CN114524756B - Hydroxy magnesium methionine and preparation method thereof - Google Patents
Hydroxy magnesium methionine and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a hydroxy methionine magnesium and a preparation method thereof, and porous particle hydroxy methionine magnesium is obtained, wherein the particle size of the porous particle is 200-600 mu m, and the porosity of the porous particle is 50-60%. The hydroxyl methionine magnesium is applied to a culture system for amplifying hematopoietic stem cells, and can obviously improve the total cell quantity obtained by in-vitro amplification of hematopoietic stem cells of fresh blood samples.
Description
Technical Field
The invention belongs to the technical field of cell biology, and particularly relates to a magnesium hydroxy methionine and a preparation method thereof.
Background
Stem cells (StemCell) are cells that have self-renewing capacity and multipotent differentiation potential. Stem cells can be classified into three major categories according to differentiation potential: totipotent stem cells, pluripotent stem cells, and monopotent stem cells. Stem cells can be further divided into, according to developmental stage: embryonic stem cells and adult stem cells. Stem cells used in clinical research and applications are now mostly adult stem cells due to ethical limitations. Adult stem cells are derived from many tissues and organs of adult animals, including hematopoietic stem cells, neural stem cells, adipose stem cells, bone marrow mesenchymal stem cells, umbilical cord stem cells, cord blood stem cells, and the like.
Hematopoietic stem cells are the only stem cell type widely used in clinical therapy to date. The hematopoietic stem cell transplantation technology can treat various blood diseases such as leukemia, lymphoma and the like, and even can produce curative effects on metabolic diseases, congenital immunodeficiency, diabetes and the like; statistically, there are over 40000 cases of hematopoietic stem cell transplantation treatment each year worldwide, wherein the majority of hematopoietic stem cell donors are derived from donor bone marrow and mobilized peripheral blood stem cells; despite the great success of this technique, 70% of patients cannot obtain a suitable donor due to the stringent pairing requirements of Human Leukocyte Antigen (HLA) pairing.
The umbilical cord blood hematopoietic stem cells have relatively low requirement on HLA (high level architecture) matching, low immunogenicity, convenient acquisition and abundant sources, and become a large source of hematopoietic stem cell transplantation donors. After the first umbilical cord blood hematopoietic stem cell transplantation operation successfully cures Faconi boy patients in the 80 s of the 20 th century, the umbilical cord blood hematopoietic stem cell transplantation cases increase year by year. It is counted that umbilical cord blood hematopoietic stem cell transplantation cases have exceeded 30000 cases worldwide and are increasing. At present, the bottleneck of the cord blood hematopoietic stem cell transplantation technology is that the cell content is low, and the number of hematopoietic stem cells and progenitor cells contained in one cord blood is insufficient to quickly restore the immune system of an adult patient, so that the opportunistic infection mortality rate is increased. At present, a temporary strategy is double cord blood transplantation, namely, one patient receives two cord blood transplantation after marrow removal, but the HLA matching difficulty of a donor is increased, so that a method for amplifying cord blood hematopoietic stem cells is needed to obtain sufficient hematopoietic stem cells for transplantation.
Numerous attempts have been made to expand cord blood hematopoietic stem cells in vitro, but none have achieved the desired results. Early days, cytokines in the blood were used to culture hematopoietic stem cells, resulting in cell differentiation and reduced transplantation. Later, it was found that Wnt signaling molecules, notch ligands, retinoic acid antagonists, etc. in the bone marrow hematopoietic stem cell microenvironment were effective in expanding cd34+ hematopoietic stem/progenitor cells. Activating Wnt signaling pathway with CHIR99021 or BIO to maintain the transplantation ability of hematopoietic stem cells cultured in vitro; in contrast, when DLL1, DSL1, or the like is added to the culture system of hematopoietic stem cells, the hematopoietic stem cells can be moderately expanded by activating Notch signals. In another study, PTN secreted by bone marrow endothelial stromal cells was also found to slightly expand hematopoietic stem cells. Under physiological conditions, hematopoietic stem cells are under hypoxia, and oxygen stress generated by in vitro culture impairs hematopoietic stem cell self-renewal and transplantation by increasing ROS levels; it was found that the addition of antioxidants and inhibition of mTOR counteracts these lesions.
Hydroxy methionine is also called methionine hydroxy analogue, is an important industrial raw material and medical intermediate, is widely applied to aspects of organic synthesis, drug synthesis, biosynthesis and the like, can be converted into L-methionine in animals, plays a biological function of methionine, is a most direct, cheap and effective active methionine source for animals, and is a more effective and economic substitute product of methionine. Magnesium hydroxy methionine has been used in animal husbandry as early as fifty in the last century and has become the feed industry, and animal husbandry and pet manufacturers have proven to be a source of methionine with excellent performance.
Currently, the prior art does not allow for significant expansion of cord blood hematopoietic stem cells. It was found by accident that the copper ion chelating agent TEPA, SIRT inhibitor Nicotinamide, was able to significantly increase hematopoietic stem cell transplantation levels and showed preliminary efficacy in clinical trials, but the in vivo survival time of the expanded cells was not long enough and the differentiation lineage was not complete. In recent years, high-throughput screening of chemical small molecules finds that an azacyclic compound SR1 and an indole analogue UM171 can more effectively amplify hematopoietic stem cells with long-term transplantation capability. Clinical experiments show that SR1 amplified hematopoietic stem cells have the ability to reconstitute the immune system of a patient, but they still do not get rid of the dependence on double cord blood transplantation. Overall, limited knowledge of hematopoietic stem cells, methods of modulation for a single signaling pathway may be detrimental to overall modulation of HSC physiological status.
The existing research of the hydroxyl methionine magnesium develops from solid particles to porous particles, and if the porous particle-shaped hydroxyl methionine magnesium can be applied to a culture system for amplifying hematopoietic stem cells, the research has a good market prospect.
Disclosure of Invention
Aiming at the problems that in the existing hematopoietic stem cell transplantation, the survival time of the amplified cells in vivo is not long enough and the differentiation lineage is not complete enough, the invention provides the hydroxyl methionine magnesium and the preparation method thereof, which are used for preparing the porous granular hydroxyl methionine magnesium, and the obtained porous granular hydroxyl methionine magnesium is applied to a culture system for amplifying hematopoietic stem cells, so that the total cell quantity obtained by in-vitro amplification of hematopoietic stem cells of fresh blood samples can be obviously improved.
The aim of the invention is achieved by the following technical scheme:
the preparation method of the hydroxyl magnesium methionine is a preparation method of porous particle hydroxyl magnesium methionine, and comprises the following steps:
1) Mixing hydroxy methionine, magnesium carbonate and water in a closed container, adding glycerol stearate accounting for 0.2-0.5% of the water mass, and performing high-pressure reaction, wherein the molar ratio of the hydroxy methionine to the magnesium carbonate is 2 (1.1-1.2), and the adding amount of the water is 3-8% of the total mass of the hydroxy methionine and the magnesium carbonate; the pressure of the high-pressure reaction is controlled to be 0.2-0.5MPa, the reaction temperature is controlled to be 100-150 ℃, and the reaction time is controlled to be 0.5-1.0h;
2) After the high-pressure reaction in the previous step is completed, the pressure of the closed container for the high-pressure reaction is released instantaneously for 1-3s;
3) And (3) after the pressure relief in the step, carrying out vacuum drying on the obtained material to obtain porous particle magnesium hydroxy methionine, wherein the particle size of the porous particle is 200-600 mu m, and the porosity of the porous particle is 50-60%.
In the invention, the following components are added:
the molar ratio of the hydroxy methionine to the magnesium carbonate in the step 1) is 2 (1.1-1.2), and the magnesium carbonate is excessive, so that the hydroxy methionine acid is promoted to react more thoroughly, the utilization rate of the hydroxy methionine is improved, the production cost of the hydroxy methionine magnesium is reduced, and meanwhile, the viscosity of a product can be reduced due to high viscosity of the hydroxy methionine and proper excessive magnesium carbonate.
The water addition amount in the step 1) is 3-8% of the total mass of the hydroxy methionine and the magnesium carbonate, preferably the water addition amount is 5% of the total mass of the hydroxy methionine and the magnesium carbonate, and a proper amount of water is added, so that on one hand, the viscosity of the reactant is reduced, the reactant is uniformly mixed, the yield is improved, on the other hand, the reaction in the closed container provides enough vapor pressure, and the formation of mesopores is facilitated; if the addition amount of water is too low, the mixing effect is reduced, and sufficient steam cannot be provided, and if the addition amount of water is too high, the concentration of reactants is reduced, the reaction speed is reduced, and moreover, the water is too much, the generated steam is too much, which is not beneficial to the control of the reaction pressure and is more beneficial to the subsequent instant pressure relief.
The pressure of the high-pressure reaction in the step 1) is controlled to be 0.2-0.5MPa, the proper pressure is controlled to increase the reaction speed, meanwhile, the water in the container is favorable for forming steam and entering into the product particles, the pressure is too low, the steam cannot smoothly enter into the particles, the pressure is too high, the formation of the particles is unfavorable, the particle size of the product particles is reduced, the performance of the product is reduced, and the pressure is preferably controlled to be 0.2, 0.3, 0.4 or 0.5MPa.
And step 2), the pressure in the closed container for high-pressure reaction is instantaneously relieved, steam in the reaction product particles is instantaneously flushed out by instantaneously relieving the pressure in the closed container, and the steam positions in the particles are not filled to form mesopores due to the instantaneous pressure relief, so that porous particles are obtained, if the pressure relief time is too long, the steam in the particles cannot instantaneously overflow, and the pore structures in the particles collapse along with the slow reduction of the pressure and the temperature in the reaction container, so that the porous particles cannot be obtained.
And 3) vacuum drying, wherein the vacuum degree is 0.04-0.08MPa, the temperature is 80-120 ℃, and the vacuum drying time is 1-2h.
The porous particle hydroxyl methionine magnesium of the step 3) improves the particle size of the product particles by controlling the particle size of the porous particles to be 200-600 mu m, is beneficial to improving the fluidity of the product and reducing the wall adhesion of the product, and can effectively reduce the dust amount in the production and use processes and provide clean labor environment; further preferably, the particle diameter is 200 to 400 μm; still more preferably, the particle size is 250-350 μm.
The invention also relates to the hydroxy methionine magnesium obtained by the preparation method of the hydroxy methionine magnesium, which is porous particle hydroxy methionine magnesium, wherein the particle size of the porous particle is 200-600 mu m, and the porosity of the porous particle is 50-60%.
Meanwhile, the invention also relates to application of the hydroxyl magnesium methionine, in particular to application in a culture system for amplifying hematopoietic stem cells, and the obtained porous granular hydroxyl magnesium methionine is applied to the culture system for amplifying hematopoietic stem cells, so that the total cell quantity obtained by in-vitro amplification of hematopoietic stem cells of fresh blood samples can be obviously improved.
Compared with the prior art, the invention has the following advantages:
1. according to the preparation method of hydroxyl magnesium methionine, the pressure in the closed reaction vessel is instantaneously removed, so that steam in reaction product particles is instantaneously flushed out, and because of instantaneous pressure relief, the steam positions in the particles are not filled to form mesopores, so that porous particles are obtained, if the pressure relief time is too long, the steam in the particles cannot instantaneously overflow, and the pore structure in the particles collapses along with slow reduction of the pressure and the temperature in the reaction vessel, so that the porous particles cannot be obtained.
2. According to the preparation method of the hydroxyl magnesium methionine, the glycerol stearate is added in the high-pressure reaction process, steam is formed along with water and enters the product particles, the glycerol stearate enters the particles in the subsequent instant pressure relief process, and the characteristics of good heat resistance, high viscosity and good hydrolysis resistance of the glycerol stearate are utilized, so that the preparation method has strong emulsifying property and special stability, can support the formation of mesoporous particles of the product, prevent the collapse of the mesoporous structure in the particles, and further obtain the porous particles.
3. The magnesium hydroxy methionine obtained by the invention is porous particle magnesium hydroxy methionine, and when the magnesium hydroxy methionine is applied to a culture system for amplifying hematopoietic stem cells, the contact area between a magnesium hydroxy methionine product and a solution is increased, the dissolution speed of the magnesium hydroxy methionine is promoted, and the absorption and utilization rate of the magnesium hydroxy methionine is effectively improved; however, when the porosity is too high, the dissolution rate of the product is too high, and the product is absorbed in a large amount in a short time, however, the organism has a limited utilization rate of hydroxy methionine magnesium in a short time, hydroxy methionine magnesium is not utilized and is wasted, and simultaneously, a great burden is caused to cells, so that the culture system suitable for expanding hematopoietic stem cells is obtained by keeping a certain porosity and controlling the porosity of the porous particles to be 50-60%.
Drawings
FIG. 1 is an enlarged view of the mesopores of the magnesium hydroxymethionine particles prepared in example 1 of the present invention;
FIG. 2 is an enlarged view of the mesopores of the magnesium hydroxymethionine particles prepared in example 2 of the present invention.
Detailed Description
The present invention is described in further detail by the following examples, which should not be construed as limiting the invention.
Example 1:
the preparation method of the hydroxy methionine magnesium comprises the following steps:
1) Mixing hydroxy methionine, magnesium carbonate and water in a closed container, adding glycerol stearate accounting for 0.3% of the water mass, and performing high-pressure reaction, wherein the molar ratio of the hydroxy methionine to the magnesium carbonate is 2:1.2, and the adding amount of the water is 5% of the total mass of the hydroxy methionine and the magnesium carbonate; the pressure of the high-pressure reaction is controlled to be 0.3MPa, the reaction temperature is controlled to be 120 ℃, and the reaction time is 1.0h;
2) After the high-pressure reaction in the previous step is completed, the pressure of the closed container for the high-pressure reaction is released instantaneously for 1-3s;
3) And (3) after the pressure relief of the step, carrying out vacuum drying on the obtained material, wherein the vacuum degree is 0.06MPa, the temperature is 100 ℃, the vacuum drying time is 1.5h, and the porous particle magnesium hydroxy methionine is obtained, the particle size of the porous particle is 250-350 mu m, and the porosity of the porous particle is 60.05%.
Example 2:
the preparation method of the hydroxy methionine magnesium comprises the following steps:
1) Mixing hydroxy methionine, magnesium carbonate and water in a closed container, adding glycerol stearate accounting for 0.2% of the water mass, and performing high-pressure reaction, wherein the molar ratio of the hydroxy methionine to the magnesium carbonate is 2:1.1, and the adding amount of the water is 3% of the total mass of the hydroxy methionine and the magnesium carbonate; the pressure of the high-pressure reaction is controlled to be 0.2MPa, the reaction temperature is controlled to be 100 ℃, and the reaction time is 1.0h;
2) After the high-pressure reaction in the previous step is completed, the pressure of the closed container for the high-pressure reaction is released instantaneously for 1-3s;
3) And (3) after the pressure relief of the step, carrying out vacuum drying on the obtained material, wherein the vacuum degree is 0.04MPa, the temperature is 80 ℃, the vacuum drying time is 1.5h, and the porous particle magnesium hydroxy methionine is obtained, the particle size of the porous particle is 200-400 mu m, and the porosity of the porous particle is 57.22%.
Example 3:
the preparation method of the hydroxy methionine magnesium comprises the following steps:
1) Mixing hydroxy methionine, magnesium carbonate and water in a closed container, adding glycerol stearate accounting for 0.5% of the water mass, and performing high-pressure reaction, wherein the molar ratio of the hydroxy methionine to the magnesium carbonate is 2:1.2, and the adding amount of the water is 8% of the total mass of the hydroxy methionine and the magnesium carbonate; the pressure of the high-pressure reaction is controlled to be 0.4MPa, the reaction temperature is controlled to be 130 ℃, and the reaction time is 0.6h;
2) After the high-pressure reaction in the previous step is completed, the pressure of the closed container for the high-pressure reaction is released instantaneously for 1-3s;
3) And (3) after the pressure relief of the step, carrying out vacuum drying on the obtained material, wherein the vacuum degree is 0.08MPa, the temperature is 120 ℃, the vacuum drying time is 2 hours, and the porous particle magnesium hydroxy methionine is obtained, the particle size of the porous particle is 200-600 mu m, and the porosity of the porous particle is 55.69%.
Example 4:
the preparation method of the hydroxy methionine magnesium comprises the following steps:
1) Mixing hydroxy methionine, magnesium carbonate and water in a closed container, adding glycerol stearate accounting for 0.4% of the water mass, and performing high-pressure reaction, wherein the molar ratio of the hydroxy methionine to the magnesium carbonate is 2:1.1, and the adding amount of the water is 6% of the total mass of the hydroxy methionine and the magnesium carbonate; the pressure of the high-pressure reaction is controlled to be 0.5MPa, the reaction temperature is controlled to be 150 ℃, and the reaction time is 0.5h;
2) After the high-pressure reaction in the previous step is completed, the pressure of the closed container for the high-pressure reaction is released instantaneously for 1-3s;
3) And (3) after the pressure relief of the step, carrying out vacuum drying on the obtained material, wherein the vacuum degree is 0.05MPa, the temperature is 110 ℃, the vacuum drying time is 1h, and the porous particle magnesium hydroxy methionine is obtained, the particle size of the porous particle is 200-600 mu m, and the porosity of the porous particle is 50.37%.
Comparative example 1:
conventional magnesium hydroxymethionine (solid particles having a particle size of 200-600 μm) is available.
Comparative example 2:
in comparison with example 1, no glycerol stearate was added in step 1), otherwise the same as in example 1.
Experimental example:
first, the English vocabulary appearing in the embodiment of the present invention and the related reagent materials are described:
stemspan SFEM II is serum-free medium, manufacturer StemCell Technologies, product 09655;
recombinant human stem cell factor rhSCF (recombined human stem cell factor), manufacturer is Stemimmu LLC, and product number is HHM-SF-1000;
recombinant human thrombopoietin rhTPO (recombined human thrombopoietin), manufacturer is Stemimhune LLC, and product number is HHM-TP-0100;
recombinant human FMS-like tyrosine kinase 3ligand rhFLT3L (recombined human FMS-like tyrosine kinase 3 ligand), stemimmune LLC, product number HHM-FT-1000;
magnesium hydroxymethionine obtained in example 1, conventional magnesium hydroxymethionine of comparative example 1, magnesium hydroxymethionine obtained in comparative example 2;
peripheral blood mononuclear cells PBMC (peripheral blood mononuclear cell)
MACS: sorting magnetic beads;
DMSO: dimethyl sulfoxide;
PBS phosphate buffer;
methocultttm GF H4435, a semi-solid medium;
CFU-E is known in full name Conoly Forming Unit of Erythrocyte, chinese is known as erythrocyte colony forming unit;
CFU-G is known in full name Conoly Forming Unit of Granulocyte, chinese by the name granulocyte colony forming unit;
CFU-M is called Conoly Forming Unit of Macrophage, chinese is called macrophage colony forming unit;
CFU-GM is known as Conoly Forming Unit of Granulocyte-Macrophage, chinese name is granulocyte-Macrophage colony forming unit;
CFU-GEMM full scale ConolyForming Unit of Granulocyte, erythrocyte,
macrotage/monocyte, megakaryocyte, mixed colonies, wherein the names are granulocyte, erythrocyte, megaphagy/monocyte, megakaryocyte colony forming units;
BFU-E is commonly known as Burst Forming Unit of Erythrocyte, chinese is known as the explosive erythrocyte colony forming unit.
The experimental method comprises the following steps:
StemSpan SFEM II medium was purchased from StemCell Technologies, rhSCF, rhTPO, rhFLT L from Stemimhune LLC; although the invention is preferred for use in humans, it may also be used in laboratory animals, such as mice and the like; human hematopoietic stem cells can be derived from bone marrow, peripheral blood, umbilical cord blood and placental blood, in the present invention, umbilical cord blood hematopoietic stem cells are taken as an example, wherein umbilical cord blood is collected from pregnant women and infants of healthy women, and tested for negative hepatitis B, hepatitis C, syphilis, AIDS, cytomegalovirus, TORCH test, mycoplasma, chlamydia, G-6PD and thalassemia, human umbilical cord blood hematopoietic stem cells express several membrane molecules: leukocyte differentiation antigen CD45, leukocyte differentiation antigen CD34, leukocyte differentiation antigen CD90, and leukocyte differentiation antigen CD49f.
1) Obtaining peripheral blood mononuclear cells;
(1) Collecting 80-120ml of umbilical cord blood with a disposable blood bag (containing anticoagulant such as heparin sodium) and transferring the umbilical cord blood from the blood bag to a 500ml culture flask, diluting with physiological saline for 2-3 times, mixing, and dropwise adding into 0.4 times volume of lymphocyte separation solution without damaging interface;
(2) Centrifugation was performed for 20min at 1500-2000rpm/min, and four layers were separated from top to bottom in centrifuge tubes with different densities: the first layer is a plasma layer, the second layer is a ring-shaped milky mononuclear cell layer (PBMC), the third layer is a transparent separated liquid layer, and the fourth layer is a red blood cell layer;
(3) Carefully sucking the second annular milky mononuclear cell layer (PBMC) into another 50ml centrifuge tube with a suction tube, supplementing normal saline, and centrifuging at 1500-2000rpm/min for 5-10min;
(4) And (3) discarding the supernatant, adding normal saline for resuspension, and finally centrifuging for 5-10min at 1500-2000rpm/min, and discarding the supernatant again to obtain PBMC cell aggregates.
2) Obtaining cd34+ cord blood hematopoietic stem cells from the PBMCs using MACS;
(1) Each cord blood PBMC was resuspended with a mixture of 50ul human cd34+ magnetic beads and 50ul FcR blocker reagent and 150ul 0.5% bsa, and incubated at 4 ℃ for 30min;
(2) Simultaneously, placing the magnet and the magnetic frame in an ultra-clean bench for irradiation of ultraviolet rays for 30min;
(3) Adding 10ml of sterile PBS, mixing, centrifuging at 1500-2000rpm/min for 5-10min, and discarding supernatant;
(4) Placing the MACS special adsorption column into a magnet, adding 500ul of 0.5% BSA for rinsing, and catching the discharged liquid with 15ml of buffer;
(5) Re-suspending 500ul of 0.5% BSA to obtain PBMC (peripheral blood mononuclear cells) in the step 3), uniformly mixing, transferring to a MACS (micro-cell-specific adsorption) column, and completely flowing out the liquid;
(6) 500ul of 0.5% BSA was washed 3 times, the column was removed and placed in 15ml tube;
(7) 1ml of 0.5% BSA was added, and the liquid, namely, CD34+ cord blood hematopoietic stem cells, was pushed into 15ml tube with a piston;
(8) The above were frozen in liquid nitrogen with a cryoprotectant DMSO, if necessary, by dilution, counting.
3) Inoculating the CD34+ cord blood hematopoietic stem cells in suspension into the cell culture medium obtained in the step 2 for culturing, adopting a StemSpan SFEM II serum-free culture medium, adding SCF to the concentration of 80ng/ml, adding FLT3 to the concentration of 90ng/ml, and adding TPO to the concentration of 30ng/ml; cell seeding density in 24 well plates was 1x10 4 Each well was filled with 50. Mu.M of the magnesium hydroxymethionine obtained in example 1, 50. Mu.M of the conventional magnesium hydroxymethionine obtained in comparative example 1, and 50. Mu.M of the magnesium hydroxymethionine obtained in comparative example 2, and the mixture was left at 37℃with 5% CO 2 Culturing in an incubator.
4) According to the cell culture state, 500 μl of the cell culture medium obtained in step 2 is supplemented every 2 days, and a large number of hematopoietic stem cells can be obtained in 7-10 days, with an expansion factor of about 4-20 times.
Cord blood hematopoietic stem cells cultured in the above experimental examples were counted for each of the cells cultured with magnesium hydroxymethionine obtained in example 1 at different concentrations on day 7.
Table 1: statistical table of the number of conditioned cells from each group on day 7 of CB CD34+ cell culture
Results:
1. the comparison of the embodiment 1 and the comparative example 1 shows that the hydroxy methionine magnesium obtained by the invention is porous particle hydroxy methionine magnesium, when the hydroxy methionine magnesium is applied to a culture system for amplifying hematopoietic stem cells, the contact area between a hydroxy methionine magnesium product and a solution is increased, the dissolution rate of the hydroxy methionine magnesium is promoted, the absorption and utilization rate of the hydroxy methionine magnesium is effectively improved, the total cell quantity obtained by in-vitro amplification of umbilical cord blood hematopoietic stem cells is further remarkably improved, and the amplified hematopoietic stem cells contain a higher proportion of CD34+CD90+ cell population, so that the hematopoietic stem cells are more primitive, have stronger differentiation potential for reconstructing a blood system, and can more effectively support clinical treatment needs.
1. By comparing the embodiment 1 with the comparative example 2, the invention shows that in the process of preparing the hydroxyl methionine magnesium, the glycerol stearate can form steam along with water and enter product particles in the process of subsequent instant pressure relief, and the glycerol stearate can enter the particles, so that the invention has strong emulsifying property and special stability by utilizing the characteristics of the glycerol stearate, such as good heat resistance, high viscosity and good hydrolysis resistance, can support the formation of mesoporous particles of the product, prevent the collapse of pore structures in the particles, and further obtain porous particles, and can obviously improve the total cell quantity obtained by in vitro expansion of the hematopoietic stem cells of fresh blood samples by applying the obtained hydroxyl methionine magnesium as a raw material to a culture system for amplifying the hematopoietic stem cells.
The preparation method of the examples is obviously superior to that of the comparative examples by comparing the basic properties of the examples and the comparative examples.
Claims (6)
1. The preparation method of the hydroxy methionine magnesium is characterized by comprising the following steps: the method comprises the following steps:
1) Mixing hydroxy methionine, magnesium carbonate and water in a closed container, adding glycerol stearate accounting for 0.2-0.5% of the water mass, and performing high-pressure reaction, wherein the molar ratio of the hydroxy methionine to the magnesium carbonate is 2 (1.1-1.2), and the adding amount of the water is 3-8% of the total mass of the hydroxy methionine and the magnesium carbonate; the pressure of the high-pressure reaction is controlled to be 0.2-0.5MPa, the reaction temperature is controlled to be 100-150 ℃, and the reaction time is controlled to be 0.5-1.0h;
2) After the high-pressure reaction in the previous step is completed, the pressure of the closed container for the high-pressure reaction is released instantaneously for 1-3s;
3) And (3) after the pressure relief in the step, carrying out vacuum drying on the obtained material to obtain porous particle magnesium hydroxy methionine, wherein the particle size of the porous particle is 200-600 mu m, and the porosity of the porous particle is 50-60%.
2. The method for producing magnesium hydroxymethionine according to claim 1, wherein: the addition amount of the water in the step 1) is 5% of the total mass of the hydroxy methionine and the magnesium carbonate.
3. The method for producing magnesium hydroxymethionine according to claim 1, wherein: the high pressure reaction in step 1) is carried out under a pressure of 0.2, 0.3, 0.4 or 0.5MPa.
4. The method for producing magnesium hydroxymethionine according to claim 1, wherein: and 3) vacuum drying, wherein the vacuum degree is 0.04-0.08MPa, the temperature is 80-120 ℃, and the vacuum drying time is 1-2h.
5. The method for producing magnesium hydroxymethionine according to claim 1, wherein: the porous particle hydroxy methionine magnesium of step 3) has a particle size of 200-400 μm.
6. The method for producing magnesium hydroxymethionine according to claim 5, wherein: the porous particle magnesium hydroxy methionine in the step 3) has the particle size of 250-350 μm.
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