Method for preparing mucoprotein, mucoprotein, antibacterial composition, drug delivery system and application thereof

Abstract

The invention discloses a method for preparing mucoprotein, the mucoprotein, an antibacterial composition, a drug delivery system and application thereof. The method comprises the following steps: 1) collecting mucus of animals, wherein the mucus contains mucoprotein; (2) mixing the mucus with a diluent, so that the mucus is diluted and homogenized; wherein the diluent comprises a buffer solution with a salt concentration of 20-190 mM; 3) filtering the mixed solution obtained in the step 2) by using a screen, wherein the aperture of the screen is 100 [mu] m-1 mm; and 4) dialyzing the filtered liquid in an alkaline buffer solution, and dialyzing with ultrapure water or deionized water to obtain mucoprotein. The method is simple in step and mild in condition, the obtained mucoprotein retains natural biological activities such as pH responsiveness, bacterial growth inhibition and bacterial biofilm damage, the molecular weight of the obtained mucoprotein is far larger than that of commercially sourced mucoprotein, and the obtained mucoprotein can be further processed into a high-grade biological material to play a role in inhibiting bacterial growth.

Description

Technical Field

[0001] The present invention belongs to the fields of engineering materials and molecular biology, and more particularly, relates to methods for preparing mucins, mucins, antibacterial compositions, drug delivery systems, and their applications. Background Art

[0002] Traditional dressings for skin and mucosal and wound care, such as gauze, films, foams, hydrogels (such as alginates, chitosans, and collagens), and hydrophilic colloids, all have some drawbacks, such as requiring trauma and mechanical debridement, causing vasoconstriction and tissue hypoxia, being cytotoxic, producing a large amount of exudate, being susceptible to enzymatic degradation, requiring frequent replacement, being occlusive, and being susceptible to bacterial contamination.

[0003] Bioactive and functional components, such as antibacterial agent compounds, are used to overcome these problems. Some of these bioactive dressings contain natural polymers, such as alginates, chitosans, and collagens, which have been reported to promote tissue regeneration. In addition, antimicrobial peptides have been incorporated into dressings and creams, and several clinical trials are currently underway. Drug-loaded dressings with antibacterial properties have been widely developed, resulting in a variety of commercially available products, including gauze, foams, hydrophilic colloids, and hydrogels. These dressings contain silver ions as antibacterial agents, and several brands offer such products. Although the use of silver ions is an active area of research, there are concerns about its cytotoxic effects, and the results of the in vivo release rate of silver ions are still controversial. In addition, the application of iodine in wound dressings is not yet fully mature, as the safety and mode of action of iodophors (such as povidone iodine) are still unclear. Antibiotics (such as ciprofloxacin and streptomycin) can also be included in dressings. However, due to concerns about antibiotic resistance, the long-term use of antibiotics is not encouraged. Therefore, there is an urgent need to develop biocompatible dressings that can promote wound healing, inhibit bacterial growth, and deliver active molecules on demand.

[0004] Mucins are a class of glycoproteins with a brush-like structure. Their structure can be simply described as having two hydrophobic protein termini, with glycans grafted onto the protein backbone between the two ends (as shown in Figure 2 A). Natural mucins, as a highly biocompatible compound, exhibit various bioactivities such as lubrication, pH responsiveness, and biofilm regulatory activity, and at the same time, they have good stability under extreme pH and bactericidal conditions. This can overcome the above problems and develop functional dressings and creams for skin and muscle and wound care. Mucins, as a bioactive compound, have been widely used for various purposes, and most of them are commercially available mucin products based on animal sources. However, due to the harsh extraction methods during large-scale production, commercially available mucin products lose most of their bioactivities, which limits their application in scenarios where mucin properties are required. Summary of the Invention

[0005] To solve the problems existing in the above-mentioned prior art, the present invention provides a method for preparing mucin, mucin, an antibacterial composition, a drug delivery system and their applications.

[0006] Specifically, the present invention provides:

[0007] (1) A method for preparing mucin, comprising the following steps:

[0008] 1) Collect the mucus of an animal, wherein the mucus contains mucin;

[0009] 2) Mix the mucus with a diluent to dilute and homogenize the mucus; wherein the diluent comprises a buffer solution with a salt concentration of 20 - 190 mM;

[0010] 3) Filter the mixture obtained in step 2) through a sieve, and the aperture of the sieve used is 100 μm - 1 mm;

[0011] 4) Dialyze the filtered liquid in an alkaline buffer solution, and then dialyze it with ultrapure water or deionized water to obtain mucin.

[0012] (2) The method according to (1), wherein the buffer solution in step 2) is selected from phosphate buffer solution, 4-morpholineethanesulfonic acid buffer solution (MES), 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid buffer solution (HEPES), tris(hydroxymethyl)aminomethane hydrochloride buffer solution (Tris-HCL) and 3-morpholinopropanesulfonic acid buffer solution (MOPS), and the buffer solution further contains a metal salt additionally, so that the total salt concentration of the buffer solution is 20 - 190 mM; the metal salt is selected from sodium salt, potassium salt, magnesium salt and calcium salt.

[0013] (3) The method according to (1), wherein the buffer solution in step 2) is a 10 - 20 mM phosphate buffer solution, and further contains 10 - 170 mM of a metal salt additionally; preferably, the phosphate buffer solution is selected from sodium phosphate buffer solution, potassium phosphate buffer solution, magnesium phosphate buffer solution and calcium phosphate buffer solution, and the metal salt is selected from sodium salt, potassium salt, magnesium salt and calcium salt.

[0014] (4) The method according to (1), wherein the pH of the buffer solution in step 2) is less than or equal to 7; for example, the pH value is below 6.

[0015] (5) The method according to (1), wherein the diluent in step 2) further comprises a microbicide and / or a preservative; preferably, the microbicide and / or the preservative comprise 2-methyl-4-isothiazolin-3-one and 5-chloro-2-methyl-4-isothiazolin-3-one; preferably, the concentration of the microbicide and / or the preservative in the diluent is 0.05% (v / v) - 0.1% (v / v).

[0016] (6) The method according to (1), wherein in step 2), the mucus is diluted 5-fold by the diluent; preferably, the mixing is carried out at 1 - 10 °C for 8 - 48 hours.

[0017] (7) The method according to (1), wherein the screen filtration in step 3) is carried out at pH 8 - 10, or at pH 12 - 13.

[0018] (8) The method according to (1), wherein the screen filtration in step 3) is carried out in a 5 - 12 mM buffer additionally containing 170 mM - 200 mM metal salt; preferably, the buffer is selected from phosphate buffer, 4-morpholineethanesulfonic acid buffer (MES), 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid buffer (HEPES), tris(hydroxymethyl)aminomethane hydrochloride buffer (Tris-HCL), and 3-morpholinopropanesulfonic acid buffer (MOPS); preferably, the metal salt is selected from sodium salt, potassium salt, magnesium salt, and calcium salt.

[0019] (9) The method according to (8), wherein the buffer further comprises a microbicide and / or a preservative; preferably, the microbicide and / or the preservative comprise 2-methyl-4-isothiazolin-3-one and 5-chloro-2-methyl-4-isothiazolin-3-one; preferably, the concentration of the microbicide and / or the preservative in the buffer is 0.05% (v / v) - 0.1% (v / v).

[0020] (10) The method according to (1), wherein the screen filtration in step 3) is carried out at least twice. The pore size of the first screen used for the first filtration is 200 μm - 1 mm, and the pore size of the second screen used for the second filtration is 100 - 500 μm, and the pore size of the first screen is larger than that of the second screen.

[0021] (11) The method according to (1), wherein the salt concentration of the buffer in step 4) is 50 mM - 200 mM; preferably, the buffer in step 4) is selected from carbonate-bicarbonate buffer, ammonium hydroxide-ammonium chloride buffer, and tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetic acid buffer.

[0022] (12) The method according to (1), wherein the pH value of the buffer solution in step 4) is 8-10 or pH 12-13.

[0023] (13) The method according to (1), wherein in step 4), the dialysis is carried out using a dialysis device with a cut-off molecular weight of 14-100 kDa.

[0024] (14) The method according to (1), wherein in step 4), the dialysis comprises: using a dialysis device with a cut-off molecular weight of 14-100 kDa, placing the filtered liquid in the buffer solution at 1-10 °C for 8-24 hours, and then placing it in ultrapure water or deionized water at 4 °C for 24-72 hours.

[0025] (15) The method according to (1), wherein the animal is a mammal, and the mucus is collected from saliva or the digestive tract.

[0026] (16) The method according to (1), wherein the method further comprises: 5) freeze-drying the obtained mucin; preferably, the freeze-drying is carried out at -50 °C to -80 °C for 48-96 hours.

[0027] (17) The method according to any one of (1)-(16), wherein the obtained mucin is stored at 0 to 10 °C or -10 to -30 °C.

[0028] (18) A mucin prepared by the method according to any one of (1)-(17).

[0029] (19) The mucin according to (18), wherein the molecular weight of the mucin is greater than 40 kDa, preferably greater than 50 kDa, and more preferably greater than 270 kDa.

[0030] (20) An antibacterial composition comprising the mucin according to (18) or (19).

[0031] (21) The antibacterial composition according to (20), wherein the composition comprises 20 (weight)% - 80 (weight)% of the mucin.

[0032] (22) The antibacterial composition according to (20), wherein the composition further comprises an antibacterial compound and / or a hydrophobic therapeutic agent; preferably, the antibacterial compound includes antibiotics, sterilizing agents, and antibacterial peptides; preferably, the hydrophobic therapeutic agent has an anti-reactive oxygen function; preferably, the hydrophobic therapeutic agent includes curcumin and total saponins.

[0033] (23) The antibacterial composition according to (20), wherein the composition is prepared in the form of an aqueous solution or a suspension.

[0034] (24) The antibacterial composition according to claim 23, wherein the aqueous solution or suspension is prepared as a nasal drop, hand sanitizer, cream or artificial scaffold treatment agent.

[0035] (25) The antibacterial composition according to (20), wherein the composition is prepared as an ointment, gel or paste.

[0036] (26) The antibacterial composition according to (25), wherein the ointment, gel or paste is prepared as a wound dressing, antibacterial and anti-biofilm coating, artificial scaffold or pH-responsive substrate.

[0037] (27) The antibacterial composition according to (20), wherein the composition is prepared as a spray or composite nanofiber membrane.

[0038] (28) The antibacterial composition according to (27), wherein the spray or composite nanofiber membrane is prepared as textiles, masks and wound dressings.

[0039] (29) The antibacterial composition according to (23), wherein the aqueous solution or suspension contains 0.1 - 2 (weight)% of the mucin.

[0040] (30) The antibacterial composition according to (23), wherein the aqueous solution or suspension further contains a buffer, viscosity modifier or wetting agent; preferably, the buffer includes phosphate and carbonate-bicarbonate buffer solutions, and the viscosity modifier or wetting agent includes glycerol and silicone oil.

[0041] (31) The antibacterial composition according to (25), wherein the ointment, gel or paste contains more than 20 (weight)% of the mucin.

[0042] (32) The antibacterial composition according to (25), wherein the ointment, gel or paste further contains an antibiotic and / or a poorly soluble drug.

[0043] (33) The antibacterial composition according to (27), wherein the spray or composite nanofiber membrane contains 1 - 5 (weight)% of the mucin.

[0044] (34) A drug delivery system comprising the mucin according to (18) or (19) and a hydrophobic therapeutic agent.

[0045] (35) The drug delivery system according to (34), further comprising methacrylic anhydride.

[0046] (36) Use of the mucin according to (18) or (19) in antibacterial, anti-biofilm, drug delivery and pH monitoring, wherein the use is not for diagnostic and therapeutic purposes.

[0047] The present invention has the following advantages and positive effects compared with the prior art:

[0048] Based on the structural characteristics of mucin, the present invention ingeniously designs a mild method for preparing mucin that does not require large-scale instruments. This method protects the polysaccharide molecules grafted on the mucin molecule from being destroyed as much as possible and preserves the structural integrity of the mucin molecule, retains the natural protective effect of the polysaccharide molecule on the protein chain, greatly reduces the probability of mucin being degraded during the preparation process, and maintains the interaction connection between mucin molecules. Therefore, the mucin prepared by the present invention retains the structural characteristics such as pH responsiveness of mucin and other natural biological activities such as inhibiting bacterial growth and destroying bacterial biofilms, and the molecular weight is much larger than that of commercially available mucin. In addition, compared with common proteins, since the mucin prepared by the present invention retains the natural protective effect of the polysaccharide molecule on the protein chain, it can be stored at a relatively high temperature.

[0049] The above advantages enable the mucin obtained by the present invention to be further processed into different forms of advanced biomaterials to play the role of inhibiting bacterial growth. In addition, the present invention utilizes the pH responsiveness of mucin to load active molecules onto the mucin-based material, realizing the composite function of drug delivery. Therefore, the preparation method of the present invention and the obtained biomaterials can not only overcome the disadvantage of the lack of biocompatibility of artificial polymers, but also provide various biological activities such as inhibiting bacterial growth, destroying biofilms, delivering active molecules, and promoting wound healing.

[0050] The method for preparing mucin of the present invention has simple steps and mild conditions. Only by collecting, homogenizing, filtering, and dialysis desalting steps can the mucin with excellent biological activity be obtained, without the need for complex purification and separation steps such as centrifugation and column chromatography (such as size exclusion chromatography (SEC)). BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 Photographs showing the process of preparing mucin of the present invention.

[0052] Figure 2 Schematic diagrams showing the structure of mucin (A), the connection between mucin molecules (B), and the aggregated state (C) and relaxed state (D) between mucin molecules.

[0053] Figure 3 Showing the mucin prepared by the present invention ( Figure 3 A) and commercially available mucin ( Figure 3 B) atomic force microscopy photographs.

[0054] Figure 4 Showing the mucin prepared by the present invention ( Figure 4 A) and commercially available mucin (Figure 4 B) SEM image after lyophilization.

[0055] Figure 5 Shows the polyacrylamide gel electrophoresis results of the mucin prepared by the present invention and commercially available mucin before and after treatment with dithiothreitol (DTT). Lane M is the molecular weight marker; lanes 1 and 3 are the mucin prepared by the present invention before and after DTT treatment, respectively; lanes 2 and 4 are the commercially available mucin before and after DTT treatment, respectively.

[0056] Figure 6 Shows the particle size (A) of the mucin prepared by the present invention and commercially available mucin measured by a 90Plus particle size / Zeta analyzer, compares the total protein content (B) and quantitative standard curve (D) of the mucin prepared by the present invention and commercially available mucin by the bicinchoninic acid (BCA) method, and compares the polysaccharide content (C) by the periodic acid Schiff staining (PAS) method. The data in the figure are expressed as mean ± standard deviation. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001, ns: not significant.

[0057] Figure 7 Shows the infrared spectrum (IR) of the mucin prepared by the present invention.

[0058] Figure 8 A shows the concept and principle schematic diagram of preparing a drug delivery system by mixing the mucin prepared by the present invention with curcumin particles. Figure 8 B shows the color change presented by the drug delivery system at different pH values.

[0059] Figure 9 Shows that the mucin prepared by the present invention has the effect of inhibiting bacterial growth and destroying bacterial biofilms. Figure 9 A shows the schematic diagram of doping mucin on nanofibers by spray electrospinning method and its effect. Figure 9 B left shows the schematic diagram of the bacterial growth curve experiment; Figure 9 B right shows the growth curve of Escherichia coli added with mucin at different time points. Figure 9 C shows the antibacterial effect results of comparing the mucin extract of the present invention with commercially available mucin by the MIC method. Figure 9 D shows the antibacterial effect results of detecting kanamycin by the MIC method.

[0060] Figure 10 A shows the circular dichroism spectra of the mucin prepared by the present invention (upper figure) and commercially available mucin (lower figure) at pH 4 (blue line) and pH 8 (red line). Figure 10B and C respectively show the structures of mucin observed by transmission electron microscopy at pH 4 (B) and pH 8 (C); the scale bars of the transmission electron microscopy are 100 nm (left) and 50 nm (right). Figure 10 D and E respectively show the structures of mucin observed by atomic force microscopy at pH 4 (D) and pH 8 (E); the scale bar of the atomic force microscopy is 100 nm.

[0061] Figure 11 Shows the analysis diagram of Figure 7 the shown FTIR results; wherein Figure 11 A is the diagram after adjusting the y-axis of Figure 7 to display the absorption value; Figure 11 B is the enlarged FTIR diagram in the range of 2800 - 3100 cm-1; Figure 11 C is the enlarged FTIR diagram in the range of 1200 - 1700 cm-1; Figure 11 D is the enlarged FTIR diagram in the range of 900 - 1200 cm-1.

[0062] Figure 12 Shows the results of size exclusion gel chromatography for the mucin extract prepared by the present invention ( Figure 12 A) and the commercially available mucin ( Figure 12 B) respectively.

[0063] Figure 13 Shows the effect of the mucin of the present invention on destroying bacterial biofilms.

[0064] Figure 14 Shows the effects of the mucin extract of the present invention, commercially available mucin, and bovine serum albumin at different concentrations on cell viability.

[0065] Figure 15 Shows the turbidity changes of the mucin extract of the present invention under different concentrations and pH conditions. The data are presented in the form of mean ± standard deviation. Detailed Embodiments

[0066] The present invention will be further described below through the description of specific embodiments and with reference to the accompanying drawings. However, this is not a limitation to the present invention. Those skilled in the art can make various modifications or improvements according to the basic idea of the present invention, but as long as they do not depart from the basic idea of the present invention, they are within the scope of the present invention.

[0067] Mucins, as glycoproteins, are involved in a variety of biological recognition processes, such as pathogen recognition, barrier and bacterial virulence regulation. Mucins are the main components of mucus and can be extracted from the surfaces of the lungs, stomach, and intestines. By reconstructing mucins in vitro while preserving their structure and natural properties, they can be used as the cornerstone of engineering biomaterials, which have the potential to be translated into commercial products. Their applications in antifouling coatings, artificial saliva, and cosmetic lubricants have been reported. Different from most proteins, mucins in the stomach remain stable under extreme pH values and bactericidal conditions. In addition, they also play a role in promoting the proliferation of intestinal probiotics and maintaining the stability of the intestinal flora. However, due to the extreme extraction conditions experienced by commercially available sources of gastric mucins, these proteins have lost many functional structural properties, making it difficult to process them into bioactive biomaterials.

[0068] The present invention provides a mild preparation method that can retain the natural biological activities of mucins, such as pH responsiveness, inhibition of bacterial growth, and disruption of bacterial biofilms.

[0069] Specifically, the present invention provides a method for preparing mucins, comprising the following steps:

[0070] 1) Collect mucus from an animal, wherein the mucus contains mucins;

[0071] 2) Mix the mucus with a diluent to dilute and homogenize the mucus; wherein the diluent comprises a buffer solution with a salt concentration of 20 - 190 mM;

[0072] 3) Filter the mixture obtained in step 2) through a sieve with a pore size of 100 μm - 1 mm;

[0073] 4) Dialyze the filtered liquid in an alkaline buffer solution, and then dialyze it with ultrapure water or deionized water to obtain mucins.

[0074] The animal includes mammals, including but not limited to pigs, cows, horses, sheep, monkeys, mice, rats, etc. The mucus can be collected from saliva or the digestive tract. The digestive tract includes the stomach and intestines.

[0075] In some embodiments, collecting mucus from an animal includes obtaining mucus from the surface of the digestive tract (such as the stomach and intestines).

[0076] In step 2) of the present invention, the mucus is diluted and homogenized by mixing it with a diluent. Preferably, the mucus is diluted 5 times by the diluent. More preferably, the mixing is carried out at 1 - 10 °C for 8 - 48 hours, such as stirring overnight at 4 °C or stirring overnight at room temperature.

[0077] The diluent contains a buffer solution with a low salt concentration. The present invention proposes that a low salt concentration can avoid the reduction of the Debye screening effect, protect the electrostatic interaction of charged mucins, and thus prevent the separation of aggregated mucins (i.e., in a relaxed state). Premature separation of aggregated mucins during the preparation process will increase the probability of protein degradation. In addition, a low salt concentration will not damage the polysaccharide molecules grafted on the mucins, maintaining the natural protective effect of the polysaccharide molecules on the protein chain, thereby protecting the protein backbone from degradation. Therefore, the present invention selects to use a buffer solution with a low salt concentration.

[0078] The low salt concentration means that the salt concentration of the buffer solution for dilution is 20 - 190 mM. For example, the salt concentration is 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM.

[0079] In addition, the present invention preferably also uses a pH value that makes the mucin molecules in an aggregated state in step 2).

[0080] The terms "aggregated state" and "relaxed state" used in the present invention mean that under different pH conditions, the structure of the mucins of the present invention will change, and the molecules will convert between the aggregated state and the relaxed state. At low pH values, the interaction between mucin molecules is enhanced, especially at the N-terminus, and thus they tend to aggregate together (as shown in Figure 2 Figure C); at higher pH values, the aggregates formed between mucin molecules will stretch and be loosely connected together by the C-terminus (as shown in Figure 2 Figure D). This is the pH responsiveness described herein.

[0081] Preferably, the pH of the buffer solution in step 2) is less than or equal to 7. In some embodiments, the pH value of the buffer solution is less than 7, for example, below 6. For gastric mucin, a more preferred pH value for making the mucin molecules in an aggregated state is 2 - 4; for intestinal mucin, a more preferred pH value for making the mucin molecules in an aggregated state is 6 - 7.

[0082] The buffer solution can be selected from phosphate buffer solution, 4-morpholineethanesulfonic acid buffer solution (MES), 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid buffer solution (HEPES), tris(hydroxymethyl)aminomethane hydrochloride buffer solution (Tris-HCL), and 3-morpholinopropanesulfonic acid buffer solution (MOPS), and the buffer solution further contains a metal salt additionally, such that the total salt concentration of the buffer solution is 20 - 190 mM. The metal salt is selected from sodium salt, potassium salt, magnesium salt, and calcium salt.

[0083] The phosphate buffer is preferably selected from sodium phosphate buffer, potassium phosphate buffer, magnesium phosphate buffer, and calcium phosphate buffer. The metal salt is preferably selected from sodium salts, potassium salts, magnesium salts, and calcium salts. Preferably, the buffer is a 10 - 20 mM phosphate buffer, and further contains 10 - 170 mM of the metal salt therein, for example, further contains 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, or 170 mM of the metal salt.

[0084] In a more specific embodiment, the buffer is a sodium phosphate - sodium chloride buffer.

[0085] It is known that the pH value of the commonly used phosphate buffer is acidic or neutral, for example, 5.8 - 7.2.

[0086] The diluent in step 2) may further contain a microbicide and / or a preservative. The microbicide and / or preservative includes but is not limited to 2 - methyl - 4 - isothiazolin - 3 - one and 5 - chloro - 2 - methyl - 4 - isothiazolin - 3 - one.

[0087] In some embodiments, the concentration of the microbicide and / or preservative in the diluent is 0.05% (v / v) - 0.1% (v / v).

[0088] Preferably, the pH of the solution system filtered through the screen in step 3) is adjusted to 8 - 10, or 12 - 13. Under alkaline conditions, the mucin molecules will be in a relaxed state. A more preferred pH value for gastric mucin molecules to be in a relaxed state is 8 - 10; a more preferred pH value for intestinal mucin molecules to be in a relaxed state is pH 12 - 13.

[0089] Preferably, the screen filtration in step 3) is carried out in a buffer with a low salt concentration.

[0090] In some preferred embodiments, the screen filtration is carried out in a 5 - 12 mM buffer additionally containing 170 mM - 200 mM of the metal salt.

[0091] Preferably, the buffer used in step 3) is selected from phosphate buffer, 4 - morpholineethanesulfonic acid buffer (MES), 2 - [4 - (2 - hydroxyethyl)piperazin - 1 - yl]ethanesulfonic acid buffer (HEPES), tris(hydroxymethyl)aminomethane hydrochloride buffer (Tris - HCL), and 3 - morpholinopropanesulfonic acid buffer (MOPS).

[0092] Preferably, the metal salt is selected from sodium salts, potassium salts, magnesium salts, and calcium salts. In some specific embodiments, the buffer in this step is a sodium phosphate - sodium chloride buffer.

[0093] The buffer solution used in step 3) may further contain a microbicide and / or a preservative. The microbicide and / or preservative includes but is not limited to 2-methyl-4-isothiazolin-3-one and 5-chloro-2-methyl-4-isothiazolin-3-one.

[0094] In some embodiments, the concentration of the microbicide and / or preservative in the buffer solution used in step 3) is 0.05% (v / v) - 0.1% (v / v).

[0095] In some preferred embodiments, the mesh filtration in step 3) is performed at least twice. The pore size of the first sieve used in the first filtration is 200 μm - 1 mm, and the pore size of the second sieve used in the second filtration is 100 - 500 μm, and the pore size of the first sieve is larger than that of the second sieve.

[0096] After the mesh filtration, the present invention performs dialysis at a low salt concentration and a pH value that relaxes the intermolecular structure of mucin (i.e., alkaline pH, preferably pH 8 - 10 or pH 12 - 13), and then performs dialysis in ultrapure water.

[0097] Preferably, the salt concentration of the buffer solution in step 4) is 50 mM - 200 mM, more preferably 200 mM. Preferably, the buffer solution is selected from carbonate-bicarbonate buffer solution, ammonium hydroxide-ammonium chloride buffer solution, and tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetic acid buffer solution.

[0098] It is known that the pH value of the commonly used carbonate-bicarbonate buffer solution is alkaline, such as pH 8.2 - 9.2, pH 8 - 10.

[0099] Since the preparation method of the present invention protects mucin from being degraded as much as possible during the preparation process, and protects the polysaccharide molecules grafted on the mucin molecules from being destroyed, retaining the natural protective effect of the polysaccharide molecules on the protein chain, greatly reducing the probability of mucin being degraded during the preparation process; moreover, the mild preparation method of the present invention can maintain the complete structure of mucin, so it can form intermolecular terminal connections like in the natural state in vivo, forming a conjugate. Therefore, the molecular weight of the mucin of the present invention is greater than that of the vast majority of macromolecular proteins. Therefore, by selecting a suitable cut-off molecular weight, substances such as lipids, microparticles, cell debris, unwanted proteins, polymers, fungicides, salts, etc. can be removed in the dialysis step, thereby realizing the purification of mucin.

[0100] The term "conjugate" means that multiple mucin molecules are connected together through the ends of the protein backbone, even in the relaxed state, such as Figure 2 shown in D. Natural mucin molecules form such connections in the natural state in vivo.

[0101] Preferably, in step 4), the dialysis is carried out using a dialysis device with a cut-off molecular weight of 14-100 kDa. Preferably, the dialysis is carried out using a dialysis device with a cut-off molecular weight of 14-50 kDa.

[0102] In addition, since the dialysis of the present invention is carried out at a pH that relaxes the mucin structure, the apparent size of the relaxed mucin increases, making it easier to retain in the dialysis tube / bag.

[0103] In some specific preferred embodiments, in step 4), the dialysis includes placing the filtered liquid in a buffer at 1-10 °C for 8-24 hours using a dialysis tube with a cut-off molecular weight of 14-100 kDa, and then placing it in ultrapure water or deionized water at 4 °C for 24-72 hours.

[0104] The method of the present invention may further include: 5) freeze-drying the obtained mucin. Preferably, the freeze-drying is carried out at -50 °C to -80 °C (for example, -50 °C to -60 °C) for 48-96 hours.

[0105] The mucin obtained by the present invention can be stored at 0 to 10 °C (for example, 0 to 8 °C) or -10 to -30 °C.

[0106] The method of the present invention retains structural features such as the pH responsiveness of mucin, as well as other natural biological activities such as inhibiting bacterial growth and destroying bacterial biofilms, and can obtain mucin with a large molecular weight. Moreover, compared with common proteins, since the mucin prepared by the present invention retains the natural protective effect of polysaccharide molecules on protein chains, it can be stored at a relatively high temperature. However, for common proteins, the preservation of purified proteins is very difficult, and generally they need to be placed at -80 °C because proteins are easily decomposed by enzymes, resulting in a decrease in quality.

[0107] Therefore, another aspect of the present invention provides a mucin prepared by the method described in the present invention.

[0108] The mucin of the present invention is a high-molecular-weight O-glycosylated linear protein with a molecular weight greater than 40 kDa, preferably greater than 50 kDa, preferably greater than 270 kDa; among them, the molecular weight of a part of the mucin of the present invention is in the range of 52-270 kDa, and the molecular weight of most mucin is above 270 kDa.

[0109] Another aspect of the present invention also provides an antibacterial composition comprising the mucin described in the present invention.

[0110] In some embodiments, the antibacterial composition comprises 20 (weight)% - 80 (weight)% of the mucin.

[0111] In some embodiments, the antibacterial composition further comprises an antibacterial compound and / or a hydrophobic therapeutic agent. Preferably, the antibacterial compound includes an antibiotic, a bactericide, and an antibacterial peptide. Preferably, the hydrophobic therapeutic agent has an anti-reactive oxygen function; preferably, the hydrophobic therapeutic agent includes curcumin and total saponins.

[0112] In some embodiments, the antibacterial composition of the present invention is prepared in the form of an aqueous solution or a suspension. Furthermore, the aqueous solution or suspension can be prepared as a nasal drop, a hand sanitizer, a cream, or an artificial scaffold treatment agent.

[0113] Preferably, the aqueous solution or suspension contains 0.1-2 (weight)% of the mucin of the present invention.

[0114] The aqueous solution or suspension may further contain a buffer, a viscosity improver, or a wetting agent. Preferably, the buffer includes, but is not limited to, phosphate and carbonate-bicarbonate buffer solutions, and the viscosity improver or wetting agent includes, but is not limited to, glycerol and silicone oil.

[0115] In other embodiments, the antibacterial composition of the present invention is prepared as an ointment, a gel, or a paste. Furthermore, the ointment, gel, or paste can be prepared as a wound dressing, an antibacterial and anti-biofilm coating, an artificial scaffold, or a pH-responsive substrate.

[0116] Preferably, the ointment, gel, or paste contains more than 20 (weight)% of the mucin.

[0117] The ointment, gel, or paste may further contain an antibiotic and / or a poorly soluble drug (hydrophobic drug), such as curcumin.

[0118] In other embodiments, the antibacterial composition of the present invention is prepared as a spray or a composite nanofiber membrane. Furthermore, the spray or composite nanofiber membrane is prepared as a textile, a mask, and a wound dressing.

[0119] Preferably, the spray or composite nanofiber membrane contains 1-5 (weight)% of the mucin.

[0120] Another aspect of the present invention also provides a drug delivery system comprising the mucin and the hydrophobic therapeutic agent of the present invention. The hydrophobic therapeutic agent includes curcumin and total saponins.

[0121] In some embodiments, the drug delivery system further comprises methacrylic anhydride.

[0122] The anti-inflammatory and antioxidant properties of curcumin have been studied. Recent studies have shown that curcumin can restore the imbalance between the production of reactive oxygen species (ROS) and antioxidant activity, shorten the inflammatory period of wounds, and accelerate the wound healing process. However, curcumin has poor water solubility (0.6 μg / mL) and extremely low bioavailability, which limits its application.

[0123] The mucin prepared by the method of the present invention retains the structural integrity and natural activity of mucin as much as possible. Its backbone has a block polymer structure, which can improve the solubility and dispersibility of curcumin in water through hydrophobic interaction. In addition, there are various chemical groups in the mucin prepared by the present invention, such as disulfide groups, amino groups, carboxyl groups, hydroxyl groups, etc. Therefore, mucin can be easily functionalized. For example, mucin can be polymerized to form a network after being connected with methacrylic anhydride through an amide bond, prompting curcumin to be wrapped in mucin molecules, thereby prolonging the local action time of curcumin. Since the mucin prepared by the present invention well retains the pH responsiveness, the molecules can transform between the aggregated state and the relaxed state according to the change of the environmental pH value. In the wound, when the wound deteriorates, the pH value of the skin microenvironment increases, which causes the mucin to exist in a relaxed state. As a result, curcumin particles are released, thus realizing the function of drug delivery. In addition, the release of curcumin will cause a color change. By visually observing the color change of the local wound, the condition of the wound can be understood and corresponding measures can be taken in a timely manner.

[0124] Another aspect of the present invention also provides the application of the mucin described in the present invention in antibacterial, anti-biofilm, drug delivery and pH monitoring, and the application is not for the purpose of diagnosis and treatment.

[0125] The content of the present invention is further explained or illustrated by the following examples, but these examples should not be construed as limiting the protection scope of the present invention.

[0126] Examples

[0127] Unless otherwise specified, the experimental methods used in the following examples are carried out using the conventional experimental procedures, operations, materials and conditions in the art.

[0128] The sources of the reagents used in the examples are as follows:

[0129] Commercially available mucin (porcine gastric mucin type III, combined sialic acid 0.5 - 1.5%, partially purified powder), dithiothreitol (DTT), periodic acid Schiff (PAS) staining kit (CatNo.395B), isopropyl β-D-1-thiogalactopyranoside (IPTG) were purchased from Sigma-Aldrich. 0.1M 2-(N-morpholino)ethanesulfonic acid (MES) buffer (pH 4) was purchased from Thermo Scientific TMCompany. 0.1 M hydrochloric acid - potassium chloride buffer (pH 2), 0.2 M carbonate - bicarbonate (CB) buffer (pH 8), and 10 mM phosphate buffer (1×PBS) were prepared by conventional methods.

[0130] Example 1: Preparation of mucin

[0131] Porcine stomachs were collected from the abattoir. The stomachs were cut open and rinsed with tap water at a low flow rate. From this point on, all operations were carried out on ice. Then, the raw mucus was gently scraped using a spoon - shaped tool and placed in a beaker. Homogenization: The collected mucus was diluted 5 - fold with 20 mM sodium phosphate buffer (containing 70 mM NaCl, 0.0075% 2 - methyl - 4 - isothiazolin - 3 - one, and 0.0025% 5 - chloro - 2 - methyl - 4 - isothiazolin - 3 - one), pH 7), and stirred overnight at room temperature. Then, filtration was carried out successively using 200 μm and 125 μm sieves. Using a dialysis tube with a cut - off molecular weight of 20 kDa, the filtered liquid was dialyzed at 4°C in 175 mM sodium carbonate - 25 mM sodium bicarbonate (pH 8) for 24 hours, and then dialyzed in ultrapure water at 4°C for 72 hours. Then, the mucin solution was lyophilized at - 50°C to - 80°C for 72 hours and stored in a drying oven at 10°C for future use. The above extraction process was as Figure 1 shown.

[0132] Example 2: Preparation of mucin

[0133] Mucin was prepared by the method of Example 1, with the differences being: The collected mucus was diluted 5 - fold with 10 mM sodium phosphate buffered saline (containing 100 mM NaCl, 0.0075% 2 - methyl - 4 - isothiazolin - 3 - one, and 0.0025% 5 - chloro - 2 - methyl - 4 - isothiazolin - 3 - one, pH 4), and stirred overnight at 4°C. Then, filtration was carried out successively using 1 mm and 500 μm sieves, and the filtration was carried out in 10 mM sodium phosphate buffer (pH 9, containing 180 mM sodium chloride). Using a dialysis tube with a cut - off molecular weight of 100 kDa, the filtered liquid was placed at 4°C in 87.5 mM sodium carbonate - 12.5 mM sodium bicarbonate (pH 8) for 24 hours, and then placed in ultrapure water at 4°C for 72 hours. Then, the mucin solution was lyophilized at - 50°C to - 80°C for 72 hours and stored in a drying oven at 4°C for future use.

[0134] Example 3: Preparation of mucin

[0135] Mucin was prepared by the method of Example 1, with the difference that: the collected mucus was diluted 5-fold with 10 mM sodium phosphate buffered saline (containing 10 mM NaCl, 0.0075% 2-methyl-4-isothiazolin-3-one and 0.0025% 5-chloro-2-methyl-4-isothiazolin-3-one, pH 6), and stirred overnight at 4 °C. Then, filtration was carried out successively using 1 mm and 500 μm sieves, and the filtration was carried out in 5 mM sodium phosphate buffer (pH 10, containing 170 mM sodium chloride). Using a dialysis tube with a cut-off molecular weight of 100 kDa, the filtered liquid was placed at 4 °C in 43.75 mM sodium carbonate - 6.248 mM sodium bicarbonate (pH 10) for 24 hours, and then placed in ultrapure water at 4 °C for 72 hours. Then, the mucin solution was lyophilized at -50 °C to -80 °C for 72 hours and stored in a drying oven at 4 °C for future use.

[0136] The mucins prepared in the above Examples 1-3 of the present invention were characterized, and the pH responsiveness, the effects of inhibiting bacterial growth and disrupting bacterial biofilms, and biocompatibility were verified, and a drug delivery system was developed. The experimental results of the mucin prepared in Example 1 are shown below as an example. The results of the mucins prepared in other examples are comparable to those of the mucin in Example 1 and will not be elaborated.

[0137] Experimental Example 1: Characterization of the mucin prepared by the present invention

[0138] Identification of the preparation :

[0139] It was identified that the mucins prepared in Examples 1-3 are high molecular weight O-glycosylated linear proteins and are the main components of the mucin extract. The experimental results of the mucin prepared in Example 1 are shown below as an example.

[0140] 1. FTIR analysis

[0141] The components of the mucin extract of the present invention were analyzed using Fourier transform infrared spectroscopy (FTIR). The Fourier transform infrared spectrum (FTIR) of the sample was measured in the range of 400 - 4000 cm -1 using the attenuated total reflection (ATR) technique with a Vertex 70 FTIR spectrometer (Bruker). The infrared spectrum was confirmed by measuring three different batches of porcine stomach extract samples prepared according to the method of Example 1.

[0142] As Figure 7As shown, the strong absorption bands at 3249, 2960 - 2851, 1625, 1531, and 1033 cm-1 in the figure can be attributed to the O-H stretching, C-H stretching, C=O stretching of amide I, N-H bending and C-N stretching of amide II, and C-O stretching in the protein, respectively.

[0143] Comparing the spectral analysis of the mucin extract of the present invention with a commercially available mucin (purchased from Sigma-Aldrich) ( Figure 11 ) shows that the main component of the mucin extract prepared in the present invention is mucin.

[0144] In addition, the analysis of the FTIR results ( Figure 11 ) shows that the absorption bands of C-H stretching, C=O stretching (amide I), N-H bending, and C-N stretching (amide II), as well as those of carbohydrate derivatives, have changed, revealing the structural differences between the commercially available mucin (purchased from Sigma-Aldrich) and the extraction sample of the present invention. To better compare the differences between different samples, the y-axis was adjusted to show the absorption values. The blue region is the "fingerprint" region of mucin, which is highly correlated with the key structural changes of proteins and glycosylation (see the literature "Pritchard, M.F., Oakley, J.L., Brilliant, C.D., Rye, P.D., Forton, J., Doull, I.J.M., Ketchell, I., Hill, K.E., Thomas, D.W., & Lewis, P.D. (2019). Mucin structural interactions with an alginate oligomer mucolytic in cystic fibrosis sputum. Vibrational Spectroscopy, 103, 102932 - 102932." and "Lewis, A.T., Jones, K., Lewis, K.E., Jones, S., & Lewis, P.D. (2013). Detection of Lewis antigen structural change by FTIR spectroscopy. Carbohydrate Polymers, 92(2), 1294–1301.") ( Figure 11 A). Figure 11 B is an enlarged FTIR view in the range of 2800 - 3100 cm -1 Within the range. At 2915 cm -1 And 2851 cm -1The band at [frequency] is a typical feature of the asymmetric and symmetric stretching vibrations of the C-H groups in CH and CH2 groups in mucin. Compared with commercially available mucin (black line), an additional band was observed at [frequency] in the extract of the present invention (brown line), and this band was also found in the FTIR results of the mucus secretion of annelid worms in another study (see the literature "Lalonde, S.V., Dafoe, L.T., Pemberton, S.G., Gingras, M.K., & Konhauser, K.O. (2010). Investigating the geochemical impact of burrowing animals: Proton and cadmium adsorption onto the mucus lining of Terebellid polychaete worms. Chemical Geology, 271(1), 44–51."). However, this band was not observed in commercially available mucin in this study or other studies (PETRASH, D.A., LALONDE, S.V., GINGRAS, M.K., & KONHAUSER, K.O. (2011). A SURROGATE APPROACH TO STUDYING THE CHEMICAL REACTIVITY OF BURROW MUCOUS LININGS IN MARINE SEDIMENTS. Palaios, 26(9), 594–600.). -1 Figure [number] is an enlarged FTIR image in the range of 1200 - 1700 cm-1 for C. Bands at 1625 cm-1 (C=O stretching of amide I), 1531 cm-1 (N-H bending and C-N stretching of amide II), 1448 cm-1 and 1397 cm-1 (C-H bending), and 1230 cm-1 (amide III) were observed in the mucin extract. Compared with the extract of the present invention (brown line), commercially available mucin (black line) showed a shift in the bands for the chemical groups of C=O, C-N, and N-H, indicating a difference in the main chain conformation of mucin. Figure 11 Figure 11 ​Figure D is an enlarged view of the FTIR "fingerprint" region in the range of 900 - 1200 cm-1, which is related to the glycosylation of mucin. 968 cm-1, 1033 cm-1, and 1074 cm-1 are characteristic bands of carbohydrate derivatives such as sialic acid, N-acetylglucosamine, and N-acetylgalactosamine (see the literature "Lewis, A. T., Jones, K., Lewis, K. E., Jones, S., & Lewis, P. D. (2013). Detection of Lewis antigen structural change by FTIR spectroscopy. Carbohydrate Polymers, 92(2), 1294–1301."). The peak between 1000 - 1080 cm-1 of the mucin extract of the present invention is wider than that of the commercially available mucin, indicating that the carbohydrate derivatives in the extract are exposed to a more complex chemical environment.

[0145] 2. Atomic Force Microscopy (AFM) and Scanning Electron Microscopy (SEM) Detection

[0146] In the air scanning mode, the morphology of mucin under different concentrations and pH conditions was observed using an atomic force microscope AFM (model Dimension ICON, Bruker). The SCANASYST-AIR-HPI probe (Bruker) was used. The mucin of the present invention (prepared through Example 1) and commercially available mucin samples were prepared at concentrations of 1.5 μg / mL, 5 μg / mL, and 14 mg / mL in 0.1 M 2-morpholinoethanesulfonic acid (MES) buffer (pH 4) and 0.2 M carbonate-bicarbonate (CB) buffer (pH 8), respectively. In all measurements, mica was freshly prepared and placed in the vapor of (3-aminopropyl)triethoxysilane (APTES) for 30 minutes. 20 μL of the sample solution was dropped on the surface of APTES-treated mica and left standing for 1 minute. Then it was rinsed three times with 1 mL of Milli-Q water, and the mica sheet was air-dried overnight. All operations on the equipment followed the manufacturer's manual. The scanning area was 100 nm or 400 nm.

[0147] The freeze-dried mucin extract prepared through Example 1 and the commercially available mucin powder were placed on a conductive tape and treated with gold coating using K575xd (purchased from Emitech Ltd) to increase the conductivity of the sample. After the SEM sample preparation was completed, the scanning electron microscope (JEOL-6390 and JEOL-6700F) was used to observe the microstructure of the mucin extract and the commercially available mucin.

[0148] Figure 3A shows the linear rigid structure of the mucin prepared by the present invention (1.5 μg / mL, pH 8) under an atomic force microscope (AFM). Its length is between 210 - 285 nm, and connections can be observed between the ends of the mucin molecules. The scanning electron microscope (SEM) image ( Figure 4 A) shows that the connections between the mucin molecules prepared by the present invention are retained and increase with the removal of water, thus forming a foamy structure of the freeze-dried product. However, as Figure 3 shown in Figures 4B, the proteins in the commercially available mucin are fragmented and the connections between them are less obvious.

[0149] Verification of molecular weight :

[0150] The molecular weight of the preparation (hereinafter also referred to as "extract") was verified by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE). An aqueous solution of the mucin extract of the present invention (prepared in Example 1) and an aqueous solution of commercially available mucin at 5 mg / mL were prepared and treated with dithiothreitol (DTT) at a final concentration of 0.9 mM. The reduced and non-reduced samples of the mucin extract of the present invention and the commercially available mucin samples were loaded into the gel wells. They were run at 120 V for 1 hour.

[0151] As Figure 5 shown. The proteins in the mucin extract of the present invention (lane 1) mainly migrated in the range greater than 50 kDa (at least greater than 40 kDa), and most remained in the gel wells. The proteins in the extract after treatment with dithiothreitol (DTT) migrated in a similar range, but the proportion of proteins remaining in the gel wells decreased significantly (lane 3). This indicates the presence of intra- and intermolecular disulfide bonds in the mucin extract. After DTT treatment, the broad band near the 175 - 270 KDa range became lighter, and several bands appeared in the 50 - 95 KDa range. SDS-PAGE analysis showed that the extract is a mixture of high molecular weight proteins linked by disulfide bonds. In contrast, no bands higher than 50 kDa were observed in the commercially available mucin samples with and without DTT treatment (lanes 2 and 4, respectively).

[0152] This result shows that the molecular weight of the mucin prepared by the present invention is greater than that of the commercially available mucin.

[0153] In addition, size exclusion gel chromatography was also performed to verify the molecular weight distribution. The mucin extract of the present invention (prepared in Example 1) and commercially available mucin were respectively dissolved in 10 mM sodium phosphate buffer (pH 7.2) to obtain a sample of 10 mg / mL. The sample solution was left overnight at 4 °C to ensure complete hydration (the commercially available mucin can be directly used after being dissolved in the buffer and mixed evenly). Then, size exclusion gel chromatography (HiLoad 16 / 600 Superose 6 pg preparative SEC column, Cytiva) was performed for separation, with a sample loading volume of 5 mL and a flow rate of 1 mL / min. According to the principle, substances with larger molecular weights are eluted from the column first, while substances with smaller molecular weights are eluted later. As can be seen from Figure 12 the left figure, most of the mucin extract of the present invention was eluted from the column before 35 minutes, and a small part of the extract was eluted at 40 - 50 minutes. Finally, the sample with small molecular weight was completely eluted at 90 - 115 minutes. As can be seen from Figure 12 the right figure, the commercially available mucin was mainly eluted from the column at 90 - 115 minutes. Generally speaking, in the mucin extract of the present invention, substances with larger molecular weights are significantly more than those of the commercially available source. Combining with the SDS-PAGE result figure, the molecular weight of the substances eluted at 90 - 115 minutes should be less than 52 KDa. The molecular weight of the substances eluted before 35 minutes should be greater than 270 KDa, and the molecular weight of the substances eluted at 40 - 50 minutes should be in the range of 50 - 270 KDa.

[0154] It can be seen therefrom that the molecular weight of the mucin extract of the present invention is greater than 50 kDa, most of which are greater than 270 kDa, and a part is in the range of 52 - 270 kDa.

[0155] Total protein content, particle size of mucin and polysaccharide content of the extract

[0156] The total protein content of the mucin prepared in the present invention (Example 1) and commercially available mucin was measured by the bicinchoninic acid (BCA) method. After weighing the mucin extract and commercially available mucin, they were respectively dissolved in 10 mM sodium phosphate buffer (pH 7.2), and the final concentration of the sample was 10 mg / mL. The sample solution was left overnight at 4 °C to ensure complete hydration. After the sample was hydrated, it was diluted to 1 mg / mL with 10 mM sodium phosphate buffer. 25 μL of each sample solution was taken, and Pierce TM BCA Protein Assay Kit (23225, Thermo Scientific TM ) was used to perform the measurement according to the manufacturer's instructions. Before detection, a standard curve ( Figure 6 D) was established with the dilution solution of bovine serum albumin (BSA) standard, and the concentration gradient was 2, 1.5, 1, 0.5, and 0 (blank) mg / mL, R 2= 0.9856. The absorbance at 562 nm was measured using a Varioskan TM LUX multimode microplate reader (purchased from Thermo Scientific TM Company).

[0157] The content of polysaccharides was compared by periodic acid Schiff staining (PAS) method. The mucin extracts (Example 1) and commercially available mucin were weighed and dissolved in 10 mM sodium phosphate buffer (pH 7.2) respectively, and the final concentration of the samples was 10 mg / mL. The sample solutions were placed at 4 °C overnight to ensure complete hydration. Detection was carried out using a periodic acid Schiff (PAS) staining kit according to the manufacturer's instructions. After the reaction, the absorbance of each sample at 550 nm was measured using a microplate reader.

[0158] For the determination of the total protein and polysaccharide contents, the measurements of each sample were repeated 3 times, and the data were expressed as mean ± standard deviation.

[0159] The mucin extracts (Example 1) and commercial mucin were weighed and dissolved in Milli-Q water respectively, and the final concentration of the samples was 5 mg / mL. The sample solutions were placed at 4 °C overnight to ensure complete hydration. After complete hydration, the samples were diluted to a final concentration of 0.4 mg / mL with 0.1 M 2-(N-morpholino)ethanesulfonic acid buffer (pH 4) or 0.2 M carbonate-bicarbonate buffer (pH 8), and thoroughly mixed and then placed at 4 °C overnight. Particle size measurements were carried out using a 90Plus Particle Size / Zeta Analyzer (purchased from Brookhaven Instruments Corporation), and the laser wavelength was 633 nm. Each sample was repeated 5 times at room temperature, and care was taken to avoid air bubbles and particle precipitation during sample loading.

[0160] The results are as Figure 6 shown. The particle size of the mucin extracted by the present invention was 551.02 nm, which was significantly larger than that of the commercially available mucin at 207.32 nm( Figure 6 A). In addition, as confirmed by BCA determination, the protein content and purity of the extract of the present invention were more than 1.5 times those of the commercially available samples( Figure 6 B). Similarly, the periodic acid Schiff (PAS) staining method showed that the polysaccharide content in the mucin of the present invention was significantly higher( Figure 6 C).

[0161] Experimental Example 2: The mucin prepared by the present invention has pH responsiveness

[0162] As Figure 2 shown in A, the monomer structure of mucin consists of D1, D2, D3 assembly domains, a CysD domain, and glycans grafted in the middle of the protein backbone. This structure further elucidates the potential of mucin to respond to changes in the wound microenvironment through the dynamic adaptation of hydrophilic and hydrophobic domains.

[0163] Circular dichroism (CD) spectroscopy, transmission electron microscopy (TEM) and atomic force microscopy (AFM)

[0164] In this experimental example, the secondary and tertiary structures of mucin under different pH conditions were characterized using circular dichroism (CD) spectroscopy. Before the characterization, the mucin samples were hydrated overnight at 4 °C in the corresponding buffer to minimize experimental variations. The CD spectra were measured using a JASCO J-810 spectrophotometer in a 0.1 mm quartz cuvette (0.35 mL) with a step resolution of 0.1 nm, a scanning speed of 100 nm / min, a bandwidth of 1 nm, and a response time of 0.5 s. The data acquisition wavelength range was 200 - 280 nm. The average value was calculated from 10 accumulations of each sample analysis. The mucin prepared in the present invention (Example 1) and commercially available mucin were separately dissolved in 0.1 M MES buffer (pH 4) and 0.2 M carbonate-bicarbonate buffer (pH 8) to prepare 0.5 mg / mL solutions. The CD spectra of the proteins under different pH conditions were further analyzed using BeStSel.

[0165] In the air-scanning mode, the morphology of mucin under different concentrations and pH conditions was observed using an atomic force microscope AFM (model Dimension ICON, Bruker). The SCANASYST-AIR-HPI probe (Bruker) was used. Samples of the mucin prepared in the present invention (Example 1) and commercially available mucin were prepared at concentrations of 1.5 μg / mL, 5 μg / mL, and 14 mg / mL in 0.1 M 2-(N-morpholino)ethanesulfonic acid (MES) buffer (pH 4) and 0.2 M carbonate-bicarbonate (CB) buffer (pH 8), respectively. In all measurements, mica was freshly prepared and placed in the vapor of (3-aminopropyl)triethoxysilane (APTES) for 30 minutes. 20 μL of the sample solution was dropped onto the surface of the APTES-treated mica and left standing for 1 minute. Then it was gently rinsed three times with 1 mL of Milli-Q water, and finally the mica sheet was air-dried overnight. All operations on the equipment followed the manufacturer's manual. The scanning area was 100 nm or 400 nm.

[0166] The sample solution for TEM detection was prepared in the same way as for AFM. The solution was dropped onto a copper grid and gently blotted with a filter paper after standing for three seconds. The sample was air-dried overnight and observed under a TEM JEM 2010 (JEOL). The average cavity area of the mucin extract at pH 8 was measured using ImageJ.

[0167] The circular dichroism (CD) spectroscopy used to study the changes in the secondary structure of proteins revealed significant changes in the secondary structure of the mucin of the present invention at different pH values ( Figure 10A, upper panel). Analysis using BeStSel showed that when the pH value changed from 4 to 8, the predicted secondary structure changed from 42.9% anti-parallel structure, 17.7% turn structure, and 39.4% random coil structure to 24.9%, 30.8%, and 44.3%, respectively. It is worth noting that the increase in pH led to a 13.1% increase in the content of the turn structure, indicating that the proline structure inside the mucin might be enhanced, and it plays the role of a proton acceptor in the protein. Generally speaking, the domain of the mucin of the present invention changed from an anti-parallel structure to a random coil structure with the increase in pH value. In contrast, there were no obvious structural changes in the CD spectra and structure predictions of commercially available mucin under different pH conditions ( Figure 10 A, lower panel).

[0168] Next, transmission electron microscopy (TEM) and atomic force microscopy (AFM) were used to observe the conformational changes of the mucin. The TEM image showed that the mucin prepared in the present invention (5 μg / mL) formed larger filamentous aggregates at pH 4 ( Figure 10 B), showing the aggregation of hydrophobic ends, which was consistent with the previously reported full-length mucin simulation model. At pH 8, the terminal aggregation was replaced by a more extended backbone, leaving cavities with an average area of about 0.015 μm 2 . Figure 10 C).

[0169] Similarly, in acidic and alkaline environments, larger aggregates and extended conformations were respectively observed in the AFM images of the mucin prepared in the present invention (5 μg / mL) ( Figure 10 D and E).

[0170] Turbidity change

[0171] The turbidity of the mucin at different pH values and concentrations was measured. An increase in turbidity indicates the aggregation of the mucin. The mucin extract (prepared in Example 1) was dissolved in 0.1 mol / L MES (2-(N-morpholino)ethanesulfonic acid) solution (pH 4) and 0.2 mol / L sodium carbonate-sodium bicarbonate aqueous solution (pH 8) at concentrations of 0, 2, 4, 7, and 14 mg / mL, and left at 4 °C overnight to fully hydrate. Then, 200 μL of the solution was transferred to a 96-well microplate, and the turbidity was measured at a wavelength of 400 nm using a Varioskan TM LUX multi-functional microplate reader (Thermo Scientific TM ). All measurements were performed with five technical replicates, and the results are shown in Figure 15 A.

[0172] To observe the turbidity of mucin extracts at pH values from 2 to 8, the pH of MES buffer (pH 4) was adjusted to pH 3 with 1 M hydrochloric acid; MES buffers at pH 5 and pH 6 were obtained by adding different amounts of 1 M sodium hydroxide. The mucin extract (prepared in Example 1) was dissolved in hydrochloric acid - potassium chloride buffer (pH 2), MES buffers at different pH values (pH 3 - 6), sodium phosphate buffer (pH 7), and sodium carbonate - sodium bicarbonate aqueous solution (pH 8) at a concentration of 10 mg / mL. Then, the turbidity of the mucin solution at different pH values was observed.

[0173] As Figure 15 shown in Figure 15 A, at pH 4, the turbidity of the mucin solution prepared in the present invention is higher than that at pH 8. At a concentration of 7 mg / mL and higher, the turbidity of the mucin solution at pH 4 is significantly higher than that at pH 8. As

[0174] Experimental Example 3: Development of a drug delivery system using the mucin prepared in the present invention

[0175] In this example, a pH - induced, mucin - based drug delivery system was developed. This system has visual monitoring and reactive oxygen species scavenging properties and is used to promote wound healing.

[0176] Prepare an aqueous solution of 10 mg / mL mucin (Example 1) and place it at 4 °C overnight for sufficient hydration. Before the reaction, adjust the pH of the mucin aqueous solution to 8 - 9 with 1 mol / L sodium hydroxide, then place the mucin aqueous solution in a 100 mL two - necked flask. While purging with nitrogen and under strong stirring, dropwise add methacrylic anhydride until its final concentration is 0.5% (w / v). After the addition is completed, place the flask in an ice bath and stir continuously for 4 hours. After the reaction is completed, use a 12 - 14 kDa dialysis membrane to remove the excess unreacted methacrylic anhydride. The dialysis lasts for 48 hours, and the water should be changed frequently to fully remove methacrylic anhydride. Then, freeze - dry the product (-60 °C to -80 °C, 72 hours) and store it at 4 °C for later use.

[0177] Prepare an aqueous solution of 10 mg / mL mucin-methacrylic anhydride and place it at 4 °C overnight for sufficient hydration. Then adjust the pH to 8 using 0.2 mol / L sodium carbonate-sodium bicarbonate aqueous solution. Under strong stirring, add a 10% Irgacure 2595 solution (Sigma-Aldrich) to a final concentration of 0.1% (v / v). After thorough mixing, also under strong stirring, slowly add dropwise a solution of 5% curcumin nanoparticles (purchased from Huzhou Pureway Biopharmaceutical Technology Co., Ltd.) dissolved in 80% glycerol solution to a final concentration of 0.5% (v / v). Then pour the solution into a 24-well plate and expose the plate to ultraviolet light (365 nm) for 5 minutes to form a gel.

[0178] When the curcumin ethanol solution is added to buffer solutions of different pH values, its color will change ( Figure 8 B, upper figure). Through the above operations, curcumin nanoparticles can be evenly dispersed in the mucin hydrogel. When 0.1 mol / L MES (2-(N-morpholino)ethanesulfonic acid) solution (pH 4) is added dropwise, the color of the hydrogel is yellow. When 0.2 mol / L sodium carbonate-sodium bicarbonate aqueous solution (pH 8) is added, the surface of the hydrogel turns orange. As the solution is continuously added, more and more solution penetrates into the hydrogel, and finally the whole hydrogel turns orange ( Figure 8 B lower figure).

[0179] Experimental Example 4: The mucin prepared by the present invention has the effect of inhibiting bacterial growth and destroying bacterial biofilms

[0180] Biofilms and bacterial infections are two main factors leading to the failure of wound healing. The mucin prepared by the present invention can inhibit bacterial growth by destroying the bacterial cell membrane and biofilm. Mucin can fold into a granular state (aggregated state) at low pH and be mixed with the nanofiber precursor solution. Under optimized parameters such as working distance and voltage, nanofiber membranes are prepared by electrospinning ( Figure 9 A). Such nanofibers can prevent bacteria in the air from attacking the wound and causing more serious bacterial infections. At the same time, the wound maintains the exchange of external gas and water vapor. Protected by the nanofibers, the mucin of the present invention inhibits bacterial growth and reduces the formation of biofilms.

[0181] The Escherichia coli used in the experiments related to this experimental example was purchased from the Beijing Bioresource Collection Center. Escherichia coli BL21(DE3) contains the plasmid pET28a inserted with the green fluorescent protein (GFP) gene.

[0182] Inhibition of bacterial growth

[0183] This experimental example utilizes the growth curve of bacteria ( Figure 9B) Evaluate the antibacterial efficacy of the mucin extract of the present invention, and compare the antibacterial effects of the mucin extract of the present invention and commercially available mucin using the minimum inhibitory concentration (MIC) determination method. Figure 9 C).

[0184] The specific operation of the bacterial growth curve experiment (as shown in Figure 9 Figure B on the left) is as follows: Escherichia coli (BL21) was cultured in LB medium (Sigma-Aldrich) and LB agar plates (Sigma-Aldrich) containing 50 μg / mL and 100 μg / mL kanamycin (Sigma-Aldrich), respectively. The experimental method referred to previous studies. Briefly, a single colony was selected from the LB agar plate and inoculated into 3 mL of LB medium. Incubate overnight at 37 °C and 200 rpm. The overnight culture was diluted 1:1000 into 250 mL of freshly prepared LB medium and cultured at 37 °C and 200 rpm for 12 hours. The absorbance at a wavelength of 600 nm (OD600) was measured every 30 minutes. To study the effect of the mucin extract on bacterial growth, the mucin extract (Example 1) was added to the bacterial medium at a final concentration of 0.1 wt.% during the lag phase (Group A), logarithmic phase (Group B), or stationary phase (Group C) (i.e., 1 hour of culture, 4.5 hours of culture, and 8 hours of culture). A negative control group without adding the mucin extract was set. 200 μL of the culture solution was taken every hour and the OD600 was measured, and the measurement continued for 12 hours. Use Varioskan TM LUX multi-functional microplate reader (Thermo Scientific TM ) to perform three technical replicates to measure OD600, and use the average value to plot the growth curve and calculate the standard error. The results are shown in Figure 9 Figure B.

[0185] The minimum inhibitory concentration (MIC) determination can be used to determine the lowest dilution of the test suspension that inhibits bacterial growth. First, a single colony was collected from the agar plate and cultured overnight at 37 °C and 200 rpm in Mueller-Hinton broth (MH, Sigma-Aldrich) medium. After overnight culture, the bacterial culture solution was diluted to 10 6CFU / mL. The mucin extract of the present invention (Example 1) and commercially available mucin were exposed to ultraviolet light for 30 minutes, and then 10 mM sodium phosphate buffer was added respectively to make the final concentration 5% (w / v). Then, 100 μL of 2X MH medium (Sigma-Aldrich) and 100 μL of the test sample were added to each well in the first column of a 96-well plate. Then, 100 μL of 1X MH medium was added to the 2nd to 11th columns. 100 μL of the solution was aspirated from the first column and added to the second column, 100 μL of the solution was aspirated from the second column and added to the third column... This operation was repeated up to the 10th column until there was 200 μL of solution in the 10th column. After that, 100 μL of the solution was removed from the 10th column and discarded. Then, 100 μL of the diluted bacterial solution (10 6 CFU / mL) was added to the 1st to 11th columns. The 11th column did not contain the test sample and was regarded as the positive control group. Finally, 100 μL of the medium was added to the 12th column as the negative control group. In addition, kanamycin at 50 mg / mL (Sigma-Aldrich) and polymyxin B at 20,000 U / mL (P4932-1 MU, Bioreagent, Sigma-Aldrich) were prepared as test samples, and the MIC determination was carried out as described above. The results are as shown in Figure 9 C and D, and the data are expressed as mean ± standard deviation (n = 8).

[0186] As Figure 9 shown by the green curve in the right figure of B, adding the mucin extract of the present invention (0.1 wt.%) during the lag phase significantly delayed the start of the logarithmic growth phase, that is, the bacteria began to grow only after 10 hours of culture. After adding the extract during the logarithmic growth phase, compared with the control group (negative control without mucin extract), the bacterial growth trend was in a stagnant state, indicating that the bacterial growth was restricted during the subsequent culture period ( Figure 9 the pink curve in B). Although the optical density in group C increased slightly after adding the mucin extract, no significant increase or decrease was observed during the subsequent culture time ( Figure 9 the blue curve in B). This finding is similar to the antibacterial growth inhibition effect found in snail mucus.

[0187] The toxicity of mucin to bacteria was evaluated by the minimum inhibitory concentration (MIC) experiment, and the bacteria were statically cultured in a 96-well microplate. The results showed that the mucin extract of the present invention could inhibit the growth of Escherichia coli, and the antibacterial effect of 0.16 wt.% mucin was equivalent to that of kanamycin at 390.7 μg / mL to 781.3 μg / mL (considering the factor of the positive control), as shown in Figure 9 the left of C and Figure 9as shown in D. At the same time, a commercially available mucin solution with the same concentration was prepared, and it was found that as the concentration increased, the number of bacteria decreased ( Figure 9 right in C). However, the antibacterial effect of the commercially available mucin at the same concentration was lower than that of the mucin extract of the present invention.

[0188] Disruption of bacterial biofilms

[0189] In addition, in this experimental example, the efficacy of the mucin extract in destroying bacterial biofilms was evaluated through the following experiment, as Figure 13 shown in A. Escherichia coli (BL21) was cultured overnight in LB medium at 37 °C and 200 rpm. Subsequently, the overnight culture solution was diluted to an optical density (OD600) of approximately 0.1. Sterilized 24-mm round glass cover slips (ordinary cover slips without any special surface treatment) were placed in 12-well plates. The culture solution containing the diluted bacteria was added to the cover slips (radius 12 mm), and static culture was carried out at 37 °C for 24 hours or 48 hours. After the culture was completed, the supernatant in the well plate was carefully aspirated, and 1 mL of sterile water was gently added to each well using an insulin needle, taking care not to damage the bacterial biofilms growing on the glass cover slips. Subsequently, the following different solutions were carefully added: (A) LB medium containing 1 mM isopropyl β-D-thiogalactoside (IPTG, Sigma-Aldrich); LB medium containing 1 mM IPTG, to which (B) a 0.5 wt.% solution of the mucin extract of the present invention (Example 1) or (C) a 0.5 wt.% polymyxin B solution (Sigma-Aldrich) was added. Then, the culture was carried out at 37 °C for 24 hours. After the culture was completed, the supernatant was carefully collected, and the biomass in the supernatant was evaluated at a wavelength of 600 nm using a Varioskan LUX multimode microplate reader (Thermo ScientificTM). Each cover slip was gently washed with 1 mL of sterile water to remove non-specific adherents, and then carefully removed with forceps and placed on a glass slide for observation. The morphology and thickness of the biofilms were examined using a confocal microscope (Nikon C2) at a laser excitation wavelength of 488 nm with 10x and 20x objective lenses. The images were analyzed using NIS-Elements confocal software (Nikon).

[0190] To promote the formation of Escherichia coli biofilms and minimize other possible bacterial contaminations that may occur during static culture, 50 μg / mL of a sub-inhibitory concentration of kanamycin (Sigma-Aldrich) was added, and this group served as the control group. The results are shown in Figure 13 the left of B. After the initial 24-hour culture ( Figure 13 left in B), the biofilms appeared thin and loose, indicating that they were in the early formation stage and immature state. Subsequently, after culturing for another 24 hours, the thickness of the biofilms increased significantly, asFigure 13 As shown on the right of B. With extended cultivation under nutrient deprivation, the biofilm matures and is able to withstand adverse conditions, such as exposure to antibiotics.

[0191] Polymyxin belongs to antimicrobial peptides and is the last resort antibiotic for multi-drug resistant bacterial infections. However, due to its neurotoxicity and nephrotoxicity, it needs to be used with caution. Polymyxin B is used to treat Gram-negative bacterial infections, such as multi-drug resistant Escherichia coli. As Figure 13 As shown on the left of C, after treatment with polymyxin B, the immature biofilm at 24 hours fragmented into several pieces, and compared with the kanamycin treatment group ( Figure 13 left of B), the thickness decreased slightly. However, it failed to eliminate the mature biofilm at 48 hours ( Figure 13 right of C), and its morphology did not change significantly. This highlights the challenge of treating bacterial infections associated with mature biofilms. When the biofilm was treated with the mucin extract of the present invention at the same concentration as polymyxin B, significant damage to the immature biofilm (24 hours) was observed within 24 hours ( Figure 13 left of D). The bacterial density decreased and dispersed from the biofilm, and the biofilm exhibited a filamentous structure. The mucin extract of the present invention also had an obvious destructive effect on the mature biofilm at 48 hours ( Figure 13 right of D), but similar to the treatment with polymyxin B, the anti-biofilm activity of the mucin extract of the present invention against the mature biofilm ( Figure 13 right of D) was not as strong as that against the immature biofilm ( Figure 13 left of D).

[0192] To study the morphological changes of the biofilm, the incubation time of the immature biofilm with the mucin extract was shortened (shortened to 12 hours). As Figure 13 shown in E, the biofilm was significantly damaged, and bacteria were released from the ruptured structure. In addition, the mucin extract significantly reduced the thickness of the mature biofilm, and its morphology was significantly different from that of the antibiotic treatment group. To better show the morphological changes, confocal images with z-axis mirroring were used. From this perspective, the biofilm treated with the mucin extract of the present invention appeared loose, and dispersed bacteria were observed above the biofilm, indicating that the structure was more unstable or more sensitive to external forces than the previous two groups. In addition, the enlarged image showed two distinct layers of biological distribution, as Figure 13 shown in F.

[0193] After treating the mature biofilm with the mucin extract or antibiotics, the biomass in the supernatant was collected and further quantified by measuring the optical density at a wavelength of 600 nm. As Figure 13As shown in Fig. G, the biomass in the supernatant after treatment with the mucin extract of the present invention was significantly higher than that in the two antibiotic treatment groups. These results support the ability of the mucin extract in this study to disrupt biofilms, thereby promoting the escape of bacteria into the supernatant rather than attachment to the coverslip surface.

[0194] Experimental Example 5: Biocompatibility of the mucin prepared by the present invention

[0195] Biocompatibility evaluation is crucial for ensuring the safety of materials in protecting vulnerable tissues such as wounds. In this experimental example, the in vitro biocompatibility of the mucin extract was evaluated by measuring the metabolic activity of NIH / 3T3 fibroblasts using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) method.

[0196] Specifically, DMEM (normal medium) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin was used as the medium. The cytocompatibility of the mucin extract was evaluated using the MTT method. The MTT method is an index for measuring cell viability by determining cell metabolism. First, the mucin extract was sterilized according to the method reported previously to eliminate potential contamination. Considering the weak penetrability of ultraviolet light to the extract, each side of the test tube containing the test sample was exposed to ultraviolet irradiation for 30 min in a biosafety cabinet, and then the test sample was dissolved in the medium and incubated for 1 day. The test samples included the mucin extract of Example 1, commercially available mucin, and bovine serum albumin (which can promote cell growth and was used as a positive control).

[0197] NIH / 3T3 fibroblasts (200 μL, 10 4 cells per well) were seeded in a 96-well plate and cultured at 37 °C and 5% CO2 for 24 h. Then the medium was replaced with normal medium (blank control group) or a mixed medium containing 2 mg / mL or 10 mg / mL of the test sample. Next, the cells were cultured for another 24 h under the same conditions. After culturing, the medium was replaced with 20 μL of MTT solution (5 mg / mL) and incubated at 37 °C and 5% CO2 for 4 h. Subsequently, the solution in the wells was carefully removed, and 200 μL of dimethyl sulfoxide was added to each well. The absorbance at a wavelength of 570 nm was measured using a Varioskan LUX multimode microplate reader. The percentage of cell viability was calculated using the formula.

[0198] % Cell viability = average ODsample / average ODblank × 100

[0199] wherein, the average ODsample is the average absorbance of the sample treatment group, and the average ODblank is the average absorbance of the group treated with only the medium.

[0200] Biocompatibility was determined with 3 technical replicates. Data are presented as mean ± standard deviation.

[0201] As Figure 14 shown, at low and high concentrations (2 mg / mL and 10 mg / mL, respectively), cells treated with the mucin extract of the present invention exhibited higher viability compared to the blank control group. Additionally, the results showed that the mucin extract of the present invention had a better promoting effect on cell growth than bovine serum albumin. However, compared to the blank control group, cells treated with 10 mg / mL commercially available mucin showed a decrease in viability ( Figure 14 ), which may be related to the residue of toxic protease inhibitors, such as benzamidine HCl and phenylmethylsulfonyl fluoride, which are commonly used in the production of commercially available proteins. These results demonstrated the excellent cytocompatibility of the mucin extract of the present invention, indicating its safety as a component of wound dressings in contact with the skin.

Claims

1. A method for preparing mucin, comprising the following steps: 1) Collecting the mucus of an animal, wherein the mucus contains mucin; 2) Mixing the mucus with a diluent to dilute and homogenize the mucus; wherein the diluent comprises a buffer solution with a salt concentration of 20 - 190 mM; 3) Subjecting the mixture obtained in step 2) to sieve filtration, and the aperture of the sieve used is 100 μm - 1 mm; 4) Dialyzing the filtered liquid in an alkaline buffer solution, and then dialyzing it with ultrapure water or deionized water to obtain mucin.

2. The method according to claim 1, wherein the buffer solution in step 2) is selected from phosphate buffer solution, 4 - morpholineethanesulfonic acid buffer solution (MES), 2 - [4 - (2 - hydroxyethyl)piperazin - 1 - yl]ethanesulfonic acid buffer solution (HEPES), tris(hydroxymethyl)aminomethane hydrochloride buffer solution (Tris - HCL), and 3 - morpholinopropanesulfonic acid buffer solution (MOPS), and the buffer solution further contains a metal salt additionally, such that the total salt concentration of the buffer solution is 20 - 190 mM; the metal salt is selected from sodium salt, potassium salt, magnesium salt, and calcium salt.

3. The method according to claim 1, wherein the buffer solution in step 2) is a phosphate buffer solution of 10 - 20 mM, and further contains a metal salt of 10 - 170 mM additionally; preferably, the phosphate buffer solution is selected from sodium phosphate buffer solution, potassium phosphate buffer solution, magnesium phosphate buffer solution, and calcium phosphate buffer solution, and the metal salt is selected from sodium salt, potassium salt, magnesium salt, and calcium salt.

4. The method according to claim 1, wherein the pH of the buffer solution in step 2) is less than or equal to 7; for example, the pH value is below 6.

5. The method according to claim 1, wherein the diluent in step 2) further contains a microbicide and / or a preservative; preferably, the microbicide and / or the preservative includes 2 - methyl - 4 - isothiazolin - 3 - one and 5 - chloro - 2 - methyl - 4 - isothiazolin - 3 - one; preferably, the concentration of the microbicide and / or the preservative in the diluent is 0.05% (v / v) - 0.1% (v / v).

6. The method according to claim 1, wherein in step 2), the mucus is diluted 5 - fold by the diluent; preferably, the mixing is carried out at 1 - 10 °C for 8 - 48 hours.

7. The method according to claim 1, wherein the sieve filtration in step 3) is carried out at pH 8 - 10, or at pH 12 - 13.

8. The method according to claim 1, wherein the sieve filtration in step 3) is carried out in a 5 - 12 mM buffer solution additionally containing 170 mM - 200 mM metal salt; preferably, the buffer solution is selected from phosphate buffer solution, 4 - morpholineethanesulfonic acid buffer solution (MES), 2 - [4 - (2 - hydroxyethyl)piperazin - 1 - yl]ethanesulfonic acid buffer solution (HEPES), tris(hydroxymethyl)aminomethane hydrochloride buffer solution (Tris - HCL), and 3 - morpholinopropanesulfonic acid buffer solution (MOPS); preferably, the metal salt is selected from sodium salt, potassium salt, magnesium salt, and calcium salt.

9. The method according to claim 8, wherein the buffer further comprises a microbicide and / or a preservative; preferably, the microbicide and / or the preservative comprises 2-methyl-4-isothiazolin-3-one and 5-chloro-2-methyl-4-isothiazolin-3-one; preferably, the concentration of the microbicide and / or the preservative in the buffer is 0.05% (v / v) - 0.1% (v / v).

10. The method according to claim 1, wherein the sieving filtration in step 3) is performed at least twice. The pore size of the first sieve used for the first filtration is 200 μm - 1 mm, and the pore size of the second sieve used for the second filtration is 100 - 500 μm, and the pore size of the first sieve is larger than that of the second sieve.

11. The method according to claim 1, wherein the salt concentration of the buffer in step 4) is 50 mM - 200 mM; preferably, the buffer in step 4) is selected from carbonate-bicarbonate buffer, ammonium hydroxide-ammonium chloride buffer, and tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetic acid buffer.

12. The method according to claim 1, wherein the pH value of the buffer in step 4) is 8 - 10 or pH 12 - 13.

13. The method according to claim 1, wherein in step 4), the dialysis is carried out using a dialysis device with a cut-off molecular weight of 14 - 100 kDa.

14. The method according to claim 1, wherein in step 4), the dialysis comprises using a dialysis device with a cut-off molecular weight of 14 - 100 kDa, placing the filtered liquid in the buffer at 1 - 10 °C for 8 - 24 hours, and then placing it in ultrapure water or deionized water at 4 °C for 24 - 72 hours.

15. The method according to claim 1, wherein the animal is a mammal, and the mucus is collected from saliva or the digestive tract.

16. The method according to claim 1, wherein the method further comprises: 5) Lyophilize the obtained mucin; preferably, the lyophilization is carried out at -50 °C to -80 °C for 48 - 96 hours.

17. The method according to any one of claims 1 - 16, wherein the obtained mucin is stored at 0 to 10 °C or -10 to -30 °C.

18. Mucin prepared by the method according to any one of claims 1 - 17.

19. The mucin according to claim 18, wherein the molecular weight of the mucin is greater than 40 kDa, preferably greater than 50 kDa, and more preferably greater than 270 kDa.

20. An antibacterial composition comprising the mucin according to claim 18 or 19.

21. The antibacterial composition according to claim 20, wherein the composition comprises 20 (weight)% - 80 (weight)% of the mucin.

22. The antibacterial composition according to claim 20, wherein the composition further comprises an antibacterial compound and / or a hydrophobic therapeutic agent; preferably, the antibacterial compound comprises an antibiotic, a sterilizing agent, and an antibacterial peptide; preferably, the hydrophobic therapeutic agent has an anti-reactive oxygen function; preferably, the hydrophobic therapeutic agent comprises curcumin and total saponins.

23. The antimicrobial composition according to claim 20, wherein the composition is prepared in the form of an aqueous solution or a suspension.

24. The antimicrobial composition according to claim 23, wherein the aqueous solution or suspension is prepared as a nasal drop, hand sanitizer, cream or artificial scaffold treatment agent.

25. The antimicrobial composition according to claim 20, wherein the composition is prepared as an ointment, gel or paste.

26. The antimicrobial composition according to claim 25, wherein the ointment, gel or paste is prepared as a wound dressing, antimicrobial and anti-biofilm coating, artificial scaffold or pH-responsive substrate.

27. The antimicrobial composition according to claim 20, wherein the composition is prepared as a spray or composite nanofiber membrane.

28. The antimicrobial composition according to claim 27, wherein the spray or composite nanofiber membrane is prepared as textiles, masks and wound dressings.

29. The antimicrobial composition according to claim 23, wherein the aqueous solution or suspension contains 0.1-2% (by weight) of the mucin.

30. The antimicrobial composition according to claim 23, wherein the aqueous solution or suspension further contains a buffer, a viscosity modifier or a wetting agent; preferably, the buffer includes phosphate and carbonate-bicarbonate buffer solutions, and the viscosity modifier or wetting agent includes glycerol and silicone oil.

31. The antimicrobial composition according to claim 25, wherein the ointment, gel or paste contains more than 20% (by weight) of the mucin.

32. The antimicrobial composition according to claim 25, wherein the ointment, gel or paste further contains an antibiotic and / or a poorly soluble drug.

33. The antimicrobial composition according to claim 27, wherein the spray or composite nanofiber membrane contains 1-5% (by weight) of the mucin.

34. A drug delivery system comprising the mucin according to claim 18 or 19 and a hydrophobic therapeutic agent.

35. The drug delivery system according to claim 34, further comprising methacrylic anhydride.

36. Use of the mucin according to claim 18 or 19 in antimicrobial, anti-biofilm, drug delivery and pH monitoring, wherein the use is not for diagnostic and therapeutic purposes.

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