Glycosylated donkey-hide gelatin with vascular endothelial cell damage repairing effect and preparation method thereof

Abstract

The present invention relates to a glycosylated donkey-hide gelatin having a vascular endothelial cell damage repairing effect and a preparation method, comprising the following steps: S1, soaking donkey skin and rinsing with clean water until the donkey skin fully absorbs water, is white in color, soft in texture and the soaking water is clean; S2, removing impurities from the donkey skin cleaned in the step S1; S3, weighing the clean donkey skin in step S2 and putting it into a steam spheronization machine for collagen extraction and obtaining a collagen solution; S4, the collagen solution in step S3 forms a concentrated gelatin solution; S5, compounding the concentrated gelatin solution in step S4 with a syrup solution, reacting to obtain a glycosylated gelatin solution; S6, placing the glycosylated gelatin in step S5 in a concentration tank for secondary concentration to obtain a glycosylated donkey-hide gelatin finished product. The advantages of the present invention are: obtaining a glycosylated donkey-hide gelatin solution with mild preparation conditions, high safety and good vascular endothelial cell damage repairing activity in the present invention. The glycosylated donkey-hide gelatin prepared by the method is significantly better than its substrate in terms of the damage repairing effect on vascular endothelial cells HUVEC, and is also significantly better than the donkey-hide gelatin product obtained by conventional production processes.

Description

Technical Field

[0001] The invention relates to glycosylated donkey-hide gelatin with vascular endothelial cell damage repairing function and a preparation method thereof, belonging to the technical field of collagen preparation. Background Art

[0002] With the increasing aging of the population in today's society, the incidence of vascular diseases such as coronary heart disease and hypertension is increasing year by year. Therefore, the research on damage to vascular endothelial cell function, which is closely related to diseases such as hypertension, hyperlipidemia, and aging, has gradually attracted people's attention.

[0003] Endothelial cells are a layer of flat polymorphic cells attached to the inner wall of blood vessels. They can form a new vascular network through budding, migration, proliferation, lumen formation and mutual fusion. They are the main participants in blood vessel formation. Vascular endothelial cells participate in regulating a variety of vascular biological functions, such as regulating blood pressure and inflammatory response, participating in the formation of thrombosis and atherosclerosis, etc. Endothelial cell damage can not only promote the formation of local vascular thrombosis, but also release vasoactive molecules such as angiotensin-converting enzyme, endothelin, endothelial cell growth factor, adhesion molecules, etc., promote vascular contraction, and then initiate and cause a series of post-injury reactions, leading to the occurrence of cardiovascular diseases such as coronary heart disease and hypertension. Endothelial cell injury factors mainly include lipopolysaccharide, inflammatory mediators, free radicals, etc. These factors can directly cause dysfunction and aggravate leukocyte-mediated inflammatory pathological damage when acting on endothelial cells. Therefore, it is of great significance to exert a protective effect on endothelial cells that are about to be damaged.

[0004] At present, human umbilical vein endothelial cells (HUVECs) are one of the most important tool cells for cardiovascular diseases. H2O2 is a type of reactive oxygen species that can directly oxidize lipids and proteins on the cell membrane, and react with intracellular iron ions through the cell membrane to generate more active free radicals, leading to a series of oxidative damage reactions.

[0005] As the main structural component of ECM, collagen participates in various biological processes such as tissue and organ morphogenesis, metabolic renewal, and injury repair. The triple helix structure of collagen molecules denatures under the action of factors such as enzymatic hydrolysis and heat, and the molecular structure changes and depolymerizes, thereby exposing the biologically active sites and hidden sites inside the molecule and generating new functions. Studies have reported that denatured type I collagen can promote endothelial cell proliferation and migration, and promote the release of angiogenesis-related enzymes and cytokines. Collagen is also one of the main components of donkey-hide gelatin. At different stages of the extraction process of donkey-hide gelatin, the molecular weight and molecular structure of collagen in donkey-hide gelatin solution are constantly changing with processing, and its effect on endothelial cells may also change accordingly.

[0006] Therefore, in order to verify the role of the prepared animal collagen and its complexes in protecting vascular endothelial cells, their effects on HUVECs cell proliferation activity and cell migration ability were studied, and an oxidative damage HUVECs cell model was established. The MDA, LDH, GSH-Px, SOD and CAT enzyme activities, and the expression level of vascular endothelial growth factor (VEGF) protein were detected to explore their effects on vascular endothelial cell function, thereby providing a basis for further studying the role of donkey-hide gelatin in improving microcirculation, preventing hypertension, coronary heart disease and other vascular diseases. Summary of the invention

[0007] In order to overcome the defects of the prior art, the present invention provides a method for preparing glycosylated donkey-hide gelatin having the function of repairing vascular endothelial cell damage and the glycosylated donkey-hide gelatin. The technical scheme of the present invention is:

[0008] A method for preparing glycosylated donkey-hide gelatin having the function of repairing vascular endothelial cell damage comprises the following steps:

[0009] S1. The donkey hide is completely immersed in a 1% sodium carbonate aqueous solution and rinsed with clean water until the donkey hide fully absorbs water, becomes white, soft, and the soaking water is clean;

[0010] S2, the donkey skin after cleaning in described step S1 is subjected to impurity removal treatment to obtain clean donkey skin;

[0011] S3, weighing the clean donkey hide in step S2 and putting it into a spheronization machine to extract collagen and obtain a collagen solution;

[0012] S4, filtering and centrifuging the collagen solution in step S3 and transferring it into a foaming machine for collagen purification to form a concentrated collagen solution;

[0013] S5, compounding the concentrated gelatin solution in step S4 with the syrup solution, and reacting them to obtain a glycosylated gelatin solution;

[0014] S6, placing the saccharified gelatin solution in step S5 in a concentration tank for secondary concentration to obtain the saccharified donkey-hide gelatin product.

[0015] The step S1 is specifically as follows: the donkey skin is completely immersed in a 1% sodium carbonate aqueous solution at 15-25° C. for 6-10 hours, and then soaked in clean water for 4-8 days, during which the water is changed once every 12 hours, until the donkey skin fully absorbs water, becomes white in color, becomes soft in texture, and the pH value of the soaking water does not exceed 8.0.

[0016] The step S2 is specifically as follows: firstly, the neck, legs and tail of the donkey skin are removed to obtain the belly and back skin, and then the grease and donkey meat impurities attached to the belly and back skin are removed by a meat remover; then, the processed belly and back skin is cut into leather pieces with a width of 10-15 cm by a skin cutter, and 3-5 times the amount of clean water is added to rinse 3-5 times, each time washing the skin for 30-40 minutes, until the turbidity value of the washing water does not exceed 240NTU, and the leather pieces are taken out and the water is removed by a water squeezer.

[0017] The step S3 is specifically as follows: placing a donkey hide block in a spheronizer, adding a 2 mol / L Na2CO3 solution preheated at 60°C in a ratio of 1:3-1:5 (m / w) to the weight of the donkey hide, adjusting the temperature of the spheronizer to 60°C-80°C, blanching for 60min-90min to fully remove fat from the donkey hide, discarding the blanching water, and then rinsing the donkey hide with drinking water to fully remove residual alkali solution, each rinsing temperature is 50-60°C, the rinsing time is 10-15min, and the rinsing is repeated at least 5 times until the water is clear after rinsing and the pH is not higher than 8.0.

[0018] The step S4 is specifically as follows: adding drinking water according to a volume ratio of 1:2-1:4 (m / v) of the weight of the clean donkey hide block to water, and maintaining a steam pressure of 0.1-0.2MPa for slight boiling extraction for 18-20h; after collecting the first glue solution, adding water according to a ratio of 1:2-1:3 (m / v) of the weight of the donkey hide block, and maintaining a steam pressure of 0.1-0.15MPa for slight boiling extraction for 3-5h; collecting the glue solution for the second time, adding water according to a ratio of 1:1-1:2 (m / v) of the weight of the donkey hide block, and maintaining a steam pressure of 0.1-0.15MPa for slight boiling extraction for 1-3h; collecting the glue solution for the third time, extracting by stepwise pressure reduction three times, so that the collagen in the donkey hide is fully dissolved in batches; combining the glue solutions collected three times to obtain a mixed glue solution.

[0019] The step S5 is specifically as follows: filtering the mixed glue solution with a 120-mesh sieve, placing the filtrate in a foaming machine for boiling and concentration, during which insoluble proteins and impurities are removed with a foaming hook, until the moisture content of the glue solution is 50%-60%, thereby obtaining a concentrated glue solution.

[0020] The step S6 is specifically as follows: the syrup is prepared by mixing rock sugar and fructose in any proportion, precipitating for about 2 hours, and filtering through a 200-mesh sieve; compounding according to a mass ratio of concentrated gum to syrup of 10:1-12:1, stirring evenly, undergoing Maillard reaction at 80-90°C for 2-4 hours, and filtering through a 120-mesh sieve to obtain a glycosylated gum.

[0021] The step S7 is specifically as follows: the glycosylated gelatin solution is placed in a concentration tank for boiling and dynamic concentration is continued for 1-3 hours until the water content reaches 20%-40%, thereby obtaining a glycosylated donkey-hide gelatin product.

[0022] A glycosylated donkey-hide gelatin is prepared by a method for preparing glycosylated donkey-hide gelatin with vascular endothelial cell damage repairing function.

[0023] The advantages of the present invention are as follows: the method first processes the donkey skin, and through condition control, the directional extraction preparation process of the collagen is screened, and after batch extraction, a glycosylated donkey-hide gelatin solution is obtained, and then the glycosylated donkey-hide gelatin solution is used as a substrate to carry out a glycosylation reaction, and the vascular endothelial cell damage repair effect is used as a screening index to optimize the glycosylation preparation process, thereby obtaining the glycosylated donkey-hide gelatin solution with mild preparation conditions, high safety, and good vascular endothelial cell damage repair activity in the present invention. The glycosylated donkey-hide gelatin prepared by the method is significantly better than its substrate in terms of the damage repair effect on vascular endothelial cells HUVEC, and is also significantly better than products obtained by other glycosylation processes. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a schematic diagram comparing the transflectance of the glycosylated donkey-hide gelatin of the present invention and commercially available donkey-hide gelatin.

[0025] Figure 2 This is a graph showing the effect of the glycosylated donkey-hide gelatin solution of the present invention on the migration rate of HUVECs cells. DETAILED DESCRIPTION

[0026] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0027] Example 1: A method for preparing glycosylated donkey-hide gelatin having the function of repairing vascular endothelial cell damage, comprising the following steps:

[0028] S1. The donkey hide is completely immersed in a 1% sodium carbonate aqueous solution and rinsed with clean water until the donkey hide fully absorbs water, becomes white, soft, and the soaking water is clean;

[0029] S2, the donkey skin after cleaning in described step S1 is subjected to impurity removal treatment to obtain clean donkey skin;

[0030] S3, weighing the clean donkey hide in step S2 and putting it into a spheronization machine to extract collagen and obtain a collagen solution;

[0031] S4, filtering and centrifuging the collagen solution in step S3 and transferring it into a foaming machine for collagen purification to form a concentrated collagen solution;

[0032] S5, compounding the concentrated gelatin solution in step S4 with the syrup solution, and reacting them to obtain a glycosylated gelatin solution;

[0033] S6, placing the saccharified gelatin solution in step S5 in a concentration tank for secondary concentration to obtain the saccharified donkey-hide gelatin product.

[0034] The step S1 is specifically as follows: the donkey skin is completely immersed in a 1% sodium carbonate aqueous solution at 15° C. for 10 hours, and then soaked in clean water for 8 days, during which the water is changed once every 12 hours, until the donkey skin fully absorbs water, becomes white, and becomes soft, and the pH value of the soaking water does not exceed 8.0;

[0035] The step S2 is specifically as follows: first, the neck, legs and tail of the donkey skin are removed to obtain the belly and back skin, and then the grease and donkey meat impurities attached to the belly and back skin are removed by a fleshing machine; the processed belly and back skin is cut into leather pieces with a width of 10 cm by a skin cutting machine, and washed 3-5 times with 3 times the amount of clean water, each washing time is 30-40 minutes, until the turbidity value of the washing water does not exceed 240NTU, the leather pieces are taken out and the water is removed by a water squeezer.

[0036] The step S3 is specifically as follows: placing a donkey hide block in a spheronizer, adding a 2 mol / L Na2CO3 solution preheated at 60°C in a ratio of 1:3 (m / w) to the weight of the donkey hide, adjusting the temperature of the spheronizer to 60°C, blanching for 60 minutes to fully remove fat from the donkey hide, discarding the blanching water, and then rinsing the donkey hide with drinking water to fully remove residual alkali solution, each rinsing temperature is 50°C, the rinsing time is 10 minutes, and the rinsing is repeated 6 times until the water is clear after rinsing and the pH is not higher than 8.0.

[0037] The step S4 is specifically as follows: adding drinking water according to the volume ratio of the clean donkey hide block to water of 1:2 (m / v), and maintaining the steam pressure of 0.1 MPa for micro-boiling extraction for 18 hours; after collecting the first glue solution, adding water according to the ratio of 1:2 (m / v) of the weight of the donkey hide block, and maintaining the steam pressure of 0.1 MPa for micro-boiling extraction for 3 hours; collecting the glue solution for the second time, adding water according to the ratio of 1:1 (m / v) of the weight of the donkey hide block, and maintaining the steam pressure of 0.1 MPa for micro-boiling extraction for 1 hour; collecting the glue solution for the third time, and extracting by stepwise pressure reduction three times so that the collagen in the donkey hide is fully dissolved in batches; combining the glue solutions collected three times to obtain a mixed glue solution.

[0038] The step S5 is specifically as follows: filtering the mixed glue solution with a 120-mesh sieve, placing the filtrate in a foaming machine for boiling and concentration, during which insoluble proteins and impurities are removed with a foaming hook, until the moisture content of the glue solution reaches 50%, thereby obtaining a concentrated glue solution.

[0039] The step S6 is specifically as follows: the syrup is prepared by mixing rock sugar and fructose in any proportion, precipitating for about 2 hours, and filtering through a 200-mesh sieve; compounding according to a mass ratio of concentrated gum to syrup of 10:1, stirring evenly, undergoing Maillard reaction at 80°C for 2 hours, and filtering through a 120-mesh sieve to obtain a glycosylated gum.

[0040] The step S7 is specifically as follows: the glycosylated gelatin solution is placed in a concentration tank for boiling and dynamic concentration is continued for 1 hour until the water content reaches 20%, thereby obtaining a glycosylated donkey-hide gelatin product.

[0041] Example 2: A method for preparing glycosylated donkey-hide gelatin having the function of repairing vascular endothelial cell damage, comprising the following steps:

[0042] S1. The donkey hide is completely immersed in a 1% sodium carbonate aqueous solution and rinsed with clean water until the donkey hide fully absorbs water, becomes white, soft, and the soaking water is clean;

[0043] S2, the donkey skin after cleaning in described step S1 is subjected to impurity removal treatment to obtain clean donkey skin;

[0044] S3, weighing the clean donkey hide in step S2 and putting it into a spheronization machine to extract collagen and obtain a collagen solution;

[0045] S4, filtering and centrifuging the collagen solution in step S3 and transferring it into a foaming machine for collagen purification to form a concentrated collagen solution;

[0046] S5, compounding the concentrated gelatin solution in step S4 with the syrup solution, and reacting them to obtain a glycosylated gelatin solution;

[0047] S6, placing the glycosylated gelatin in step S5 in a concentration tank for secondary concentration to obtain a glycosylated donkey-hide gelatin product.

[0048] The step S1 is specifically as follows: the donkey skin is completely immersed in a 1% sodium carbonate aqueous solution at 25° C. for 6 hours, and then soaked in clean water for 4 days, during which the water is changed once every 12 hours, until the donkey skin fully absorbs water, becomes white in color, becomes soft in texture, and the pH value of the soaking water does not exceed 8.0.

[0049] The step S2 is specifically as follows: first, the neck, legs and tail of the donkey skin are removed to obtain the belly and back skin, and then the grease and donkey meat impurities attached to the belly and back skin are removed by a fleshing machine; the processed belly and back skin is cut into leather blocks with a width of 15 cm by a skin cutting machine, and washed with 5 times the amount of clean water for 5 times, each time for 40 minutes, until the turbidity value of the washing water does not exceed 240NTU, the leather blocks are taken out and the water is removed by a water squeezer.

[0050] The step S3 is specifically as follows: placing a donkey hide block in a spheronizer, adding a 2 mol / L Na2CO3 solution preheated at 60°C in a ratio of 1:5 (m / w) to the weight of the donkey hide, adjusting the temperature of the spheronizer to 80°C, blanching the donkey hide for 60-90 minutes to fully remove fat from the donkey hide, discarding the blanching water, and then rinsing the donkey hide with drinking water to fully remove residual alkali solution, each rinsing temperature is 50°C, the rinsing time is 15 minutes, and the rinsing is repeated 8 times until the water is clear after rinsing and the pH is not higher than 8.0.

[0051] The step S4 is specifically as follows: adding drinking water according to the volume ratio of the weight of the clean donkey hide block to water of 1:4 (m / v), and maintaining the steam pressure of 0.2MPa for micro-boiling extraction for 20 hours; after collecting the first glue solution, adding water according to the ratio of 1:3 (m / v) of the weight of the donkey hide block, and maintaining the steam pressure of 0.15MPa for micro-boiling extraction for 5 hours; collecting the glue solution for the second time, adding water according to the ratio of 1:2 (m / v) of the weight of the donkey hide block, and maintaining the steam pressure of 0.15MPa for micro-boiling extraction for 3 hours; collecting the glue solution for the third time, and extracting by stepwise pressure reduction three times so that the collagen in the donkey hide is fully dissolved in batches; combining the glue solutions collected three times to obtain a mixed glue solution.

[0052] The step S5 is specifically as follows: filtering the mixed glue solution with a 120-mesh sieve, placing the filtrate in a foaming machine for boiling and concentration, during which insoluble proteins and impurities are removed with a foaming hook, until the moisture content of the glue solution reaches 55%, thereby obtaining a concentrated glue solution.

[0053] The step S6 is specifically as follows: the syrup is prepared by mixing rock sugar and fructose in any proportion, precipitating for about 2 hours, and filtering through a 200-mesh sieve; compounding according to a mass ratio of concentrated gum to syrup of 12:1, stirring evenly, undergoing Maillard reaction at 90°C for 4 hours, and filtering through a 120-mesh sieve to obtain a glycosylated gum.

[0054] The step S7 is specifically as follows: the glycosylated gelatin solution is placed in a concentration tank for boiling and dynamic concentration is continued for 3 hours until the water content reaches 40%, thereby obtaining a glycosylated donkey-hide gelatin product.

[0055] Example 3: A method for preparing glycosylated donkey-hide gelatin having the function of repairing vascular endothelial cell damage, comprising the following steps:

[0056] S1. The donkey hide is completely immersed in a 1% sodium carbonate aqueous solution and rinsed with clean water until the donkey hide fully absorbs water, becomes white, soft, and the soaking water is clean;

[0057] S2, the donkey skin after cleaning in described step S1 is subjected to impurity removal treatment to obtain clean donkey skin;

[0058] S3, weighing the clean donkey hide in step S2 and putting it into a spheronization machine to extract collagen and obtain a collagen solution;

[0059] S4, filtering and centrifuging the collagen solution in step S3 and transferring it into a foaming machine for collagen purification to form a concentrated collagen solution;

[0060] S5, compounding the concentrated gelatin solution in step S4 with the syrup solution, and reacting them to obtain a glycosylated gelatin solution;

[0061] S6, placing the saccharified gelatin solution in step S5 in a concentration tank for secondary concentration to obtain the saccharified donkey-hide gelatin product.

[0062] The step S1 is specifically as follows: the donkey skin is completely immersed in a 1% sodium carbonate aqueous solution at 15-25° C. for 8 hours, and then soaked in clean water for 6 days, during which the water is changed once every 12 hours, until the donkey skin fully absorbs water, becomes white and soft, and the pH value of the soaking water does not exceed 8.0.

[0063] The step S2 is specifically as follows: first, the neck, legs and tail of the donkey skin are removed to obtain the belly and back skin, and then the grease and donkey meat impurities attached to the belly and back skin are removed by a fleshing machine; the processed belly and back skin is cut into leather blocks with a width of 12 cm by a skin cutting machine, and washed with 4 times the amount of clean water for 4 times, each time for 35 minutes, until the turbidity value of the washing water does not exceed 240NTU, the leather blocks are taken out and the water is removed by a water squeezer.

[0064] The step S3 is specifically as follows: placing a donkey hide block in a spheronizer, adding a 2 mol / L Na2CO3 solution preheated at 60°C in a ratio of 1:4 (m / w) to the weight of the donkey hide, adjusting the temperature of the spheronizer to 70°C, blanching for 80 minutes at a constant temperature to fully remove fat from the donkey hide, discarding the blanching water, and then rinsing the donkey hide with drinking water to fully remove residual alkali solution, each rinsing temperature is 55°C, the rinsing time is 13 minutes, and the rinsing is repeated 5 times until the water is clear after rinsing and the pH is not higher than 8.0.

[0065] The step S4 is specifically as follows: adding drinking water according to the volume ratio of the clean donkey hide block weight to water of 1:3 (m / v), and maintaining the steam pressure of 0.2MPa for slight boiling extraction for 18-20h; after collecting the first glue solution, adding water according to the ratio of 1:2.5 (m / v) of the hide weight, and maintaining the steam pressure of 0.1-0.15MPa for slight boiling extraction for 3-5h; collecting the glue solution for the second time, adding water according to the ratio of 1:1.5 (m / v) of the donkey hide block weight, and maintaining the steam pressure of 0.12MPa for slight boiling extraction for 2h; collecting the glue solution for the third time, and extracting by stepwise pressure reduction three times so that the collagen in the donkey hide is fully dissolved in batches; combining the glue solutions collected three times to obtain a mixed glue solution.

[0066] The step S5 is specifically as follows: filtering the mixed glue solution with a 120-mesh sieve, placing the filtrate in a foaming machine for boiling and concentration, during which insoluble proteins and impurities are removed with a foaming hook, until the moisture content of the glue solution is 50%-60%, thereby obtaining a concentrated glue solution.

[0067] The step S6 is specifically as follows: the syrup is prepared by mixing rock sugar and fructose in any proportion, precipitating for about 2 hours, and filtering through a 200-mesh sieve; compounding according to a mass ratio of concentrated gum to syrup of 11:1, stirring evenly, undergoing Maillard reaction at 85°C for 3 hours, and filtering through a 120-mesh sieve to obtain a glycosylated gum.

[0068] The step S7 is specifically as follows: the glycosylated gelatin solution is placed in a concentration tank for boiling and dynamic concentration is continued for 2 hours until the water content reaches 35%, thereby obtaining a glycosylated donkey-hide gelatin product.

[0069] Example 4: A method for preparing glycosylated donkey-hide gelatin having the function of repairing vascular endothelial cell damage, comprising the following steps:

[0070] S1. The donkey hide is completely immersed in a 1% sodium carbonate aqueous solution and rinsed with clean water until the donkey hide fully absorbs water, becomes white, soft, and the soaking water is clean;

[0071] S2, the donkey skin after cleaning in described step S1 is subjected to impurity removal treatment to obtain clean donkey skin;

[0072] S3, weighing the clean donkey hide in step S2 and putting it into a spheronization machine to extract collagen and obtain a collagen solution;

[0073] S4, filtering and centrifuging the collagen solution in step S3 and transferring it into a foaming machine for collagen purification to form a concentrated collagen solution;

[0074] S5, compounding the concentrated gelatin solution in step S4 with the syrup solution, and reacting them to obtain a glycosylated gelatin solution;

[0075] S6, placing the saccharified gelatin solution in step S5 in a concentration tank for secondary concentration to obtain the saccharified donkey-hide gelatin product.

[0076] The step S1 is specifically as follows: the donkey skin is completely immersed in a 1% sodium carbonate aqueous solution at 23° C. for 9 hours, and then soaked in clean water for 7 days, during which the water is changed once every 12 hours, until the donkey skin fully absorbs water, becomes white in color, becomes soft in texture, and the pH value of the soaking water does not exceed 8.0.

[0077] The step S2 is specifically as follows: first, the neck, legs and tail of the donkey skin are removed to obtain the belly and back skin, and then the grease and donkey meat impurities attached to the belly and back skin are removed by a fleshing machine; the processed belly and back skin is cut into leather pieces with a width of 10-15 cm by a skin cutting machine, and washed with 5 times the amount of clean water for 5 times, each time for 36 minutes, until the turbidity value of the washing water does not exceed 240NTU, the leather pieces are taken out and the water is removed by a water squeezer.

[0078] The step S3 is specifically as follows: placing a donkey hide block in a spheronizer, adding a 2 mol / L Na2CO3 solution preheated at 60°C in a ratio of 1:3 (m / w) to the weight of the donkey hide, adjusting the temperature of the spheronizer to 75°C, blanching for 85 minutes to fully remove fat from the donkey hide, discarding the blanching water, and then rinsing the donkey hide with drinking water to fully remove residual alkali solution, each rinsing temperature is 57°C, the rinsing time is 12 minutes, and the rinsing is repeated at least 5 times until the water is clear after rinsing and the pH is not higher than 8.0.

[0079] The step S4 is specifically as follows: adding drinking water according to the volume ratio of the clean donkey hide block weight to water of 1:2 (m / v), and maintaining the steam pressure of 0.2MPa for micro-boiling extraction for 20 hours; after collecting the first glue solution, adding water according to the ratio of 1:3 (m / v) of the weight of the donkey hide block, and maintaining the steam pressure of 0.1-0.15MPa for micro-boiling extraction for 5 hours; collecting the glue solution for the second time, adding water according to the ratio of 1:2 (m / v) of the weight of the donkey hide block, and maintaining the steam pressure of 0.15MPa for micro-boiling extraction for 3 hours; collecting the glue solution for the third time, and extracting by stepwise pressure reduction three times so that the collagen in the donkey hide is dissolved in batches; combining the glue solutions collected three times to obtain a mixed glue solution.

[0080] The step S5 is specifically as follows: filtering the mixed glue solution with a 120-mesh sieve, placing the filtrate in a foaming machine for boiling and concentration, during which insoluble proteins and impurities are removed with a foaming hook, until the moisture content of the glue solution reaches 57%, thereby obtaining a concentrated glue solution.

[0081] The step S6 is specifically as follows: the syrup is prepared by mixing rock sugar and fructose in any proportion, precipitating for about 2 hours, and filtering through a 200-mesh sieve; compounding according to a mass ratio of concentrated gum to syrup of 12:1, stirring evenly, undergoing Maillard reaction at 86°C for 3 hours, and filtering through a 120-mesh sieve to obtain a glycosylated gum.

[0082] The step S7 is specifically as follows: the glycosylated gelatin solution is placed in a concentration tank for boiling and dynamic concentration is continued for 3 hours until the water content reaches 20%, thereby obtaining a glycosylated donkey-hide gelatin product.

[0083] The present invention also relates to a glycosylated donkey-hide gelatin, which is prepared by a method for preparing a glycosylated donkey-hide gelatin having a vascular endothelial cell damage repairing function.

[0084] Analysis of component differences: The spectrum of donkey-hide gelatin was scanned by near-infrared spectrometer, and the similarity of the components of glycosylated donkey-hide gelatin and commercially available donkey-hide gelatin was evaluated based on the similarity of the spectra. The scanning conditions are as follows: Equipment: Antaris II FT-NIR spectrometer (Thermo Fisher, USA); Test conditions: 128 scans in the optical fiber probe transflection spectrum acquisition mode, resolution: 4cm-1, spectral range: 4000-10000cm-1, spectrum acquisition temperature: 25°C. Sample preparation: Take a sample of donkey-hide gelatin block and use water to prepare a gelatin solution with a solid content concentration of 40%. Using 10 batches of glycosylated donkey-hide gelatin samples, the transflection spectra were collected and combined with discriminant analysis to establish a qualitative analysis model, see Figure 1 .

[0085] Comparative determination of glycosylated donkey-hide gelatin and commercial donkey-hide gelatin, see Figure 2 As shown in the figure, the similarity of glycosylated donkey-hide gelatin (sample 1) is greater than 90%, and the quality is stable and reliable, while the similarity of commercial donkey-hide gelatin (sample 2) is less than 80%, and its composition is different from that of glycosylated donkey-hide gelatin.

[0086] Basic component analysis: The material basis of donkey-hide gelatin was studied: the water content was determined according to the national standard GB 5009.3-2016, and the remaining substances were the content in the dry basis. In the dry basis, the protein content was determined according to the national standard GB 5009.5-2016 Kjeldahl nitrogen method, the fat was determined according to the national standard GB 5009.6-2016 acid hydrolysis method, the ash content was determined according to the national standard GB 5009.4-2016, and the sugar content was determined according to the phenol sulfuric acid method. The results are shown in Table 1.

[0087]

[0088] Table 1

[0089] Minerals were determined using an inductively coupled plasma emission spectrometer and an atomic fluorescence spectrometer. The contents of potassium, sodium, calcium, magnesium, zinc, iron, barium, and strontium were determined according to the second method of national standard GB 5009.268-2016, and the content of selenium was determined according to the first method of national standard GB5009.93-2017. The results are shown in Table 2.

[0090]

[0091] Table 2

[0092] Molecular weight distribution analysis of donkey-hide gelatin: The molecular weight distribution of the sample was determined by high performance liquid chromatography using a TSK GEL G3000PWXL column. Gel exclusion chromatography chromatographic conditions: chromatograph: Agilent 1260, equipped with UV detector and GPC software processing system; chromatographic column: TSKgel G2000SWXL (7.8mm*30cm, particle size 5μm); mobile phase: acetonitrile: water (PH2.0) 3:7; flow rate: 1.0ml / min; injection volume: 10μl column temperature: 30℃; detection wavelength: 220nm. Standards used for sample collection and relative molecular weight calibration curve: uracil (MW112), glutathione (MW307), bacitracin (MW1422), aprotinin (MW6511), cytochrome C (MW12384), bovine serum albumin V (MW68000), 1mg each, dissolved in 1mL of pure water, filtered with a 0.45nm microporous filter membrane and injected. The results were analyzed by GPC software.

[0093]

[0094] Table 3

[0095] Among them, Table 3 is the molecular weight distribution of two donkey-hide gelatin samples in each section; Determination of hydroxyproline content: Referring to the method of YY / T1511-2017, the results of hydroxyproline content determination are shown in Table 3. The L-hydroxyproline content of the two donkey-hide gelatin samples is between 11.2% and 11.9%, and the content difference is not large.

[0096]

[0097] Table 4

[0098] Table 4 is a table of hydroxyproline content analysis. Amino acid composition analysis: The amino acid composition of donkey-hide gelatin products was determined according to the method specified in GB5009.124-2016 "Determination of Amino Acids in Foods". The sample weight was 30 mg, and the volume was adjusted to 50 ml after digestion. Then 2 ml was taken and dried with N2. After drying, the volume was adjusted to 5 ml. After membrane treatment, 20 μl was taken for injection, and the amino acid composition was analyzed using Hitachi L-8900 fully automatic amino acid analyzer.

[0099]

[0100]

[0101] Table 5

[0102] The amino acid composition results are shown in Table 5. The analysis results of 16 amino acids show that there is no significant difference between the two donkey-hide gelatin samples. When the raw materials are the same, the types and quantities of amino acids will be consistent.

[0103] Efficacy evaluation: HUVEC cell proliferation activity detection: CCK-8 method was used to detect cell proliferation activity. After HUVECs were subcultured, ECM complete culture medium (500ml ECM+25ml FBS+5ml ECGS+5ml P / S) was used to prepare endothelial cell single cell suspension and adjusted to 3×10 4 / mL, and set up a blank group, a negative control group and different concentrations of donkey-hide gelatin groups respectively. Add 100 μL of cell suspension to each sample well plate. After culturing in a 5% CO2, 37°C incubator for 48 h, add 10 μL of CCK-8 reagent to each well and mix thoroughly with the culture medium. After 1 h, measure the absorbance at a wavelength of 450 nm.

[0104]

[0105] Table 6

[0106] First, the effect of donkey-hide gelatin products on the proliferation rate of HUVEC cells was detected by CCK-8 method, and the results are shown in Table 6. When the added concentration was in the range of 50-500ug / ml, the proliferation rate of HUVECs cells was higher than 80%, indicating that the four samples were non-cytotoxic. When the added concentration of the four samples was 200ug / ml, the cell proliferation rate was the highest; compared with the negative control, the addition of 500ug / ml and 200ug / ml of glycosylated donkey-hide gelatin liquid had a very significant promoting effect on the survival of HUVEC cells (P<0.01). Therefore, in the experiment of the effect of donkey-hide gelatin on oxidative damage of HUVEC cells, the addition amount was 100ug / ml and 200ug / ml. Among them, * is compared with the negative control group, p<0.05; ** is compared with the negative control group, p<0.01.

[0107] HUVEC cell migration ability detection: scratch test to observe cell migration ability. After HUVEC subculture, endothelial cell single cell suspension was prepared and adjusted to 3×10 4 / mL, and set up blank group and negative control group, as well as different glycosylated donkey-hide gelatin sample groups, add 1mL of cell suspension to each well of a 6-well plate containing each sample, and culture in a 5% CO2, 37℃ incubator for 48h. After the cells adhere to the wall and completely cover the culture plate, use a 10μL sterile pipette tip to slide quickly vertically on the bottom of the 6-well plate to make scratches. Replace the cell culture medium and remove the vascular endothelial cells that fall off during the scratching. Continue to culture in a 5% CO2, 37℃ incubator, and take pictures and observe at 0h, 4h and 8h. Figure 2 As shown, S1 is commercially available donkey-hide gelatin; S2 is Example 1; S3 is Example 2; S4 is Example 3; S5 is Example 4; C: blank control group.

[0108] The results of the effect of glycosylated donkey-hide gelatin on the migration rate of HUVEC cells are shown in Table 7. After 4 hours of streaking, the migration rate of HUVEC cells in the group with the addition of three glycosylated donkey-hide gelatin solutions was significantly higher than that of cells in the ordinary donkey-hide gelatin and control groups. Endothelial cells are the main participants in angiogenesis, which can form a new vascular network through proliferation, migration, lumen formation and mutual fusion. Studies have shown that collagen substances can participate in various biological processes such as tissue and organ morphogenesis, metabolic renewal, and damage repair. This study also further proves that glycosylated donkey-hide gelatin can promote the proliferation and migration of vascular endothelial cells.

[0109]

[0110] Table 7

[0111] As shown in Table 8, the effect of glycosylated donkey-hide gelatin on the proliferation rate of oxidatively damaged HUVEC cells. After HUVECs were subcultured, endothelial cell single-cell suspensions were prepared, and 1×105 / mL endothelial cell suspensions were inoculated in 6-well culture plates containing different donkey-hide gelatin samples at 1 mL / well, and cultured in a 37°C cell culture incubator for 24 hours. After the cells were tightly attached to the wall and completely covered the bottom of the plate, the supernatant was discarded and 600 μmol / L H2O2 was added to each well. After 4 hours, PBS was used to wash, and DMEM culture medium without fetal bovine serum was added to each well for a further 24 hours to determine the survival rate of oxidatively damaged HUVEC cells. Among them, # indicates P<0.05 compared with the negative control group, ## indicates P<0.01 compared with the negative control group; * indicates P<0.05 compared with the model group, and ** indicates P<0.01 compared with the model group.

[0112]

[0113]

[0114] Table 8

[0115] The results of the effects on cell survival inhibition rate are shown in Table 8. Compared with the normal control group, the cells in the H2O2 injury model group showed obvious growth inhibition, and the cell proliferation rate reached 62.12% at 48h. Compared with the model group, the cell survival rates of the groups adding the four glycosylated donkey-hide gelatin solutions were significantly higher than those of the model group (P<0.01), while there was no significant difference in the commercial donkey-hide gelatin group. Among them, # indicates P<0.05 compared with the negative control group, ## indicates P<0.01 compared with the negative control group, * indicates P<0.05 compared with the model group, and ** indicates P<0.01 compared with the model group.

[0116]

[0117]

[0118] Table 9

[0119] Among them, Table 9 shows the effect of glycosylated donkey-hide gelatin on the oxidative indexes of oxidatively damaged HUVEC cells. Compared with the normal control group without oxidative damage, the SOD enzyme activity of the oxidatively damaged HUVEC cells in the model group was significantly reduced; after adding glycosylated donkey-hide gelatin, the SOD enzyme activity of the oxidatively damaged HUVEC cells increased to varying degrees, and there was a significant difference in the SOD enzyme activity of the glycosylated donkey-hide gelatin compared with the model group (P<0.05). Among them,

[0120] # indicates P<0.05 compared with the negative control group, ## indicates P<0.01 compared with the negative control group, * indicates P<0.05 compared with the model group, and ** indicates P<0.01 compared with the model group.

[0121] Compared with the negative control group, the LDH enzyme activity of HUVEC in the oxidative damage model group was significantly increased. LDH is a cytoplasmic enzyme that is stable in the cytoplasm. When the cell membrane is damaged by oxidation, LDH is released. The expression of LDH in the cell culture supernatant increases, indicating that the cell membrane is damaged. After adding glycosylated donkey-hide gelatin solution, the LDH enzyme activity of oxidatively damaged HUVEC cells decreased, but when the added concentration was 100ug / ml, there was no significant difference between the donkey-hide gelatin group and the model group (P>0.05), while the LDH enzyme activity of the glycosylated donkey-hide gelatin solution groups 1 and 2 was significantly different from the model group (P<0.01).

[0122] Compared with the negative control group, the MDA content of HUVEC cells in the oxidative damage model group increased significantly, that is, when the body is damaged by oxidative stress, the expression of MDA increases. The MDA content of the cells in the ordinary donkey-hide gelatin group was not significantly lower than that in the model group, but the MDA content of the other three glycosylated gelatin groups was significantly lower than that in the model group (P<0.01).

[0123] The cell culture medium was centrifuged at 1500r / min for 10 minutes, and the supernatant of oxidatively damaged HUVEC cells treated with different donkey-hide gelatin was collected and diluted 1:1 for reaction. The enzyme-linked immunosorbent assay was used in combination with the standard curve. The operation steps were according to the instructions of the kit to detect the expression level of vascular endothelial growth factor (VEGF) protein and evaluate the functional activity of donkey-hide gelatin samples with different processing methods in protecting vascular endothelial cells.

[0124]

[0125] Table 10

[0126] Table 10 shows the effect of glycosylated donkey-hide gelatin on VEGF in HUVEC cells damaged by oxidation. The negative control group HUVEC cells expressed the least VEGF. The model group HUVEC cells were stimulated by oxidative damage, and the VEGF expression was significantly higher than that in the control group. After adding three kinds of glycosylated donkey-hide gelatin, the VEGF expression of glycosylated donkey-hide gelatin cells increased significantly compared with the model group (P>0.01).

[0127] # indicates P<0.05 compared with the negative control group, ## indicates P<0.01 compared with the negative control group, * indicates P<0.05 compared with the model group, and ** indicates P<0.01 compared with the model group.

[0128] The above are all preferred embodiments of the present invention, and are not intended to limit the protection scope of the present invention. Therefore, any equivalent changes made based on the structure, shape, and principle of the present invention should be included in the protection scope of the present invention.

[0129] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for preparing glycosylated donkey-hide gelatin having the function of repairing vascular endothelial cell damage, characterized in that: The following steps are involved: S1. The donkey hide is completely immersed in a 1% sodium carbonate aqueous solution and rinsed with clean water until the donkey hide fully absorbs water, becomes white, soft, and the soaking water is clean; S2, the donkey skin after cleaning in described step S1 is subjected to impurity removal treatment to obtain clean donkey skin; S3, weighing the clean donkey hide in step S2 and putting it into a spheronization machine to extract collagen and obtain a collagen solution; S4, filtering and centrifuging the collagen solution in step S3 and transferring it into a foaming machine for collagen purification to form a concentrated collagen solution; S5, compounding the concentrated gelatin solution in step S4 with the syrup solution, and reacting them to obtain a glycosylated gelatin solution; S6, placing the saccharified gelatin solution of step S5 in a concentration tank for secondary concentration to obtain a saccharified donkey-hide gelatin product; The step S3 is specifically as follows: placing the donkey hide block in a spheronizer, adding a 2 mol / L Na2CO3 solution preheated at 60°C in a ratio of 1:3-1:5 m / w to the weight of the donkey hide, adjusting the temperature of the spheronizer to 60°C-80°C, blanching for 60min-90min to fully remove fat from the donkey hide, discarding the blanching water, and then rinsing the donkey hide with drinking water to fully remove residual alkali solution, each rinsing temperature is 50-60°C, the rinsing time is 10-15min, and the rinsing is repeated at least 5 times until the water is clear after rinsing and the pH is not higher than 8.0; The step S4 is specifically as follows: adding drinking water according to a volume ratio of 1:2-1:4 m / v of the weight of the clean donkey hide block to water, and maintaining a steam pressure of 0.1-0.2 MPa for slight boiling extraction for 18-20 hours, collecting the first glue solution, adding water according to a ratio of 1:2-1:3 m / v of the weight of the donkey hide block, and maintaining a steam pressure of 0.1-0.15 MPa for slight boiling extraction for 3-5 hours, collecting the glue solution for the second time, adding water according to a ratio of 1:1-1:2 m / v of the weight of the donkey hide block, and maintaining a steam pressure of 0.1-0.15 MPa for slight boiling extraction for 1-3 hours, collecting the glue solution for the third time, extracting by stepwise depressurization three times, so that the collagen in the donkey hide is fully dissolved in batches, and combining the glue solutions collected three times to obtain a mixed glue solution; The step S5 is specifically as follows: filtering the mixed glue solution with a 120-mesh sieve, placing the filtrate in a foam extractor for boiling and concentration, during which insoluble proteins and impurities are removed with a foaming hook, until the moisture content of the glue solution is 50%-60%, thereby obtaining a concentrated glue solution; The step S6 is specifically as follows: the syrup is prepared by mixing rock sugar and fructose in any proportion, precipitating for about 2 hours, and filtering through a 200-mesh sieve; compounding according to a mass ratio of concentrated gum to syrup of 10:1-12:1, stirring evenly, performing Maillard reaction at 80-90°C for 2-4 hours, and filtering through a 120-mesh sieve to obtain a glycosylated gum; Step S7 specifically comprises: placing the glycosylated gelatin solution in a concentration tank for boiling and continuously dynamically concentrating for 1-3 hours until the water content reaches 20%-40%, thereby obtaining a glycosylated donkey-hide gelatin product; The step S1 is specifically as follows: completely immersing the donkey skin in a 1% sodium carbonate aqueous solution at 15-25° C. for 6-10 hours, and then continuing to soak in clean water for 4-8 days, during which the water is changed once every 12 hours, until the donkey skin fully absorbs water, becomes white, and becomes soft, and the pH value of the soaking water does not exceed 8.0; The step S2 is specifically as follows: firstly, the neck, legs and tail of the donkey skin are removed to obtain the belly and back skin, and then the grease and donkey meat impurities attached to the belly and back skin are removed by a meat remover; then, the processed belly and back skin is cut into leather pieces with a width of 10-15 cm by a skin cutter, and washed 3-5 times with 3-5 times the amount of clean water, each time washing the skin for 30-40 minutes, until the turbidity value of the washing water does not exceed 240NTU, and the leather pieces are taken out and the water is removed by a water squeezer.

2. A glycosylated donkey-hide gelatin having the function of repairing vascular endothelial cell damage, characterized in that: The invention discloses a glycosylated donkey-hide gelatin having the function of repairing vascular endothelial cell damage according to claim 1.

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