Preparation method of high-stability potassium aspartate based on enzyme membrane reaction

Abstract

The invention discloses a preparation method of high-stability potassium aspartate based on enzyme membrane reaction. The preparation method comprises the following steps: firstly, carrying out fermentation culture and immobilization treatment on enzyme-producing thalli; then, the immobilized microspheres are put into a conversion solution for low-temperature enzymatic reaction, a reaction system intercepts a target product in real time through membrane separation, and finally, stability enhancement and drying forming of the product are achieved under mild conditions through biological active molecule wrapping treatment. According to the process, the defects of high energy consumption, thermosensitive degradation and stability of a traditional method are overcome, and a closed-loop manufacturing system from biotransformation and resource recovery to product stability is formed.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a method for preparing highly stable potassium aspartate based on enzyme membrane reaction. Background Technology

[0002] Potassium aspartate has a high affinity for cell membranes, serving as an effective carrier of potassium ions and facilitating their efficient entry into cells, thereby rapidly correcting intracellular hypokalemia. By promoting potassium ion influx, it can restore the normal polarization state of cardiomyocytes, activate the sodium-potassium pump function, optimize cellular energy metabolism (especially enhancing mitochondrial oxidative phosphorylation efficiency), and reduce myocardial oxygen consumption. Under conditions of myocardial hypoxia or ischemia, potassium aspartate helps maintain myocardial contractility, improve cardiac output, and stabilize heart rhythm. Therefore, it is mainly used clinically as an electrolyte supplement, exerting a powerful cardioprotective effect for the prevention and treatment of coronary heart disease, heart failure, and arrhythmias caused by digitalis poisoning.

[0003] However, potassium aspartate itself has a low melting point and is somewhat hygroscopic, with an industry-common loss on drying exceeding 5%. When the storage environment is humid, the product further absorbs moisture, easily forming eutectic mixtures in localized areas or dissolving in its own water of crystallization. This can cause the product to "melt" at temperatures far below its melting point, manifesting as a damp surface, clumping, or even liquefaction. Once the product clumps or liquefies, it becomes unusable, causing not only economic losses but also potential safety hazards (especially critical for pharmaceuticals).

[0004] Unfortunately, most existing synthesis processes not only fail to effectively address the core issue of poor stability of potassium aspartate, but their production processes themselves often involve high-temperature reactions or drying (70-240℃), leading to the risk of degradation of heat-sensitive substances. The potassium content typically decreases by more than 3% over 6 months. Freeze-drying (-30℃) or ultra-low temperature crystallization (-30℃) processes are extremely energy-intensive. Problems such as the introduction of organic solvents, fluidized bed drying, and high-temperature wall adhesion during spray drying further contribute to solvent residues, crystal damage, equipment contamination, and deterioration of production economics, creating a vicious cycle of dual losses in physical stability and economic efficiency. Therefore, it is necessary to develop an environmentally friendly and highly stable process for preparing potassium aspartate. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide a method for preparing highly stable potassium aspartate based on an enzyme membrane reaction. This invention uses a bio-enzymatic method to prepare potassium aspartate under mild conditions, yielding a product with high purity and stability, effectively solving the problem of the extremely short shelf life of potassium aspartate.

[0006] The technical solution adopted by the present invention to solve the above-mentioned technical problems is: a method for preparing highly stable potassium aspartate based on enzyme membrane reaction, characterized by comprising the following steps:

[0007] (1) Optimization of fermentation of transformant bacteria: Escherichia coli was fermented in fermentation medium; the fermentation broth was centrifuged to obtain wet cells;

[0008] (2) Whole-cell immobilization: The wet bacterial cells prepared in step (1) were prepared into a suspension and mixed with 1% to 3% gel matrix; 0.5% nano TiO2 was added to prevent bacterial film contamination; after full dispersion, 0.3% polydopamine was added to enhance the binding force between bacterial cells and gel; after solidification, the microspheres were transferred into a 0.3% polydopamine / ethanol solution and shaken at 25°C for 2 hours.

[0009] (3) Continuous conversion of enzyme membrane reaction: Add the solidified microspheres described in step (2) to the conversion solution and react the enzyme membrane at 25-55℃ for 1 hour; after the reaction, remove the bacterial cells by centrifugation and separate potassium aspartate in real time using a 300Da nanofiltration membrane.

[0010] (4) Biomolecular encapsulation: The potassium aspartate obtained in step (3) is added to the coating solution at a mass ratio of 1:0.5 to 0.8 into a mixing tank, stirred at room temperature for 30 minutes, and then spray dried.

[0011] In the above technical solution, the preparation method of the culture medium in step (1) is as follows: glucose 15g / L, yeast powder 10g / L, tryptone 10g / L, KH2PO4 3g / L, MgSO4 0.5g / L, dissolve the above raw materials in an appropriate amount of water, and then adjust the pH to 7.0-8.0 with sodium hydroxide.

[0012] In the above technical solution, in step (2), the suspension preparation method is as follows: take 30g of centrifuged wet bacterial cells and add 50mM Tris-HCl buffer (pH 7.5) to prepare 200mL of suspension.

[0013] In the above technical solution, in step (2), the gel matrix includes one or more of the following: agar, carrageenan, konjac glucosamine, gelatin, chitosan, xanthan gum, hydroxypropyl methylcellulose, poloxamer, etc.

[0014] In the above technical solution, in step (3), the conversion solution is: 1.2M fumaric acid, 2.4M potassium hydroxide, 0.5M ammonia source, 0.5mM manganese chloride, and 0.1M Tris-HCl (pH 7.5).

[0015] HOOC-CH=CH-COOH+2KOH→KOOC-CH=CH-COOK+2H2O

[0016] KOOC-CH=CH-COOK+NH3→KOOC-CH2-CH(NH2)-COOK

[0017] The ammonia source used in the conversion solution is NH3·H2O. Using this ammonia source to replace NH4Cl can reduce the competition between ammonia and potassium, and improve purity and potassium ion utilization.

[0018] KOOC-CH=CH-COOK+NH4 + → + H3N-CH2COO - +K +

[0019] In the above technical solution, in step (4), the coating solution is: 0.5% alginate + 0.3% copper lysine.

[0020] In the above technical solution, in step (4), the spray drying conditions are: inlet air temperature 85~125℃, outlet air temperature 40~80℃.

[0021] In this invention, "%" generally represents a mass percentage.

[0022] Compared with the prior art, the beneficial effects of the present invention are:

[0023] (1) This invention integrates enzyme membrane reaction and bio-encapsulation technology. First, the enzyme-producing bacteria are fermented and immobilized, and the mechanical and antibacterial properties of the gel microspheres are enhanced by modification with nanomaterials. Then, the immobilized microspheres are added to the conversion solution for low-temperature enzymatic reaction, and the target product is retained in real time by membrane separation. Finally, the product is encapsulated with bioactive molecules to achieve enhanced stability and drying under mild conditions. This process overcomes the high energy consumption, heat-sensitive degradation, and stability defects of traditional methods, forming a closed-loop manufacturing system from bioconversion and resource recovery to product stability.

[0024] (2) The enzyme membrane reaction process and the introduction of the compound coating solution in this invention both contribute to improving the bioavailability of the product. Based on this, the potassium aspartate product prepared by using a combination of alginate and copper lysine for micro-encapsulation has a purity of 99.79% to 99.88%. This not only effectively solves the problem of easy liquefaction in high temperature and high humidity environments, but also avoids the problem of decreased bioavailability that often accompanies the encapsulation process, demonstrating good comprehensive performance. Attached Figure Description

[0025] Figure 1 The digestibility and absorption rate of potassium aspartate obtained under different processing methods. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the described embodiments of the present invention without creative effort are within the protection scope of the present invention.

[0027] The terms “comprising,” “including,” “having,” “containing,” or any other variations thereof, as used herein, are intended to cover non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such composition, step, method, article, or apparatus.

[0028] When a quantity, concentration, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pair of any upper or preferred value with any lower or preferred value, regardless of whether the range is disclosed individually. For example, when the range "1 to 5" is disclosed, the described range should be interpreted as including ranges "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5", etc. When numerical ranges are described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within that range.

[0029] Furthermore, the technical features involved in the various embodiments of the present invention can be combined with each other as long as they do not conflict with each other.

[0030] Example 1: A method for preparing highly stable potassium aspartate based on enzyme membrane reaction, comprising the following steps:

[0031] (1) Optimization of fermentation of transforming bacteria: Escherichia coli was fermented in a fermentation medium (15 g / L glucose, 10 g / L yeast powder, 10 g / L tryptone, 3 g / L KH2PO4, 0.5 g / L MgSO4. The above raw materials were dissolved in an appropriate amount of water and then the pH was adjusted to 7.0-8.0 with sodium hydroxide); the fermentation broth was centrifuged to obtain wet cells;

[0032] (2) Whole-cell immobilization: 30g of the wet bacterial cells prepared in step (1) were added to 50mM Tris-HCl buffer (pH 7.5) to prepare a 200mL suspension. After mixing with 3% sodium alginate, the mixture was stirred at room temperature for 30min; 0.5% nano TiO2 was added and dispersed thoroughly, followed by 0.3% polydopamine; after solidification, the microspheres were transferred to 0.3% polydopamine and shaken at 25℃ for 2h.

[0033] (3) Continuous conversion via enzyme membrane reaction: The solidified microspheres described in step (2) were added to the conversion solution (1.2M fumaric acid, 2.4M potassium hydroxide, 0.5M ammonia source, 0.5mM manganese chloride, 0.1M Tris-HCl (pH 7.5)) and enzymatically reacted at 37℃ for 1 hour; after the reaction was completed, the microspheres were centrifuged at 4℃, 4000r / min, for 15min to remove bacterial cells, and then potassium aspartate was separated using a 300Da nanofiltration membrane.

[0034] (4) Biomolecular encapsulation: The potassium aspartate obtained in step (3) and the coating solution (0.5% alginate + 0.3% copper lysine) were added to a mixing tank at a mass ratio of 1:0.5. After stirring at room temperature for 30 minutes, spray drying was carried out (inlet air temperature 100℃, outlet air temperature 60℃).

[0035] Example 2: A method for preparing highly stable potassium aspartate based on enzyme membrane reaction, comprising the following steps:

[0036] (1) Optimization of fermentation of transforming bacteria: Escherichia coli was fermented in a fermentation medium (15 g / L glucose, 10 g / L yeast powder, 10 g / L tryptone, 3 g / L KH2PO4, 0.5 g / L MgSO4. The above raw materials were dissolved in an appropriate amount of water and then the pH was adjusted to 7.0-8.0 with sodium hydroxide); the fermentation broth was centrifuged to obtain wet cells;

[0037] (2) Whole-cell immobilization: 30g of the wet bacterial cells prepared in step (1) were added to 50mM Tris-HCl buffer (pH 7.5) to prepare a 200mL suspension. After mixing with 1% xanthan gum, the mixture was stirred at room temperature for 30min; 0.5% nano TiO2 was added and dispersed thoroughly, followed by 0.3% polydopamine; after solidification, the microspheres were transferred to 0.3% polydopamine and shaken at 25℃ for 2h.

[0038] (3) Continuous conversion via enzyme membrane reaction: The solidified microspheres described in step (2) were added to the conversion solution (1.2M fumaric acid, 2.4M potassium hydroxide, 0.5M ammonia source, 0.5mM manganese chloride, 0.1M Tris-HCl (pH 7.5)) and enzymatically reacted at 45℃ for 1 hour; after the reaction was completed, the microspheres were centrifuged at 4℃, 5000r / min, for 10min to remove bacterial cells, and then potassium aspartate was separated using a 300Da nanofiltration membrane.

[0039] (4) Biomolecular encapsulation: The potassium aspartate obtained in step (3) and the coating solution (0.5% alginate + 0.3% copper lysine) were added to a mixing tank at a mass ratio of 1:0.6. After stirring at room temperature for 30 minutes, spray drying was carried out (inlet air temperature 110℃, outlet air temperature 70℃).

[0040] Comparative Example 1: Preparation of potassium aspartate using conventional crystallization process, including the following steps:

[0041] Dissolve 50g of aspartic acid in 300g of purified water, add 25g of potassium hydroxide, and after the solution becomes clear, continue the reaction at 90℃ for 4 hours; then turn on the cooling to crystallize, filter and dry the crystals to obtain potassium aspartate.

[0042] Comparative Example 2: It is basically the same as Example 1, except that only the enzyme membrane reaction in steps (1) to (3) is performed, and the obtained potassium aspartate is not subjected to the biomolecule encapsulation treatment in step (4).

[0043] Comparative Example 3: The potassium aspartate prepared by the crystallization process in Comparative Example 1 was subjected to the same biomolecule encapsulation treatment as step (4) in Example 1.

[0044] Comparative Example 4: It is basically the same as Example 1, except that the "encapsulation solution" mentioned in the biomolecule encapsulation treatment in step (4) is 0.8% alginate.

[0045] Comparative Example 5: It is basically the same as Example 1, except that the "encapsulation solution" mentioned in the biomolecule encapsulation treatment in step (4) is 0.8% copper lysine.

[0046] Effect Experiment Example

[0047] In Example 1, the purity of the prepared potassium aspartate product was 99.79%, and in Example 2, the purity of the prepared potassium aspartate product was 99.88%. The high-temperature stability study data and accelerated stability study data are shown in Tables 1-2 and 8-9.

[0048] The data in Tables 1-2 and 8-9 show that the drying weight loss of potassium aspartate remained stable at less than 2% in a high humidity environment of 75% (temperature 40℃) and a high temperature environment of 60℃ (humidity 60%), confirming that the bio-coating layer (alginic acid + copper lysine) effectively blocked the penetration of water molecules; the potassium content decay rate was <0.5%, proving that the low-temperature enzyme membrane reaction + coating technology avoided the instability problem of potassium aspartate during storage; and there was no clumping / liquefaction throughout the process, completely solving the industry problem of "chemical processing" of potassium aspartate.

[0049] Table 1. High-temperature stability study data from Example 1

[0050]

[0051] Table 2. High-temperature stability study data from Example 2

[0052]

[0053]

[0054] Table 3. High-temperature stability study data for Comparative Example 1

[0055]

[0056] Table 4. High-temperature stability study data for Comparative Example 2

[0057]

[0058]

[0059] Table 5. High-temperature stability study data for Comparative Example 3

[0060]

[0061] Table 6. High-temperature stability study data for Comparative Example 4

[0062]

[0063] Table 7. High-temperature stability study data for Comparative Example 5

[0064]

[0065]

[0066] Table 8. Accelerated Stability Study Data from Example 1

[0067]

[0068] Table 9. Accelerated Stability Study Data from Example 2

[0069]

[0070]

[0071] Table 10 Comparative Example 1 Accelerated Stability Study Data

[0072]

[0073] Table 11 Comparative Example 2 Acceleration Stability Study Data

[0074]

[0075] Table 12 Comparative Example 3 Acceleration Stability Study Data

[0076]

[0077] Table 13 Comparative Example 4 Accelerated Stability Study Data

[0078]

[0079] Table 14 Comparative Example 5 Accelerated Stability Study Data

[0080] Tables 1 to 14 show the high-temperature stability and accelerated stability data of potassium aspartate obtained under different treatment methods. Figure 1 The digestibility and absorption rate of potassium aspartate obtained under different processing methods.

[0081] Comparative stability data shows that the conventional crystallization process in the industry (Comparative Example 1) has the worst stability. Under a high temperature environment of 60℃ (60% humidity), the sample melted on the 5th day of storage; under a high humidity environment of 75% (40℃), the sample also liquefied in the first month. This indicates that its stability in actual storage and application is poor, which seriously limits the application range of potassium aspartate.

[0082] The product prepared using the enzyme membrane reaction process (Comparative Example 2) showed the highest in vitro digestibility and absorption rate, and its stability was slightly better than that of the crystallization process. However, it still could not effectively solve the problem of rapid liquefaction of the sample under high temperature and high humidity conditions. Further encapsulation of potassium aspartate with alginate (Comparative Example 4) showed a significant improvement in sample stability. No liquefaction was observed in the high temperature stability test and the accelerated stability test over a period of 6 months, and all performance indicators remained stable. However, this treatment resulted in a decrease in in vitro digestibility and absorption rate compared to Comparative Examples 1 and 2.

[0083] When lysine copper, which has high bioavailability, was selected as the encapsulation solution (Comparative Example 5), the bioavailability of the sample was significantly affected, but its stability was lower than that of the alginate-encapsulated sample (Comparative Example 4).

[0084] Furthermore, using a combination of alginate and copper lysine as the encapsulation solution (Examples 1 and 2), the stability of the resulting product was superior to that treated with either alginate or copper lysine alone. In stability tests, all performance parameters were the most stable among all methods, indicating a synergistic effect between the two. Simultaneously, its bioavailability was significantly higher than that of products obtained through commonly used crystallization processes in the industry.

[0085] As a control, potassium aspartate obtained from the crystallization process was encapsulated using the same encapsulation solution (Comparative Example 3). Although its stability was improved, it was significantly lower than that of the enzyme membrane reaction sample after encapsulation treatment (Examples 1 and 2), and its bioavailability was also much lower than the latter.

[0086] In summary, both the enzyme membrane reaction process and the introduction of the lysine copper encapsulation solution contribute to improving the bioavailability of the product. Furthermore, the encapsulation treatment using a combination of alginate and lysine copper not only effectively solves the problem of easy liquefaction of potassium aspartate in high-temperature and high-humidity environments but also avoids the bioavailability decline often associated with encapsulation treatment, demonstrating excellent overall performance.

[0087] The above embodiments are merely preferred embodiments of the present invention and are only used to explain the present invention, not to limit the present invention. Any changes, substitutions, modifications, etc., made by those skilled in the art without departing from the spirit and essence of the present invention should be within the protection scope of the present invention.

Claims

1. A method for preparing highly stable potassium aspartate based on enzyme membrane reaction, characterized in that, Includes the following steps: (1) Optimization of fermentation of transformant bacteria: Escherichia coli was fermented in fermentation medium; the fermentation broth was centrifuged to obtain wet cells; (2) Whole cell immobilization: The wet bacterial cells prepared in step (1) were prepared into a suspension and mixed with 1% to 3% gel matrix; 0.5% nano TiO2, 0.3% polydopamine, and 0.3% polydopamine / ethanol solution were added and shaken at 25°C for 2 hours. (3) Continuous conversion of enzyme membrane reaction: The solidified microspheres described in step (2) are added to the conversion solution and the enzyme reaction is carried out at 25-55℃ for 1 hour; after the reaction is completed, the bacterial cells are removed by centrifugation and potassium aspartate is separated in real time using a 300Da nanofiltration membrane. (4) Biomolecular encapsulation: The potassium aspartate obtained in step (3) is added to the coating solution at a mass ratio of 1:0.5 to 0.8 into a mixing tank, stirred at room temperature for 30 minutes, and then spray dried.

2. The method for preparing highly stable potassium aspartate based on enzyme membrane reaction as described in claim 1, characterized in that, In step (1), the culture medium is prepared as follows: glucose 15g / L, yeast powder 10g / L, tryptone 10g / L, KH2PO4 3g / L, MgSO4 0.5g / L. The above raw materials are dissolved in an appropriate amount of water, and then the pH is adjusted to 7.0-8.0 with sodium hydroxide.

3. The method for preparing highly stable potassium aspartate based on enzyme membrane reaction as described in claim 1, characterized in that, In step (2), the suspension is prepared by taking 30g of centrifuged wet bacterial cells and adding 50mM Tris-HCl buffer (pH 7.5) to prepare 200mL of suspension.

4. The method for preparing highly stable potassium aspartate based on enzyme membrane reaction as described in claim 1, characterized in that, In step (2), the gel matrix includes one or more of the following: agar, carrageenan, konjac glucosamine, gelatin, chitosan, xanthan gum, hydroxypropyl methylcellulose, poloxamer, etc.

5. The method for preparing highly stable potassium aspartate based on enzyme membrane reaction as described in claim 1, characterized in that, In step (3), the conversion solution is: 1.2M fumaric acid, 2.4M potassium hydroxide, 0.5M ammonia source, 0.5mM manganese chloride, and 0.1M Tris-HCl (pH 7.5).

6. The method for preparing highly stable potassium aspartate based on enzyme membrane reaction as described in claim 5, characterized in that, The ammonia source used in the conversion solution is NH3·H2O.

7. The method for preparing highly stable potassium aspartate based on enzyme membrane reaction as described in claim 1, characterized in that, In step (4), the coating solution is: 0.5% alginate + 0.3% copper lysine.

8. The method for preparing highly stable potassium aspartate based on enzyme membrane reaction as described in claim 1, characterized in that, In step (4), the spray drying conditions are: inlet air temperature 85-125℃, outlet air temperature 40-80℃.

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