Soluble collagen peptide, preparation method thereof and cosmetic with tightening effect

Abstract

The invention discloses a soluble collagen peptide and a preparation method thereof, and a cosmetic with a tightening effect. After collagen containing a short peptide sequence with a tightening effect is subjected to enzymolysis, a large amount of soluble collagen peptides with relatively small molecular weight and relatively high hydroxyproline content are formed. Good skin infiltration capacity and skin tightening and anti-wrinkle effects are shown. The preparation method is simple in process and short in period, the compact and anti-wrinkle polypeptide can be efficiently prepared, and the bioavailability and the biological activity of collagen are remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioactive substances, and specifically relates to a soluble collagen peptide and a preparation method thereof, and cosmetics with firming effect. Background Art

[0002] Collagen is a structural protein widely found in animals, accounting for more than 25% of the total protein in organisms. Collagen plays a vital role in maintaining skin elasticity, bone strength, tissue repair, etc. Therefore, it is widely used in many fields such as food, medicine, tissue engineering, cosmetics, etc.

[0003] Currently, most collagen products on the market are derived from animal tissues such as pigs, cattle, and fish; although these animal-derived collagens have certain structural similarities with human-derived collagens, they are also potentially immunogenic and may even carry viruses. Humanized collagen, on the other hand, has a structure and properties that are highly similar to human collagen, which reduces the risk of immune rejection and viral hazards, and improves biocompatibility, biological activity, and application safety. Therefore, human-derived collagen is gradually gaining high recognition and attention in the industry.

[0004] In the field of skin care, human collagen is widely used. There are many types of collagen, with type I, type III and type IV being the most common, and their molecular weights generally exceed 40kD. In particular, human collagen type III is a homotrimer composed of three collagen chains, and its single-chain molecular weight can be as high as 180kDa. This molecule contains multiple repeated helical domains, which not only provides excellent support functions, but also has the characteristics of high temperature resistance and high stability. However, due to its large molecular weight, the transdermal absorption of collagen faces challenges.

[0005] Enzymatic hydrolysis is a gentle method that uses enzyme catalysis to hydrolyze collagen into peptides. Specific enzymes can cut collagen molecules into bioactive peptides based on their specific cleavage sites. This method can maximize the biological activity of collagen peptides and help improve their application effects in skin care products. Summary of the invention

[0006] In order to overcome the shortcomings of the prior art, the first purpose of the present invention is to provide a method for preparing soluble collagen peptides, by constructing a collagen with a short peptide sequence with a firming effect, and forming a polypeptide sequence with a firming effect after enzymatic cleavage, thereby realizing the efficient preparation of functional small molecule polypeptides.

[0007] The second object of the present invention is to provide a soluble collagen peptide prepared by the above method, which has a small molecular weight and good permeability.

[0008] The third object of the present invention is to provide a cosmetic having a firming effect.

[0009] The first object of the present invention can be achieved by adopting the following technical solutions:

[0010] A method for preparing a soluble collagen peptide comprises enzymatically hydrolyzing a collagen having an amino acid sequence shown in SEQ ID NO.1 and / or SEQ ID NO.2 to obtain the soluble collagen peptide.

[0011] Furthermore, the enzyme is one or a combination of two or more of trypsin, pepsin, proteinase K or neutral protease.

[0012] Furthermore, the collagen is obtained by the following method:

[0013] Constructing an amino acid sequence as shown in SEQ ID NO.1 or SEQ ID NO.2;

[0014] constructing a recombinant plasmid for expressing collagen having the amino acid sequence;

[0015] The recombinant plasmid is transferred into a host cell, and fermentation culture and induced expression are performed to obtain the collagen.

[0016] Furthermore, the host cell is any one of Lactococcus lactis, Escherichia coli, Saccharomyces cerevisiae or Bacillus subtilis.

[0017] Furthermore, an inducer is used to induce expression, and the conditions for inducing expression are: induction at 25-35°C for 16-30h.

[0018] Furthermore, after the induction expression is completed, a separation process is also included: separating the bacterial cells from the fermentation liquid, washing and then homogenizing;

[0019] Use purified water for washing, the amount of water used is 16-32 times the mass of the bacteria;

[0020] The homogenization uses a sterile liquid; a homogenizer is used to homogenize 1-4 times at 600-1200 bar.

[0021] Furthermore, the enzymatic hydrolysis process is specifically as follows: centrifuging after homogenization, and adding the enzyme to the supernatant for enzymatic hydrolysis.

[0022] Furthermore, the amount of the enzyme added is 0.2-2% of the collagen protein mass; the enzymatic hydrolysis conditions are 30-35° C. for 0.5-2 h.

[0023] Furthermore, the enzyme is a combination of trypsin and papain, wherein the amount of trypsin added is 0.6% of the collagen mass; the amount of papain added is 0.4% of the collagen mass, the enzyme cleavage temperature is 35° C., and the enzyme cleavage time is 1 hour.

[0024] The second object of the present invention can be achieved by adopting the following technical solutions:

[0025] A soluble collagen peptide is prepared by the above-mentioned method for preparing the soluble collagen peptide.

[0026] The third object of the present invention can be achieved by adopting the following technical solutions:

[0027] A cosmetic with firming effect, comprising the soluble collagen peptide described above.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] 1. The present invention discloses a method for preparing a soluble collagen peptide. On the basis of a commonly used collagen sequence, a short peptide sequence with a firming effect is modified and added. Different from the commonly used collagen, the polypeptides formed after enzymatic cleavage have insufficient activity. The collagen of the present application forms a large number of polypeptides with a firming effect after enzymatic cleavage, thereby improving the bioavailability and biological activity of collagen.

[0030] 2. The soluble collagen peptide of the present invention has a smaller molecular weight and a higher hydroxyproline content, and exhibits better skin penetration ability and skin tightening and anti-wrinkle effects.

[0031] 3. The present invention provides a cosmetic with firming effect, comprising the soluble collagen peptide of the present application, which can achieve better skin penetration ability and firming and anti-wrinkle effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is the total ion current chromatogram of the sample (positive ion mode);

[0033] Figure 2 is the total ion current chromatogram of the sample (negative ion mode);

[0034] Figure 3 This is a small molecule gel electrophoresis analysis diagram of soluble collagen in this application;

[0035] Figure 4 The graph shows the results of elastin secretion by human skin fibroblasts in different treatment groups. DETAILED DESCRIPTION

[0036] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are only partial embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0037] Collagen has excellent biological activity. In order to promote the transdermal absorption of collagen and exert its skin care effect, a specific enzymatic hydrolysis technology is used to decompose the macromolecular collagen into small molecular, soluble collagen peptides. This method has a simple process, low cost, and improves the permeability of the molecule, thereby enhancing its bioavailability in skin care. However, most of the peptides formed after the enzymatic hydrolysis of the commonly used collagen have low activity, and only a very small part can have a firming and anti-wrinkle effect, and the bioavailability is low.

[0038] This application modifies and adds a short peptide sequence with firming effect on the basis of the commonly used collagen sequence, and can use the host cell chassis for high-density fermentation. The obtained collagen produces a large number of peptides with firming effect under specific enzymatic hydrolysis, thereby improving the bioavailability and biological activity of collagen. The preparation method has a simple process and a short cycle, and the obtained collagen peptide maintains high biological activity, significantly improving the bioavailability of collagen.

[0039] Therefore, the present application provides a method for preparing a soluble collagen peptide, wherein the soluble collagen peptide is obtained by enzymatically hydrolyzing the collagen having the amino acid sequence shown in SEQ ID NO.1 and / or SEQ ID NO.2.

[0040] Among them, the collagen with the amino acid sequence shown in SEQ ID NO.1 is type III collagen, and SEQ ID NO.1 is as follows:

[0041] SEQ ID NO.1:

[0042] MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLLFKNGEVAATKVGALSKGQDANLAGSGSGHMHHHHH HSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKACSSSPSKHCGGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGPMGP RGAPGERGRPGLPGAAGARGNDGARGSDGQPGPPGPPGTAGFPGSPGAKGQGCCNGPKGCSSKWCRDHARCCGEV.

[0043] The collagen with the amino acid sequence shown in SEQ ID NO.2 is type I collagen, and SEQ ID NO.2 is as follows:

[0044] SEQ ID NO.2:

[0045] MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMHHHHHHSSGLVPRGSGMKETAAA KFERQHMDSPDLGTDDDDKGKPGRPGERGPPGPQGARGLPGTAGLPGMKGHRGFSGLDGAKGDAGPAGPKGEPGSPGENGAPGQMGPRGLPGERGRPGAPGPAGARGNDGAATGAAGPGPTGPAGPPGFPGAVGAKGEA.

[0046] The collagen sequence of the present application is based on the commonly used type III collagen and type I collagen, and is recombinantly modified to add a short peptide sequence with a firming effect. Different from the existing collagen protein, most of the peptides produced after enzymatic cleavage do not have the effect of firming the skin. The recombinant collagen protein of the present application will form a peptide sequence with a firming effect after enzymatic cleavage, which greatly improves the bioavailability of collagen. The method of enzymatically hydrolyzing collagen into peptides using the catalytic action of enzymes is gentle, and can specifically cut collagen, fully expose the active groups, and obtain peptide segments with biological activity, which maximizes the biological activity of collagen peptides and improves bioavailability.

[0047] As one embodiment, the enzyme is one or a combination of two or more of trypsin, pepsin, proteinase K or neutral protease.

[0048] Specific enzymes can cut collagen molecules into biologically active peptides based on their specific cleavage sites. Hydroxyproline (Hyp) is a key active ingredient in collagen, and its content is related to the biological activity of collagen. In collagen, the content of hydroxyproline is relatively high, accounting for about 12-14% of the total amino acids, and it is relatively rare in other types of proteins. Therefore, it has become an important biomarker for measuring collagen activity. Through enzymatic hydrolysis, hydroxyproline in collagen can be released more effectively, thereby improving its bioavailability and biological activity. The determination of hydroxyproline content is a key indicator for evaluating product activity. As the hydroxyproline content increases, the activity of collagen also increases.

[0049] The collagen of the present application can be cleaved by one or a combination of two or more of trypsin, pepsin, proteinase K or neutral protease to effectively release hydroxyproline in the collagen. Preferably, the enzyme is a combination of trypsin and papain; the combination of trypsin and papain can release hydroxyproline in the collagen of the present application with the greatest probability.

[0050] As one embodiment thereof, the collagen is obtained by the following method:

[0051] Constructing an amino acid sequence as shown in SEQ ID NO.1 or SEQ ID NO.2;

[0052] constructing a recombinant plasmid for expressing collagen having the amino acid sequence;

[0053] The recombinant plasmid is transferred into a host cell, and fermentation culture and induced expression are performed to obtain the collagen.

[0054] Constructing collagen sequences and expressing them through host cells is a conventional technical means in the art for preparing recombinant collagen. This method can be used to perform high-density fermentation through the host cell chassis to obtain a large amount of high-purity target recombinant collagen.

[0055] The amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2 is based on human type III collagen and type I collagen, and is modified with a short peptide sequence with a firming effect. The resulting collagen has a structure and properties that are highly similar to the body's own collagen, reduces the risks of immune rejection and viral hazards, improves biocompatibility, biological activity and application safety; and also increases the firming effect.

[0056] A recombinant plasmid expressing the collagen having the amino acid sequence is constructed, and a corresponding nucleotide sequence is obtained according to the amino acid sequence of the constructed collagen. The nucleotide sequence is connected to the plasmid to obtain a recombinant plasmid expressing the corresponding collagen.

[0057] The expression of the recombinant plasmid is carried out in a host cell. The recombinant plasmid is transferred into the host cell, and fermentation culture and induced expression are carried out.

[0058] As one embodiment, the host cell is any one of Lactococcus lactis, Escherichia coli, Saccharomyces cerevisiae or Bacillus subtilis.

[0059] As one embodiment, the specific method of fermentation culture and induced expression is:

[0060] The host cells were inoculated into the optimized culture medium for fermentation and the feeding conditions were optimized to increase the host cell fermentation density by 10-20 times. 600 Reach 50-60;

[0061] Add inducer to the culture medium after fermentation, adjust the temperature to 25-35°C and induce for 16-30 hours.

[0062] By culturing and inducing expression, high-expression bacteria can be obtained.

[0063] In one embodiment, the step of separating and washing the bacterial cells is further included: collecting the fermentation liquid and centrifuging the bacterial cells;

[0064] The bacteria were washed with purified water, the ratio of purified water added was 1:1-32, and the bacteria were collected by centrifugation after washing.

[0065] In one embodiment, the collected cells are homogenized. The washed cells are added with a cell disruption solution, homogenized 1-4 times with a high pressure homogenizer at 600-1200 bar, and the supernatant is collected by centrifugation. The obtained supernatant contains the collagen.

[0066] In one embodiment, the enzymatic hydrolysis process is specifically as follows: centrifugation is performed after homogenization, and the enzyme is added to the supernatant for enzymatic hydrolysis.

[0067] The supernatant is directly enzymatically hydrolyzed, and the resulting collagen peptides not only maintain high biological activity, but are also rich in natural metabolites and cell wall components produced during the fermentation process, significantly improving the bioavailability of collagen peptides and their permeability to the skin.

[0068] In one embodiment, the amount of the enzyme added is 0.2-2% of the collagen protein mass; the enzymatic hydrolysis conditions are 30-35° C. for 0.5-2 h.

[0069] In one embodiment, the enzyme is a combination of trypsin and papain, wherein the amount of trypsin added is 0.6% of the mass of collagen; the amount of papain added is 0.4% of the mass of collagen, the enzyme cleavage temperature is 35° C., and the enzyme cleavage is for 1 hour.

[0070] The following is a further description of a specific implementation method.

[0071] Example 1

[0072] 1. Construction of engineered bacteria

[0073] (1) Collagen amino acid sequence

[0074] Type III collagen sequence:

[0075] MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLLFKNGEVAATKVGALSKGQDANLAGSGSGHMHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKA CSSSPSKHCGGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSDGQPGPPGPPGTAGFPGSPGAKGQGCCNGPKGCSSKWCRDHARCCGEV.

[0076] (2) Vector construction and transformation

[0077] According to the reverse design of the amino acid sequence and codons of the type III collagen sequence, a nucleotide sequence for encoding the type III collagen sequence is obtained; the nucleotide sequence is inserted into the pET28a plasmid to construct a recombinant vector, which is introduced into Escherichia coli BL21 and the recombinant engineered bacteria are screened.

[0078] 2. High-density fermentation of host cells

[0079] (1) Host cell culture medium fermentation

[0080] The recombinant engineered bacteria were inoculated into LB medium and cultured at 37°C for 10-12 hours to increase the density of host cell fermentation cells by 10-20 times.600 Reach 50-60.

[0081] (2) Inducible expression

[0082] In step (1) after fermentation, IPTG was added at a final concentration of 1 mM, and the expression was induced at 30°C for 25 h to obtain high-expression bacteria.

[0083] (3) Bacterial centrifugation

[0084] The fermentation broth was collected and centrifuged at 3000 rps, 4°C, for 30 min to collect the bacterial precipitate.

[0085] (4) Washing of bacteria

[0086] The bacteria were washed with purified water, with the ratio of purified water added being 1:16 or 1:32 for bacteria: purified water, and the bacteria were collected by centrifugation after washing.

[0087] (5) Bacterial homogenization

[0088] Take the washed cells, add the bacterial lysis solution, homogenize twice with a high-pressure homogenizer at 1000 bar, and collect the supernatant by centrifugation.

[0089] (6) Enzymatic hydrolysis

[0090] The supernatant of the centrifuged homogenate is enzymatically hydrolyzed to obtain soluble collagen peptides.

[0091] (7) Membrane filtration

[0092] The enzymatic hydrolyzate is passed through a ceramic membrane and the filtrate is collected to obtain a soluble collagen solution.

[0093] Example 2

[0094] 1. Construction of engineered bacteria

[0095] (1) Collagen amino acid sequence

[0096] Type I collagen sequence:

[0097] MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMHHHHHHSSGLVPRGSGMKETAAA KFERQHMDSPDLGTDDDDKGKPGRPGERGPPGPQGARGLPGTAGLPGMKGHRGFSGLDGAKGDAGPAGPKGEPGSPGENGAPGQMGPRGLPGERGRPGAPGPAGARGNDGAATGAAGPGPTGPAGPPGFPGAVGAKGEA.

[0098] (2) Vector construction and transformation

[0099] According to the reverse design of the amino acid sequence and codons of the type I collagen, a nucleotide sequence for encoding the type I collagen is obtained; the nucleotide sequence is inserted into the pET28a plasmid to construct a recombinant vector, which is introduced into Escherichia coli BL21 to screen and obtain recombinant engineered bacteria.

[0100] 2. High-density fermentation of host cells

[0101] (1) Host cell culture medium fermentation

[0102] The recombinant engineered bacteria were inoculated into LB medium and cultured at 37°C for 10-12 hours to increase the density of host cell fermentation cells by 10-20 times. 600 Reach 50-60.

[0103] (2) Inducible expression

[0104] In step (1) after fermentation, IPTG was added at a final concentration of 1 mM, and the expression was induced at 30°C for 25 h to obtain high-expression bacteria.

[0105] (3) Bacterial centrifugation

[0106] The fermentation broth was collected and centrifuged at 3000 rps, 4°C, for 30 min to collect the bacterial precipitate.

[0107] (4) Washing of bacteria

[0108] The bacteria were washed with purified water, with the ratio of purified water added being 1:16 or 1:32 for bacteria: purified water, and the bacteria were collected by centrifugation after washing.

[0109] (5) Bacterial homogenization

[0110] Take the washed cells, add the bacterial lysis solution, homogenize twice with a high-pressure homogenizer at 1000 bar, and collect the supernatant by centrifugation.

[0111] (6) Enzymatic hydrolysis

[0112] The supernatant of the centrifuged homogenate is enzymatically hydrolyzed to obtain soluble collagen peptides.

[0113] (7) Membrane filtration

[0114] The enzymatic hydrolyzate is passed through a ceramic membrane and the filtrate is collected to obtain a soluble collagen solution.

[0115] Taking the collagen of Example 1 as an example, the optimal conditions under different enzyme reaction conditions were studied. Trypsin, pepsin, neutral protease and papain were used for enzymolysis under different conditions, and the hydroxyproline content in the enzymolysis products was shown in Tables 1 to 4:

[0116] Table 1 Trypsin enzymolysis conditions test

[0117]

[0118] According to the test results in Table 1, under different trypsin addition amounts, different temperatures and enzymatic hydrolysis times, the best enzymatic hydrolysis effect was achieved when trypsin addition amount was 1%, enzymatic hydrolysis temperature was 35°C, and enzymatic hydrolysis time was 1h; the obtained hydroxyproline concentration could reach 836.32ppm.

[0119] Table 2 Pepsin enzymolysis conditions test

[0120]

[0121] According to the test results in Table 2, the enzymatic hydrolysis effect was best when the amount of pepsin added was 0.5%, the enzymatic digestion temperature was 35°C, and the enzymatic digestion time was 2h; however, the concentration of hydroxyproline was only 593.83 ppm.

[0122] Table 3 Neutral protease enzymatic hydrolysis conditions test

[0123]

[0124] According to the test results in Table 3, when the addition amount of neutral protease is 1%, the enzyme digestion temperature is 30°C, and the enzyme digestion time is 2h, the enzymatic hydrolysis effect of neutral protease is the best, and the concentration of hydroxyproline obtained is 568.26ppm.

[0125] Table 4 Papain enzymolysis conditions test

[0126]

[0127] According to the test results in Table 4, papain was used for enzymolysis, the amount of papain added was 0.5%, the enzymolysis temperature was 30°C, the enzymolysis was 1h, the concentration of hydroxyproline could reach 716.23ppm, and the enzymolysis effect was the best.

[0128] Therefore, considering the amount of enzyme added to the enzymatic reaction, the enzymatic temperature and time, and the yield of hydroxyproline, trypsin and papain have the best enzymatic cleavage effect; the combination of the two has a better enzymatic effect.

[0129] The orthogonal experimental method was used to determine the optimal enzymatic digestion conditions for trypsin and papain: trypsin and papain were taken and tested by adding 0.5% of each, with enzymatic digestion temperatures of 30°C and 35°C and enzymatic digestion time of 1h. The results are shown in Table 5.

[0130] Table 5

[0131]

[0132] According to the test results in Table 5, the best enzymatic effect was achieved when the two enzymes were added in an amount of 0.5% each and mixed together, the enzyme digestion temperature was 35°C, and the enzyme digestion time was 1 hour.

[0133] Trypsin and papain were tested at different addition ratios, with an enzyme cleavage temperature of 35°C and a cleavage time of 1 h. The concentration of hydroxyproline is shown in Table 6:

[0134] Table 6

[0135]

[0136] Therefore, combined with the results in Table 6, the enzyme digestion conditions finally determined were: 0.6% trypsin, 0.4% papain, 35°C enzyme digestion temperature, 1h enzyme digestion, the best enzymatic effect, and a higher hydroxyproline content.

[0137] According to the above enzyme digestion conditions, the digestion products were tested by liquid chromatography-high resolution mass spectrometry at Guangzhou Quality Supervision and Inspection Institute:

[0138] Instruments: Thermo Scientific Q Exactive high-resolution mass spectrometer, electronic balance, ultrasonic oscillator, vortex oscillator.

[0139] Reagents: acetonitrile (chromatographic grade); methanol (chromatographic grade); formic acid (chromatographic grade); pure water (18.2 MΩ·cm).

[0140] Sample processing

[0141] Weigh 0.2024 g of the sample into a 10 mL stoppered colorimetric tube, dilute to 10 mL with 50% methanol aqueous solution, mix after ultrasonication for 15 min, filter through a 0.22 μm microporous membrane, and place in a sample bottle for testing for polypeptide amino acid composition analysis.

[0142] Liquid chromatography instrument conditions

[0143] a) Chromatographic column: Hypersil GOLD (1.9 μm, 2.1 mm × 50 mm);

[0144] b) Column temperature: 30°C;

[0145] c) Mobile phase: A: acetonitrile, B: 0.01% formic acid water, gradient elution program see Table 7;

[0146] d) Flow rate: 0.3 mL / min;

[0147] e) Injection volume: 1 μL

[0148] Table 7 Mobile phase composition

[0149]

[0150]

[0151] High resolution mass spectrometry instrument conditions

[0152] a) Ion source: electrospray ion source (H-ESI source);

[0153] b) Monitoring mode: positive ion Full MS-ddMS2 mode and negative ion Full MS-ddMS2 mode are collected once each;

[0154] c) ESI spray voltage: positive ion +3000V, negative ion -2500V;

[0155] d) Sheath gas: 45Arb;

[0156] e) Auxiliary gas: 12Arb;

[0157] f) Purge gas: 0Arb;

[0158] g) Ion transfer tube temperature: 325°C;

[0159] h) Atomizing gas temperature: 350°C;

[0160] i) Primary scanning range: 100-1500 m / z;

[0161] j) Secondary scanning range: automatic.

[0162] Liquid chromatography-high resolution mass spectrometry sample spectrum Figure 1 and Figure 2 The results are shown in Table 8 and Table 9.

[0163] Table 8 Sample chromatographic peak matching results in positive ion mode

[0164]

[0165]

[0166] Table 9 Sample chromatographic peak matching results in negative ion mode

[0167] Serial number Matching compound results Retention time area 1 L-Hydroxyproline / L-Hydroxyproline 0.488 <![CDATA[8.75×10 8 <!-- 10 -->]]> 2 Adenine 0.617 <![CDATA[1.14×10 8 ]]> 3 Adenosine 0.616 <![CDATA[7.07×10 7 ]]> 4 Leu-Ala / Leucine-Ala 0.515 <![CDATA[8.55×10 6 ]]> 5 Ala-Gly-Ala / Alanine-Glycine-Alanine 0.478 <![CDATA[6.41×10 6 ]]> 6 Ile-Gln / Isoleucine-Glutamine 0.509 <![CDATA[6.34×10 6 ]]> 7 Ala-Ala / Alanine-Alanine 0.476 <![CDATA[4.18×10 6 ]]> 8 Ala-Gly / Alanine-Glycine 0.476 <![CDATA[4.03×10 6 ]]> 9 Gly-Met / Glycine-Methionine 0.523 <![CDATA[2.91×10 6 ]]>

[0168] From Tables 8 and 9 and Figure 1-2 It can be seen that the polypeptide amino acids formed after enzymatic hydrolysis of the collagen of the present application contain a large amount of L-hydroxyproline, adenosine, adenine, and L-proline, glycine-alanine-arginine, glycine-leucine, glycine-phenylalanine, leucine-alanine, alanine-glycine-alanine, isoleucine-glutamine, alanine-alanine, alanine-glycine and glycine-methionine. It has a small molecular weight and a high hydroxyproline content, and has good application in skin penetration, skin firming and anti-wrinkle.

[0169] The soluble collagen solution prepared in Example 1 was subjected to small molecule gel electrophoresis (SDS-PAGE) analysis. The results are as follows: Figure 3 shown.

[0170] from Figure 3 It can be seen that the molecular weight of the final product of the collagen in Example 1 after enzymatic hydrolysis is between 6.5 kD and 30 kD, and is mainly concentrated at 6.5 kD. Therefore, it can be known that the collagen has formed small molecular polypeptides, and the molecular weight is significantly reduced.

[0171] The soluble collagen solution prepared in Example 1 was subjected to quantitative analysis of its elastin secretion level to evaluate its skin tightening and anti-wrinkle effects.

[0172] Effect of soluble collagen peptides on elastin secretion of human skin fibroblasts (BJ): The fibroblasts obtained from different treatment groups were cultured in a 37°C, 5% CO2 incubator for 24 h, and the supernatant was collected and measured according to the instructions of the elastin detection kit. The results are shown in Table 9 and Figure 4 As shown:

[0173] Table 9

[0174] Group Elastin (pg / mL) Blank control group 0.59±0.22 Control group (no enzymatic treatment) 1.08±0.19 Experimental group (0.625 mg / mL) 2.11±0.01 Experimental group (1.250 mg / mL) 2.99±0.39 Experimental group (2.500 mg / mL) 3.08±0.19

[0175] According to the data in Table 9 and Figure 4 It can be seen that at concentrations of 0.625 mg / mL, 1.250 mg / mL and 2.500 mg / mL in the experimental group, the elastin content in the cell supernatant increased linearly, and the difference was significant compared with the blank control group (P < 0.01), indicating that the product prepared by this method has a significant firming and anti-wrinkle effect, and the results are statistically significant.

[0176] In summary, the preparation method of a soluble collagen peptide of the present application is modified and added with a short peptide sequence with a firming effect on the basis of a commonly used collagen sequence. Different from the commonly used collagen, which has insufficient activity in the polypeptides formed after enzymatic cleavage, the collagen of the present application forms a large number of polypeptides with a smaller molecular weight and a higher hydroxyproline content after enzymatic cleavage, which show better skin penetration ability, firming skin and anti-wrinkle effects; and significantly improves the bioavailability and biological activity of collagen.

[0177] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A method for preparing soluble collagen peptide, characterized in that: The soluble collagen peptide is obtained by enzymatically hydrolyzing the collagen having the amino acid sequence shown in SEQ ID NO.1 and / or SEQ ID NO.

2.

2. The method for preparing a soluble collagen peptide according to claim 1, characterized in that: The enzyme used for enzymolysis is one or a combination of two or more of trypsin, pepsin, proteinase K or neutral protease.

3. The method for preparing a soluble collagen peptide according to claim 1 or 2, characterized in that: The collagen is obtained by the following method: Constructing an amino acid sequence as shown in SEQ ID NO.1 or SEQ ID NO.2; constructing a recombinant plasmid for expressing collagen having the amino acid sequence; The recombinant plasmid is transferred into a host cell, and fermentation culture and induced expression are performed to obtain the collagen.

4. The method for preparing a soluble collagen peptide according to claim 3, characterized in that: The host cell is any one of Lactococcus lactis, Escherichia coli, Saccharomyces cerevisiae or Bacillus subtilis; Inducing expression uses an inducing agent, and the inducing expression conditions are: 25-35°C for 16-30h.

5. The method for preparing a soluble collagen peptide according to claim 3, characterized in that: After the induction expression is completed, the method further includes a separation process: separating the bacterial cells from the fermentation liquid, washing the cells and then homogenizing the cells; Use purified water for washing, the amount of water used is 16-32 times the mass of the bacteria; The homogenization uses a sterile liquid; a homogenizer is used to homogenize 1-4 times at 600-1200 bar.

6. The method for preparing a soluble collagen peptide according to claim 3, characterized in that: The enzymatic hydrolysis process is specifically as follows: centrifugation is performed after homogenization, and the enzyme is added to the supernatant for enzymatic hydrolysis.

7. The method for preparing a soluble collagen peptide according to claim 1 or 2, characterized in that: The amount of the enzyme added is 0.2-2% of the collagen protein mass; the enzymatic hydrolysis conditions are 30-35° C. for 0.5-2 h.

8. The method for preparing a soluble collagen peptide according to claim 1 or 2, characterized in that: The enzyme is a combination of trypsin and papain, wherein the amount of trypsin added is 0.6% of the collagen protein; the amount of papain added is 0.4% of the collagen protein, the enzyme cleavage temperature is 35°C, and the enzyme cleavage is for 1 hour.

9. A soluble collagen peptide, characterized in that: The soluble collagen peptide is prepared by the preparation method of any one of claims 1 to 8.

10. A cosmetic with firming effect, characterized in that: The cosmetic comprises the soluble collagen peptide according to any one of claims 1 to 9.

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