CN112063676B - Casein purification method and preparation method of iron protein succinate - Google Patents
Casein purification method and preparation method of iron protein succinate Download PDFInfo
- Publication number
- CN112063676B CN112063676B CN202010908958.6A CN202010908958A CN112063676B CN 112063676 B CN112063676 B CN 112063676B CN 202010908958 A CN202010908958 A CN 202010908958A CN 112063676 B CN112063676 B CN 112063676B
- Authority
- CN
- China
- Prior art keywords
- casein
- iron
- protein
- succinate
- protein succinate
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/02—Nutrients, e.g. vitamins, minerals
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4732—Casein
Abstract
The invention belongs to the field of biological medicines, and particularly relates to a casein purification method and a preparation method of iron protein succinate. A method for the purification of casein, the method comprising the steps of: 1) moderately hydrolyzing casein by adopting acid protease or neutral protease; 2) hydrolyzing impurity proteins and polypeptides by combining aminopeptidase and carboxypeptidase; 3) amino acids and oligopeptides produced after hydrolysis are removed according to an isoelectric precipitation method, and the molecular weight of amino acids and oligopeptides having bitter taste is controlled. The casein purified by the method has high utilization rate and reduced bitter taste, and can relieve gastrointestinal discomfort and rapidly release iron. In addition, the purified casein is applied to the preparation method of the iron protein succinate, the protein is acylated to obtain succinyl protein, and the appropriate complexation constant K for combining the succinyl protein and ferric iron is obtained by controlling the appropriate acylation degree, so that the aims of releasing the effective dose of the iron and controlling the cost are fulfilled.
Description
Technical Field
The invention belongs to the field of biological medicines, and particularly relates to a casein purification method and a preparation method of iron protein succinate.
Background
Iron deficiency is a widely prevalent worldwide nutritional deficiency, seriously threatens human health, normal people maintain the balance of iron metabolism in vivo, 1-1.5 mg of iron needs to be taken from the outside every day, and pregnant women and lactating women need to take 2-4 mg of iron every day, so that the pregnant women and the lactating women are more likely to cause iron deficiency.
The most representative iron deficiency disease is iron deficiency anemia, and according to the statistics of the World Health Organization (WHO), one third of people all over the world have iron deficiency anemia, and the proportion of iron deficiency anemia patients in developed countries and developing countries is high. Statistics show that the incidence of iron deficiency anemia in men is 10%, that in women is 20%, the incidence of iron deficiency anemia in pregnant women can reach 40%, and the incidence of iron deficiency anemia in children exceeds 50%.
Iron supplementation treatment began in the uk of the 17 th century, the first generation of iron supplementation preparations represented by ferrous sulfate. Inorganic iron salt preparations including ferrous carbonate, sodium ferric phosphate, ferrous chloride, ferric pyrophosphate, ferric phosphate, etc. are developed as iron supplements. However, the iron supplement preparations of these inorganic salts use iron ions to supplement iron directly, have serious adverse reactions and are not easy to absorb.
The second generation iron supplement agent is small molecular organic acid iron salt, and the representative medicines comprise ferrous lactate, ferrous succinate, ferrous gluconate, ferrous fumarate (ferrous fumarate) and the like. The second generation of iron supplement solves the problem of low iron absorption rate, so that the iron absorption rate is generally improved, but the defects still exist, such as strong iron taste of ferrous lactate, and serious gastrointestinal adverse reactions of nausea, vomiting, epigastric pain and the like can be caused; the ferrous gluconate is astringent in taste and caramel odor, the side effect is similar to that of ferrous lactate, and other second-generation iron supplement agents also have similar adverse reaction.
The third generation of iron supplement represented by sodium ferric ethylenediamine tetraacetate is on the market at the end of the 20 th century, and the iron supplement does not exist in the form of ions any more, but iron and small molecular organic matters are complexed to form a chelate. The iron supplement removes rust smell of the traditional iron supplement, has less obvious stimulation to gastrointestinal tract and has relatively stable property. Representative drugs also include ferric glycinate, ferric threonine, and the like. The third generation iron supplement agent has higher cost and can not be popularized at present, and the iron absorption rate is improved but is still not satisfactory.
In recent years, macromolecular iron supplement preparations mainly comprising polysaccharide iron complex and protein iron complex are developed, and the polysaccharide iron complex is often administered in the form of injection, so that the production cost is high and the use of patients is inconvenient. The iron succinate protein has good antianemic effect, and has good treatment effect on absolute and relative anaemia caused by insufficient iron intake, acute or chronic blood loss, dominant or recessive blood loss caused by infection of various age groups and the like.
Iron (ISP) succinate is a polypeptide iron supplement drug, which was developed in 80 s of mak pharmaceutical factory, italy, and was marketed in italy in 1987 and in more than 20 countries including spain, portugal, greece, argentina, korea, etc. in the world.
The iron protein succinate is reddish brown powder, has no special odor, is a product obtained by acylation of casein and iron ion complexation, wherein the iron ion is combined with an acylation site of the casein through intermolecular acting force, is wrapped in a casein molecular structure, can be dissolved under an alkaline condition and is separated out under an acidic condition.
The iron protein succinate is not digested by pepsin, but is hydrolyzed by trypsin at neutral pH. Because of the properties of the iron protein succinate, the iron contained in the iron protein succinate is protected by a protein membrane and reacts with hydrochloric acid and pepsin in different gastric juice, and the iron ion-free gastrointestinal tract irritation reaction is avoided after the iron protein succinate is taken, so that the gastric mucosa cannot be damaged. The iron protein succinate keeps the sensitivity of casein to pH value, so that the iron protein succinate has intestinal targeting property. Iron in iron protein succinate is initially released in the duodenum, and in particular should be released in the jejunum, as the normal increase in pH causes this compound to become soluble again and the protein membrane to be digested by trypsin. Such iron is very beneficial to the physiological absorption of the organism, but cannot form too high absorption peaks; in fact, it presents a constant tendency to absorb, gradually reaching an optimal plateau of absorption and storage in various parts of the body. Thus, iron protein succinates generally do not create gastrointestinal tolerance problems. In a word, the absorptivity of the iron protein succinate is not influenced by gastric acid reduction and food components, and has no stimulation and corrosion to gastrointestinal mucosa, thereby avoiding adverse reaction of the digestive tract. Is especially suitable for treating anemia of pregnant and lactating women. Is also suitable for patients with digestive tract ulcer and iron deficiency anemia.
The protein constituting the iron protein succinate is mainly casein. Casein has a complex structural composition, including four types of α s1-, α s2-, β -and κ/pseudo- κ -casein. However, due to the complexity of the casein source and purification process, food grade casein contains no more than 20% of contaminating proteins or polypeptides in addition to the four types of casein described above. Due to the existence of the impurities, the casein has bad flavors such as bitter taste, astringent taste, spicy taste, ammonia taste and the like, and the patient compliance is poor after the casein is prepared into a solution for direct administration, so that the long-term taking willingness of the infant patient is particularly seriously influenced.
Patent CN107090011A states that a surprising advantage can be obtained in the production of iron caseinsuccinylate if dietary casein, which has been subjected to a purification process aimed at removing protein impurities, inorganic impurities and/or increasing the phosphorus/nitrogen ratio (P/N ratio), is used as starting material. However, this patent only considers that the amount of said protein impurities is below about 15%, preferably below about 10%, and reduces the protein impurities by means of physical purification, but does not eliminate them completely or reduce the impurity content to very low levels.
At present, the ferric protein succinate oral solution is on the market at home, but related patents mostly focus on preparation development, the research on the preparation method and the representation of the ferric protein succinate is less, and the cognition on the microstructure of the ferric protein succinate is very little. Therefore, the development of a preparation method of the proper iron protein succinate and the quality control have practical significance.
Disclosure of Invention
In order to solve the above technical problems, an object of the present invention is to provide a method for purifying casein, and another object is to use the purified casein for the preparation of iron protein succinate, and characterization of the iron protein succinate, especially the characterization of microstructure. The purification method mainly adopts an enzymolysis method to purify casein, has mild conditions, high safety, low cost and easy control of hydrolysis, improves the utilization rate of the casein purified by the method, reduces the bitter taste, relieves the discomfort of gastrointestinal tracts and can quickly release iron. In addition, the preparation method of the iron protein succinate adopts purified casein to be acylated to obtain succinyl protein, and obtains the appropriate complexation constant K for combination of the succinyl protein and ferric iron by controlling the appropriate acylation degree, thereby achieving the effective dosage of releasing iron and controlling the cost. The method has the advantages of simple preparation method and low production cost, and can be well applied to the preparation of the protein iron-supplementing medicaments.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for the purification of casein, the method comprising the steps of:
1) adopting acid or neutral protease to moderately hydrolyze casein;
2) hydrolyzing impurity proteins and polypeptides by combining aminopeptidase and carboxypeptidase;
3) removing amino acids and oligopeptides generated after hydrolysis according to isoelectric precipitation method, and controlling molecular weight of amino acids and oligopeptides with bitter taste.
Preferably, the protease used in the hydrolysis in step 1) is alpha-chymotrypsin.
Preferably, the content of α s 1-casein in the casein composition after hydrolysis in step 1) is less than 40%, preferably less than 30%; the beta-casein content is less than 40%, preferably less than 30%.
Preferably, the molecular weight of the bitter amino acid and oligopeptide in the step 3) is controlled within the range of 500-3000 Da, and the content of the bitter amino acid and oligopeptide is reduced to below 0.5%, preferably below 0.3% after precipitation and purification by an isoelectric point precipitation method.
Preferably, the pH of the product obtained in step 3) is 4.0-5.0 by isoelectric precipitation.
Preferably, the weight average molecular weight MW of the finally obtained casein is 10,000-30,000 Da, preferably 15,000-25,000 Da.
Further, the application provides casein prepared by the method.
Further, the application provides an application of the casein in preparation of iron-supplementing protein medicines.
Preferably, the protein iron-supplementing medicine in the application is iron protein succinate.
Further, the present application also provides a method for preparing iron proteinsuccinate, comprising the following steps:
1) weighing the casein, and adding alkali to dissolve the casein;
2) after the protein is completely dissolved, adding succinic anhydride, and acylating to obtain a crude product of the succinic protein, wherein the acylation degree of casein is 80-100%, and the complex constant K of the succinyl protein is 0.93-1.87;
3) adding hydrochloric acid into the crude succinyl protein solution, slowly acidifying until the pH value is 2.0-4.5 and the precipitate is 2.0-2.5 near the isoelectric point, redissolving, and repeating for multiple times until refined succinyl protein is obtained;
4) adding alkali into the refined succinyl protein to dissolve the refined succinyl protein, adding a solution containing ferric ions, and reacting to obtain a crude product of the iron protein succinate;
5) adding hydrochloric acid into the crude iron protein succinate solution, slowly acidifying until the pH value is 2.0-4.5 and the isoelectric point is 2.0-2.5, precipitating, redissolving, and repeating for multiple times until refined iron protein succinate is obtained;
6) the iron protein succinate is dried by a suitable method.
As a specific embodiment, a method for preparing iron protein succinate comprising casein purified by the method comprises the following steps:
1) weighing a certain amount of casein, and adding a proper amount of alkali to dissolve the casein;
2) heating to 45 ℃ after the protein is completely dissolved, adding a proper amount of succinic anhydride, adding a proper amount of sodium carbonate solution, maintaining the pH value at about 8.0, and acylating for 2-3 hours to obtain a crude product of the succinic acid protein;
3) adding hydrochloric acid into the crude succinyl protein solution, slowly acidifying to a certain pH value, separating out a white precipitate, separating out the precipitate in a filtering mode, washing the precipitate with water and redissolving the precipitate with a sodium carbonate solution, adding hydrochloric acid, acidifying to a certain pH value, separating out a white precipitate, and repeating for multiple times until refined succinyl protein is obtained;
4) dissolving refined succinyl protein with appropriate amount of alkali, heating to 45 deg.C, adding appropriate amount of ferric trichloride, adding appropriate amount of alkali, maintaining pH at about 8.0, and reacting for 3 hr to obtain crude product of iron protein succinate;
5) adding hydrochloric acid into the crude iron protein succinate product, slowly acidifying to a certain pH value, separating out a white precipitate, separating the precipitate in a filtering mode, washing the precipitate with water and redissolving the precipitate with a sodium carbonate solution, adding hydrochloric acid, acidifying to a certain pH value, separating out a white precipitate, and repeating for multiple times until refined iron protein succinate is obtained;
6) the iron protein succinate is dried by a suitable method.
Preferably, the base in step 1) and step 4) comprises one or more of sodium hydroxide, sodium carbonate, sodium bicarbonate, potassium hydroxide, potassium carbonate and potassium bicarbonate.
Preferably, the succinyl protein obtained in the step 2) has a complexation constant K of 0.93-1.87, preferably 1.12-1.75.
Preferably, the acylation degree of the casein in the step 2) is 80-100%, preferably 85-95%.
Preferably, hydrochloric acid is added in the step 3) to slowly acidify to pH 2.0-4.5, and the isoelectric point is preferably 2.0-2.5, and the precipitation is complete under the condition.
Preferably, hydrochloric acid is added in the step 5) to slowly acidify to pH 2.0-4.5, and the isoelectric point is preferably 2.5-3.0, and the precipitation is complete under the condition.
Preferably, the viscosity of the iron protein succinate obtained in the step 5) is 10-20 cP, and preferably 10-15 cP under the conditions of the concentration of 5% and the temperature of 25 ℃.
Preferably, the method for drying the iron protein succinate in the step 6) is a spray drying method, a freeze drying method and a spray freeze drying method, preferably the spray drying method, and the particle size of the iron protein succinate obtained by the method is 100-300 nm, preferably 150-250 nm.
Further, the application provides the iron protein succinate prepared by the method.
Further, the application provides an application of the preparation method of the iron protein succinate in preparation of an iron protein succinate solution.
In the present application alpha-chymosin is used to hydrolyse casein. As the casein products produced abroad all use milk as raw materials, the casein is obtained by purification. The purification methods mainly include enzymatic hydrolysis, chemical purification (acid hydrolysis, alkaline hydrolysis) and physical purification (heat, high pressure). Physical means is generally used as an auxiliary means to chemical means because precipitation is incomplete. Since the alkaline purification product has peculiar smell, while the acid purification is easy to denature the protein, generate toxic substances, seriously damage the safety of the protein, and have great difficulty in controlling degradation conditions and products, the chemical purification can only be used as a primary treatment mode. Compared with a material flow and chemical purification mode, the enzymolysis method has the advantages of mild production conditions, high safety, low cost and easy control of hydrolysis conditions, and can carry out positioning hydrolysis to produce specific protein or polypeptide under specific conditions. On the other hand, after the α s 1-casein enters the stomach, it is easy to form a non-digestible clot in the stomach under the action of rennet and gastric acid, which affects the digestive absorption of protein and further causes stomach discomfort, abdominal distension, etc. In addition, beta-lactoglobulin is liable to cause allergic reactions in infants. The utilization rate of protein can be improved by adopting protease to carry out moderate enzymolysis on cow milk protein, and adverse reaction of infants is avoided. Therefore, the casein used by the invention is casein which is subjected to enzyme hydrolysis after being primarily purified by adopting a physical and chemical mode.
Proteases capable of hydrolyzing casein include alkaline proteases (Alcalase, trypsin), acid proteases (pepsin, chymotrypsin) and neutral proteases (papain). When the alkaline protease hydrolyzes the protein, deamidation reaction is easy to occur, so that the product has ammonia taste or spicy taste. The casein used in the present invention is therefore preferably casein after hydrolysis by an acid protease or a neutral protease.
The study showed that casein was suspended in the aqueous system in the form of micelles. Regarding the external shape of the micelle, electron microscope observation showed that the casein micelle had a roughly spherical structure. The "Waugh model" considers that the casein micelle exhibits a core-shell pattern and has the presence of a subgolloid structure, i.e. the subgolloid structure consisting of only alpha and beta-casein constitutes the core, the shellConsisting of a kappa-casein molecule and a phosphoserine residue (Ca)2+Tightly bonded) to form a protective layer. The chymosin mainly has the functions of decomposing peptide bonds of casein in milk, partially decomposing casein layer on the surface of casein micelle to form pseudo kappa-casein and glycomacropeptide, losing stability of casein micelle and using Ca2+In the process, protein is coagulated to form curd protein. The alpha-chymotrypsin is used for decomposing milk protein, so that the bitterness of the product is reduced.
In the application, the protease is adopted to carry out enzymolysis on the milk protein properly, otherwise, after the kappa-casein layer is completely decomposed, the alpha-casein and the beta-casein are continuously decomposed, so that the casein is degraded into the polypeptide. Research shows that the particle size distribution of the alpha-casein zymolyte is more and more concentrated, and the particle size is smaller and smaller. Within 30min of enzymolysis, the particle size distribution of beta-casein zymolyte is not obviously changed, and the particle size of kappa-casein is obviously changed within the first 10min and then has a similar change trend with alpha-casein. That is, the ratio of α s 1-and β -casein in the casein should not be too high for the purpose of avoiding gastric discomfort and for the purpose of rapid iron release in the intestine.
In the present application, the combination of aminopeptidase and carboxypeptidase is also used to hydrolyze contaminating proteins or polypeptides. This is because, in general, the more hydrophobic amino acid residues in a protein or peptide chain, the more likely the bitter taste of the substance is, and the more bitter taste is significantly enhanced by amino acids having aromatic side chains (phenylalanine Phe, tryptophan Trp, tyrosine Tyr). In addition, some basic amino acid residues such as Arg (arginine) also contribute to bitter taste, especially when the basic amino acid residue is located at the N-terminus, the bitter taste is stronger. Because free amino acids and oligopeptides have much less bitter taste than proteins and polypeptides, the hydrolysis product is facilitated to generate free amino acids by the action of exoproteases (aminopeptidase and carboxypeptidase), and the bitter taste can be reduced by cutting off hydrophobic amino acids from the tail end by exoproteases. Aminopeptidases can selectively release amino acids from the N-terminus of polypeptides and proteins by hydrolysis. Carboxypeptidases are capable of selectively releasing amino acids by hydrolysis of the C-terminus of polypeptides and proteins.
The present application employs isoelectric precipitation to remove amino acids and oligopeptides because of their minimal solubility near their isoelectric point. The protein hydrolysate is adjusted to the vicinity of isoelectric point, so that the bitter and astringent substances are reduced in water solubility to form precipitate, and then removed by centrifugation or filtration. However, some of low molecular weight amino acids and oligopeptides produced by hydrolysis of foreign proteins or polypeptides are also responsible for bitterness and astringency. However, not all amino acids and oligopeptides have bitter and astringent taste. On the other hand, even when isoelectric precipitation is used, amino acids and oligopeptides cannot be completely removed, and thus, it is necessary to control the content of bitter and astringent amino acids and oligopeptides to a suitable range. Finally, the casein obtained should have a certain molecular weight. Too high a molecular weight means incomplete hydrolysis of the milk protein, low casein content, high impurity protein content. An excessively low molecular weight means that casein is excessively hydrolyzed to produce a large amount of polypeptides, and the polypeptides are difficult to form the microstructure of iron protein succinate, so that the therapeutic effect is not achieved.
The binding of ferric iron to casein in this application depends on its nature (α s1-, α s2-, β -and κ -casein) and the form of the iron. It is estimated that only 7 iron ions can be bound per 1 beta-casein molecule, and that several other structures have limited ability to bind iron. By succinylation of casein, the complexing sites of protein and iron can be greatly improved. However, too high a complexation constant means that succinyl protein binds tightly to iron, which is detrimental to iron release; too low a complexation constant means that succinyl protein is not tightly bound to iron, and the amount of iron bound to the protein is low, which means that the amount of iron proteinsuccinate needs to be increased in order to achieve an effective iron dose, which will result in the possibility of dissolution during formulation production and drive up costs; meanwhile, iron is easy to separate out in the storage process of the iron protein succinate, so that the free iron exceeds the standard and the drug effect is reduced. On the other hand, the presence of the complexation constant K also places a limit on the degree of succinylation of casein. The excessively high acylation rate improves the combination rate of protein and iron, which means that the iron content in the iron protein succinate is increased and the protein content is reduced, so that the instability of the iron protein succinate molecule is increased, the protein membrane is easy to break, and partial iron is released to cause the excessive free iron, thereby not only influencing the drug effect, but also influencing the taste. Too low an acylation rate will reduce the amount of iron bound to the protein, which means that to achieve an effective iron dose, the amount of iron protein succinate needs to be increased, which will lead to the possibility of dissolution during formulation production and drive up costs. The acylation degree of casein is determined by adopting an o-phthalaldehyde method, and the acylation degree can be more than 100% in theory because succinic acid is dibasic acid.
The reaction time in the present application may be extended due to the expansion of the production lot, and therefore, the reaction time should not be strictly limited. And in order to achieve the filtration step in the iron protein succinate oral solution production process, the iron protein succinate must have a low viscosity.
The beneficial effect of this application lies in: 1) the alpha-chymotrypsin is adopted to decompose the milk protein, so that the bitterness and astringency of the product are reduced; 2) the method is characterized in that the method adopts a mode of combining aminopeptidase and carboxypeptidase to hydrolyze impurity protein or polypeptide, removes protein or polypeptide impurities which bring bad flavor, and improves the taste of the product; 3) the appropriate casein molecular weight can form the microstructure of the iron protein succinate, thereby achieving better treatment effect; 4) by succinylation of casein, the appropriate acylation degree and the complexing constant K can greatly improve the complexing site of protein and iron, increase the stability of iron protein succinate molecules, facilitate the release of iron, reduce the cost and improve the drug effect.
Drawings
FIG. 1: release of iron protein succinate in different media.
FIG. 2: releasing iron protein succinate made of casein with the same proportion of protein.
FIG. 3: composition of iron protein succinate (GPC).
FIG. 4: release of iron protein succinates of different molecular weights.
FIG. 5: release of iron protein succinates with different complexation constants.
FIG. 6: release of iron protein succinates of different viscosities.
FIG. 7: release of iron protein succinates of different particle sizes.
Figure 8a, 8b particle size distribution of iron protein succinate.
Figure 9a microscopic morphology (SEM) of iron protein succinate after freeze-drying (20% mannitol contained) and 9b microscopic morphology (SEM) of iron protein succinate (solution, 1%).
Detailed Description
The invention is further illustrated by the following specific examples.
EXAMPLE 1 extraction and purification of Casein
(1) Taking a proper amount of milk, cooling to about 4 ℃, centrifuging for 10min at 3000r/min, and removing an upper fat layer;
(2) the lower layer emulsion is put into a beaker and heated to about 40 ℃, a proper amount of hydrochloric acid is slowly added under stirring, the pH is adjusted to about 4.7, and a large amount of floccule precipitate is generated. Cooling to room temperature, centrifuging at 3000r/min for 10min, and discarding the supernatant.
(3) Washing the precipitate with purified water for three times, centrifuging at 3000r/min for 10min, and discarding supernatant to obtain crude casein.
(4) Mashing the precipitate, adding a proper amount of ethanol, stirring, standing for a moment, and transferring the suspension to a Buchner funnel for suction filtration; the filter cake is continuously mashed, a proper amount of ether is added, the mixture is stirred and then stands for a moment, and the suspension is transferred to a Buchner funnel for suction filtration.
(5) Mashing the filter cake, and adding sodium hydroxide for dissolving; then slowly adding hydrochloric acid until precipitation is generated, continuously adding hydrochloric acid until the pH value is about 2.0, stirring and dissolving, and continuously adding hydrochloric acid during the period to keep the pH value about 2.0; heating to 45 deg.C, adding alpha-chymotrypsin (enzyme to substrate ratio of 0.3), and hydrolyzing for 40 min; when the degree of hydrolysis is about 10%, the hydrolysis is stopped. The casein composition obtained as described above is α s 1: α s 2: beta: kappa. apprxeq.3: 4: 3.
(6) Adding sodium hydroxide into the casein solution to adjust the pH value to 7.5-8.0, heating to 45 ℃, adding aminopeptidase and carboxypeptidase, and hydrolyzing for 60min to obtain casein with the molecular weight of 10,000-30,000 Da.
(7) Adding appropriate amount of hydrochloric acid into the casein hydrolysate solution, adjusting to the isoelectric point (pH 4.2), and removing amino acids and oligopeptides (500-3000 Da) as hydrolysate by precipitation, wherein the final content is less than 0.3%. The molecular weight of the finally obtained casein is 10,000-30,000 Da.
Example 2 preparation of iron protein succinate
(1) Weighing a certain amount of casein, and adding a proper amount of sodium carbonate solution to dissolve the casein.
(2) And after the protein is completely dissolved, heating to 45 ℃, adding a proper amount of succinic anhydride, adding a proper amount of sodium carbonate solution, maintaining the pH value at about 8.0, and acylating for 2-3 hours (the acylation degree is 90%) to obtain a crude product of the succinic acid protein.
(3) Adding hydrochloric acid into the crude succinate protein solution, slowly acidifying to pH2.2, separating out white precipitate, and separating out precipitate by using a filtration mode; washing the precipitate with water, redissolving with sodium carbonate solution, adding hydrochloric acid, acidifying to pH2.2, and separating out white precipitate; repeating the steps for multiple times until refined succinic acid protein is obtained.
(4) Adding a proper amount of sodium carbonate solution into the refined succinic acid protein to dissolve the succinic acid protein, heating to 45 ℃, adding a proper amount of ferric trichloride into the succinic acid protein, adding a proper amount of sodium carbonate solution, maintaining the pH value to be about 8.0, and reacting for 3 hours to obtain a crude product of the iron protein succinate;
(5) adding hydrochloric acid into the crude product of the iron protein succinate, slowly acidifying until the pH value is 2.7, separating out a white precipitate, and separating the precipitate by using a filtration mode; washing the precipitate with water, redissolving with sodium carbonate solution, adding hydrochloric acid, acidifying to pH2.7, and separating out white precipitate; repeating the steps for multiple times until refined iron protein succinate is obtained.
(6) Drying the iron protein succinate in a spray drying mode, wherein the drying parameters are as follows: the spraying pressure is 0.2MPa, the air inlet temperature is 120 ℃, the air outlet temperature is 90 ℃, and the air inlet quantity is 25m 3/h.
The iron protein succinate is not digested by pepsin, but is hydrolyzed by trypsin at neutral pH. Therefore, the iron contained in the iron protein succinate is protected by the protein membrane and is not reacted with hydrochloric acid and pepsin in gastric juice to be released. As iron protein succinate enters the intestine, it becomes soluble again as a result of the increase in pH and the protein film is digested by trypsin, the patch containing iron protein succinate begins to be released in the duodenum, in particular in the jejunum.
Example 3 iron protein succinate Release Medium
According to the special properties of the product, the release degree of the product is an important standard for distinguishing the quality of the product. The release of iron from iron proteinsuccinate in different media was examined (see figure 1), and the dissolution conditions were as follows: paddle method, 50rpm, 900ml medium.
Wherein, hydrochloric acid solution (pH1.2) is adopted for 0-2 h of the blue curve, phosphate solution (pH6.8) is adopted for 2-4 h, and the iron protein succinate is almost not released under the condition.
And (3) changing the investigation conditions, wherein the red curve is artificial gastric juice for 0-2 h, and the artificial intestinal juice for 2-4 h, so that the iron protein succinate is almost not released in the artificial gastric juice and is rapidly released in the artificial intestinal juice. Therefore, the artificial intestinal juice can be used as a release medium of the iron protein succinate.
Example 4 quality control of iron protein succinate
4.1 degree of Release of iron protein succinate from Casein consisting of proteins of different ratios
The release of iron protein succinate made from casein composed of different ratios of proteins was examined (see figure 1). With α s 1: α s 2: beta: the release speed is gradually increased by the change of the kappa composition ratio (the ratios of alpha s1 and beta are reduced, and the ratio of alpha s2 is increased). α s 1: α s 2: when the beta composition ratio is 3:3:3 to 4:1:4, the release speed is reasonable, and the iron cannot be absorbed in time due to too fast release or insufficient iron absorption amount due to too slow release can be avoided. On the other hand, since the initial ratio of α s1 to β in casein is relatively high, complete elimination thereof is technically difficult and unnecessary.
From fig. 3, it can be found that the prepared iron protein succinate mainly contains 3 components. The proportion of kappa-casein in conventional casein is about 10%, and since kappa-casein is the outermost layer, enzymatic hydrolysis is most rapid, so that the practically obtained iron protein succinate does not contain kappa-casein.
The above results confirm that the α s 1-casein content in the casein composition obtained in example 1 is less than 40%, preferably less than 30%; the beta-casein content is less than 40%, preferably less than 30%.
4.2 Release Rate of iron protein succinate from Casein of different molecular weight
The release of iron protein succinate from casein of different molecular weights was examined (see figure 4). From the release profile, it can be seen that iron protein succinate prepared with too low a molecular weight has a serious burst release phenomenon, because too low a molecular weight means that casein is hydrolyzed too much, more polypeptides are produced, and the polypeptides are difficult to form a protein film and completely wrap iron. Whereas too high a molecular weight produces iron protein succinate that is released too slowly, since high molecular weight means a larger particle size, while a larger particle size means that the iron is embedded deeper and thus more difficult to release. The above results confirm that the casein obtained in example 1 should have a molecular weight of 10,000-30,000 Da, preferably 15,000-25,000 Da.
4.3 Release Rate of iron protein succinate with different complexation constants
The release of iron-protein succinate from casein with different complexation constants was examined (see figure 5). From the release profile it can be seen that there are slow release situations for iron proteinsuccinate made with too small a complexation constant, since a lower complexation constant means that less iron is embedded, causing this part of the iron to be embedded deep in the protein and thus more difficult to release. And the ferric protein succinate prepared by excessively high complexation constant is released too fast and is not beneficial to absorption. The results confirm that the iron complexometric constant K of the protein succinate obtained in example 2 should be 0.93-1.87, preferably 1.12-1.75.
4.4 Release of iron protein succinates of varying viscosity
The release rates of iron protein succinate at different viscosities were examined (see figure 6). The release rate decreases sharply with increasing viscosity of the iron protein succinate solution. The lower viscosity can realize the possibility of filtration in the production process of the iron protein succinate oral solution and ensure the absorption of iron. The above results confirm that the solution viscosity (5%, 25 ℃) of the iron protein succinate obtained in example 2 should be 10 to 20cP, preferably 10 to 15 cP.
4.5 Release of iron protein succinates of different particle size
The release rates of iron protein succinate at different viscosities were examined (see figure 7). Iron proteinsuccinates with too low a particle size are released too quickly to facilitate iron absorption. Iron proteinsuccinate of too large a particle size means that iron is embedded deep in the protein and thus is more difficult to release. The above results confirm that the solution viscosity (5%, 25 ℃) of the iron-protein succinate obtained in example 2 should be 100 to 300nm, preferably 150 to 250 nm.
Example 5 microstructure of iron protein succinate
5.1 particle size distribution of iron protein succinate
The average particle size of the sample was around 225nm (see FIGS. 8a, 8 b). The sample has peaks at about 30nm, 150nm and 1000 nm; the peak height at 30nm is very low, and the influence on the product quality is small and can be ignored. The larger peak at 1000nm indicates that larger size particles are present in the sample, possibly due to agglomeration of small particles. In fact, the data above are derived from the iron protein succinate solution after a period of standing.
However, the iron protein succinate just prepared has the particle size of 150-250 nm, and no large particles exist. This indicates that the iron protein succinate is easily polymerized in solution, so the product should be dried in time after being prepared, and should not be stored in solution for a long time.
5.2 microscopic morphology of iron protein succinates
The microscopic morphology (scanning electron microscopy, SEM) of iron proteinsuccinate is shown in fig. 9. The iron protein succinate after freeze-drying appeared more rounded (see figure 9 a). In the solution, the iron protein succinate is in a micelle state; part of the micelles have the phenomena of adhesion and polymerization, and the correctness of the particle size distribution of 5.1 is also verified.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention, including any reference to the above-mentioned embodiments. Various modifications to these embodiments will be readily apparent to those skilled in the art. The general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (9)
1. A method for purifying casein, comprising the steps of:
1) hydrolyzing casein by adopting alpha-chymotrypsin, wherein the content of alpha s 1-casein in the hydrolyzed casein is less than 40 percent, and the content of beta-casein is less than 40 percent;
2) hydrolyzing impurity proteins and polypeptides by adopting a mode of combining aminopeptidase and carboxypeptidase, wherein the molecular weight range of bitter amino acid and oligopeptide is controlled to be 500-3000 Da;
3) removing amino acids and oligopeptides generated after hydrolysis according to an isoelectric point precipitation method, and reducing the content of the amino acids and oligopeptides to be below 0.5 percent after precipitation and purification by the isoelectric point precipitation method; the casein weight average molecular weight M finally obtainedWThe temperature is 10,000-30,000 Da; the pH value of the isoelectric precipitation method is 4.0-5.0.
2. The method for purifying casein according to claim 1, wherein the casein composition after hydrolysis in step 1) has an α s 1-casein content of less than 30%; the content of beta-casein is less than 30%.
3. The method of claim 1, wherein the casein has a weight average molecular weight MWThe range of 12,500-25,000 Da.
4. Casein prepared by the process of any one of claims 1 to 3.
5. A method for preparing iron protein succinate, which is characterized by comprising the following steps:
1) weighing the casein of claim 4, and adding an alkali to dissolve the casein;
2) after the protein is completely dissolved, adding succinic anhydride, and acylating to obtain a crude product of the succinic protein, wherein the acylation degree of casein is 80-100%, and the complex constant K of the succinyl protein is 0.93-1.87;
3) adding hydrochloric acid into the crude succinyl protein solution, slowly acidifying until the pH value is 2.0-4.5 and the precipitate is 2.0-2.5 near the isoelectric point, redissolving, and repeating for multiple times until refined succinyl protein is obtained;
4) adding alkali into the refined succinyl protein to dissolve the refined succinyl protein, adding a solution containing ferric ions, and reacting to obtain a crude product of the iron protein succinate;
5) adding hydrochloric acid into the crude iron protein succinate solution, slowly acidifying until the pH value is 2.0-4.5 and the isoelectric point is 2.0-2.5, precipitating, redissolving, and repeating for multiple times until refined iron protein succinate is obtained;
6) the iron protein succinate is dried by a suitable method.
6. The method of preparing iron protein succinate according to claim 5, wherein the alkali in step 1) and step 4) comprises one or more of sodium hydroxide, sodium carbonate, sodium bicarbonate, potassium hydroxide, potassium carbonate and potassium bicarbonate.
7. The method for preparing iron protein succinate according to claim 5, wherein the succinyl protein obtained in the step 2) has a complexation constant K of 1.12-1.75; the acylation degree of casein should be 85-95%.
8. The method according to claim 5, wherein the viscosity of the iron protein succinate obtained in step 5) is 10-20 cP at a concentration of 5% and a temperature of 25 ℃.
9. The method according to claim 5, wherein the method for drying the iron protein succinate in step 6) comprises spray drying, freeze drying and spray freeze drying, and the particle size of the iron protein succinate obtained by the method is 100-300 nm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010908958.6A CN112063676B (en) | 2020-09-02 | 2020-09-02 | Casein purification method and preparation method of iron protein succinate |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010908958.6A CN112063676B (en) | 2020-09-02 | 2020-09-02 | Casein purification method and preparation method of iron protein succinate |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112063676A CN112063676A (en) | 2020-12-11 |
| CN112063676B true CN112063676B (en) | 2021-11-12 |
Family
ID=73665780
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010908958.6A Active CN112063676B (en) | 2020-09-02 | 2020-09-02 | Casein purification method and preparation method of iron protein succinate |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112063676B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114632143A (en) * | 2020-12-16 | 2022-06-17 | 武汉科福新药有限责任公司 | Protein iron succinate oral liquid and preparation method thereof |
| CN114634562B (en) * | 2020-12-16 | 2023-09-29 | 武汉科福新药有限责任公司 | Preparation method of protein iron succinate |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1057127C (en) * | 1993-04-23 | 2000-10-04 | 北京大学 | All-hydrolytic method for fixed composite exopeptidase and bitter-removing method thereof |
| PT2588488T (en) * | 2010-06-30 | 2017-06-26 | Italfarmaco Spa | Iron (iii) caseinsuccinylate and method for the preparation thereof |
| CN102379359B (en) * | 2010-08-30 | 2013-09-25 | 内蒙古伊利实业集团股份有限公司 | Casein enzymatic hydrolysis method and hydrolysis product |
| RU2581911C2 (en) * | 2014-03-20 | 2016-04-20 | Федеральное государственное бюджетное образовательное учреждение высшего образования "Кемеровский государственный сельскохозяйственный институт" (ФГБОУ ВО Кемеровский ГСХИ) | Method of producing functional additive based on casein hydrolysate |
| CN106589103A (en) * | 2016-12-21 | 2017-04-26 | 武汉华科大生命科技有限公司 | Preparation method of iron proteinsuccinylate |
-
2020
- 2020-09-02 CN CN202010908958.6A patent/CN112063676B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN112063676A (en) | 2020-12-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112063676B (en) | Casein purification method and preparation method of iron protein succinate | |
| JP5619014B2 (en) | High calorie enteral preparation | |
| JPH0790002A (en) | Polysaccharide complex formed by cation | |
| US20080299285A1 (en) | Process For The Preparation Of Ferri-Succinylcasein | |
| PT86357B (en) | METHOD FOR PREPARING A MIXTURE OF RICH PEPTIDES IN DI-E TRI-PEPTIDOS, USEFULALLY USED IN ARTIFICIAL NUTRITION AND DIETETIC, AND INSTALLATION FOR THE EXECUTION OF THE REFERENCE PROCESS | |
| JP2000063284A (en) | Inhibitor against recurrence of inflammatory enteritis | |
| CN1570127A (en) | High F value oligopeptide production method by corn protein enzymolysis | |
| JP3691685B2 (en) | Blood sugar level rise inhibitor | |
| JP2737790B2 (en) | Food containing silk protein hydrolyzate and method for producing the same | |
| JP3425813B2 (en) | Gelled food composition | |
| WO2006093166A1 (en) | Enzyme-treated product comprising rice peptide and composition for improving physiological activity comprising the product | |
| JPH07255398A (en) | Oral and perrectal nutrient composition having high preservative stability and palatability | |
| RU2270576C2 (en) | Biological method for production of food-grade preparation based on haem iron and food-grade preparation obtained thereby | |
| JP6347954B2 (en) | Thorough nutritional composition | |
| EP0665012B1 (en) | Antiallergy agent and nutritional composition containing glutamine | |
| JP4191261B2 (en) | Iron casein complex and its production method | |
| CN1204651A (en) | Preparation method for spirulina polypeptide and medicament containing these polypeptide and application | |
| JP3410235B2 (en) | Novel protein composition and nutritional composition | |
| JP3397258B2 (en) | Calcium absorption promoting water-soluble fraction, composition containing the same, and calcium absorption promoting additive | |
| JP5312780B2 (en) | Food / drink and pharmaceutical composition for reducing blood ammonia concentration | |
| JPH0515339A (en) | Protein-like composition, nourishing medicine for serious stress and nourishing medicine for hepatic disease | |
| CN1215774C (en) | Albumen oligo peptide and preparing method thereof | |
| JP2001233783A (en) | Preoperative nutrient agent | |
| JPH04182435A (en) | Composition for patient with hepatopathy | |
| JP3655732B2 (en) | Food containing plant germ extract |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |