Collagen freeze-dried powder and preparation method thereof

Abstract

The invention discloses collagen freeze-dried powder and a preparation method thereof, and belongs to the technical field of cosmetics. The collagen freeze-dried powder comprises collagen and water, the water content of the collagen freeze-dried powder is 4-5wt%, and the balance is collagen. According to the collagen freeze-dried powder product, the product stability is enhanced while the properties of the collagen freeze-dried powder product are improved.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetics, and particularly relates to a collagen lyophilized powder and a preparation method thereof. Background Art

[0002] In daily life, the skin is easily affected by external and internal factors, resulting in insufficient biosynthesis and loss of structural collagen in the skin, causing structural collapse, showing wrinkles, and often accompanied by pigmentation caused by oxidation reactions, presenting signs of aging. People have always used various means to cover up and treat this aging.

[0003] As a new type of biological gene beauty product, the lyophilized powder containing collagen has bioactive collagen as its active ingredient, which can achieve beauty effects such as anti-wrinkle, repair, and lightening of spots. In order to maintain the biological activity of collagen in the lyophilized powder to the greatest extent, the existing technology often uses a segmented freeze-drying technique to produce the lyophilized powder, and requires that the internal crystallization of the freeze-dried product be dense, the appearance be beautiful, and there be no obvious collapse, shrinkage, bubbling, etc. Therefore, traditional freeze-dried collagen products usually contain skeleton raw materials such as mannitol and trehalose to maintain the stability of the product.

[0004] However, these additives may cause skin irritation or allergic reactions, limiting the wide application of the product. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a collagen lyophilized powder and a preparation method thereof, which are used to overcome the problem that the lyophilized powder in the existing technology usually contains skeleton raw materials and is prone to cause skin irritation or allergies.

[0006] In the first aspect, the present invention provides a collagen lyophilized powder, which includes collagen and water. The water content of the collagen lyophilized powder is 4wt% - 5wt%, and the balance is collagen.

[0007] Compared with the existing technology, the collagen lyophilized powder of the present invention only contains collagen and water, does not contain skeleton raw materials such as mannitol and trehalose, reduces the irritation reaction to the skin during use, and the collagen is more easily absorbed by the skin. In addition, the surface of the lyophilized powder of the present invention is flat, delicate, without debris, without broken powder or cracking, the powder is closely attached to the bottle wall, and after knocking and shaking the bottle body 2 - 5 times, the powder does not break or disperse.

[0008] In the second aspect, the present invention also provides a preparation method of the collagen lyophilized powder, which is applied to the preparation of the above-mentioned collagen lyophilized powder, and includes the following steps:

[0009] Prepare a collagen freeze-drying stock solution, which includes collagen and water;

[0010] Gradually cool the collagen freeze-dried stock solution to -40°C to -45°C and pre-freeze it for 4 to 6 hours.

[0011] Perform N times of sublimation drying on the pre-frozen collagen freeze-dried stock solution, where N is an integer greater than 1. The temperature of the sublimation drying increases gradually from the 1st time to the Nth time, and the vacuum degree of the sublimation drying decreases gradually from the 1st time to the Nth time.

[0012] Perform M times of desorption drying on the collagen freeze-dried stock solution after sublimation drying, where M is an integer greater than 1. The temperature of the desorption drying increases gradually from the 1st time to the Mth time, and the vacuum degree of the desorption drying decreases gradually or remains unchanged from the 1st time to the Mth time to obtain collagen freeze-dried powder.

[0013] Compared with the prior art, in the method for preparing the collagen freeze-dried powder of the present invention, by selecting specific and reasonable pre-freezing temperature and pre-freezing time, first pre-freeze the collagen freeze-dried stock solution, and then perform N times of sublimation drying and M times of desorption drying on the pre-frozen collagen freeze-dried stock solution. At the same time, by limiting that the temperature of the sublimation drying increases gradually, the vacuum degree decreases gradually, the temperature of the desorption drying increases gradually, and the vacuum degree of the desorption drying decreases gradually or remains unchanged, the freeze-drying curve is optimized. Through the above fine control, it can be ensured that the collagen can form a stable morphology in each sublimation drying process and desorption drying process, avoiding phenomena such as collapse, shrinkage, and bubbling. While improving the product properties of the collagen freeze-dried powder, the product stability is enhanced, and the quality standard of the freeze-dried product meets the requirements specified by the national drug standard.

[0014] Furthermore, the temperature of each sublimation drying is independently selected from -35°C to -2°C, the vacuum degree is independently selected from 10 Pa to 20 Pa, and the total time of N times of sublimation drying is 10 hours to 54 hours.

[0015] Furthermore, the temperature difference between two adjacent sublimation dryings is 2°C to 16°C.

[0016] Furthermore, during the sublimation drying, the temperature rising time between two adjacent sublimation dryings is 0.5 h to 1 h.

[0017] Furthermore, the vacuum degree difference between two adjacent sublimation dryings is 1 Pa to 3 Pa.

[0018] Furthermore, the temperature of each desorption drying is independently selected from 0°C to 30°C, the vacuum degree is independently selected from 10 Pa to 18 Pa, and the total time of M times of desorption drying is 10 hours to 30 hours.

[0019] Furthermore, the temperature difference between two adjacent desorption dryings is 5°C to 15°C; and / or,

[0020] During the analytical drying, the temperature rising time between two adjacent analytical dryings is 0.5 h to 1 h; and / or,

[0021] During the analytical drying, the difference in vacuum degree between two adjacent analytical dryings is 0 Pa to 3 Pa.

[0022] Furthermore, the content of collagen in the freeze-dried collagen stock solution is 0.1 wt% - 1.0 wt%, and the balance is water; and / or

[0023] 1 < N ≤ 7; and / or

[0024] 1 < M ≤ 7.

[0025] Furthermore, after the analytical drying is completed, a pressure rise test is also included, and the set pressure rise limit value in the pressure rise test is 3 Pa / min. Description of the Drawings

[0026] Figure 1 It is a graph of the bioactivity test results with keratinocytes as the experimental object;

[0027] Figure 2 It is a graph of the bioactivity test results with fibroblasts as the experimental object. Detailed Embodiments

[0028] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0029] In the prior art, traditional freeze-dried collagen products usually need to add skeleton raw materials, such as mannitol, trehalose, etc., during the preparation process to maintain the structural stability of the freeze-dried collagen products. However, as additives, these skeleton raw materials may, on the one hand, cause skin irritation or allergic reactions during use, and on the other hand, due to the existence of the skeleton structure, they will also affect the absorption and utilization of collagen by the skin.

[0030] To solve the above technical problems, on the one hand, an embodiment of the present invention provides a freeze-dried collagen powder, which includes collagen and water, and the water content of the freeze-dried collagen powder is 4 wt% to 5 wt%, and the balance is collagen.

[0031] With the above technical solution, the collagen lyophilized powder of the present invention only contains collagen and water, and does not contain framework raw materials such as mannitol and trehalose, reducing the irritation reaction to the skin during use. Although the collagen lyophilized powder of the present invention does not contain a framework structure, it still has a good morphology. Through experiments, it is observed that its appearance surface is flat, delicate, without debris, no broken powder or cracking phenomenon, and the powder tightly adheres to the bottle wall. After knocking and shaking the bottle body 2 to 5 times, the powder does not break or disperse, and the activity of collagen molecules is fully guaranteed. In addition, in the lyophilized powder of the present invention, the content of collagen is as high as 95% - 96%. And because it only contains collagen and does not contain a framework structure, when used, it can not only reduce the irritation to the skin, but also greatly improve the absorption and utilization rate of collagen by the skin, greatly improving the use performance of the product.

[0032] In a second aspect, the embodiment of the present invention also provides a preparation method of collagen lyophilized powder, which is applied to the preparation of the above-mentioned collagen lyophilized powder, and includes the following steps:

[0033] S1. Prepare a collagen freeze-drying stock solution, and the collagen freeze-drying stock solution includes collagen and water; by way of example, in this step, the content of collagen in the collagen freeze-drying stock solution is 0.1 wt% - 1.0 wt%, and the balance is water.

[0034] S2. Gradually cool the collagen freeze-drying stock solution to -40°C to -45°C and pre-freeze it for 4 to 6 hours; in this step, the gradual cooling time is 20 minutes to 40 minutes.

[0035] S3. Perform N sublimation drying operations on the pre-frozen collagen freeze-drying stock solution, where N is an integer greater than 1. The temperature of the sublimation drying gradually increases from the 1st to the Nth time, and the vacuum degree of the sublimation drying gradually decreases from the 1st to the Nth time;

[0036] S4. Perform M desorption drying operations on the collagen freeze-drying stock solution after sublimation drying, where M is an integer greater than 1. The temperature of the desorption drying gradually increases from the 1st to the Mth time, and the vacuum degree of the desorption drying gradually decreases or remains unchanged, to obtain the collagen lyophilized powder.

[0037] Adopting the above technical solution, the inventor found that the preparation method of the collagen lyophilized powder of the present invention pre-freezes the collagen lyophilized stock solution by selecting a reasonable pre-freezing temperature and pre-freezing time, and then performs N times of sublimation drying and M times of desorption drying on the pre-frozen collagen lyophilized stock solution under specific conditions. In particular, by controlling the temperature to gradually increase and the vacuum degree to gradually decrease during sublimation drying, it can be ensured that the skeleton structure of the collagen itself as the lyophilized powder is not damaged after each sublimation drying. At the same time, the inventor also found that by further controlling the temperature to gradually increase and the desorption drying vacuum degree to gradually decrease or remain unchanged during desorption drying, during this process, the structural stability of the dried collagen can be further ensured during the process of removing the remaining moisture in the collagen lyophilized stock solution. In summary, by controlling the temperature and vacuum degree of sublimation drying and desorption drying, the technical solution of the present invention further optimizes the lyophilization curve. Through the above fine control, it can be ensured that the collagen can form a stable morphology during each sublimation drying process and desorption drying process, avoiding phenomena such as collapse, shrinkage, and bubbling. While improving the product properties of the collagen lyophilized powder of the present invention, the product stability is enhanced, and the quality standard of the lyophilized product meets the requirements specified by the national drug standard.

[0038] Combined with the above embodiments, in the embodiments of the present invention, the temperature of each sublimation drying is further independently selected from -35°C to -2°C, and the vacuum degree is independently selected from 10 Pa to 20 Pa, and the total time of N times of sublimation drying is 10 hours to 54 hours.

[0039] It should be understood that the temperature selected for each sublimation drying in this embodiment can be any temperature value between -35°C and -2°C, as long as it is ensured that the sublimation drying temperature increases gradually. The vacuum degree selected for each sublimation drying in this embodiment is any vacuum degree value between 10 Pa and 20 Pa, as long as it is ensured that the vacuum degree of sublimation drying decreases gradually. In addition, the total time in this embodiment is the total time maintained during the N times of sublimation drying, which is 10 hours to 54 hours.

[0040] Due to the multiple sublimation dryings performed on the collagen lyophilized stock solution in the present invention, combined with the above embodiments, within the above sublimation drying temperature range, vacuum degree range, and total sublimation drying time, the embodiments of the present invention further limit the temperature difference between two adjacent sublimation dryings to 2°C to 16°C.

[0041] In some of these embodiments, the temperature increase time between two adjacent sublimation dryings is 0.5 h to 1 h.

[0042] In some of these embodiments, the vacuum degree difference between two adjacent sublimation dryings is 1 Pa to 3 Pa.

[0043] By limiting the temperature difference, heating-up time, and vacuum degree difference between two adjacent sublimation drying processes, it can be ensured that the sublimation drying process is precisely controlled under various process conditions, enabling collagen to maintain the integrity of its structure during sublimation drying and avoiding the collapse and deformation of collagen molecules, etc.

[0044] In some of these embodiments, the content of collagen in the collagen freeze-drying stock solution is 0.1 wt% - 1.0 wt%, and the balance is water.

[0045] In some of these embodiments, 1 < N ≤ 7; optionally, N can be 2, 3, 4, 5, 6, or 7.

[0046] For example, taking sublimation drying five times as an example, in the first stage of sublimation drying, the heat transfer oil temperature reaches -35°C to -33°C in 0.5 hours to 1 hour, the vacuum degree is maintained at 18 Pa to 20 Pa, and it is maintained for 15 hours to 20 hours; in the second stage, the heat transfer oil temperature reaches -31°C to -28°C in 0.5 hours to 1 hour, the vacuum degree is maintained at 15 Pa to 17 Pa, and it is maintained for 10 hours to 15 hours; in the third stage, the heat transfer oil temperature reaches -25°C to -20°C in 0.5 hours to 1 hour, the vacuum degree is maintained at 13 Pa to 14 Pa, and it is maintained for 5 hours to 8 hours; in the fourth stage, the heat transfer oil temperature reaches -18°C to -15°C in 0.5 hours to 1 hour, the vacuum degree is maintained at 11 Pa to 12 Pa, and it is maintained for 2 hours to 3 hours; in the fifth stage, the heat transfer oil temperature reaches -6°C to -2°C in 0.5 hours to 1 hour, the vacuum degree is maintained at 10 Pa to 11 Pa, and it is maintained for 5 hours to 8 hours.

[0047] It should be noted that in the above-mentioned sublimation drying stages, when specifically selecting the temperature and vacuum degree, ensure that the temperature of sublimation drying increases gradually from the first time to the fifth time, the vacuum degree of sublimation drying decreases gradually from the first time to the fifth time, and the temperature difference between two adjacent times is 2°C to 16°C and the vacuum degree difference is 1 Pa to 3 Pa.

[0048] Combined with the above embodiments, in the embodiments of the present invention, the temperature of each desorption drying is further independently selected from 0°C to 30°C, the vacuum degree is independently selected from 10 to 18 Pa, and the total time of M times of desorption drying is 10 to 30 hours.

[0049] It should be understood that in this embodiment, the temperature selected for each desorption drying can be any temperature value between 0°C and 30°C, as long as it is ensured that the desorption drying temperature increases gradually. The vacuum degree selected for each desorption drying in this embodiment is any vacuum degree value between 10 Pa and 18 Pa, as long as it is ensured that the vacuum degree of desorption drying decreases gradually or remains unchanged. The total time is 10 hours to 30 hours during the M times of desorption drying.

[0050] By limiting the temperature difference, heating-up time, and vacuum degree difference between two adjacent analytical drying processes, the analytical drying process can be further precisely controlled under various process conditions, further ensuring that the collagen can maintain the integrity of its structure during the analytical drying process, further avoiding the collapse and deformation of collagen molecules, etc., so that the finally prepared collagen lyophilized powder has a stable morphology, avoiding phenomena such as collapse, shrinkage, and bubbling, improving the product properties of the collagen lyophilized powder while enhancing the product stability, and the quality standard of the lyophilized product meets the requirements specified in the national drug standard.

[0051] Since the collagen lyophilized stock solution after sublimation drying in the present invention is further subjected to multiple analytical drying processes, in combination with the above embodiments, within the above analytical drying temperature range, vacuum degree range, and total analytical drying time, the present invention further limits the temperature difference between two adjacent analytical drying processes to be 5°C to 15°C.

[0052] In some embodiments, the heating-up time between two adjacent analytical drying processes is 0.5 h to 1 h.

[0053] In some embodiments, the vacuum degree difference between two adjacent analytical drying processes is 0 Pa to 3 Pa.

[0054] In some embodiments, 1 < M ≤ 7; optionally, M can be 2, 3, 4, 5, 6, or 7.

[0055] For example, taking three times of analytical drying as an example, the temperature of the heat transfer oil in the first stage of analytical drying reaches 0°C to 5°C in 0.5 h to 1 h, the vacuum degree is maintained at 15 Pa to 18 Pa, and it is maintained for 5 h to 8 h; the temperature of the heat transfer oil in the second stage reaches 10°C to 15°C in 0.5 h to 1 h, the vacuum degree is maintained at 12 Pa to 15 Pa, and it is maintained for 4 h to 6 h; the temperature of the heat transfer oil in the third stage reaches 20°C to 25°C in 0.5 h to 1 h, the vacuum degree is maintained at 10 Pa to 14 Pa, and it is maintained for 10 h to 13 h.

[0056] Furthermore, in combination with the above embodiments, after the analytical drying is completed in the embodiments of the present invention, a pressure rise test is further included, and the set pressure rise limit value in the pressure rise test is 3 Pa / min. Specifically, the pressure rise is set at 3 Pa / min, the pressure rise is qualified, the lyophilization ends, and the moisture content of the lyophilized powder is 4% to 5%.

[0057] To further illustrate the collagen lyophilized powder and its preparation method of the present invention, the present invention also provides the following specific embodiments. It should be understood that the raw materials used in the following embodiments are all commercially available raw materials unless otherwise specified, specifically as follows:

[0058] The collagen is selected as type A type III collagen.

[0059] Example 1

[0060] In this example, the preparation method of the collagen skin care lyophilized powder includes the following steps:

[0061] S1. Raw material preparation:

[0062] Place the collagen skin care lyophilized powder stock solution in a freeze-drying device. The content of collagen in the collagen skin care lyophilized powder stock solution is 0.1 wt%.

[0063] S2. Pre-freezing:

[0064] The temperature of the heat transfer oil reaches -40°C in 20 minutes and is maintained for 5 hours.

[0065] S3. Sublimation drying:

[0066] In the first stage, the temperature of the heat transfer oil reaches -35°C in 0.5 hours, the vacuum degree is maintained at 20 Pa, and it is maintained for 15 hours.

[0067] In the second stage, the temperature of the heat transfer oil reaches -31°C in 0.5 hours, the vacuum degree is maintained at 18 Pa, and it is maintained for 10 hours.

[0068] In the third stage, the temperature of the heat transfer oil reaches -25°C in 0.5 hours, the vacuum degree is maintained at 16 Pa, and it is maintained for 5 hours.

[0069] In the fourth stage, the temperature of the heat transfer oil reaches -18°C in 0.5 hours, the vacuum degree is maintained at 14 Pa, and it is maintained for 2 hours.

[0070] In the fifth stage, the temperature of the heat transfer oil reaches -6°C in 0.5 hours, the vacuum degree is maintained at 12 Pa, and it is maintained for 5 hours.

[0071] Step 3. Desorption drying:

[0072] In the first stage, the temperature of the heat transfer oil reaches 0°C in 0.5 hours, the vacuum degree is maintained at 18 Pa, and it is maintained for 5 hours.

[0073] In the second stage, the temperature of the heat transfer oil reaches 10°C in 0.5 hours, the vacuum degree is maintained at 15 Pa, and it is maintained for 4 hours.

[0074] In the third stage, the temperature of the heat transfer oil reaches 20°C in 0.5 hours, the vacuum degree is maintained at 12 Pa, and it is maintained for 10 hours.

[0075] Step 4. Pressure rise test:

[0076] Set the pressure rise to 3 Pa / min. The pressure rise is qualified, the freeze-drying is completed, and a white lyophilized powder product is obtained. The moisture content is 4.67%. The internal crystals of the product are relatively dense, the appearance is beautiful, and there are no obvious phenomena such as collapse, shrinkage, and bubbling.

[0077] Example 2

[0078] The preparation method of the collagen skin care lyophilized powder in this example includes the following steps:

[0079] S1. Raw material preparation:

[0080] Place the collagen skin care lyophilized powder stock solution in a freeze-drying device, and the collagen content in the collagen skin care lyophilized powder stock solution is 0.5 wt%.

[0081] S2. Pre-freezing:

[0082] The temperature of the heat transfer oil reaches -45°C in 40 minutes and is maintained for 4 hours;

[0083] S3. Sublimation drying:

[0084] In the first stage, the temperature of the heat transfer oil reaches -35°C in 1 hour, the vacuum degree is maintained at 18 Pa, and it is maintained for 10 hours;

[0085] In the second stage, the temperature of the heat transfer oil reaches -30°C in 1 hour, the vacuum degree is maintained at 15 Pa, and it is maintained for 15 hours;

[0086] In the third stage, the temperature of the heat transfer oil reaches -20°C in 1 hour, the vacuum degree is maintained at 12 Pa, and it is maintained for 8 hours;

[0087] In the fourth stage, the temperature of the heat transfer oil reaches -18°C in 1 hour, the vacuum degree is maintained at 11 Pa, and it is maintained for 3 hours;

[0088] In the fifth stage, the temperature of the heat transfer oil reaches -2°C in 1 hour, the vacuum degree is maintained at 10 Pa, and it is maintained for 4 hours;

[0089] Step 3. Desorption drying:

[0090] In the first stage, the temperature of the heat transfer oil reaches 0°C in 1 hour, the vacuum degree is maintained at 14 Pa, and it is maintained for 9 hours;

[0091] In the second stage, the temperature of the heat transfer oil reaches 20°C in 1 hour, the vacuum degree is maintained at 13 Pa, and it is maintained for 15 hours;

[0092] In the third stage, the temperature of the heat transfer oil reaches 30°C in 1 hour, the vacuum degree is maintained at 12 Pa, and it is maintained for 6 hours;

[0093] Step 4. Pressure rise test:

[0094] Set the pressure rise to 3 Pa / minute. The pressure rise is qualified, the freeze-drying is completed, and a white lyophilized powder product is obtained. The moisture content is 4.12%. The internal crystals of the product are relatively dense, the appearance is beautiful, and there are no obvious phenomena such as collapse, shrinkage, and bubbling.

[0095] Example 3

[0096] The preparation method of the collagen skin care lyophilized powder in this example includes the following steps:

[0097] S1. Raw material preparation:

[0098] Place the collagen skin care freeze-dried powder stock solution in a freeze-drying device. The collagen content in the collagen skin care freeze-dried powder stock solution is 0.3 wt%.

[0099] S2. Pre-freezing:

[0100] The temperature of the heat transfer oil reaches -40°C in 20 minutes and is maintained for 6 hours.

[0101] S3. Sublimation drying:

[0102] In the first stage, the temperature of the heat transfer oil reaches -33°C in 1 hour, the vacuum degree is maintained at 16 Pa, and it is maintained for 20 hours.

[0103] In the second stage, the temperature of the heat transfer oil reaches -28°C in 1 hour, the vacuum degree is maintained at 15 Pa, and it is maintained for 15 hours.

[0104] In the third stage, the temperature of the heat transfer oil reaches -20°C in 1 hour, the vacuum degree is maintained at 14 Pa, and it is maintained for 7 hours.

[0105] In the fourth stage, the temperature of the heat transfer oil reaches -15°C in 1 hour, the vacuum degree is maintained at 13 Pa, and it is maintained for 3 hours.

[0106] In the fifth stage, the temperature of the heat transfer oil reaches -4°C in 1 hour, the vacuum degree is maintained at 12 Pa, and it is maintained for 5 hours.

[0107] Step 3. Desorption drying:

[0108] In the first stage, the temperature of the heat transfer oil reaches 5°C in 1 hour, the vacuum degree is maintained at 14 Pa, and it is maintained for 8 hours.

[0109] In the second stage, the temperature of the heat transfer oil reaches 15°C in 1 hour, the vacuum degree is maintained at 14 Pa, and it is maintained for 6 hours.

[0110] In the third stage, the temperature of the heat transfer oil reaches 25°C in 1 hour, the vacuum degree is maintained at 14 Pa, and it is maintained for 13 hours.

[0111] Step 4. Pressure rise test:

[0112] Set the pressure rise to 3 Pa / minute. The pressure rise is qualified, the freeze-drying is completed, and a white freeze-dried powder product is obtained. The moisture content is 4.32%. The internal crystallization of the product is relatively dense, the appearance is beautiful, and there are no obvious phenomena such as collapse, shrinkage, and bubbling.

[0113] Example 4

[0114] The preparation method of the collagen skin care freeze-dried powder in this example includes the following steps:

[0115] S1. Raw material preparation:

[0116] Place the collagen skin care freeze-dried powder stock solution in a freeze-drying device. The content of collagen in the collagen skin care freeze-dried powder stock solution is 1.0 wt%.

[0117] S2. Pre-freezing:

[0118] The temperature of the heat-conducting oil reaches -42°C in 30 minutes and is maintained for 5 hours.

[0119] S3. Sublimation drying:

[0120] In the first stage, the temperature of the heat-conducting oil reaches -35°C in 0.5 hour, the vacuum degree is maintained at 16 Pa, and it is maintained for 25 hours.

[0121] In the second stage, the temperature of the heat-conducting oil reaches -20°C in 0.5 hour, the vacuum degree is maintained at 14 Pa, and it is maintained for 15 hours.

[0122] In the third stage, the temperature of the heat-conducting oil reaches -5°C in 0.5 hour, the vacuum degree is maintained at 12 Pa, and it is maintained for 10 hours.

[0123] Step 3. Desorption drying:

[0124] In the first stage, the temperature of the heat-conducting oil reaches 2°C in 0.5 hour, the vacuum degree is maintained at 12 Pa, and it is maintained for 8 hours.

[0125] In the second stage, the temperature of the heat-conducting oil reaches 20°C in 0.5 hour, the vacuum degree is maintained at 10 Pa, and it is maintained for 12 hours.

[0126] Step 4. Pressure rise test:

[0127] Set the pressure rise to 3 Pa / minute. The pressure rise is qualified, the freeze-drying is completed, and a white freeze-dried powder product is obtained. The moisture content is 4.89%. The internal crystallization of the product is relatively dense, the appearance is beautiful, and there are no obvious phenomena such as collapse, shrinkage, and bubbling.

[0128] Comparative Example 1

[0129] Compared with Example 1, in Comparative Example 1, the vacuum degree in each stage during sublimation drying is maintained at 16 Pa unchanged, and other conditions are the same as those in Example 1, and a freeze-dried powder E is obtained.

[0130] Comparative Example 2

[0131] Compared with Example 1, in Comparative Example 1, sublimation drying is carried out in four stages, and the vacuum degree in each stage increases successively. The vacuum degrees are as follows: the vacuum degree in the first stage is the ultimate vacuum degree, the vacuum degree in the second stage is maintained at 30 Pa, the vacuum degree in the third stage is maintained at 40 Pa, and the vacuum degree in the fourth stage is maintained at 50 Pa. Other conditions are the same as those in Example 1, and a freeze-dried powder F is obtained.

[0132] Comparative Example 3

[0133] Compared with Example 1, in Comparative Example 1, the analytical drying was carried out in three stages, and the vacuum degree increased successively in each stage. The vacuum degrees were as follows: the vacuum degree in the first stage was 12 Pa, the vacuum degree in the second stage was maintained at 15 Pa, and the vacuum degree in the third stage was maintained at 18 Pa. Other conditions were the same as those in Example 1, and the freeze-dried powder G was obtained.

[0134] Comparative Example 4

[0135] The components of the freeze-dried powder in this comparative example were as follows, by mass percentage, including 12% mannitol, 1% type A and type III collagen, and the balance was water.

[0136] S1. Heat the water to 120 °C and keep it warm for 30 min;

[0137] S2. Cool the water in S1 to 45 °C, and successively add mannitol and type A and type III collagen, and stir evenly to obtain a mixture.

[0138] S3. Take a sample of the mixture obtained in S2 for testing. After passing the test, fill it into a 5 mL vial and cover the vial cap as required.

[0139] S4. Put the vials obtained in S3 into the freeze dryer in turn, start freeze drying, and the freeze drying temperature program is as follows:

[0140] The first step: freeze at -40 °C for 4 h; the second step: evacuate to a vacuum degree of 40 mPa and maintain for 1 h; the third step: maintain at -30 °C for 1 h; the fourth step: maintain at -10 °C for 4 h; the fifth step: maintain at 10 °C for 4 h; the sixth step: maintain at 35 °C for 8 h.

[0141] S5. After the freeze drying is completed, press the inner plug tightly, take out the vial, and press the cap to obtain the components of the freeze-dried powder.

[0142] Test Example

[0143] 1. Sensory indicators

[0144] Take 2 g of the freeze-dried powder obtained in Examples 1 to 4 and Comparative Examples 1 to 4 respectively and dissolve them with 5 g of the solvent component, where the solvent component is mainly sterile water, and perform a cold and heat cycle stability test. First, perform a low-temperature test at (-8 ± 2) °C and keep it for 12 h, then heat it to (40 ± 1) °C for a high-temperature test and keep it for 12 h, and return to room temperature to complete one cycle. A total of 3 cycles are performed, and the state of the essence before and after the cycle is observed with the eyes. After returning to room temperature, no obvious change in properties compared with before the test is recorded as A; observing a small amount of turbidity is recorded as B, and observing partial turbidity or even the appearance of precipitates is recorded as C. The test results are shown in Table 1.

[0145] Table 1 Cold and heat cycle stability test results of freeze-dried powder

[0146]

[0147] 2. Collagen bioactivity test

[0148] (A) Using keratinocytes as the experimental subjects, the bioactivity of collagen in the freeze-dried powder was investigated. The specific experimental steps are as follows:

[0149] (1) Take human epidermal keratinocytes in good growth condition, digest the human epidermal keratinocytes with trypsin solution to prepare a cell suspension. The mass-volume concentration of the trypsin solution is 0.25% (indicating that 0.25 grams of trypsin is contained in every 100 milliliters of aqueous solution). Count the cells with a cell counting plate and adjust the cell density to 104 cells / mL. Uniformly inoculate the cell suspension into a 96-well cell culture plate, add 100 μL of the cell suspension to each well, place the culture plate in a cell culture incubator, and culture for 24 hours to allow the cells to adhere to the wall.

[0150] (2) Prepare the keratinocyte detection solution: Dilute the samples with sterile PBS in equal amounts to prepare the culture medium models for each group. Among them, the blank control group BC is a keratinocyte model without drug treatment and without specific culture medium; the positive control group PC is a keratinocyte culture medium without drug treatment and with a culture condition of containing 1 ng / mL of EGF; the sample group A is administered with the freeze-dried powder A of Example 1, with an administration concentration of 0.0025 mg / mL and a culture condition of a keratinocyte culture medium without EGF; the sample group B is administered with the freeze-dried powder A of Example 1, with an administration concentration of 0.025 mg / mL and a culture condition of a keratinocyte culture medium without EGF. Aspirate the culture medium in the 96-well plate, add 100 microliters of the above sample solution to each well, set 6 replicates for each sample, and place them in a 37°C, 5% CO2 incubator for continuous incubation for 24 h, 48 h, and 72 h.

[0151] (3) At each time point, add 20 μL of MTT solution (prepared with PBS at pH = 7.4, 5 mg / mL) to the corresponding wells, continue to incubate for 4 h, and terminate the culture.

[0152] (4) After the incubation is completed, discard the supernatant, add 150 μL of DMSO to each well, and shake for 10 min to completely dissolve the MTT blue-violet crystals.

[0153] (5) Use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance value (OD value) of each well at 490 nm, and use the absorbance value of the well with only the culture medium without inoculated cells as the blank control for zero adjustment. Take the average value of 6 wells. According to the OD values of each well, calculate and analyze the relative viability of keratinocytes in different samples with reference to the MTT colorimetric method to evaluate the bioactivity of collagen. Generally speaking, the higher the relative viability value, the stronger the cell proliferation ability and the higher the bioactivity of collagen. The cell proliferation results obtained from the test are shown in Table 2 and as Figure 1 shown.

[0154] Table 2 Bioactivity test results with keratinocytes as experimental subjects

[0155]

[0156] Note: When using the t-test method for statistical analysis, compared with the BC group, the significance in the PC group and the sample groups is indicated by *, where P-value < 0.05 is indicated by *, and P-value < 0.01 is indicated by **.

[0157] From Table 2 and Figure 1 the results of the line graph, it can be seen that: compared with the BC group, the cell proliferation rate of the PC group increased significantly, indicating that the positive control in this test was effective. While compared with BC, after culturing for 24 h, 48 h, and 72 h, the viability of keratinocytes in sample groups A and B showed a significant growth trend, almost comparable to that of the PC group, indicating that the collagen lyophilized powder of this application still maintained significant bioactivity.

[0158] (B) Using fibroblasts as experimental subjects, the bioactivity of collagen in the lyophilized powder was investigated. The specific experimental steps refer to the above (A), with the differences being: the blank control group BC was not medicated, and the culture condition was a culture medium containing 2% serum; the positive control group PC was not medicated, and the culture condition was a culture medium containing 10% serum; sample group C was administered with the lyophilized powder A of Example 1, with a dosing concentration of 0.00025 mg / mL and a culture condition of a culture medium containing 2% serum; sample group D was administered with the lyophilized powder A of Example 1, with a dosing concentration of 0.0025 mg / mL and a culture condition of a culture medium containing 2% serum; sample group E was administered with the lyophilized powder A of Example 1, with a dosing concentration of 0.0025 mg / mL and a culture condition of a culture medium containing 2% serum. Other conditions refer to step (A). The cell proliferation results obtained from the test are shown in Table 3 and Figure 2 as follows.

[0159] Table 3 Bioactivity test results with fibroblasts as experimental subjects

[0160]

[0161] Note: When using the t-test method for statistical analysis, compared with the BC group, the significance in the PC group and the sample groups is indicated by *, where P-value < 0.05 is indicated by *, and P-value < 0.01 is indicated by **.

[0162] From Table 3 and Figure 2It can be seen from the line chart that compared with the BC group, the cell proliferation rate of the PC group increased significantly, indicating that the positive control of this test was effective. Compared with the BC group, after 72 hours of culture, the fibroblast viability of sample group C was significant. After 48 hours of culture, the fibroblast viability of sample group D was significant. After 24 hours of culture, the fibroblast viability of sample group E was significant, indicating that the collagen freeze-dried powder of this application still maintained significant biological activity.

[0163] 3. Efficacy test

[0164] (1) Selection and grouping of volunteers

[0165] 180 healthy volunteers meeting the skin aging criteria were selected, including 18 males and 162 females, aged between 35 and 49 years old, with an average age of 40.05 years. None of them were allergic to cosmetics, had no acute facial inflammation, had not used hormonal drugs or immunosuppressants, and had not applied other topical preparations.

[0166] These 180 people were randomly divided into 9 groups, with 20 people in each group, and there were two males in each group. Among them, groups 1 to 8 were experimental groups, and group 9 was the control group. The main manifestations of skin aging were: dryness, roughness, desquamation, obvious wrinkle formation, and reduced elasticity.

[0167] (2) Test method

[0168] Experimental groups: Before use, 1 g of the freeze-dried powder of Examples 1-4 and Comparative Examples 1-4 was mixed evenly with 10 mL of solvent respectively, where the solvent component was sterile water. After complete dissolution, skin topical preparations were obtained respectively. After cleansing the face in the morning and evening, the prepared skin topical preparations were evenly applied to the facial skin and massaged for an appropriate time. The treatment course was 2 months.

[0169] Control group: Only the solvent was applied in the control group, and the usage method of the solvent was the same as that described for the experimental groups.

[0170] After continuous use for 2 months, a professional person scored the symptoms before and after treatment to evaluate the usage effect.

[0171] (3) Scoring criteria

[0172] 1) Wrinkles: The skin texture before and after using the product was measured by the fast optical imaging method for the skin (FOITS). Those with shallow fine crow's feet at the corners of the eyes and shallow wrinkles on the forehead were counted as 1 point; those with thick crow's feet at the corners of the eyes, on the forehead, and on the cheeks were counted as 2 points; those with obvious wrinkles at the corners of the eyes, on the forehead, on the cheeks, and around the mouth were counted as 3 points.

[0173] 2) Skin elasticity: 1 point if the skin can recover quickly after pressing but more skin wrinkles are produced around the pressed part; 2 points if the skin has poor elasticity and can recover within 1-2 seconds after pressing; 3 points if the skin has poor elasticity and can recover within more than 2 seconds after pressing.

[0174] 3) Roughness and dryness: If the skin of the face, forehead, or periorbital area is not smooth to the touch at one point, it will be scored as 1 point; if the skin of the cheek, forehead, or periorbital area is not smooth to the touch at two points, or if the skin of the cheek, forehead, or periorbital area is rough to the touch at one point, or if the skin of the cheek, forehead, or periorbital area is rough to the touch at one point, or if the skin of the cheek, forehead, or periorbital area is rough to the touch at more than one point, it will be scored as 3 points.

[0175] (4) Efficacy evaluation criteria

[0176] Symptom score improvement rate = (symptom score before treatment - symptom score after treatment) / score before treatment × 100%. Significantly effective: symptom score reduction ≥ 70%; effective: symptom score reduction ≥ 50%; ineffective: symptoms are not significantly improved or worsen, symptom score reduction < 50%. The results are shown in Table 4.

[0177] (5) Evaluation results

[0178] Table 4 Comparison of clinical symptom scores in different groups before and after treatment

[0179] Group Number of people Integral before use Integral after use Improvement rate Example 1 20 96 16 83.3％ Example 2 20 99 13 86.9％ Example 3 20 97 14 85.5％ Example 4 20 92 10 89.1％ Comparative example 1 20 92 45 51.1％ Comparative example 2 20 93 60 35.5％ Comparative example 3 20 94 43 54.2％ Comparative example 4 20 91 30 67.0％ Control group 20 97 83 14.4％

[0180] From the test results in Table 4, it can be seen that the trial use of the collagen freeze-dried powder in Examples 1 to 4 has obvious effects on improving skin wrinkles, skin elasticity, roughness and dryness, with the improvement rate reaching more than 80%, and the effect is obvious.

[0181] The improvement rate of the collagen freeze-dried powder in Comparative Example 1 is only 51.12%, and the improvement rate of the collagen freeze-dried powder in Comparative Example 3 is only 54.2%. Compared with Examples 1 to 4, the improvement rate is significantly reduced. The reason may be that during the sublimation drying or analytical drying process, there is no skeleton support in the raw materials for the preparation of the collagen freeze-dried powder, and the vacuum degree remains unchanged or increases gradually, which will cause the prepared collagen freeze-dried powder to collapse and deform, affecting its use effect.

[0182] The improvement rate of the collagen freeze-dried powder in Comparative Example 2 was only 35.5%, and there was no obvious improvement in the symptoms. The reason may be that the vacuum degree of sublimation drying in Comparative Example 2 was gradually increased from the ultimate vacuum degree to 50 Pa. The excessive vacuum degree affected the molecular structure of the collagen freeze-dried powder, thereby affecting its molecular activity and greatly reducing its use effect.

[0183] In Comparative Example 4, the freeze-dried collagen powder prepared by the preparation process in the prior art may cause the skin's absorption and utilization rate of the active ingredient collagen because its composition contains the skeletal structure mannitol. Its skin improvement rate can only reach nearly 67%, which is much lower than the skin improvement rate of the freeze-dried collagen powder in Examples 1 to 4 of this application.

[0184] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or perform equivalent replacements on some of the technical features. Any modifications, equivalent replacements, improvements, 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 freeze-dried powder, characterized in that: The invention comprises collagen and water, wherein the water content of the collagen freeze-dried powder is 4wt% to 5wt%, and the remainder is collagen.

2. A method for preparing freeze-dried collagen powder, applied to the preparation of freeze-dried collagen powder in claim 1, characterized in that: The following steps are involved: preparing a lyophilized collagen solution, wherein the lyophilized collagen solution comprises collagen and water; The freeze-dried collagen stock solution is gradually cooled to -40°C to -45°C and pre-frozen for 4 to 6 hours; Sublimation drying is performed N times on the pre-frozen collagen freeze-dried stock solution, wherein N is an integer greater than 1, the temperature of the sublimation drying is gradually increased from the first time to the Nth time, and the vacuum degree of the sublimation drying is gradually decreased from the first time to the Nth time; The collagen freeze-dried stock solution after sublimation drying is subjected to M times of analytical drying, wherein M is an integer greater than 1, the temperature of the analytical drying is gradually increased from the first time to the Mth time, and the vacuum degree of the analytical drying is gradually decreased or remains unchanged from the first time to the Mth time, so as to obtain collagen freeze-dried powder.

3. The preparation method according to claim 2, characterized in that: The temperature of each sublimation drying is independently selected from -35°C to -2°C, the vacuum degree is independently selected from 10 Pa to 20 Pa, and the total time of N times of sublimation drying is 10 hours to 54 hours.

4. The preparation method according to claim 2 or 3, characterized in that: The temperature difference between two adjacent sublimation dryings is 2°C to 16°C.

5. The preparation method according to any one of claims 2 to 4, characterized in that: During the sublimation drying, the temperature rising time between two adjacent sublimation dryings is 0.5h to 1h.

6. The preparation method according to any one of claims 2 to 5, characterized in that: The difference in vacuum between two adjacent sublimation dryings is 1 Pa to 3 Pa.

7. The preparation method according to claim 2, characterized in that: The temperature of each analytical drying is independently selected from 0°C to 30°C, the vacuum degree is independently selected from 10 Pa to 18 Pa, and the total time of M analytical dryings is 10 hours to 30 hours.

8. The preparation method according to claim 7, characterized in that: The temperature difference between two adjacent drying steps is 5°C to 15°C; and / or, During the analytical drying, the temperature rise time between two adjacent analytical dryings is 0.5h to 1h; and / or, During the analytical drying, the difference in vacuum between two adjacent analytical dryings is 0 Pa to 3 Pa.

9. The preparation method according to claim 2, characterized in that: The content of collagen in the freeze-dried collagen stock solution is 0.1wt%-1.0wt%, and the balance is water; and / or 1<N≤7; and / or 1＜M≤7。 10. The preparation method according to claim 2, characterized in that: After the analytical drying is completed, a pressure rise test is also performed. If the pressure rise is qualified, the freeze drying is completed. The pressure rise limit value set in the pressure rise test is 3 Pa / min.

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