CN115226725B - Chimeric silver-based nano lysozyme, preparation method and application thereof - Google Patents

Chimeric silver-based nano lysozyme, preparation method and application thereof Download PDF

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Publication number
CN115226725B
CN115226725B CN202210907650.9A CN202210907650A CN115226725B CN 115226725 B CN115226725 B CN 115226725B CN 202210907650 A CN202210907650 A CN 202210907650A CN 115226725 B CN115226725 B CN 115226725B
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solution
lysozyme
silver
nano
chimeric
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CN115226725A (en
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何正有
蒋用
李天林
何艳凤
汪静
杨雨婷
黄巧凤
田玉婷
郭欣玲
鄢丹
毕建军
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Sichuan Yijieshi Medical Technology Co ltd
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Sichuan Yijieshi Medical Technology Co ltd
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Abstract

The invention belongs to the technical field of nano lysozyme preparation, and in particular relates to a preparation method, a product and application of chimeric silver-based nano lysozyme, which comprise the following steps: s1, taking a silver nitrate solution and a high molecular dispersing agent solution for reaction, and adjusting the pH value to 6-12 by using ammonium hydroxide, wherein the reaction end point is the color of the reacted solution changing into deep yellow; s2, uniformly mixing the deep yellow solution and the natural small organic molecule compound solution to obtain the solution containing the chimeric silver-based nano lysozyme. The chimeric silver-based nano lysozyme prepared by the invention has a composite structure of 'template polymer-nano silver-natural small organic molecule', and has good killing effect on bacteria, fungi and viruses, especially drug-resistant bacteria. Compared with biological lysozyme, the chimeric silver-based nano lysozyme prepared by the method has the advantages of wider microorganism killing spectrum, high biocompatibility, strong biological activity, high stability, modularized design and the like.

Description

Chimeric silver-based nano lysozyme, preparation method and application thereof
Technical Field
The invention belongs to the technical field of nano lysozyme preparation, and particularly relates to chimeric silver-based nano lysozyme, a preparation method and application thereof.
Background
In recent years, the problem of drug resistance is becoming severe, and has been developed into a serious public health safety problem in the global area, seriously endangering human health and survival. Currently, clinically common important resistant gram-positive bacteria are methicillin-resistant Lin Putao cocci (MRSA), penicillin-resistant pneumococci (PRSP) and vancomycin-resistant enterococci (VRE). The incidence rate of the infection of the methicillin-resistant staphylococcus aureus (MRSA) is reported to be 20-80 percent in various places in China, and the infection caused by the methicillin-resistant coagulase negative staphylococcus (MRCNS) is also obviously increased. The drug-resistant gram-negative bacilli are mainly: klebsiella pneumoniae and E.coli producing ultra-broad spectrum beta lactamase (extended-spectrum betalactamases, ESBLs), pseudomonas aeruginosa, acinetobacter and Acinetobacter maltophilia with multi-drug resistance, and furthermore, the proportion of candida species resistant to fluconazole and tubercle bacillus resistant to drugs is also increasing. In addition, the animal industry uses glycopeptides in large quantities, which allows a variety of animals to produce VRE, indirectly affecting human health. In order to solve the problem of drug-resistant bacteria, a more effective way is as follows: the novel sanitary disinfection product is developed, the cleanness and sanitation of the environment, object surfaces, air, skin, hands and the like are enhanced, and pathogenic microorganisms are isolated from the reach of human bodies. The existing disinfectant has the problems of strong stimulation, instability, high toxicity and the like, so that the development of novel products which are economical, efficient, safe and environment-friendly is urgently needed, and one important research and development direction is to develop novel antibacterial and antiviral lysozyme.
Lysozyme is a glycoside hydrolase that catalyzes the hydrolysis of the beta-1, 4 glycosidic bond between the N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) of glycosaminoglycans. Researches show that part of nano-enzymes have a certain similar inhibition effect on pathogenic microorganisms, and can be used for preventing and controlling pathogenic and drug-resistant microorganisms in the fields of biomedicine, public health and the like. In 2007, the group Yan Xiyun of the institute of biophysics of the chinese academy of sciences first proposed the concept of nanoenzyme (Nanozyme) in the Nature Nanotechnology journal, indicating that nanoenzymes are a novel class of enzymatically active nanomaterials with catalytic activity and enzymatic reaction kinetics similar to those of natural enzymes. Wherein, nano lysozyme (nanomlyzyme) is a functional material with inhibition or killing activity to pathogenic microorganism, with lysozyme enzymatic property and brand new quasi-enzymatic structure. The antibacterial mechanism is as follows: the nano enzyme with reasonable structural design has the capability of regulating and controlling ROS Reactive Oxygen Species (ROS) in the environment, damages a refractory biomembrane of the ROS reactive oxygen species, and can kill various pathogenic gram-positive bacteria and gram-negative bacteria with high efficiency, thereby achieving the anti-infection effect. Therefore, the nano lysozyme can be used for killing and preventing infection of infectious and pathogenic microorganisms. CN113975459A discloses a preparation method of a nano-enzyme hydrogel sheet and application of the nano-enzyme hydrogel sheet in a band-aid, wherein a core material has a composite structure of TA-CDs/AgNPs nano-enzyme and Cu, fe-N-C nano-enzyme, and the nano-enzyme with the structure shows high antibacterial catalytic activity and can be used for preparing band-aid products. CN111742941a discloses a nano enzyme preparation-rare earth compound antibacterial agent and application thereof, and the nano enzyme and biological enzyme cooperate to effectively improve rooting effect of plants, and can kill growing substrate strains on the premise of ensuring plant growth, thereby providing good environment for plant growth. Therefore, compared with the natural lysozyme, the nano lysozyme has the advantages of simple preparation, large-scale production, strong environmental tolerance, low preparation and storage cost, reusability and the like.
At present, part of patents utilize natural products to prepare nano lysozyme, the synthetic reaction speed is high, the synthetic condition is simple, and the method is suitable for industrial production. However, the chemical components of the natural products comprise organic acid, alkaloid, polysaccharide, protein, flavonoid, terpenes, steroid, saponin, tannins and other substances, the composition is quite complex, and the obtained nano silver particles, namely AgNPs nano lysozyme, have poor structure controllability. The correlation analysis is as follows:
CN109702218A discloses a method for preparing nano silver particles by using phyllanthus emblica extract; utilizing phyllanthus emblica fruit extract and AgNO 3 The solution reacts under the condition of heating and stirring to prepare nano silver particles, and the prepared nano silver particles have stable structure and strong antibacterial effect on the bacterial brown stripe disease pathogen RS-2 of rice.
CN107671305a discloses a method for rapidly preparing nano silver bacteriostatic agent by using ligustrum japonicum fruit extract, comprising collecting mature ligustrum japonicum fruit, preparing ligustrum japonicum fruit extract by steps of heating and refluxing, etc.; making ligustrum quihoui fruit extract and AgNO 3 And (3) carrying out ultrasonic treatment after mixing the solutions, and simultaneously carrying out sunlight irradiation to obtain the nano silver bacteriostatic agent.
The method needs plant extracts in the process of synthesizing AgNPs nano lysozyme, and the plant extracts have the defects of complex influencing factors, complex operation process and the like, and the antibacterial effect of the prepared nano silver is unstable. In addition, the plant species disclosed in the prior art which can be used for synthesizing the nano silver particles are few, and in addition, the plant extract has the problems of poor batch reproducibility, various reaction mechanisms, difficulty in mass production and the like. In order to solve the problem, a simple and convenient synthesis method of AgNPs nano lysozyme with excellent effect of killing pathogenic microorganisms is urgently needed to be developed.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a preparation method, a product and application of chimeric silver-based nano lysozyme.
The aim of the invention is realized by the following technical scheme: the preparation method of the chimeric silver-based nano lysozyme comprises the following steps:
s1, taking a silver nitrate solution and a high molecular dispersing agent solution for reaction, and adjusting the pH value to be 6-12 by using ammonium hydroxide before the reaction, wherein the reaction end point is the color of the solution changed into deep yellow; wherein the reaction temperature is 0-100 ℃; continuous stirring is required in the reaction process;
wherein the polymer dispersing agent in the polymer dispersing agent solution comprises one or more of tannic acid, alpha-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin, hydroxypropyl-beta-cyclodextrin, xanthan gum, tragacanth gum, guar gum, acacia, peach gum, sodium carboxymethyl cellulose and sodium alginate;
s2, uniformly mixing the dark yellow solution obtained in the step S1 with a natural small organic molecule compound solution to obtain a solution containing the chimeric silver-based nano lysozyme. The method for uniformly mixing is preferably as follows: stirring for 4-48 hours at room temperature.
Further, in step S1, the molar ratio of the silver nitrate in the silver nitrate solution to the polymeric dispersant is 1:0.01 to 10;
and/or, in the step S1, the concentration of the silver nitrate solution is 0.01-5%;
and/or, in the step S1, the concentration of the polymer dispersing agent solution is 0.01-10%.
In step S1, the ammonium hydroxide is used in an amount of 0.01-5% of the volume of the silver nitrate solution and the polymeric dispersant solution.
Further, in step S1, the ammonium hydroxide includes one or more of dodecyl trimethyl ammonium hydroxide, hexadecyl trimethyl ammonium hydroxide, dodecyl dimethyl benzyl ammonium hydroxide, didecyl dimethyl ammonium hydroxide, and didecyl ammonium hydroxide.
Further, in step S2, the natural small organic molecule compound in the natural small organic molecule compound solution includes one or more of artemisinin, baicalin, andrographolide, benzoic acid, salicylic acid, fumaric acid, abietic acid, protocatechuic acid, gallic acid, caffeic acid, cinnamic acid, chlorogenic acid, rosmarinic acid, glycyrrhetinic acid, thymol, carvacrol and luteolin.
Further, in step S2, the molar ratio of the natural small organic molecule compound to the silver nitrate in the natural small organic molecule compound solution and the silver nitrate solution is 1:0.01 to 10;
and/or, in the step S2, the concentration range of the natural small organic molecule compound solution is 0.01-10%.
The invention also provides a solution containing the chimeric silver-based nano lysozyme, which is prepared by the preparation method, wherein the particle size of the chimeric silver-based nano lysozyme in the solution is 10-30 nm.
The invention also provides an application of the solution containing the chimeric silver-based nano lysozyme as a disinfectant, an antibacterial agent or in preparing a disinfectant and antibacterial material.
Furthermore, the disinfectant and the antibacterial agent can be used for disinfecting various parts or organs of a human body, medical and health instruments in hospitals, daily necessities, public environments and biosafety.
Further, the concentration of the chimeric silver-based nano lysozyme in the disinfectant and the antibacterial agent is 5-200 mg/L.
The basic principle of the invention is as follows:
in general, after silver ions are reduced in a solution, large-particle silver simple substance particles are often aggregated, and it is difficult to obtain a uniform dispersion tyndall sol with high stability. In order to promote the system to convert to the tyndall sol and prevent the aggregation of silver simple substance particles, the polymer dispersing agents with different structures are added into the original system, thereby being beneficial to forming the metal silver nano particles with controllable structure, high stability and uniform dispersion. Because the macromolecular dispersing agent has different hydrophilic parts and hydrophobic parts, the structural unit is basically clear, and the space structure has the characteristic of designability, thereby being beneficial to forming nano silver particles with different space structures in different spaces, and the hydrophobic groups of the macromolecules are utilized to mutually isolate the nano silver particles in the space structure, so that the macromolecular dispersing agent has a good molecular impression effect. Therefore, according to the rationalized design of different macromolecule templates and spaces thereof, the nano material with different catalytic properties, namely nano enzyme, can be obtained, can be used for a plurality of organic synthesis reactions or biochemical reactions in industry, and has wide application prospect in the fields of clinical medical diagnosis and the like. In general, nano-enzymes made of silver ions generally have activity against pathogenic microorganisms, on the basis of which the structure is subjected to chimeric design of molecular targets, and natural small organic molecules with different structures are assembled on the parent structure, so that the active attack capability of the nano-enzymes on pathogenic microorganisms is improved, and the antimicrobial activity of the nano-enzymes is greatly improved. Because the natural-source small molecular compound has the characteristics of natural affinity with a living body, small toxic and side effects, reversible combination and the like, the natural-source small molecular compound has great promotion effect on the activity of the original nano enzyme. Therefore, the silver-based nano lysozyme designed by the scheme has the advantages of wider microorganism killing spectrum, high biocompatibility, strong biological activity, high stability, modularization design and the like compared with the common lysozyme on pathogenic microorganisms.
The beneficial effects of the invention are as follows:
the AgNPs nano lysozyme (AgNPs nanomlysozyme) shows excellent antibacterial activity and has industrial application prospect. The AgNPs nano lysozyme with different structures and catalytic activities can be prepared by adopting a proper process flow. Specifically, the invention designs the silver-based nano lysozyme embedded with the natural organic small molecular compound. The chimeric nano lysozyme has better activity of resisting pathogenic microorganisms, is a sanitary disinfection material with great potential, and can be used in the fields of medical treatment and sanitation, environmental protection, medical diagnosis, chemical synthesis and the like.
1. The preparation method has the advantages of simple process, low energy consumption, easily available raw materials, no need of adding a reducing agent and a catalyst, and suitability for large-scale production.
2. The chimeric AgNPs nano lysozyme obtained by the invention has the structural characteristics of 'template macromolecule-nano silver cavity-natural organic micromolecule', the average grain diameter is between 10 and 30nm, and the chimeric AgNPs nano lysozyme has the advantages of structural design, stable space structure, uniform average grain diameter and the like, and is used for influenza virus H 3 N 2 The antibacterial agent has high killing rate on hand-foot-mouth virus (enterovirus 71), methicillin-resistant staphylococcus aureus, vancomycin-resistant enterococci, enteropathogenic bacteria, suppurative coccus, pathogenic microzyme and common bacteria of nosocomial infection.
3. The chimeric AgNPs nano lysozyme product prepared by the invention has the advantages of high environmental compatibility, high safety and remarkable effect, can be used as a sanitary disinfection or antibacterial material for preventing and treating pathogenic and infectious diseases, and has wide application and popularization prospects in the production fields of medical treatment, household disinfectants and sanitary products.
Drawings
FIG. 1 is an assembly flow diagram of HP-beta-CD AgNPs nanolysozyme chimeric to baicalin;
FIG. 2 is a TEM and particle size distribution diagram of baicalin-chimeric HP-beta-CD AgNPs nanolysozyme;
FIG. 3 is a TEM image of GT AgNPs lysozyme of chimeric fumaric acid;
FIG. 4 is an XRD pattern of baicalin-chimeric HP-beta-CD AgNPs nanolysozyme;
FIG. 5 is a UV-Vis absorption curve of baicalin-chimeric HP-beta-CD AgNPs nanolysozyme.
Detailed Description
The technical solution of the present invention will be described in further detail with reference to the accompanying drawings, but the scope of the present invention is not limited to the following description.
Example 1
Taking 0.8g of hydroxypropyl-beta-cyclodextrin, adding 100mL of deionized water, stirring at room temperature for 24 hours to fully dissolve the hydroxypropyl-beta-cyclodextrin, thus obtaining the hydroxypropyl-beta-cyclodextrin solution with the concentration of 8 mg/mL. And adding 100mL of deionized water into 0.18g of silver nitrate solid, and stirring to dissolve the silver nitrate solid to obtain a silver nitrate solution with the concentration of 1.8 mg/mL. The above silver nitrate solution (1.8 mg/ml) was added to the hydroxypropyl-beta-cyclodextrin solution in equal volume (1:1 v/v), the pH was adjusted to 7.26 with 10% aqueous dodecyltrimethylammonium hydroxide, heated to 60℃and stirred at 200rpm for 2 hours. The color of the solution is changed from colorless to light yellow and then to dark yellow, and the nano silver particle solution wrapped by the hydroxypropyl-beta-cyclodextrin, namely HP-beta-CD AgNPs, is obtained. The formation of HP-beta-CD AgNPs was confirmed by UV-visible spectrophotometry to determine its surface plasmon resonance signal. Adding 10mL of baicalin solution with the concentration of 1.0mg/mL into the HP-beta-CD AgNPs solution, and stirring at 200rpm for 24 hours at room temperature to obtain the HP-beta-CD AgNPs lysozyme embedded with the baicalin.
Example two
0.8g of acacia is taken, 100mL of deionized water is added, and the mixture is stirred for 24 hours at room temperature to be fully dissolved, insoluble matters are removed by filtration, and thus, the acacia solution with the concentration of 8mg/mL is obtained. And adding 100mL of deionized water into 0.18g of silver nitrate solid, and stirring to dissolve the silver nitrate solid to obtain a silver nitrate solution with the concentration of 1.8 mg/mL. The above silver nitrate solution (1.8 mg/ml) was added to the gum arabic solution in an equal volume (1:1 v/v), the pH was adjusted to 8.90 with 10% aqueous cetyltrimethylammonium hydroxide, heated to 60℃and stirred at 200rpm for 2 hours. The color of the solution is changed from colorless to light yellow, and then the solution is changed into dark yellow, so that the Arabic gum coated nano silver particle solution, namely GA AgNPs, is obtained. The formation of GA AgNPs was confirmed by measuring its surface plasmon resonance by uv-vis spectrophotometry. And adding 10mL of benzoic acid solution with the concentration of 1.0mg/mL into the GA AgNPs solution, and stirring at 200rpm for 24 hours at room temperature to obtain the GA AgNPs lysozyme embedded with benzoic acid.
Example III
Taking 0.8g of guar gum, adding 100mL of deionized water, stirring at room temperature for 24 hours to fully dissolve the guar gum, and filtering to remove insoluble substances to obtain the guar gum solution with the concentration of 8 mg/mL. And adding 100mL of deionized water into 0.18g of silver nitrate solid, and stirring to dissolve the silver nitrate solid to obtain a silver nitrate solution with the concentration of 1.8 mg/mL. The above silver nitrate solution (1.8 mg/ml) was added to the guar gum solution in equal volumes (1:1 v/v), the pH was adjusted to 11.23 with 10% aqueous dodecyldimethylbenzyl ammonium hydroxide, heated to 60℃and stirred at 200rpm for 2 hours. The color of the solution is changed from colorless to light yellow, and then the solution is changed into dark yellow, so that the guar gum coated nano silver particle solution, namely GG AgNPs, is obtained. The formation of GG AgNPs was confirmed by measuring its surface plasmon resonance by uv-vis spectrophotometry. And adding 10mL of chlorogenic acid solution with the concentration of 1.0mg/mL into the GG AgNPs solution, and stirring at 200rpm for 24 hours at room temperature to obtain the GG AgNPs lysozyme embedded with chlorogenic acid.
Example IV
Taking 0.8g of tragacanth, adding 100mL of deionized water, stirring at room temperature for 24 hours to fully dissolve, and filtering to remove insoluble substances to obtain the tragacanth solution with the concentration of 8 mg/mL. And adding 100mL of deionized water into 0.18g of silver nitrate solid, and stirring to dissolve the silver nitrate solid to obtain a silver nitrate solution with the concentration of 1.8 mg/mL. The above silver nitrate solution (1.8 mg/ml) was added to the tragacanth solution in an equal volume (1:1 v/v), the pH was adjusted to 6.92 with 10% aqueous cetyltrimethylammonium hydroxide, heated to 60℃and stirred at 200rpm for 2 hours. The color of the solution is changed from colorless to light yellow, and then the solution is changed into dark yellow, thus obtaining the nano silver particle solution coated by the tragacanth, namely GT AgNPs. The formation of GT AgNPs was confirmed by measuring its surface plasmon resonance by uv-vis spectrophotometry. And adding 10mL of a fumaric acid solution with the concentration of 1.0mg/mL into the GT AgNPs solution, and stirring at 200rpm for 24 hours at room temperature to obtain the GT AgNPs lysozyme embedded with the fumaric acid.
Example five
Taking 0.8g of xanthan gum, adding 100mL of deionized water, stirring at room temperature for 24 hours to fully dissolve the xanthan gum, and filtering to remove insoluble substances to obtain a xanthan gum solution with the concentration of 8 mg/mL. And adding 100mL of deionized water into 0.18g of silver nitrate solid, and stirring to dissolve the silver nitrate solid to obtain a silver nitrate solution with the concentration of 1.8 mg/mL. The above silver nitrate solution (1.8 mg/ml) was added to the xanthan gum solution in equal volume (1:1 v/v), the pH was adjusted to 7.59 with 10% aqueous cetyltrimethylammonium hydroxide, heated to 60℃and stirred at 200rpm for 2 hours. The color of the solution is changed from colorless to light yellow, and then the solution is changed into dark yellow, and the nano silver particle solution wrapped by the xanthan gum, namely GGAgNPs, is obtained. The formation of GG AgNPs was confirmed by measuring its surface plasmon resonance by uv-vis spectrophotometry. And adding 10mL of gallic acid solution with the concentration of 1.0mg/mL into the GT AgNPs solution, and stirring at 200rpm for 24 hours at room temperature to obtain the GG AgNPs lysozyme embedded with the gallic acid.
Example six
The nano lysozyme solution of each embodiment can be respectively used for preparing new products such as medical supplies, household disinfection supplies, daily chemical supplies such as laundry detergent and the like with disinfection functions.
Such as: a plant moxa nano silver disinfection spray containing nano lysozyme and a preparation method thereof.
Taking a certain amount of nano lysozyme solution prepared in example 1; adding herba Rosmarini officinalis (ROSMARINUS OFFICINALIS) extract, cortex Magnolia officinalis (MAGNOLIA OFFICINALIS) bark extract, folium Artemisiae Argyi (ARTEMISIA ARGYI) extract, tea (CAMELLIA SINENSIS) polyphenols, and fructus Rhodomyrti (GARCINIA MANGOSTANA) pericarp extract; placing into a batching container, adding deionized purified water to make the final concentration of silver element in nano lysozyme be 50ppm, stirring to make the silver element fully dissolved to obtain clear solution, detecting the content of nano lysozyme, and canning after the nano lysozyme is qualified.
Such as: an Ijieshi brand plant moxa pet degerming and mite-killing spray containing nano lysozyme and a preparation method thereof.
Taking a certain amount of nano lysozyme solution prepared in example 4; adding herba Taraxaci (TARAXACUM MONGOLICUM) extract, flos Chrysanthemi (CHRYSANTHEMUM MORIFOLIUM) extract, folium Artemisiae Argyi (ARTEMISIA ARGYI) extract, tea (CAMELLIA SINENSIS) polyphenol, fructus Rhodomyrti (GARCINIA MANGOSTANA) pericarp extract, and plant mite-repellent extract; placing into a batching container, adding deionized purified water to make the final concentration of silver element in nano lysozyme be 50ppm, stirring to make the silver element fully dissolved to obtain clear solution, detecting the content of nano lysozyme, and canning after the nano lysozyme is qualified.
Experimental example 1
Taking HP-beta-CD AgNPs lysozyme embedded with baicalin and prepared in example 1, carrying out TEM analysis to obtain a graph 2, taking GT AgNPs lysozyme embedded with fumaric acid and prepared in example 4, carrying out TEM analysis to obtain a graph 3, and can be seen from the graph: the particle size of the chimeric AgNPs lysozyme is distributed between 10 and 20nm, the average particle size is 16nm, and the chimeric AgNPs lysozyme is spherical, which proves that the AgNPs lysozyme prepared by the method can stably exist in aqueous solution and accords with the expected design.
Experimental example 2
The HP-beta-CD AgNPs lysozyme embedded with baicalin, which is prepared in the embodiment 1, is taken and analyzed by XRD to obtain a graph in which 20-23 degrees are dispersed diffraction peaks of PVP, 2 theta = 38 degrees, 44 degrees, 64.3 degrees and 77.3 degrees are diffraction peaks corresponding to face-centered cubic (fcc) crystal faces of silver phases (111), (200), (220) and (311) respectively, so that the spatial structure of silver particles in the nano-enzyme is a face-centered cubic structure, no other obvious characteristic peaks in the graph are shown, the purity of the obtained silver particles is high, and the performance is complete.
Experimental example 3
The baicalin-chimeric HP-beta-CD AgNPs lysozyme prepared in example 1 was subjected to UV-Vis analysis to obtain FIG. 5. The analysis in the figure shows that the color of the solution is gradually deepened from pale yellow, which indicates that nano silver is formed and is embodied on an ultraviolet absorption curve of the solution, and the maximum absorption wavelength of the nano silver is about 420 nm; according to Mie theory, the absorption peak is the ultraviolet characteristic absorption of typical spherical nano silver, and the peak shape of the peak is gradually narrowed along with the formation of nano silver particles; the half-width is reduced, which indicates that the size of the nano silver particles is more uniform, and HP-beta-CD plays roles of dispersing, buffering and space architecture in the solution, and the obtained nano lysozyme is not easy to agglomerate and is more stable in the solution.
Experimental example 4
The sterilizing spray of the Jieshi brand and Jieshi brand plant moxa nano silver containing 50ppm of nano lysozyme, which is prepared in the example 6, is taken for carrying out a suspension quantitative sterilization test, and the sterilizing effect of the sterilizing spray on vancomycin-resistant enterococci (ATCC 51299) and methicillin-resistant staphylococcus aureus (ATCC 43300) is tested.
The test basis is as follows: 2.1.1.5.5 and 2.1.1.7.4 of the 2002 edition of the disinfection Specification
Evaluation basis: 2002 edition of sterilizing technical Specification
Inspection conclusion:
(1) Quantitative identification test of neutralizing agent suspension: PBS solution containing 3% Tween-80, 0.5% sodium thiosulfate, 0.5% L-histidine, 0.5% peptone, 0.85% sodium chloride, 1.43% lecithin and 0.1% cysteine can effectively neutralize the killing effect of the 'sterilizing spray of the moxa nano silver of the Jieshi brand plant' stock solution, and the neutralizing agent and the neutralizing product have no adverse effect on methicillin-resistant staphylococcus aureus.
(2) Quantitative sterilization test of suspension: the sample stock solution acts for 15min, the test is repeated for 3 times, the killing logarithmic values of the vancomycin-resistant enterococci (ATCC 51299) and methicillin-resistant staphylococcus aureus (ATCC 43300) are both more than 5.00, the sample stock solution meets the evaluation rule of 2002 edition-2.1.1.7.7 of disinfection technical Specification (the killing logarithmic value is more than or equal to 5.00), and the sample is qualified for disinfection of the tested strain.
And the experimental result is verified by a microbiological analysis detection center in Guangdong province.
Experimental example 5
The sterilizing spray of the Jieshi brand plant moxa nano silver containing 50ppm of nano lysozyme prepared in the example 6 is taken, and the sterilizing activity of the sterilizing spray on enterovirus 71 type and influenza virus (H3N 2) is detected.
The test basis is as follows: the examination was carried out with reference to items 2.1.1.10.5 and 2.1.1.10.7 of the disinfection technical Specification (2002 edition).
Evaluation basis: evaluation was performed according to the "disinfection technical Specification" (2002 edition).
Inspection conclusion:
1. through 3 repeated tests, the neutralizer solution of the D/E neutralization broth can effectively neutralize the residual effect of the sterilizing spray of the ideas plant ideas nano silver of the ideas to enterovirus 71 and influenza virus (H3N 2), and the neutralizer and the neutralization product have no influence on the enterovirus 71 inactivation test and the growth of cells; has no influence on the inactivation test of influenza virus (H3N 2) and the growth of chicken embryo.
2. Through 3 repeated tests, under the constant temperature test condition of 20 ℃, the sterilizing and spraying effect of the Ijieshi brand and the Ijieshi brand plant moxa nano silver is applied for 10.0min, and the enterovirus 71 and the influenza virus (H) 3 N 2 ) Average inactivation log value of (C)>4.00, which meets the specification of disinfection qualification in the technical Specification of disinfection (2002 edition).
The experimental result is verified by "Zhongguancun International pharmaceutical inspection and certification science and technology Co., ltd.
Experimental example 6
The sterilizing spray of the Jieshi brand plant moxa nano silver containing 50ppm nano lysozyme prepared in the example 6 is taken, and the common physical and chemical indexes and the killing effect on common pathogenic bacteria are detected.
The test basis is as follows: chapter 1.6 of cosmetic safety Specification (2015 edition); disinfection technical Specification (2002 edition) 2.2.1.4; disinfection technical Specification (2002 edition) 2.2.3; disinfection technical Specification (2002 edition) 2.2.4; 2.1.1.5.5 of the disinfection Specification (2002 edition); 2.1.1.7 of the disinfection Specification (2002 edition); 2.1.1.9 of the disinfection Specification (2002 edition); 2.1.2.10 of the disinfection Specification (2002 edition); 2.1.3.4 of the disinfection Specification (2002 edition); 2.1.3.5 of the disinfection Specification (2002 edition); disinfection technical Specification (2002 edition) 2.3.1; disinfection technical Specification (2002 edition) 2.3.2; disinfection technical Specification (2002 edition) 2.3.3; disinfection technical Specification (2002 edition) 2.3.4; 2.3.8.4 to the disinfection Specification (2002 edition).
Evaluation basis: disinfection technical Specification (2002 edition)
Inspection conclusion:
(1) pH value test: the pH (25 ℃) of the sample, "Ijieshi brand plant moxa nano silver disinfection spray" was 3.56.
(2) Silver content test: the silver content of the sample 'Yijieshi plant moxa nano silver disinfection spray' is 53.1mg/kg.
(3) Stability test (silver content): the initial silver content of the sample was measured to be 5.59mg/kg. The silver content of the sample is 52.8mg/kg after the sample is stored for 3 months at 37 ℃, the reduction rate is 0.6% compared with the reduction rate before the sample is stored, the sample meets the requirement that the reduction rate of the effective components in the disinfection technical Specification (2002 edition) -2.2.3.2.1 is not more than 10%, and the storage effective period of the sample can be set to be 2 years.
(4) Metal corrosiveness experiment: the average corrosion rate of the spray stock solution for sterilizing the Ijieshi plant moxa nano silver is 0.0042mm/a (basically no corrosion), the average corrosion rate of the spray stock solution for sterilizing the moxa nano silver is 0.0016mm/a (basically no corrosion), the average corrosion rate of the spray stock solution for sterilizing the Ijieshi plant moxa nano silver is 0.0301mm/a (slightly corrosion), and the average corrosion rate of the spray stock solution for sterilizing the moxa nano silver is 0.0092mm/a (basically no corrosion).
(5) Neutralizer identification assay (bacteria): the neutralizing agent identification test shows that the D/E neutralization broth can effectively neutralize the sterilizing effect of the ideas plant moxa nano silver disinfection spray stock solution on escherichia coli (8099), and the neutralizing agent and the neutralizing product have no adverse effect on the escherichia coli (8099) and a culture medium, so that the neutralizing agent is applicable to quantitative killing tests of escherichia coli (8099), staphylococcus aureus (ATCC 6538) and pseudomonas aeruginosa (ATCC 15442).
(6) Quantitative bacterial suspension killing test: according to the disinfection technical Specification (2002 edition), under experimental conditions, the average killing logarithmic values of the sterilizing spray stock solution of the Aijieshi plant moxa nano silver for 15min on staphylococcus aureus (ATCC 6538), escherichia coli (8099) and pseudomonas aeruginosa (ATCC 15442) are all more than 5.00, and the sterilizing spray stock solution meets the standard requirements of the disinfection technical Specification (2002 edition).
(7) Neutralizer identification assay (fungi): the neutralizing agent identification test shows that the D/E neutralization broth can effectively neutralize the sterilizing effect of the ideas plant moxa nano silver disinfection spray stock solution on candida albicans (ATCC 10231), and the neutralizing agent and the neutralization product have no adverse effect on candida albicans (ATCC 10231) and a culture medium, so that the neutralizing agent is suitable for the candida albicans (ATCC 10231) quantitative killing test.
(8) Quantitative killing test of candida albicans suspension: according to the disinfection technical Specification (2002 edition), under experimental conditions, the average killing log value of the Yijieshi plant moxa nano silver disinfection spray stock solution acting for 15min on candida albicans (ATCC 10231) is more than 4.00, and meets the standard requirements of the disinfection technical Specification (2002 edition).
(9) In-situ identification test of disinfectants to object surface disinfection (wooden surface): the on-site test of the surface disinfection of the wooden object shows that under the experimental condition, the average sterilization logarithmic value of natural bacteria on the surface of the wooden object is more than 1.00 after 15 minutes of the action of the nano silver disinfection spray stock solution of the Ijieshi brand plant meets the standard requirement of the disinfection technical Specification (2002 edition).
(10) Air disinfection effect simulation site: the simulation field test result of the air disinfection effect shows that in a 20m3 closed space, under the experimental condition, the spray stock solution of the moxa nano silver disinfection of the Yijieshi plant is sprayed in the closed space for 15min at the concentration of 10mL/m3, and the killing rate of the staphylococcus albus is more than 99.90% in 3 test results, thereby meeting the requirements of disinfection technology Specification (2002 edition) on disinfection qualification.
(11) Air disinfection effect field test: in a 30m3 room, under experimental conditions, the spray stock solution for sterilizing the ideas plant moxa nano silver is atomized into the air at the concentration of 10mL/m3, and the extinction rate of the spray stock solution for sterilizing the ideas plant moxa nano silver for 15min to air natural bacteria is more than 90% as the test results, and meets the standard requirements of the sterilizing technical Specification (2002 edition).
(12) Acute oral toxicity test: under the experimental condition, acute oral toxicity LD of the sterilizing spray stock solution of the Aijieshi brand plant nano silver on KM mice 50 >5000 mg/kg.bw, the acute oral toxicity test belongs to actual innocuity, and meets the requirements of the disinfection technical Specification (2002 edition).
(13) Acute inhalation toxicity test: under the experimental conditions, the acute inhalation toxicity LC50 of the spray stock solution of the Ijieshi brand plant moxa nano silver disinfection to KM mice is more than 10000mg/m3, and the acute inhalation toxicity test is practically nontoxic and meets the requirements of disinfection technical Specification (2002 edition).
(14) One complete skin irritation test: under the experimental conditions, the stimulation strength of the Ijieshi plant moxa nano silver disinfection spray stock solution to a once complete skin stimulation test of New Zealand rabbits belongs to no stimulation, and meets the requirements of disinfection technical Specification (2002 edition).
(15) Acute eye irritation test: under the experimental conditions, the damage type of the nano silver disinfection spray stock solution of the Ijieshi brand plant on the eye irritation of New Zealand rabbits is non-irritating, and meets the requirements of the disinfection technical Specification (2002 edition).
(16) Mouse bone marrow multipotent erythrocyte micronucleus assay: under the experimental conditions, the spray stock solution for sterilizing the ideas plant moxa nano silver can not cause the increase of the bone marrow micronucleus rate of KM mice, so the spray stock solution has no in-vivo chromosome damage effect, belongs to mutation negativity, and meets the requirements of the sterilizing technical Specification (2002 edition).
And the experimental result is verified by Qingdao scientific quality detection limited company.
The foregoing is merely a preferred embodiment of the invention, and it is to be understood that the invention is not limited to the form disclosed herein but is not to be construed as excluding other embodiments, but is capable of numerous other combinations, modifications and environments and is capable of modifications within the scope of the inventive concept, either as taught or as a matter of routine skill or knowledge in the relevant art. And that modifications and variations which do not depart from the spirit and scope of the invention are intended to be within the scope of the appended claims.

Claims (7)

1. The preparation method of the chimeric silver-based nano lysozyme is characterized by comprising the following steps of:
s1, taking a silver nitrate solution and a high-molecular dispersing agent solution for reaction, and adjusting the pH value to 6-12 by using ammonium hydroxide before the reaction, wherein the reaction end point is the color of the solution changed into deep yellow;
wherein the macromolecular dispersing agent in the macromolecular dispersing agent solution is hydroxypropyl-beta-cyclodextrin or tragacanth; the ammonium hydroxide is one or more of dodecyl trimethyl ammonium hydroxide, hexadecyl trimethyl ammonium hydroxide, dodecyl dimethyl benzyl ammonium hydroxide, bisdodecyl dimethyl ammonium hydroxide and bisdecyl ammonium hydroxide;
s2, uniformly mixing the deep yellow solution obtained in the step S1 with a natural small organic molecule compound solution to obtain a solution containing the chimeric silver-based nano lysozyme;
wherein the natural small organic molecule compound in the natural small organic molecule compound solution is baicalin or fumaric acid.
2. The preparation method according to claim 1, wherein in the step S1, a molar ratio of the silver nitrate in the silver nitrate solution to the polymeric dispersant in the polymeric dispersant solution is 1: 0.01-10;
and/or, in the step S1, the concentration of the silver nitrate solution is 0.01-5%;
and/or, in the step S1, the concentration of the polymer dispersing agent solution is 0.01-10%.
3. The method according to claim 1, wherein in the step S1, the ammonium hydroxide is used in an amount of 0.01 to 5% by volume of the silver nitrate solution and the polymeric dispersant solution.
4. The preparation method according to claim 1, wherein in step S2, the molar ratio of the natural small organic molecule compound to the silver nitrate in the natural small organic molecule compound solution and the silver nitrate solution is 1: 0.01-10;
and/or in the step S2, the concentration range of the natural small organic molecule compound solution is 0.01-10%.
5. The solution containing the chimeric silver-based nano-lysozyme prepared by the preparation method of any one of claims 1 to 4, wherein the particle size of the chimeric silver-based nano-lysozyme in the solution is 10 to 30nm.
6. Use of the solution containing said chimeric silver-based nano-lysozyme according to claim 5 for the preparation of a disinfectant.
7. The use according to claim 6, wherein the concentration of the chimeric silver-based nano-lysozyme in the disinfectant is 5-200 mg/L.
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