Recombinant mussel mucoprotein preparation for repairing mucosal lesion and preparation method thereof

Abstract

The invention discloses a recombinant mussel mucoprotein preparation for repairing mucosal lesion and a preparation method thereof. The recombinant mussel mucin preparation is prepared from recombinant mussel mucin and pharmaceutic adjuvants, the pharmaceutical composition comprises the following components in percentage by weight: 0.5-12.5% of recombinant mussel mucin and the balance of pharmaceutic adjuvants, the pharmaceutic adjuvant is trehalose, lactose or mannitol or a mixture thereof. While the protein activity is maintained, the microspheres with the aerodynamic particle size of about 2.6 microns and good inhalability are successfully prepared, effective retention and long-acting slow release of drugs in local mucous membranes can be realized, and a technical model is provided for industrialization of bioadhesive preparations. The preparation process is simple and stable, the obtained powder mist preparation can effectively promote repair of mechanical injury of the nasal mucosa and has good biological safety and stability, and an innovative solution is provided for clinical treatment of acute and chronic mucosa (including nasal mucosa) injury.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a recombinant mussel mucin preparation, a preparation method thereof, and an application thereof in repairing mucosal damage. Background Art

[0002] As the first physical and immune barrier of the respiratory tract, the integrity of the nasal mucosa is crucial for maintaining the homeostasis of the nasal microenvironment. However, according to clinical data, approximately 15%-25% of the world's population suffers from nasal mucosal damage due to various factors, leading to a high incidence of nasal inflammatory diseases; the incidence of mucosal adhesions caused by poor mucosal healing after nasal surgery is as high as 28.6%. Nasal mucosal damage and its complications seriously affect the patient's postoperative and quality of life, and its clinical and scientific research value has received widespread attention in recent years. Currently, common clinical treatments for nasal mucosal damage mainly include glucocorticoids, antibiotics, nasal packing, nasal irrigation, etc. However, these methods are prone to cause multi-dimensional side effects and are easily affected by the special moist environment of the nasal cavity.

[0003] Mussel adhesive protein (MAP) is a novel biomaterial extracted from marine mussels. It possesses exceptionally strong wetting and adhesive properties, as well as diverse biological activities (wound repair, antibacterial, and anti-inflammatory). It can form waterproof, breathable, and insulating nanoscale mesh microstructures at various interfaces. In the biomedical field, natural mussel-derived MAP has achieved industrial application. Using genetic engineering techniques and synthetic biology strategies, recombinant expression and large-scale production of MAP are now possible. Studies have shown that recombinant MAP retains the biological properties of natural mussel MAP and can absorb moisture and adhere to damaged, moist nasal mucosal surfaces, forming a protective film. This MAP not only recruits cells, promotes cell growth, and accelerates wound healing, but also blocks pathogen invasion and alleviates various nasal discomfort symptoms.

[0004] Currently, mussel mucin has been used to treat nasal diseases. However, the common mussel mucin nasal products on the market are all liquid preparations, and there are a series of challenges, specifically: the use of natural mussels, which has a limited source, low yield, and high cost; liquid dosage forms, complex ingredients, poor stability, easy pH changes, easy loss of protein activity, little nasal deposition and retention, and low bioavailability. The above conditions can easily affect the product appearance, safety of use, and efficacy stability, ultimately leading to a decline in quality and reduced repair effect.

[0005] Nasal powder inhalers, as solid nasal preparations, are made by combining the drug substance with appropriate excipients to form a powder that is then sprayed into the nasal cavity using an appropriate drug delivery device. Compared to traditional liquid sprays, solid powder inhalers offer advantages such as simple ingredients, ease of use, and long-lasting stability, and are gaining increasing attention both domestically and internationally. Currently available nasal powder inhalers, such as budesonide nasal powder inhaler, dexamethasone fenpropiate nasal powder inhaler, epinephrine nasal dry powder, insulin nasal powder inhaler, and curcumin nasal powder inhaler, are mostly hormonal or herbal extract powder inhalers, primarily used for allergic rhinitis or the treatment of systemic or neurological diseases. There is a lack of protein-based nasal powder inhalers for the treatment of acute or chronic nasal mucosal damage, particularly recombinant mussel mucin nasal powder inhalers prepared using spray-drying technology, which exhibits excellent biocompatibility, long-lasting stability, and high nasal deposition rate.

[0006] Therefore, based on the shortcomings of existing nasal powder mist preparations and their preparation processes, as well as mussel mucin nasal liquid preparations, the purpose of the present invention is to utilize the super strong wet adhesion film-forming properties and wound repair function of recombinant mussel mucin, combined with the advantages of nasal administration of solid powder mist preparations, to develop a recombinant mussel mucin preparation for the treatment of acute and chronic mucosal (including nasal mucosal) injuries. Summary of the Invention

[0007] The present invention aims to overcome the shortcomings of existing products and technologies and provide a recombinant mussel mucin preparation and its preparation method that can quickly absorb moisture in a humid environment (including the nasal cavity), adhere and retain in situ, prolong the retention time of active proteins in the mucosa, improve the bioavailability of active proteins, and promote the repair of mucosal (including nasal mucosa) damage.

[0008] In order to achieve the above object of the invention, the present invention adopts the following technical solutions:

[0009] A recombinant mussel mucin preparation consists of recombinant mussel mucin and pharmaceutical excipients. The preparation contains 0.5%-12.5% ​​of the recombinant mussel mucin by weight, and the remainder is the pharmaceutical excipients. The pharmaceutical excipients are trehalose, lactose or mannitol or a mixture thereof.

[0010] Preferably, in the above-mentioned recombinant mussel mucin preparation, the pharmaceutical excipient is trehalose.

[0011] The preparation method of the recombinant mussel mucin preparation comprises the following steps:

[0012] (1) mixing the recombinant mussel mucin and pharmaceutical excipients in proportion, adding ultrapure water to dissolve, and ultrasonically dispersing at 24-27° C. and 80-100 kPa for 10-30 minutes to obtain a uniformly dispersed precursor solution;

[0013] (2) The precursor liquid was spray-dried with the following parameters: inlet air temperature 110-130°C, injection rate 1.0-2.5 mL / min, fan frequency 4.0-6.0 Hz, atomizing air pressure 0.05-0.09 MPa, and the powder was collected and sealed for storage.

[0014] The recombinant mussel mucin is the one disclosed in patent publication number CN117986340A, CN120040572A or CN116693692A, or a polypeptide having more than 85% identity with the amino acid sequence of the protein.

[0015] Compared with the prior art, the present invention has the following advantages:

[0016] (1) The present invention combines the wet adhesion properties of recombinant mussel mucin with the need for mucosal (including nasal mucosa) repair for the first time, and develops a recombinant mussel mucin powder mist preparation that can quickly absorb moisture and adhere to the damaged area and form a protective film in situ, thereby achieving synergistic repair of physical barriers and biological activities.

[0017] (2) The present invention optimizes excipient molecules through molecular dynamics modeling and screens the formulations and compositions of different powders to effectively improve the thermal stability of recombinant mussel mucin, increase the bioavailability of the protein, and improve its mucosal (including nasal mucosal) retention performance. As a preferred embodiment, trehalose can protect the recombinant mussel mucin molecules through stronger hydrogen bonding and glass transition effect, effectively enhancing the structural and functional stability of the recombinant mussel mucin molecules.

[0018] (3) The present invention uses spray drying technology to prepare the powder, thereby achieving long-term maintenance of protein activity and suitable aerodynamic properties. The established spray drying process is highly operable and reproducible, providing a standardized production path for the nasal delivery system of biomacromolecules. Its mass median aerodynamic particle diameter (MMAD) is 2.6-12.9μm. It can quickly absorb moisture and adhere to surfaces such as plastics, glass, metals, and biological tissues, and solidify into a film.

[0019] (4) The recombinant mussel mucin preparation prepared by the present invention can effectively promote the repair of nasal mucosal damage. It has been proven by in vitro and in vivo experiments that it has good biosafety and stability. Its "ready-to-use" administration characteristics and nasal adaptability provide an innovative solution for the clinical treatment of acute and chronic mucosal (including nasal mucosal) damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly describe the embodiments of the present invention, the accompanying drawings related to the embodiments are annotated below:

[0021] Figure 1 The results are the molecular interactions between recombinant mussel mucin and various pharmaceutical excipients;

[0022] Figure 2 The figures are scanning electron microscopy results of the recombinant mussel mucin preparations in various embodiments and comparative examples;

[0023] Figure 3 This is the appearance morphology of the recombinant mussel mucin preparation at 40°C;

[0024] Figure 4 This is a diagram of the moisture absorption and film-forming properties of the recombinant mussel mucin preparation after compounding with different excipients;

[0025] Figure 5 The diagram shows the moisture absorption and adhesion of the recombinant mussel mucin preparation on the subcutaneous mucosal surface after compounding with different excipients.

[0026] Figure 6 This is a picture of the recombinant mussel mucin preparation after mixing with different excipients, which absorbs moisture and dissolves before solidifying into a film on the surface of the fragment carrier;

[0027] Figure 7 It is the time it takes for the recombinant mussel mucin preparation mixed with different excipients to solidify and form a film on the surface of the fragment carrier after absorbing moisture and dissolving;

[0028] Figure 8 The results of cell compatibility-live-dead staining of recombinant mussel mucin preparations;

[0029] Figure 9 This is a diagram of the cell compatibility-cell viability test of the recombinant mussel mucin preparation;

[0030] Figure 10 The toxicity of recombinant mussel mucin preparation to nasal septum mucosa and cilia is shown;

[0031] Figure 11 This is a diagram showing the effect of recombinant mussel mucin preparation in promoting the regeneration and repair of nasal septum mucociliary cells. DETAILED DESCRIPTION

[0032] The present invention will be further explained below with reference to the examples. For those skilled in the art, it should be understood that the following examples are only used to illustrate the present invention and do not limit the scope of protection of the present invention. Under the guidance of the present invention, the contents adjusted according to the current technical means are within the scope of protection of the present invention.

[0033] Example 1: Screening for the best excipient based on computer simulation prediction

[0034] The structural modeling of each protein was performed using Alphafold2, SWISS-MODEL, and I-TASSER homology modeling platforms, with a focus on retaining the conformation of the adhesion domain (tyrosine-rich, DOPA residue region) for binding to excipient molecules. Pymol software was used for pre-processing, and the surface structure-related primary sequence characteristics and three-dimensional structural characteristics and molecular evolution site distribution were predicted and analyzed based on the docking algorithm, and the structural data were processed. Chitosan, trehalose, lactose, mannitol, etc. were selected as candidate excipients, and the molecular interaction relationship between the candidate excipients and the recombinant mussel mucin was predicted using Autodock vina. The results are as follows: Figure 1 shown.

[0035] Experimental results showed that trehalose had the strongest binding effect with recombinant mussel mucin, with an intermolecular binding energy of -6.5. This was achieved by forming hydrogen bonds with the lysine and proline amino acids in the recombinant mussel mucin, thereby improving its thermal stability. Mannitol and lactose, however, had intermolecular binding energies of -4.8 and -6.3, respectively, demonstrating strong binding effects, but weaker than trehalose.

[0036] Example 2: Preparation of recombinant mussel mucin preparation

[0037] The formula components of each embodiment and comparative example are shown in Table 1

[0038] Table 1. Formulas of Examples and Comparative Examples

[0039]

[0040]

[0041] The recombinant mussel mucin (lyophilized sample) and the excipients were added to a clean beaker in proportion, and 25-50 mL of ultrapure water was added to completely dissolve the mixture. The mixture was then ultrasonically dispersed at 80-100 kPa at 24° C.-27° C. for 10-30 min to obtain a clear, translucent, and evenly dispersed spray drying precursor solution. The precursor solution was spray dried (the spray dryer operating parameters were set as follows: inlet temperature: 110-130° C.; injection rate: 1.0-2.5 mL / min; fan frequency: 4.0-6.0 Hz; atomizing air pressure: 0.05-0.09 MPa). The powder at the sample outlet was collected to obtain a recombinant mussel mucin preparation, and the resulting powder mist preparation was stored in a drying oven.

[0042] Particle morphology observation: Take a small amount of dry powder sample and spread it on a clean and dry weighing paper. Use a small copper table (with conductive glue of appropriate size on the surface) to gently pick up the powder sample. Use an ear-cleaning ball to blow off the excessive sample powder accumulated on the surface of the copper table so that the powder sample is dispersed as evenly as possible on the conductive glue of the copper table. Then spray the sample with gold. Finally, transfer the sample to a scanning electron microscope and observe and take pictures at different magnifications to record the particle morphology of the sample. Figure 2 As shown, the unique properties of recombinant mussel mucin (lysine and tyrosine), due to its rich lysine and tyrosine content (cationic, hydrophobic, π-π stacking, and hydrogen bonding), endow the peptide with strong amphiphilicity and self-assembly tendencies, driving it to form spherical nanoparticles or aggregates in solution with a hydrophobic (tyrosine) core and a hydrophilic (lysine) shell. After formula optimization and spray drying, the powder particles of the nasal powder spray formulation are spherical or hemispherical, with a smooth surface and uniform particle size. Formula screening combined the respective advantages of trehalose and mannitol to balance the "glass formation rate" (mannitol) with the "amorphous stability" (trehalose), achieving a more uniform spherical structure.

[0043] Example 3: Particle size and particle size distribution detection of recombinant mussel mucin preparation

[0044] The particle size and distribution of recombinant mussel mucin powder aerosol preparations were determined using a fully automated dry powder, aerosol, and powder mist laser particle size analyzer (HELOS, H4508 & RODOS / L, R4). 0.3-0.5 g of sample particles were weighed and placed on the stage. Samples were injected at 3.5 bar pressure, a 50% injection rate, and a 15 mm lift. The results were analyzed using the instrument's built-in software, PAQXOS 5.2. Each sample was analyzed three times, and the average values ​​of D10, D50, and D90 were calculated. As shown in Table 2, the powder aerosol preparations prepared with recombinant mussel mucin combined with a single excipient exhibited smaller and more uniform particles, with the majority sized around 6 μm, the largest particles remaining around 13 μm, and the smallest particles ranging from 2 to 3 μm. In contrast, the powder aerosol preparations prepared with recombinant mussel mucin combined with multiple excipients exhibited a broad particle size distribution, making them less suitable for topical nasal administration.

[0045] Table 2. Particle size distribution of recombinant mussel mucin preparations of Examples 1-7

[0046]

[0047]

[0048] Example 4: Evaluation of thermal stability of recombinant mussel mucin preparations

[0049] The samples of the embodiment and the comparative example were placed in a drug stability test chamber, set at 40°C, and the appearance changes of the test samples were observed on the 60th day. Figure 3 As shown, Examples 1-3 maintained a loose powder form after prolonged storage at 40°C, indicating better thermal stability, while the remaining Examples and Comparative Examples all exhibited varying degrees of aggregate agglomeration. This indicates that the use of a single pharmaceutical excipient does not result in aggregate agglomeration, while compounding excipients is more susceptible to this phenomenon.

[0050] Example 5: Hygroscopic Adhesion of Recombinant Mussel Mucin Preparation

[0051] 10 / 50 mg of the powder mist sample of Example 1-3 was dispersed on a glass slide / wet mouse subcutaneous mucosa surface, and 30 / 150 μL of PBS solution was added dropwise to the powder particle surface. The absorption of the liquid by the powder particles and the changes in the overall morphology of the powder were observed at different time points. The results are as follows: Figure 4 and 5 As shown, compared to mannitol and lactose, trehalose dihydrate has a larger molecular space, more hydrogen-bonded water, and a greater volume exclusion effect. Therefore, at the same dose, the solubility of the recombinant mussel mucin in the dissolved state of the powder aerosol formulation sample of Example 2 is greater, and more recombinant mussel mucin molecules are released, allowing them to exert their superior adhesion (wet adhesion) function at interfaces or mucosal surfaces. Therefore, compared to Examples 1 and 3, the powder aerosol formulation of Example 2 can absorb liquid faster, achieving in situ hygroscopic adhesion.

[0052] Example 6: Interfacial film-forming properties of recombinant mussel mucin preparations

[0053] 2 μL, 10 μL, and 50 μL of the prepared powder mist solution of Examples 1-3 were added dropwise to the surface of the cover glass, and allowed to air dry at room temperature. The solidification and adhesion of the sample solution were observed, and the time required for solidification was recorded. After the solution was completely dry, the morphology change was observed using a scanning electron microscope. The results are as follows: Figure 6 As shown in Figure 7, when the droplet volume was 2 μL, the solidification time was approximately 20 minutes, with no significant differences between the groups of examples. When the droplet volume was 10 μL, Example 2 had the shortest solidification time, approximately 25 minutes, and when the droplet volume was 50 μL, Example 2 had the shortest solidification time, approximately 71 minutes. After solidification, it was observed that the powder mist sample of Example 2 formed a sticky film on the surface of the glass slide, while Examples 1 and 3 only showed crystallization / uneven film formation at the periphery. Therefore, as a preferred method, the powder mist preparation prepared using trehalose as an excipient in combination with recombinant mussel mucin can quickly absorb water on the mucosal surface to form a thin film that blocks external stimuli and facilitates cell adhesion and crawling.

[0054] Example 7: Evaluation of the biocompatibility of recombinant mussel mucin preparations (1) Cytocompatibility of powder aerosol preparations

[0055] ① Preparation of test sample: Take 100 mg of the preferred recombinant mussel mucin preparation and completely dissolve it in 1 mL of ultrapure water to obtain a 100 mg / mL powder aerosol solution. Then filter it three times using a 0.22 μm filter membrane and store it at -20°C until use.

[0056] ② Plate coating: In a clean bench, take 20 μL of the filtered and sterilized powder mist preparation solution sample and slowly drip it into the center of the bottom of a 96-well plate (the normal group was not treated, and the solvent group was coated with an equal amount of ultrapure water). The solution was gradually covered with the solution at the bottom of the well. The plate was covered and the edges were sealed with sealing film. The plate was then moved to 4°C for overnight coating. The next day, the plate was transferred to a clean bench, the 3-stage exhaust was turned on, and the plate was air-dried for 2 hours. Finally, the plate was washed with 100 μL / well PBS for later use.

[0057] ③ Preparation of cell suspension: Mouse skin fibroblast L929 cells in the logarithmic growth phase with a cell confluence of more than 90% were rinsed with sterile PBS. After removing the PBS, trypsin was added and digested in a 37°C, 5% CO2 incubator. Digestion was terminated by adding serum-containing medium (MEM). The cells were centrifuged at 1000 rpm for 3 minutes, the supernatant was removed, and the cells were fully resuspended in serum-containing medium (MEM) to prepare a cell suspension (100,000 cells / mL).

[0058] ④ Inoculation: Take out the 96-well plate to be used, inoculate 10,000 cells / well, and then transfer to a 37°C, 5% CO2 incubator for further incubation for 48 hours;

[0059] ⑤ CCK-8 assay for cell viability: After 48 h of culture, the cell wells were removed from the plate, and the cell growth status was observed under an inverted microscope and photographed. The plates were then transferred to a clean bench, and 10 μL of CCK-8 reagent was added to each well. The plates were then transferred to a 37°C, 5% CO2 incubator and incubated for 2.5 h. Finally, the absorbance at 450 nm was measured using an automatic microplate reader to calculate the cell viability.

[0060] ⑥ Live cell staining: Stain the cells in the 96-well plate using the acridine orange staining kit and take fluorescent photos of the live cells using a fluorescence microscope. The results are as follows: Figure 8 、 9 As shown, compared with the normal group and the solvent group, there was no significant change in cell morphology and cell activity in the Example group, indicating that within the test concentration range, the recombinant mussel mucin preparation of the Example had no significant cytotoxicity to L929 cells.

[0061] (2) Safety of topical nasal administration of powder aerosol preparations

[0062] ① Nasal administration: Rats were anesthetized with an intraperitoneal injection of 3% sodium pentobarbital. A normal group and a recombinant mussel mucin preparation group (Example), with 6 animals in each group, were administered 1.5-3.0 mg of the drug slowly into each nostril using a powder spray device, once daily for 7 days.

[0063] ②Observation record: Take photos and record the rats' mental state, nasal appearance and weight changes every other day after administration: whether there is nasal redness and swelling, bleeding, runny nose, curling up, slowed movement, weight loss, etc.

[0064] ③Indicator examination: On the 8th day, rats were anesthetized by intraperitoneal injection of 3% sodium pentobarbital and humanely killed. The nasal septum (including mucosa) was separated and fixed. Scanning electron microscopy observation: The complete nasal septum (including mucosa) fixed in 2.5% glutaraldehyde was taken out and washed 3 times with PBS, 15 minutes each time, and then dehydrated with 50%-100% ethanol gradient, 15 minutes each time. The complete nasal septum was supercritically dried, and then the nasal septum sample was metal-coated and finally observed by scanning electron microscopy. Figure 10 As shown, after 7 days of continuous administration, the animals ate a normal diet, were in good spirits, and gained weight slowly. No adverse reactions such as redness, swelling, bleeding, and ulceration of the nasal septum mucosa were observed. Scanning electron microscopy observation of the nasal cilia structure showed no significant differences among the groups. The cilia were dense, stretched, without defects, and arranged in the same direction, indicating that the recombinant mussel mucin preparation of the example did not cause obvious nasal mucosal toxicity.

[0065] Example 8: Repair effect of recombinant mussel mucin preparation on mechanical damage to nasal mucosa

[0066] (1) Model establishment: After guinea pigs were anesthetized by intraperitoneal injection of 3% sodium pentobarbital (50 mg / kg), an interdental brush (maximum diameter 2 mm, tooth length 1.5 cm) was used to brush the nasal septum in the middle and anterior part of the right nasal cavity of the guinea pig in a counterclockwise rotation for 10-15 times to damage the nasal septum mucosa. The brush was applied until slight blood streaks appeared on the surface of the interdental brush and the outer edge of the nostril. The guinea pigs were placed in a supine position for 10 minutes to reduce the backflow of blood into the lungs, thereby establishing a guinea pig nasal mucosal mechanical injury model.

[0067] (2) Group administration: 4 groups were set up, including normal group, model group, commercial product group, and recombinant mussel mucin preparation group, with 10 animals in each group. The guinea pigs were fixed in a supine position, and the recombinant mussel mucin preparation was slowly delivered to the right nasal cavity using a powder mist delivery device (the operation was repeated 3-4 times, and the final dosage was controlled at 1.5-3.0 mg). The normal group was not treated with injury, and the model group was not treated with medication after injury; the commercial product group was given 50 μL of sample solution using a microsyringe; the treatment was given once a day for 6 consecutive days, and the animals' mental state, eating behavior, weight changes, and local hygiene of the nostrils were recorded.

[0068] (3) Guinea pigs were humanely killed on the 7th and 14th days and the nasal septum mucosa was isolated for SEM observation. Figure 11 As shown, SEM images of the normal group revealed intact nasal cilia, with neatly arranged and uniformly oriented cilia. Seven days after surgery, the model group had severe mucosal damage, covered by a thick mucus blanket, and no cilia could be observed. In the recombinant mussel mucin preparation group, the nasal septum was intact, with numerous regenerated cilia distributed on the mucosal surface, and the number of cilia increased compared to the group receiving the lower concentration. In the commercial product group, partial mucosal damage was observed, accompanied by a small amount of mucus blanket, with short regenerated cilia appearing in some areas. Fourteen days after surgery, the model group still had significant mucosal damage, with a disorganized, short structure of cilia observed on the surface. In the recombinant mussel mucin preparation group, a dense, long structure of cilia of uniform length and relatively neat arrangement was observed on the mucosal surface, similar to the structure of the normal group. In the commercial product preparation group, the nasal septum healed, with accelerated cilia growth and the appearance of cilia of varying lengths. A small amount of secretions remained on the mucosal surface, indicating that the recombinant mussel mucin preparation can promote the repair of nasal mucosal damage, and this is related to the dosage and duration of treatment.

Claims

1. A recombinant mussel mucin preparation, characterized in that The invention is composed of recombinant mussel mucin and pharmaceutical excipients; in terms of weight percentage, it contains 0.5%-12.5% ​​of recombinant mussel mucin, and the remainder is pharmaceutical excipients; the pharmaceutical excipients are trehalose, lactose or mannitol or a mixture thereof.

2. The recombinant mussel mucin preparation according to claim 1, characterized in that The pharmaceutical excipient is trehalose.

3. The method for preparing the recombinant mussel mucin preparation according to claim 1, characterized in that: The following steps are involved: (1) mixing the recombinant mussel mucin and pharmaceutical excipients in proportion, adding ultrapure water to dissolve, and ultrasonically dispersing at 24-27° C. and 80-100 kPa for 10-30 minutes to obtain a uniformly dispersed precursor solution; (2) The precursor liquid was spray-dried with the following parameters: inlet air temperature 110-130°C, injection rate 1.0-2.5 mL / min, fan frequency 4.0-6.0 Hz, atomizing air pressure 0.05-0.09 MPa, and the powder was collected and sealed for storage.

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