A natural elastic collagen dipeptide and its preparation method

Abstract

The present invention belongs to the technical field of active substance extraction, and particularly relates to a natural elastic collagen dipeptide and a preparation method thereof. While preparing elastin peptides, the present invention retains the original collagen components in tissues, avoiding waste of protein resources. The raw material pretreatment conditions of the present invention are mild, the concentration of lye is reduced, the amount of waste liquid generated is reduced, and it is more environmentally friendly. The present invention applies ultra-high pressure technology to perform protein modification and solubilization treatment on the raw materials, so that the tertiary and quaternary structures of elastin and collagen are stretched, increasing the solubility and extraction rate of proteins. The protein modification treatment exposes more internal cleavage sites of proteins, enhances the sensitivity of proteins to enzymes, improves the enzymatic hydrolysis efficiency, reduces the amount of enzyme used, and lowers the enzyme cost. The natural elastic collagen dipeptide prepared by the present invention has a ratio closer to the original ratio in tissues and has a more efficient utilization effect on improving skin health after oral administration.

Description

Technical Field

[0001] The present invention belongs to the technical field of active substance extraction, and particularly relates to a natural elastic collagen dipeptide and a preparation method thereof. Background Art

[0002] Elastin is the main component of elastic fibers and exists in connective tissues combined with collagen, endowing tissues with elasticity and tensile strength, and playing an important role in maintaining the shape and function of various organs. The content of elastin in tissues varies depending on the degree of elastic function required by the tissues. Calculated by dry weight, the content of elastin is the highest in ligaments, accounting for about 78 - 80%, followed by about 50% in the aorta and about 20% in the lungs. The content of elastin in the skin is relatively low, about 2 - 4%, while the content of collagen in the skin exceeds 70%. Although elastin has a low content in the skin, it plays an important role in maintaining the skin state and function. It provides the tensile strength and lasting elasticity of the skin, is responsible for the support of the skin, and works together with collagen responsible for skin filling and fullness to keep the skin smooth, with a high water content and firm. That is to say, in the skin, ligaments and other tissues of the human body, elastin usually coexists with collagen, and they jointly play important physiological functions. The ratio between them has an important impact on the mechanical properties of cells and tissues.

[0003] However, at present, people extract and enzymatically hydrolyze elastin peptides from tissues such as animal ligaments, lungs, cartilage, and arterial bulbs. The existing preparation processes usually remove the collagen in the tissues during the raw material pretreatment stage (for example, through concentrated alkali high-temperature treatment) to prepare elastin peptides with a relatively high purity. This not only causes waste of collagen resources but also destroys the natural ratio of elastin and collagen in the raw materials. The existing extraction process of elastin is complex, uses concentrated alkali in pretreatment, requires a large amount of washing water, and generates a large amount of wastewater, which is not friendly to the environment. Elastin and collagen are insoluble in water and have a tight structure and are not easily digested by enzymes, resulting in low efficiency of the existing extraction process. In addition, more importantly, the existing extraction process cannot achieve the synchronous extraction of elastin peptides and collagen peptides in a natural ratio, and thus currently products containing the above two active ingredients are all obtained by artificially combining the two raw materials of elastin peptides and collagen peptides. On the one hand, this makes the operation steps more cumbersome, and on the other hand, it cannot ensure that the two active ingredients play their roles in the natural ratio in the tissue.

[0004] Therefore, how to retain the collagen component during the preparation of elastin peptides and enzymatically prepare elastic collagen dipeptides with a natural ratio is an urgent problem to be solved. Summary of the Invention

[0005] In view of the defects and deficiencies in the prior art, the present invention provides a method for preparing natural elastic collagen dipeptide. The natural elastic collagen dipeptide prepared by the method of the present invention can significantly improve skin elasticity and moisture content, improve the functional barrier of the skin cutin layer and reduce wrinkles, and has a more efficient utilization effect on improving skin health.

[0006] The object of the present invention can be achieved by the following technical solutions:

[0007] In the first aspect, the present invention provides a method for preparing natural elastic collagen dipeptide, and the preparation method includes the following steps:

[0008] (1) Pretreatment: The cleaned animal tissue rich in elastin is minced into small pieces, 6-10 times the weight of alkaline solution is added, and it is soaked at room temperature for 3-5 h to remove fat and impurities. The precipitate is washed with distilled water until neutral to obtain a pretreated elastin tissue containing collagen, wherein the alkaline solution is sodium hydroxide solution with a mass concentration of 0.1%-0.3%;

[0009] (2) Protein modification and solubilization treatment: The pretreated elastin tissue containing collagen is packed into a polyethylene packaging bag, evacuated and sealed, and then placed in the high-pressure chamber of a high-pressure device. Using water as the pressure-transmitting medium, high-pressure treatment is carried out, wherein the treatment pressure is set to 200-400 MPa, the treatment time is 5-10 min, the pressure increase rate is 5-7 MPa / s, and the pressure decrease rate is 15-20 MPa / s;

[0010] (3) Protein extraction: The elastin tissue containing collagen after modification and solubilization treatment is mixed with pure water at a mass ratio of 1:2-6, and heated at 85-100 °C for 1-3 h to obtain an elastic collagen extract;

[0011] (4) Enzymolysis: The pH value of the elastic collagen extract is adjusted to 7.5-9.0, protease accounting for 0.5-2.5% of the raw material mass is added, the temperature is adjusted to 50-60 °C, and enzymolysis is carried out for 3-8 h to obtain an elastic collagen dipeptide enzymolysis solution;

[0012] (5) Enzyme inactivation: The enzymolysis solution is heated to 85-95 °C and kept warm for 10-20 min;

[0013] (6) Static settlement and solid-liquid separation: The enzymolysis solution is placed for 2-5 h, the liquid material is stratified up and down, and then the upper and lower layers are filtered respectively, and the filtrates are combined to obtain a mixed clear liquid;

[0014] (7) Decoloration: The mixed clear liquid is decolorized through an activated carbon fiber membrane;

[0015] (8) Purification and concentration: The decolorized solution is purified through a nanofiltration membrane to remove salt ions and free amino acids, and part of the water is removed for concentration;

[0016] (9) Drying: Freeze-drying to obtain elastic collagen dipeptide powder.

[0017] As an alternative, in the above preparation method, in step (1), the animal tissue rich in elastin is one or more of large arteries, ligaments, or the bulbus arteriosus of fish hearts.

[0018] Preferably, the animal tissue rich in elastin is the bulbus arteriosus of fish hearts.

[0019] As an alternative, in the above preparation method, in step (4), the protease is one or a combination of several of alkaline protease, papain, bromelain, neutral protease, flavor protease, trypsin, or pepsin.

[0020] Preferably, the protease is a combination of alkaline protease and neutral protease.

[0021] More preferably, the mass ratio of the alkaline protease to the neutral protease is 2:1.

[0022] As an alternative, in the above preparation method, in step (6), the liquid material is stratified up and down. The upper clear liquid passes through a cotton cake filter press, and the lower wet residue precipitate is subjected to solid-liquid separation using a ceramic membrane. The obtained clear liquids are mixed.

[0023] As an alternative, in the above preparation method, the pore size of the ceramic membrane is 50 - 100 nm.

[0024] As an alternative, in the above preparation method, in step (8), the pore size of the nanofiltration membrane is 200 - 300 Da.

[0025] In a preferred embodiment, the preparation method includes the following steps:

[0026] (1) Pretreatment: The cleaned bulbus arteriosus of fish hearts is minced into small pieces, 6 - 10 times the weight of an alkaline solution is added, and it is soaked at room temperature for 3 - 5 h to remove fat and impurities. The precipitate is washed with distilled water until neutral to obtain pretreated bulbus arteriosus of fish hearts. Among them, the alkaline solution is a sodium hydroxide solution with a mass concentration of 0.1% - 0.3%;

[0027] (2) Protein modification and solubilization treatment: The pretreated bulbus arteriosus of fish hearts is placed in a polyethylene packaging bag, vacuum-sealed and then put into the high-pressure chamber of a high-pressure device. Using water as the pressure-transmitting medium, ultra-high pressure treatment is carried out. Among them, the treatment pressure is set to 200 - 400 MPa, the treatment time is 5 - 10 min, the pressure increase rate is 5 - 7 MPa / s, and the pressure decrease rate is 15 - 20 MPa / s;

[0028] (3) Protein extraction: The bulbus arteriosus of fish heart after modified solubilization treatment is mixed with pure water at a mass ratio of 1:2 - 6, and heated at 85 - 100 °C for 1 - 3 h to obtain an elastic collagen extraction solution;

[0029] (4) Enzymolysis: The pH value of the elastic collagen extraction solution is adjusted to 7.5 - 9.0, protease accounting for 0.5 - 2.5% of the raw material mass is added, the temperature is adjusted to 50 - 60 °C, and enzymolysis is carried out for 3 - 8 h to obtain an enzymolysis solution of elastic collagen dipeptide. The protease is a combination of alkaline protease and neutral protease, and the mass ratio of the alkaline protease to the neutral protease is 2:1;

[0030] (5) Enzyme inactivation: The enzymolysis solution is heated to 85 - 95 °C and kept warm for 10 - 20 min;

[0031] (6) Standing and solid-liquid separation: The enzymolysis solution is placed for 2 - 5 h, the material liquid is stratified up and down, the upper clear liquid passes through a cotton cake filter press, and the lower wet residue precipitate is subjected to solid-liquid separation using a ceramic membrane (50 - 100 nm), and the obtained clear liquid is mixed;

[0032] (7) Decolorization: The mixed clear liquid is decolorized through an activated carbon fiber membrane;

[0033] (8) Purification and concentration: The decolorized solution is purified through a nanofiltration membrane (200 - 300 Da) to remove salt ions and free amino acids, and part of the water is removed for concentration;

[0034] (9) Drying: Freeze-drying to obtain elastic collagen dipeptide powder.

[0035] In the second aspect, the present invention provides a natural elastic collagen dipeptide prepared by the preparation method described in the first aspect above.

[0036] The present invention has the following beneficial effects compared with the prior art:

[0037] (1) The present invention uses large arteries, ligaments rich in elastin, and the bulbus arteriosus of fish heart, etc. as raw materials. While preparing elastin peptides, the original collagen components in the tissues are retained, avoiding waste of protein resources.

[0038] (2) The raw material pretreatment conditions are mild, the concentration of alkali solution is reduced, the amount of waste liquid generated is reduced, and it is more environmentally friendly.

[0039] (3) The present invention applies ultra-high pressure technology to carry out protein modification and solubilization treatment on the raw materials, so that the tertiary and quaternary structures of elastin and collagen are stretched, increasing the solubility and extraction rate of proteins. The protein modification treatment exposes more internal cleavage sites of proteins, enhancing the sensitivity of proteins to enzymes, improving the enzymolysis efficiency, reducing the amount of enzyme used, and lowering the enzyme cost.

[0040] (4) The natural elastic collagen dipeptide prepared by the present invention has a ratio closer to the original ratio in tissues and has a more efficient utilization effect on improving skin health after oral administration. Description of the Drawings

[0041] Figure 1 : Comparison of the molecular weight distributions of the samples prepared in each example and comparative example. Detailed Description of the Invention

[0042] The present invention will be further described below with reference to specific examples. It should be understood that the specific examples described herein are only used to explain the present invention and are not used to limit the scope of the present invention.

[0043] For those technical or conditions not specified in the examples, they shall be in accordance with the techniques or conditions described in the literature in this field or in accordance with the product specifications. For the reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained through regular channels.

[0044] In the following examples, the experimental methods, unless otherwise specified, are all conventional methods. The test materials used in the following examples, unless otherwise specified, are all commercially available products.

[0045] As used herein, "elastin peptide" refers to a small molecule peptide hydrolyzate prepared by enzymatic hydrolysis and separation and purification of tissues rich in elastin.

[0046] As used herein, "collagen peptide" refers to a small molecule peptide hydrolyzate prepared by enzymatic hydrolysis and separation and purification of tissues rich in collagen.

[0047] As used herein, "natural elastic collagen dipeptide" refers to a mixture of elastin peptide and collagen peptide with a natural ratio prepared by enzymatic hydrolysis using raw materials rich in elastin and retaining the original collagen component therein.

[0048] As used herein, the content of desmosine (%) = mass of desmosine in the sample / mass of the sample × 100.

[0049] As used herein, the content of isodesmosine (%) = mass of isodesmosine in the sample / mass of the sample × 100.

[0050] As used herein, the content of hydroxyproline (%) = mass of hydroxyproline in the sample / mass of the sample × 100.

[0051] Preparation Example:

[0052] Example 1

[0053] (1) Pretreatment: Add 8 times the weight of the alkaline solution to the cleaned fish heart arterial bulb, soak it at room temperature for 5 h, and wash it with distilled water until neutral to obtain the pretreated heart arterial bulb. Among them, the alkaline solution is a sodium hydroxide solution with a mass concentration of 0.1%.

[0054] (2) Protein modification and solubilization treatment: Put the pretreated heart arterial bulb into a polyethylene packaging bag, evacuate and seal it, and then put it into the high-pressure chamber of a high-pressure device. Use water as the pressure-transmitting medium for high-pressure treatment. Among them, the treatment pressure is set at 200 MPa, the treatment time is 10 min, the pressure increase rate is 7 MPa / s, and the pressure decrease rate is 20 MPa / s.

[0055] (3) Protein extraction: Mix the heart arterial bulb after modification and solubilization treatment with pure water at a mass ratio of 1:5, and heat it at 90 °C for 3 h to obtain an elastin and collagen extraction solution.

[0056] (4) Enzymolysis: Adjust the pH value of the elastin and collagen extraction solution to 7.5, add 1.0% alkaline protease and 0.5% neutral protease based on the mass of the raw material, adjust the temperature to 55 °C, and carry out enzymolysis for 6 h to obtain an elastin and collagen dipeptide enzymolysis solution.

[0057] (5) Enzyme inactivation: Raise the temperature of the enzymolysis solution to 90 °C and keep it warm for 15 min.

[0058] (6) Standing and solid-liquid separation: Let the enzymolysis solution stand for 3 h, and the material liquid is stratified up and down. The upper clear liquid passes through a cotton cake filter press to obtain the first-stage clear liquid. The lower wet residue precipitate is subjected to solid-liquid separation using a ceramic membrane (50 nm - 100 nm) to obtain the second-stage clear liquid. Combine the first-stage clear liquid and the second-stage clear liquid.

[0059] (7) Decolorization: Pass the mixed clear liquid through an activated carbon fiber membrane for decolorization.

[0060] (8) Purification and concentration: The decolorized solution is purified through a nanofiltration membrane (200 - 300 Da) to remove salt ions and free amino acids, and part of the water is removed for concentration.

[0061] (9) Drying: Freeze-dry to obtain elastin and collagen dipeptide powder.

[0062] Example 2

[0063] (1) Pretreatment: Add 8 times the weight of the alkaline solution to the cleaned fish heart arterial bulb, soak it at room temperature for 3 h, and wash it with distilled water until neutral to obtain the pretreated heart arterial bulb. Among them, the alkaline solution is a sodium hydroxide solution with a mass concentration of 0.3%.

[0064] (2) Protein modification and solubilization treatment: The pretreated cardiac arterial bulb is packed in a polyethylene packaging bag, vacuum-sealed and then placed in the high-pressure chamber of a high-pressure equipment. Using water as the pressure-transmitting medium, high-pressure treatment is carried out. The treatment pressure is set at 200 MPa, the treatment time is 10 min, the pressure increase rate is 7 MPa / s, and the pressure decrease rate is 20 MPa / s.

[0065] (3) Protein extraction: The cardiac arterial bulb after modification and solubilization treatment is mixed with pure water at a mass ratio of 1:5, and heated at 90 °C for 3 h to obtain an elastin and collagen extraction solution.

[0066] (4) Enzymatic hydrolysis: The pH value of the elastin and collagen extraction solution is adjusted to 7.5, 1.0% of alkaline protease and 0.5% of neutral protease based on the mass of the raw material are added, the temperature is adjusted to 55 °C, and enzymatic hydrolysis is carried out for 6 h to obtain an elastin and collagen double-protein peptide enzymatic hydrolysis solution.

[0067] (5) Enzyme inactivation: The enzymatic hydrolysis solution is heated to 90 °C and kept warm for 15 min.

[0068] (6) Static settling and solid-liquid separation: The enzymatic hydrolysis solution is left standing for 3 h, the liquid material is stratified up and down. The upper clear liquid passes through a cotton cake filter press to obtain a first-stage clear liquid. The lower wet residue precipitate is subjected to solid-liquid separation using a ceramic membrane (50 nm - 100 nm) to obtain a second-stage clear liquid. The first-stage clear liquid and the second-stage clear liquid are combined.

[0069] (7) Decolorization: The mixed clear liquid is decolorized through an activated carbon fiber membrane.

[0070] (8) Purification and concentration: The decolorized solution is purified through a nanofiltration membrane (200 - 300 Da) to remove salt ions and free amino acids, and part of the water is removed for concentration.

[0071] (9) Drying: Freeze-drying is carried out to obtain elastin and collagen double-protein peptide powder.

[0072] Example 3

[0073] (1) Pretreatment: The cleaned cardiac arterial bulb of fish is added with an alkaline solution 8 times its weight, soaked at room temperature for 5 h, and washed with distilled water until neutral to obtain a pretreated cardiac arterial bulb. Among them, the alkaline solution is a sodium hydroxide solution with a mass concentration of 0.1%.

[0074] (2) Protein modification and solubilization treatment: The pretreated cardiac arterial bulb is packed in a polyethylene packaging bag, vacuum-sealed and then placed in the high-pressure chamber of a high-pressure equipment. Using water as the pressure-transmitting medium, high-pressure treatment is carried out. The treatment pressure is set at 300 MPa, the treatment time is 5 min, the pressure increase rate is 7 MPa / s, and the pressure decrease rate is 20 MPa / s.

[0075] (3) Protein extraction: The modified and solubilized cardiac arterial bulb was mixed with pure water at a mass ratio of 1:5 and heated at 90 °C for 3 h to obtain an elastin and collagen extraction solution.

[0076] (4) Enzymatic hydrolysis: The pH value of the elastin and collagen extraction solution was adjusted to 7.5, 1.0% alkaline protease and 0.5% neutral protease based on the mass of the raw material were added, the temperature was adjusted to 55 °C, and enzymatic hydrolysis was carried out for 6 h to obtain an elastin and collagen double protein peptide enzymatic hydrolysis solution.

[0077] (5) Enzyme inactivation: The enzymatic hydrolysis solution was heated to 90 °C and kept warm for 15 min.

[0078] (6) Static settling and solid-liquid separation: The enzymatic hydrolysis solution was left standing for 3 h, the liquid material was stratified up and down, the upper clear liquid was filtered through a cotton cake filter press to obtain a first-stage clear liquid, and the lower wet residue precipitate was subjected to solid-liquid separation using a ceramic membrane (50 nm - 100 nm) to obtain a second-stage clear liquid. The first-stage clear liquid and the second-stage clear liquid were combined.

[0079] (7) Decolorization: The mixed clear liquid was decolorized through an activated carbon fiber membrane.

[0080] (8) Purification and concentration: The decolorized solution was purified through a nanofiltration membrane (200 - 300 Da) to remove salt ions and free amino acids, and part of the water was removed for concentration.

[0081] (9) Drying: Freeze-drying was carried out to obtain elastin and collagen double protein peptide powder.

[0082] Example 4

[0083] (1) Pretreatment: The cleaned cardiac arterial bulb of fish was added to an alkaline solution with 8 times its weight and soaked at room temperature for 5 h, and then washed with distilled water until neutral to obtain a pretreated cardiac arterial bulb. Among them, the alkaline solution was a sodium hydroxide solution with a mass concentration of 0.1%.

[0084] (2) Protein modification and solubilization treatment: The pretreated cardiac arterial bulb was put into a polyethylene packaging bag, evacuated and sealed, and then placed in the high-pressure chamber of a high-pressure device. Using water as the pressure-transmitting medium, high-pressure treatment was carried out. Among them, the treatment pressure was set to 200 MPa, the treatment time was 10 min, the pressure increase rate was 7 MPa / s, and the pressure decrease rate was 20 MPa / s.

[0085] (3) Protein extraction: The modified and solubilized cardiac arterial bulb was mixed with pure water at a mass ratio of 1:5 and heated at 90 °C for 3 h to obtain an elastin and collagen extraction solution.

[0086] (4) Enzymatic hydrolysis: The pH value of the elastin and collagen extraction solution was adjusted to 7.5, 1.0% alkaline protease and 0.5% papain based on the mass of the raw material were added, the temperature was adjusted to 55 °C, and enzymatic hydrolysis was carried out for 6 h to obtain an elastin and collagen double protein peptide enzymatic hydrolysis solution.

[0087] (5) Enzyme inactivation: Heat the enzymolysis solution to 90 °C and keep it warm for 15 min.

[0088] (6) Standing and solid-liquid separation: Let the enzymolysis solution stand for 3 h. The liquid and solid are stratified. The supernatant on the upper layer passes through a cotton cake filter press to obtain the first-stage clear liquid. The wet residue precipitate on the lower layer is subjected to solid-liquid separation using a ceramic membrane (50 nm - 100 nm) to obtain the second-stage clear liquid. The first-stage clear liquid and the second-stage clear liquid are combined.

[0089] (7) Decoloration: Decolorize the mixed clear liquid through an activated carbon fiber membrane.

[0090] (8) Purification and concentration: The decolorized liquid is purified through a nanofiltration membrane (200 - 300 Da) to remove salt ions and free amino acids, and part of the water is removed for concentration.

[0091] (9) Drying: Freeze-dry to obtain the elastic collagen dipeptide powder.

[0092] Comparative Example 1

[0093] In step (1) of Example 1, change it to adding 8 times the weight of an alkaline solution to the clean fish heart arterial bulb and soaking it at 85 °C for 1 h, where the mass concentration of the alkaline solution is 0.5%, and other conditions are the same as in Example 1.

[0094] Comparative Example 2

[0095] Delete step (1) in Example 1. The fish heart arterial bulb is only washed without pre-treatment by soaking in an alkaline solution, and other conditions are the same as in Example 1.

[0096] Comparative Example 3

[0097] Delete step (2) in Example 1, and other conditions are the same as in Example 1.

[0098] Comparative Example 4

[0099] Delete step (2) in Example 1, and change the enzyme addition amount in step (4) of Example 1 to 2.0% of alkaline protease and 1.0% of neutral protease based on the mass of the raw material, and other conditions are the same as in Example 1.

[0100] Effect Example:

[0101] Effect Example 1

[0102] For the test indexes and their detection and analysis methods involved in each example and comparative example prepared in the above preparation examples of the present invention are as follows:

[0103] Contents of desmosine and isodesmosine: The sample was hydrolyzed under acidic conditions and determined by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS). The retention time and mass-to-charge ratio of desmosine and isodesmosine standards were used for qualitative and confirmatory analysis, and the external standard method was used for quantification. Chromatographic conditions: Atlantis dC18 (3μm, 150mm×2.1mm), column temperature 30°C, flow rate 0.3mL / min, mobile phase A: aqueous solution (containing 7mM heptafluorobutyric anhydride, 5mM ammonium acetate), mobile phase B: 80% acetonitrile (containing 7mM heptafluorobutyric anhydride, 5mM ammonium acetate); elution program: from 0 to 14min, mobile phase A was gradient eluted from 100% to 30%, and from 14 to 22min, it was eluted to 100% mobile phase A. Mass spectrometry conditions: electrospray ionization source, positive ion mode, ion source spray voltage 3.5kV, capillary temperature 320°C, collision energy 35eV, desmosine parent ion (m / z) 526.3, daughter ion (m / z) 481.3, isodesmosine parent ion (m / z) 526.3, daughter ion (m / z) 397.3.

[0104] Content of hydroxyproline: It was determined using an acid hydrolysis method hydroxyproline kit.

[0105] Molecular weight distribution: It was analyzed by high performance liquid chromatography (HPLC). Chromatographic conditions: chromatographic column TSKgel G2000 SWXL 300mm×7.8mm, column temperature 30°C, mobile phase: aqueous solution containing 40% acetonitrile and 0.05% trifluoroacetic acid, detection wavelength 220nm, flow rate 0.5mL / min, injection volume 10μL. By analyzing and plotting the chromatograms of a series of standards, a relative molecular mass calibration curve and its equation were obtained. The chromatograms and data of the samples were calculated and processed using GPC data processing software to obtain the relative molecular weight and distribution range of the samples.

[0106] The results comparison of the total content of desmosine and isodesmosine, hydroxyproline content, and yield in the samples prepared in Examples 1-4 and Comparative Examples 1-4 is shown in Table 1.

[0107] Table 1: Comparison of the total content of desmosine and isodesmosine, hydroxyproline and yield in the samples prepared in each example and comparative example

[0108]

[0109] As shown in Table 1, the total content of desmosine and isodesmosine in the elastic collagen dipeptide prepared in the examples is higher than 0.3%, and the hydroxyproline content is higher than 3%, indicating that the samples contain relatively high contents of both elastin peptide and collagen peptide components, and the yields are all higher than 11%.

[0110] In Comparative Example 1, the heart arterial bulb raw material was treated by heating with strong alkali solution, resulting in a significant decrease in the yield and a marked decrease in the hydroxyproline content, indicating that this pretreatment condition led to a large loss of collagen in the raw material. Compared with Example 1, in Comparative Example 2, the yield increased, but the total content of desmosine and isodesmosine and the hydroxyproline content both decreased, indicating that impurities could be removed by pretreatment of the raw material, improving the purity of the elastic collagen dipeptide. Compared with Example 1, in Comparative Example 3 and Comparative Example 4, the original enzyme dosage and double the enzyme dosage were used respectively, and the yields both decreased, indicating that protein modification solubilization treatment of the raw material could improve the enzymatic hydrolysis efficiency and reduce the enzyme dosage.

[0111] The comparison of the molecular weight distribution of the samples prepared in the examples and comparative examples is shown in Figure 1 . From Figure 1 it can be seen that compared with Example 1, the proportion of small molecule peptides in Comparative Example 3 and Comparative Example 4 decreased significantly, indicating that protein modification solubilization treatment could improve the enzymatic hydrolysis effect and increase the proportion of small molecule peptides.

[0112] Effect Example 2: Skin test

[0113] Test method: A total of 120 healthy female volunteers aged 35 - 45 years, with a BMI in the range of 18.5 - 27.9 kg / m 2 and a wrinkle severity rating scale (WSRS) score ≥ 2 for facial wrinkles were recruited. They were randomly divided into three groups: Control Group 1, Control Group 2, and Experimental Group, with 40 people in each group, and a double-blind randomized trial was conducted. Each person in the Experimental Group orally took a product containing 3 g of the natural elastic collagen dipeptide prepared in Example 1 every day. Each person in Control Group 1 orally took a product containing 3 g of artificially formulated elastic and collagen peptides (including 0.3 g of the elastic protein peptide prepared in Comparative Example 1 and 2.7 g of commercially available collagen peptide) every day. Each person in Control Group 2 orally took a product containing 3 g of the elastic collagen dipeptide prepared in Comparative Example 3 every day. The trial lasted for 12 weeks in total. On the 0th day of the trial and the 90th day of the trial end. A CORTEX ( Combo) skin physiological index tester was used to detect the water content of the skin stratum corneum, transepidermal water loss, and skin elasticity of the volunteers' skin. A multi-spectral dermoscope image processing workstation (Cloudscope, CBS - 2021, Wuhan Boshi Electronics Co., Ltd.) was used for the test and collection of skin images, and the wrinkles were scored.

[0114] The SPSS 2.0 statistical analysis software was used for data statistical analysis. Paired sample statistical analysis before and after the experiment was performed on the experimental group and the control group respectively, that is, the subjects were paired with themselves before and after. All outcome variables were continuous variables. For measurement data where the difference before and after the intervention conformed to a normal distribution or an approximately normal distribution, paired sample T-tests were used, and the results were expressed as (mean ± standard deviation); for measurement data where the difference did not conform to a normal distribution, paired sample rank sum tests were used, and the results were described using the median and interquartile range (the distance between the 25th percentile and the 75th percentile, that is, P25 and P75).

[0115] The test results of skin moisture content are shown in Table 2.

[0116] Table 2: Changes in the water content of the stratum corneum of the skin before and after the experiment in the experimental group and the control group

[0117]

[0118] The results showed that the change rates of the average water content of the stratum corneum at 8 sites on the facial skin of Control Group 1 and Control Group 2 before and after the experiment were 17.39% and 25.60% respectively, and the change rate of the average water content of the stratum corneum in the experimental group was 36.52%. The statistical results showed significant differences. The experimental results indicate that the natural elastic collagen dipeptide prepared by the method of the present invention has a more significant effect on improving skin moisture content.

[0119] The test results of transepidermal water loss are shown in Table 3.

[0120] Table 3: Changes in transepidermal water loss before and after the experiment in the experimental group and the control group

[0121]

[0122] The results showed that the transepidermal water loss on both cheeks of the experimental group decreased significantly by 28.34% before and after the experiment, and the transepidermal water loss in Control Group 1 and Control Group 2 decreased by 11.99% and 19.94% respectively, indicating that the natural elastic collagen dipeptide prepared by the method of the present invention has a more significant effect on improving the barrier function of the facial stratum corneum.

[0123] The test results of skin elasticity are shown in Table 4.

[0124] Table 4: Changes in skin viscoelasticity values before and after the experiment in the experimental group and the control group

[0125]

[0126]

[0127] The results showed that the average viscoelasticity of the left and right cheeks in the experimental group increased by 9.33% after the 90-day experiment, which was higher than the elastic change rates of Control Group 1 and Control Group 2, indicating that the natural elastic collagen dipeptide prepared by the method of the present invention helps to improve and enhance the elasticity of the skin.

[0128] The results of the skin wrinkle test are shown in Table 5.

[0129] Table 5: Changes in skin wrinkles before and after the experiment of the experimental group and the control groups

[0130]

[0131] The results showed that the index values of superficial wrinkles, fine lines and deep wrinkles of the skin of the volunteers in the experimental group decreased significantly after the 90-day experiment, and the degree of decrease was higher than that of Control Group 1 and Control Group 2, indicating that the natural elastic collagen dipeptide prepared by the method of the present invention has a more significant effect on improving skin wrinkles.

[0132] In summary, for the natural elastic collagen peptide prepared according to the method of the present invention, the ratio of elastin to collagen is closer to the original ratio in the tissue. After 90 days of oral administration, it has a significant effect on improving skin elasticity and moisture content, improving the functional barrier of the skin cutin layer and reducing wrinkles, and has a more efficient utilization effect on improving skin health.

[0133] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these changes and modifications.

Claims

1. A method for preparing a natural elastic collagen dual protein peptide, characterized in that: The steps of the preparation method are as follows: (1) Pretreatment: Add 8 times the weight of alkaline solution to the cleaned fish cardiac arterial bulb, soak it at room temperature for 3-5 hours, and wash it with distilled water until it is neutral, thereby obtaining a pretreated cardiac arterial bulb, wherein the alkaline solution is a sodium hydroxide solution with a mass concentration of 0.1-0.3%; (2) Protein modification and solubilization treatment: The pretreated cardiac arterial bulbs were placed in a polyethylene packaging bag, which was sealed by vacuum evacuation and placed in the high-pressure chamber of an ultrahigh-pressure device. Water was used as the pressure transmission medium for ultrahigh-pressure treatment, where the treatment pressure was set to 200 MPa, the treatment time was 5-10 min, the pressure increase rate was 7 MPa / s, and the pressure reduction rate was 20 MPa / s; (3) Protein extraction: The modified and solubilized cardiac arterial bulb was mixed with purified water at a mass ratio of 1:5 and heated at 90 °C for 3 h to obtain elastin and collagen extracts; (4) Enzymatic hydrolysis: The pH value of the elastin and collagen extract was adjusted to 7.5, 1.0% alkaline protease and 0.5% neutral protease were added, the temperature was adjusted to 55°C, and the enzymatic hydrolysis was performed for 6 h to obtain the elastin collagen dual protein peptide enzymatic hydrolyzate; (5) Inactivate enzyme: Heat the enzymatic solution to 90°C and keep warm for 15 min; (6) Standing and solid-liquid separation: The enzymatic hydrolyzate was allowed to stand for 3 h, and the feed liquid was separated into upper and lower layers. The upper clear liquid was filtered through a cotton cake filter press to obtain a first-level clear liquid. The lower wet residue precipitate was separated into a solid and liquid using a ceramic membrane of 50-100 nm to obtain a second-level clear liquid. The first-level clear liquid and the second-level clear liquid were combined to obtain a mixed clear liquid. (7) Decolorization: The mixed clear liquid is decolorized by passing it through an activated carbon fiber membrane to obtain a decolorized liquid; (8) Purification and concentration: The decolorized liquid is purified by passing through a nanofiltration membrane of 200-300 Da to remove salt ions and free amino acids, and to remove part of the water for concentration; (9) Drying: Freeze drying to obtain elastic collagen dual protein peptide powder.

2. A method for preparing a natural elastic collagen dual protein peptide, characterized in that: The steps of the preparation method are as follows: (1) Pretreatment: Add 8 times the weight of alkaline solution to the cleaned fish cardiac arterial bulb, soak it at room temperature for 5 h, and wash it with distilled water until it is neutral to obtain the pretreated cardiac arterial bulb, wherein the alkaline solution is sodium hydroxide solution with a mass concentration of 0.1%; (2) Protein modification and solubilization treatment: The pretreated cardiac arterial bulb was placed in a polyethylene packaging bag, which was sealed by vacuum evacuation and placed in the high-pressure chamber of an ultrahigh-pressure device. Water was used as the pressure transmission medium for ultrahigh-pressure treatment. The treatment pressure was set to 200 MPa, the treatment time was 10 min, the pressure increase rate was 7 MPa / s, and the pressure reduction rate was 20 MPa / s. (3) Protein extraction: The modified and solubilized cardiac arterial bulb was mixed with purified water at a mass ratio of 1:5 and heated at 90 °C for 3 h to obtain elastin and collagen extracts; (4) Enzymatic hydrolysis: The pH value of the elastin and collagen extract was adjusted to 7.5, 1.0% alkaline protease and 0.5% papain by weight of the raw materials were added, the temperature was adjusted to 55°C, and the enzymatic hydrolysis was performed for 6 h to obtain the elastin collagen dual protein peptide enzymatic hydrolyzate; (5) Inactivate enzyme: Heat the enzymatic solution to 90°C and keep warm for 15 min; (6) Standing and solid-liquid separation: The enzymatic hydrolyzate was allowed to stand for 3 h, and the feed liquid was separated into upper and lower layers. The upper clear liquid was filtered through a cotton cake filter press to obtain a first-level clear liquid. The lower wet residue precipitate was separated into a solid and liquid using a ceramic membrane of 50-100 nm to obtain a second-level clear liquid. The first-level clear liquid and the second-level clear liquid were combined to obtain a mixed clear liquid. (7) Decolorization: The mixed clear liquid is decolorized by passing it through an activated carbon fiber membrane to obtain a decolorized liquid; (8) Purification and concentration: The decolorized liquid is purified by passing through a nanofiltration membrane of 200-300 Da to remove salt ions and free amino acids, and to remove part of the water for concentration; (9) Drying: Freeze drying to obtain elastic collagen dual protein peptide powder.

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