Collagen freeze-dried product and preparation method thereof

Abstract

The invention provides a collagen freeze-dried product and a preparation method thereof, and belongs to the technical field of biology. In the formula design, through the synergistic effect of the arginine (salt) and the surfactant, denaturation and aggregation of the collagen freeze-dried product in the production process and the storage period are inhibited, the stability of the product is improved, and the prepared collagen freeze-dried product is high in activity, purity and safety. In the freeze-drying process, pre-freezing parameters are optimized, and the nucleation effect of ice crystals in the freezing process of the collagen mixed solution is controlled by regulating and controlling parameters such as temperature and vacuum degree in the freeze-drying process, so that the shapes and the sizes of the crystals in the collagen freeze-dried product are more uniform, the drying time is shortened, and the production efficiency is improved. And the collagen freeze-dried product with uniform pore diameter and excellent appearance is obtained, and the uniformity and stability of batch and inter-batch products are improved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a freeze-dried collagen product and a preparation method thereof. Background Art

[0002] Collagen is the main component of the dermis, especially type I and type III collagen are the most abundant. In different regions of the dermis, the forms of collagen are diverse. In the reticular layer of the dermis, collagen fibers and elastic fibers together form a network structure, which not only supports the overall structure of the skin but also endows the skin with necessary elasticity and mobility. With age, the collagen in the dermis gradually loses, resulting in a thinner layer thickness, which is an important factor in the formation and deepening of wrinkles. Collagen is widely used in functional skin care products, medical dressings, conventional skin care products and aesthetic injection products, aiming to improve the skin quality by using its powerful moisturizing, nourishing, hemostatic and repair capabilities, and can also play a certain filling role, so it is often used as a biomedical material for implantable medical devices.

[0003] Recombinant collagen freeze-dried fibers have developed rapidly as a new technology in the field of medical aesthetics. However, during the preparation and storage of recombinant collagen freeze-dried fibers, there are problems such as collagen degradation or aggregation. For example, in the production process of recombinant collagen preparations, the sterilization filtration process will subject the protein to the combined effects of high shear and adsorption, that is, the solid-liquid interface effect leads to changes in the conformation of collagen and the formation of multimers. Stirring, pumping, and filling are all indispensable processes in the production of collagen preparations, and the irreversible adsorption of collagen on the stainless steel surface will also induce the aggregation of collagen. During the freeze-drying process, the continuous change in concentration during the pre-freezing stage may lead to changes in pH value or phase separation, which may also cause collagen degradation. In the drying stage, water molecules are removed from the protein surface, and the loss of its hydration shell may lead to instability of the native protein structure, resulting in its inactivation, protein conformational changes leading to protein aggregation, etc. During the use of freeze-drying stabilizers, there are also problems such as the freeze-drying stabilizer being too large to hydrogen bond with collagen proteins, unable to replace the water lost during dehydration, or the freeze-drying stabilizer forming a separate amorphous phase with the protein, affecting the stability of collagen. In addition, even in the state of freeze-dried powder (fibers), all these effects may damage the effectiveness and shelf-life stability of recombinant collagen proteins. In addition, freeze-drying is a commonly used technique to maintain the chemical stability and physical properties of proteins during storage. The pre-freezing stage of freeze-drying has an important impact on the physicochemical properties and appearance of the product. Pre-freezing mainly cools the solution, and after reaching the supercooling point, ice crystals begin to form and crystallize; general pre-freezing crystallization is spontaneous nucleation, which is random and uncontrolled, and its duration is very long, resulting in large differences in the morphology and size of ice crystals in each bottle. This not only affects the subsequent drying rate but also the uniformity of the product appearance. Therefore, the development of methods to maintain the stability of freeze-dried fibers during preparation and storage has become a challenging task.

[0004] Therefore, there is an urgent need for a preparation method to improve the stability of collagen freeze-dried products during production and storage. Summary of the Invention

[0005] For this reason, the technical problem to be solved by the present invention is to overcome the problem in the prior art of lacking a preparation method to improve the freeze-drying stability of collagen.

[0006] To solve the above technical problems, the present invention provides a freeze-dried collagen product and a preparation method thereof. In the present invention, the synergistic effect of a surfactant and arginine (salt) is utilized in the formulation. The surfactant inhibits the adsorption of recombinant collagen at the interface during the production of the collagen preparation, thereby preventing the aggregation of recombinant collagen induced by the interface (such as interface effects occurring during membrane filtration, stirring, mixing, pumping, and filling). During the freeze-drying process, arginine (arginine salt) acts as a protein stabilizer to prevent the structural damage and aggregation of collagen during freeze-drying. At the same time, similar to the effective prevention of protein aggregation in aqueous solutions by amino acids, it can also prevent the aggregation of the collagen fiber preparation during the storage period. By controlling the temperature and vacuum degree in the pre-freezing stage of the freeze-drying process, a freeze-dried collagen product with good stability is obtained. The freeze-dried collagen product of the present invention has uniform pore sizes, excellent appearance, and extremely low water content, and still maintains good purity after undergoing an accelerated aging experiment at 40°C.

[0007] The first object of the present invention is to provide a preparation method of a freeze-dried collagen product, comprising the following steps:

[0008] S1. Mix collagen with a surfactant and an arginine derivative to obtain a collagen mixture;

[0009] S2. Subject the collagen mixture to freeze-drying treatment to obtain the freeze-dried collagen product;

[0010] Wherein, the freeze-drying treatment includes a pre-freezing stage, a primary drying stage, and a secondary drying stage.

[0011] In the pre-freezing stage, the temperature is lowered to 0 to -15°C and maintained for 30 - 50 min, then the temperature is lowered to -50 to -30°C and maintained for 3 - 6 h;

[0012] In the primary drying stage, the temperature is raised to -30 to -5°C and maintained for 10 - 30 h;

[0013] In the secondary drying stage, the temperature is raised to 0 - 25°C and maintained for 4 - 6 h.

[0014] Further, in the pre-freezing stage, the temperature is reduced to 0 to -15°C and maintained for 30 - 50 min, then the vacuum degree is set to 0.1 - 1 mbar and maintained for 1 - 10 min, and then the temperature is reduced to -50 to -30°C and maintained for 3 - 6 h. During the freeze-drying process, in the pre-freezing stage, first, the freeze-drying solution is balanced at a temperature lower than the crystallization temperature, then vacuum is introduced, and the evaporation of the solvent promotes the reduction of the liquid surface temperature, thereby generating crystallization. The vacuum degree in the pre-freezing stage affects the formation of ice crystals. When the vacuum degree is low, the evaporation rate of water molecules slows down, which may cause the growth rate of ice crystals to slow down, and rapid nucleation crystallization cannot be achieved, thus reducing the optimization effect. If the ice crystals grow too fast, it may increase the time for the protein to be exposed to a higher concentration of solutes, thereby increasing the risk of protein denaturation. In addition, if the vacuum degree is too high, there may also be a phenomenon of spraying bottles.

[0015] Further, the surfactant is selected from one or more of polysorbate 20, polysorbate 80, poloxamer 188, polyoxyethylene octylphenyl ether, and sodium dodecyl sulfate.

[0016] Further, the arginine derivative is selected from one or more of arginine, arginine citrate, arginine phosphate, arginine succinate, arginine hydrochloride, and arginine lactate, and its chemically acceptable salts.

[0017] Further, the chemically acceptable salts of the arginine derivative include arginine hydrochloride, arginine citrate hydrochloride, arginine phosphate hydrochloride, arginine succinate hydrochloride, and arginine lactate hydrochloride.

[0018] Further, in step S1, the mass ratio of the collagen to the arginine derivative is (1 - 20):1.

[0019] Further, in step S1, the mass ratio of the collagen to the surfactant is (1 - 20):(0.01 - 0.02). The mass ratio of arginine, surfactant, and collagen affects the stability of collagen.

[0020] During the freeze-drying process, the mixture of arginine (salt), surfactant and collagen can protect proteins from unstable stresses during the freezing and drying stages, reduce the interactions between protein molecules, and thus maintain the molecular structure of proteins. Meanwhile, on the premise of ensuring the stability of collagen, it can also regulate the glass transition temperature (Tg’) of the freeze-drying solution and the glass transition temperature (Tg) of the freeze-dried solid, increase the drying temperature, and shorten the drying time, which is of great value to freeze-dried preparations. In the production process of protein preparations, surfactants can inhibit the adsorption of recombinant collagen at the interface and thus prevent the aggregation of recombinant collagen induced by the interface (such as interface effects will occur during membrane filtration, stirring, mixing, pumping and filling). Similar to the effective prevention of protein aggregation in aqueous solutions by amino acids (salts), it can also prevent the aggregation of collagen fiber preparations during the storage period. The synergistic effect of arginine (salt) and surfactant improves the stability of recombinant collagen freeze-dried preparations and reduces or inhibits their degradation or aggregation. The sum of the above characteristics makes the synergistic effect of arginine (salt) and surfactant play an important role in the production of collagen freeze-dried preparations. The synergistic effect of arginine (salt) and surfactant is concentration-sensitive. With the increase of collagen concentration, its stabilizing effect will decrease. However, when the collagen concentration is low and the stabilizer concentration is high, it may cause collagen denaturation. The stability of the protein is the best under the conditions of high protein concentration and high stabilizer, but this will lead to a high solid concentration in the whole formula and cause difficulties in freeze-drying.

[0021] Further, the pH of the collagen mixture described in step S1 is 6-8.

[0022] Further, the collagen mixture described in step S1 is sterilized.

[0023] Further, the sterilization treatment is to filter and sterilize the collagen mixture through a bacteria-reducing filter and a sterilizing filter.

[0024] The second object of the present invention is to provide a collagen freeze-dried product prepared by the above preparation method.

[0025] The beneficial effects of the present invention:

[0026] The present invention provides a freeze-dried collagen product and a preparation method thereof. In the formulation design of the present invention, through the synergistic effect of arginine derivatives and surfactants on collagen, the denaturation and aggregation of the freeze-dried collagen product during the production process and storage period are inhibited, the stability of the product is improved, and the prepared freeze-dried collagen product has high activity, high purity and high safety. In the freeze-drying process, the pre-freezing parameters are optimized, and by controlling the temperature and vacuum degree during the freeze-drying process, the nucleation effect of the crystallization of the collagen mixture during the freeze-drying process is controlled, so that the internal crystal shape and size of the freeze-dried collagen product are more uniform, which can not only shorten the primary drying time and reduce the moisture content of the product, but also obtain a freeze-dried collagen product with uniform pore size and excellent appearance, and improve the uniformity and stability of the products within batches and between batches. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to make the content of the present invention easier to be clearly understood, the following further details the present invention according to the specific embodiments of the present invention in combination with the accompanying drawings, wherein

[0028] Figure 1 is a photograph of the appearance of Example 1;

[0029] Figure 2 is a photograph of the appearance of Comparative Example 3;

[0030] Figure 3 is a scanning electron microscope photograph of Example 1;

[0031] Figure 4 is a scanning electron microscope photograph of Comparative Example 3. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0032] The following further illustrates the present invention in combination with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the embodiments cited do not limit the present invention.

[0033] Example 1: Preparation of a freeze-dried recombinant type III humanized collagen product

[0034] (1) Preparation of the freeze-drying feed liquid

[0035] Take 10 mg / mL of recombinant type III humanized collagen raw material, 1 mg / mL of arginine hydrochloride and 0.01 mg / mL of Tween 80, and stir evenly at room temperature; adjust the pH value of the system to 7 with sodium hydrogen phosphate / sodium dihydrogen phosphate buffer solution, and stir evenly at room temperature to obtain a collagen mixture; filter the above-mentioned collagen mixture through a 0.45 μm bacteria-reducing filter and a 0.22 μm sterilizing filter for sterilization.

[0036] (2) Freeze-drying

[0037] The filtered liquid material is filled into vials at a filling volume of 2.5 mL / vial, and then transferred to the shelves of a freeze dryer for freeze-drying. The freeze-drying process is set as shown in Table 1.

[0038] Table 1 Freeze-drying process settings for Example 1

[0039]

[0040] After the freeze-drying program is completed, nitrogen is flushed into the chamber and capping is carried out inside the chamber. After capping, the vials are taken out of the chamber for crimping to obtain the freeze-dried recombinant type III collagen product.

[0041] Example 2

[0042] (1) Preparation of the freeze-drying liquid material

[0043] Take 10 mg / mL of recombinant type III humanized collagen raw material, 1 mg / mL of lactic arginine, and 0.01 mg / mL of Tween 80, and stir evenly at room temperature; adjust the pH value of the system to 7 with sodium hydrogen phosphate / sodium dihydrogen phosphate buffer, and stir evenly at room temperature to obtain a collagen mixture; filter and sterilize the above collagen mixture through a 0.45 μm bacteria-reducing filter and a 0.22 μm bacteria-removing filter.

[0044] (2) Freeze-drying

[0045] The filtered liquid material is filled into vials at a filling volume of 2.5 mL / vial, and then transferred to the shelves of a freeze dryer for freeze-drying. The freeze-drying process is set as shown in Table 1.

[0046] After the freeze-drying program is completed, nitrogen is flushed into the chamber and capping is carried out inside the chamber. After capping, the vials are taken out of the chamber for crimping to obtain the freeze-dried recombinant type III collagen product.

[0047] Comparative Example 1

[0048] Comparative Example 1 is the same as the steps of Example 1, except that Tween 80 is not added in Comparative Example 1. The specific steps are as follows:

[0049] (1) Preparation of the freeze-drying liquid material:

[0050] Take 10 mg / mL of recombinant type III humanized collagen and 1 mg / mL of arginine hydrochloride, and stir evenly at room temperature. Adjust the pH value of the system to 7 with sodium hydrogen phosphate / sodium dihydrogen phosphate buffer, and stir evenly at room temperature to obtain a collagen mixture. Filter and sterilize the above collagen mixture through a 0.45 μm bacteria-reducing filter and a 0.22 μm bacteria-removing filter.

[0051] (2) Freeze-drying

[0052] The filtered feed liquid above is filled into vials at a filling volume of 2.5 mL / vial, and then transferred to the shelves of a freeze dryer for freeze drying. The freeze drying process is set as shown in Table 2.

[0053] Table 2 Setting of the freeze drying process for Comparative Example 1

[0054]

[0055] After the freeze drying program is completed, nitrogen is introduced into the chamber and stopper pressing is carried out inside the chamber. After stopper pressing, the vials are taken out of the chamber for capping to obtain the freeze-dried product of recombinant type III humanized collagen.

[0056] Comparative Example 2

[0057] Comparative Example 2 is the same as the steps of Example 1, except that arginine hydrochloride is not added in Comparative Example 2. The specific steps are as follows:

[0058] (1) Preparation of the freeze-drying feed liquid

[0059] Take 10 mg / mL of recombinant type III humanized collagen raw material and 0.01 mg / mL of Tween 80 and stir evenly at room temperature. Adjust the pH value of the system to 7 with sodium hydrogen phosphate / sodium dihydrogen phosphate buffer solution and stir evenly at room temperature to obtain a collagen mixture. The above collagen mixture is filtered and sterilized through a 0.45 μm bacteria-reducing filter and a 0.22 μm sterilizing filter.

[0060] (2) Freeze drying

[0061] The filtered feed liquid above is filled into vials at a filling volume of 2.5 mL / vial, and then transferred to the shelves of a freeze dryer for freeze drying. The freeze drying process is set as shown in Table 3.

[0062] Table 3 Setting of the freeze drying process for Comparative Example 2

[0063]

[0064] After the freeze drying program is completed, nitrogen is introduced into the chamber and stopper pressing is carried out inside the chamber. After stopper pressing, the vials are taken out of the chamber for capping to obtain the freeze-dried fibers of recombinant type III collagen.

[0065] Comparative Example 3

[0066] Comparative Example 3 is the same as the steps of Example 1, except that the freeze drying steps in Comparative Example 3 are changed. The specific operations are as follows:

[0067] (1) Preparation of the freeze-drying feed liquid:

[0068] Take 10 mg / mL of recombinant humanized type III collagen raw material, 1 mg / mL of arginine hydrochloride, and 0.01 mg / mL of Tween 80, and stir evenly at room temperature. Adjust the pH value of the system to 7 with sodium hydrogen phosphate / sodium dihydrogen phosphate buffer, and stir evenly at room temperature to obtain a collagen mixture. Filter and sterilize the above-mentioned collagen mixture through a 0.45 μm bacteria-reducing filter and a 0.22 μm bacteria-removing filter.

[0069] (2) Freeze-drying

[0070] Fill the above freeze-dried liquid material into vials at a filling volume of 2.5 mL / vial, then transfer it to the shelf of a freeze-dryer for freeze-drying, and the freeze-drying process is set as shown in Table 4.

[0071] Table 4 Freeze-drying process settings for Comparative Example 3

[0072] Stage Parameter Pre-freezing stage At a rate of 1 °C / min, the shelf temperature is lowered to -40 °C and kept for 5 h for pre-freezing Primary drying stage At a rate of 1 °C / min, the shelf temperature is raised to -20 °C and kept for 20 h for sublimation drying Secondary drying stage At a rate of 1 °C / min, the shelf temperature is raised to 10 °C and kept for 5 h for desorption drying

[0073] After the freeze-drying program is completed, fill the chamber with nitrogen and perform plugging inside the chamber. After plugging, take out the chamber and crimp the vials to obtain freeze-dried recombinant humanized type III collagen fibers.

[0074] Comparative Example 4

[0075] Comparative Example 4 is the same as the steps of Example 1, except that in Comparative Example 4, arginine hydrochloride is replaced with lysine hydrochloride, and the specific operation is as follows:

[0076] (1) Preparation of freeze-dried liquid material

[0077] Take 10 mg / mL of recombinant humanized type III collagen raw material, 1 mg / mL of lysine hydrochloride, and 0.01 mg / mL of Tween 80, and stir evenly at room temperature; adjust the pH value of the system to 7 with sodium hydrogen phosphate / sodium dihydrogen phosphate buffer, and stir evenly at room temperature to obtain a freeze-dried liquid material; filter and sterilize the above freeze-dried liquid material through a 0.45 μm bacteria-reducing filter and a 0.22 μm bacteria-removing filter.

[0078] (2) Freeze-drying

[0079] Fill the above filtered liquid material into vials at a filling volume of 2.5 mL / vial, then transfer it to the shelf of a freeze-dryer for freeze-drying, and the freeze-drying process is set as shown in Table 5.

[0080] Table 5 Freeze-drying process settings for Comparative Example 4

[0081]

[0082] After the freeze-drying program is completed, fill the chamber with nitrogen and perform plugging inside the chamber. After plugging, take out the chamber and crimp the vials to obtain a freeze-dried recombinant type III collagen product.

[0083] Comparative Example 5

[0084] The steps of Comparative Example 5 were the same as those of Example 1, except that the ratio of arginine hydrochloride to Tween 80 in Comparative Example 5 was changed. The specific operations were as follows:

[0085] (1) Preparation of the freeze-dried feed solution

[0086] Take 10 mg / mL of recombinant type III humanized collagen raw material, 0.1 mg / mL of arginine hydrochloride, and 0.5 mg / mL of Tween 80, and stir evenly at room temperature; adjust the pH value of the system to 7 with sodium hydrogen phosphate / sodium dihydrogen phosphate buffer, and stir evenly at room temperature to obtain the freeze-dried feed solution; filter and sterilize the above freeze-dried feed solution through a 0.45 μm bacteria-reducing filter and a 0.22 μm sterilizing filter.

[0087] (2) Freeze-drying

[0088] Fill the filtered feed solution into vials at a filling volume of 2.5 mL / vial, and then transfer it to the shelf of the freeze-dryer for freeze-drying. The freeze-drying process is set as shown in Table 6.

[0089] Table 6 Freeze-drying process settings for Comparative Example 5

[0090]

[0091] After the freeze-drying program is completed, fill the chamber with nitrogen and perform plugging inside the chamber. After plugging, take out the chamber and crimp the vials to obtain the recombinant type III collagen freeze-dried product.

[0092] Test Example

[0093] (1) Test the appearance, scanning electron microscopy observation, and reconstitution time of the samples prepared in Example 1 and Comparative Example 3.

[0094] Appearance: Observe the appearance of the freeze-dried fibers in a bright place.

[0095] Scanning electron microscopy: Take a small amount of sample / bulk / thin film sample and directly stick it to the conductive adhesive, and use a Quorum SC7620 sputtering coater to spray gold for 45 s (the specific gold spraying time is determined according to the sample / test requirements), and the gold spraying current is 10 mA; then use a scanning electron microscope to take pictures of the sample morphology, and the detector is an SE2 secondary electron detector. It can be seen from the comparison that in Example 1, the vacuum-controlled nucleation technology was used in the pre-freezing stage of the freeze-drying process, while Comparative Example 3 was a common single-step pre-freezing. The appearance of the freeze-dried fiber product in Example 1 was excellent, and the pore size distribution was relatively uniform. However, in Comparative Example 3, the appearance of the product showed shrinkage and bottle dropping, and the appearance was very rough and the internal pore sizes were uneven. In addition, the moisture content of the freeze-dried fibers in Example 1 was 0.82%, and the moisture content of the freeze-dried fibers in Comparative Example 3 was 5.1%.

[0096] Redissolution time: Randomly select 3 vials of recombinant collagen freeze-dried products, add 2 mL of normal saline, and measure the time required for complete dissolution. The redissolution time of the freeze-dried fibers obtained in Example 1 was 8 s, while the redissolution times of Comparative Examples 1-5 were all greater than 13 s. It is speculated that the changes in the freeze-drying process and the freeze-drying stabilizer may have led to the crystallinity and pore size of the recombinant collagen freeze-dried products, thereby affecting the infiltration efficiency of normal saline and resulting in an extended redissolution time.

[0097] Table 7 Redissolution times of the recombinant collagen freeze-dried fibers obtained in Examples 1-2 and Comparative Examples 1-5

[0098]

[0099]

[0100] (2) Stability experiments of Examples 1-2 and Comparative Examples 1-5

[0101] The freeze-dried fibers prepared in Examples 1-2 and Comparative Examples 1-5 were subjected to an accelerated aging experiment at 40 °C to verify their stability.

[0102] Table 8 Results of the stability experiments of the collagen freeze-dried products prepared in Examples 1-2 and Comparative Examples 1-5

[0103]

[0104] From the above results, it can be seen that after 6 months of accelerated aging at 40 °C, the purity of the collagen in Examples 1 and 2 did not decrease much, while in Comparative Examples 1, 2, 4, and 5, there was a significant decrease in purity due to degradation or aggregation. This indicates that adding a freeze-drying stabilizer to the recombinant collagen freeze-dried product formula can improve the stability of recombinant collagen during manufacturing and storage, and using different freeze-drying stabilizers will result in significant differences in the stability of collagen. Comparing Example 1 and Comparative Example 3, by using optimized freeze-drying parameters, not only can a fiber structure with uniform pores be prepared, but the moisture content of the product can also be controlled at a relatively low level. The lower the moisture content, the more stable the collagen structure. Comparing Example 1 with Comparative Example 4, it can be found that different amino acids and Tween 80 have different effects on maintaining the stability of collagen. The difference in charges between different amino acids may affect their interaction with collagen, ultimately affecting the freeze-drying stability of collagen.

[0105] Obviously, the above embodiments are merely examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. And the obvious changes or modifications derived therefrom still fall within the protection scope of the present invention.

Claims

1. A preparation method of a freeze-dried collagen product, characterized in that, It includes the following steps: S1. Mix collagen with a surfactant and an arginine derivative to obtain a collagen mixture; S2. Subject the collagen mixture to freeze-drying treatment to obtain the freeze-dried collagen product; Among them, the freeze-drying treatment includes a pre-freezing stage, a primary drying stage, and a secondary drying stage. The pre-freezing stage is to cool down to 0 to -15°C, hold for 30 - 50 min, then cool down to -50 to -30°C, and hold for 3 - 6 h; The primary drying stage is to heat up to -30 to -5°C and hold for 10 - 30 h; The secondary drying stage is to heat up to 0 - 25°C and hold for 4 - 6 h.

2. The preparation method according to claim 1, wherein The surfactant is selected from one or more of polysorbate 20, polysorbate 80, poloxamer 188, polyethylene glycol monooctyl phenyl ether, and sodium dodecyl sulfate.

3. The preparation method according to claim 1, wherein, The arginine derivative is selected from one or more of arginine, arginine citrate, arginine phosphate, arginine succinate, arginine lactate, and its chemically acceptable salts.

4. The preparation method according to claim 1, characterized in that, The pre-freezing stage is to cool down to 0 to -15°C, hold for 30 - 50 min, then set the vacuum degree to 0.1 - 1 mbar and hold for 1 - 10 min, and then cool down to -50 to -30°C and hold for 3 - 6 h.

5. The preparation method according to claim 1, wherein In step S1, the mass ratio of the collagen to the arginine derivative is (1 - 20):

1.

6. The preparation method according to claim 1, characterized in that, In step S1, the mass ratio of the collagen to the surfactant is (1 - 20):(0.01 - 0.02).

7. The preparation method according to claim 1, characterized in that, The pH of the collagen mixture in step S1 is 6 - 8.

8. The preparation method according to claim 1, characterized in that, The collagen mixture in step S1 is subjected to sterilization treatment.

9. The preparation method according to claim 8, characterized in that The sterilization treatment is to filter and sterilize the collagen mixture through a bacteria-reducing filter and a sterilizing filter.

10. A freeze-dried collagen product prepared by the preparation method according to any one of claims 1 - 9.

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