Lysozyme composition microparticles and methods of making and using the same

Abstract

The present application relates to a lysozyme composition microparticle and its preparation method and application, and relates to the technical field of protein medicine preparation. Specifically, the present application uses trehalose as an excipient, and after being combined with lysozyme, the lysozyme composition microparticle is prepared by a low-temperature spray drying method, including a dissolving and low-temperature spray drying step, wherein the inlet temperature is 40-48 DEG C, and the outlet temperature is 20-38 DEG C. The preparation method of the present application has simple steps, controllable conditions, high repeatability, and the prepared product has good uniformity. The lysozyme composition microparticle is spherical or spheroidal, has a high drug loading capacity of more than 75%, an average geometric size of less than 5 mu m, good fluidity, a water content of less than 5% w / w, a surface hydrophobicity of not less than 0.60, a biological activity of more than 95%, good long-term stability, high bioavailability in medical use, and improved antibacterial capacity.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of protein drug preparation, in particular to a lysozyme composition microparticle and a preparation method and application thereof. BACKGROUND

[0002] Bio-macromolecular drugs such as polypeptides, proteins, monoclonal antibodies and recombinant vaccines have the characteristics of high activity, strong specificity, low toxicity, clear biological function and being conducive to clinical application compared with small molecule drugs, and are increasingly concerned in the field of medical preparations. Lysozyme, also known as muramidase or N-acetylmuramidase, is a protein that can hydrolyze bacterial mucopolysaccharides. Lysozyme mainly breaks down the insoluble mucopolysaccharides in the cell wall by destroying the β-1, 4 glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in the cell wall, so that the cell wall is broken and the contents escape, resulting in the lysis of bacteria. Lysozyme as a natural antibacterial agent is concerned in the field of biological medicine. According to its source, it can be divided into four categories, namely plant lysozyme, animal lysozyme, microbial lysozyme and egg white lysozyme. Compared with the above, egg white lysozyme is widely used in medicine, and its physicochemical properties and enzyme activity are relatively stable. However, the stability of lysozyme extracted from animals and plants and microorganisms is poor, and the technology is difficult and the cost is high. In order to improve the stability and functionality of lysozyme, the existing technology adopts powder preparation process to prepare it into spherical and / or spherical-like microparticles, which can reduce the surface exposure, so that the lysozyme molecules are wrapped inside the microparticles, reducing the direct contact with the external environment (such as high temperature, pH change or protease), at the same time, the nano / micron spherical particles are more easily endocytosed by phagocytes or target cells, thereby effectively improving the bioavailability of lysozyme drugs. The commonly used preparation process of lysozyme-containing microparticles includes spray drying, freeze drying and spray freeze drying, etc. Lysozyme is loaded with excipients (such as sugars, polyols or amino acids) to form protective microparticles. However, the traditional spray drying technology is carried out at high temperature, and the lysozyme microparticles are easily affected by high temperature stress, resulting in loss of enzyme activity; the traditional freeze drying technology can retain high biological activity, but the solid obtained is usually in the form of blocks or irregular fragments, and has poor fluidity; the spray freeze drying technology can prepare porous lysozyme-containing microparticles with good fluidity, but the equipment running cost is high, and the microparticles are easy to absorb moisture, which is not conducive to long-term stability. Therefore, the present application is proposed. SUMMARY

[0003] To solve the above technical problems, the application determines the process capable of preparing the spherical lysozyme composition microparticle with good fluidity, high biological activity and good morphology through screening of excipients and preparation methods, and then optimizes the temperature of the preparation method, i.e., the low-temperature spray drying process, to find the optimal condition for preparing the lysozyme composition microparticle. The lysozyme composition microparticle prepared by the application can achieve a biological activity of more than 97%, and can still maintain a biological activity of at least 92% after being stored for 90 days, thereby having good stability and being conducive to realizing high-efficiency antibiosis.

[0004] The first object of the application is to provide a preparation method of a lysozyme composition microparticle, comprising the following steps:

[0005] S1, mixing lysozyme and trehalose to prepare a precursor solution;

[0006] S2, performing low-temperature spray drying treatment on the precursor solution to obtain the lysozyme composition microparticle; the conditions of the low-temperature spray drying treatment include: the inlet temperature is 40-48℃, and the outlet temperature is 20-38℃.

[0007] Further, the lysozyme is egg white lysozyme.

[0008] Further, in step S1,

[0009] (1) the mass ratio of the lysozyme to the trehalose is (1-5):1;

[0010] (2) in the precursor solution, the total mass percentage of the lysozyme and the trehalose (solid substance) is 1%-5%;

[0011] (3) the mixing condition is a 30-40℃ water bath;

[0012] (4) the precursor solution is an aqueous solution.

[0013] The advantage of the application lies in high drug loading and small trehalose dosage. The content of the traditional excipient is more than 700%-50%, the drug loading of the application is more than 50% (the excipient is less than 50%), such as about 67% (2:1), 75% (3:1), more than 80% (4:1 or 5:1).

[0014] Further, in step S2,

[0015] (1) the conditions of the low-temperature spray drying treatment include: the flow rate of the dispersion wind (atomization wind) is 2-6 m 3 / h;

[0016] (2) the conditions of the low-temperature spray drying treatment include: the feeding flow rate is 300-1200 mL / h;

[0017] (3) the conditions of the low-temperature spray drying treatment include that the spray pressure is 0.2-0.6 MPa;

[0018] (4) the conditions of the low-temperature spray drying treatment include that the hot air (dry air) flow rate is 120-300 m 3 / h.

[0019] Most preferably, in the low-temperature spray drying treatment, the inlet temperature is 42℃ and the outlet temperature is 28℃.

[0020] Further, the lysozyme composition microparticles are spherical microparticles; and / or the lysozyme composition microparticles are microparticles.

[0021] Currently, there is no related report on the preparation of lysozyme microparticles by low-temperature spray drying (40-48℃) technology. The prior art uses complex gel method and pectin as an excipient to prepare lysozyme composition microparticles by spray drying, which has the following problems: first, the traditional spray drying technology is used, the inlet temperature is set to 150-180℃, in the extremely high temperature environment, the structure of lysozyme will be damaged, and the production energy consumption is increased, and in the absence of pectin, the biological activity of lysozyme is only about 63%; second, the loss of biological activity of lysozyme can be alleviated only when the pectin concentration is increased to a certain range, but the highest activity value is not more than 80%. At the same time, too high concentration of pectin will increase the interaction force and destroy the stability of the structure of lysozyme, thereby reducing its activity and poor long-term stability. In addition, some technologies use four non-ionic surfactants (polysorbate 80, poloxamer 188, poloxamer 407 and sucrose stearate) as excipients to prepare lysozyme-loaded liposome dry powder microparticles by spray drying technology. The problem is that although the drying temperature is reduced (80-90℃), the biological activity of lysozyme is only about 60%, and only a few formulations (mass ratio of lysozyme to poloxamer 188 is 1:5) maintain about 88% of the biological activity after the spray drying process, and the biological activity of the lysozyme-containing microparticles prepared by the rest of the formulations under this process is not significantly improved, and the drug loading is reduced and the drug administration frequency is increased. Some technologies prepare pure lysozyme microparticles by improving the spray drying process parameters, which has the following problems: although the inlet temperature is reduced, the lowest temperature is only reduced to 70℃, the biological activity of the prepared lysozyme microparticles is only about 80%, and the yield is only 40%, which still has a certain gap compared with the low-temperature spray drying technology of the present application.

[0022] The application discloses a preparation method of a high-biological-activity lysozyme composition microparticle, the composition comprising lysozyme and excipients, and the preparation method is a low-temperature spray drying technology, the inlet temperature of the low-temperature spray drying technology is 40-48 DEG C, the outlet temperature is 20-38 DEG C, the drug loading of the obtained microparticle is above 50% (preferably above 75%), the average geometric size of the prepared lysozyme composition microparticle is less than 5 microns, and the activity of the prepared lysozyme composition microparticle is greater than 95%. The composition comprises at least 75% of lysozyme in mass fraction; and the composition comprises a trehalose excipient. As comparison, the activity of the lysozyme microparticle prepared by a traditional spray drying technology is extremely low, the spray freezing drying technology and the vacuum freeze drying technology are not conducive to the preparation of microparticles with good fluidity, the morphology and size of the microparticles are uncontrollable, the average geometric size of the lysozyme microparticle prepared by the spray freezing drying technology is relatively large, the aggregation is serious, and the water content is relatively high; meanwhile, the protection effect of the added trehalose on the biological activity of lysozyme is the best in the low-temperature spray drying process.

[0023] The application fully dissolves lysozyme and excipients in water, and prepares microparticles by a low-temperature spray drying technology, so that the lysozyme microparticle with high biological activity is obtained. The solid content is 1-5% (w / w), preferably 4% (w / w). The inlet temperature used is 40-48 DEG C; the outlet temperature is 20-38 DEG C; the hot air speed is 120-300 m 3 / h; and the precursor liquid feeding flow is 300-1200 mL / h. The lysozyme composition microparticle has an average geometric size of less than 5 microns, a water content of less than 5% (w / w), a surface hydrophobicity of not less than 0.60, and a relative biological activity of greater than 95%.

[0024] The second object of the application is to provide the lysozyme composition microparticle prepared by the preparation method.

[0025] The third object of the application is to provide the application of the lysozyme composition microparticle in the preparation of antibacterial drugs.

[0026] Further, the antibacterial drug is used for inhibiting gram-positive bacteria, and the antibacterial drug prepared by the application has more excellent effects in inhibiting staphylococcus (such as Staphylococcus aureus), streptococcus (such as Streptococcus pyogenes and Streptococcus pneumoniae), and bacillus (such as Bacillus subtilis).

[0027] The fourth object of the application is to provide an antibacterial drug prepared by taking the lysozyme composition microparticle as an effective component.

[0028] The application has the following beneficial effects:

[0029] (1) The present application provides a preparation method of lysozyme composition microparticles with high biological activity, the lysozyme composition comprising lysozyme and trehalose, the preparation method being a low-temperature spray drying technology, comprising three unit operations of dissolution, atomization and drying, wherein the inlet temperature of the low-temperature spray drying technology is 40-48℃, the outlet temperature is 20-38℃, the prepared lysozyme composition microparticles with high biological activity have a drug loading of at least 50%, an average geometric size of less than 5 μm, a water content of less than 5% w / w, a surface hydrophobicity of not less than 0.60, and a biological activity of greater than 95%.

[0030] (2) The microparticles prepared by the present application have a spherical and / or spheroid-like morphology, and have great potential in the application in the medical field. Specifically, the present application prepares dense spherical or spheroid-like microparticles with small size, good flowability and stable structure, the obtained microparticles have high biological activity, the dry powder form can improve the storage stability of lysozyme, reduce the transportation cost, and the spherical structure is more convenient for drug filling, so it is more beneficial to practical application to fully mix lysozyme with functional excipients to prepare composition microparticles with high biological activity. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 The macroscopic and microscopic morphology diagrams of the lysozyme microparticles prepared by the three drying technologies in Example 1.

[0032] Figure 2 The macroscopic and microscopic morphology diagrams of the lysozyme and lysozyme composition microparticles prepared by the low-temperature spray drying in Example 2.

[0033] Figure 3 The macroscopic and microscopic morphology diagrams of the lysozyme microparticles prepared by the vacuum freeze drying technology in Example 2. DETAILED DESCRIPTION

[0034] The present application will be further described below in combination with the drawings and specific examples, so that those skilled in the art can better understand the present application and implement it, but the examples are not limiting to the present application.

[0035] The technical solutions involved in the present application are as follows:

[0036] Traditional spray drying technology is usually carried out at an inlet temperature higher than 120℃, which easily causes the structural loss of lysozyme, resulting in the breakage of disulfide bond and the loss of biological activity of lysozyme. Although traditional freeze drying technology is beneficial to protect the activity of lysozyme, it is difficult to control the particle size distribution of microparticles, and it is easy to form uneven or oversized microparticles with poor flowability. Spray freeze drying technology combines traditional spray drying and freeze drying technology, and is generally considered to have advantages in the preparation of heat-sensitive protein microparticles. However, when it is applied to the preparation of lysozyme microparticles, it is found that the rapid freezing process will cause the structural change of lysozyme, the porous structure will make the microparticles easy to absorb moisture, and thus cause the loss of biological activity. At the same time, it cannot avoid the defects of large energy consumption and long time period of freeze drying process. The low-temperature spray drying process is used to prepare spherical and / or spheroid lysozyme microparticles with good flowability. The prepared microparticles have small size and dense surface, can better protect lysozyme from the negative effects of high temperature, and have high production efficiency. Therefore, it has certain advantages in the preparation of lysozyme composition microparticles with high biological activity. At the same time, mixing lysozyme with trehalose excipient can effectively improve the physicochemical stability of lysozyme in the process of atomization, drying and storage, and fully protect the biological activity of lysozyme.

[0037] Specifically, the present application provides a preparation method of lysozyme or lysozyme composition microparticles with high biological activity. Lysozyme or lysozyme composition is stirred at a speed of 300-1000 rpm under water bath at 30-40℃, and then low-temperature spray drying treatment is carried out after complete dissolution, to obtain lysozyme composition microparticles with high biological activity. The lysozyme composition includes lysozyme and excipient, and the obtained product is high-biological-activity microparticles containing lysozyme and excipient.

[0038] In the present application, lysozyme and lysozyme composition microparticles are prepared by low-temperature spray drying process technology. The inlet temperature of the low-temperature spray drying technology is 40-48℃, and the outlet temperature is 20-38℃. The microparticles contain not less than 50% mass fraction of lysozyme, have an average geometric size of less than 5 μm, have a spherical and / or spheroid macroscopic morphology, have a water content of less than 5% w / w, have a surface hydrophobicity of 0.60, and have a biological activity of more than 95%.

[0039] The present application relates to a preparation method of composition particles with lysozyme as active pharmaceutical ingredient and combined with different excipients. The raw materials used in the present application are existing products, and the specific preparation operation and test method are conventional technologies.

[0040] The lysozyme used in the present application is chicken egg white lysozyme, which is purchased from Shanghai Yuan Ye Biological Technology Co., Ltd. with the product number S10038.

[0041] Example 1: Selection of process

[0042] Accurately weigh the lysozyme, and fully stir in a 37°C constant temperature water bath at a speed of 500 rpm. After the lysozyme is completely dissolved, a precursor solution is prepared with a solid content of 4%. Lysozyme microparticles are prepared using low-temperature spray drying technology, with a feed flow rate of 720 mL / h; an atomization pressure of 0.5 MPa; a dispersion air flow rate of 6 m 3 / h; an inlet temperature of 42°C, an outlet temperature of 28°C; and a hot air flow rate of 210 m 3 / h. After drying with hot air, product 1 is obtained. Lysozyme microparticles are prepared using low-temperature spray drying technology, with an inlet temperature of 55°C and an outlet temperature of 40°C, and the same conditions as product 1, to obtain product 2. Lysozyme microparticles are prepared using low-temperature spray drying technology, with an inlet temperature of 48°C and an outlet temperature of 32°C, and the same conditions as product 1, to obtain product 3. Lysozyme microparticles are prepared using low-temperature spray drying technology, with an inlet temperature of 30°C and an outlet temperature of 21°C, and the same conditions as product 1, to obtain product 4. Lysozyme microparticles are prepared using high-temperature spray drying technology, with an inlet temperature of 150°C and an outlet temperature of 130°C, and the same conditions as product 1, to obtain product 5. Lysozyme microparticles are prepared using spray freeze drying technology, with the same atomization unit operation as the spray drying process of product 1. The atomized droplets enter a cold air medium to be frozen into ice balls, with a freezing temperature of -50°C. The ice balls are collected by a collection device and further transferred to a vacuum freeze dryer for vacuum freeze drying to obtain product 6. The vacuum freeze drying conditions are as follows: primary drying at -40, -20, and -10°C for 12 h, and secondary drying at 0, 10, and 20°C for 8 h. The macroscopic and microscopic morphologies of the obtained lysozyme microparticles are shown in Figure 1 Table 1.

[0043] Table 1. Particle size and water content of lysozyme microparticles prepared by three drying technologies

[0044]

[0045] It can be seen that the lysozyme microparticles prepared by low-temperature spray drying at three temperatures of 42℃, 48℃ and 55℃ are all spherical structures with dense surfaces. With the increase of drying temperature, the surface of the microparticles gradually appears collapsed holes, and the aggregation and adhesion between the microparticles are aggravated. There is no significant difference in particle size, moisture content, etc. among the three products, but the span value of the lysozyme microparticles prepared at 48℃ is higher than that of the other two, and the particle size distribution is wider. This can also be observed from the morphology diagram. The product 1 prepared at 42℃ has relatively better uniformity. When low-temperature spray drying is carried out at a lower temperature (such as 30℃), the droplets cannot be fully dried, so the corresponding lysozyme dry powder microparticles cannot be prepared. In addition, compared with the lysozyme microparticles prepared by low-temperature spray drying technology, the lysozyme microparticles prepared by traditional spray drying technology are spherical structures, and the surface collapse is deeper. This may be due to the fact that the drying temperature is too high, the solvent evaporation rate is extremely fast, and the shell formation rate is also accelerated, resulting in that the average geometric size of the microparticles is slightly higher than that of the microparticles prepared by low-temperature spray drying technology; the lysozyme microparticles prepared by spray freeze-drying technology are spherical / spherical structures, the surface is covered with a thin film and attached with fine pores, the aggregation between the microparticles is serious, the average geometric size is large, and at the same time, due to the porous surface, it is easier to absorb moisture, so the moisture content is higher. Compared with traditional spray drying and spray freeze-drying technology, low-temperature spray drying technology is more suitable for preparing lysozyme microparticles with small size, high dispersity and low moisture content.

[0046] The density, surface hydrophobicity, biological activity and yield data of the lysozyme microparticles prepared by low-temperature spray drying, traditional spray drying and spray freeze-drying respectively are shown in Table 2.

[0047] Table 2 Density, surface hydrophobicity, biological activity and yield of lysozyme microparticles prepared by three drying technologies

[0048]

[0049] It can be seen that there is no obvious difference in the flowability of the lysozyme microparticles prepared by low-temperature spray drying and traditional spray drying, and the Carr index is basically the same. The lower the Carr index, the better the flowability of the microparticles. However, as the drying temperature increases, the lysozyme undergoes a significant change in the tertiary structure, and the surface hydrophobicity gradually deteriorates. Even at 55°C, the spray drying only retains about 78% of the biological activity, and at 150°C, the activity decreases to only about 76%. In addition, the flowability of the lysozyme microparticles prepared by spray freeze drying is poorer than that of low-temperature spray drying, and the Carr index is greater than 50%. Without the addition of excipients, the shear stress generated during atomization and the rapid freezing process can cause changes in the tertiary structure of pure lysozyme, deteriorate the surface hydrophobicity, and reduce the biological activity to only about 79%. Moreover, due to the porous surface of the lysozyme microparticles prepared by spray freeze drying, they are prone to moisture absorption. After 90 days of storage under long-term conditions (25°C, 60% RH, sealed), the water content increases to 13.57%, and the activity decreases to only about 68%. However, sample 1 still retains 83% of the biological activity after storage under the same conditions. In addition, compared with traditional spray drying and spray freeze drying, the yield of the products prepared by low-temperature spray drying at three temperatures is significantly higher, all above 78%, and the yield of product 1 is the highest. From the test results of the lysozyme microparticles prepared by the three processes, it can be seen that the pure microparticles prepared by low-temperature spray drying at an inlet temperature of 42°C have better flowability, better protect the tertiary structure of lysozyme, and retain the highest biological activity and yield.

[0050] Example 2: Screening of excipients (excipients)

[0051] (1) Add the materials according to the formulation in Table 3 into a beaker, and stir thoroughly in a 37°C constant-temperature water bath at a speed of 500 rpm. After the lysozyme and excipients are completely dissolved, a clear and transparent lysozyme precursor solution is obtained. The lysozyme precursor solution is subjected to low-temperature spray drying, with a precursor feed flow rate of 720 mL / h; an atomization pressure of 0.5 MPa; a dispersion air flow rate of 6 m 3 / h; an inlet temperature of 42°C, an outlet temperature of 28°C; and a hot air flow rate of 210 m 3 / h.

[0052] Table 3 Formulation table of lysozyme and lysozyme composition solutions

[0053]

[0054] The macroscopic and microscopic morphology of the lysozyme composition microparticles prepared by low-temperature spray drying are shown in Figure 2The particle size data and water content are shown in Table 4. It can be seen that the spray-dried lysozyme microparticles at a lower temperature (42°C) are spherical or spheroidal, and have fine pores on the surface due to the collapse caused by the shell formation during solvent evaporation. The addition of trehalose and leucine makes the pore size relatively larger and the degree of collapse increases. The addition of mannitol intensifies the aggregation between microparticles, and the microparticles no longer maintain a spherical structure, but form agglomerates in the form of wrinkled blocks. The addition of sucrose as an excipient, although maintaining the spherical structure of the microparticles and a smooth surface, causes serious adhesion between the microparticles, and also forms agglomerates. Since glycine is prone to crystallization, it precipitates on the surface of the microparticles, resulting in poor uniformity of the composition microparticle surface, and forming irregular microparticles in the form of half spherical and half block crystals. In addition, isoleucine has strong hydrophobicity, and has a very fast migration rate during microparticle formation, intensifying the degree of surface wrinkling of the microparticles. Although the dispersibility between the microparticles is good, it also leads to strong electrostatic interaction. The addition of excipients causes the average geometric size of the microparticles to increase, but the average geometric size of the seven microparticles is less than 3 μm as a whole (except for formulation 6 with glycine). The water content of formulation 2 with trehalose is the lowest at 4.58%, and the water content of formulation 5 with sucrose is the highest. The easily crystallizable mannitol and glycine also cause the water content of the composition microparticles to increase.

[0055] Table 4 Particle size and water content of low-temperature spray-dried lysozyme-containing microparticles

[0056]

[0057] The density, surface hydrophobicity, bioactivity and yield data of the lysozyme-containing microparticles prepared by low-temperature spray drying are shown in Table 5. It can be seen that the four formulations, Formulation 1 to Formulation 4, all maintain good flowability during low-temperature spray drying, and the Carr index of the four lysozyme microparticles is about 30% to 40%. The addition of trehalose and leucine can improve the flowability of the microparticles, especially leucine. However, the shear stress generated during atomization can cause changes in the tertiary structure of the microparticles, reducing their surface hydrophobicity, so the bioactivity of Formulation 1 without excipients is reduced to about 88%. The addition of excipients can effectively improve this situation, and the bioactivity of the lysozyme microparticles can be increased to about 98% by adding only 25% w / w trehalose. After 90 days of storage under the long-term storage conditions in Example One, the activity is maintained at 92.58%, which greatly improves the production efficiency, treatment effect and drug stability while maintaining a high drug loading. However, mannitol and leucine are not as effective as trehalose in protecting the activity of lysozyme. The bioactivity of the lysozyme composition microparticles prepared with sucrose as an excipient is only about 82%, which may be due to the high water content of the lysozyme composition microparticles prepared, causing the lysozyme molecular chain segments to arrange more disorderly. The bioactivity of the composition microparticles prepared with glycine as an excipient is only about 86%, which may be because glycine is prone to crystallization and phase separation with lysozyme during microparticle formation, which cannot effectively protect the bioactivity of lysozyme. Due to the strong hydrophobicity of isoleucine, the dissolution speed in the preparation of the precursor solution is relatively slow, increasing the granulation time. Meanwhile, during low-temperature spray drying, isoleucine is easily exposed on the surface of lysozyme due to its strong hydrophobicity, intensifying the hydrophobic interaction of protein molecules and the change in the tertiary structure of the protein (surface hydrophobicity of about 0.51), which is not conducive to the protection of the bioactivity of lysozyme (about 81%). Therefore, the excipient protection effect of Formulations 5 to 7 is far inferior to that of trehalose, and even worse than that of no excipient. In addition, Formulation 2 with trehalose as an excipient maintains the highest yield. The strong electrostatic interaction between microparticles caused by leucine and isoleucine with strong hydrophobicity results in a large amount of deposition in the low-temperature spray drying tower, making it difficult to collect and having the most obvious negative impact on the yield.

[0058] Table 5 Density, surface hydrophobicity, bioactivity and yield of low-temperature spray-dried lysozyme-containing microparticles

[0059]

[0060] In summary, trehalose is the optimal excipient, and the subsequent experiments are set up with this optimal choice.

[0061] (2) According to the same proportions as Formulation 1, Formulation 2, Formulation 3 and Formulation 4 in this example, lysozyme precursor solutions were prepared respectively, 10 mL of each formulation was accurately transferred into a sample bottle and vacuum freeze-drying was performed to prepare microparticles. The sample bottle was placed in a vacuum freeze-drying machine, the pre-freezing temperature was set to -40℃, the pre-freezing time was 3 hours, the first drying was kept at -40, -20 and -10℃ for 12 hours respectively, the second drying was kept at 0, 10 and 20℃ for 8 hours respectively, and product 7, product 8, product 9 and product 10 were prepared. The macroscopic and microscopic morphology of the obtained lysozyme and lysozyme composition microparticles are shown in FIG. 6. It can be seen that the lysozyme microparticles after vacuum freeze-drying present large irregular fragments, the surface is smooth, the surface of the microparticles of Formulation 3 containing leucine has wrinkles and fine pores, and the surface of the microparticles of Formulation 4 containing mannitol has obvious wrinkles due to the growth of mannitol crystals, and fine crystals can be observed. Figure 3 The water content of the four formulation samples was 5.03%, 5.33%, 5.18% and 5.24% w / w respectively, and there was no significant difference.

[0062] The density, surface hydrophobicity and biological activity data of the lysozyme-containing microparticles prepared by vacuum freeze-drying are shown in Table 6. It can be seen that compared with low-temperature spray drying, the four lysozyme-containing microparticles prepared by vacuum freeze-drying have a Karl index of about 55% or more, and the flowability is poor. In addition, during the vacuum freeze-drying process, the mechanical stress generated by the slow growth of ice crystals during the low-temperature process of the four formulations lasts for a long time, which destroys the tertiary structure of lysozyme, resulting in a lower surface hydrophobicity, especially for mannitol which is easy to crystallize. Therefore, the biological activity is also reduced, and the biological activity of Formulation 2 containing trehalose is the highest (92%). The freeze-drying technology is not conducive to the preparation of lysozyme spherical microparticles with good flowability and adjustable size and morphology, which increases the energy consumption and time cost of the instrument, and also increases the action time of cold stress on lysozyme. Therefore, the low-temperature spray drying technology is more suitable for preparing lysozyme microparticles with high biological activity.

[0063] Table 6 Density, surface hydrophobicity and biological activity of lysozyme-containing microparticles prepared by vacuum freeze-drying

[0064]

[0065] The above examples are only preferred examples for fully illustrating the present application, and the protection scope of the present application is not limited thereto. Any equivalent replacement or transformation made by those skilled in the art on the basis of the present application is within the protection scope of the present application. The protection scope of the present application is subject to the claims.

Claims

1. Use of a particulate lysozyme composition for the manufacture of an antibacterial medicament, characterized in that, The preparation method of the lysozyme composition microparticle is: S1, mixing lysozyme and trehalose to prepare a precursor solution; the mass ratio of the lysozyme to the trehalose is 3:1, and the precursor solution is an aqueous solution; S2, performing low-temperature spray drying treatment on the precursor solution to obtain the lysozyme composition microparticle; The conditions of the low-temperature spray drying process include: an inlet temperature of 42°C, an outlet temperature of 28°C, a dispersed air flow rate of 6 m 3 / h, a feed flow rate of 720 mL / h, a spray pressure of 0.5 MPa, and a hot air flow rate of 210 m 3 / h. The lysozyme is egg white lysozyme, the drug loading of the lysozyme composition microparticle is 75%, the relative biological activity is greater than 95%, the average geometric size is less than 5 μm, and the yield of the preparation method is 86%.

2. Use according to claim 1, characterized in that, In step S1, at least one of the following features is included: (1) in the precursor solution, the total mass percentage of the lysozyme and the trehalose is 1%-5%; (2) the mixing condition is a 30-40℃ water bath.

3. Use according to claim 1, characterized in that, The lysozyme composition microparticle is a spherical microparticle or a spheroid microparticle.

4. Use according to claim 1, characterized in that, The use of the antibacterial drug includes inhibiting gram-positive bacteria.

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