorbance value. Further purification was performed using high-performance liquid chromatography (HPLC). The chromatographic conditions were as follows: a C18 column; mobile phase A was water containing 0.06-0.08% trifluoroacetic acid (v / v); mobile phase B was acetonitrile; and gradient elution conditions were: 0-15 min, 3% B; 15-20 min, 3% B-10% B; 20-30 min, 10% B-20% B; 30-40 min, 10% B-20% B. The concentration was 20%B-35%B; the flow rate was 0.8-1.0 mL / min; the detection wavelength was 280 nm; the chromatographic peaks with a retention time of 26 minutes were collected; and the chromatographic peaks were concentrated and freeze-dried to obtain small molecule active peptides of fish maw.

[0032] Thirdly, the present invention provides a method for preparing acne-removing handmade soap using the above-mentioned acne-removing composition, which specifically includes the following steps: (1) Preparation of alkaline solution: Add sodium hydroxide to deionized water, dissolve it completely and let it stand at room temperature; (2) Oil and fat classification: The oil and fat composition is classified into hard oil, soft oil and superfatting oil; (3) The soft oil and alkaline solution are mixed and stirred until a slight trace state is reached to obtain the first mixture; (4) Add hard oil to the first mixture and continue stirring until the medium trace state is reached to obtain the second mixture; (5) Add small molecule active peptides of fish maw, anti-inflammatory and soothing extract, essential oil and superfatting oil to the second mixture, stir thoroughly to obtain soap solution; (6) Molding and curing: Pour the soap liquid into the mold, keep it warm for 24-48 hours, then unmold it and let it air dry for 4-6 weeks under ventilation conditions to obtain acne-removing handmade soap.

[0033] According to a preferred embodiment of the present invention, in step (5), the anti-inflammatory and soothing extract, essential oil, and superfatting oil can be added using a separate cooking method. Specifically, the second mixture that has reached a moderate trace state is first divided into a main component and a secondary component. Then, the small molecule active peptides of fish maw are added to the main component. Finally, the anti-inflammatory and soothing extract, essential oil, and superfatting oil are added to the secondary component. After being thoroughly stirred and mixed, the mixture is then combined and injected into a mold to form a pattern.

[0034] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0035] Example 1

[0036] 1. This embodiment prepares a small molecule active peptide from fish maw, and the specific preparation steps are as follows: (1) Preparation of fish maw oligopeptide powder; After cleaning and crushing, the raw fish maw (yellow croaker) was mixed with 20 times its volume of water and homogenized. A stepwise enzymatic hydrolysis was then performed using a compound enzyme consisting of alkaline protease, cellulase, and trypsin in a mass ratio of 4:3:1. The amount of the compound enzyme added was 5% of the raw fish maw mass. Hydrolysis was carried out at 45℃ and pH 8.0 for 3 hours with stirring. After hydrolysis, the temperature was raised to 90℃ for 10 minutes to inactivate the enzyme, yielding the enzymatic hydrolysate. The hydrolysate was centrifuged at 8000 rpm for 10 minutes to remove particulate matter. Membrane separation technology was then used to separate the peptides, with a molecular weight cutoff of 3000 Da. The membrane solution was spray-dried to obtain fish maw oligopeptide powder. The obtained fish maw oligopeptide powder contained 92.5% peptides with a molecular weight less than 1000 Da.

[0037] (2) Preparation of small molecule active peptides from fish maw; The fish maw oligopeptide powder obtained in step (1) was dissolved in water to prepare a solution with a concentration of 100 mg / mL. Separation and purification were performed using Sephadex LH-20 column chromatography (3.0 × 100 cm) with 40% methanol as the mobile phase and a flow rate of 0.4 mL / min. Absorbance was measured at 280 nm, and the desired peak was collected based on the absorbance value. Further purification was performed using high-performance liquid chromatography (HPLC) under the following conditions: Elite C18 column (4.6 mm × 250 mm, 5 µm); mobile phase A was 0.08% trifluoroacetic acid in water (v / v); mobile phase B was acetonitrile; and gradient elution conditions were: 0-15 min, 3% B; 15-20 min, 3% B-10% B; 20-30 min, 10% B-20% B; 30-40 min, 10% B-20% B; 30-40 min, 10% B-20% B. The concentration was 20% B-35% B; the flow rate was 0.8 mL / min; the detection wavelength was 280 nm; and the chromatographic peak with a retention time of 26 minutes was collected. After concentration and freeze-drying, small molecule active peptides of fish maw were obtained. The small molecule active peptides of fish maw were detected as a single peak by liquid chromatography, and their structure was determined by high performance liquid chromatography-mass spectrometry. The amino acid sequence was Tyr-Gly-Arg-Gly-Pro.

[0038] 2. This embodiment provides an acne-removing composition containing small molecule active peptides from fish maw, comprising 200g coconut oil, 150g palm oil, 400g olive oil, 150g jojoba oil, 100g unrefined avocado oil, 130g sodium hydroxide, 200g deionized water, 15g small molecule active peptides from fish maw, 120g honeysuckle extract, 120g centella asiatica extract, and 50g tea tree essential oil.

[0039] 3. This embodiment prepares a handmade acne-removing soap containing small molecule active peptides from fish maw. The specific preparation steps are as follows: In a well-ventilated area, slowly add sodium hydroxide to deionized water, stirring until completely dissolved. Cool to room temperature to obtain the lye solution. Heat coconut oil and palm oil together to 60°C until completely melted; this is the hard oil group. Mix olive oil and jojoba oil, stirring until smooth; this is the soft oil group. Slowly pour the cooled lye solution into the soft oil group, stirring continuously with an electric mixer for about 15 minutes until the mixture thickens and reaches a light trace; this is the first mixture. Slowly pour the hard oil group into the first mixture, continuing to stir for about 30-40 minutes until the soap reaches a medium trace; this is the second mixture. Divide the second mixture into approximately 70% (main component). Two portions, 30% (secondary portion) and 30% (secondary portion), are prepared. In the primary portion of the soap mixture, dissolved small-molecule active peptides from fish maw, honeysuckle extract, centella asiatica extract, and unrefined avocado oil are added and thoroughly mixed. In the secondary portion of the soap mixture, 20g of activated charcoal powder (first mixed with a small amount of soap mixture to form a paste) is added and thoroughly mixed. Tea tree essential oil is added to both the primary and secondary portions of the soap mixture and quickly mixed. The primary portion of the soap mixture is poured into a mold, followed by the secondary portion, which is then slowly poured on top. A marble pattern is gently drawn with a toothpick. The mold is covered with plastic wrap and placed in an insulated box for 48 hours before unmolding. After cutting the soap, it is left to dry in a cool, ventilated place for 4 weeks. The pH value is then checked and found to be below 9, yielding the acne-removing handmade soap.

[0040] Example 2

[0041] 1. This embodiment prepares a small molecule active peptide from fish maw, and the specific preparation steps are as follows: (1) Preparation of fish maw oligopeptide powder; After cleaning and crushing, the fish maw raw material (yellow croaker) was mixed with 18 times its volume of water and homogenized. A stepwise enzymatic hydrolysis was then performed using a compound enzyme consisting of alkaline protease, cellulase, and trypsin in a mass ratio of 5:3:2. The amount of the compound enzyme added was 4% of the fish maw raw material mass. Hydrolysis was carried out at 50℃ and pH 7.5 for 4 hours with stirring. After hydrolysis, the temperature was raised to 90℃ for 10 minutes to inactivate the enzyme, yielding the enzymatic hydrolysate. The hydrolysate was centrifuged at 8000 rpm for 10 minutes to remove particulate matter. Membrane separation technology was then used to separate the peptides, with a molecular weight cutoff of 3000 Da. The membrane solution was spray-dried to obtain fish maw oligopeptide powder. The obtained fish maw oligopeptide powder contained 91.2% peptides with a molecular weight less than 1000 Da.

[0042] (2) Preparation of small molecule active peptides from fish maw; The fish maw oligopeptide powder obtained in step (1) was dissolved in water to prepare a solution with a concentration of 100 mg / mL. Separation and purification were performed using Sephadex LH-20 column chromatography (3.0 × 100 cm) with 40% methanol as the mobile phase and a flow rate of 0.5 mL / min. The absorbance was measured at 280 nm, and the desired peak was collected based on the absorbance value. Further purification was performed using high-performance liquid chromatography (HPLC) under the following conditions: Elite C18 column (4.6 mm × 250 mm, 5 µm); mobile phase A was 0.07% trifluoroacetic acid water (v / v); mobile phase B was acetonitrile; and the gradient elution conditions were: 0-15 min, 3% B; 15-20 min, 3% B-10% B; 20-30 min, 10% B-20% B; 30-40 min, 10% B-20% B; 30-40 min, 10% B-20% B. The concentration was 20% B-35% B; the flow rate was 0.8 mL / min; the detection wavelength was 280 nm; and the chromatographic peak with a retention time of 26 minutes was collected. After concentration and freeze-drying, small molecule active peptides of fish maw were obtained. The small molecule active peptides of fish maw were detected as a single peak by liquid chromatography, and their structure was determined by high performance liquid chromatography-mass spectrometry. The amino acid sequence was Tyr-Gly-Arg-Gly-Pro.

[0043] 2. This embodiment provides an acne-removing composition containing small molecule active peptides from fish maw, comprising 200g coconut oil, 200g palm oil, 100g shea butter, 300g olive oil, 150g castor oil, 50g rice bran oil, 140g sodium hydroxide, 240g deionized water, 15g small molecule active peptides from fish maw, 120g forsythia extract, 120g aloe vera leaf extract, and 50g lavender essential oil.

[0044] 3. This embodiment prepares a handmade acne-removing soap containing small molecule active peptides from fish maw. The specific preparation steps are as follows: In a well-ventilated area, slowly add sodium hydroxide to deionized water, stirring until completely dissolved. Cool to room temperature to obtain the lye solution. Heat coconut oil, palm oil, and shea butter to 60°C until completely melted; this is the hard oil group. Mix olive oil and castor oil, stirring until smooth; this is the soft oil group. Slowly pour the cooled lye solution into the soft oil group, stirring continuously with an electric mixer for about 15 minutes until the mixture thickens and reaches a light trace; this is the first mixture. Slowly pour the hard oil group into the first mixture, continuing to stir for about 30-40 minutes until the soap reaches a medium trace; this is the second mixture. Divide the second mixture into approximately 70 portions. Divide the soap into two portions: 30% (main portion) and 30% (secondary portion). Add dissolved fish maw small-molecule active peptides, forsythia extract, aloe vera leaf extract, and rice bran oil to the main portion of the soap mixture and stir thoroughly. Add 20g of turmeric powder to the secondary portion of the soap mixture (first mix with a small amount of soap mixture to form a paste) and stir thoroughly. Add lavender essential oil to both the main and secondary portions of the soap mixture and stir quickly until well combined. Pour the main portion of the soap mixture into a mold, then slowly pour the secondary portion on top, gently drawing a marble pattern with a toothpick. Cover the mold with plastic wrap and place it in an insulated box for 48 hours before unmolding. After cutting the soap, allow it to air dry in a cool, ventilated place for 4 weeks. Test the pH value until it drops below 9 to obtain the acne-removing handmade soap.

[0045] Example 3

[0046] 1. This embodiment prepares a small molecule active peptide from fish maw, and the specific preparation steps are as follows: (1) Preparation of fish maw oligopeptide powder; After cleaning and crushing, the fish maw raw material (yellow croaker) was mixed with 16 times its volume of water and homogenized. A stepwise enzymatic hydrolysis was then performed using a compound enzyme consisting of alkaline protease, cellulase, and trypsin in a mass ratio of 6:4:2. The amount of the compound enzyme added was 5% of the mass of the fish maw raw material. Hydrolysis was carried out at 55℃ and pH 8.5 with stirring for 5 hours. After hydrolysis, the temperature was raised to 90℃ for 10 minutes to inactivate the enzyme, yielding the enzymatic hydrolysate. The hydrolysate was centrifuged at 8000 rpm for 10 minutes to remove particulate matter. Membrane separation technology was then used to separate the oligopeptides, with a molecular weight cutoff of 3000 Da. The membrane solution was spray-dried to obtain fish maw oligopeptide powder. The obtained fish maw oligopeptide powder contained 90.2% peptides with a molecular weight less than 1000 Da.

[0047] (2) Preparation of small molecule active peptides from fish maw; The fish maw oligopeptide powder obtained in step (1) was dissolved in water to prepare a solution with a concentration of 100 mg / mL. Separation and purification were performed using Sephadex LH-20 column chromatography (3.0 × 100 cm) with 40% methanol as the mobile phase and a flow rate of 0.6 mL / min. The absorbance was measured at 280 nm, and the desired peak was collected based on the absorbance value. Further purification was performed using high-performance liquid chromatography (HPLC) under the following conditions: Elite C18 column (4.6 mm × 250 mm, 5 µm); mobile phase A was 0.07% trifluoroacetic acid in water (v / v); mobile phase B was acetonitrile; and the gradient elution conditions were: 0-15 min, 3% B; 15-20 min, 3% B-10% B; 20-30 min, 10% B-20% B; 30-40 min, 10% B-20% B; 30-40 min, 10% B-20% B. The concentration was 20% B-35% B; the flow rate was 0.8 mL / min; the detection wavelength was 280 nm; and the chromatographic peak with a retention time of 26 minutes was collected. After concentration and freeze-drying, small molecule active peptides of fish maw were obtained. The small molecule active peptides of fish maw were detected as a single peak by liquid chromatography, and their structure was determined by high performance liquid chromatography-mass spectrometry. The amino acid sequence was Tyr-Gly-Arg-Gly-Pro.

[0048] 2. This embodiment provides an acne-removing composition containing small molecule active peptides from fish maw, comprising 350g coconut oil, 150g palm oil, 300g olive oil, 150g evening primrose oil, 50g walnut oil, 154g sodium hydroxide, 309g deionized water, 15g small molecule active peptides from fish maw, 50g purple gromwell extract, 100g verbena extract, 80g menthol extract, and 20g peppermint essential oil.

[0049] 3. This embodiment prepares a handmade acne-removing soap containing small molecule active peptides from fish maw. The specific preparation steps are as follows: In a well-ventilated area, slowly add sodium hydroxide to deionized water, stirring until completely dissolved. Cool to room temperature to obtain the lye solution. Mix coconut oil and palm oil and heat to 60°C until completely melted to obtain the hard oil group. Mix olive oil and evening primrose oil and stir well to obtain the soft oil group. Slowly pour the lye solution cooled to room temperature into the soft oil group and stir continuously with an electric mixer for about 15 minutes until the mixture thickens and reaches a slightly traced state to obtain the first mixture. Slowly pour the hard oil group into the first mixture and continue stirring for about 30-40 minutes until the soap reaches a moderate traced state to obtain the second mixture. Add small molecule active peptides of fish maw, comfrey extract, verbena extract, menthol extract, peppermint essential oil, and walnut oil to the second mixture and stir thoroughly to obtain the soap solution. Pour the soap solution into a mold, cover the mold surface with plastic wrap, and place it in an insulated box to keep warm for 48 hours before unmolding. After cutting the soap, let it air dry in a cool, ventilated place for 4 weeks. Check that the pH value drops below 9 to obtain the acne-removing handmade soap.

[0050] Comparative Example 1

[0051] 1. This embodiment prepares a fish maw peptide powder, and the specific preparation steps are as follows: The raw materials of fish maw (yellow croaker, cod, grouper, sea bass, sturgeon) were pulverized in a pulverizer for 5 minutes and passed through an 80-mesh sieve to obtain fish maw peptide powder.

[0052] 2. This embodiment provides a composition containing fish maw peptide powder, comprising 200g coconut oil, 200g palm oil, 400g olive oil, 150g sweet almond oil, 50g aloe vera oil, 136g sodium hydroxide, 200g deionized water, 15g fish maw peptide powder, 120g forsythia extract, 120g aloe vera leaf extract, and 50g lavender essential oil.

[0053] 3. This embodiment prepares a handmade soap containing fish maw peptide powder. The specific preparation steps are as follows: In a well-ventilated area, slowly add sodium hydroxide to deionized water, stirring until completely dissolved. Cool to room temperature to obtain the lye solution. Mix coconut oil and palm oil and heat to 60°C until completely melted to obtain the hard oil group. Mix olive oil and sweet almond oil and stir well to obtain the soft oil group. Slowly pour the lye solution cooled to room temperature into the soft oil group and stir continuously with an electric mixer for about 15 minutes until the mixture thickens and reaches a slightly traced state to obtain the first mixture. Slowly pour the hard oil group into the first mixture and continue stirring for about 30-40 minutes until the soap reaches a moderate traced state to obtain the second mixture. Add fish maw peptide powder, forsythia extract, aloe vera leaf extract, aloe vera oil, lavender essential oil, and 20g of turmeric powder to the second mixture and stir thoroughly to obtain the soap solution. Pour the soap solution into a mold, cover the mold surface with plastic wrap, and place it in an insulated box to keep warm for 48 hours before unmolding. After cutting the soap, let it air dry in a cool, ventilated place for 4 weeks. Check that the pH value drops below 9 to obtain the handmade soap.

[0054] Experimental Example 1: Antibacterial Test of Propionibacterium acnes

[0055] 1. Experimental methods; The sterilized culture medium (tryptone soy agar (TSA) + 5% defibrin sheep blood) was inoculated with the national standard strain of Propionibacterium acnes (CMCC(B) 65111) and incubated in an anaerobic incubator at 36℃ for 48 hours. An appropriate amount of sterile sodium chloride solution was added to wash the culture, and the bacterial suspension was aspirated into sterile test tubes. The bacterial count was determined using a bacterial turbidimeter to be approximately [missing information - likely a count of bacteria per ml]. Bacterial suspension.

[0056] Accurately weigh 1.0 g each of the handmade soaps prepared in Examples 1-3 and Comparative Example 1, and dissolve them separately in 10 mL of sterile physiological saline. Gently shake to ensure complete dissolution, preparing a 10% stock solution. Then, use sterile saline to serially dilute the stock solution to a series of concentrations of 5%, 2.5%, 1.25%, 0.625%, and 0.3125%. The positive control group benzoyl peroxide solution was prepared directly with PBS.

[0057] 2. Determination of antibacterial concentration; (1) Add 5% defibrinated sheep blood to brain heart infusion agar medium (BHI), autoclave at 121℃ for 20 min and set aside.

[0058] (2) In a 96-well plate, add 100 μL of BHI liquid culture medium to each well, and then add 100 μL of the test solution of a series of concentrations, so that the final concentration of the sample in the well is 5%, 2.5%, 1.25%, 0.625%, and 0.3125%. (3) 100 μL of BHI culture medium and 100 μL of bacterial solution were added to the positive control group, and 200 μL of sterile BHI culture medium was added to the negative control group.

[0059] (4) Add 10 μL of activated bacterial solution to each well (final bacterial concentration approximately (CFU / mL), gently shake to mix.

[0060] (5) After anaerobic culture at 37℃ for 48 h, the absorbance at 600 nm was measured using an ELISA reader. The lowest sample concentration with an absorbance value 50% lower than that of the positive control group is defined as the minimum inhibitory concentration (MIC). 3. Determination of bactericidal concentration; Take 10 μL of bacterial suspension from wells with no visible bacterial growth, spread it on blood agar plates, and incubate anaerobically at 37°C for 48 h. Count the colonies and take the lowest sample concentration with a colony count ≤ 10 CFU / plate as the minimum bactericidal concentration (MBC).

[0061] 4. Parallel experiments; Three parallel wells were set up for each sample concentration, and the experiment was repeated three times, with the average value taken.

[0062] 5. Test results; Table 1

[0063] As can be seen from the test results in Table 1, the MIC values ​​indicate that even at low concentrations, the acne-removing handmade soaps prepared in Examples 1-3 of this invention can still effectively inhibit the growth of Propionibacterium acnes, providing a solid theoretical basis for their acne-removing efficacy in practical use.

[0064] This invention discloses a small-molecule active peptide from fish maw, an acne-removing composition, an acne-removing handmade soap, and a method for preparing the same. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The product of this invention has been described through preferred embodiments, and those skilled in the art can clearly make modifications or appropriate alterations and combinations to the product without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

Claims

1. A small molecule active peptide of Euphausia superba, characterized in that, The amino acid sequence of the small molecule active peptide in fish maw is: Tyr-Gly-Arg-Gly-Pro.

2. The acne-removing composition comprising the small molecule active peptide of chelonian in claim 1, characterized in that, The acne-removing composition is made of the following components by weight percentage: 20-70% oil composition, 6-20% sodium hydroxide, 10-30% deionized water, 0.1-5% fish maw small molecule active peptides, 0.5-3% anti-inflammatory and soothing extract, and 0.05-5% essential oil; the sum of the weight percentages of all components in the acne-removing composition is 100%.

3. The acne treatment composition of claim 2, wherein, The oil composition includes hard oil, soft oil, and superfatting oil; the hard oil is selected from one or more of coconut oil, palm oil, and shea butter; the soft oil is selected from one or more of olive oil, grapeseed oil, castor oil, jojoba oil, and evening primrose oil; and the superfatting oil is selected from one or more of unrefined avocado oil, rice bran oil, and walnut oil.

4. The acne treatment composition of claim 2, wherein The anti-inflammatory and soothing extract is selected from one or more of the following: honeysuckle extract, centella asiatica extract, forsythia extract, aloe vera leaf extract, lithospermum erythrorhizon extract, verbena extract, menthol extract, and salvia miltiorrhiza extract.

5. The acne treatment composition of claim 2 wherein, The essential oil is selected from one or more of tea tree oil, lavender oil, and peppermint oil.

6. The acne treatment composition of claim 2 wherein, The preparation method of the small molecule active peptides from fish maw is as follows: (1) After crushing the raw fish maw, it is enzymatically hydrolyzed stepwise using a compound enzyme. The enzymatic hydrolysis pH is 7.5-9.0, the temperature is 45-55℃, and the time is 3-6 hours. After enzymatic hydrolysis, the enzyme is inactivated. After centrifugation, the hydrolysate is separated using membrane separation technology. The membrane solution is spray-dried to obtain fish maw oligopeptide powder. (2) Dissolve the fish maw oligopeptide powder in water to prepare a solution with a concentration of 10-100 mg / mL. Separate and purify the solution using Sephadex LH-20 column chromatography with a dextran gel. Measure the absorbance at 280 nm and collect the desired peak based on the absorbance value. Further purify the solution using high performance liquid chromatography with a detection wavelength of 280 nm. Collect the chromatographic peak with a retention time of 26 minutes. After concentration and freeze-drying, obtain the small molecule active peptides of fish maw.

7. The acne treatment composition of claim 6 wherein, The complex enzyme is composed of alkaline protease, cellulase, and trypsin in a mass ratio of (4-6):(3-5):(1-3).

8. The acne treatment composition of claim 6 wherein, The mobile phase for the Sephadex LH-20 column chromatography of the dextran gel was 40% methanol, and the flow rate was 0.4-0.6 mL / min. The chromatographic conditions for the high-performance liquid chromatography were as follows: C18 column, mobile phase A was water containing 0.06-0.08% trifluoroacetic acid by volume, mobile phase B was acetonitrile, and the gradient elution conditions were: 0-15 min, 3% B; 15-20 min, 3% B-10% B; 20-30 min, 10% B-20% B; 30-40 min, 20% B-35% B; and the flow rate was 0.8-1.0 mL / min.

9. The acne treatment composition of claim 6 wherein, The proportion of peptides with a molecular weight of less than 1000 Da in the fish maw oligopeptide powder is greater than 90%.

10. A method for preparing an anti-acne hand soap using the anti-acne composition according to claim 2, characterized in that, Includes the following steps: (1) Preparation of alkaline solution: Add sodium hydroxide to deionized water, dissolve it completely and let it stand at room temperature; (2) Oil and fat classification: The oil and fat composition is classified into hard oil, soft oil and superfatting oil; (3) The soft oil and alkaline solution are mixed and stirred until a slight trace state is reached to obtain the first mixture; (4) Add hard oil to the first mixture and continue stirring until the medium trace state is reached to obtain the second mixture; (5) Add small molecule active peptides of fish maw, anti-inflammatory and soothing extract, essential oil and superfatting oil to the second mixture, stir thoroughly to obtain soap solution; (6) Molding and curing: Pour the soap liquid into the mold, keep it warm for 24-48 hours, then unmold it and let it air dry for 4-6 weeks under ventilation conditions to obtain acne-removing handmade soap.

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