CN108864271B - Preparation method of iron protein succinate - Google Patents
Preparation method of iron protein succinate Download PDFInfo
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Abstract
The invention relates to the technical field of pharmacy, and particularly discloses a preparation method of high-acylation-degree protein iron succinate. On the basis of the existing preparation method of the iron protein succinate, the preparation method prepares the succinyl protein with high acylation degree (the acylation degree is more than 99.3 percent), finally prepares the iron protein succinate with high acylation degree, increases the hydrophilic group, increases the water solubility, reduces the viscosity of the water solution, and solves the problems of high viscosity and more sediments of the iron protein succinate oral solution.
Description
Technical Field
The invention relates to the technical field of pharmacy, in particular to a preparation method of high-acylation-degree protein iron succinate.
Background
The iron in the iron protein succinate is combined with the emulsion succinate protein to form an iron-protein complex, so as to treat various iron-deficiency anemia. The structure is as follows:
the iron protein succinate is in the form of precipitate when the pH value is less than 4, and is changed into a soluble substance again when the pH value is higher (pH value is 7.5-8). In addition, iron proteinsuccinate is not digestible by pepsin, but is hydrolyzed by trypsin at neutral pH. Due to these properties of iron protein succinate, iron contained therein is protected by protein membrane and reacts with hydrochloric acid and pepsin in different gastric juice, thus not causing gastric mucosa injury. The iron in the iron protein succinate is released in duodenum, because the compound becomes soluble again due to the normal pH value rise, and the protein membrane is digested by trypsin, and the released iron is very beneficial to the physiological absorption of the body, but does not form too high absorption peak; in fact, it presents a constant tendency to absorb, gradually reaching an optimal plateau of absorption and storage in various parts of the body.
In 1987, a nano-polypeptide protein iron-supplementing drug named iron-succinate (ISP) was developed by the pharmaceutical factory of pan-mark in italy, and was successfully marketed in italy and marketed in 20 countries including spain, portugal, greece, argentina, korea, china, etc. successively. The iron protein succinate oral solution has been approved by the company italfarmacaco s.a. spain at the national application import under the trade name (chinese) phenanthrepril, under the trade name (english) Ferplex.
The existing preparation route of iron protein succinate mainly takes casein as a raw material, the casein and succinic anhydride are subjected to acylation reaction to generate succinyl protein, and ferric ions and the succinyl protein are subjected to iron-carrying reaction to generate the iron protein succinate. The reaction formula is as follows:
US4493829 discloses a method for preparing iron protein succinate. Taking milk powder as a raw material, carrying out acylation reaction with succinic anhydride under an alkaline condition (pH of 7.5-8), centrifuging or filtering reaction liquid after the reaction is finished, adding hydrochloric acid into filtrate for acidification (pH of 2.5-3) to precipitate, and centrifuging or filtering to obtain the succinyl protein. Dispersing succinyl protein in water, dissolving under alkaline condition (pH7.5), adding ferric trichloride solution for iron-carrying reaction, and filtering after the reaction to obtain crude product of iron protein succinate. The crude iron protein succinate is dispersed in water, dissolved under alkaline condition (pH7.5), and filtered to obtain solution. The iron protein succinate can be prepared by the following method: 1) acidifying (pH2.5), filtering, and vacuum drying. 2) The solution is dialyzed to remove sodium chloride, and then is freeze-dried or spray-dried. The acylation degree of the product prepared by the method is 95%.
CN102838667 discloses a preparation method of iron protein succinate. Performing acylation reaction on casein serving as a raw material and succinic anhydride under an alkaline condition (pH of 9.0-10.0), after the reaction is finished, performing centrifugal filtration, adding hydrochloric acid into filtrate for acidification (pH of 1-4) to precipitate, and performing centrifugation to obtain the precipitate to obtain the succinyl protein. Dispersing succinyl protein in water, dissolving under alkaline condition (pH9.0-10.0), and centrifuging to obtain succinyl protein solution. Adding a ferric trichloride solution, simultaneously adding sodium hydroxide to keep the pH value of the reaction solution at 9.0-10.0 for carrying out iron-carrying reaction, and centrifuging after the reaction is finished to obtain a solution. And adding hydrochloric acid for acidification (pH 1-4) to precipitate, and centrifuging to obtain a filter cake. Dispersing in water, dissolving under alkaline condition (pH 9.0-10.0), acidifying (pH 1-4), and centrifuging to obtain filter cake. And dispersing in water, dissolving under an alkaline condition (pH of 9.0-10.0), centrifuging, finely filtering the obtained solution by using a 1.2-micron ceramic membrane, acidifying (pH of 1-4), centrifuging to obtain a filter cake, and freeze-drying to obtain the iron protein succinate. The acylation degree of the product prepared by the method is 95.0-96.8%.
In the process of preparing the iron protein succinate oral solution, the problems of poor solubility and slow dissolution speed of the iron protein succinate, high viscosity, more sediments and the like of the prepared oral solution generally exist. The iron protein succinate solution has high viscosity, which can cause difficult operation of removing microorganisms through microfiltration in the process of preparing oral solution.
The percentage of amino groups that the casein is acylated in an acylation reaction to the original free amino groups of the casein is called the degree of acylation. The succinyl protein with higher acylation degree has better water solubility, lower viscosity of aqueous solution and stronger iron carrying capacity, and correspondingly the finally prepared protein iron succinate has good water solubility and low viscosity of aqueous solution. On the basis of the existing preparation method of the iron protein succinate, the problems of low dissolution speed, high viscosity of oral solution and more sediments in the preparation process of the iron protein succinate oral solution can be solved by improving the acylation degree of the iron protein succinate, increasing hydrophilic groups of the iron protein succinate, increasing the water solubility of the iron protein succinate and reducing the viscosity of an aqueous solution of the iron protein succinate.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a preparation method of iron protein succinate with high acylation degree (acylation degree > 99%), good water solubility, low viscosity of an aqueous solution, good filter membrane permeability, low impurity content and stable quality, which comprises the following steps:
(1) adding casein and water with the weight 10-15 times that of the casein into a reaction container, adding 1-4 mol/L sodium hydroxide solution while stirring to adjust the pH of the reaction solution to 7.0-10.0 so as to dissolve the casein. Slowly adding succinic anhydride with the total weight and casein weight ratio of 1: 0.2-1: 0.4 in multiple batches, adding 1 mol/L-4 mol/L sodium hydroxide solution while adding succinic anhydride to keep the pH of the reaction solution to be 7.0-10.0, continuously stirring and reacting for 30-45 min after the succinic anhydride is added, adding 1 mol/L-5 mol/L hydrochloric acid into the reaction solution to adjust the pH to be 2-3, continuously stirring for 30-45 min, filtering, and washing the obtained solid until the pH of the eluate is 7 to obtain the succinyl protein wet solid.
(2) Dispersing the wet succinylated protein solid obtained in the step (1) with water in an amount which is 5-7 times the weight of the casein, and adding 1-4 mol/L sodium hydroxide solution while stirring to adjust the pH of the reaction solution to 4.1-4.8 so as to dissolve the succinylated protein. Centrifuging or filtering (the aperture of the filter material is 120-3 μm) to obtain the succinyl protein solution.
(3) Slowly adding 1-10 wt% of ferric trichloride aqueous solution into the succinyl protein solution obtained in the step (2), wherein the weight ratio of succinyl protein to iron element is 1: 0.45-1: 0.55, continuously stirring for 30-45 min after the addition is finished, filtering, and washing the obtained filter cake with water until the pH value of an eluate is 7, so as to obtain a crude product wet solid of the iron protein succinate.
(4) And (3) dispersing the wet crude iron protein succinate solid obtained in the step (3) by water with the weight 10-15 times that of casein, adding 1-4 mol/L sodium hydroxide solution while stirring to adjust the pH of the reaction solution to 7.0-10.0, and dissolving the crude iron protein succinate. And (3) primarily filtering through a 5-micron filter membrane, then finely filtering through a 1-micron filter membrane, adding 1-5 mol/L hydrochloric acid into the obtained solution to adjust the pH value to be 2-3, filtering, and washing the obtained solid with water until the pH value of an eluate is 7 to obtain the wet iron protein succinate product.
(5) And (3) granulating the wet iron protein succinate obtained in the step (4) by using a swing granulator with a mesh size of 10-50, and drying the wet iron protein succinate in a boiling dryer to obtain the iron protein succinate.
Further, the pH of the reaction solution was adjusted to 4.1 by adding 1mol/L to 4mol/L sodium hydroxide solution as described in step 2).
Further, the filter cake in the step 2) is dispersed by water with 5 times of the weight of the casein input amount.
Further, the ratio of the total weight of succinic anhydride to the weight of casein was 3: 1.
Further, in the step (1), succinic anhydride is added to the casein solution in 15 to 25 times.
Further, the specific conditions for drying in step (5) are: drying for 0.5-1 h at 20-30 ℃, then heating to 50-70 ℃, and drying for 2-3 h.
Further, the specific conditions for drying in step (5) are: drying at 30 deg.C for 0.5 hr, heating to 70 deg.C, and drying for 2 hr.
The invention provides a preparation method of iron protein succinate, which takes casein as a raw material, prepares succinyl protein after succinic anhydride acylation, prepares an iron protein succinate crude product by iron-carrying reaction with ferric iron through complexation, and finally prepares the iron protein succinate through refining. The succinyl protein prepared by the method has acylation degree higher than 99%, good water solubility, low viscosity of aqueous solution, good filter membrane permeability, low impurity content and high yield.
The characteristics of the iron protein succinate product prepared by the method greatly make up the defects of slow dissolution speed, high solution viscosity and more solution sediments in the preparation process of the iron protein succinate oral solution prepared by the existing method.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below.
In the following examples, food grade casein is used as a raw material and purchased from Gansu Hualing Casein Co.
Example 1
A preparation method of iron protein succinate comprises the following steps:
to a 5L reaction flask, 1.2Kg of purified water was added, and 100g of casein was added with stirring. After the addition, the mixture was stirred for 0.5 hour, and 2.5mol/L sodium hydroxide solution was added while stirring, and the pH was adjusted to 7.5 to dissolve casein. A total of 300g of succinic anhydride was added to the casein solution 20 times, and after completion of the addition for one hour, 2.5mol/L sodium hydroxide solution was added while adding succinic anhydride, to maintain the pH of the reaction solution at 7.5. After the succinic anhydride is added, continuously stirring for 30min, adding 3mol/L hydrochloric acid to adjust the pH value of the reaction solution to 2.5, continuously stirring for 30min, and filtering to obtain the wet succinyl protein solid. Adding the wet succinyl protein solid into a 5L reaction bottle, adding 500g of purified water, adding 2.5mol/L sodium hydroxide solution while stirring to adjust the pH value to 4.5 so as to dissolve the succinyl protein, filtering, and adding a proper amount of water into the obtained filtrate so that the total volume of the succinic acid protein solution is 600 ml. Taking a proper amount of solution, measuring the content of the succinyl protein and measuring the acylation degree of the succinyl protein.
The weight determination method of the succinyl protein comprises the following steps:
and (3) taking 10ml of the succinyl protein solution, dropwise adding 0.1mol/L hydrochloric acid to adjust the pH value to be 2.0-3.0, filtering, and drying the obtained solid at 50-55 ℃ under reduced pressure for 6 hours by using phosphorus pentoxide.
Calculating the formula: w1＝(m/10)×V；
W1: succinyl protein weight g;
v: the total volume ml of the succinyl protein solution;
m: weight g of sample obtained after treatment. The same applies to the following examples.
Adding 1.528Kg of 9.5 wt% ferric trichloride solution into the succinyl protein solution, stirring for 30 minutes after adding, and filtering to obtain wet solid of the crude iron protein succinate product.
And adding the wet solid of the crude iron protein succinate obtained in the previous step into a 5L reaction bottle, adding 1.2Kg of purified water, and stirring to obtain a suspension. 2.5mol/L sodium hydroxide aqueous solution is added under stirring to adjust the pH value to be stable at 8.5 so as to completely dissolve the sodium hydroxide. Primarily filtering the reaction solution by a 5-micron filter membrane, finely filtering by a 1-micron filter membrane, adding 3mol/L hydrochloric acid into the obtained filtrate while stirring to adjust the pH value to 2.5, continuously stirring for 30 minutes, filtering, washing the obtained solid until the pH value of an eluate is 7 to obtain a wet iron protein succinate, granulating the wet iron protein succinate by a 50-mesh swinging granulator, drying at 30 ℃ for 0.5h in a boiling dryer, and heating to 70 ℃ for drying for 2h to obtain 102.7g of iron protein succinate.
Example 2
A preparation method of iron protein succinate comprises the following steps:
to a 5L reaction flask, 1.2Kg of purified water was added, and 100g of casein was added with stirring. After the addition, the mixture was stirred for 0.5 hour, and 2.5mol/L sodium hydroxide solution was added while stirring, and the pH was adjusted to 7.5 to dissolve casein. A total of 300g of succinic anhydride was added to the casein solution 20 times, and after completion of one hour of addition, 2.5mol/L sodium hydroxide solution was added while adding succinic anhydride to maintain the pH of the reaction solution at 7.5. After the succinic anhydride is added, stirring is continued for 30min, and 3mol/L hydrochloric acid is added to adjust the pH value to 2.5. Stirring for 30min, and filtering to obtain wet succinyl protein solid. Adding the wet succinyl protein solid into a 5L reaction bottle, adding 500g of purified water, adding 2.5mol/L sodium hydroxide solution while stirring to adjust the pH value to 4.2 so as to dissolve the succinyl protein, filtering, and adding a proper amount of water into the obtained filtrate so that the total volume of the succinic acid protein solution is 600 ml. Taking a proper amount of solution, measuring the content of the succinyl protein and measuring the acylation degree of the succinyl protein.
Adding 1.528Kg of 9.5 wt% ferric trichloride solution into the succinyl protein solution, stirring for 30 minutes after adding, and filtering to obtain wet solid of the crude iron protein succinate product.
And adding the wet solid of the crude iron protein succinate obtained in the previous step into a 5L reaction bottle, adding 1.2Kg of purified water, and stirring to obtain a suspension. Adding 2.5mol/L sodium hydroxide aqueous solution while stirring to adjust the pH value to be 8.5. Primarily filtering the reaction solution by a 5-micron filter membrane, finely filtering by a 1-micron filter membrane, adding 3mol/L hydrochloric acid into the obtained filtrate while stirring to adjust the pH value to 2.5, continuously stirring for 30 minutes, filtering, washing the obtained solid until the pH value of an eluate is 7 to obtain a wet iron protein succinate, granulating the wet iron protein succinate by a 50-mesh swinging granulator, drying at 30 ℃ for 0.5h in a boiling dryer, and then heating to 70 ℃ for 2h to obtain 101.9g of iron protein succinate.
Example 3
A preparation method of iron protein succinate comprises the following steps:
to a 5L reaction flask, 1.2Kg of purified water was added, and 100g of casein was added with stirring. After the addition, the mixture was stirred for 0.5 hour, and 2.5mol/L sodium hydroxide solution was added while stirring, and the pH was adjusted to 7.5 to dissolve casein. A total of 300g of succinic anhydride was added to the casein solution 20 times, and after completion of one hour of addition, 2.5mol/L sodium hydroxide solution was added while adding succinic anhydride to maintain the pH of the reaction solution at 7.5. After the succinic anhydride is added, stirring is continued for 30min, and 3mol/L hydrochloric acid is added to adjust the pH value to 2.5. Stirring for 30min, and filtering to obtain wet succinyl protein solid. Adding the wet succinyl protein solid into a 5L reaction bottle, adding 500g of purified water, adding 2.5mol/L sodium hydroxide solution while stirring to adjust the pH value to 4.1 so as to dissolve the succinyl protein, filtering, and adding a proper amount of water into the obtained filtrate so that the total volume of the succinic acid protein solution is 600 ml. Taking a proper amount of solution, measuring the content of the succinyl protein and measuring the acylation degree of the succinyl protein.
Adding 1.528Kg of 9.5 wt% ferric trichloride solution into the succinyl protein solution, stirring for 30 minutes after adding, and filtering to obtain wet solid of the crude iron protein succinate product.
And adding the wet solid of the crude iron protein succinate obtained in the previous step into a 5L reaction bottle, adding 1.2Kg of purified water, and stirring to obtain a suspension. Adding 2.5mol/L sodium hydroxide aqueous solution while stirring to adjust the pH value to be 8.5. Primarily filtering the reaction solution by a 5-micron filter membrane, finely filtering by a 1-micron filter membrane, adding 3mol/L hydrochloric acid into the obtained filtrate while stirring to adjust the pH value to 2.5, continuously stirring for 30 minutes, filtering, washing the obtained solid until the pH value of an eluate is 7 to obtain a wet iron protein succinate, granulating the wet iron protein succinate by a 50-mesh swinging granulator, drying at 30 ℃ for 0.5h in a boiling dryer, and then heating at 70 ℃ for 2h to obtain 80.3g of iron protein succinate.
Example 4
A preparation method of iron protein succinate comprises the following steps:
to a 5L reaction flask, 1.2Kg of purified water was added, and 100g of casein was added with stirring. After the addition, the mixture was stirred for 0.5 hour, and 2.5mol/L sodium hydroxide solution was added while stirring, and the pH was adjusted to 7.5 to dissolve casein. A total of 300g of succinic anhydride was added to the casein solution 20 times, and after completion of one hour of addition, 2.5mol/L sodium hydroxide solution was added while adding succinic anhydride to maintain the pH of the reaction solution at 7.5. After the succinic anhydride is added, stirring is continued for 30min, and 3mol/L hydrochloric acid is added to adjust the pH value to 2.5. Stirring for 30min, and filtering to obtain wet succinyl protein solid. Adding the wet succinyl protein solid into a 5L reaction bottle, adding 500g of purified water, adding 2.5mol/L sodium hydroxide solution while stirring to adjust the pH value to 4.8 so as to dissolve the succinyl protein, filtering, and adding a proper amount of water into the obtained filtrate so that the total volume of the succinic acid protein solution is 600 ml. Taking a proper amount of solution, measuring the content of the succinyl protein and measuring the acylation degree of the succinyl protein.
Adding 1.528Kg of 9.5 wt% ferric trichloride solution into the succinyl protein solution, stirring for 30 minutes after adding, and filtering to obtain wet solid of the crude iron protein succinate product.
And adding the wet solid of the crude iron protein succinate obtained in the previous step into a 5L reaction bottle, adding 1.2Kg of purified water, and stirring to obtain a suspension. Adding 2.5mol/L sodium hydroxide aqueous solution while stirring to adjust the pH value to be 8.5. Primarily filtering the reaction solution by a 5-micron filter membrane, finely filtering by a 1-micron filter membrane, adding 3mol/L hydrochloric acid into the obtained filtrate while stirring to adjust the pH value to 2.5, continuously stirring for 30 minutes, filtering, washing the obtained solid until the pH value of an eluate is 7 to obtain a wet iron protein succinate, granulating the wet iron protein succinate by a 50-mesh swinging granulator, drying at 30 ℃ for 0.5h in a boiling dryer, and then heating to 70 ℃ for 2h to obtain 102.8g of iron protein succinate.
Example 5
A preparation method of iron protein succinate comprises the following steps:
to a 5L reaction flask, 1.2Kg of purified water was added, and 100g of casein was added with stirring. After the addition, the mixture was stirred for 0.5 hour, and 2.5mol/L sodium hydroxide solution was added while stirring, and the pH was adjusted to 7.5 to dissolve casein. A total of 300g of succinic anhydride was added to the casein solution 20 times, and after completion of one hour of addition, 2.5mol/L sodium hydroxide solution was added while adding succinic anhydride to maintain the pH of the reaction solution at 7.5. After the succinic anhydride is added, stirring is continued for 30min, and 3mol/L hydrochloric acid is added to adjust the pH value to 2.5. Stirring for 30min, and filtering to obtain wet succinyl protein solid. Adding the wet succinyl protein solid into a 5L reaction bottle, adding 560g of purified water, adding 2.5mol/L sodium hydroxide solution while stirring to adjust the pH value to 4.5 so as to dissolve the succinyl protein, filtering, and adding a proper amount of water into the obtained filtrate so that the total volume of the succinic acid protein solution is 700 ml. Taking a proper amount of solution, measuring the content of the succinyl protein and measuring the acylation degree of the succinyl protein.
Adding 1.528Kg of 9.5 wt% ferric trichloride solution into the succinyl protein solution, stirring for 30 minutes after adding, and filtering to obtain wet solid of the crude iron protein succinate product.
And adding the wet solid of the crude iron protein succinate obtained in the previous step into a 5L reaction bottle, adding 1.2Kg of purified water, and stirring to obtain a suspension. Adding 2.5mol/L sodium hydroxide aqueous solution while stirring to adjust the pH value to be 8.5. Primarily filtering the reaction solution by a 5-micron filter membrane, finely filtering by a 1-micron filter membrane, adding 3mol/L hydrochloric acid into the obtained filtrate while stirring to adjust the pH value to 2.5, continuously stirring for 30 minutes, filtering, washing the obtained solid until the pH value of an eluate is 7 to obtain a wet iron protein succinate, granulating the wet iron protein succinate by a 50-mesh swinging granulator, drying at 30 ℃ for 0.5h in a boiling dryer, and then heating to 70 ℃ for 2h to obtain 105.2g of iron protein succinate.
Example 6
A preparation method of iron protein succinate comprises the following steps:
to a 5L reaction flask, 1.2Kg of purified water was added, and 100g of casein was added with stirring. After the addition, the mixture was stirred for 0.5 hour, and 2.5mol/L sodium hydroxide solution was added while stirring, and the pH was adjusted to 7.5 to dissolve casein. A total of 300g of succinic anhydride was added to the casein solution 20 times, and after completion of one hour of addition, 2.5mol/L sodium hydroxide solution was added while adding succinic anhydride to maintain the pH of the reaction solution at 7.5. After the succinic anhydride is added, stirring is continued for 30min, and 3mol/L hydrochloric acid is added to adjust the pH value to 2.5. Stirring for 30min, and filtering to obtain wet succinyl protein solid. Adding the wet succinyl protein solid into a 5L reaction bottle, adding 500g of purified water, adding 2.5mol/L sodium hydroxide solution while stirring to adjust the pH value to 4.9 so as to dissolve the succinyl protein, filtering, and adding a proper amount of water into the obtained filtrate so that the total volume of the succinic acid protein solution is 600 ml. Taking a proper amount of solution, measuring the content of the succinyl protein and measuring the acylation degree of the succinyl protein.
Adding 1.528Kg of 9.5 wt% ferric trichloride solution into the succinyl protein solution, stirring for 30 minutes after adding, and filtering to obtain wet solid of the crude iron protein succinate product.
And adding the wet solid of the crude iron protein succinate obtained in the previous step into a 5L reaction bottle, adding 1.2Kg of purified water, and stirring to obtain a suspension. Adding 2.5mol/L sodium hydroxide aqueous solution while stirring to adjust the pH value to be 8.5. Primarily filtering the reaction solution by a 5-micron filter membrane, finely filtering by a 1-micron filter membrane, adding 3mol/L hydrochloric acid into the obtained filtrate while stirring to adjust the pH value to 2.5, continuously stirring for 30 minutes, filtering, washing the obtained solid until the pH value of an eluate is 7 to obtain a wet iron protein succinate, granulating the wet iron protein succinate by a 50-mesh swinging granulator, drying at 30 ℃ for 0.5h in a boiling dryer, and then heating to 70 ℃ for 2h to obtain 104.4g of iron protein succinate.
Example 7
A preparation method of iron protein succinate comprises the following steps:
to a 5L reaction flask, 1.2Kg of purified water was added, and 100g of casein was added with stirring. After the addition, the mixture was stirred for 0.5 hour, and 2.5mol/L sodium hydroxide solution was added while stirring, and the pH was adjusted to 7.5 to dissolve casein. A total of 300g of succinic anhydride was added to the casein solution 20 times, and after completion of one hour of addition, 2.5mol/L sodium hydroxide solution was added while adding succinic anhydride to maintain the pH of the reaction solution at 7.5. After the succinic anhydride is added, stirring is continued for 30min, and 3mol/L hydrochloric acid is added to adjust the pH value to 2.5. Stirring for 30min, and filtering to obtain wet succinyl protein solid. Adding the wet succinyl protein solid into a 5L reaction bottle, adding 900g of purified water, adding 2.5mol/L sodium hydroxide solution while stirring to adjust the pH value to 4.5 so as to dissolve the succinyl protein, filtering, and adding a proper amount of water into the obtained filtrate so that the total volume of the succinic acid protein solution is 1000 ml. Taking a proper amount of solution, measuring the content of the succinyl protein and measuring the acylation degree of the succinyl protein.
Adding 1.528Kg of 9.5 wt% ferric trichloride solution into the succinyl protein solution, stirring for 30 minutes after adding, and filtering to obtain wet solid of the crude iron protein succinate product.
And adding the wet solid of the crude iron protein succinate obtained in the previous step into a 5L reaction bottle, adding 1.2Kg of purified water, and stirring to obtain a suspension. Adding 2.5mol/L sodium hydroxide aqueous solution while stirring to adjust the pH value to be 8.5. Primarily filtering the reaction solution by a 5-micron filter membrane, finely filtering by a 1-micron filter membrane, adding 3mol/L hydrochloric acid into the obtained filtrate while stirring to adjust the pH value to 2.5, continuously stirring for 30 minutes, filtering, washing the obtained solid until the pH value of an eluate is 7 to obtain a wet iron protein succinate, granulating the wet iron protein succinate by a 50-mesh swinging granulator, drying at 30 ℃ for 0.5h in a boiling dryer, and then heating to 70 ℃ for 2h to obtain 104.3g of iron protein succinate.
Example 8
A preparation method of iron protein succinate comprises the following steps:
to a 5L reaction flask, 1.2Kg of purified water was added, and 100g of casein was added with stirring. After the addition, the mixture was stirred for 0.5 hour, and 2.5mol/L sodium hydroxide solution was added while stirring, and the pH was adjusted to 7.5 to dissolve casein. A total of 300g of succinic anhydride was added to the casein solution 20 times, and after completion of one hour of addition, 2.5mol/L sodium hydroxide solution was added while adding succinic anhydride to maintain the pH of the reaction solution at 7.5. After the succinic anhydride is added, stirring is continued for 30min, and 3mol/L hydrochloric acid is added to adjust the pH value to 2.5. Stirring for 30min, and filtering to obtain wet succinyl protein solid. Adding the wet succinyl protein solid into a 5L reaction bottle, adding 1.2Kg of purified water, adding 2.5mol/L sodium hydroxide solution while stirring to adjust the pH value to 7.5, dissolving the succinyl protein, filtering, and adding a proper amount of water into the obtained filtrate to make the total volume of the succinic protein solution be 1500 ml. Taking a proper amount of solution, measuring the content of the succinyl protein and measuring the acylation degree of the succinyl protein.
Adding 1.528Kg of 9.5 wt% ferric trichloride solution into the succinyl protein solution, stirring for 30 minutes after adding, and filtering to obtain wet solid of the crude iron protein succinate product.
And adding the wet solid of the crude iron protein succinate obtained in the previous step into a 5L reaction bottle, adding 1.2Kg of purified water, and stirring to obtain a suspension. Adding 2.5mol/L sodium hydroxide aqueous solution while stirring to adjust the pH value to be 8.5. Primarily filtering the reaction solution by a 5-micron filter membrane, finely filtering by a 1-micron filter membrane, adding 3mol/L hydrochloric acid into the obtained filtrate while stirring to adjust the pH value to 2.5, continuously stirring for 30 minutes, filtering, washing the obtained solid until the pH value of an eluate is 7 to obtain a wet iron protein succinate, granulating the wet iron protein succinate by a 50-mesh swinging granulator, drying at 30 ℃ for 0.5h in a boiling dryer, and then heating to 70 ℃ for 2h to obtain 106.4g of iron protein succinate.
Example 9
A preparation method of iron protein succinate comprises the following steps:
to a 5L reaction flask, 1.2Kg of purified water was added, and 100g of casein was added with stirring. After the addition, the mixture was stirred for 0.5 hour, and 2.5mol/L sodium hydroxide solution was added while stirring, and the pH was adjusted to 7.5 to dissolve casein. A total of 300g of succinic anhydride was added to the casein solution 20 times, and after completion of one hour of addition, 2.5mol/L sodium hydroxide solution was added while adding succinic anhydride to maintain the pH of the reaction solution at 7.5. After the succinic anhydride is added, stirring is continued for 30min, and 3mol/L hydrochloric acid is added to adjust the pH value to 2.5. Stirring for 30min, and filtering to obtain wet succinyl protein solid. Adding the wet succinyl protein solid into a 5L reaction bottle, adding 500g of purified water, adding 2.5mol/L sodium hydroxide solution while stirring to adjust the pH value to 7.5 so as to dissolve the succinyl protein, filtering, and adding a proper amount of water into the obtained filtrate so that the total volume of the succinic acid protein solution is 600 ml. Taking a proper amount of solution, measuring the content of the succinyl protein and measuring the acylation degree of the succinyl protein.
Adding 1.528Kg of 9.5 wt% ferric trichloride solution into the succinyl protein solution, stirring for 30 minutes after adding, and filtering to obtain wet solid of the crude iron protein succinate product.
And adding the wet solid of the crude iron protein succinate obtained in the previous step into a 5L reaction bottle, adding 1.2Kg of purified water, and stirring to obtain a suspension. Adding 2.5mol/L sodium hydroxide aqueous solution while stirring to adjust the pH value to be 8.5. Primarily filtering the reaction solution by a 5-micron filter membrane, finely filtering by a 1-micron filter membrane, adding 3mol/L hydrochloric acid into the obtained filtrate while stirring to adjust the pH value to 2.5, continuously stirring for 30 minutes, filtering, washing the obtained solid until the pH value of an eluate is 7 to obtain a wet iron protein succinate, granulating the wet iron protein succinate by a 50-mesh swinging granulator, drying at 30 ℃ for 0.5h in a boiling dryer, and then heating to 70 ℃ for 2h to obtain 103.7g of iron protein succinate.
Example 10
A preparation method of iron protein succinate comprises the following steps:
to a 5L reaction flask, 1.2Kg of purified water was added, and 100g of casein was added with stirring. After the addition, the mixture was stirred for 0.5 hour, and 2.5mol/L sodium hydroxide solution was added while stirring, and the pH was adjusted to 7.5 to dissolve casein. A total of 300g of succinic anhydride was added to the casein solution 20 times, and after completion of one hour of addition, 2.5mol/L sodium hydroxide solution was added while adding succinic anhydride to maintain the pH of the reaction solution at 7.5. After the succinic anhydride is added, stirring is continued for 30min, and 3mol/L hydrochloric acid is added to adjust the pH value to 2.5. Stirring for 30min, and filtering to obtain wet succinyl protein solid. Adding the wet succinyl protein solid into a 5L reaction bottle, adding 1.2Kg of purified water, adding 2.5mol/L sodium hydroxide solution while stirring to adjust the pH value to 4.5, dissolving the succinyl protein, filtering, adding a proper amount of water into the obtained filtrate to make the total volume of the succinic protein solution be 1500 ml. Taking a proper amount of solution, measuring the content of the succinyl protein and measuring the acylation degree of the succinyl protein.
Adding 1.528Kg of 9.5 wt% ferric trichloride solution into the succinyl protein solution, stirring for 30 minutes after adding, and filtering to obtain wet solid of the crude iron protein succinate product.
And adding the wet solid of the crude iron protein succinate obtained in the previous step into a 5L reaction bottle, adding 1.2Kg of purified water, and stirring to obtain a suspension. Adding 2.5mol/L sodium hydroxide aqueous solution while stirring to adjust the pH value to be 8.5. Primarily filtering the reaction solution by a 5-micron filter membrane, finely filtering by a 1-micron filter membrane, adding 3mol/L hydrochloric acid into the obtained filtrate while stirring to adjust the pH value to 2.5, continuously stirring for 30 minutes, filtering, washing the obtained solid until the pH value of an eluate is 7 to obtain a wet iron protein succinate, granulating the wet iron protein succinate by a 50-mesh swinging granulator, drying at 30 ℃ for 0.5h in a boiling dryer, and then heating to 70 ℃ for 2h to obtain 104.1g of iron protein succinate.
Method for measuring acylation degree in each of the above examples
The percentage of amino groups that the casein is acylated in an acylation reaction to the original free amino groups of the casein is called the degree of acylation.
The ninhydrin solution is co-heated with amino acids to produce ammonia, which reacts with ninhydrin and reducing ninhydrin to produce a purple compound. The color intensity is proportional to the amino acid content, and the amino acid content is determined by measuring the absorbance at a wavelength of 570 nm.
The determination method comprises the following steps: accurately measuring 5ml of each of the casein contrast solution and the sample solution, placing the casein contrast solution and the sample solution into a 25ml volumetric flask, adding 1ml of ninhydrin color developing agent, heating in a boiling water bath for 15 minutes, cooling in the water bath to room temperature, and then respectively metering the volume to the scale by using ethanol with the volume fraction of 60%. After leaving for 20 minutes, absorbance at a wavelength of 570nm was measured and blank correction was made. The degree of acylation is calculated as follows:
asp: absorbance of the succinyl protein test solution;
ap: absorbance of the casein control solution;
cp: casein control solution concentration;
csp: concentration of the succinyl protein test solution.
The results of the determination of the degree of acylation of the intermediate succinyl protein in the above examples are shown in the following table:
table 1: results of measuring the degree of acylation of intermediate succinyl protein in examples 1 to 10
| Examples | Example 1 | Example 2 | Example 3 | Example 4 | Example 5 |
| Degree of acylation | 99.5％ | 99.6％ | 99.7％ | 99.5％ | 99.3％ |
| Examples | Example 6 | Example 7 | Example 8 | Example 9 | Example 10 |
| Degree of acylation | 98.3％ | 96.5％ | 95.6％ | 96.8％ | 96.5％ |
Study on characteristics of aqueous solution of iron protein succinate prepared in the above examples
Solubility: dissolving a protein succinic acid iron sample in a proper amount of 2.5mol/L sodium hydroxide solution, adding water to a constant volume, and preparing a solution with the pH value of 8, wherein the ratio of the volume ml of the solution after constant volume to the mass g of the protein succinic acid iron sample is 10: 1. Then, the insoluble matter was dried and measured by vacuum filtration through a 6 μm filter. And calculating the dissolution ratio thereof according to the initial amount to be dissolved.
Viscosity: dissolving the iron protein succinate sample in a proper amount of 2.5mol/L sodium hydroxide solution, adding water for dilution, preparing solutions with pH of 8 and the mass fractions of the iron protein succinate of 5% and 6%, respectively, and then measuring the viscosity of the solutions.
Filter permeability: 5ml of iron protein succinate solution with the mass fraction of 5% under the condition of viscosity measurement is filtered through a filter membrane with the diameter of 3cm and the pore diameter of 0.45 mu m in a vacuum filtration manner.
And (3) sediment: weighing 8g of iron protein succinate sample, dissolving the iron protein succinate sample in a proper amount of 2.5mol/L sodium hydroxide solution to prepare a solution with pH of 8, and adding water to the solution to reach a constant volume of 150 ml. 60ml of the above solution was taken and placed in a centrifuge tube (tube diameter 40mm, height 115cm) and centrifuged at 4000 rpm for 10 minutes, and the volume of the bottom residue was counted.
The results of the study on the characteristics of the iron protein succinate aqueous solution prepared in the above embodiment are shown in tables 2 to 3 below:
table 2: EXAMPLES 1-10 Studies of characteristics of iron protein succinate aqueous solution
The results of the iron protein succinate related index determination prepared in the above examples are shown in the following table:
table 3: EXAMPLES 1-10 results of measurement of iron protein succinate
| Inspection item | Free iron | Iron content | Free succinic acid | Total succinic acid | Chloride compound |
| Example 1 | Not detected out | 5.1％ | 0.02％ | 9.8％ | <0.1％ |
| Example 2 | Not detected out | 4.9％ | 0.03％ | 9.6％ | <0.1％ |
| Example 3 | Not detected out | 5.0％ | 0.01％ | 9.5％ | <0.1％ |
| Example 4 | Not detected out | 5.3％ | 0.02％ | 9.6％ | <0.1％ |
| Example 5 | Not detected out | 5.2％ | 0.02％ | 9.7％ | <0.1％ |
| Example 6 | Not detected out | 4.8％ | 0.03％ | 9.2％ | <0.1％ |
| Example 7 | Not detected out | 5.1％ | 0.01％ | 9.0％ | <0.1％ |
| Example 8 | Not detected out | 5.4％ | 0.03％ | 8.8％ | <0.1％ |
| Example 9 | Not detected out | 5.3％ | 0.02％ | 8.9％ | <0.1％ |
| Example 10 | Not detected out | 5.3％ | 0.03％ | 8.6％ | <0.1％ |
Claims (1)
1. A preparation method of iron protein succinate is characterized in that: the preparation method comprises the following steps:
(1) adding 1.2kg of purified water into a 5L reaction bottle, and adding 100g of casein under stirring; after the addition, stirring for 0.5 hour, adding 2.5mol/L sodium hydroxide solution while stirring, and adjusting the pH value to be stable to 7.5 to dissolve the casein; adding 300g of succinic anhydride in total into the casein solution for 20 times, and adding 2.5mol/L sodium hydroxide solution while adding the succinic anhydride after one hour is finished to keep the pH value of the reaction solution at 7.5; after the succinic anhydride is added, continuously stirring for 30min, and adding 3mol/L hydrochloric acid to adjust the pH value to 2.5; continuously stirring for 30min, and filtering to obtain wet succinyl protein solid;
(2) adding the wet succinyl protein solid into a 5L reaction bottle, adding 500g of purified water, adding 2.5mol/L sodium hydroxide solution while stirring to adjust the pH value to 4.1 so as to dissolve the succinyl protein, filtering, adding a proper amount of water into the obtained filtrate so that the total volume of the succinic acid protein solution is 600 ml; taking a proper amount of solution, measuring the content of the succinyl protein, and measuring the acylation degree of the succinyl protein;
(3) Adding 1.528kg of 9.5 wt% ferric trichloride solution into the succinyl protein solution, continuing to stir for 30 minutes after the addition is finished, and filtering to obtain a wet solid of a crude product of the iron protein succinate;
(4) adding the wet solid of the crude iron protein succinate obtained in the previous step into a 5L reaction bottle, adding 1.2kg of purified water, and stirring to obtain a suspension; adding 2.5mol/L sodium hydroxide aqueous solution while stirring to adjust the pH value to be 8.5; primarily filtering the reaction solution by a 5-micron filter membrane, finely filtering by a 1-micron filter membrane, adding 3mol/L hydrochloric acid into the obtained filtrate while stirring to adjust the pH value to 2.5, continuously stirring for 30 minutes, filtering, washing the obtained solid until the pH value of an eluate is 7 to obtain a wet iron protein succinate, granulating the wet iron protein succinate by a 50-mesh swinging granulator, drying at 30 ℃ for 0.5h in a boiling dryer, and then heating at 70 ℃ for 2h to obtain 80.3g of iron protein succinate.
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| CN104402984A (en) * | 2014-11-11 | 2015-03-11 | 武汉科福新药有限责任公司 | Preparation method of iron-protein succinylate with high iron content |
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| CN104402984A (en) * | 2014-11-11 | 2015-03-11 | 武汉科福新药有限责任公司 | Preparation method of iron-protein succinylate with high iron content |
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