Preparation method of freeze-dried collagen fiber

Abstract

The present application provides a method for preparing freeze-dried collagen fibers, wherein the collagen is type III collagen, and the method comprises: (1) recombinant expression to produce type III collagen; (2) purifying the type III collagen produced in step (1); (3) preparing the purified type III collagen into a freeze-dried raw material solution; the freeze-dried raw material solution is an aqueous solution, which contains 0.5-2 mg / mL type III collagen, 5-10 mg / mL trehalose, 10-15 mg / mL mannitol, and 10-15 mg / mL brown algae extract; the pH of the freeze-dried raw material solution is 7.0-7.5; (4) freeze-drying the freeze-dried raw material solution obtained in step (3). The method of the present application can effectively maintain the biological activity of the freeze-dried collagen fibers using a relatively simple freeze-drying protectant formula and freeze-drying procedure, providing a good raw material for cosmetics, medical fillers, and medical excipients.

Description

Technical Field

[0001] The present application belongs to the field of biological preparations and proteins. Specifically, the present application provides a method for preparing freeze-dried collagen fibers. Background Art

[0002] Type III collagen is the main fibroblast collagen in the human body. It is formed by α1 (III) peptide chains and has the functions of promoting angiogenesis, maintaining lung function, promoting skin growth and repairing injuries. Type III collagen is a good material for preparing cosmetics, fillers and excipients with repair functions. At present, there have been a lot of studies on recombinant expression of type III collagen and its preparation into various cosmetic and medical products.

[0003] For the sake of storage and ease of use, type III collagen is best prepared in a freeze-dried form. However, how to ensure that the basic structure and effect of type III collagen are not lost during the freeze-drying process and that the function of the original protein can be restored after rehydration is still relatively scarce. Research in this field is basically focused on using common sugars, sugar alcohols, and amino acids as protective agents to ensure the basic morphology and physical properties of freeze-dried products. Summary of the invention

[0004] In view of the above situation, on the one hand, the present application provides a method for preparing freeze-dried collagen fibers, wherein the collagen is type III collagen, and the method comprises:

[0005] (1) Recombinant expression and production of type III collagen;

[0006] (2) purifying the type III collagen produced in step (1);

[0007] (3) preparing the purified type III collagen into a freeze-dried raw material solution: the pH of the freeze-dried raw material solution is 7.0-7.5, and the freeze-dried raw material solution is an aqueous solution, which contains 0.5-2 mg / mL type III collagen, 5-10 mg / mL trehalose, 10-15 mg / mL mannitol, and 10-15 mg / mL brown algae extract;

[0008] (4) Lyophilizing the lyophilized raw material solution obtained in step (3).

[0009] Furthermore, the amino acid sequence of the type III collagen is shown in SEQ ID NO.1.

[0010] Furthermore, in step (1), Pichia pastoris is used to recombinantly express and produce type III collagen.

[0011] Furthermore, in step (2), one or more methods selected from the group consisting of ammonium sulfate precipitation, affinity chromatography, ion exchange chromatography, and molecular sieve chromatography are used to purify the type III collagen produced in step (1).

[0012] Furthermore, the pH of the freeze-dried raw material solution is 7.2, and the freeze-dried raw material solution is an aqueous solution, which contains 1 mg / mL type III collagen, 8 mg / mL trehalose, 10 mg / mL mannitol, and Ascophyllum nodosum ( Ascophyllum nodosum ) extract 10mg / mL.

[0013] Further, the freeze-drying process is:

[0014] Place the lyophilized stock solution at 4°C for more than 30 minutes;

[0015] Lower the temperature from 4°C to -40°C and prefreeze at -40°C for 180 minutes;

[0016] The temperature was raised from -40°C to -20°C, and sublimation dried for 900 minutes at a vacuum of 20 Pa and a temperature of -20°C;

[0017] The temperature was raised from -20°C to -5°C, and the sample was dried under a vacuum of 20 Pa and a temperature of -5°C for 240 minutes.

[0018] Further, the freeze-drying process is:

[0019] Place the lyophilized stock solution at 4°C for more than 30 minutes;

[0020] Reduce temperature from 4°C to -40°C at a rate of 1°C / min, and prefreeze at -40°C for 180 minutes;

[0021] The temperature was raised from -40°C to -20°C at a rate of 5°C / min, and sublimation dried at a vacuum of 20 Pa and a temperature of -20°C for 900 minutes;

[0022] The temperature was raised from -20°C to -5°C at a rate of 5°C / min, and the sample was dried at -5°C under a vacuum of 20 Pa for 240 minutes.

[0023] On the other hand, the present application provides freeze-dried collagen fibers obtained by the above preparation method.

[0024] On the other hand, the present application provides the use of the above-mentioned collagen freeze-dried fiber in the preparation of cosmetics with skin repair function.

[0025] On the other hand, the present application provides the use of the above-mentioned collagen freeze-dried fibers in the preparation of medical collagen filling materials or dressings.

[0026] When preparing the freeze-dried raw material solution, various components can be directly dissolved and the pH can be adjusted to the required value; alternatively, a low concentration acetic acid solution, such as 0.5-1% w / v acetic acid solution, can be used to dissolve the collagen first, then add an appropriate amount of water and other components, and finally adjust the pH to the required value.

[0027] In the case of a known protein sequence, a person skilled in the art can query its related gene sequence, and optionally can use known codon optimization methods for further processing to obtain a gene sequence suitable for different hosts, optional hosts include but are not limited to Pichia pastoris and Escherichia coli.

[0028] The method of purifying proteins in fermentation products can be routinely selected by those skilled in the art. For example, when expressing proteins, those skilled in the art can add suitable His or streptomycin tags to further purify the proteins; or those skilled in the art can purify proteins by ammonium sulfate precipitation and subsequent ion exchange or molecular sieve chromatography steps. The degree of protein purification depends on different application requirements, such as electrophoretic purity.

[0029] Beneficial effects:

[0030] The method of the present application can effectively maintain the biological activity of freeze-dried collagen fibers using a relatively simple freeze-drying protectant formula and freeze-drying procedure, and provides good raw materials for cosmetics, medical fillers, and medical excipients. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The results of cell proliferation experiments of freeze-dried collagen fibers in each group;

[0032] Figure 2 The results of DPPH free radical scavenging experiment of each group of collagen freeze-dried fibers;

[0033] Figure 3 The results of the DPPH free radical scavenging experiment of the freeze-dried product group without collagen. DETAILED DESCRIPTION

[0034] Example 1 Related sequences and methods

[0035] sequence:

[0036] Unless otherwise specified, the type III collagen sequences used in the examples are all the type III collagen mature protein sequences shown in SEQ ID NO. 1. The protein was expressed in Pichia pastoris using the Pichia pastoris codon-optimized gene sequence shown in SEQ ID NO. 2, with a yield of about 1.1-1.2 g / L, and affinity chromatography purification with a yield of about 20%.

[0037] Human type III collagen mature peptide sequence (SEQ ID NO.1):

[0038]

[0039] Type III collagen gene sequence (SEQ ID NO.2):

[0040]

[0041] Freeze-drying method:

[0042] Since the present application does not involve a study on the parameters of the freeze-drying process, the freeze-drying procedures in the embodiments are all freeze-drying procedures commonly used in the applicant's experiments: decreasing from 4°C to -40°C at a rate of 1°C / min, and pre-freezing at -40°C for 180 minutes; increasing the temperature from -40°C to -20°C at a rate of 5°C / min, and sublimation drying for 900 minutes at a vacuum degree of 20Pa and a temperature of -20°C; increasing the temperature from -20°C to -5°C at a rate of 5°C / min, and desorption drying for 240 minutes at a vacuum degree of 20Pa and a temperature of -5°C.

[0043] Main reagents:

[0044] Trehalose, mannitol, chondroitin sulfate, and dextran are all domestically produced products of food and cosmetic grade.

[0045] Ascophyllum nodosum extract (polysaccharide content 50%), Dunaliella salina extract (polysaccharide content 60%), Delesseria Sanguinea extract (polysaccharide content 65%), Spirulina Platensis extract (polysaccharide content 75%), and Laminaria japonica extract (polysaccharide content 75%) were purchased from Qingdao Lanbao and were prepared by conventional water extraction and alcohol precipitation method.

[0046] Basic property determination:

[0047] The water content, pH value at 0.5 mg / mL concentration, and protein content were determined according to the method of the 2020 edition of the Pharmacopoeia.

[0048] Redissolution performance:

[0049] Use water for injection to prepare a 1 mg / mL (excluding other ingredients) collagen solution; after adding collagen, place it on an oscillator (amplitude 5 mm, vibration speed 300 times / min), observe the changes in the clarity of the suspension, and record the dissolution time.

[0050] DPPH free radical scavenging experiment:

[0051] Referring to previous studies, a 5 mg / mL collagen concentration (excluding other ingredients) with a more obvious antioxidant effect and not reaching the highest concentration was selected. Take 2.5 mL of sample, mix with 2.5 mL of 0.1 mM DPPH anhydrous ethanol solution or 2.5 mL of anhydrous ethanol, and react at 25°C in the dark for 30 minutes; measure the absorbance of the sample, control, and reference solution (2.5 mL of anhydrous ethanol plus 2.5 mL of 0.1 mM DPPH anhydrous ethanol solution) at 519 nm; calculate the DPPH free radical scavenging rate = (1-(A reaction-A control) / A reference) × 100%.

[0052] Cell proliferation assay:

[0053] Mouse embryonic fibroblast 3T3 cells were purchased from Shane Biotechnology and cultured in 1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2.

[0054] 3T3 cells in the logarithmic growth phase were prepared into a suspension of 1×105 cells / mL, 200 μL per well was inoculated into the well plate and cultured for 24 hours; the culture medium containing 0.5 mg / mL reconstituted collagen / normal culture medium control was replaced and cultured for 24 hours; the relative cell proliferation rate was detected by CCK method = absorbance of collagen group / absorbance of control group × 100%.

[0055] Example 2 Preparation of Type III Collagen Freeze-Dried Fibers

[0056] Type III collagen was dissolved in a 1% w / v acetic acid aqueous solution, and then trehalose and mannitol were prepared into a freeze-dried raw material solution, which contained 8 mg / mL trehalose, 10 mg / mL mannitol, and 1 mg / mL type III collagen.

[0057] Lyophilization was carried out according to the procedure described in Example 1.

[0058] The product is a white sponge / flocculated solid. Electron microscope images show that it has typical collagen fiber curling, entanglement and flocculent structure. The water content is not higher than 1.6% within a few months of storage, and the pH is maintained at 5-8. The protein content is above 96%.

[0059] The above results show that the type III collagen freeze-dried fiber has the expected structure and good storage performance. However, in further applications, it was found that the type III collagen freeze-dried fiber was lacking in the re-dissolution speed and the effect of promoting cell proliferation after re-dissolution. The freeze-drying process needs to be further improved.

[0060] Example 3 Further Improvement of Freeze-Drying Process

[0061] The freeze-dried raw material liquid ratio in Example 2 was obtained by the applicant after extensive screening of common monosaccharides, disaccharides, polysaccharides, amino acids, sugar alcohols, surfactants and other ingredients. Its ingredients are relatively simple and its effect is no less than that of the freeze-dried raw material liquid containing more than ten ingredients in the prior art. Therefore, when further improving, the applicant tried to use seaweed polysaccharides that are less used in the field. Many seaweeds are ingredients that can be used in cosmetics and food, and their safety is relatively guaranteed; its diverse monosaccharide composition, uronic acid content, and sulfate / acetyl modification bring about the diversity of sugar chain conformation, arrangement and biological function, which is expected to provide a new protection mechanism and effect for the freeze-drying process.

[0062] Based on the freeze-dried raw material solution in Example 2, the following ingredients were added:

[0063] Group 1: Ascophyllum nodosum extract (polysaccharide content 50%): 10 mg / mL;

[0064] Group 2: Dunaliella salina extract (polysaccharide content 60%): 10 mg / mL;

[0065] Group 3: Delesseria Sanguinea extract (polysaccharide content 65%): 10 mg / mL;

[0066] Group 4: Spirulina Platensis extract (polysaccharide content 75%): 10 mg / mL;

[0067] Group 5: Laminaria japonica extract (polysaccharide content 75%): 10 mg / mL;

[0068] Group 6: Chondroitin sulfate: 10 mg / mL;

[0069] Group 7: dextran: 10 mg / mL;

[0070] Control 1: freeze-dried raw material solution in Example 2 (without polysaccharide);

[0071] Control 2: non-lyophilized type III collagen.

[0072] The resolubility performance of each group is shown in Table 1

[0073] Table 1 Redissolution properties of each group of freeze-dried collagen fibers

[0074]

[0075] Notes to Table 1: As the dissolution process detected by naked eyes is not precise, the above data are the average values ​​of three parallel experiments and are only accurate to seconds. The storage condition is to seal the container and place it in a 4°C refrigerator.

[0076] The results showed that the use of polysaccharides except dextran could significantly shorten the re-dissolution time by about 5S.

[0077] The results of the cell proliferation experiments in each group are shown in Figure 1 As shown, the results indicate that the freeze-drying-reconstitution process does impair the cell proliferation-promoting effect of collagen, while the addition of two types of fucoidan helps to eliminate this impairment.

[0078] Further results of DPPH free radical scavenging experiments are shown in Figure 2 As shown, type III collagen has a certain antioxidant effect, which will also be damaged by the freeze-drying-redissolution process, but the addition of Ascophyllum nodosum extract, Kelp extract, and Red Algae extract can help restore this damage.

[0079] Considering that many seaweed extracts have antioxidant effects, in order to clarify whether the above effects are caused by the seaweed extracts themselves, we further prepared freeze-dried products containing no collagen and only protective agents, group 1-1, group 3-1, and group 5-1, which were roughly converted according to the amount used in preparing 5 mg / mL collagen solution, and added similar amounts of sugar freeze-dried products to measure the antioxidant properties. If the sum of the antioxidant properties of group 1-1, group 3-1, group 5-1 and the antioxidant properties of control 1 is roughly equal to the values ​​of group 1, group 3, and group 5, it can be considered that the improvement of the antioxidant effects of group 1, group 3, and group 5 compared with control 1 is caused by the seaweed extract itself; if the sum of the antioxidant properties of group 1-1, group 3-1, group 5-1 and the antioxidant properties of control 1 is significantly different from that of group 1, group 3, and group 5, it can be considered that the use of seaweed extracts protects the antioxidant properties of collagen.

[0080] Although it is not entirely reasonable to simply calculate the scavenging activity of DPPH free radicals, Figure 3 The results still clearly showed that compared with control 1, the recovery of the antioxidant effect in groups 3 and 5 was largely provided by red algae extract and kelp extract, while the Ascophyllum nodosum extract in group 1 did provide a protective effect against the loss of antioxidant properties of type III collagen during freeze-drying (20.5 of group 1-1 + 32.7 of control 1 was much less than 69.1 of group 1).

[0081] In summary, the lyoprotectants of type III collagen 1 mg / mL, trehalose 8 mg / mL, mannitol 10 mg / mL, and Ascophyllum nodosum extract 10 mg / mL had a good protective effect on the lyophilization process of type III collagen mature peptide.

[0082] The applicant also tried other self-assembled / truncated type III collagen α1 chains, and the results were not as significant as when used for the mature collagen peptide of SEQ ID NO.1. This may be because the truncation and the construct can form similar structures, but there are still obvious differences in the three-dimensional conformation of the collagen fibers, and other parameters such as the dosage need to be further adjusted.

Claims

1. A method for preparing collagen freeze-dried fibers, It is characterized by, The collagen is type III collagen, and the method includes: (1) Recombinant expression and production of type III collagen; (2) Purify the type III collagen produced in step (1); (3) Prepare the purified type III collagen into a freeze-dried raw material liquid; the freeze-dried raw material liquid is an aqueous solution, which contains type III collagen 1 mg / mL, trehalose 8 mg / mL, mannitol 10 mg / mL, Ascophyllum nodosum extract 10 mg / mL; the pH of the freeze-dried raw material solution is 7.2; (4) Lyophilize the freeze-dried raw material liquid obtained in step (3); The amino acid sequence of type III collagen is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he freeze-drying process of step (4) is: Place the freeze-dried raw material liquid at 4°C for more than 30 minutes; Decrease from 4°C to -40°C at a rate of 1°C / min, and pre-freeze at -40°C for 180 minutes; Raise the temperature from -40°C to -20°C at a speed of 5°C / min, and sublimate and dry for 900 minutes at a vacuum of 20Pa and a temperature of -20°C; The temperature was raised from -20°C to -5°C at a rate of 5°C / min, and analyzed and dried under a vacuum of 20 Pa and a temperature of -5°C for 240 minutes.

2. The method according to claim 1, It is characterized by, In step (1), Pichia pastoris or CHO are used to recombinantly express type III collagen.

3. The method of claim 1, It is characterized by, In step (2), one or more methods selected from ammonium sulfate precipitation, affinity chromatography, ion exchange chromatography, and molecular sieve chromatography are used to purify the type III collagen produced in step (1).

4. Type III collagen freeze-dried fiber, It is characterized by, The type III collagen freeze-dried fiber is prepared using the method according to any one of claims 1-3.

5. Application of the type III collagen freeze-dried fiber according to claim 4 in the preparation of medical collagen filling materials or dressings.

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