Preparation method and application of high-content instant small-molecule donkey-hide gelatin granules

Abstract

The present application relates to the technical field of small molecule donkey-hide gelatin granules, and in particular to a preparation method and application of high-content instant small molecule donkey-hide gelatin granules. The preparation method comprises the following steps: coarsely crushing donkey-hide gelatin blocks or donkey-hide gelatin powder; adding water to a reaction kettle, heating, and adding the donkey-hide gelatin coarse powder while stirring to obtain a donkey-hide gelatin solution; heating the reaction kettle, introducing sterile nitrogen into the bottom and top of the reaction kettle, pressurizing, and closing the valve; opening the stirring of the reaction kettle, performing a hot reaction, stopping the hot reaction after the viscosity of the reaction kettle liquid reaches the standard, and filtering the liquid; and granulating the fine powder obtained after spray drying of the filtrate and the binder by using a fluidized bed boiling granulator, and drying, to obtain the granules. The prepared donkey-hide gelatin granules have more than 90% of high-activity small molecule polypeptides (molecular weight ≤3000 Da), no exogenous impurities are introduced, and have the characteristics of fast dispersion speed, good efficacy, and easy absorption. The preparation process has the characteristics of high intelligent degree, green environmental protection, and low cost.

Description

Technical Field

[0001] This invention relates to the field of small molecule donkey-hide gelatin granule preparation technology, specifically to a method for preparing high-content, fast-dissolving small molecule donkey-hide gelatin granules and its application. Background Technology

[0002] Donkey-hide gelatin (Ejiao) is a solid gelatin made from the skin of donkeys (Equus equina) through boiling and concentration. Its main component is collagen, accounting for about 80%. Donkey-hide gelatin is revered by physicians throughout history as a "holy medicine" for replenishing blood, nourishing yin, moistening dryness, and stopping bleeding. Small-molecule donkey-hide gelatin is a low-molecular-weight protein (or polypeptide) substance obtained by processing the collagen in donkey-hide gelatin. It has the effects of replenishing blood, beautifying the complexion, and anti-aging, and is more easily absorbed compared to traditional donkey-hide gelatin.

[0003] Currently, the mainstream process for preparing small-molecule donkey-hide gelatin both domestically and internationally is enzymatic hydrolysis. For example, Chinese invention patent application CN117448405A discloses a method for producing donkey-hide gelatin peptides. This method involves mixing donkey-hide gelatin powder with water, adjusting the pH to 6.5-7, adding neutral protease, trypsin, and flavor protease for enzymatic hydrolysis, and obtaining the product after decolorization and drying. This method is characterized by its simple and easy-to-control process, strong stability, and the resulting small-molecule donkey-hide gelatin contains >90% oligopeptides (on a dry basis) and >96% small-molecule peptides with a molecular weight ≤3000 Da. Chinese invention patent application CN118932003A provides a donkey-hide gelatin peptide and its preparation method. This method includes pretreatment of donkey-hide gelatin raw materials, preparation of primary and secondary enzymatic hydrolysates, followed by boiling inactivation, fat absorption, ultrafiltration fractionation, and drying to obtain the donkey-hide gelatin peptides. The above enzymatic hydrolysis process requires the addition of a large amount of water for dilution. After enzymatic hydrolysis, it needs to be further heated for sterilization, and then filtered to remove impurities such as high molecular weight proteases and inorganic salts. At the same time, the enzymatic hydrolysis process requires precise control of multiple parameters such as pH and temperature, and repeated adjustments are required. This not only increases the operating cost, but also easily causes the generation of impurities.

[0004] Therefore, there is an urgent need to develop a new preparation method to increase the content of low molecular weight proteins (or peptides) in donkey-hide gelatin, while simplifying the process, reducing production costs, and ensuring the quality and activity of the product. Summary of the Invention

[0005] Based on the technical problems existing in the current technical solutions, this invention provides a method for preparing high-content, fast-dissolving small-molecule donkey-hide gelatin granules and its application. The method adopts a physical pyrolysis method, which does not introduce exogenous impurities, eliminates the need to consider the risks of exogenous substances, and eliminates enzyme inactivation and impurity removal processes. The technology is simple to operate and environmentally friendly. The molecular weight distribution of proteins (or peptides) in the small-molecule donkey-hide gelatin is tested, and the proportion of proteins (or peptides) with a molecular weight ≤3000 Da is more than 90%, while the proportion of proteins (or peptides) with a molecular weight greater than 3000 Da and the sum of amino acids is less than 10%. In the granulation process, water or water and one of the following: sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose, gum arabic, xanthan gum, carrageenan, starch, and modified starch are used as binders. The amount of excipients used is small, and the dissolution rate is within 10 seconds.

[0006] To achieve the above-mentioned objectives, the technical solution of the present invention is as follows: The first aspect of this invention relates to a method for preparing high-content, fast-dissolving donkey-hide gelatin granules, the method comprising the following steps: (1) Coarse grinding; coarsely grind the donkey-hide gelatin blocks or donkey-hide gelatin cubes to obtain coarse donkey-hide gelatin powder; (2) Melting: Add water to a high-pressure heating reactor for donkey-hide gelatin, heat it, and add coarse donkey-hide gelatin powder while stirring to obtain a donkey-hide gelatin solution; (3) Adding nitrogen: Heat the reactor, introduce sterile nitrogen from the bottom of the reactor until it is oxygen-free, close the bottom gas valve, stop stirring, then introduce sterile nitrogen from the top of the reactor, pressurize it, and close all valves; (4) Thermal reaction: The reactor is stirred, heated and reacted. The viscosity of the liquid in the reactor is monitored. When the viscosity of the liquid in the reactor is 10-120 Pa·s, the thermal reaction is stopped, the temperature is reduced, the liquid is released, and the filtrate is obtained after filtration through 200-800 mesh. (5) Drying: The filtrate is spray-dried to obtain a fine powder; (6) Granulation: Fine powder and binder are granulated by fluidized bed granulation machine, and the resulting particles after drying have a particle size range of 180-850μm, which is the donkey-hide gelatin granules.

[0007] Preferably, in step (2), the amount of water added is 3-8 times the amount of donkey-hide gelatin used, the temperature is heated to 40-60℃, and the stirring time is 0.5-2h.

[0008] Preferably, in step (3), the reactor is heated to 40-60°C, the amount of sterile nitrogen gas introduced into the bottom of the reactor is 2-5 reactor volumes, and the pressurization is to make the pressure inside the reactor 0.14-0.48 MPa.

[0009] Preferably, in step (4), the temperature inside the vessel is heated to 110-150°C and the reaction is carried out for 3-10 hours.

[0010] Preferably, in step (4), a filter aid is added during filtration, and the filter aid is selected from at least one of activated clay, diatomaceous earth, magnesium oxide, aluminum oxide and activated carbon.

[0011] Preferably, in step (5), the inlet air temperature during spray drying is 150-210℃ and the outlet air temperature is 70-105℃.

[0012] Preferably, the adhesive in step (6) is purified water or purified water and one or two of sodium carboxymethyl cellulose (CMC-Na), hydroxypropyl methyl cellulose (HPMC), gum arabic, xanthan gum, carrageenan, starch and modified starch.

[0013] More preferably, the prepared small molecule donkey-hide gelatin particles, after testing of the molecular weight distribution of proteins (or peptides), have a proportion of more than 90% with a molecular weight ≤3000Da, and a proportion of less than 10% of the total amount of proteins (or peptides) and amino acids with a molecular weight greater than 3000Da.

[0014] The second aspect of this invention relates to the application of the small molecule donkey-hide gelatin particles prepared by the above-described preparation method in the preparation of donkey-hide gelatin products.

[0015] Preferably, the product is a pharmaceutical, food, or health food.

[0016] The beneficial effects of the present invention are as follows: (1) The method provided by the present invention adopts physical pyrolysis, which will not introduce exogenous impurities, does not need to consider the risk of exogenous substances, and has no enzyme inactivation, impurity removal and other processes. The technical process is simple to operate and is green and environmentally friendly; (2) By using oxygen isolation, the reaction temperature during thermal reaction is ≥110℃, the reaction vessel is heated evenly, the reaction efficiency is high, the protein in donkey-hide gelatin is fully pyrolyzed and will not be over-degraded or charred. According to the test of the molecular weight distribution of protein (or polypeptide), the proportion of the molecular weight ≤3000Da is more than 90%, and the proportion of protein (or polypeptide) with a molecular weight greater than 3000Da and the sum of amino acids is less than 10%; (3) Water or water and one of sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose, gum arabic, xanthan gum, carrageenan, starch and modified starch are used as binders in granulation. The amount of auxiliary materials is small, the content of small molecule protein (or polypeptide) is high, and the dissolution rate is within 10s. Detailed Implementation

[0017] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0018] Example 1 A method for preparing high-content, fast-dissolving, small-molecule donkey-hide gelatin granules, the preparation method being as follows: (1) Coarse crushing; take the donkey-hide gelatin block and coarsely crush it, then pass it through a 40-mesh sieve to obtain coarse donkey-hide gelatin powder; (2) Melting: Take a high-pressure heating reactor for donkey-hide gelatin, add drinking water with 5 times the amount of donkey-hide gelatin, heat to 50°C, turn on the stirring, and add the coarse powder of donkey-hide gelatin to the hot water while stirring for 1 hour to make the donkey-hide gelatin powder fully dissolved; (3) Adding nitrogen: Heat the reactor to 70°C, and slowly introduce sterile nitrogen from the bottom of the reactor while stirring. The gas flow rate is 3 reactor volumes. The sterile nitrogen replaces the air in the reactor with oxygen-free air by passing through the donkey-hide gelatin solution. Close the bottom gas valve, stop stirring, and then introduce sterile nitrogen from the top of the reactor. Pressurize the reactor to a pressure of 0.26 MPa, and then close all valves. (4) Thermal reaction: The reactor is stirred and heated to 130°C. The reaction is carried out continuously for 5 hours. The viscosity of the liquid in the reactor is monitored in real time using an online rotational viscometer. The initial viscosity of the liquid is ≥500 Pa·s. When the viscosity of the liquid in the reactor is 60 Pa·s, the thermal reaction is stopped, the temperature is reduced, the liquid is released, and the liquid is filtered through a 400-mesh filter. The filter aid is diatomaceous earth. The filtrate is kept for later use. (5) Drying: The above filtrate is spray-dried with an inlet air temperature of 180°C and an outlet air temperature of 95°C to obtain a dry fine powder; (6) Granulation: Fine powder and water are granulated by a fluidized bed granulator with an air inlet temperature of 60℃. After drying, the particle size range of the granules is 180-850μm (20 mesh-80 mesh), which is small molecule donkey-hide gelatin granules.

[0019] Tests showed that 94.3% of the small-molecule donkey-hide gelatin granules contained proteins (or peptides) with a molecular weight ≤3000 Da, the dispersion time was 9.1 s, and the yield was 92.5%.

[0020] Example 2: A method for preparing high-content, fast-dissolving, small-molecule donkey-hide gelatin granules, the preparation method being as follows: (1) Coarse crushing; take the donkey-hide gelatin block and coarsely crush it, then pass it through a 40-mesh sieve to obtain coarse donkey-hide gelatin powder; (2) Melting: Take a high-pressure heating reactor for donkey-hide gelatin, add drinking water with a dosage of 3 times that of donkey-hide gelatin, heat to 60°C, turn on the stirring, and add the coarse powder of donkey-hide gelatin to the hot water while stirring for 0.5 hours to fully dissolve the donkey-hide gelatin powder; (3) Adding nitrogen: Heat the reactor to 80°C, and slowly introduce sterile nitrogen from the bottom of the reactor while stirring. The gas flow rate is 2 reactor volumes. The sterile nitrogen replaces the air in the reactor with oxygen-free air through the donkey-hide gelatin solution. Close the bottom gas valve, stop stirring, and then introduce sterile nitrogen from the top of the reactor. Pressurize the reactor to a pressure of 0.14 MPa, and then close all valves. (4) Thermal reaction: The reactor is stirred and heated to 110°C. The reaction is carried out continuously for 10 hours. The viscosity of the liquid in the reactor is monitored in real time using an online rotational viscometer. The initial viscosity of the liquid is ≥500 Pa·s. When the viscosity of the liquid in the reactor is 120 Pa·s, the thermal reaction is stopped, the temperature is reduced, the liquid is released, and the liquid is filtered through a 200-mesh filter. The filter aid is diatomaceous earth. The filtrate is kept for later use. (5) Drying: The above filtrate is spray-dried with an inlet air temperature of 150°C and an outlet air temperature of 105°C to obtain a dry fine powder; (6) Granulation: Fine powder and water are granulated by a fluidized bed granulator with an air inlet temperature of 45℃. After drying, the particle size range of the granules is 180-850μm (20 mesh-80 mesh), which is the granules of donkey-hide gelatin.

[0021] Tests showed that 93.7% of the proteins (or peptides) in the small molecule donkey-hide gelatin granules had a molecular weight of ≤3000 Da, the dispersion time was 9.3 s, and the yield was 91.8%.

[0022] Example 3: A method for preparing high-content, fast-dissolving, small-molecule donkey-hide gelatin granules, the preparation method being as follows: (1) Coarse crushing; take the donkey-hide gelatin block and coarsely crush it, then pass it through a 40-mesh sieve to obtain coarse donkey-hide gelatin powder; (2) Melting: Take a high-pressure heating reactor for donkey-hide gelatin, add drinking water with 8 times the amount of donkey-hide gelatin, heat to 40°C, turn on the stirring, and add the coarse powder of donkey-hide gelatin to the hot water while stirring for 1 hour to make the donkey-hide gelatin powder fully dissolved; (3) Adding nitrogen: Heat the reactor to 60°C, and slowly introduce sterile nitrogen from the bottom of the reactor while stirring. The gas flow rate is 5 reactor volumes. The sterile nitrogen replaces the air in the reactor with oxygen-free air by passing through the donkey-hide gelatin solution. Close the bottom gas valve, stop stirring, and then introduce sterile nitrogen from the top of the reactor. Pressurize the reactor to a pressure of 0.48 MPa, and then close all valves. (4) Thermal reaction: The reactor is stirred and heated to 150°C. The reaction is carried out continuously for 3 hours. The viscosity of the liquid in the reactor is monitored in real time using an online rotational viscometer. The initial viscosity of the liquid is ≥500 Pa·s. When the viscosity of the liquid in the reactor is 20 Pa·s, the thermal reaction is stopped, the temperature is reduced, the liquid is released, and the liquid is filtered through an 800-mesh filter. The filter aid is diatomaceous earth. The filtrate is kept for later use. (5) Drying: The above filtrate is spray-dried with an inlet air temperature of 210°C and an outlet air temperature of 70°C to obtain a dry fine powder; (6) Granulation: Fine powder, water and hydroxypropyl methylcellulose (0.5% of the fine powder) are granulated by a fluidized bed granulator with an inlet air temperature of 80℃. After drying, the particle size range of the granules is 180-850μm (20 mesh-80 mesh), which is the granules of donkey-hide gelatin.

[0023] Tests showed that 92.5% of the small-molecule donkey-hide gelatin granules contained proteins (or peptides) with a molecular weight ≤3000 Da, the dispersion time was 8.8 s, and the yield was 95.7%.

[0024] Comparative Example 1 The difference from Example 1 is that step (3) is omitted in Comparative Example 1, that is, the process of adding nitrogen to remove oxygen is not included before the thermal reaction, and the rest is the same as in Example 1.

[0025] Tests showed that 71.2% of the small-molecule donkey-hide gelatin granules contained proteins (or peptides) with a molecular weight ≤3000 Da, the dispersion time was 15.2 s, and the yield was 91.4%.

[0026] Comparative Example 2 The difference from Example 1 is that the thermal reaction temperature in step (4) of Comparative Example 2 is 80°C, and the rest is the same as in Example 1.

[0027] Tests showed that 62.7% of the small-molecule donkey-hide gelatin granules contained proteins (or peptides) with a molecular weight ≤3000 Da, the dispersion time was 21.9 s, and the yield was 92.7%.

[0028] Comparative Example 3 The difference from Example 1 is that the thermal reaction temperature in step (4) of Comparative Example 3 is 180°C, and the rest is the same as in Example 1.

[0029] Tests showed that 96.33% of the small molecule donkey-hide gelatin granules contained proteins (or peptides) with a molecular weight ≤3000 Da. Due to excessive reaction, the sample was dark brown in color and failed to dissolve completely within 5 minutes, resulting in a large amount of precipitation; the yield was 74.4%.

[0030] Comparative Example 4 The preparation of small molecule donkey-hide gelatin peptides was carried out according to Example 5 of Chinese Invention Patent Application CN118932003 A, as follows: Raw material pretreatment: Crush 500g of donkey-hide gelatin blocks and pass them through a 100-mesh sieve to obtain donkey-hide gelatin powder. Add 4 times the amount of purified water to the donkey-hide gelatin powder, stir and heat to boiling, continue stirring until completely dissolved, and then cool to 45-55℃.

[0031] Preparation of primary donkey-hide gelatin enzymatic hydrolysate: Adjust the pH value to 7.5-8.5 using sodium hydroxide, add 5.00g of alkaline protease (enzyme to donkey-hide gelatin mass ratio of 1:100) and 5.00g of bromelain (enzyme to donkey-hide gelatin mass ratio of 1:100), and incubate in a water bath at 45-55℃ for 10h to obtain primary donkey-hide gelatin enzymatic hydrolysate.

[0032] Preparation of secondary donkey-hide gelatin enzymatic hydrolysate: Adjust the pH value to 7.5-8.5, add 5.00g of neutral protease (enzyme to donkey-hide gelatin mass ratio of 1:100) and 5.00g of flavor protease (enzyme to donkey-hide gelatin mass ratio of 1:100), and incubate in a water bath at 45-55℃ for 6h to obtain secondary donkey-hide gelatin enzymatic hydrolysate. Inactivation treatment: Boil the secondary donkey-hide gelatin enzymatic hydrolysate for 10 minutes to inactivate it, and then cool it to room temperature.

[0033] Liposuction treatment: Add 2% activated carbon by volume to the secondary enzymatic hydrolysate of inactivated donkey-hide gelatin, stir for 10-20 minutes, let stand for 2 hours, and remove the activated carbon.

[0034] Ultrafiltration fractionation: Polyethersulfone filter media is used to fractionate the filtrate according to molecular weight 10kDa, and the filtrate with a molecular weight below 10kDa is collected.

[0035] Drying process: Spray drying was used to obtain powder, which is called donkey-hide gelatin peptide, denoted as donkey-hide gelatin peptide five, with a yield of 62.4%.

[0036] Tests showed that 94.7% of the proteins (or peptides) in the small molecule donkey-hide gelatin granules had a molecular weight of ≤3000 Da, the dispersion time was 9.0 s, and the yield was 61.2%.

[0037] The above detailed description is a specific description of one of the feasible embodiments of the present invention. This embodiment is not intended to limit the patent scope of the present invention. All equivalent implementations or modifications that do not depart from the present invention should be included within the scope of the technical solution of the present invention.

Claims

1. A method for preparing high-content, fast-dissolving, small-molecule donkey-hide gelatin granules, characterized in that, The preparation method comprises the following steps: (1) Coarse grinding; coarsely grind the donkey-hide gelatin blocks or donkey-hide gelatin cubes to obtain coarse donkey-hide gelatin powder; (2) Melting: Add water to a high-pressure heating reactor for donkey-hide gelatin, heat it, and add coarse donkey-hide gelatin powder while stirring to obtain a donkey-hide gelatin solution; (3) Adding nitrogen: Heat the reactor, introduce sterile nitrogen from the bottom of the reactor until it is oxygen-free, close the bottom gas valve, stop stirring, then introduce sterile nitrogen from the top of the reactor, pressurize it, and close all valves; (4) Thermal reaction: The reactor is stirred, heated and reacted. The viscosity of the liquid in the reactor is monitored. When the viscosity of the liquid in the reactor is 10-120 Pa·s, the thermal reaction is stopped, the temperature is reduced, the liquid is released and filtered through 200-800 mesh to obtain the filtrate. (5) Drying: The filtrate is spray-dried to obtain a fine powder; (6) Granulation: The fine powder and binder are granulated by a fluidized bed granulator, and the resulting granules, after drying, have a particle size range of 180-850 μm, thus obtaining the small molecule donkey-hide gelatin granules; wherein In step (3), the amount of sterile nitrogen gas introduced into the bottom of the reactor is 2-5 reactor volumes, and the pressurization is to make the pressure inside the reactor 0.14-0.48 MPa; in step (4), the reactor is heated to 110-150℃ and reacted for 3-10 hours. In step (6), the adhesive is water or water and hydroxypropyl methylcellulose, and when the adhesive is water and hydroxypropyl methylcellulose, the amount of hydroxypropyl methylcellulose used is 0%-0.5% of the amount of fine powder in step (5).

2. The preparation method according to claim 1, characterized in that, In step (2), the amount of water added is 3-8 times the amount of donkey-hide gelatin used, and the temperature is heated to 40-60℃ with a stirring time of 0.5-2h.

3. The preparation method according to claim 1, characterized in that, In step (4), a filter aid is added during filtration. The filter aid is selected from at least one of activated clay, diatomaceous earth, magnesium oxide, aluminum oxide, and activated carbon.

4. The preparation method according to claim 1, characterized in that, In step (5), the inlet air temperature during spray drying is 150-210℃ and the outlet air temperature is 70-105℃.

5. The preparation method according to any one of claims 1-4, characterized in that, The prepared small molecule donkey-hide gelatin granules, after testing, contain more than 90% highly active small molecule polypeptides with a molecular weight ≤3000Da, and the proportion of proteins or polypeptides with a molecular weight greater than 3000Da and the sum of amino acids is less than 10%.