Antibacterial compound as well as preparation method and application thereof

Abstract

The invention relates to an antibacterial compound and a preparation method and application thereof, the antibacterial compound comprises enzyme, metal and polymer, the existence form of the metal comprises metal ions and metal nanoparticles, and the metal nanoparticles account for more than 50% of all metal elements. According to the antibacterial compound, the enzyme-metal-polymer compound constructed by taking enzyme protein and polymer molecules as templates can keep the conformation of enzyme stable, and meanwhile, the effect of reducing aggregation of metal particles can be achieved, so that the antibacterial effect of the compound is more lasting, and the biotoxicity is lower.

Description

Technical Field

[0001] The present application generally relates to an antibacterial compound and a preparation method and application thereof. Background Art

[0002] Most of the bad smells in life are caused by the growth of bacteria and fungi, which can decompose organic matter and produce volatile compounds. For example, sweat contains water, inorganic salts, fatty acids, urea, ammonia and other components, which will emit unpleasant odors after being decomposed by bacteria. The acid odor of sweat and the odor of shoes and socks are mainly caused by bacteria decomposing organic compounds in sweat to produce substances such as butyric acid, valeric acid, and isovaleric acid. The various unpleasant odors caused by bacteria make our home environment no longer fresh and clean.

[0003] At present, the common deodorization treatment methods are mainly divided into three categories: physical method, chemical method and biological method. The physical method uses porous materials to absorb odor substances, or uses aromatic smells to cover up the odor. The odor is absorbed or covered up, which does not really solve the odor problem, and the effect is not good; the chemical method converts the odor components into odorless substances through chemical reactions such as oxidation, reduction, addition, and condensation, and completely decomposes the odor components; the biological method uses the inhibitory effect of deodorizing microorganisms on corrupt bacteria to prevent nitrogen-containing organic matter from being converted into odorous substances and inhibit the production of odorous substances. Although the current deodorization methods can effectively eliminate the odor in the odor source and kill live bacteria by covering, adsorbing and decomposing, their deodorization durability and ability to kill live bacteria need to be improved.

[0004] Chinese patent application CN111484421A discloses an antibacterial silver ion compound, wherein the cation in the compound is a coordinated cation formed by silver ions and amino acid ligands, and the stability of the silver ions can be improved through such coordination. The silver ion concentration in the compound is 10 to 6500 ppm, and has certain biological toxicity.

[0005] US patent application US20120076942A1 discloses an antibacterial fabric finishing agent, comprising water, metal and polymer complex, which can be used in low pH scenarios to improve the sustained antibacterial performance. However, the metal concentration in the finishing agent is as high as 50 to 1000 ppm. Under the condition of high metal concentration, the antibacterial fabric finishing agent has certain biological toxicity and poor environmental friendliness.

[0006] Chinese patent application CN116043528 A discloses a method for manufacturing a fabric antibacterial finishing working solution and an antibacterial fabric, which solves the problem that the silver ion fabric antibacterial finishing working solution used for fabric antibacterial finishing causes unstable fabric color, but its composition contains ammonia and has a pungent odor. In view of the problems of poor color stability and strong odor of fabrics finished with silver antibacterial agents at present, the antibacterial agent containing biological enzymes proposed in the present invention is an enzyme-silver-polymer ternary composite antibacterial complex, which has no pungent odor, can be used for fabric finishing to maintain the original properties of the fabric, has good color stability, and the antibacterial fabric has excellent antibacterial properties and water washability.

[0007] Therefore, it is necessary to provide an antibacterial preparation that can reduce biological toxicity and improve antibacterial durability while ensuring its antibacterial effect.

[0008] Based on the above technical problems, the present invention is proposed. Summary of the invention

[0009] In one embodiment, the present invention discloses an antibacterial complex, which comprises an enzyme, a metal, and a polymer. The metal exists in the form of metal ions and metal nanoparticles, and the proportion of metal nanoparticles in all metal elements exceeds 50%.

[0010] Furthermore, the polymer is selected from one or more polymers having amino, pyridyl, pyrrolyl, imidazole, thiazolyl, hydroxyl and the like groups on the side groups or main chains.

[0011] Furthermore, the polymer is selected from cationic polymers containing polyamino groups.

[0012] Furthermore, the metal is a metal with antibacterial effect, and the metal with antibacterial effect is selected from one or more of copper, zinc, silver, aluminum, vanadium, gold, tin, nickel, etc.; the metal forms a ligand complex with the polymer / enzyme.

[0013] Furthermore, the metal is selected from silver.

[0014] Furthermore, the enzyme, metal, and polymer form coordination through self-assembly, and one coordination mode includes: the polymer is connected to the surface of the enzyme, and the metal is connected to the surface of the polymer and / or the enzyme.

[0015] Furthermore, the mass fraction of the metal in the antibacterial composite is 0.2% to 0.6%.

[0016] Furthermore, the mass fraction of the polymer in the antimicrobial composite is 1% to 10%;

[0017] The number of groups provided on the polymer side groups or main chain exceeds the number of metal atoms.

[0018] Furthermore, the mass fraction of the polymer in the antibacterial composite is 3% to 6%.

[0019] Furthermore, the mass fraction of the enzyme in the antibacterial complex is 0.2% to 1%.

[0020] Furthermore, the average particle size of the antibacterial complex is 80 to 200 nm.

[0021] Furthermore, the method for preparing the antibacterial complex includes: adding a raw material solution containing metal ions after adding the raw material enzyme.

[0022] Furthermore, the method of using the antimicrobial compound to finish fabrics includes: using two rollers arranged opposite to each other to squeeze the fabric moistened with the antimicrobial compound solution, heating the rollers on both sides, and the temperature of the rollers is 50-100°C.

[0023] Furthermore, the method of using the antimicrobial compound to finish fabrics includes: immersing the fabric in an immersion tank configured with an antimicrobial compound finishing liquid, the immersion tank being a sealed container, and evacuating the immersion tank to make the air pressure in the immersion tank lower than the atmospheric pressure.

[0024] The above summary does not include an exhaustive enumeration of all aspects of the invention. It is contemplated that the present invention includes all systems and methods that can be implemented by all suitable combinations of the various aspects summarized above and disclosed in the detailed description below and specifically pointed out in the claims filed with this patent application. Such combinations have specific advantages not specifically set forth in the above summary. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Embodiments are illustrated by way of example and not limitation in the figures of the accompanying drawings in which like reference numerals indicate similar elements. It should be noted that references to "an" or "an" embodiment in this disclosure are not necessarily to the same embodiment.

[0026] Figure 1 A schematic diagram of the enzyme, silver, and polymer binding structure of an embodiment of the present invention is shown;

[0027] Figure 2 A schematic diagram showing the ratio of the interaction forces between the enzyme, silver and the polymer in an embodiment of the present invention is shown;

[0028] Figure 3 A schematic diagram showing the biological toxicity of the embodiments of the present invention to different cells is shown. DETAILED DESCRIPTION

[0029] In this section, we will explain several embodiments of the present invention with reference to the accompanying drawings. Whenever the composition, structure and other aspects of the composite described in the embodiments are not clearly defined, the scope of the present invention is not limited to the composition and structure shown. The content shown is only for illustrative purposes. In addition, although many details are described, it should be understood that some embodiments of the present invention can be implemented without these details. In other cases, well-known structures and technologies are not shown in detail to avoid blurring the understanding of this description.

[0030] As used herein, the singular forms "a", "an", "the", etc. are intended to include the plural forms as well, unless the context indicates otherwise. It should be further understood that the terms "include" and / or "comprise" define the presence of features, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, steps, operations, elements, ingredients, components and / or their collections.

[0031] As used herein, the terms "or" and "and / or" should be interpreted as inclusive or meaning any one or any combination. Thus, "A, B, or C" or "A, B, and / or C" means "any of the following: A; B; C; A and B; A and C; B and C; A, B, and C." Exceptions to this definition will only occur when a combination of elements, functions, steps, or acts are inherently mutually exclusive to some extent.

[0032] The present invention relates to a composite material. Generally speaking, a composite material refers to a new material formed by combining two or more materials with different properties. This new material can usually retain the performance of a part of each component material, but this composite material may not necessarily have more advantageous performance in all dimensions than its component materials. The composite material described in the present invention not only maintains the advantages of the performance of each component material, but also can obtain comprehensive performance that cannot be achieved by a single component material through the complementarity and correlation of the performance of each component, and achieves unexpected material performance, especially excellent antibacterial performance and low biological toxicity.

[0033] The composite material referred to in the present invention includes enzymes, metals, and polymers. These components are compounded in a specific ratio, combination mode, etc., and the obtained antibacterial composite has relatively excellent comprehensive performance.

[0034] Enzymes

[0035] Enzymes are a class of biological macromolecules composed of amino acids that have catalytic properties and can accelerate specific chemical reactions.

[0036] The enzyme in the antibacterial complex of the present invention is selected from oxidative antibacterial enzymes that catalyze oxidative antibacterial effects. As an optional embodiment, the oxidative antibacterial enzyme in the complex of the present invention is selected from one or more of glucose oxidase, lactoperoxidase, catalase, horseradish peroxidase, cytochrome c, alcohol dehydrogenase, superoxide dismutase, tyrosinase, carbonic anhydrase, and laccase.

[0037] As a further optional embodiment, the enzyme in the complex of the present invention is selected from one or more of lysozymes. Lysozyme is a hydrolase that can catalyze the dissolution of bacterial surface polymers or cell walls, such as lysostaphylococcal enzyme, egg white lysozyme, bacterial lysozyme (such as N-acetylhexosaminidase, amidase), fungal lysozyme (such as chitinase, β-glucanase), phage lytic enzyme, etc. Lysozyme can effectively hydrolyze the peptidoglycan of bacterial cell walls. In addition, it can perforate the negatively charged bacterial cell membrane to form regular ion channels, thereby causing a large amount of K in the cell. + and the outflow of contents, eventually leading to the death of bacteria.

[0038] Enzyme protein and enzyme described in this specification have the same meaning.

[0039] Antimicrobial Metal

[0040] A variety of metal ions and single substances have effective antibacterial effects, and their antibacterial mechanisms are similar, such as destroying bacterial cell membranes, interfering with bacterial metabolism, and affecting bacterial DNA replication. As an optional embodiment, the antibacterial metal of the present invention can be selected from one or more of copper, zinc, silver, aluminum, vanadium, gold, tin, nickel, etc.; as an embodiment, the metal in the composite of the present invention is selected from copper, zinc, and silver.

[0041] As a further embodiment, the metal in the complex of the present invention is selected from silver. Metallic silver can specifically bind to the negatively charged thiol group (-SH) in the protease in the bacteria, thereby piercing the cell wall and cell membrane, making it impossible for the bacteria to breathe, metabolize or reproduce, and playing a sterilizing role. In addition, silver can also achieve antibacterial effects through oxidation, destruction of cell DNA replication and other ways, and is a widely used antibacterial metal.

[0042] Silver in all its forms has antimicrobial properties.

[0043] As an optional embodiment, the silver element in the antibacterial composite of the present invention mainly exists in the form of silver nanoparticles. The steps of preparing silver nanoparticles are: silver ions in the solution are reduced to silver atoms under the action of a reducing agent, the silver atoms begin to generate silver nuclei, the silver atoms continue to grow on the surface of the nuclei, and finally grow into silver particles of a certain particle size, which are called silver nanoparticles. However, due to the large specific surface area of ​​silver nanoparticles, they are easy to agglomerate and it is difficult to stably exist in the solution for a long time.

[0044] As another optional embodiment, the silver element in the antibacterial complex of the present invention exists mainly in the form of silver ions. Silver ions refer to ions formed when silver loses one or more electrons. The most common valence of silver is +1, and silver also has +2 and +3 valences. After the silver in the complex of the present invention is coordinated with the polymer / enzyme protein, it exists in the form of complexed silver ions, and more of them exist in +1 valence. The bactericidal effect and stability of silver ions are better than those of silver nanoparticles. This is because the negatively charged lipids on the bacterial cell membrane are more likely to capture positively charged silver ions through electrostatic attraction, thereby significantly improving the antibacterial properties of silver ions. However, free silver ions are easily reduced by light to form silver nanoparticles (AgNPs), which then react with oxygen or other oxidizing substances in the air to be oxidized to form AgNPs. 2 O and other precipitations make the antibacterial effect of the silver-containing antibacterial agent worse, and make the silver-containing antibacterial preparation and even the carrier sprayed with it turn yellow or black. This is also the reason why many silver-containing antibacterial materials, such as silver-containing antibacterial plastics and silver-containing antibacterial fabrics, turn yellow or black after being used for a period of time.

[0045] polymer

[0046] A polymer is a compound with a large molecular mass formed by many identical, simple structural units repeatedly linked by covalent bonds.

[0047] The polymer in the present invention is selected from polymers having amino, pyridyl, pyrrolyl, imidazole, thiazolyl, hydroxyl and the like groups on the side groups or main chains, such as one or more of xanthan gum, ε-polylysine, polyaspartic acid, Pluronic F127, polyether, polyvinyl alcohol, polyvinyl imidazole, polyethyleneimine and its derivatives, polyoxyethylene-polyoxypropylene copolymers, polyvinyl pyrrolidone and its derivatives.

[0048] The amino, pyridine, pyrrolyl, imidazole, thiazolyl, hydroxyl and other groups on the side groups or main chains of the polymer selected from the composite of the present invention can "capture" nano-metal particles. Since the molecular weight of the polymer itself is very large, it can provide a sufficient number of the above groups to fully combine the nano-metal particles and avoid the nano-metal particles from aggregating with each other to produce agglomeration and precipitation. Therefore, the polymer in the composite of the present invention can avoid the mutual agglomeration of nano-metals, thereby controlling the size of the nano-metal particles and fully ensuring the antibacterial effect of the nano-metal component. As an optional embodiment, when the antibacterial metal component in the antibacterial composite of the present invention is silver, compared with the technical means of forming silver-ammonia complex ions with ammonia water and silver nitrate to stabilize the silver element, the antibacterial composite of the present invention has no ammonia odor and is more suitable for use in daily life.

[0049] As a further optional embodiment, the polymer of the present invention is a cationic polymer containing multiple amino groups, such as polyethyleneimine, polyacrylamide, chitosan, ε-polylysine, polydopamine, etc. This type of polymer is selected because, on the one hand, it contains abundant amino groups and can fully capture and bind nano-metal particles. On the other hand, the amino groups in the polymer can bind protons in water to carry positive charges (also known as "protonation"), and can interact with bacterial cell membranes or cell walls to have a certain antibacterial effect.

[0050] Antimicrobial complex

[0051] The antibacterial complex of the present invention includes enzyme, metal and polymer. The above three components form an enzyme-metal-polymer ternary complex through self-assembly. There is sufficient "metal-nitrogen" coordination, "metal-oxygen" coordination and "metal-sulfur" coordination between the metal and the polymer / enzyme protein to form stable complexed metal nanoparticles. Figure 1 Shown is a microscopic embodiment of the complex of the present invention, in which the largest enzyme serves as a template or core, and the polymer serves as an intermediate structure connecting the enzyme and the nanometal particles. Specifically, the polymer is tightly connected to the enzyme surface to form a stable composite structure, and at the same time, the metal is bound to the side group or main chain group of the polymer.

[0052] Self-assembly refers to a way in which the components of a complex spontaneously form an ordered structure. In the process of self-assembly, the basic structural units spontaneously organize or aggregate into a stable structure with a certain regular geometric appearance under the interaction based on non-covalent bonds.

[0053] As an optional embodiment, the metal in the antibacterial composite of the present invention mainly exists in the form of metal ions, and the proportion of metal ions compared to all metal elements exceeds 50%. Taking metallic silver as an example, the silver ions in the antibacterial composite of the present invention coordinate with polymers / enzyme proteins to form a stable silver complex, and the silver ions in the silver complex are mainly +1 valence silver ions. The higher the silver valence, the better the bactericidal effect, so for the same amount of silver, silver ions have a higher bactericidal effect than silver nanoparticles.

[0054] In order to make the silver element in the final system mainly exist in the form of silver ions, in the step of preparing the antibacterial composite of the present invention, the order of adding the materials is controlled so that the raw material solution containing silver ions and the polymer raw material are added before the raw material enzyme, for example, the antibacterial composite is prepared in the order of adding polymer, silver-containing solution, enzyme or silver-containing solution, polymer, enzyme. By adopting such a raw material addition order, on the one hand, it is possible to avoid the reducing amino acids contained on the surface of the enzyme from reducing the silver ions to nano-silver, and on the other hand, since the silver element has coordinated with the polymer to form a complexed silver, the influence of silver on the enzyme conformation can be reduced, thereby ensuring that the enzyme activity is not affected or is less affected.

[0055] As another optional embodiment, the metal in the antibacterial composite of the present invention exists in the form of a metal element, and the proportion of the metal element to the total metal elements exceeds 50%. Taking silver as an example, the silver element in the composite of the present invention mainly exists in the form of a complex of nanoparticles (AgNPs), and the size of the silver particles is not greater than 5nm, and further, the size of the silver particles is not greater than 2nm. For silver nanoparticles (AgNPs), their antibacterial activity is size-dependent. The smaller the size of the silver nanoparticles, the larger the specific surface area of ​​the particles, and the easier it is to bind to bacteria, thereby showing higher antibacterial activity. In order to make the silver in the solution exist mainly in the form of silver nanoparticles, the order of adding the substances is controlled so that the raw enzyme is added before the raw solution containing silver ions, and the silver ions are reduced to nanosilver particles by utilizing the reducing properties of the enzyme.

[0056] The form of the metal element in the composite can be detected by X-ray photoelectron spectroscopy (XPS) or other methods.

[0057] Silver complex refers to a compound formed by silver ions and ligands, also known as silver compounds. Free silver ions are sensitive to light and oxygen, and silver ions are easily reduced by light to form silver nanoparticles (AgNPs). The silver nanoparticles formed in this way will further grow to form large particles and precipitate, and their chemical properties are relatively unstable, which will cause the nanosilver particles to be oxidized to produce Ag. 2 O, causing antibacterial preparations to turn yellow or black.

[0058] The silver (including silver ions and nano silver particles) used in the antibacterial composite of the present invention forms a ligand complex with a polymer / enzyme protein. Compared with silver ions in a free state and nano silver particles of uncontrollable size, the silver complex in the antibacterial composite of the present invention has stable chemical properties and a slow silver loss rate, making the antibacterial effect more durable. At the same time, since the silver is in a complexed state, the influence of silver on the conformation of the biological enzyme can be reduced, thereby ensuring the enzyme activity.

[0059] As an embodiment, the polymer in the complex of the present invention is selected from polyvinyl pyrrolidone (PVP), the antibacterial metal is selected from silver, and the enzyme is selected from lysozyme. The enzyme-metal-polymer ternary complex in this embodiment is referred to as Ag / LPV.

[0060] As another embodiment, the polymer in the complex of the present invention is selected from branched polyethyleneimine (BPEI), the antibacterial metal is selected from silver, and the enzyme is selected from lysozyme. The enzyme-metal-polymer ternary complex in this embodiment is referred to as Ag / LBP.

[0061] Figure 2 The test results of the interaction force between lysozyme and polymer are shown. The non-bonded force is mainly calculated by the crystal structure of the material components using explicit solvent all-atom molecular dynamics (MD) simulation. Other measurement or calculation methods can also be used. The results show that the interaction between enzymes, metals, and polymers is mainly based on van der Waals interactions and electrostatic interactions. Specifically, for a specific enzyme-metal-polymer complex, the ratio of the sum of the energy of van der Waals interactions and the energy of electrostatic interactions to the total interaction energy is defined as "non-bonded interaction ratio". For the complex of the present invention, the lower limit of the non-bonded interaction ratio is selected from 80%, 85%, 90%, and 95%. If the non-bonded interaction ratio of the complex is less than the above lower limit, then in the enzyme-metal-polymer ternary complex, the metal has a greater influence on the enzyme protein conformation, and the metal may destroy the enzyme active center, resulting in enzyme inactivation or reduced activity, thereby reducing the stability of the entire complex system, reducing the antibacterial effect and affecting the antibacterial persistence.

[0062] In addition, the interaction forces between the components in the composite of the present invention also include hydrogen bonding forces, hydrophobic forces, and the like.

[0063] In the antibacterial composite of the present invention, the mass fraction of the metal is 0.01% to 1%. As a further embodiment, the mass fraction of the metal is 0.1% to 0.8%. Furthermore, the mass fraction of the metal is 0.2% to 0.6%.

[0064] If the mass fraction of the metal exceeds the upper limit, in the solution for preparing the antibacterial complex, in addition to the complexed metal state, the free metal ions in the solution are easily reduced by light to form nano-metal particles of uncontrollable size, and the nano-metal particles further grow to form larger particles, and finally aggregate at the bottom of the system. In other words, the template effect of the polymer and enzyme protein cannot achieve sufficient coordination and binding with the excess metal, and the system is prone to discoloration and precipitation, affecting the application of the antibacterial complex. In addition, when the mass fraction of the metal is too high, it is easy to change the conformation of the enzyme protein and thus inactivate the enzyme, affecting the antibacterial effect of the system.

[0065] As an optional embodiment, the metal in the complex of the present invention is selected from silver, and the antibacterial complex of the present invention can be used after dilution. In the diluted solution, the minimum inhibitory concentration (MIC) of silver in the antibacterial complex of the present invention is 6ppm, 8ppm, 10ppm, 12ppm, and 15ppm. The minimum inhibitory concentration (MIC) refers to the lowest concentration of silver in a solution containing an antibacterial agent for inhibiting the growth of bacteria such as Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa in a suspension. In traditional silver-containing antibacterial agents, since silver has certain biological toxicity, but at the same time silver is the most important element that exerts antibacterial effects in this type of antibacterial agent, for this type of silver-containing antibacterial agent, there is a contradictory relationship between its antibacterial performance and cytotoxicity, that is, the stronger the antibacterial performance, the higher its cytotoxicity; if it is necessary to reduce cytotoxicity, the antibacterial performance will inevitably be sacrificed.

[0066] Since the antibacterial effect of the antibacterial complex of the present invention is not only exerted by metals, but also by enzymes and even optional polymers, the antibacterial complex of the present invention can still exert better antibacterial performance under the condition of low metal concentration. Taking silver as the metal as an implementation method, the antibacterial complex of the present invention is subjected to an in vitro cytotoxicity test to test the minimum bactericidal concentration of the antibacterial complex product of the present invention. The minimum bactericidal concentration (minimumbactericidal concentration, MBC) refers to the lowest concentration of the sample at which no bacteria grow on the bacterial growth plate after the bacteria and the sample have been incubated for a period of time. Human cervical cancer cells HeLa and mouse embryonic fibroblasts NIH3T3 were selected for the experiment. Figure 3As shown, when the concentration of the complex is less than 1 μg / mL, both HeLa and NIH3T3 cells can maintain a survival rate of more than 90%, which means that when the concentration of the complex is greater than 1 μg / mL, cytotoxicity will be generated. The minimum bactericidal concentration (MBC) of the antibacterial complex of the embodiment of the present invention is measured to be 0.07 μg / mL, which is nearly 15 times lower than the concentration that produces cytotoxicity (greater than 1 ug / mL). According to this test, it can be concluded that the minimum bactericidal concentration (MBC) of the antibacterial complex of the present invention is much lower than the concentration that produces cytotoxicity, which further proves that the antibacterial complex of the present invention has low biological toxicity.

[0067] In the antibacterial composite of the present invention, the mass fraction of the polymer is 1% to 10%. As a further embodiment, the mass fraction of the polymer is 2% to 7%. Furthermore, the mass fraction of the polymer is 3% to 6%.

[0068] The antibacterial complex of the present invention provides a certain amount of polymer, which can effectively regulate the interaction between metal and enzyme protein and the size of metal, and reduce the influence of the combination of enzyme and metal on the conformation and activity of the enzyme. At the same time, the mass fraction of the polymer in the antibacterial complex of the present invention is greater than the lower limit value and is in an excessive state. The "excessive state" referred to in the present invention refers to the state in which there are nitrogen-containing or oxygen-containing groups that have not reacted with silver ions on the polymer after the nitrogen atoms or oxygen atoms or two atoms provided by the side chains or main chain groups of the polymer in the system act simultaneously to completely complex the silver ions, which is also called the polymer excess state. For example, when branched polyethyleneimine (BPEI) is used as a polymer, after the silver ions and the N atoms of BPEI are coordinated and combined at a ratio of 1:3, the remaining amino groups are in an excessive state; after the silver ions and the N or O atoms of ε-polylysine are combined at a ratio of 1:2, the remaining nitrogen-containing or oxygen-containing groups are excessive; after the silver ions and the O atoms of PVP are combined at a ratio of 1:1, the remaining oxygen-containing groups are excessive. In an excess state, the number of groups such as amino, pyridyl, pyrrolyl, imidazole, thiazolyl, hydroxyl, etc. on the side groups or main chains of the polymer used to "capture" metal ions or single substances exceeds the number required by the metal mass fraction. Through such a composition ratio, the antibacterial composite of the present invention can be fully combined with the carrier component through the groups on the side chains or main chains of the polymer when used on the carrier, thereby improving the binding strength and the antibacterial long-term effect. The polymer content in the antibacterial composite of the present invention is less than the upper limit value, because if the polymer content is too high, on the one hand, it is difficult for the high concentration of polymer to be completely dissolved in the solvent, and on the other hand, the high concentration of polymer will also affect the pH value in the solution. The change in pH value will change the dissociation degree of amino acids in the enzyme protein, affecting the formation and stability of non-bonded interactions between ternary complexes in the system, thereby affecting the stability of the composite.

[0069] In the complex of the present invention, the mass fraction of the enzyme protein is 0.01% to 1%. As a further embodiment, the mass fraction of the enzyme protein is 0.03% to 0.7%. Further, the mass fraction of the enzyme protein is 0.05% to 0.5%.

[0070] If the enzyme protein concentration is less than the lower limit, the antibacterial effect of the enzyme protein cannot be fully exerted. If the enzyme protein concentration is too high, the non-bonded force between proteins and between complexes will change, making the particle size of the formed complex uneven, thereby increasing the aggregation tendency of the complex or causing aggregation, reducing the antibacterial effect.

[0071] As an optional embodiment, in order to make the metal in the complex exist mainly in the form of ions, the mass fraction of the enzyme protein is 0.01% to 0.2%. The enzyme surface generally contains reducing amino acids, which will accelerate the reduction of metal ions and cause the metal ions to form metal nanoparticles. The enzyme content is controlled within the above range, while ensuring the stability of the ternary complex, the metal in the ionic state is reduced to metal elements, so that the metal in the system exists mainly in the form of complexed metal ions, and an antibacterial complex with high activity, high stability and uniform particle size is synthesized.

[0072] Accordingly, as another optional embodiment, in order to make the metal in the complex exist mainly in the form of nanoparticles, the mass fraction of the enzyme protein is 0.2% to 1%. The reducing amino acids contained on the surface of a large number of enzyme proteins can play a reducing role, so that the metal in the extraction system exists mainly in the form of nanoparticles.

[0073] The solvent used in the complex of the present invention can be selected from inorganic solvents, such as Tris-HCl, PBS and HEPES, etc., and can also be selected from organic solvents, such as alcohols, esters, acids, etc. As an optional embodiment, the solvent used in the complex of the present invention is selected from water, which is the most common inorganic solvent, non-toxic, non-polluting, cheap, easily available, safe and environmentally friendly.

[0074] The antibacterial composite provided by the present invention has an average particle size of 5 nm to 500 nm. As a further embodiment, the average particle size of the antibacterial composite is 50 nm to 300 nm. Furthermore, the average particle size of the antibacterial composite is 80 nm to 200 nm. The particle size of the antibacterial composite can ensure that it has a highly effective antibacterial effect.

[0075] The antibacterial complex provided by the present invention uses enzyme molecules and high molecular polymers as stabilizers at the same time, and the enzyme protein confines the metal to its surface area, effectively regulating the size of metal particles and preventing the aggregation of metal particles. The antibacterial complex of the present invention also includes a polymer, which is combined with the enzyme and the metal respectively, further stabilizing the enzyme-metal binding effect, and the enzyme, metal and polymer are self-assembled through non-bonded interactions such as mainly van der Waals forces and electrostatic effects to form a stable ternary antibacterial complex. The enzyme-metal-polymer complex constructed with the enzyme protein and the polymer molecule as a template can maintain the conformation of the enzyme stable, while reducing the aggregation of metal particles, making the antibacterial effect of the complex more durable.

[0076] The product of the antimicrobial complex of the present invention can be in liquid or solid form. As an optional embodiment, the antimicrobial complex product of the present invention is in liquid form, and the antimicrobial complex in liquid form can be used by processes such as spraying, dipping and padding. The spraying process is a common surface treatment technology, which forms a uniform coating by spraying an appropriately diluted antimicrobial complex on the surface of an object to achieve the effect of antibacterial and deodorizing the surface of the object. The dipping process is to soak the antimicrobial complex solution for a period of time, and then naturally dry or bake to obtain an antibacterial fabric. The padding process is to place the object to be treated in the antimicrobial complex solution for dipping, and form an antibacterial coating on the fiber after high-temperature setting.

[0077] As another optional embodiment, the antibacterial composite product of the present invention is in solid form, such as antibacterial masterbatch. Specifically, after a certain amount of antibacterial masterbatch and corresponding resin particles are mixed, an antibacterial fiber with antibacterial effect is prepared according to the fiber processing and molding method.

[0078] The antimicrobial composite of the present invention can be physically adsorbed onto the surface of an object, or embedded into the interior of an object, such as being bonded to fabric fibers via chemical bonds or non-chemical bonds.

[0079] The antibacterial complex of the present invention adsorbs and tightly wraps pathogens through electrostatic and van der Waals force interactions. The metals, enzymes and even polymers as antibacterial components synergistically destroy cell walls or cell membranes, promote the metal to destroy the cell membrane and penetrate into the bacteria. The complex induces bacteria to produce oxidative stress reactions. Excessive reactive oxygen species (ROS) will damage bacterial lipids and proteins, causing internal disorders in the bacteria and ultimately causing bacterial death.

[0080] The two or more antibacterial components in the antibacterial complex of the present invention increase the destruction of bacteria and viruses through synergistic effect, thereby achieving the purpose of killing bacteria and viruses in a broad spectrum.

[0081] Antimicrobial fabric finishing

[0082] As one of the application scenarios of the antibacterial composite of the present invention, the antibacterial composite of the present invention can be applied to fabric finishing, so that the antibacterial composite is combined with fabric fibers to produce fabrics with antibacterial properties.

[0083] As an optional embodiment, the fabric can be selected from natural fibers, chemical fibers or composite fibers, such as plant fibers, animal fibers, cellulose fibers, protein fibers, polyester fibers, polyurethane fibers, polyamide fibers, polyacrylonitrile fibers, polyvinyl chloride fibers, polyolefin fibers and the like. The present invention does not limit the material of the fabric.

[0084] As an optional process for fabric finishing, the padding process generally refers to a process of soaking or wetting a fabric containing an antibacterial complex solution by roller squeezing. As an implementation method of the padding process of the present invention, first, an antibacterial finishing solution is prepared in a water trough, and then the fabric is passed through the water trough at a speed of 10 to 30 m / min, and then the fabric is squeezed by two rollers arranged opposite to each other, and at least one roller is heated at a temperature of 50 to 100°C. Next, an oven setting machine is used to dry and set the fabric at a temperature of 60 to 120°C, a speed of 10 to 30 m / min, and a time of 1 to 3 minutes. Using two rollers arranged opposite to each other to squeeze the wet fabric can increase the pressure, so that the antibacterial complex is fully combined with the fabric fibers, and the antibacterial effectiveness and long-term effect are improved. Heating the rollers can, on the one hand, make the antibacterial compound and the fabric fibers more tightly bonded. On the other hand, since the antibacterial compound of the present invention contains enzymes, in order to ensure that the enzymes can exert their antibacterial activity, the drying temperature needs to be controlled within a certain range. However, if the temperature is too low, the drying speed will be affected, reducing production efficiency. Therefore, heating the rollers while rolling can achieve a certain drying effect and improve the production efficiency of fabric finishing.

[0085] As another optional process for fabric finishing, the impregnation process generally refers to a process in which the fabric is immersed in an impregnation tank prepared with antibacterial fabrics, and then the fabric is taken out for drying and finalization. As a specific example of an embodiment, the impregnation process of the present invention immerses the fabric in an impregnation tank configured with the antibacterial finishing liquid of the antibacterial composite of the present invention at a bath ratio (ratio of the weight of the dry weight of the fabric to the antibacterial finishing liquid of the fabric) of 1:5 to 30. The impregnation tank is a sealed container, and the impregnation tank is evacuated to make the air pressure in the impregnation tank lower than the atmospheric pressure. The impregnation is carried out for a total of 10 to 60 minutes, and then the clothes are taken out and placed in a drying box for drying at a drying temperature of 60 to 100°C. Making the air pressure in the impregnation tank lower than the atmospheric pressure can allow the microbubbles in the fibers in the fabric to escape, allowing the solution configured with the antibacterial composite to fully infiltrate the fabric, thereby improving the utilization rate of the antibacterial composite.

[0086] The nanometer-scale size of the antibacterial composite of the present invention enables it to penetrate into the interior of the fabric fiber more evenly, thereby improving the binding force with the fabric fiber. At the same time, the hydroxyl, amino, carboxyl and other groups provided by the enzyme protein in the antibacterial composite of the present invention are connected with the fabric fiber through covalent bonds, electrostatic effects and van der Waals forces, so that a uniform antibacterial film layer is formed on the fabric, thereby improving the binding strength between the antibacterial composite and the fabric fiber, and while ensuring the antibacterial performance, the loss of antibacterial components is reduced, making the antibacterial performance more long-lasting.

[0087] In particular, an excess of polymer is provided in the antibacterial composite component of the present invention. The long-chain dispersing effect of the polymer, on the one hand, makes the enzyme and the antibacterial metal bind more tightly. On the other hand, the groups provided by the excess polymer can form a good match with the fabric fibers, thereby improving the bonding strength between the antibacterial composite and the fabric and enabling the antibacterial composite to be evenly dispersed on the fabric.

[0088] Examples of implementation of antimicrobial complexes

[0089] Implementation Example 1:

[0090] Under light-proof conditions, weigh 5g of polyvinyl pyrrolidone (PVP) and add it to a flask, and add 32.5mL of ultrapure water, and stir and dissolve at a rate of 300rpm at 25°C; weigh 0.32g of silver nitrate and dissolve it in 50mL of ultrapure water and sonicate it. The concentration during dissolution is 6.4mg / mL, and the drop rate is controlled to 0.5mL~1mL / min. Stir while adding. After the drop addition is completed, continue the reaction under stirring for 30min; add 4.6mg / mL lysozyme solution to the flask 17.5mL, stirring while adding; using a pH adjuster to adjust the pH of the system to 6.0-8.0, and then stirring the mixed solution at a rate of 300rpm at a reaction temperature of 45°C for 3 hours to prepare an enzyme-silver-polymer ternary complex (abbreviated as: Ag / LPV), the concentration of polyvinyl pyrrolidone (PVP) in the final system is 49.8mg / mL, the concentration of lysozyme is 0.8mg / mL, and the concentration of silver element is 2.1mg / mL (mainly in the form of silver ions). The calculated non-bonded interaction ratio is 90%.

[0091] Implementation Example 2:

[0092] Under light-proof conditions, weigh 5g of branched polyethyleneimine (BPEI) and add it to a flask, and add 32.5mL of ultrapure water, and stir and dissolve at a rate of 300rpm at 25°C; weigh 0.32g of silver nitrate and dissolve it in 50mL of ultrapure water and sonicate it. The concentration during dissolution is 6.4mg / mL, and the drop rate is controlled to 0.5mL~1mL / min. Stir while adding. After the drop addition is completed, continue the reaction under stirring for 30min; add 4.6mg / mL lysozyme solution to the flask 17.5mL, stirring while adding; using a pH adjuster to adjust the pH of the system to 6.0-8.0, and then stirring the mixed solution at a rate of 300rpm at a reaction temperature of 45°C for 3 hours to prepare an enzyme-silver-polymer ternary complex (abbreviated as: Ag / LBP), the concentration of branched polyethyleneimine (BPEI) in the final system is 50.6mg / mL, the concentration of lysozyme is 0.8mg / mL, and the concentration of silver element is 2.0mg / mL (mainly in the form of silver ions). The calculated non-bonded interaction ratio is 92%.

[0093] Implementation Example 3:

[0094] Under light-proof conditions, 5 g of polyvinyl pyrrolidone (PVP) was weighed and added to a flask, and 32.5 mL of ultrapure water was added, and the mixture was stirred at 300 rpm at 25 °C to dissolve; 0.32 g of silver nitrate was weighed and dissolved in 50 mL of ultrapure water and ultrasonicated to a concentration of 6.4 mg / mL during dissolution, and the dropwise addition rate was controlled to 0.5 mL to 1 mL / min, with stirring while adding. After the dropwise addition was completed, the reaction was continued for 30 min under stirring; 4.6 mg / mL laccase solution was added to the flask for 1 h. 7.5mL, stirring while adding; using a pH adjuster to adjust the pH of the system to 6.0-8.0, and then stirring the mixed solution at a rate of 300rpm at a reaction temperature of 45°C for 3 hours to prepare an enzyme-silver-polymer ternary complex (abbreviated as: Ag / LaPV), the concentration of polyvinyl pyrrolidone (PVP) in the final system is 49.9mg / mL, the concentration of laccase is 0.8mg / mL, and the concentration of silver element is 2.2mg / mL (mainly in the form of silver ions). The calculated non-bonded interaction ratio is 82%.

[0095] Implementation Example 4:

[0096] Under light-proof conditions, weigh 5g of polyvinyl pyrrolidone (PVP) and add it to a flask, and add 32.5mL of ultrapure water, and stir and dissolve at a rate of 300rpm at 25°C; weigh 0.32g of silver nitrate and dissolve it in 50mL of ultrapure water and sonicate it. The concentration during dissolution is 6.4mg / mL, and the drop rate is controlled to 0.5mL~1mL / min. Stir while adding. After the drop addition is completed, continue the reaction under stirring for 30min; add 4.6mg / mL horseradish peroxidase solution to the flask 17.5mL, stirring while adding; using a pH adjuster to adjust the pH of the system to 6.0-8.0, and then stirring the mixed solution at a rate of 300rpm at a reaction temperature of 45°C for 3 hours to prepare an enzyme-silver-polymer ternary complex (abbreviated as: Ag / HPV), the concentration of polyvinyl pyrrolidone (PVP) in the final system is 49.7mg / mL, the concentration of horseradish peroxidase is 0.8mg / mL, and the concentration of silver element is 2.1mg / mL (mainly in the form of silver ions). The calculated non-bonded interaction ratio is 84%.

[0097] Implementation Example 5:

[0098] Under light-proof conditions, weigh 5 g of ε-polylysine (ε-PL) and add it to a flask, and add 32.5 mL of ultrapure water, and stir and dissolve at 300 rpm at 25 ° C; add 17.5 mL of 4.6 mg / mL lysozyme solution to the flask, stirring while adding; weigh 0.32 g of silver nitrate and dissolve it in 50 mL of ultrapure water and ultrasonicate it. The concentration during dissolution is 6.4 mg / mL, and the drop speed is controlled to 0.5 mL to 1 mL / min, stirring while adding. After the drop addition is completed, under the stirring condition, The reaction was continued for 30 minutes under the conditions; the pH of the system was adjusted to 6.0-8.0 using a pH adjuster, and then the mixed solution was stirred at a rate of 300 rpm and a reaction temperature of 45°C for 3 hours to prepare an enzyme-silver-polymer ternary complex (abbreviated as: Ag / LPL). The ε-polylysine (ε-PL) concentration in the final system was 49.5 mg / mL, the lysozyme concentration was 0.8 mg / mL, and the silver element concentration was 2.0 mg / mL (mainly in the form of nanosilver particles). The calculated non-bonded interaction ratio was 80%.

[0099] Implementation Example 6:

[0100] Under light-proof conditions, weigh 5 g of polydopamine (PDA) and add it to a flask, and add 32.5 mL of ultrapure water, and stir and dissolve at 300 rpm at 25 ° C; add 17.5 mL of 4.6 mg / mL lysozyme solution to the flask, stirring while adding; weigh 0.32 g of silver nitrate and dissolve it in 50 mL of ultrapure water and ultrasonicate it. The concentration during dissolution is 6.4 mg / mL, and the drop speed is controlled to 0.5 mL to 1 mL / min, stirring while adding. After the drop addition is completed, under the stirring condition, The reaction was continued for 30 minutes under the conditions; the pH of the system was adjusted to 6.0-8.0 using a pH adjuster, and then the mixed solution was stirred at a reaction temperature of 45°C at a rate of 300 rpm for 3 hours to prepare an enzyme-silver-polymer ternary complex (abbreviated as: Ag / LPD). The concentration of polydopamine (PDA) in the final system was 49.4 mg / mL, the concentration of lysozyme was 0.8 mg / mL, and the concentration of silver element was 2.1 mg / mL (mainly in the form of nanosilver particles). The calculated non-bonded interaction ratio was 91%.

[0101] Comparative Example 7 (excess metal):

[0102] Under light-proof conditions, 5 g of polyvinyl pyrrolidone (PVP) was weighed and added to a flask, and 32.5 mL of ultrapure water was added, and the mixture was stirred and dissolved at a rate of 300 rpm at 25°C; 1.75 g of silver nitrate was weighed and dissolved in 50 mL of ultrapure water and ultrasonicated, and the concentration during dissolution was 6.4 mg / mL, and the dropping speed was controlled to be 0.5 mL-1 mL / min, and stirring was performed while adding. After the dropwise addition was completed, the reaction was continued for 30 minutes under stirring conditions; 17.5 mL of 4.6 mg / mL lysozyme solution was added to the flask, and stirring was performed while adding; a pH regulator was used to adjust the pH of the system to 6.0-8.0, and then the mixed solution was stirred and reacted at a rate of 300 rpm at a reaction temperature of 45°C for 3 hours to prepare an enzyme-silver-polymer ternary complex (abbreviated as: Ag / LPV), and the concentration of polyvinyl pyrrolidone (PVP) in the final system was 49.8 mg / mL, the concentration of lysozyme was 0.7 mg / mL, and the concentration of silver element was 11.6 mg / mL.

[0103] Comparative Example 8 (excess enzyme):

[0104] Under light-proof conditions, 5 g of polyvinyl pyrrolidone (PVP) was weighed and added to a flask, and 32.5 mL of ultrapure water was added, and the mixture was stirred and dissolved at a rate of 300 rpm at 25°C; 0.32 g of silver nitrate was weighed and dissolved in 50 mL of ultrapure water and ultrasonicated, and the concentration during dissolution was 6.4 mg / mL, and the dropwise addition speed was controlled to be 0.5 mL-1 mL / min, and stirring was performed while adding. After the dropwise addition was completed, the reaction was continued for 30 min under stirring conditions; 17.5 mL of 68.6 mg / mL lysozyme solution was added to the flask, and stirring was performed while adding; a pH regulator was used to adjust the pH of the system to 6.0-8.0, and then the mixed solution was stirred and reacted at a rate of 300 rpm at a reaction temperature of 45°C for 3 hours to prepare an enzyme-silver-polymer ternary complex (abbreviated as: Ag / LPV), and the concentration of polyvinyl pyrrolidone (PVP) in the final system was 49.9 mg / mL, the concentration of lysozyme was 12.2 mg / mL, and the concentration of silver element was 2.0 mg / mL.

[0105] Comparative Example 9 (excess polymer):

[0106] Under light-proof conditions, 12 g of polyvinyl pyrrolidone (PVP) was weighed and added to a flask, and 32.5 mL of ultrapure water was added, and the mixture was stirred and dissolved at a rate of 300 rpm at 25°C; 0.32 g of silver nitrate was weighed and dissolved in 50 mL of ultrapure water and ultrasonicated, and the concentration during dissolution was 6.4 mg / mL, and the dropwise addition speed was controlled to be 0.5 mL-1 mL / min, and stirring was performed while adding. After the dropwise addition was completed, the reaction was continued for 30 min under stirring conditions; 17.5 mL of 4.6 mg / mL lysozyme solution was added to the flask, and stirring was performed while adding; a pH regulator was used to adjust the pH of the system to 6.0-8.0, and then the mixed solution was stirred and reacted at a rate of 300 rpm at a reaction temperature of 45°C for 3 hours to prepare an enzyme-silver-polymer ternary complex (abbreviated as: Ag / LPV), and the concentration of polyvinyl pyrrolidone (PVP) in the final system was 122.7 mg / mL, the concentration of lysozyme was 0.8 mg / mL, and the concentration of silver element was 2.0 mg / mL.

[0107] Comparative Example 10 (no polymer):

[0108] Under light-proof conditions, 0.32 g of silver nitrate was weighed and added to a flask, and 82.5 mL of ultrapure water was added to dissolve and ultrasonicated, and the concentration during dissolution was 3.87 mg / mL; 17.5 mL of 4.6 mg / mL lysozyme solution was added to the flask while stirring; a pH adjuster was used to adjust the pH of the system to 6.0-8.0, and then the mixed solution was stirred at a reaction temperature of 45°C at a rate of 300 rpm for 3 hours, and the lysozyme concentration in the final system was 0.8 mg / mL and the silver element concentration was 2.2 mg / mL.

[0109] Comparative Example 11 (no enzyme):

[0110] Under light-proof conditions, 5 g of polyvinyl pyrrolidone (PVP) was weighed and added to a flask, and 32.5 mL of ultrapure water was added, and the mixture was stirred and dissolved at a rate of 300 rpm at 25 ° C; 0.32 g of silver nitrate was weighed and dissolved in 50 mL of ultrapure water and ultrasonicated, and the concentration during dissolution was 6.4 mg / mL, and the dropping speed was controlled to 0.5 mL-1 mL / min, and stirring was performed while adding. After the dropwise addition was completed, the reaction was continued for 30 minutes under stirring conditions; 17.5 mL of ultrapure water was added to the flask, and stirring was performed while adding; a pH adjuster was used to adjust the pH of the system to 6.0-8.0, and then the mixed solution was stirred and reacted at a rate of 300 rpm at a reaction temperature of 45 ° C for 3 hours. The concentration of polyvinyl pyrrolidone (PVP) in the final system was 49.6 mg / mL, and the concentration of silver element was 2.1 mg / mL.

[0111] Comparative Example 12 (silver-free):

[0112] Under light-proof conditions, 5 g of polyvinyl pyrrolidone (PVP) was weighed and added to a flask, and 82.5 mL of ultrapure water was added, and the mixture was stirred at 300 rpm at 25 ° C to dissolve; 17.5 mL of 4.6 mg / mL lysozyme solution was added to the flask while stirring; a pH adjuster was used to adjust the pH of the system to 6.0-8.0, and then the mixed solution was stirred at 300 rpm at a reaction temperature of 45 ° C for 3 hours. The concentration of polyvinyl pyrrolidone (PVP) in the final system was 49.8 mg / mL, and the concentration of lysozyme was 0.8 mg / mL.

[0113] As an optional embodiment, the preparation of the antibacterial composite of the present invention is carried out under light-proof conditions. The solution containing silver ions is easily reduced under light, especially ultraviolet light, resulting in large deviations in the existence form and mass fraction of the silver element that actually participates in the formation of the composite in the solution, making the quality of the final antibacterial product uncontrollable.

[0114] As an optional embodiment, the lower limit of the time of stirring the reaction at a rate of 300rpm after adding enzyme, metal and polymer to the reaction system is 2 hours, 2.5 hours, 3 hours and 3.5 hours, and the upper limit of the reaction time is 4 hours, 4.5 hours and 5.5 hours. If the reaction time is less than the lower limit, the enzyme, metal and polymer cannot be fully combined to form a stable and effective complex. If the reaction time exceeds the upper limit, the metal is oxidized in contact with air, resulting in yellowing and discoloration of the complex product, and affecting the antibacterial performance.

[0115] Enzyme activity test

[0116] For Examples 1 to 6 and Comparative Examples 7 to 9, the lysozyme activity was determined by turbidimetry:

[0117] Preparation of substrate suspension: Weigh 7.5 mg of Micrococcus lysodeikticus, add a little phosphate buffer, grind in a mortar for 3 minutes, and then add an appropriate amount of phosphate buffer to make the total volume about 25 mL. The absorbance of the suspension measured at 25°C ± 0.1°C at a wavelength of 450nm is 0.70 ± 0.05. Place the enzyme solution and substrate suspension in a 25°C water bath at 25°C for 10-15 minutes, draw 950 μL of substrate into a cuvette, add 50 μL of enzyme solution and mix evenly. Use a UV spectrophotometer to measure the dynamic changes of absorbance at 450nm within 30 seconds of the reaction. Take the enzyme activity of the single, unmixed lysozyme as a reference, set the reference value as 100%, and calculate the relative enzyme activity of the above test object relative to the single lysozyme.

[0118] Implementation Example 3 Laccase enzyme activity determination:

[0119] 7.48 mg of ABTS and 30 mL of 10 mM phosphate buffer (pH 7.4) were added to a 50 mL beaker to prepare a substrate solution. 950 μL of substrate solution and 50 μL of enzyme solution were added to a cuvette and mixed evenly, and the change in absorbance of the reaction system at 420 nm was measured using an ultraviolet spectrophotometer. The relative enzyme activity of the laccase in the complex of Example 3 relative to the free laccase was calculated with the enzyme activity of the free laccase alone and unmixed as a reference, and the reference value was set as 100%.

[0120] Implementation Example 4 Horseradish peroxidase activity assay:

[0121] (1) Prepare solution A: 0.1mol / L pH7.0 phosphate buffer; solution B: 20mmol / L guaiacol aqueous solution; solution C: 8mmol / L hydrogen peroxide aqueous solution. All reagents and sample solutions were incubated in a constant temperature box at 25℃±2℃ for 30min before measurement. (2) In a 1cm cuvette, add 2.8mL solution A, 0.1mL solution B, 0.05mL solution C and 0.05mL enzyme solution in sequence, mix quickly, and use a UV spectrophotometer to measure the change in absorbance of the reaction system at 436nm. Using the enzyme activity of the single, unmixed free horseradish peroxidase as a reference, assuming the reference value to be 100%, calculate the relative enzyme activity of the horseradish peroxidase in the complex of Example 4 relative to the free enzyme.

[0122] Table 1 Relative enzyme activity test results of the examples and comparative examples

[0123] Serial number Non-bonded force ratio Relative enzyme activity Implementation example (1) 90％ 87％ Implementation Example (2) 92％ 91％ Implementation Example (3) 82％ 84％ Implementation Example (4) 84％ 87％ Implementation Example (5) 80％ 81％ Implementation Example (6) 91％ 89％ Comparative Example (7) 90％ 65％ Comparative Example (6) 90％ 73％ Comparative Example (6) 90％ 86％

[0124] According to the above test data, using the material ratio of enzyme, metal and polymer of the present invention, the metal has less effect on the enzyme conformation, and the relative enzyme activity can be maintained at a higher value (>80%). If the content of enzyme and metal is too high, the impact on the relative enzyme activity is greater. In addition, the higher the proportion of non-bonded forces, the smaller the effect of metal on the enzyme conformation.

[0125] Antibacterial performance test

[0126] According to WS / T 650-2019 5.2.1 suspension quantitative bactericidal test, the antibacterial effect of the examples and comparative examples was evaluated. The experimental steps are as follows:

[0127] (1) Take a 24-hour fresh slant culture of the test bacteria (Escherichia coli) and wash it with PBS. Dilute it to 5.0x10 5 CFU / mL~4.5x10 6 CFU / mL bacterial suspension is ready for use;

[0128] (2) Take a sterile test tube, first add 5.0 mL of the antibacterial diluent (silver concentration is 12 ppm, except for Comparative Example 12) diluted by the same multiple, place it in a 20°C ± 1°C water bath for 5 minutes, then add 0.1 mL of the test bacterial suspension, quickly mix and immediately start timing. The reaction time between Escherichia coli and the antibacterial diluent is 20 minutes;

[0129] (3) Take 0.5 mL of the mixture of E. coli and antibacterial diluent and add it to 4.5 mL of neutralizer and mix well;

[0130] (4) After the mixture of the test bacteria and the antibacterial diluent in each tube was treated with the neutralizer for 10 min, 1.0 mL of the sample solution was taken out from each tube and the number of surviving bacteria was determined by the live bacteria culture counting method. After each tube of sample solution was diluted to an appropriate multiple, two plates were inoculated;

[0131] (5) At the same time, PBS was used instead of antibacterial diluent as a positive control group;

[0132] (6) All experimental and control groups were cultured at 36°C±1°C, and E. coli was cultured for 48 h and the final results were observed.

[0133] (7) Calculation of sterilization rate

[0134] Sterilization rate (%) = (AB) / A×100%

[0135] Where:

[0136] A—The amount of bacteria recovered in the positive control group, in CFU / mL;

[0137] B—The amount of bacteria recovered in the test group, in CFU / mL.

[0138] Table 2 Sterilization rate test results of the examples and comparative examples

[0139]

[0140] According to the above test data, the present invention uses enzyme, metal and polymer complexes for bactericidal and antibacterial effects, which can achieve a bactericidal effect of more than 99% and has excellent bactericidal performance.

[0141] Antibacterial long-term effect test

[0142] Refer to the continuous antibacterial experiment of "Antibacterial and Antibacterial Effect Evaluation Method" WS / T 650-2019 to test the long-term antibacterial performance of the compound. Specifically including:

[0143] (1) The examples and comparative examples were diluted by the same multiples to prepare a dilution solution with a silver concentration of 50 μg / mL (except comparative example 12), and 0.5 mL of the dilution solution was evenly applied to the surface of a 5 cm×5 cm PE plastic plate sample, and dried at room temperature to prepare an antibacterial sample, which was used as the experimental group; and the sample without any treatment was used as the control group;

[0144] (2) The antibacterial performance of the experimental and control samples was tested after being placed at room temperature for 0 days and 60 days. Fresh Escherichia coli suspension was added to the samples to ensure that the contamination level was 1.25×10 7 cfu / sample~1.25×10 8 cfu / sample, after the plastic samples of the experimental group and the control group were exposed to E. coli for 20 minutes, samples were taken and diluted, and spread on the plate. Incubated in a 37℃ constant temperature incubator for 18-24 hours, the formation of colonies was observed, and the long-term bactericidal rate of the experimental group and the control group was calculated.

[0145] (3) Calculation of sterilization rate

[0146] Sterilization rate (%) = (AB) / A×100%

[0147] Where:

[0148] A—The amount of bacteria recovered from the control group samples, in CFU / sample;

[0149] B—The amount of bacteria recovered from the experimental group samples, in CFU / sample.

[0150] Table 3 Bactericidal long-term test results of examples and comparative examples

[0151]

[0152] The results of the sustained antibacterial experiment of the antibacterial compound show that the antibacterial compound of the present invention has a sterilization rate of ≥90% within 60 days and has a sustained antibacterial effect within this period of time.

[0153] Preparation of antibacterial finishing liquid and preparation of antibacterial fabric

[0154] The antibacterial compound of Examples 1 to 6 and Comparative Examples 7 to 12 is used to prepare a fabric antibacterial finishing liquid. 5 parts of the antibacterial compound are taken, diluted with water to make the total number of the fabric antibacterial finishing working liquid 1000 parts, stirred and mixed evenly for use. Then, according to a bath ratio (ratio of the dry weight of the fabric to the antibacterial finishing liquid of the fabric) of 1:10, a polyester fabric (polyester fiber) is added to the antibacterial finishing liquid at room temperature and immersed for 20 to 30 minutes, and the excess antibacterial finishing liquid is squeezed out to make the liquid carrying rate of the fabric 80% to 100%, and then the fabric is dried at 80°C to obtain an antibacterial fabric.

[0155] Fabric antibacterial performance test

[0156] According to the standard "T / SZTIA 001-2020 Antibacterial Fibers and Textiles", the antibacterial composites prepared in the examples and comparative examples were used for antibacterial finishing of fabrics, and then the 5A antibacterial level test was performed. The fabrics were washed in a household double-tub washing machine in accordance with GB / T 20944.3-2008 Evaluation of antibacterial properties of textiles Part 3: Oscillation method, washed for 20 cycles (equivalent to 100 washes) and dried, and the test strains of Escherichia coli, Staphylococcus aureus, and Candida albicans were selected for antibacterial effect testing, and the results are as follows:

[0157]

[0158] According to the above test data, the fabric treated with the antibacterial composite of the present invention still reaches the 5A antibacterial grade after being washed 100 times.

[0159] Dissolution testing of antimicrobial compounds

[0160] The fabrics treated with the antibacterial compounds of the examples and comparative examples were tested for dissolution of the antibacterial compounds according to the halo method in Appendix E of FZ / T73023-2006. The dissolution index of the antibacterial substances used in antibacterial knitwear is: after the antibacterial fabric is washed once, the width of the inhibition zone D ≤ 5mm. The antibacterial fabric samples after washing once were tested, and the results are as follows:

[0161]

[0162] According to the test results, the antibacterial composite of the present invention, as an antibacterial substance of antibacterial knitwear, meets the dissolution index of antibacterial knitwear, and the antibacterial fabric is judged to be a non-dissolution type antibacterial fabric (inhibition zone width D≤1mm).

[0163] Stability test of antibacterial fabrics

[0164] With reference to the stability test method in Appendix C of GB 15979-2002 National Standard of the People's Republic of China for Hygiene Standards of Disposable Sanitary Products, the antibacterial composites prepared in the examples and comparative examples were used for antibacterial finishing of fabrics, and then the antibacterial fabrics were stored in a constant temperature box at 37±0.5℃ and kept at a relative humidity of >75% for 3 months. The samples were subjected to an accelerated test at 37℃, and then the antibacterial fabrics were subjected to a 5A antibacterial level test according to the standard "T / SZTIA 001-2020 Antibacterial Fibers and Textiles". The fabrics were washed in a household double-barrel washing machine in accordance with GB / T 20944.3-2008 Evaluation of Antibacterial Properties of Textiles Part 3: Oscillation Method, washed for 20 cycles (equivalent to 100 washes) and dried, and the test strains of Escherichia coli, Staphylococcus aureus, and Candida albicans were selected for antibacterial effect tests, and the results are as follows:

[0165]

[0166] According to the test results, the 5A antibacterial grade of the antibacterial fabric is maintained at room temperature for at least two years, and the antibacterial fabric has excellent antibacterial durability. In the comparative examples (Comparative Examples 7 and 10) in which the polymer is not excessive compared to the metal, the antibacterial performance is significantly attenuated after being stored for 3 months or after being washed 100 times. In addition, during the stability test, the antibacterial composite of the present invention is used for fabrics, and the antibacterial fabrics maintain their original color, while the antibacterial fabrics prepared in Comparative Examples 7 and 10 have yellowing. The possible reason is that the silver ions in the comparative composites fail to obtain sufficient complexation, and the silver ions react with substances such as oxygen, hydrogen sulfide, and sulfur ions in the air, causing the fabric to turn yellow and gradually black.

[0167] The present invention is not limited to the specific structure and arrangement shown. As long as a similar technical solution to the present invention is adopted and a similar effect can be achieved, it should be considered to belong to the protection scope of the present invention.

Claims

1. An antibacterial complex, comprising an enzyme, a metal, and a polymer, wherein the metal exists in the form of metal ions and metal nanoparticles, and the proportion of the metal nanoparticles in all metal elements exceeds 50%.

2. The antimicrobial compound according to claim 1, Features: The polymer is selected from one or more polymers having amino, pyridyl, pyrrolyl, imidazole, thiazolyl, hydroxyl and the like groups on the side groups or main chains.

3. The antibacterial compound according to claim 2, Features: The polymer is selected from cationic polymers containing polyamino groups.

4. The antimicrobial compound according to claim 1, Features: The metal is a metal with antibacterial effect, and the metal with antibacterial effect is selected from one or more of copper, zinc, silver, aluminum, vanadium, gold, tin, nickel, etc.; The metal forms a ligand complex with the polymer / enzyme.

5. The antimicrobial compound according to claim 4, Features: The metal is selected from silver.

6. The antimicrobial compound according to claim 5, Features: The enzyme, metal and polymer form coordination through self-assembly, and one of the coordination modes includes: the polymer is connected to the surface of the enzyme, and the metal is connected to the surface of the polymer and / or the enzyme.

7. The antimicrobial compound according to claim 5, Features: The mass fraction of metal in the antibacterial composite is 0.2% to 0.6%.

8. The antimicrobial compound according to claim 6, Features: The mass fraction of the polymer in the antibacterial composite is 1% to 10%; The number of groups provided on the side groups or main chain of the polymer exceeds the number of metal atoms.

9. The antimicrobial compound according to claim 8, Features: The mass fraction of the polymer in the antibacterial composite is 3% to 6%.

10. The antimicrobial compound according to claim 6, Features: The mass fraction of the enzyme in the antibacterial complex is 0.2% to 1%.

11. The antimicrobial composite according to any one of claims 1 to 10, Features: The average particle size of the antibacterial complex is 80-200 nm.

12. A method for preparing the antimicrobial compound according to any one of claims 1 to 10, Features: The method comprises: The raw material solution containing metal ions is added after the raw material enzyme is added.

13. A method for finishing fabrics using the antimicrobial compound according to any one of claims 1 to 10, Features: The method comprises: The fabric moistened with the antibacterial complex solution is squeezed by using two rollers arranged opposite to each other, and at least one of the rollers on both sides is heated, and the temperature of the rollers is 50-100°C.

14. A method for finishing fabrics using the antimicrobial compound according to any one of claims 1 to 10, Features: The method comprises: The fabric is immersed in an immersion tank configured with the antibacterial composite finishing liquid, wherein the immersion tank is a sealed container, and the immersion tank is evacuated to make the air pressure in the immersion tank lower than the atmospheric pressure.

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