Fish collagen peptide with skin-lightening and freckle-removing effects, preparation method, and application thereof

Abstract

The present invention provides a fish collagen peptide with skin-lightening and freckle-removing effects, as well as a preparation method and application thereof, and belongs to the technical field of protein peptides. The present invention uses fish skin and / or fish scales as raw materials, and then performs heat treatment, homogenization treatment, enzymatic hydrolysis treatment and enzyme inactivation purification in sequence after degreasing and deodorizing treatment to obtain fish collagen peptide. In the fish collagen peptide prepared by the present invention, polypeptides of 189~3000Da account for more than 90%, and the protein content is as high as more than 99.23%. Experiments show that the fish collagen peptide can effectively reduce the proportion of skin spots on the subjects, and at the same time increase the L value and ITA value of the subjects' skin. It can be seen that the fish collagen peptide prepared by the present invention has good skin-lightening and freckle-removing effects, and provides a new way to prepare related products.

Description

Technical Field

[0001] The present invention belongs to the technical field of protein peptide preparation, and particularly relates to a fish collagen peptide with skin-lightening and freckle-removing effects, and a preparation method and application thereof. Background Art

[0002] Collagen is primarily found in the human skin, bones, eyes, teeth, tendons, and internal organs (including the heart, stomach, intestines, and blood vessels). It maintains the morphology and structure of the skin and other tissues and organs, and is also a crucial raw material for repairing damaged tissues. Collagen makes up 70% of human skin. When collagen is deficient, skin problems can easily develop. When collagen in the dermis oxidizes and breaks down, its support for the epidermis disappears, causing uneven skin collapse and wrinkles. Collagen is now gradually entering the beauty and skincare market, with supplementation through external means to reduce collagen loss.

[0003] Fish skin and scales are rich in collagen and are therefore commonly used to produce fish collagen peptides. Currently, fish collagen peptides produced using existing methods are used for anti-aging, improving skin moisturizing properties, and reducing wrinkles. However, no reports have been found on methods for producing fish collagen peptides with skin-brightening and freckle-removing properties. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide a method for preparing fish collagen peptide, so that the prepared fish collagen peptide has good skin lightening and freckle removal effects.

[0005] The present invention provides a method for preparing fish collagen peptide, comprising the following steps:

[0006] The defatted and deodorized fish scales are subjected to heat treatment and homogenization treatment to obtain a pretreated material;

[0007] The fish scale degreasing and deodorizing method comprises simultaneously soaking with an oxidizing agent and an acid or alkali;

[0008] The pretreated material is subjected to protease enzymatic hydrolysis under vortex conditions, and after the enzyme is inactivated, an enzymatic hydrolysis product is obtained;

[0009] The protease comprises at least two of alkaline protease, neutral protease, bromelain and papain;

[0010] The liquid phase is separated from the enzymatic hydrolysis product, and impurities are removed to obtain fish collagen peptide.

[0011] Preferably, the pH value of the defatted and fishy fish scales is 6.5 to 7.5;

[0012] The electrical conductivity of the defatted and deodorized fish scales is less than 500 μS / cm.

[0013] Preferably, the oxidant comprises any one of the following working concentrations: 0.6% to 2.0% by volume of chlorine dioxide preparation, 0.1% to 0.5% by volume of hydrogen peroxide, and 0.1 to 0.15 ppm of ozone;

[0014] The acid includes a citric acid aqueous solution or a malic acid aqueous solution with a working concentration of 0.9% to 1.5% by mass, or a hydrochloric acid aqueous solution with a working concentration of 0.1% to 0.3% by volume;

[0015] The alkali includes a sodium hydroxide aqueous solution with a working concentration of 0.1% to 0.3% by mass.

[0016] Preferably, the heat treatment temperature is 55-100° C., and the heat treatment time is 30-90 min.

[0017] Preferably, the homogenization treatment includes high-speed shearing, emulsification, homogenization and homogenization;

[0018] The high-speed shearing speed is 10000-18000 rpm; the high-speed shearing time is 30-60 s;

[0019] The emulsification speed is 2000-2500 r / min, and the emulsification time is 20-30 min;

[0020] The homogenization speed is 20000-25000 r / min, and the homogenization time is 30-60 s;

[0021] The homogenization pressure is 1000~1500 bar, and the homogenization is two-stage.

[0022] Preferably, in the enzymatic hydrolysis system, the mass percentage of the alkaline protease is 0.1‰~1.5‰;

[0023] The mass percentage of the neutral protease is 0.4‰~1.2‰;

[0024] The mass percentage of the bromelain is 0.1% to 0.5%;

[0025] The mass percentage of the papain is 0.3‰~1.0‰;

[0026] The temperature of the protease hydrolysis is 55-65°C;

[0027] The pH value of the system during the enzymatic hydrolysis of the protease is 7.0-8.5;

[0028] The reaction time of the protease hydrolysis is 1 to 3 hours.

[0029] Preferably, the impurity removal includes decolorization and further fishy removal;

[0030] The decolorization includes decolorization by filtration with a filter membrane; the pore size of the filter membrane is 5000~20000Da;

[0031] The method for removing the fishy smell again includes activated carbon adsorption.

[0032] The present invention provides fish collagen peptides prepared by the preparation method. In the fish collagen peptides, the mass percentage of peptides greater than 10,000 Da is 0.01% to 0.02%, the mass percentage of peptides between 5,000 and 10,000 Da is 0.04% to 0.83%, the mass percentage of peptides between 3,000 and 5,000 Da is 0.68% to 4.58%, and the mass percentage of peptides between 1,000 and 3,000 Da is 13.82% to 34.42%; the mass percentage of peptides between 500 and 1,000 Da is 31.96% to 32.76%, the mass percentage of peptides between 189 and 500 Da is 24.15% to 47.42%, and the mass percentage of peptides less than 189 Da is 3.24% to 5.89%.

[0033] The present invention provides the use of the fish collagen peptide in preparing medicines or skin care products for lightening and removing spots, or in preparing health care products, foods or food additives.

[0034] The present invention provides a method for preparing fish collagen peptides, comprising the following steps: subjecting defatted and deodorized fish scales to heat treatment and homogenization treatment to obtain a pretreated material; the method for defatting and deodorizing the fish scales comprises simultaneously soaking the scales with an oxidant and an acid or alkali; subjecting the pretreated material to protease hydrolysis under vortex conditions, inactivating the enzyme, and obtaining an enzymatic product; the protease comprises at least two of alkaline protease, neutral protease, bromelain, and papain; separating the liquid phase from the enzymatic product, removing impurities, and obtaining fish collagen peptides. Compared with using an oxidant or an acid or alkali alone for defatting and deodorizing treatment, the simultaneous soaking of the oxidant and the acid or alkali in the present invention exerts a synergistic effect, which can greatly reduce the content of lipid components in the fish scales, so that no fat is detected in the prepared fish collagen peptides, while ensuring that the prepared product has no fishy smell, thereby improving the consumer experience. At the same time, the fish collagen peptide prepared by the present invention has been clinically verified to have good skin lightening and freckle removal effects. The subjects were tested for 28 days, and the results showed that the area of ​​the spots was reduced by 15.34% compared with before the test, which was a significant difference. At the same time, the results of the skin L value test showed that the skin L value increased by 2.19% compared with before the test, and there was a significant difference. The present invention also tested the skin ITA value of the subjects after 28 days, and the results showed that the skin ITA value increased by 9.19% compared with before the test, which was a significant difference. It can be seen that the fish collagen peptide prepared by the method of the present invention has the effect of improving skin brightness and removing skin spots, and can be used to prepare products for lightening and freckle removal. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The results of the measurement of the percentage of pigmentation area of ​​the subjects before and after taking fish collagen peptides; Note: ## indicates a significant difference between the two groups P <0.05;

[0036] Figure 2 The results of the skin L value test of the subjects before and after taking fish collagen peptide; Note: ## indicates a significant difference between the two groups P <0.05;

[0037] Figure 3 The results of the ITA value measurement of the subjects' skin before and after taking fish collagen peptides; Note: ## indicates a significant difference between the two groups P <0.05. DETAILED DESCRIPTION

[0038] The present invention provides a method for preparing fish collagen peptide, comprising the following steps:

[0039] The defatted and deodorized fish scales and / or fish skin are subjected to heat treatment and homogenization treatment to obtain a pretreated material;

[0040] The pretreated material is subjected to protease enzymatic hydrolysis under vortex conditions, and after the enzyme is inactivated, an enzymatic hydrolysis product is obtained;

[0041] The protease comprises at least two of alkaline protease, neutral protease, bromelain and papain;

[0042] The liquid phase is separated from the enzymatic hydrolysis product, and impurities are removed to obtain fish collagen peptide.

[0043] The present invention performs heat treatment and homogenization treatment on defatted and deodorized fish scales and / or fish skin to obtain pretreated materials.

[0044] In the present invention, the fish scales or fish skin are preferably derived from freshwater or marine fish. The fish species from which the fish scales are derived include at least one of tilapia, grass carp, and snakehead carp. In a specific embodiment of the present invention, grass carp scales and snakehead carp scales are used as examples to illustrate the method for preparing fish collagen peptides. The fish skin is preferably derived from at least one of cod, tilapia, and basa. The fish scales or fish skin may include fresh, dried, or frozen fish scales or skin. The fish scales or fish skin are preferably rinsed to remove impurities and blood. The rinsing method preferably involves washing the fish skin or fish scales with 2 to 4 times the amount of water. The number of rinses is determined by ensuring that the washing water is clear and free of visible blood or impurities. When the fish scales or fish skin are dried, they are preferably pre-soaked in water for 12 to 15 hours, or 13 to 14 hours. When the fish scales or fish skin are frozen, they are preferably pre-soaked in water for 18 to 24 hours, or 20 to 22 hours.

[0045] In the present invention, the method for degreasing and removing fishy smell of the fish scales and / or fish skin preferably comprises treatment with an oxidant and acid or alkali soaking treatment in sequence. The oxidant preferably comprises any one of the following working concentrations: a chlorine dioxide preparation with a volume concentration of 0.6% to 2.0%, a hydrogen peroxide with a volume concentration of 0.1% to 0.5%, and ozone with a volume concentration of 0.1 to 0.15 ppm; or a chlorine dioxide preparation with a volume concentration of 0.8% to 1.0%, a hydrogen peroxide with a volume concentration of 0.2% to 0.4%, and ozone with a volume concentration of 0.12 to 0.14 ppm. In order to further improve the healthiness of the product, the oxidant adopts a food-grade preparation product. The acid comprises a citric acid aqueous solution or a malic acid aqueous solution with a working concentration of 0.9% to 1.5% by mass, or a hydrochloric acid aqueous solution with a working concentration of 0.1% to 0.3% by volume, or a citric acid aqueous solution or a malic acid aqueous solution with a working concentration of 1.2% by mass, or a hydrochloric acid aqueous solution with a working concentration of 0.2% by volume. The alkali includes a sodium hydroxide aqueous solution with a working concentration of 0.1% to 0.3% by mass, which can be 0.2%. The degreasing and fishy removal is preferably accompanied by stirring. The stirring condition is to stir for 2 to 5 minutes every 2 to 6 hours. After the degreasing and fishy removal, it is preferred to rinse the degreasing and fishy removal fish scales and / or fish skin with clean water. After rinsing, the pH value of the degreasing and fishy removal fish scales and / or fish skin is 6.5 to 7.5, which can be 6.8 to 7.2, or even 7.0. The electrical conductivity of the degreasing and fishy removal fish scales and / or fish skin is <500μS / cm, and can also be 386 to 479μS / cm.

[0046] In the present invention, the heat treatment temperature is preferably 55-100°C, 58-80°C, or 60-68°C; the heat treatment time is preferably 30-90 minutes, 40-80 minutes, or 60-70 minutes. The heat treatment moderately softens the fish scales, facilitating subsequent processing. The homogenization treatment preferably includes high-speed shearing, emulsification, homogenization, and homogenization. The rotation speed of the high-speed shear is preferably 10,000-18,000 rpm, and can be 15,000 rpm; the time of the high-speed shear is preferably 30-60 s, and can be 45 s; the rotation speed of the emulsification is preferably 2,200-2,400 r / min, and can be 2,400 r / min; the time of the emulsification is preferably 20-30 min, and can be 25 min; the homogenization condition is preferably 22,000-24,000 r / min, and can be 24,000 r / min; the homogenization time is preferably 35-45 s, and can be 35 s; the homogenization condition is preferably 1,200-1,400 Bar, and can be 1,300-1,400 Bar.

[0047] After obtaining the pretreated material, the present invention performs protease enzymatic hydrolysis on the pretreated material under vortex conditions, and after inactivating the enzyme, obtains an enzymatic hydrolysis product.

[0048] In the present invention, the mass percentage of the alkaline protease is preferably 0.1‰~1.5‰, can be 0.2‰~0.4‰, and can also be 0.3‰; the mass percentage of the neutral protease is preferably 0.4‰~1.2‰, can be 0.6‰~1.2‰, and can be 1.0‰; the mass percentage of the bromelain is preferably 0.1%~0.5%, can be 0.2%; the mass percentage of the papain is preferably 0.3‰~1.0‰, and can be 0.6‰. In an embodiment of the present invention, 0.4‰ alkaline protease is compounded with 1.2‰ neutral protease for enzymatic hydrolysis; 0.3‰ alkaline protease is compounded with 1.0‰ neutral protease and 0.2‰ bromelain for enzymatic hydrolysis; and 0.2‰ alkaline protease, 0.6‰ neutral protease and 0.6‰ papain are also included for compound enzymatic hydrolysis. The alkaline protease is derived from Bacillus subtilis and has an activity of 300,000 U / ml. The neutral protease is derived from Bacillus subtilis and has an activity of 120,000 U / ml. Papain is 800,000 U / g. Bromelain is 600,000 U / g.

[0049] In the present invention, the temperature for protease hydrolysis is preferably 55-65°C, and can also be 58-62°C, or even 60°C. The pH value of the system during protease hydrolysis is preferably 7.0-8.5, and can be 7.5-8.0, or even 7.8. The reaction time for protease hydrolysis is 1-3 hours, or 2-2.5 hours. The vortex rotation speed is preferably 60 rpm. The enzyme inactivation method preferably includes adjusting the pH to an acidic range (4.5-4.8) and inactivating the enzyme using ultrahigh pressure treatment or pulsed intense light treatment.

[0050] Obtain an enzymatic hydrolysis product, and the present invention separates a liquid phase from the enzymatic hydrolysis product, removes impurities, and obtains fish collagen peptide.

[0051] In the present invention, the method for separating the liquid phase from the enzymatic hydrolyzate preferably utilizes a membrane press plate-frame filtration device for solid-liquid separation. The impurity removal preferably includes decolorization, secondary fishy smell removal, concentration, and clarification. The decolorization preferably includes membrane filtration for decolorization. The pore size of the filter membrane is preferably 5,000 to 20,000 Da, and may be 10,000 Da. The secondary fishy smell removal method preferably includes activated carbon adsorption. During the activated carbon adsorption, the working mass percentage of the activated carbon is preferably 2% to 4%, and may be 3%. During the activated carbon adsorption, the system temperature is preferably 55 to 65°C, and may be 60°C. The activated carbon adsorption time is preferably 35-45 minutes, and may be 40 minutes. The concentration method preferably includes concentrating the first concentrate using a 300 Da1 nanofiltration membrane to 1 / 5 of the original volume, and then re-concentrating the obtained first concentrate using rotary evaporation to 35% to 38% of the volume of the first concentrate to obtain a second concentrate. The clarification method includes filtering the second concentrate using multiple layers of fine cardboard and a PP filter element to collect the polypeptide concentrate.

[0052] In the present invention, after the impurity removal, the obtained polypeptide concentrate is preferably sterilized. The sterilization method preferably includes membrane filtration. The large pore size of the filter membrane is preferably 0.22 μm. The sterilized fish collagen peptide preferably includes drying. The drying method includes spray drying. The inlet air temperature of the spray drying is preferably 160~70°C, and can be 165°C. The outlet air temperature of the spray drying is preferably 90~95°C, and can be 92°C. The atomization frequency of the spray drying is preferably 20~25Hz, and can be 22Hz.

[0053] The present invention provides fish collagen peptides prepared by the preparation method. In the fish collagen peptides, the mass percentage of peptides greater than 10,000 Da is 0.01%-0.02%, the mass percentage of peptides between 5,000 and 10,000 Da is 0.04%-0.83%, the mass percentage of peptides between 3,000 and 5,000 Da is 0.68%-4.58%, and the mass percentage of peptides between 1,000 and 3,000 Da is 13.82%-34.42%; the mass percentage of peptides between 500 and 1,000 Da is 31.96%-32.76%, the mass percentage of peptides between 189 and 500 Da is 24.15%-47.42%, and the mass percentage of peptides less than 189 Da is 3.24%-5.89%.

[0054] In the present invention, among the fish collagen peptides, the mass percentage of peptides greater than 10,000 Da is 0.01%, the mass percentage of peptides between 5,000 and 10,000 Da is 0.67%, the mass percentage of peptides between 3,000 and 5,000 Da is 4.25%, and the mass percentage of peptides between 1,000 and 3,000 Da is 32.76%; the mass percentage of peptides between 500 and 1,000 Da is 32.76%, the mass percentage of peptides between 189 and 500 Da is 26.58%, and the mass percentage of peptides less than 189 Da is 3.77%.

[0055] In the present invention, fish scales and skins from different fish are used as materials, and alkaline protease and neutral protease or a combination thereof with bromelain or papain are used for enzymatic hydrolysis to prepare fish collagen peptides with a molecular weight mainly ranging from 189 to 3000 Da. The protein content of the fish collagen peptides prepared by the present invention is above 99.23%, which is higher in purity than fish collagen peptides extracted by conventional methods, and the molecular weight of the peptides is larger than that of fish collagen peptides extracted by conventional methods.

[0056] The fish collagen peptide has the effect of brightening the skin and removing freckles. The present invention provides the use of the fish collagen peptide in preparing medicines or skin care products for brightening the skin and removing freckles, or in preparing health care products, foods or food additives.

[0057] In the present invention, the types of spots included in the freckle removal include freckles, chloasma, etc. The freckle removal includes lightening spots and reducing their area. The skin lightening includes improving dull skin and brightening the complexion. In an embodiment of the present invention, the freckle removal effect is evaluated by measuring the reduction in the area of ​​the spots, while the skin lightening effect is evaluated by the percentage increase in the L value and ITA value of the skin.

[0058] In the present invention, the product preferably includes at least one of the following: medicine, skin care products, health care products, food, and food additives. Skin care products include creams, lotions, toners, facial masks, and anti-freckle essences. The mass percentage of the fish collagen peptide in the skin care product is 1% to 20%, and may be 2%, 3%, 4%, 5%, 10%, or 15%. The health care product preferably includes at least one of the following dosage forms: oral liquid, capsule, powder, granule, and chewable tablet. The mass percentage of the fish collagen peptide in the health care product is 5% to 95%, and may be 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, or 80%. The food product includes at least one of the following: beverages, meat products, fish products, egg products, pasta products, candy products, and vegetable products. The mass percentage of the fish collagen peptide in the food product is 1% to 50%, and may be 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40%.

[0059] In one embodiment of the present invention, through human body testing, taking the fish collagen peptide for 28 days can significantly improve the area of ​​facial spots of the subject, indicating that the fish collagen peptide has a freckle removal effect when used alone. At the same time, the present invention also measured the L value and ITA value of the subject's facial skin. The results showed that the fish collagen peptide can significantly increase the L value and ITA value of the subject's facial skin, indicating that the fish collagen peptide has the effect of improving skin brightness when used alone. It can be seen that the fish collagen peptide provided by the present invention has the effect of brightening the skin and removing spots, and has a good effect on improving dull skin, brightening skin gloss, inhibiting melanin deposition and reducing pigment accumulation.

[0060] The following is a detailed description of a fish collagen peptide with skin-lightening and freckle-removing effects, a preparation method thereof, and applications thereof provided by the present invention, in conjunction with examples. However, these examples should not be construed as limiting the scope of protection of the present invention.

[0061] Example 1

[0062] Preparation method of fish collagen peptide with skin-lightening and freckle-removing effects

[0063] (1) Rinse: Soak fresh frozen grass carp scales in clean water for 20 hours to thaw, drain and rinse with clean water three times;

[0064] (2) Degreasing and removing fishy smell: Add 3 times the weight of clean water to the rinsed fish scales, add 0.8% chlorine dioxide preparation (food grade, ClO2 content 48%) and 1.2% monohydrated citric acid (food grade, content >99%), and soak for 24 hours. Stir intermittently for 5 minutes every 2 hours, and rinse with clean water until the pH value is 6.8 and the conductivity is 467μS / cm.

[0065] (3) Mild heat treatment: put the cleaned fish scales into the reaction tank, add 2.5 times the mass of clean water, heat to 58 ° C, and heat treat for 40 minutes, with air blowing and stirring turned on throughout the process;

[0066] (4) Slurrying: After the heat treatment is completed, the material is sheared at 18000 r / min for 45 s and homogenized at 22000 rpm for 40 s. The homogenate is then transferred to a high-speed homogenizer and homogenized in two stages at 1250 bar to obtain fish scale slurry.

[0067] (5) Enzymatic hydrolysis: The fish scale slurry was stirred at 30 rpm in a vortex turbulent state, and alkaline protease (derived from Bacillus subtilis, activity 300,000 U / ml), neutral protease (derived from Bacillus subtilis, activity 120,000 U / ml) and papain (800,000 U / g) were added at a final concentration of 0.2‰. The enzymatic hydrolysis temperature was 58 °C, the pH value was 7.0-7.5, and the reaction was carried out for 2 h.

[0068] (6) Inactivation of enzyme: The pH value of the enzymatic hydrolysis product was adjusted to 4.5, and the protease was inactivated by pulsed strong light treatment for 5 seconds;

[0069] (7) Primary clarification: Use a diaphragm press plate-frame filter (pressure 0.02 MPa) to perform solid-liquid separation to obtain a primary polypeptide solution;

[0070] (8) Purification: The primary polypeptide solution was decolorized by passing through a 10,000 Da1 filter membrane, and activated carbon with a solid content of 3% was added to the decolorized solution. The pH value was adjusted to 4.2, and the solution was adsorbed and deodorized at 55°C for 30 minutes. After the adsorption, the activated carbon was removed to obtain a purified polypeptide solution.

[0071] (9) Concentration: The purified polypeptide solution was concentrated to 20% by a 300 Da1 nanofiltration membrane, and the obtained first concentrated solution was concentrated to 37% by a triple-effect evaporator (70-65-60°C) to obtain a second concentrated solution;

[0072] (10) Secondary clarification: The second concentrated solution was filtered through multi-layer fine paperboard and PP filter element (the pore size of the paperboard was 5 μm, and the pore size of the filter element was 0.45 and 0.22 μm) to obtain a clarified polypeptide concentrate;

[0073] (11) Sterilization: The clarified polypeptide concentrate is filtered and sterilized using a 0.22 μm microporous filter to obtain a sterilized polypeptide concentrate;

[0074] (12) Drying: The sterilized peptide concentrate was spray-dried with an inlet air temperature of 160°C, an outlet air temperature of 90°C, and an atomization frequency of 25 Hz to obtain fish collagen peptide.

[0075] The prepared fish collagen peptides were subjected to component determination, and the content ratio of peptides in each molecular weight range was determined (GB / T 31645-2008).

[0076] The product test results are shown in Table 1.

[0077] Table 1 Fish collagen peptides from grass carp scales

[0078]

[0079] Example 2

[0080] Preparation method of fish collagen peptide with skin-lightening and freckle-removing effects

[0081] (1) Rinse: Soak fresh frozen black fish scales in clean water for 18 hours to thaw, drain and rinse with clean water three times;

[0082] (2) Degreasing and removing fishy smell: Add 3 times the weight of clean water to the rinsed fish scales, then add 0.2% hydrogen peroxide (food grade, mass fraction 32-36%) and 0.3% sodium hydroxide (liquid, mass fraction 30-33%), and soak for 24 hours. Stir intermittently for 5 minutes every 2 hours. Rinse with clean water until the pH value is 7.2 and the conductivity is 386μS / cm.

[0083] (3) Mild heat treatment: put the cleaned fish scales into the reaction tank, add 2 times the mass of clean water, heat to 65℃, and heat treat for 60 minutes, with air blowing and stirring turned on throughout the process;

[0084] (4) Slurrying: After the heat treatment is completed, the material is subjected to high-speed shearing at 15,000 r / min for 50 s, emulsification at 2,500 r / min, and two-stage high-speed homogenization at a pressure of 1,400 Bar to obtain fish scale slurry;

[0085] (5) Enzymatic hydrolysis: The fish scale slurry was placed in a vortex turbulent state under ultra-high-speed stirring, and alkaline protease (derived from Bacillus subtilis, activity 350,000 U), neutral protease (derived from Bacillus subtilis, activity 150,000 U) and bromelain (600,000 U) were added at a final concentration of 0.3‰, the temperature was 60°C, the pH was controlled at 7.5-7.8, and the reaction was carried out for 2.5 h;

[0086] (6) Inactivation of enzyme: After completion, the pH value of the system was adjusted to 4.5, and the protease was inactivated by pulsed strong light treatment for 5 seconds;

[0087] (7) Primary clarification: Use a membrane press plate-frame filter to separate the solid and liquid to obtain a primary polypeptide solution;

[0088] (8) Purification: The primary polypeptide solution was decolorized by passing through a 10,000 Da1 membrane, and then activated carbon with a solid content of 2% was added. At the same time, the pH value was adjusted to 4.5. The fishy smell was removed by adsorption at 60°C for 40 minutes. After the adsorption, the activated carbon was removed to obtain a purified polypeptide solution.

[0089] (9) Concentration: The purified polypeptide solution was concentrated to 20% by a 300 Da1 nanofiltration membrane. The obtained first concentrated solution was then concentrated to 38% by a triple-effect evaporator (70-65-60°C) to obtain a second concentrated solution.

[0090] (10) Secondary clarification: The second concentrated solution was filtered through multiple layers of fine paperboard and PP filter element (paperboard pore size 5 μm, filter element pore size 0.45 and 0.22 μm) to obtain a clarified polypeptide concentrate;

[0091] (11) Sterilization: Filter and sterilize using a 0.22 μm microporous filter to obtain the sterilized polypeptide;

[0092] (12) Drying: The sterilized polypeptide is spray-dried with an air inlet temperature of 170°C, an air outlet temperature of 95°C, and an atomization frequency of 28 Hz to obtain a fish collagen peptide product derived from black fish scales.

[0093] The fish collagen peptide product derived from black fish scales was tested according to the method of Example 1. The product test results are shown in Table 2.

[0094] Table 2 Determination results of fish collagen peptides from black fish scales

[0095]

[0096] Example 3

[0097] Preparation method of fish collagen peptide with skin-lightening and freckle-removing effects

[0098] (1) Rinse: Soak fresh frozen cod skin in clean water for 24 hours to thaw, drain and rinse with clean water three times;

[0099] (2) Degreasing and removing fishy smell: Add 4 times the weight of fish skin to clean water, 2.0% chlorine dioxide preparation (food grade, ClO2 content 48%) and 2.5% malic acid (food grade, content >99%), and soak for 18 hours. Stir intermittently for 2 minutes every 6 hours. Rinse with clean water until the pH is 6.8 and the conductivity is 479μS / cm.

[0100] (3) Mild heat treatment: crush the cleaned fish skin and put it into the reaction tank, add 1.5 times the mass of clean water, heat it to 60℃, and heat it for 30min, with air blowing and stirring turned on throughout the process;

[0101] (4) Slurrying: After the heat treatment is completed, the material is emulsified by high-speed shearing (10,000 r / min) and high-pressure homogenization to obtain fish skin slurry;

[0102] (5) Enzymatic hydrolysis: The fish skin pulp was placed in a vortex turbulent state under ultra-high-speed stirring, and 0.4‰ alkaline protease (derived from Bacillus subtilis, with an activity of 250,000 U) and 1.2‰ neutral protease (derived from Bacillus subtilis, with an activity of 100,000 U) were added. The temperature was 60°C, the pH was controlled at 7.5-8.0, and the reaction was carried out for 3 h.

[0103] (6) Inactivation of enzymes: After completion, the pH of the system is adjusted to 4.8, and the protease is inactivated by ultrahigh pressure treatment;

[0104] (7) Primary clarification: Use a membrane press plate-frame filter to separate the solid and liquid to obtain a primary polypeptide solution;

[0105] (8) Purification: The polypeptide solution was decolorized by passing through a 10,000 DaI membrane, and then activated carbon with a solid content of 5% was added for adsorption and deodorization. The pH was controlled at 4.8, the temperature was 65°C, and the time was 60 min. After the end, the activated carbon was removed to obtain a purified polypeptide solution.

[0106] (9) Concentration: Concentrate to 20% using a 300 DaI nanofiltration membrane, and then concentrate to 35% using a triple-effect evaporator (70-65-60°C);

[0107] (10) Secondary clarification: Filter the concentrate through multiple layers of fine paperboard and PP filter element to obtain clarified polypeptide concentrate;

[0108] (11) Sterilization: Filter and sterilize using a 0.22 μm microporous filter;

[0109] (12) Drying: Spray drying, air inlet temperature 165°C, air outlet 92°C, atomization frequency 22 Hz, to obtain fish collagen peptides derived from cod skin.

[0110] The fish collagen peptide product was tested according to the method of Example 1. The product test results are shown in Table 3.

[0111] Table 3 Detection results of fish collagen peptides from cod skin

[0112]

[0113] Example 4

[0114] Human experiment on freckle removal and skin brightening

[0115] The product prepared in Example 1 was subjected to human trial experiments to evaluate its efficacy in removing freckles and brightening the skin.

[0116] A total of 31 subjects, numbered 1 to 31, were enrolled in this study. Subject 13 withdrew from the study for personal reasons, resulting in a total of 30 subjects with an average age of (45.0 ± 9.2) years. The human skin testing of fish collagen peptides was commissioned by Hubei Ruibang Biotechnology Co., Ltd., under the license number BSEYNNUB4537357R7. The fish collagen peptides were taken one packet at a time, mixed well with warm water, and taken twice daily, morning and evening, for 28 days. The Wilcoxon paired rank sum test was used to statistically analyze the baseline values ​​and the 28-day results.

[0117] The results are shown in Table 4 and Figure 1 Compared with the baseline value, the area of ​​the spots was significantly reduced after taking the sample for 28 days ( P =0.0013).

[0118] Table 4 Test results of the percentage of pigmentation area of ​​the subjects

[0119]

[0120] The results of the measured skin L values ​​of the subjects were statistically analyzed using the paired sample T test. The results are shown in Table 5 and Figure 2 Compared with the baseline value, the L value of the subjects' skin increased by 2.19% after taking the sample for 28 days, and there was a significant difference ( P <0.001).

[0121] Table 5 Test results of the skin L value of the subjects

[0122]

[0123] The ITA results of the subjects' skin were statistically analyzed using the paired Wilcoxon rank sum test. The results are shown in Table 6 and Figure 3 Compared with the baseline value, the ITA value of the subjects' skin increased by 9.19% after taking the sample for 28 days, and the difference was significant ( P <0.001).

[0124] Table 6 Test results of the subjects' skin ITA values

[0125]

[0126] Comparative Example 1

[0127] A preparation method of fish collagen peptide

[0128] (1) Rinse: Soak fresh frozen grass carp scales in clean water for 20 hours to thaw, drain and rinse with clean water three times;

[0129] (2) Deliming and degreasing:

[0130] Fish scale deliming: Add 4 times the weight of clean water and 5% food-grade hydrochloric acid (mass fraction 37%) to the rinsed fish scales and soak for 18 hours. This process is carried out in a dedicated fish scale deliming reactor. Stirring is turned on throughout the process. Monitor the pH value of the system (control it below 1.0). Rinse with clean water until the pH value reaches 5.5.

[0131] Degreasing and removing fishy smells: Soak fish scales in 3 times their weight of water, 8% hydrogen peroxide (food grade, 35% H2O2 content), and 5% liquid caustic soda (food grade, 32% content) for 24 hours. Stir intermittently for 5 minutes every 2 hours. Rinse with clean water until the pH reaches 8.0.

[0132] (3) Heat treatment: heat to 95°C and maintain for 2 hours;

[0133] (4) Enzymatic hydrolysis: Cool the heat-treated material to 60°C, add alkaline protease (derived from Bacillus subtilis, 350,000 U) at a final concentration of 2‰, control the pH value to 9.0-9.5, and perform enzymatic hydrolysis for 2.5 h to obtain the enzymatic hydrolysis product;

[0134] (6) Inactivation of enzyme: Heat the enzymatic hydrolysis product to 95°C for 30 min;

[0135] (7) Primary clarification: Filter through a plate and frame filter, collect the filtrate, and obtain the enzymatic hydrolyzate;

[0136] (8) Purification: Add activated carbon with a solid content of 10% to the enzymatic hydrolysate, decolorize at a pH of 4.5 and 85°C for 1.5 h to obtain a purified polypeptide solution;

[0137] (9) Concentration: The purified polypeptide solution is concentrated to 25% by nanofiltration membrane to obtain a first concentrated solution, which is then concentrated to 45% by scraper evaporator to obtain a second concentrated solution;

[0138] (10) Secondary clarification: The second concentrated liquid is filtered through cardboard to obtain a clarified liquid;

[0139] (11) Sterilization: Pass the clarified liquid through a 0.22 μm microporous filter to obtain a sterilized polypeptide solution;

[0140] (12) Drying: The sterilized polypeptide solution was spray-dried with an air inlet temperature of 195°C, an air outlet temperature of 95°C, and an atomization frequency of 25 Hz to obtain fish collagen peptides derived from grass carp scales.

[0141] The fish collagen peptides derived from grass carp scales prepared above were determined according to the method of Example 1. The determination results are shown in Table 7.

[0142] Table 7 Determination results of fish collagen peptides from grass carp scales prepared by traditional methods

[0143]

[0144] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing fish collagen peptide, characterized in that: The following steps are involved: The defatted and fishy-removed grass carp scales are subjected to heat treatment and homogenization treatment to obtain a pretreated material; The fish scale degreasing and deodorizing method comprises simultaneously soaking with an oxidizing agent and an acid; The oxidant is a chlorine dioxide preparation with a volume concentration of 0.6% to 2.0%; The acid is a citric acid aqueous solution with a mass percentage of 0.9% to 1.5%; The heat treatment temperature is 55-60°C; The heat treatment time is 30 to 60 minutes; The homogenization treatment includes high-speed shearing and homogenization; the rotation speed of the high-speed shearing includes 15000-18000 rpm; the time of the high-speed shearing is 30-45 s; the rotation speed of the homogenization is 22000-24000 r / min, and the time of the homogenization is 35-45 s; The pretreated material is subjected to protease enzymatic hydrolysis under vortex conditions, and after the enzyme is inactivated, an enzymatic hydrolysis product is obtained; The protease consists of alkaline protease, neutral protease and papain; The mass percentage of the alkaline protease is 0.1‰~1.5‰; The mass percentage of the neutral protease is 0.4‰~1.2‰; The mass percentage of the papain is 0.3‰~1.0‰; The alkaline protease is derived from Bacillus subtilis; the neutral protease is derived from Bacillus subtilis; The temperature of the protease hydrolysis is 58-60°C; The pH value of the system during the enzymatic hydrolysis of the protease is 7.0-7.5; The reaction time of the protease hydrolysis is 2 to 2.5 hours; The liquid phase is separated from the enzymatic hydrolysis product, and impurities are removed to obtain fish collagen peptide.

2. The preparation method according to claim 1, characterized in that The pH value of the defatted and fishy fish scales is 6.5 to 7.5; The electrical conductivity of the defatted and deodorized fish scales is less than 500 μS / cm.

3. The preparation method according to claim 1 or 2, characterized in that The impurity removal includes decolorization and fishy removal again; The decolorization includes decolorization by filtration with a filter membrane; the pore size of the filter membrane is 5000~20000Da; The method for removing the fishy smell again includes activated carbon adsorption.

4. The fish collagen peptide prepared by the preparation method according to any one of claims 1 to 3, characterized in that: Among the fish collagen peptides, the mass percentage of peptides greater than 10,000 Da is 0.01%~0.02%, the mass percentage of peptides between 5,000 and 10,000 Da is 0.67%~0.83%, the mass percentage of peptides between 3,000 and 5,000 Da is 4.25%~4.58%, and the mass percentage of peptides between 1,000 and 3,000 Da is 32.76%~34.42%; the mass percentage of peptides between 500 and 1,000 Da is 31.96%~32.76%, the mass percentage of peptides between 189 and 500 Da is 24.15%~26.58%, and the mass percentage of peptides less than 189 Da is 3.24%~3.77%.

5. Use of the fish collagen peptide according to claim 4 in the preparation of medicines or skin care products for brightening and removing spots or in the preparation of health care products.

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