Wrapping method and application of core-shell lysozyme particles

Abstract

The invention discloses a wrapping method and application of core-shell type lysozyme particles, and the method comprises the following steps: dropwise adding a negatively charged polypeptide solution with the pH value of 7.4 + / -0.2 into a lysozyme solution with the pH value of 7.4 + / -0.2 at 2-6 DEG C under the magnetic stirring of 200rpm-400rpm, and forming a lysozyme polypeptide core through electrostatic adsorption; mixing the lysozyme polypeptide nucleus, a calcium chloride solution and a disodium hydrogen phosphate solution under stirring at the temperature of less than or equal to 25 DEG C and at the speed of 200-400 rpm, adjusting the pH value to 8.0 + / -0.1, reacting for 1-4 hours, centrifuging, and discarding supernate to obtain lysozyme polypeptide nucleus-calcium phosphate microcapsules; and mixing the lysozyme polypeptide core-calcium phosphate microcapsules with a chitosan solution with the pH value of 5.5-6.0 at the temperature of less than or equal to 25 DEG C under the stirring of 150-250 rpm to form the core-shell type lysozyme particles. According to the method, low-temperature and low-speed stirring is adopted in the whole process, pH is close to neutral, no organic solvent is needed, and effective wrapping of lysozyme is achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of lysozyme, and particularly relates to a wrapping method and application of core-shell lysozyme particles. BACKGROUND

[0002] Lysozyme is a kind of alkaline enzyme capable of hydrolyzing bacterial mucopolysaccharide, and can be used as a natural anti-infection substance with bactericidal effect. Lysozyme has effects such as antibacterial, antiviral, hemostatic, anti-inflammatory and analgesic, and accelerating tissue recovery. Lysozyme has problems such as pH sensitivity, high temperature denaturation, solvent and metal ion influence, so lysozyme needs to be wrapped when used.

[0003] At present, common wrapping methods include liposome wrapping, nanoparticle embedding, polysaccharide / protein microcapsule and the like. Among them, the liposome wrapping method has good biocompatibility, but the encapsulation rate is low (about 20% to 40%), and the storage stability of lysozyme is insufficient; the nanoparticle embedding method can be long-acting and sustained-release, but organic solvents are used, which can easily lead to inactivation of lysozyme; the polysaccharide / protein microcapsule uses high-temperature spraying, which can easily affect the activity of lysozyme. It can be seen that the current wrapping method is difficult to be applied to lysozyme wrapping. SUMMARY

[0004] The present application provides a wrapping method and application of core-shell lysozyme particles, to solve the technical problem that the current wrapping method is difficult to be applied to lysozyme wrapping.

[0005] In a first aspect, the present application provides a wrapping method of core-shell lysozyme particles, comprising the following steps:

[0006] Step one, under the magnetic stirring at 2℃ to 6℃ and 200rpm to 400rpm, a negative polypeptide solution with a pH value of 7.4±0.2 is added dropwise into a lysozyme solution with a pH value of 7.4±0.2, to form a lysozyme polypeptide core by electrostatic adsorption;

[0007] Step two, under the stirring at ≤25℃ and 200rpm to 400rpm, the lysozyme polypeptide core, a calcium chloride solution and a sodium phosphate dibasic solution are mixed, the pH value is adjusted to 8.0±0.1 and reacted for 1h to 4h, then centrifuged, and the supernatant is discarded, to obtain a lysozyme polypeptide core-calcium phosphate microcapsule;

[0008] Step three, under the stirring at ≤25℃ and 150rpm to 250rpm, the lysozyme polypeptide core-calcium phosphate microcapsule is mixed with a chitosan solution with a pH value of 5.5 to 6.0, to form a core-shell lysozyme particle.

[0009] In some embodiments, the step one comprises:

[0010] lysozyme is added to a phosphate buffer solution with a pH value of 7.4±0.2 to obtain a lysozyme solution;

[0011] a negatively charged polypeptide is added to a phosphate buffer solution with a pH value of 7.4±0.2 to obtain a negatively charged polypeptide solution;

[0012] The negatively charged polypeptide solution is added dropwise to the lysozyme solution under magnetic stirring at 4℃ and 300 rpm, and the stirring is continued until the Zeta potential is -5mV~+5mV, to obtain a lysozyme polypeptide core.

[0013] In some embodiments thereof, the negatively charged polypeptide comprises one or both of polyglutamic acid and polyaspartic acid, and the molecular weight of the negatively charged polypeptide is 5kDa~15kDa.

[0014] In some embodiments thereof, the concentration ratio of the lysozyme solution to the negatively charged polypeptide solution is 1:(0.5~1.4), and the lysozyme solution and the negatively charged polypeptide solution are in equal volume.

[0015] In some embodiments thereof, the step two comprises:

[0016] calcium chloride is dissolved in a HEPES buffer solution with a pH value of 8.0±0.1 and pre-cooled at 4℃ to obtain a calcium chloride solution;

[0017] disodium hydrogen phosphate is dissolved in a HEPES buffer solution with a pH value of 8.0±0.1 and citric acid is added to obtain a disodium hydrogen phosphate solution;

[0018] The lysozyme polypeptide core and the calcium chloride solution are mixed in equal volume under magnetic stirring at ≤25℃ and 300 rpm for 10 min, and then the disodium hydrogen phosphate solution is added dropwise, and after 2h of reaction, EDTA is added and ice-bath cooled, and centrifuged at 4℃ and 8000 rpm for 15 min, and the supernatant is discarded, and the lysozyme polypeptide core-calcium phosphate microcapsule is obtained after washing with a phosphate buffer solution.

[0019] In some embodiments thereof, the amount of the disodium hydrogen phosphate solution added dropwise satisfies that the molar ratio of calcium ions in the calcium chloride solution to hydrogen phosphate ions in the disodium hydrogen phosphate solution is 1:(1.5~1.67).

[0020] In some embodiments thereof, the step three comprises:

[0021] chitosan powder is dissolved in an acetic acid solution and stirred in a 50℃ water bath for 1h~3h until completely dissolved, and insoluble substances are removed by membrane filtration, and the pH value is adjusted to 5.5~6.0 to obtain a chitosan solution;

[0022] The lysozyme polypeptide core-calcium phosphate microcapsule is suspended in a phosphate buffer solution, and is treated with ultrasonic waves at 50 W for 10 s to obtain a microcapsule suspension;

[0023] The chitosan solution is added dropwise to the microcapsule suspension under stirring at ≤25℃ and 200 rpm, and after stirring for 30 min, a 1% w / v TPP solution is added, and stirring is continued until the surface potential is positive and the chitosan layer thickness is 10 nm-20 nm, and centrifugation is performed at 4℃ and 8000 rpm for 15 min, the supernatant is discarded, and washing is performed with a phosphate buffer solution to obtain the core-shell lysozyme particles.

[0024] In some embodiments, the molecular weight of the chitosan powder is 50 kDa-150 kDa, the concentration ratio of the chitosan solution to the microcapsule suspension is (0.1-0.4):1, and the chitosan solution and the microcapsule suspension are in equal volume.

[0025] In a second aspect, the present application further provides a core-shell lysozyme particle prepared based on the above encapsulation method.

[0026] In a third aspect, the present application further provides use of the core-shell lysozyme particle prepared based on the above encapsulation method in preparation of an oral care product, and the oral care product includes toothpaste, mouthwash, oral spray, denture cleaner or gingival care gel.

[0027] Compared with the prior art, the present application has the following beneficial effects:

[0028] The lysozyme and the negatively charged polypeptide are electrostatically adsorbed to form a uniform complex core, thereby improving the structural stability and uniformity of the lysozyme; the lysozyme polypeptide core is reacted with calcium chloride and disodium hydrogen phosphate to generate a calcium phosphate microcapsule, thereby wrapping the lysozyme polypeptide core with an inorganic mineralization layer to enhance the mechanical stability; and then the calcium phosphate microcapsule is wrapped with chitosan to functionalize the surface of the calcium phosphate microcapsule, thereby forming a single-core double-shell structure with biocompatibility. The present application realizes effective encapsulation of lysozyme by using low-temperature, low-speed stirring and pH close to neutral throughout the process without using organic solvents. DETAILED DESCRIPTION

[0029] In order to make the above objectives, features and advantages of the present application more apparent and easy to understand, the specific embodiments of the present application are described in detail below. In the following description, a large number of specific details are set forth in order to fully understand the present application. However, the present application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without departing from the scope of the present application, so the present application is not limited to the specific embodiments disclosed below.

[0030] As used herein the term "prepared from" is synonymous with "comprising." As used herein the terms "comprise", "comprising", "include", "including", "have", "having" or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises a list of elements is not necessarily limited only to those elements but can include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0031] When expressing a range, a preferred range, or a range of upper and lower preferred values in a series, it is to be understood that the described range is intended to include all ranges formed from any pair of any upper limit or preferred value and any lower limit or preferred value, whether or not the range is expressly disclosed. For example, where a range "1 to 5" is disclosed, the described range is to be interpreted to include the range "1 to 4," "1 to 3," "1 to 2," "1 to 2 and 4 to 5," "1 to 3 and 5," etc. When numerical ranges are described herein, unless the context indicates otherwise, the range is intended to include the end values and all the integers and fractions within that range.

[0032] Further, the indefinite articles "a" and "an" preceding an element or component of the application are intended to be non-limiting regarding the number of elements or components. The recitation "a" or "an" is to be interpreted to mean "one or at least one" and the singular form is to be interpreted as including the plural form as well, unless otherwise indicated.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0034] Example 1

[0035] A method for encapsulating lysozyme particles of core-shell type, comprising the following steps:

[0036] Step one, adding lysozyme with purity ≥95% into a phosphate buffer solution with pH value of 7.4±0.2 to obtain a lysozyme solution with final concentration of 5 mg / mL; adding polyglutamic acid with molecular weight of 10 kDa into a phosphate buffer solution with pH value of 7.4±0.2 to obtain a negatively charged polypeptide solution with final concentration of 7 mg / mL; under magnetic stirring at 4℃ and 300 rpm, adding the negatively charged polypeptide solution with equal volume to the lysozyme solution into the lysozyme solution drop by drop at a rate of 0.5 mL / min, and continuously stirring until the Zeta potential is -5 mV~5 mV to obtain a lysozyme polypeptide core;

[0037] Step two, dissolve calcium chloride in HEPES buffer with pH value of 8.0±0.1 and pre-cool at 4℃ to obtain calcium chloride solution with final concentration of 10mM; dissolve disodium hydrogen phosphate in HEPES buffer with pH value of 8.0±0.1 and add citric acid to obtain disodium hydrogen phosphate solution with final concentration of 6mM; under magnetic stirring at ≤25℃, 300rpm, mix lysozyme polypeptide core with calcium chloride solution in equal volume for 10min, then add disodium hydrogen phosphate solution drop by drop at 0.5mL / min, continue to react for 2h, then add EDTA and cool in ice bath, centrifuge at 4℃, 8000rpm for 15min, discard supernatant, wash with phosphate buffer solution to obtain lysozyme polypeptide core-calcium phosphate microcapsule;

[0038] Step three, dissolve chitosan powder with molecular weight of 100kDa in acetic acid solution and stir in 50℃ water bath for 1h-3h until completely dissolved, remove insoluble substances by 0.45μm membrane filtration, adjust pH value to 5.5-6.0 to obtain chitosan solution with final concentration of 1mg / mL; suspend lysozyme polypeptide core-calcium phosphate microcapsule in phosphate buffer solution, treat with 50W ultrasonic for 10s to obtain microcapsule suspension with final concentration of 5mg / mL; under stirring at ≤25℃, 200rpm, add chitosan solution with equal volume of microcapsule suspension to microcapsule suspension drop by drop at 0.2mL / min, continue to stir for 30min, then add 1%w / v TPP solution, continue to stir until surface potential changes from negative to positive and chitosan layer thickness is 10nm-20nm, centrifuge at 4℃, 8000rpm for 15min, discard supernatant, wash with phosphate buffer solution to obtain core-shell lysozyme particle.

[0039] Example 2

[0040] A wrapping method of core-shell lysozyme particle, comprising the following steps:

[0041] Step one, add lysozyme with purity≥95%to phosphate buffer solution with pH value of 7.4±0.2 to obtain lysozyme solution with final concentration of 5mg / mL; add polyglutamic acid with molecular weight of 5kDa to phosphate buffer solution with pH value of 7.4±0.2 to obtain negatively charged polypeptide solution with final concentration of 2.5mg / mL; under magnetic stirring at 4℃, 300rpm, add negatively charged polypeptide solution with equal volume of lysozyme solution to lysozyme solution drop by drop at 0.5mL / min, continue to stir until Zeta potential is-5mV-+5mV to obtain lysozyme polypeptide core;

[0042] Step two, dissolve calcium chloride in HEPES buffer with pH value of 8.0±0.1 and pre-cool at 4℃ to obtain calcium chloride solution with final concentration of 10mM; dissolve sodium phosphate dibasic in HEPES buffer with pH value of 8.0±0.1 and add citric acid to obtain sodium phosphate dibasic solution with final concentration of 6.6mM; under magnetic stirring at ≤25℃ and 300rpm, mix the lysozyme polypeptide core with the calcium chloride solution in equal volume for 10min, then add the sodium phosphate dibasic solution drop by drop at 0.5mL / min, continue to react for 2h, then add EDTA and cool in ice bath, centrifuge at 4℃ and 8000rpm for 15min, discard the supernatant, wash with phosphate buffer solution to obtain lysozyme polypeptide core-calcium phosphate microcapsules;

[0043] Step three, dissolve chitosan powder with molecular weight of 150kDa in acetic acid solution and stir in 50℃ water bath for 1h-3h until completely dissolved, remove insoluble substances by 0.45μm membrane filtration, adjust pH value to 5.5-6.0 to obtain chitosan solution with final concentration of 0.05mg / mL; suspend the lysozyme polypeptide core-calcium phosphate microcapsules in phosphate buffer solution, treat with 50W ultrasonic for 10s to obtain microcapsule suspension with final concentration of 5mg / mL; under stirring at ≤25℃ and 200rpm, add chitosan solution with equal volume of the microcapsule suspension to the microcapsule suspension drop by drop at 0.2mL / min, continue to stir for 30min, then add 1%w / v TPP solution, continue to stir until the surface potential changes from negative to positive and the thickness of chitosan layer is 10nm-20nm, centrifuge at 4℃ and 8000rpm for 15min, discard the supernatant, wash with phosphate buffer solution to obtain core-shell lysozyme particles.

[0044] Example 3

[0045] A wrapping method of core-shell lysozyme particles, comprising the following steps:

[0046] Step one, add lysozyme with purity of ≥95% to phosphate buffer solution with pH value of 7.4±0.2 to obtain lysozyme solution with final concentration of 5mg / mL; add polyglutamic acid with molecular weight of 150kDa to phosphate buffer solution with pH value of 7.4±0.2 to obtain negatively charged polypeptide solution with final concentration of 5mg / mL; under magnetic stirring at 4℃ and 300rpm, add the negatively charged polypeptide solution with equal volume of the lysozyme solution to the lysozyme solution drop by drop at 0.5mL / min, continue to stir until the Zeta potential is-5mV-+5mV to obtain lysozyme polypeptide core;

[0047] Step two, dissolve calcium chloride in HEPES buffer with pH value of 8.0±0.1 and pre-cool at 4℃ to obtain calcium chloride solution with final concentration of 10mM; dissolve disodium hydrogen phosphate in HEPES buffer with pH value of 8.0±0.1 and add citric acid to obtain disodium hydrogen phosphate solution with final concentration of 6.3mM; under magnetic stirring at ≤25℃, 300rpm, mix the lysozyme polypeptide core with the calcium chloride solution in equal volume for 10min, then add the disodium hydrogen phosphate solution drop by drop at 0.5mL / min, continue the reaction for 2h, then add EDTA and cool in ice bath, centrifuge at 4℃, 8000rpm for 15min, discard the supernatant, wash with phosphate buffer solution to obtain lysozyme polypeptide core-calcium phosphate microcapsule;

[0048] Step three, dissolve chitosan powder with molecular weight of 50kDa in acetic acid solution and stir in 50℃ water bath for 1h-3h until completely dissolved, remove insoluble substances by 0.45μm membrane filtration, adjust pH value to 5.5-6.0 to obtain chitosan solution with final concentration of 2mg / mL; suspend the lysozyme polypeptide core-calcium phosphate microcapsule in phosphate buffer solution, treat with 50W ultrasound for 10s to obtain microcapsule suspension with final concentration of 5mg / mL; under stirring at ≤25℃, 200rpm, add chitosan solution with equal volume of the microcapsule suspension to the microcapsule suspension at 0.2mL / min drop by drop, continue stirring for 30min, then add 1%w / v TPP solution, continue stirring until the surface potential changes from negative to positive and the thickness of chitosan layer is 10nm-20nm, centrifuge at 4℃, 8000rpm for 15min, discard the supernatant, wash with phosphate buffer solution to obtain core-shell lysozyme particles.

[0049] Comparative Example 1

[0050] Comparative Example 1 removes Step one compared with Example 1, and uses the lysozyme solution of Step one instead of the lysozyme polypeptide core used in Step two.

[0051] Comparative Example 2

[0052] Comparative Example 2 removes Step two compared with Example 1, and uses the lysozyme polypeptide core of Step one instead of the lysozyme polypeptide core-calcium phosphate microcapsule used in Step three.

[0053] Comparative Example 3

[0054] Comparative Example 3 removes Step three compared with Example 1, and uses the lysozyme polypeptide core-calcium phosphate microcapsule of Step two for subsequent tests.

[0055] Comparative Example 4

[0056] Comparative Example 4 uses deionized water instead of the phosphate buffer solution of Step one compared with Example 1.

[0057] Comparative Example 5

[0058] Comparative Example 5 used deionized water instead of HEPES buffer solution in step two compared with Example 1.

[0059] Comparative Example 6

[0060] Comparative Example 6 used mixed mode instead of dropwise addition mode in steps one to three compared with Example 1.

[0061] Performance test:

[0062] The lysozyme particles obtained in Examples 1 to 3 and Comparative Examples 1 to 6 were tested for encapsulation efficiency, average particle size, zeta potential and enzyme activity retention rate, and the test results are shown in Table 1.

[0063] Table 1 Test results of Examples 1 to 3 and Comparative Examples 1 to 6

[0064] Sample Encapsulation efficiency / % Average particle size / nm Zeta potential / mV Enzyme activity retention / % Example 1 87.7 252.1 +33.64 83.6 Example 2 90.3 231.6 +42.91 92.4 Example 3 94.9 287.3 +47.68 88.9 Comparative Example 1 48.1 153.4 +5.93 45.6 Comparative Example 2 64.5 167.9 +17.36 49.2 Comparative Example 3 72.4 189.8 -16.72 67.5 Comparative Example 4 44.7 518.1 +1.28 32.7 Comparative Example 5 56.2 437.3 +13.19 51.4 Comparative Example 6 49.4 682.6 +9.65 33.2

[0065] As can be seen from Table 1, the core-shell type lysozyme particles obtained in Examples 1 to 3 have an encapsulation efficiency of 87% to 95%, an average particle size of 200 to 300 nm, a zeta potential of more than 33 mV, and an enzyme activity retention rate of more than 83.6, indicating that the core-shell type lysozyme particles obtained in Examples 1 to 3 are storage stable.

[0066] After step one was removed in Comparative Example 1, the encapsulation efficiency decreased, indicating that polyglutamic acid can improve the wrapping efficiency, avoid the loss of free lysozyme, and the polyglutamic acid core can reduce direct contact with calcium ions to protect enzyme activity. After step two was removed in Comparative Example 2, the encapsulation efficiency decreased, indicating that the calcium phosphate microcapsule can reduce lysozyme leakage, improve encapsulation stability, and the calcium phosphate layer can buffer pH changes, reduce the risk of enzyme denaturation, and the negative charge of calcium phosphate enhances the adsorption of chitosan, making the particle more positively charged. After step three was removed in Comparative Example 3, the encapsulation efficiency decreased, indicating that the chitosan layer can further fix lysozyme, reduce leakage, and the chitosan reverses the negative charge of calcium phosphate, giving the particle a positive charge, and the chitosan layer provides additional protection, reducing the negative impact of the environment on the enzyme. After deionized water was used instead of phosphate buffer in step one in Comparative Example 4, the enzyme activity retention rate decreased significantly, indicating that the buffer reduces pH fluctuations and protein deformation, ensuring efficient complexation of lysozyme-polyglutamic acid. After deionized water was used instead of HEPES buffer in step two in Comparative Example 5, the encapsulation efficiency decreased, indicating that HEPES stabilizes the pH environment, ensuring uniform deposition and efficient wrapping of calcium phosphate. After using mixed mode instead of dropwise addition mode in steps one to three in Comparative Example 6, the average particle size increased significantly, indicating that direct mixing mode can easily lead to heterogeneous aggregation and loose results, and incomplete wrapping and local environmental mutations can cause enzyme denaturation.

[0067] Application Example

[0068] The lysozyme particles prepared in Examples 1 to 3 and Comparative Examples 1 to 6 were added to a toothpaste formulation at a dosage of 0.05% to prepare a toothpaste liquid, and a toothpaste liquid to which uncoated lysozyme was added was used as a control group to test the antibacterial effect of the toothpaste: Streptococcus mutans CGMCC 12499 was inoculated in a culture dish with human saliva as a substrate, and was incubated in an anaerobic bag containing 80% N2, 10% H2, and 10% CO2 at 37°C for 48 h; after the toothpaste liquid was placed in an electric heating incubator at 45°C ± 1°C for 6 months, 100 μL of the toothpaste liquid was taken and inoculated in the above culture dish, and after incubation at 37°C for 48 h, the diameter of the inhibition zone was measured, and the test results are shown in Table 2.

[0069] Table 2 Inhibition zone results of the toothpaste liquid prepared in Examples 1 to 3 and Comparative Examples 1 to 6

[0070] Sample Streptococcus mutans inhibition zone diameter / mm Control 6.5 Example 1 25.1 Example 2 26.9 Example 3 28.7 Comparative Example 1 11.4 Comparative Example 2 8.7 Comparative Example 3 10.8 Comparative Example 4 9.5 Comparative Example 5 12.6 Comparative Example 6 8.9

[0071] As can be seen from Table 2, the inhibition zone diameter of Streptococcus mutans of Examples 1 to 3 was more than 25 mm after being placed at 45°C ± 1°C for 6 months, and it can be seen that the core-shell lysozyme particles prepared in Examples 1 to 3 have better thermal stability. The uncoated lysozyme in the control group was almost ineffective, and the lysozyme particles prepared in Comparative Examples 1 to 6 continued to be affected by high temperature, which further reduced the activity, and the structural differences of the lysozyme particles of Comparative Examples 1 to 6 resulted in different antibacterial effects, and the lysozyme particles of Example 2 were most affected after losing the calcium phosphate layer.

[0072] The technical features of the above-described embodiments can be combined in any manner, and to make the description concise, not all possible combinations of the technical features in the above-described embodiments are described, however, as long as the combinations of the technical features do not contradict each other, they should be considered within the scope of the present disclosure.

[0073] The above-described specific embodiments further illustrate the purpose, technical solutions, and beneficial effects of the present application, and it should be understood that the above-described specific embodiments are merely specific embodiments of the present application and are not intended to limit the protection scope of the present application. It is particularly pointed out that any modification, equivalent replacement, improvement, etc. made by those skilled in the art within the spirit and principles of the present application should be included within the protection scope of the present application.

Claims

1. A method of encapsulating core-shell lysozyme particles, characterized by, The method comprises the following steps: Step one, under the magnetic stirring at 2-6℃ and 200-400rpm, the negatively charged polypeptide solution with pH value of 7.4±0.2 is added dropwise into the lysozyme solution with pH value of 7.4±0.2 to form the lysozyme polypeptide core by electrostatic adsorption; Step two, under the stirring at ≤25℃ and 200-400rpm, the lysozyme polypeptide core, calcium chloride solution and sodium phosphate dibasic solution are mixed, the pH value is adjusted to 8.0±0.1 and reacted for 1-4h, then centrifuged, the supernatant is discarded, and the lysozyme polypeptide core-calcium phosphate microcapsule is obtained; Step three, under the stirring at ≤25℃ and 150-250rpm, the lysozyme polypeptide core-calcium phosphate microcapsule is mixed with the chitosan solution with pH value of 5.5-6.0 to form the core-shell lysozyme particle.

2. The method for encapsulating core-shell lysozyme particles as described in claim 1, characterized in that, The step one comprises: lysozyme is added into the phosphate buffer solution with pH value of 7.4±0.2 to obtain the lysozyme solution; the negatively charged polypeptide is added into the phosphate buffer solution with pH value of 7.4±0.2 to obtain the negatively charged polypeptide solution; under the magnetic stirring at 4℃ and 300rpm, the negatively charged polypeptide solution is added dropwise into the lysozyme solution, and the stirring is continued until the Zeta potential is -5mV-+5mV to obtain the lysozyme polypeptide core.

3. The method for encapsulating core-shell lysozyme particles as described in claim 1, characterized in that, The negatively charged polypeptide comprises one or both of polyglutamic acid and polyaspartic acid, and the molecular weight of the negatively charged polypeptide is 5-15kDa.

4. The method for encapsulating core-shell lysozyme particles as described in claim 1, characterized in that, The concentration ratio of the lysozyme solution to the negatively charged polypeptide solution is 1:(0.5-1.4), and the lysozyme solution and the negatively charged polypeptide solution are in equal volume.

5. The method for encapsulating core-shell lysozyme particles as described in claim 1, characterized in that, The step two comprises: calcium chloride is dissolved in the HEPES buffer with pH value of 8.0±0.1 and pre-cooled at 4℃ to obtain the calcium chloride solution; sodium phosphate dibasic is dissolved in the HEPES buffer with pH value of 8.0±0.1 and citric acid is added to obtain the sodium phosphate dibasic solution; under the magnetic stirring at ≤25℃ and 300rpm, the lysozyme polypeptide core is mixed with the calcium chloride solution in equal volume for 10min, then the sodium phosphate dibasic solution is added dropwise, the reaction is continued for 2h, then EDTA is added and ice-bath cooled, centrifuged at 4℃ and 8000rpm for 15min, the supernatant is discarded, washed with the phosphate buffer solution to obtain the lysozyme polypeptide core-calcium phosphate microcapsule.

6. The method for encapsulating core-shell lysozyme particles as described in claim 5, characterized in that, The dropwise amount of the sodium phosphate dibasic solution satisfies that the molar ratio of calcium ions in the calcium chloride solution to hydrogen phosphate ions in the sodium phosphate dibasic solution is 1:(1.5-1.67).

7. The method for encapsulating core-shell lysozyme particles as described in claim 1, characterized in that, The step three comprises: chitosan powder is dissolved in the acetic acid solution, stirred in the 50℃ water bath for 1-3h until completely dissolved, membrane filtered to remove the insoluble substances, the pH value is adjusted to 5.5-6.0 to obtain the chitosan solution; the lysozyme polypeptide core-calcium phosphate microcapsule is suspended in the phosphate buffer solution, and ultrasonically treated at 50W for 10s to obtain the microcapsule suspension; The chitosan solution is added dropwise to the microcapsule suspension under stirring at ≤25℃ and 200 rpm, 1% w / v TPP solution is added after stirring for 30 min, and stirring is continued until the surface potential is positive and the chitosan layer thickness is 10 nm-20 nm, centrifugation is performed at 4℃ and 8000 rpm for 15 min, the supernatant is discarded, and washing is performed with a phosphate buffer solution to obtain core-shell lysozyme particles.

8. The method for encapsulating core-shell lysozyme particles as described in claim 7, characterized in that, The chitosan powder has a molecular weight of 50 kDa-150 kDa, the concentration ratio of the chitosan solution to the microcapsule suspension is (0.1-0.4):1, and the chitosan solution and the microcapsule suspension are in equal volume.

9. A core-shell lysozyme particle characterized in that, Prepared based on the coating method as claimed in any one of claims 1 to 8.

10. Use of core-shell lysozyme particles prepared according to the process of any one of claims 1 to 8 for the manufacture of an oral care product, characterized in that, The oral care product includes toothpaste, mouthwash, oral spray, denture cleaner, or gum care gel.