Natural elastic collagen double-protein peptide and preparation method thereof

Abstract

The invention belongs to the technical field of active substance extraction, and particularly relates to a natural elastic collagen double-protein peptide and a preparation method thereof. While the elastin peptide is prepared, the original collagen component in the tissue is reserved, so that the waste of protein resources is avoided. The raw material pretreatment conditions are mild, the concentration of alkali liquor is reduced, the output of waste liquid is reduced, and the method is more environment-friendly. According to the method, the raw materials are subjected to protein modification and solubilization treatment by applying an ultrahigh pressure technology, so that three-stage and four-stage structures of elastin and collagen are stretched, the solubility and the extraction rate of protein are increased, more enzyme cutting sites are exposed in the protein through protein modification treatment, the sensitivity of the protein to enzyme is enhanced, and the enzymolysis efficiency is improved; the enzyme consumption is reduced, and the enzyme cost is reduced. According to the natural elastic collagen double-protein peptide prepared by the invention, the proportion better fits the original proportion in tissues, and the natural elastic collagen double-protein peptide has a more efficient utilization effect on improving skin health after being orally taken.

Description

Technical Field

[0001] The invention belongs to the technical field of active substance extraction, and specifically relates to a natural elastic collagen dual protein peptide and a preparation method thereof. Background Art

[0002] Elastin is the main component of elastic fibers. It exists in connective tissues combined with collagen, giving the tissue elasticity and tensile strength, and plays 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 tissue. On a dry weight basis, the content of elastin in ligaments is the highest, 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 lower, about 2-4%, while the content of collagen in the skin exceeds 70%. Although the content of elastin in the skin is low, it plays an important role in maintaining the state and function of the skin. 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 the filling and plumpness of the skin to keep the skin smooth, hydrated and firm. That is to say, in the skin, ligaments and other tissues of the human body, elastin usually coexists with collagen, and they play important physiological functions together. The ratio between them has an important influence on the mechanical properties of cells and tissues.

[0003] However, people currently extract and enzymatically prepare elastin peptides from tissues such as ligaments, lungs, cartilage and arterial bulbs of animals. The existing preparation process usually removes collagen in the tissue during the raw material pretreatment stage (for example, by concentrated alkali high temperature treatment) to prepare elastin peptides with higher purity. This not only causes a waste of collagen resources, but also destroys the natural ratio of elastin and collagen in the raw materials. The existing elastin extraction process is complicated, concentrated alkali is used for pretreatment, a lot of water is used for cleaning, and the amount of wastewater is large, which is not friendly to the environment. Elastin and collagen are insoluble in water, and their structures are compact and not easily cut by enzymes, resulting in low efficiency of the existing extraction process. In addition, more importantly, the existing extraction process cannot achieve the simultaneous extraction of elastin peptides and collagen peptides with a natural ratio, which leads to the current products containing the above two active ingredients being obtained by artificially combining the two raw materials of elastin peptides and collagen peptides, which makes the operation steps more complicated on the one hand, and on the other hand, it is impossible to ensure that the two active ingredients play a role in the original natural ratio in the tissue.

[0004] Therefore, how to retain the components of collagen when preparing elastin peptides and prepare elastin collagen dual protein peptides with natural ratios by enzymatic hydrolysis is an urgent problem to be solved. Summary of the invention

[0005] The present invention aims at the defects and deficiencies in the prior art and provides a method for preparing a natural elastic collagen dual-protein peptide. The natural elastic collagen dual-protein peptide prepared by the method of the present invention can significantly improve skin elasticity and moisture content, improve the functional barrier of the skin stratum corneum and reduce wrinkles, and has a more efficient utilization effect on improving skin health.

[0006] The purpose of the present invention can be achieved through the following technical solutions:

[0007] In a first aspect, the present invention provides a method for preparing a natural elastic collagen dual-protein peptide, the preparation method comprising the following steps:

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

[0009] (2) Protein modification and solubilization treatment: the pretreated collagen-containing elastin tissue is placed in a polyethylene packaging bag, which is vacuum-sealed and placed in a high-pressure chamber of an ultrahigh-pressure device, and ultrahigh-pressure treatment is performed using water as a pressure medium, 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 reduction rate is 15-20 MPa / s;

[0010] (3) Protein extraction: the modified solubilized elastin tissue containing collagen is mixed with purified water at a mass ratio of 1:2-6, and heated at 85-100° C. for 1-3 h to obtain an elastin collagen extract;

[0011] (4) Enzymatic hydrolysis: adjust the pH value of the elastic collagen extract to 7.5-9.0, add 0.5-2.5% of the raw material weight of protease, adjust the temperature to 50-60° C., and perform enzymatic hydrolysis for 3-8 hours to obtain elastic collagen double protein peptide enzymatic hydrolyzate;

[0012] (5) Inactivation of enzyme: Heat the enzymatic solution to 85-95°C and keep warm for 10-20 min;

[0013] (6) Standing and solid-liquid separation: The enzymatic hydrolyzate is allowed to stand for 2-5 hours, the feed liquid is separated into upper and lower layers, and then the upper and lower layers are filtered separately, and the filtrates are combined to obtain a mixed clear liquid;

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

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

[0016] (9) Drying: Freeze drying to obtain elastic collagen double protein peptide powder.

[0017] As an optional manner, in the above preparation method, in step (1), the elastin-rich animal tissue is one or more of aorta, ligament or fish heart arterial bulb.

[0018] Preferably, the animal tissue rich in elastin is fish heart bulb.

[0019] As an optional manner, in the above preparation method, in step (4), the protease is one or a combination 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 optional manner, in the above preparation method, in step (6), the feed liquid is separated into upper and lower layers, the upper clear liquid is passed through a cotton cake filter press, and the lower wet residue precipitate is subjected to solid-liquid separation using a ceramic membrane, and the prepared clear liquids are mixed.

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

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

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

[0026] (1) Pretreatment: The cleaned fish cardiac arterial bulb is crushed into small pieces, and an alkaline solution of 6-10 times its weight is added, and the pieces are soaked at room temperature for 3-5 hours to remove fat and impurities, and the precipitate is washed with distilled water until it is neutral, thereby obtaining a pretreated fish cardiac arterial bulb, wherein 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 fish cardiac arterial bulb is placed in a polyethylene packaging bag, which is vacuum-sealed and then placed in a high-pressure chamber of an ultrahigh-pressure device, and ultrahigh-pressure treatment is performed using water as a pressure transmission medium, 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 reduction rate is 15-20 MPa / s;

[0028] (3) Protein extraction: the fish cardiac arterial bulb after the modified solubilization treatment is mixed with purified water at a mass ratio of 1:2-6, and heated at 85-100°C for 1-3h to obtain an elastic collagen extract;

[0029] (4) Enzymatic hydrolysis: adjusting the pH value of the elastic collagen extract to 7.5-9.0, adding 0.5-2.5% of the raw material weight of protease, adjusting the temperature to 50-60° C., and performing enzymatic hydrolysis for 3-8 hours to obtain an elastic collagen double protein peptide hydrolyzate, wherein 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) Inactivation of enzyme: Heat the enzymatic solution to 85-95°C and keep warm for 10-20 min;

[0031] (6) Standing and solid-liquid separation: The enzymatic hydrolyzate is placed for 2-5 hours, the feed liquid is separated into upper and lower layers, the upper clear liquid is passed through a cotton cake filter press, and the lower wet residue precipitate is separated into solid and liquid using a ceramic membrane (50-100 nm), and the prepared clear liquids are mixed;

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

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

[0034] (9) Drying: Freeze drying to obtain elastic collagen double protein peptide powder.

[0035] In a second aspect, the present invention provides a natural elastic collagen dual-protein peptide prepared by the preparation method described in the first aspect.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] (1) The present invention uses elastin-rich aorta, ligaments and fish heart artery bulb as raw materials to prepare elastin peptides while retaining the original collagen components in the tissues to avoid wasting protein resources.

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

[0039] (3) The present invention uses ultrahigh 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, the solubility and extraction rate of the protein are increased, and the protein modification treatment exposes more internal enzyme cleavage sites of the protein, enhances the sensitivity of the protein to enzymes, improves the enzymatic hydrolysis efficiency, reduces the amount of enzyme used, and reduces the cost of enzyme use.

[0040] (4) The natural elastic collagen dual protein peptide prepared by the present invention has a ratio that is more in line with the original ratio in the tissue, and has a more efficient utilization effect on improving skin health after oral administration. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 : Comparison of molecular weight distribution of samples prepared in each embodiment and comparative example. DETAILED DESCRIPTION

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

[0043] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

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

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

[0046] As used herein, "collagen peptides" refer to small molecule peptide hydrolysates prepared by enzymatic hydrolysis, separation and purification of collagen-rich tissues.

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

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

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

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

[0051] Preparation Example:

[0052] Example 1

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

[0054] (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, and ultrahigh-pressure treatment was performed using water as the pressure medium. 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.

[0055] (3) Protein extraction: The modified 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.

[0056] (4) Enzymatic hydrolysis: The pH value of the elastic collagen extract was adjusted to 7.5, 1.0% alkaline protease and 0.5% neutral protease by weight of the raw material were added, the temperature was adjusted to 55° C., and the enzymatic hydrolysis was performed for 6 h to obtain the elastic collagen double protein peptide enzymatic hydrolyzate.

[0057] (5) Inactivation of enzymes: Heat the enzymatic solution to 90°C and keep it warm for 15 min.

[0058] (6) Standing and solid-liquid separation: The enzymatic hydrolyzate was allowed to stand for 3 hours, 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 from the solid by a ceramic membrane (50nm-100nm) to obtain a second-level clear liquid. The first-level clear liquid and the second-level clear liquid were combined.

[0059] (7) Decolorization: The mixed clear liquid is decolorized by passing it through an activated carbon fiber membrane.

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

[0061] (9) Drying: Freeze drying to obtain elastic collagen double protein peptide powder.

[0062] Example 2

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

[0064] (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, and ultrahigh-pressure treatment was performed using water as the pressure medium. 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.

[0065] (3) Protein extraction: The modified 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.

[0066] (4) Enzymatic hydrolysis: The pH value of the elastic collagen extract was adjusted to 7.5, 1.0% alkaline protease and 0.5% neutral protease by weight of the raw material were added, the temperature was adjusted to 55° C., and the enzymatic hydrolysis was performed for 6 h to obtain the elastic collagen double protein peptide enzymatic hydrolyzate.

[0067] (5) Inactivation of enzymes: Heat the enzymatic solution to 90°C and keep it warm for 15 min.

[0068] (6) Standing and solid-liquid separation: The enzymatic hydrolyzate was allowed to stand for 3 hours, 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 from the solid by a ceramic membrane (50nm-100nm) to obtain a second-level clear liquid. The first-level clear liquid and the second-level clear liquid were combined.

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

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

[0071] (9) Drying: Freeze drying to obtain elastic collagen double protein peptide powder.

[0072] Example 3

[0073] (1) Pretreatment: Add 8 times the weight of alkaline solution to the cleaned fish heart artery bulb, soak it at room temperature for 5 hours, and wash it with distilled water until it becomes neutral, thereby obtaining the pretreated heart artery bulb. The alkaline solution is sodium hydroxide solution with a mass concentration of 0.1%.

[0074] (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, and ultrahigh-pressure treatment was performed using water as the pressure medium. The treatment pressure was set to 300 MPa, the treatment time was 5 min, the pressure increase rate was 7 MPa / s, and the pressure reduction rate was 20 MPa / s.

[0075] (3) Protein extraction: The modified 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.

[0076] (4) Enzymatic hydrolysis: The pH value of the elastic collagen extract was adjusted to 7.5, 1.0% alkaline protease and 0.5% neutral protease by weight of the raw material were added, the temperature was adjusted to 55° C., and the enzymatic hydrolysis was performed for 6 h to obtain the elastic collagen double protein peptide enzymatic hydrolyzate.

[0077] (5) Inactivation of enzymes: Heat the enzymatic solution to 90°C and keep it warm for 15 min.

[0078] (6) Standing and solid-liquid separation: The enzymatic hydrolyzate was allowed to stand for 3 hours, 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 from the solid by a ceramic membrane (50nm-100nm) to obtain a second-level clear liquid. The first-level clear liquid and the second-level clear liquid were combined.

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

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

[0081] (9) Drying: Freeze drying to obtain elastic collagen double protein peptide powder.

[0082] Example 4

[0083] (1) Pretreatment: Add 8 times the weight of alkaline solution to the cleaned fish heart artery bulb, soak it at room temperature for 5 hours, and wash it with distilled water until it becomes neutral, thereby obtaining the pretreated heart artery bulb. The alkaline solution is sodium hydroxide solution with a mass concentration of 0.1%.

[0084] (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, and ultrahigh-pressure treatment was performed using water as the pressure medium. 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.

[0085] (3) Protein extraction: The modified 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.

[0086] (4) Enzymatic hydrolysis: The pH value of the elastic collagen extract was adjusted to 7.5, 1.0% alkaline protease and 0.5% papain were added, the temperature was adjusted to 55° C., and the enzymatic hydrolysis was performed for 6 h to obtain the elastic collagen double protein peptide enzymatic hydrolyzate.

[0087] (5) Inactivation of enzymes: Heat the enzymatic solution to 90°C and keep it warm for 15 min.

[0088] (6) Standing and solid-liquid separation: The enzymatic hydrolyzate was allowed to stand for 3 hours, 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 from the solid by a ceramic membrane (50nm-100nm) to obtain a second-level clear liquid. The first-level clear liquid and the second-level clear liquid were combined.

[0089] (7) Decolorization: The mixed clear liquid is decolorized by passing it through an activated carbon fiber membrane.

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

[0091] (9) Drying: Freeze drying to obtain elastic collagen double protein peptide powder.

[0092] Comparative Example 1

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

[0094] Comparative Example 2

[0095] Step (1) in Example 1 was deleted, and the fish cardiac arterial bulb was only cleaned without being pretreated by soaking in alkali solution. Other conditions were 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] Step (2) in Example 1 was deleted, and the amount of enzyme added in step (4) in Example 1 was changed to 2.0% alkaline protease and 1.0% neutral protease by weight of the raw material. Other conditions were the same as in Example 1.

[0100] Effect embodiment:

[0101] Effect Example 1

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

[0103] Content of desmosin and isodesmosin: The sample was hydrolyzed under acidic conditions and determined using high performance liquid chromatography-mass spectrometry (HPLC-MS / MS). The retention time and mass-to-charge ratio of desmosin and isodesmosin standards were used for qualitative and confirmation, and the external standard method was used for quantitative analysis. Chromatographic conditions: Atlantis dC18 (3 μm, 150 mm×2.1 mm), column temperature 30°C, flow rate 0.3 mL / min, mobile phase A: aqueous solution (containing 7 mM heptafluorobutyric anhydride, 5 mM ammonium acetate), mobile phase B: 80% acetonitrile (containing 7 mM heptafluorobutyric anhydride, 5 mM ammonium acetate); elution program: 0-14 min mobile phase A gradient elution from 100% to 30%, 14-22 min elution to 100% mobile phase A. Mass spectrometry conditions: electrospray ion source, positive ion mode, ion source spray voltage 3.5 kV, capillary temperature 320°C, collision energy 35 eV, 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] Hydroxyproline content: The hydroxyproline content was determined using an acid hydrolysis method hydroxyproline kit.

[0105] Molecular weight distribution: Analyzed using high performance liquid chromatography (HPLC). Chromatographic conditions: chromatographic column TSKgelG2000 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. The relative molecular mass calibration curve and its equation were obtained by analyzing and plotting the chromatograms of the series of standard products, and 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 in the samples.

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

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

[0108]

[0109] As shown in Table 1, the total content of desmosin and isodesmosin in the elastic collagen dual-protein peptide prepared in the example was higher than 0.3%, and the hydroxyproline content was higher than 3%, indicating that the sample contained relatively high content of elastin peptide and collagen peptide components, and the yield was higher than 11%.

[0110] Comparative Example 1 uses concentrated alkali solution to heat the heart artery bulb raw material, and the yield is greatly reduced, and the hydroxyproline content is significantly reduced, indicating that this pretreatment condition leads to a large loss of collagen in the raw material. Compared with Example 1, Comparative Example 2 has an improved yield, but the total content of desmosin and isodesmosin and the hydroxyproline content are reduced, indicating that the raw material can be pretreated to remove impurities and improve the purity of elastic collagen dual protein peptide. Compared with Example 1, Comparative Examples 3 and Comparative Examples 4 use the original enzyme amount and double the enzyme amount, respectively, and the yield is reduced, indicating that the raw material is treated with protein modification and solubilization, which can improve the enzymatic efficiency and reduce the amount of enzyme.

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

[0112] Effect example 2: Skin test

[0113] Test method: Recruitment age is between 35-45 years old, BMI is between 18.5-27.9 kg / m 2 A total of 120 healthy female volunteers with a facial wrinkle severity rating scale (WSRS) score ≥ 2 within the range were randomly divided into three groups: control group 1, control group 2 and experimental group, with 40 people in each group, for a double-blind randomized trial. In the experimental group, each person orally added 3g of the natural elastic collagen dual protein peptide product prepared in Example 1 every day, in the control group 1, each person orally added 3g of the artificially formulated elastic and collagen peptide product (containing 0.3g of the elastic peptide prepared in Comparative Example 1 and 2.7g of commercially available collagen peptide) every day, and in the control group 2, each person orally added 3g of the elastic collagen dual protein peptide product prepared in Comparative Example 3 every day. The experiment lasted for a total of 12 weeks. On the 0th day from the start of the experiment to the 90th day at the end of the experiment. Use CORTEX ( The skin physiological index tester (Combo) was used to detect the moisture content of the stratum corneum, transepidermal water loss and skin elasticity of the volunteers' skin. The multispectral dermatoscope image processing workstation (Yunjing, CBS-2021, Wuhan Bosi Electronics Co., Ltd.) was used to test and collect skin images and score wrinkles.

[0114] SPSS2.0 statistical analysis software was used for statistical analysis of the data. Paired sample statistical analysis was performed before and after the experiment in the experimental group and the control group, that is, the subjects were paired before and after. All outcome variables were continuous variables. The difference before and after the intervention was in accordance with the normal distribution or approximately normal distribution of the measurement data. The paired sample T test was used, and the results were expressed as (mean ± standard deviation); the difference did not conform to the normal distribution of the measurement data, and the paired sample rank sum test was used. The results were described by the median and interquartile range (the distance between the 25th percentile and the 75th percentile, that is, P25 and P75).

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

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

[0117]

[0118] The results showed that the average moisture content of the stratum corneum at eight sites of the facial skin of the control group 1 and the control group 2 before and after the test was 17.39% and 25.60% respectively, and the average moisture content of the stratum corneum of the experimental group was 36.52%, and the results were statistically significant. The experimental results show that the natural elastic collagen dual protein peptide prepared by the method of the present invention has a more significant effect on improving the moisture content of the skin.

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

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

[0121]

[0122] The results showed that the transepidermal water loss of both cheeks in the test group decreased significantly by 28.34% before and after the test, and the transepidermal water loss of control group 1 and control group 2 decreased by 11.99% and 19.94%, respectively, indicating that the natural elastic collagen dual protein peptide 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 results of the skin elasticity test 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 of the experimental group increased by 9.33% after the 90-day experiment, which was higher than the elasticity change rate of control group 1 and control group 2, indicating that the natural elastic collagen dual protein peptide prepared by the method of the present invention is helpful 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 in the experimental group and the control group before and after the experiment

[0130]

[0131] The results showed that after 90 days of the experiment, the superficial wrinkles, fine lines and deep wrinkles index values ​​of the experimental group volunteers were significantly reduced, and the degree of reduction was higher than that of control group 1 and control group 2, indicating that the natural elastic collagen dual protein peptide prepared by the method of the present invention has a more significant effect on improving skin wrinkles.

[0132] In summary, the natural elastic collagen peptide prepared according to the method of the present invention has a ratio of elastin to collagen that is closer to the original ratio in the tissue. After oral administration for 90 days, it has significant effects on improving skin elasticity and moisture content, improving the functional barrier of the skin stratum corneum, 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 equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for preparing a natural elastic collagen dual protein peptide, Features: The preparation method comprises the following steps: (1) Pretreatment: The cleaned elastin-rich animal tissue is crushed into small pieces, and 6-10 times the weight of an alkaline solution is added, and the pieces are soaked at room temperature for 3-5 hours to remove fat and impurities, and the precipitate is washed with distilled water until it is neutral, thereby obtaining a pretreated collagen-containing elastin tissue, 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 collagen-containing elastin tissue is placed in a polyethylene packaging bag, which is vacuum-sealed and placed in a high-pressure chamber of an ultrahigh-pressure device, and ultrahigh-pressure treatment is performed using water as a pressure medium, 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 reduction rate is 15-20 MPa / s; (3) Protein extraction: the modified solubilized collagen-containing elastin tissue is mixed with purified water at a mass ratio of 1:2-6, and heated at 85-100° C. for 1-3 h to obtain an elastin collagen extract; (4) Enzymatic hydrolysis: adjust the pH value of the elastic collagen extract to 7.5-9.0, add 0.5-2.5% of the raw material weight of protease, adjust the temperature to 50-60° C., and perform enzymatic hydrolysis for 3-8 hours to obtain elastic collagen double protein peptide enzymatic hydrolyzate; (5) Inactivation of enzyme: Heat the enzymatic solution to 85-95°C and keep warm for 10-20 min; (6) Standing and solid-liquid separation: The enzymatic hydrolyzate is allowed to stand for 2-5 hours, the feed liquid is separated into upper and lower layers, and then the upper and lower layers are filtered separately, and the filtrates are combined to obtain a mixed clear liquid; (7) Decolorization: The mixed clear liquid is decolorized by passing it through an activated carbon fiber membrane; (8) Purification and concentration: The decolorized liquid is purified by a nanofiltration membrane 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 double protein peptide powder.

2. The preparation method according to claim 1, Features: In step (1), the elastin-rich animal tissue is one or more of aorta, ligament or bulbus arteriosus of fish heart.

3. The preparation method according to claim 1, Features: In step (4), the protease is one or a combination of alkaline protease, papain, bromelain, neutral protease, flavor protease, trypsin or pepsin.

4. The preparation method according to claim 1, Features: In step (6), the feed liquid is separated into upper and lower layers, the upper clear liquid is passed through a cotton cake filter press, and the lower wet residue precipitate is subjected to solid-liquid separation using a ceramic membrane, and the prepared clear liquids are mixed.

5. The preparation method according to claim 4, Features: The pore size of the ceramic membrane is 50-100 nm.

6. The preparation method according to claim 1, Features: In step (8), the pore size of the nanofiltration membrane is 200-300 Da.

7. The natural elastic collagen dual protein peptide prepared by the preparation method according to any one of claims 1 to 6.

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