Collagen-calcium chelate as well as preparation method and application thereof

Abstract

The invention discloses a collagen-calcium chelate as well as a preparation method and application thereof, the collagen-calcium chelate is prepared by taking collagen with a specific sequence and soluble calcium salt as raw materials and adopting a water system synthesis method. The effects of locking water, improving skin quality, fading pigment, enhancing skin elasticity and whitening of collagen and the effects of promoting skin tissue regeneration, maintaining skin integrity, strengthening skin barrier, promoting epidermal cell proliferation and differentiation, remodeling skin extracellular matrix, whitening, relieving and the like of calcium ions are combined; the collagen-calcium chelate has the advantages of good stability and high biological value, and can achieve the purposes of whitening, moisturizing, repairing and soothing.

Description

Technical Field

[0001] The present invention relates to the technical field of synthetic biology, and in particular to a collagen-calcium chelate and a preparation method and application thereof. Background Art

[0002] Calcium is one of the most abundant mineral elements in the human body, with a total amount of 1300g, which is about 1.5-2.0% of body weight. Calcium ions promote platelet aggregation, accelerate the synthesis of thrombin, and promote the early formation of fibrin. Calcium ions help stimulate the synthesis of collagen and the formation of blood vessels. It can increase the metabolic activity of fibroblasts, promote migration, and regulate the release of MMP and cytokines. In addition, calcium ions play an important role in skin tissue regeneration, maintaining skin integrity, strengthening skin barriers, promoting epidermal cell proliferation and differentiation, remodeling skin extracellular matrix, as well as whitening, soothing and many other efficacy areas.

[0003] The main function of collagen is to lock in moisture in the skin, improve skin texture, reduce skin pigmentation, enhance skin elasticity and whiten the skin. Many existing studies only focus on the whitening and moisturizing effects of simple collagen or metal ions, which is not only troublesome to use, but also very likely to cause collagen to degrade easily and the actual use effect is not ideal. Chelating collagen with metals to prepare chelates can not only greatly improve the stability of collagen, but also accumulate their respective effects to achieve the effect of 1 plus 1 being greater than 2. In addition, it is convenient for people to use.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide a collagen-calcium chelate and a preparation method and application thereof. The collagen-calcium chelate obtained by the preparation method of the present invention has the advantages of good stability and high biological efficacy, and can achieve the purposes of whitening, moisturizing, repairing and soothing.

[0006] The present invention is achieved in that:

[0007] In a first aspect, the present invention provides a collagen-calcium chelate, wherein the collagen of the collagen-calcium chelate is derived from type I collagen, and the amino acid sequence thereof is shown in SEQ ID NO:1.

[0008] In a second aspect, the present invention provides a method for preparing the above-mentioned collagen-calcium chelate, which comprises: using the above-mentioned collagen and soluble calcium salt as raw materials, and adopting a water system synthesis method to prepare the collagen-calcium chelate.

[0009] In some embodiments, the above collagen is obtained by high-density fermentation production of recombinant Escherichia coli that is capable of expressing the collagen.

[0010] In some embodiments, the soluble calcium salt is selected from at least one of calcium chloride and calcium acetate.

[0011] In some embodiments, the mass ratio of the above collagen to the soluble salt is 10:1 to 3:1.

[0012] In some embodiments, the reaction conditions are: stirring the reaction at 20-50° C. for 0.5-4 h, and controlling the pH to 4.5-6.5.

[0013] In some embodiments, the alkaline solution for adjusting the pH in the above reaction is selected from at least one of a sodium hydroxide solution, a potassium hydroxide solution, and a barium hydroxide solution.

[0014] In some embodiments, the preparation method further comprises: after the reaction is completed, dialyzing or ultrafiltration treatment is performed on the reaction solution, and then vacuum freeze drying treatment is performed.

[0015] In a third aspect, the present invention also provides the use of the above collagen-calcium chelate in the field of cosmetics.

[0016] In a fourth aspect, the present invention also provides the use of the above collagen-calcium chelate in the field of food or medicine.

[0017] The present invention has the following beneficial effects:

[0018] The present invention uses collagen of a specific sequence and soluble calcium salt as raw materials, and adopts a water system synthesis method to prepare collagen-calcium chelate, which combines the effects of collagen in locking moisture, improving skin quality, lightening pigments, enhancing skin elasticity and whitening with the effects of calcium ions in promoting skin tissue regeneration, maintaining skin integrity, strengthening skin barriers, promoting epidermal cell proliferation and differentiation, remodeling skin extracellular matrix, and whitening and soothing, so that the collagen-calcium chelate has the advantages of good stability and high biological efficacy, and can achieve the purposes of whitening, moisturizing, repairing and soothing. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0020] Figure 1It is the infrared spectrum of collagen-calcium chelate, in which the horizontal axis is wave number (cm-1), the vertical axis is transmittance (%), curve 1 is the collagen curve, and curve 2 is the collagen-calcium chelate curve. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0022] In order to obtain safer, more stable and more effective cosmetics, the present invention combines collagen with calcium and prepares collagen-calcium chelate by a water system synthesis method. The specific method is as follows:

[0023] Collagen and soluble calcium salt are used as raw materials, mixed and reacted; after the reaction, the reaction liquid is dialyzed or ultrafiltered, and then vacuum freeze-dried.

[0024] The collagen used is derived from type I collagen, and its amino acid sequence is shown in SEQ ID NO: 1:

[0025] 。

[0026] During the experiment, it was found that compared with full-length type I collagen, the collagen fragment in the present invention not only has the effects of full-length type I collagen, but is also easier to be absorbed and utilized due to its smaller molecular weight, and thus has stronger whitening, moisturizing, repairing and soothing effects when prepared as collagen-calcium chelate.

[0027] In the present invention, the collagen used is produced by high-density fermentation of genetically engineered bacteria BL21 (DE3). Specifically, the construction method of the genetically engineered bacteria BL21 (DE3) is as follows:

[0028] The gene of the collagen fragment with the amino acid sequence shown in SEQ ID NO: 1 is inserted into an expression vector to obtain a recombinant vector, and then the recombinant vector is introduced into the starting strain BL21 (DE3) to obtain the above-mentioned genetically engineered bacteria.

[0029] The method of producing collagen by high-density fermentation of genetically engineered bacteria is a conventional method in the art and is not limited thereto in the present invention, as long as the collagen can be obtained.

[0030] The soluble calcium salt in the above preparation method includes but is not limited to calcium chloride and calcium acetate, and may also be other soluble calcium salts available in the art.

[0031] The collagen and soluble calcium salt are synthesized by a water system to obtain collagen-calcium chelate. In the reaction system, the mass ratio of collagen to soluble calcium salt is 10:1 to 3:1. Specifically, the mass ratio of collagen to soluble calcium salt can be 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1 or 3:1, or any other ratio between 10:1 and 3:1.

[0032] When the collagen and soluble calcium salt are reacted, the reaction conditions are: stirring the reaction at 20-50° C. for 0.5-4 hours, and controlling the pH to 4.5-6.5.

[0033] Specifically, the reaction temperature can be 20°C, 25°C, 30°C, 35°C, 40°C, 45°C or 50°C, or any other value between 20 and 50°C. The reaction time can be 0.5h, 1h, 2h, 2.5h, 3h, 3.5h or 4h, or any other value between 0.5 and 4h. The pH can be 4.5, 5, 5.5, 6 or 6.5, or any other value between 4.5 and 6.5.

[0034] In the above process, for adjusting the pH, the alkaline solution includes but is not limited to sodium hydroxide solution, potassium hydroxide solution and barium hydroxide solution, and may also be other alkaline solutions available in the art.

[0035] After the reaction is completed, the reaction solution needs to be separated and purified to separate the collagen-calcium chelate in the reaction solution. Optionally, the separation and purification operation includes dialysis or ultrafiltration treatment of the reaction solution, followed by vacuum freeze drying. The specific operation method and steps are not limited in the present invention, as long as the collagen-calcium chelate can be separated from the reaction solution.

[0036] Through the above preparation method, the present invention can obtain a collagen-calcium chelate, which has the advantages of good stability and high biological efficacy, and can achieve the purposes of whitening, moisturizing, repairing and soothing. Therefore, the chelate can be applied in the field of cosmetics or medicines.

[0037] Specifically, the products prepared from the collagen-calcium chelate of the present invention include cosmetics, foods or medicines.

[0038] The above-mentioned cosmetics include facial masks, essences, facial cleansers and the like. The collagen-calcium chelate of the present invention can be added to different types of cosmetics alone, or the collagen-calcium chelate of the present invention can be compounded with other active ingredients and added to different types of cosmetics.

[0039] Alternatively, the collagen-calcium chelate can be added to different types of food alone, or the collagen-calcium chelate of the present invention can be compounded with other active ingredients and added to different types of food.

[0040] Alternatively, the above-mentioned collagen-calcium chelate can be combined with pharmaceutically acceptable excipients to prepare a medicine with whitening, moisturizing, repairing and soothing effects. The above-mentioned collagen-calcium chelate can also be compounded with other active ingredients to prepare a medicine with whitening, moisturizing, repairing and soothing effects.

[0041] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0042] Example 1

[0043] This embodiment is a method for preparing collagen-calcium chelate, and the specific steps are as follows:

[0044] (1) Weigh 100 mg of collagen and dissolve it in 10 mL of double distilled water. Then, add 10 mg / mL calcium chloride solution to the collagen solution. The reaction temperature is 20° C. and the mixture is stirred for 1.5 hours. At the same time, a 0.5 mol / L sodium hydroxide solution is added to control the pH value of the reaction solution to 4.0.

[0045] The amino acid sequence of collagen is shown in SEQ ID NO:1.

[0046] (2) After the above reaction is completed, the reaction solution is dialyzed and then vacuum freeze-dried to obtain collagen-calcium chelate.

[0047] Example 2

[0048] This embodiment is a method for preparing collagen-calcium chelate, and the specific steps are as follows:

[0049] (1) Weigh 100 mg of collagen and dissolve it in 10 mL of double distilled water. Then, add 10 mg / mL calcium acetate solution to the collagen solution. The reaction temperature is 20°C. The mixture is stirred for 1.5 hours. At the same time, a 0.5 mol / L sodium hydroxide solution is added to control the pH value of the reaction solution to 4.0.

[0050] The amino acid sequence of collagen is shown in SEQ ID NO:1.

[0051] (2) After the above reaction is completed, the reaction solution is dialyzed and then vacuum freeze-dried to obtain collagen-calcium chelate.

[0052] Example 3

[0053] This embodiment is a method for preparing collagen-calcium chelate, and the specific steps are as follows:

[0054] (1) Weigh 30 mg of collagen and dissolve it in 10 mL of double distilled water. Then, add 10 mg / mL calcium chloride solution to the collagen solution. The reaction temperature is 20°C. The mixture is stirred for 1.5 hours. At the same time, a 0.5 mol / L sodium hydroxide solution is added to control the pH value of the reaction solution to 4.0.

[0055] The amino acid sequence of collagen is shown in SEQ ID NO:1.

[0056] (2) After the above reaction is completed, the reaction solution is dialyzed and then vacuum freeze-dried to obtain collagen-calcium chelate.

[0057] Comparative Example 1

[0058] This comparative example is collagen with an amino acid sequence as shown in SEQ ID NO: 1.

[0059] Experimental Example 1

[0060] The collagen-calcium chelate obtained in Example 1 and the collagen in Comparative Example 1 were subjected to infrared spectroscopy analysis. The analysis results are as follows: Figure 1As shown, 1 is collagen and 2 is collagen-calcium chelate.

[0061] The positions and intensities of amide I and amide II bands in collagen-calcium chelates have changed, indicating that the positions of the absorption peaks of the functional groups of the protein molecules will shift when the chelate is formed, indicating that there is an interaction between collagen and calcium, which is beneficial to enhancing the stability of collagen.

[0062] Experimental Example 2

[0063] This experiment compares the effects of the collagen-calcium chelate obtained in Example 1, the collagen of Comparative Example 1, and type I collagen (Sequence ID XP_063455318.1, total length 1507aa) on in vitro tyrosinase inhibition. The test method refers to the requirements of the Guangdong Cosmetics Association Group Standard "T / GDCA 0062021 Test Method for Tyrosinase Activity Inhibition of Cosmetic Raw Materials (In Vitro Method)". The collagen-calcium chelate, collagen, and the above-mentioned type I collagen (Sequence ID XP_063455318.1, total length 1507aa) obtained in Comparative Examples 1 and 2 were used to detect the in vitro tyrosinase inhibition rate. The specific method is as follows:

[0064] In a 96-well plate, set up solvent background wells (Ta), solvent reaction wells (Tb), sample background wells (Tc), and sample reaction wells (Td). Among them, the Ta group is a solvent background group, without adding substrate L-tyrosine solution and sample solution; the Tb group is a solvent reaction group, with substrate L-tyrosine solution but no sample solution; the Tc group is a sample background group, without adding substrate L-tyrosine solution but adding sample solution; the Td group is a sample reaction group, with both substrate L-tyrosine solution and sample solution. Three replicate wells are made for each group.

[0065] Experimental steps:

[0066] As shown in Table 1, add L-tyrosine solution, sample solution / solvent, and PBS buffer to each well in turn, mix thoroughly, incubate at 37°C for 10 min, then add 20 μL of tyrosinase solution to each well in turn, mix and react at 37°C for 5 min±5 s, then put into the microplate reader and detect at 475 nm. The concentration of the samples is 0.2 mg / mL.

[0067] Note: The time from adding tyrosinase solution to measuring absorbance in each well should be kept consistent (5min±5s).

[0068] Table 1 Sample addition table for tyrosinase activity inhibition test

[0069]

[0070] Calculation of tyrosinase activity inhibition rate:

[0071] Y=(1-(Ad-Ac) / (Ab-Aa))×100%

[0072] Wherein: Y is the tyrosinase activity inhibition rate; Ad is the absorbance of the sample reaction well; Ac is the absorbance of the sample background well; Ab is the absorbance of the solvent reaction well; Aa is the absorbance of the solvent background well.

[0073] Table 2 Tyrosinase activity inhibition results of Example 1, Comparative Example 1 and Type I collagen

[0074] experiment Tyrosinase activity inhibition rate (%) Example 1 (Collagen-Calcium Chelate) 43.18±0.34 Comparative Example 1 (Collagen) 34.48±0.27 Type I collagen 5.98±0.25

[0075] According to the test results in Table 2, it can be seen that compared with the same concentration of collagen used in the present invention and type I collagen (Sequence ID is XP_063455318.1, full length 1507aa), the inhibition rate of collagen-calcium chelate on tyrosinase activity can be increased by 8.7% and 37.2%, respectively. This shows that collagen-calcium chelate has a better effect than collagen used in the present invention in inhibiting tyrosinase activity. At the same time, it can be seen that the collagen used in the present invention and collagen-calcium chelate have a more excellent effect than type I collagen (Sequence ID is XP_063455318.1, full length 1507aa) in inhibiting tyrosinase activity.

[0076] Experimental Example 3

[0077] This experiment compares the effects of the collagen-calcium chelate obtained in Example 1, the collagen of Comparative Example 1, and type I collagen (Sequence ID is XP_063455318.1, full length 1507aa) on the inhibition rate of zebrafish melanin production.

[0078] Experimental method: The zebrafish were divided into 4 groups, including a normal control group and an experimental group. The normal control group was not treated, and the experimental group was incubated with the collagen-calcium chelate obtained in Example 1, the collagen of Comparative Example 1, and type I collagen (Sequence ID is XP_063455318.1, total length 1507aa) at a concentration of 0.1 mg / mL, respectively. After incubation for 48 hours, the zebrafish were photographed and the melanin signal intensity of the zebrafish head was analyzed to evaluate its whitening effect.

[0079] Table 3 Melanin production inhibition results of Example 1, Comparative Example 1 and Type I collagen

[0080]

[0081]

[0082] According to the test results in Table 3, it can be seen that compared with the same concentration of collagen (Sequence ID is XP_063455318.1, full length 1507aa) and full sequence type I collagen, the inhibition rate of melanin production in zebrafish by collagen-calcium chelate can be increased by 8.7% and 19.20%, respectively. This indicates that collagen-calcium chelate has a better effect than collagen used in the present invention in inhibiting melanin production in zebrafish, and also indicates that collagen-calcium chelate and collagen used in the present invention have a more excellent effect than type I collagen (Sequence ID is XP_063455318.1, full length 1507aa) in inhibiting melanin production in zebrafish.

[0083] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A collagen-calcium chelate, characterized in that: The collagen of the collagen-calcium chelate is derived from type I collagen, and its amino acid sequence is shown in SEQ ID NO:

1.

2. The method for preparing the collagen-calcium chelate according to claim 1, characterized in that: include: The collagen-calcium chelate is prepared by using the collagen and soluble calcium salt as raw materials and adopting a water system synthesis method.

3. The preparation method according to claim 2, characterized in that: The collagen is obtained by high-density fermentation production of a recombinant Escherichia coli capable of expressing the collagen.

4. The preparation method according to claim 2, characterized in that: The soluble calcium salt is selected from at least one of calcium chloride and calcium acetate.

5. The preparation method according to claim 2, characterized in that: The mass ratio of the collagen to the soluble salt is 10:1 to 3:

1.

6. The preparation method according to claim 2, characterized in that: The collagen and the soluble salt are mixed and reacted under the following conditions: stirring the reaction at 20-50° C. for 0.5-4 hours and controlling the pH value to 4.5-6.

5.

7. The preparation method according to claim 6, characterized in that: The alkaline solution for adjusting pH in the reaction is selected from at least one of a sodium hydroxide solution, a potassium hydroxide solution and a barium hydroxide solution.

8. The preparation method according to claim 7, characterized in that: The preparation method further comprises: after the reaction is completed, dialyzing or ultrafiltration treatment is performed on the reaction solution, and then vacuum freeze drying treatment is performed.

9. Use of the collagen-calcium chelate according to claim 1 or the collagen-calcium chelate obtained by the preparation method according to any one of claims 2 to 8 in the field of cosmetics.

10. Use of the collagen-calcium chelate according to claim 1 or the collagen-calcium chelate obtained by the preparation method according to any one of claims 2 to 8 in the field of food or medicine.

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