CN109336124B

CN109336124B - Modified clay and preparation method and application thereof

Info

Publication number: CN109336124B
Application number: CN201811325799.6A
Authority: CN
Inventors: 夏庆银; 董海良; 王曦
Original assignee: China University of Geosciences Beijing
Current assignee: China University of Geosciences Beijing
Priority date: 2018-11-08
Filing date: 2018-11-08
Publication date: 2021-01-05
Anticipated expiration: 2038-11-08
Also published as: CN109336124A

Abstract

The invention discloses a modified clay, a preparation method and application, wherein the modified clay is obtained by mixing natural clay containing iron with an intercalation agent and a reducing agent in sequence; also discloses a sterilization composition and a preparation method thereof, the main component of the sterilization composition is modified clay, and the sterilization composition also comprises a buffering agent for maintaining the pH value of the composition. The modified clay has broad-spectrum antibacterial performance, is mild to skin, and has good sterilization effect and high efficiency; the preparation method of the modified clay has the advantages of simple operation, easily controlled conditions and low requirement on equipment.

Description

Modified clay and preparation method and application thereof

Technical Field

The invention belongs to the field of preparation of wound sterilization materials, and particularly relates to modified clay and a preparation method and application thereof.

Background

The main approaches to skin infection treatment are topical or oral administration of various antibiotics, such as ticarcillin-clavulanic acid (ticarcillin-clavulanic acid), ceftazidime (ceftazidime), minocycline (minocycline), levofloxacin (levoflloxacin), chloramphenicol (chloramphenicol) beta-lactams (beta-lactams), quinolones (quinolones), aminoglycosides (aminoglycosides), tetracyclines (tetracyclines), etc. (paracyanine, xujin. quinolone antibiotics's progress in skin infectious diseases [ J ] world medicine, 2007 (543) 545; zhan, lie, wang. traditional Chinese medicine for external application to treat skin and soft tissue infection-related research [ J ] jejun, 2011.69-72. Antibiotic bactericidal materials mainly act on specific substances and structural compositions in pathogenic bacteria cells to block the synthesis of specific structures, for example, beta-lactam antibiotics can block the synthesis of cell wall mucopeptide to cause the cell wall defect (Wang Xiaoling. beta-lactam antibiotics are used in clinical rational application [ J ]. Chinese sanitation, 2003(12): 63-63); or block the synthesis of specific intracellular active components, such as tetracycline antibiotics specifically bind to the A position of the bacterial ribosome 30S subunit, prevent the connection of aminoacyl-tRNA at that position, thereby inhibiting the growth of peptide chain and affecting the synthesis of bacterial protein (Zhangjun. pathogenic bacteria resistance mechanism to antibacterial drugs [ J ]. Natl. Acad. Med. antibiotic Manual, 2002,23(5): 206-). 214.).

Antibiotic bactericidal material acts on specific intracellular components or structures, so that the applicability of the antibiotic bactericidal material is generally limited, and related research shows that various antibiotics have certain side effects on organisms, for example, tetracycline antibiotics have toxic property on liver and viscera of human bodies and have adverse effects on development of teeth and bones (Xunian flowers, forest life, Pajie, and the like. Along with the wide application of antibiotics, a large number of resistant strains and super bacteria emerge continuously, the existing antibiotics are more and more insufficient, the research and development period of new antibiotics is extremely long, the manufacturing cost is high, and the defect of great antibiotic medicines is highlighted.

In recent years, related scholars find that natural clay can be used as a substitute of an antibacterial material, and existing clay minerals collected in nature are directly applied to affected parts, so that a certain antibacterial effect is achieved. However, natural clay is used for sterilization, and the following problems are involved:

(1) the sterilization effect is poor, the dosage is large, and the application amount is more than 90g/L to play the sterilization role;

(2) the sterilization needs longer time and low efficiency, and generally the affected part can be cured after about 2 months of application;

(3) the effective bacteriostasis time is short, and is generally within 24 hours;

(4) the sterilization pH is less than 4 or more than 9, so that secondary damage to human skin is easily caused;

(5) has no broad-spectrum bactericidal performance and extremely limited bactericidal effect on partial strains (such as staphylococcus aureus).

Therefore, there is a need to provide a clay with high-efficiency broad-spectrum antibacterial property, mild property to skin, no side effect, long antibacterial time and low cost.

Disclosure of Invention

In order to overcome the above problems, the present inventors have conducted intensive studies and, as a result, found that: sequentially carrying out intercalation and reduction modification on the iron-containing natural clay to obtain modified clay with high-efficiency broad-spectrum antibacterial performance under a near-neutral condition (pH is 6-8); meanwhile, the buffering agent is added into the clay, so that the optimum sterilization pH value of the system can be maintained, and the sterilization efficiency is improved, thereby completing the invention.

Specifically, the present invention aims to provide the following:

in a first aspect, there is provided a modified clay, wherein the modified clay is obtained by modifying an iron-containing natural clay by intercalation with an intercalating agent and reducing with a reducing agent.

In a second aspect, there is provided a germicidal composition comprising the modified clay of the first aspect, and further comprising a buffering agent,

the buffer is one or more of MES, PIPES, HEPES, Bis-Tris, MOPS, Tricine, TEA or disodium hydrogen phosphate-sodium dihydrogen phosphate buffer.

In a third aspect, there is provided a process for preparing the modified clay of the first aspect, wherein the process comprises the steps of:

step 1, mixing iron-containing natural clay with an intercalation agent to obtain intercalation modified clay;

and 2, mixing the intercalation modified clay prepared in the step 1 with a reducing agent to obtain the modified clay.

In a fourth aspect, a method for preparing the bactericidal composition of the second aspect is provided, wherein the method comprises the following steps:

step I, cleaning modified clay;

step II, preparing mother liquor of the modified clay, and adjusting the pH value;

and III, mixing the modified clay mother liquor prepared in the step II with a buffering agent to prepare the bactericidal composition.

In a fifth aspect, there is provided a use of the modified clay of the first aspect in the preparation of a biocidal material.

The invention has the advantages that:

(1) the modified clay provided by the invention has the advantages of cheap and easily available raw materials, no allergen and universality;

(2) the modified clay provided by the invention acts on cardiolipin generally existing on the surface of prokaryotic bacteria, has a spectrum bacteriostasis effect on the prokaryotic bacteria, and does not generate a chemical toxic effect on eukaryotic organisms;

(3) the modified clay provided by the invention plays a role under a near-neutral condition, is mild to skin and cannot generate secondary damage;

(4) the modified clay provided by the invention has a positively charged surface, is mutually attracted with bacteria, and has good sterilization effect and high efficiency;

(5) according to the modified clay provided by the invention, the organic intercalation agent is adopted for intercalation, so that the reduction rate of iron in the clay structure is greatly improved, the oxidation rate of ferrous iron is reduced, and the bacteriostasis time is prolonged;

(6) the modified clay provided by the invention adopts a chemical reducing agent, so that the reduction degree of divalent iron in the structure in the clay is improved, and the sterilization efficiency of the clay is improved;

(7) the sterilization composition provided by the invention can maintain the moderate and most suitable sterilization pH value of the system to the organism in the using process, and has high sterilization efficiency;

(8) the preparation method of the modified clay provided by the invention is simple to operate, easy to control conditions, low in equipment requirement and especially suitable for being applied to areas with poor medical conditions.

Drawings

Fig. 1 shows XRD patterns of samples prepared in example 1 of the present invention, comparative examples 1, 9, 12 and 15;

FIG. 2 shows results of killing Escherichia coli by the samples prepared in examples 1 to 3 of the present invention and comparative examples 1 to 3;

FIG. 3 shows the results of killing Escherichia coli by the samples prepared in examples 1,4 and 5 of the present invention and comparative examples 1,4, 5, 9 to 14;

FIG. 4 is a graph showing comparison of E.coli-killing ability and efficiency of samples prepared in example 1 of the present invention and comparative example 1;

FIG. 5 shows the results of killing stenotrophomonas maltophilia of the samples prepared in examples 5 to 7 of the present invention and comparative examples 5 to 7;

FIG. 6 shows the results of killing E.coli in 48 hours for the samples prepared in example 8 of the present invention and comparative example 8.

Detailed Description

The present invention will be described in further detail below with reference to preferred embodiments and examples. The features and advantages of the present invention will become more apparent from the description.

The word "exemplary" is used exclusively herein to mean "serving as an example, embodiment, or illustration. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.

In a first aspect, the present invention provides a modified clay obtained by modifying an iron-containing natural clay by intercalation with an intercalating agent and reducing with a reducing agent.

According to a preferred embodiment of the invention, the iron-containing natural clay is selected from one or more of the group consisting of nontronite, montmorillonite, illite, chlorite, mica, rectorite, and illite smectite layered minerals.

In a further preferred embodiment, the iron-containing natural clay is selected from one or more of nontronite, montmorillonite, illite, or chlorite, preferably nontronite.

In a still further preferred embodiment, the particle size of the iron-containing natural clay is less than 5 μm, preferably less than 3 μm, more preferably less than 2 μm.

According to a preferred embodiment of the invention, the intercalating agent is an organic intercalating agent and/or an inorganic intercalating agent, preferably an organic intercalating agent.

The inventor of the invention researches and discovers that the surfaces of natural clay containing iron are negatively charged, and the surfaces of bacteria are also negatively charged, so that the clay and the bacteria cannot be in close contact with each other due to the action of coulomb repulsion force when the clay is used for sterilization; and active oxygen generated by oxidizing structural iron in the clay is extremely unstable and is quenched, so that bacteria and the clay repel each other, the action difficulty of the active oxygen is increased, and the sterilization efficiency is reduced.

Therefore, in the invention, the natural clay containing iron is preferably modified by using the intercalation agent, so that the surface of the modified clay is positively charged, and can be tightly combined with bacteria with negatively charged surfaces, and the sterilization capability and efficiency of the intercalated modified clay are greatly improved.

In a further preferred embodiment, the organic intercalating agent is selected from one or more of chitosan, dimethyl sulfoxide, a silane coupling agent, acrylamide, cetyltrimethylammonium bromide, sodium dodecylsulfonate, polydiallyldimethylammonium chloride or octadecyltrimethylammonium chloride. In a further preferred embodiment, the organic intercalant is selected from one or more of chitosan, silane coupling agents, acrylamide, cetyltrimethylammonium bromide, sodium dodecylsulfonate.

In a still further preferred embodiment, the organic intercalant is selected from one or more of chitosan, acrylamide or cetyltrimethylammonium bromide, such as chitosan.

The inventor researches and finds that the chitosan is cheap and easy to obtain and has higher cost performance; the intercalation operation by using the method is simple, and the requirement on equipment is low; the success rate of intercalation is high and the sterilization effect of the obtained modified clay is good. Therefore, in the present invention, chitosan is preferably selected as the organic intercalating agent.

According to a preferred embodiment of the present invention, the inorganic intercalant is an inorganic metal salt solution, preferably selected from one or more of iron, calcium, magnesium, aluminum, zinc, potassium or sodium metal salt solutions. In a further preferred embodiment, the inorganic metal salt solution is selected from one or more of iron, calcium, magnesium or aluminium metal salt solutions.

The inventor researches and discovers that the following differences are mainly found between the intercalation by using the organic intercalation agent and the intercalation by using the inorganic intercalation agent: (1) the period of the inorganic intercalation is longer, generally 10 days, and the period of the organic intercalation is only 1-2 days; (2) the inorganic intercalation needs high-temperature calcination, the requirement on equipment is high, the preparation method is complex and the safety is low; (3) the success rate of inorganic intercalation is lower than that of organic intercalation; (4) the sterilization effect of the modified clay after the inorganic intercalation is poorer than that of the organic intercalation. Therefore, in the present invention, it is preferable to perform intercalation modification using an organic intercalant.

According to a preferred embodiment of the invention, the weight ratio of the intercalation agent to the iron-containing natural clay is (0.05-30): 1, preferably (0.08-25): 1, more preferably (0.1 to 20): 1.

the inventor finds that when the weight ratio of the intercalation agent to the iron-containing natural clay is less than 0.05:1, the electronegativity of the iron-containing natural clay cannot be completely twisted, the Zeta potential is still negative, and during sterilization, repulsive force still exists between bacteria and clay, so that the sterilization effect is not obviously improved; when the weight ratio of the intercalating agent to the natural clay containing iron is more than 30:1, the intercalation modification is saturated, the Zeta potential is not improved along with the increase of the intercalation dosage any more, the sterilization effect is not obviously improved, and the proportion of the intercalating agent is increased, so that only the resource is wasted.

According to a preferred embodiment of the invention, the reducing agent is a chemical reducing agent and/or a biological reducing agent, preferably a chemical reducing agent.

The inventor of the invention discovers that when the ferrous iron with the structure is oxidized under the mild regulation of near-neutral skin, a large amount of Reactive Oxygen Species (ROS) can be released, and the Reactive Oxygen Species (ROS) even taking hydroxyl free radicals (. OH) as the active oxygen species attack cells, oxidize lipid molecules on the surface of cell membranes, expand the permeability of the cell membranes, further enable metal ions (Fe) in clay to enter the cells and catalyze intracellular O₂And H produced by cellular respiration₂O₂And the like, and active oxygen is continuously generated to further attack intracellular components and structures, thereby killing bacteria.

However, iron oxides and iron-containing clay minerals widely distributed in the natural deposition environment mainly exist in a valence state of fe (iii), and therefore, in the present invention, it is preferable to perform a reduction treatment on the intercalated clay so as to reduce trivalent iron in the clay structure to divalent iron, thereby improving the sterilization efficiency.

The present inventors have also found that the reductive iron-containing clay is not toxic to eukaryotes because active oxygen acts mainly by attacking cardiolipin on cell membranes, whereas the cardiolipin of eukaryotes is mainly distributed in mitochondria, stored intracellularly, and cannot act as external active oxygen.

In a further preferred embodiment, the chemical reducing agent is a strong reducing agent, preferably selected from one or more of sodium dithionite, sodium hypophosphite, vitamin C, hydrazine hydrate or sodium bisulfite.

In a still further preferred embodiment, the chemical reducing agent is one or more of sodium dithionite, sodium hypophosphite or sodium bisulfite, preferably sodium dithionite.

According to a preferred embodiment of the present invention, the bioreductive agent is Iron Reducing Bacteria (IRB), such that the structural trivalent iron is reduced by the action of a catalyst with sodium lactate as a carbon source.

Wherein the catalyst is an electron shuttle (AQDS).

The inventor finds that the reduction rate of the structural iron in the clay reduced by the chemical reducing agent is obviously higher than that of the biological reducing agent. Therefore, in the present invention, it is preferable to reduce trivalent iron in clay with a chemical reducing agent.

According to a preferred embodiment of the present invention, the weight ratio of the clay after intercalation modification to the reducing agent is 1: (2-6), preferably 1: (3-5), more preferably 1: 4.

the inventor researches and discovers that the modified clay after intercalation and reduction has good bactericidal effect under near-neutral conditions and is mild to the skin.

In a second aspect, the present invention provides a germicidal composition comprising the modified clay of the first aspect, and further comprising a buffering agent.

The present inventors have found that the modified clay has a good bactericidal effect under near-neutral skin temperature conditions and does not cause secondary damage to living skin, and therefore, in the present invention, it is preferable to add a buffer to the bactericidal composition in order to prevent sweat and the like from affecting the pH of the system while maintaining the optimum pH for sterilization by the modified clay.

According to a preferred embodiment of the present invention, the pH of the germicidal composition is maintained at 6-8 by adjusting the pH with a buffer.

The inventor researches and discovers that the pH value of the sterilization composition is kept between 6 and 8 under the regulation of a buffering agent, ferrous iron in the modified clay can be promoted to be oxidized, active oxygen is released, and the sterilization effect is improved.

In a further preferred embodiment, the buffer is one or more of MES, PIPES, HEPES, Bis-Tris, MOPS, Tricine, TEA or disodium hydrogen phosphate-sodium dihydrogen phosphate buffer.

Wherein, the buffer is a common buffer in the market.

In a still further preferred embodiment, the buffer is one or more of MES, PIPES, or HEPES.

Wherein the buffering pH range of MES (2- (N-morpholino) ethanesulfonic acid) is 5.5-6.7, the buffering pH range of PIPES (piperazine-1, 4-diethylsulfonic acid) is 6.1-7.5, and the buffering pH range of HEPES (4-hydroxyethyl piperazine ethanesulfonic acid) is 6.8-8.2. In the invention, different buffers can be selected to adjust the pH according to the actual sterilization needs.

Preferably, the buffer is MES.

In the invention, the bactericidal effect of the bactericidal composition is mainly ferrous iron (Fe (II)) in the structure of the modified clay, and the content of the modified clay is generally calculated by the content of the ferrous iron.

According to a preferred embodiment of the present invention, in the bactericidal composition, the molar ratio of the modified clay (calculated as ferrous iron) to the buffer is (0.5-10): 5, preferably (2-6): 5, more preferably 4: 5.

wherein the molar ratio of the modified clay (calculated by ferrous iron) to the buffering agent is (0.5-10): 5, preferably (2-6): 5, more preferably 4:5, namely: the molar ratio of the ferrous iron (Fe (II)) to the buffer is (0.5-10): 5, preferably (2-6): 5, more preferably 4: 5. for example: the modified clay in the bactericidal composition contains 4 mol of ferrous iron (Fe (II)) and 5 mol of buffering agent.

In a further preferred embodiment, the concentration of divalent iron in the modified clay is greater than 0.08mM, preferably greater than 0.1mM, during use of the germicidal composition;

the concentration of the buffer is greater than 0.8mM, preferably greater than 1 mM.

In a still further preferred embodiment, the modified clay has a divalent iron concentration of 4mM and the buffer has a concentration of 5mM during use of the germicidal composition.

In a third aspect, the present invention provides a process for preparing a modified clay according to the first aspect, comprising the steps of:

step 1, mixing the natural clay containing iron with an intercalation agent to obtain the intercalation modified clay.

According to a preferred embodiment of the present invention, when the intercalating agent is an organic intercalating agent, the intercalation comprises the following steps:

step 1-1, preparing a solution of natural clay containing iron and organic intercalation agent with a certain granularity.

According to a preferred embodiment of the present invention, the particle size of the prepared iron-containing natural clay is less than 5 μm, preferably less than 3 μm, and more preferably less than 2 μm.

The preparation method of the iron-containing natural clay with certain granularity mainly comprises the following steps:

(1) grinding the iron-containing natural clay with a mortar to particles having a particle size of 200 mesh (<74 μm);

(2) taking ground natural clay containing iron, and soaking in NaCl solution for 24h to make the concentration of the natural clay containing iron be 50 g/L;

(3) uniformly stirring the clay and NaCl solution mixed system in the step (2) by using a magnetic stirrer, wherein ultrasonic and stirring methods (stirring for 1 hour every 2 hours of ultrasonic) can be simultaneously adopted;

(4) centrifuging the uniformly mixed clay solution at 4000G for 5min, discarding the supernatant, cleaning, adding Milli-Q Water (ultrapure Water prepared by a Milli-Q system) to constant volume, and centrifuging at 50G for 6min to obtain the supernatant, namely clay particles smaller than 2 μm;

(5) packaging the centrifuged supernatant into dialysis bags, sealing, adding a certain amount of Milli-Q water into a large beaker, and dialyzing the packaged and sealed dialysis bags in the beaker to remove residuesRemaining Cl^-Dialyzing for 12-18h, and replacing Milli-Q Water in the beaker every 2-3 h;

(6) by AgNO₃Solution detection of Cl^-Whether the removal is complete or not;

(7) filling the dialyzed clay into 50ml centrifuge tubes, freezing at-80 deg.C, removing the cover, wrapping the centrifuge tube with sealing film and rubber band, and freeze-drying with a freeze dryer.

According to a preferred embodiment of the present invention, the organic intercalating agent is selected from one or more of chitosan, dimethyl sulfoxide, a silane coupling agent, acrylamide, cetyltrimethylammonium bromide, sodium dodecylsulfate, polydiallyldimethylammonium chloride or octadecyltrimethylammonium chloride.

In a further preferred embodiment, the organic intercalant is selected from one or more of chitosan, a silane coupling agent, acrylamide, cetyltrimethylammonium bromide or sodium dodecyl sulphate.

Preferably, the organic intercalant is selected from one or more of chitosan, acrylamide or cetyltrimethylammonium bromide, such as chitosan.

Step 1-2, mixing the organic intercalation agent solution with the natural clay containing iron according to a preset proportion.

According to a preferred embodiment of the present invention, the predetermined ratio is (0.05-30) of the weight ratio of the organic intercalant solution to the iron-containing natural clay: 1, preferably (0.08-25): 1, more preferably (0.1 to 20): 1, such as 1: 1.

And (1) oscillating and washing the mixed system in the step (1-2).

According to a preferred embodiment of the present invention, the mixed system is shaken for 20 to 30 hours, preferably 22 to 26 hours, such as 24 hours.

In a further preferred embodiment, the washing is washing the intercalated clay with water, preferably with deionized water.

In a further preferred embodiment, the number of washing is 2 to 5, preferably 3.

According to another preferred embodiment of the present invention, when the intercalating agent is an inorganic intercalating agent, the intercalation comprises the steps of:

and step i, hydrolyzing the inorganic metal salt solution.

According to a preferred embodiment of the present invention, the inorganic metal salt solution is selected from one or more of iron, calcium, magnesium, aluminum, zinc, potassium or sodium metal salt solutions.

In a further preferred embodiment, the inorganic metal salt solution is selected from one or more of iron, calcium, magnesium or aluminium metal salt solutions.

In the invention, the inorganic metal salt solution to be intercalated needs to be fully hydrolyzed, preferably, oscillation hydrolysis dissociation is carried out, and the oscillation dissociation time is 6-10 days, preferably 8 days.

And ii, mixing the hydrolyzed inorganic metal salt with the natural clay containing iron.

In the invention, the dissociated inorganic metal salt solution is mixed with the iron-containing clay, and the weight ratio of the inorganic metal salt solution to the iron-containing clay is (0.05-30): 1, preferably (0.08-25): 1, more preferably (0.1 to 20): 1.

and iii, drying the mixed system in the step ii, and then performing burning treatment.

Wherein, the drying is freeze drying treatment, and the burning is carried out in a muffle furnace.

According to a preferred embodiment of the present invention, the burning temperature is 400 to 600 ℃, preferably 450 to 550 ℃, and more preferably 500 ℃.

In a further preferred embodiment, the burning time is 1 to 3 hours, preferably 1.5 to 2.5 hours, and more preferably 2 hours.

In the invention, the intercalating agent can also be an inorganic organic mixed intercalating agent, and preferably, the clay intercalated by an inorganic metal salt solution is mixed with organic intercalating agents with different concentrations for intercalation.

In the invention, the clay modified by intercalation can slow down the oxidation rate of ferrous iron and effectively prolong the sterilization time.

The inventor of the invention discovers that the effective reduction rate (ferrous concentration/total iron concentration) of the intercalated clay is obviously improved compared with that of the non-intercalated clay when the intercalated clay is reduced under the same reduction treatment condition.

According to a preferred embodiment of the present invention, the reducing agent is a chemical reducing agent selected from one or more of sodium dithionite, sodium hypophosphite, vitamin C, hydrazine hydrate or sodium bisulfite.

Preferably, the chemical reducing agent is one or more of sodium dithionite, sodium hypophosphite or sodium bisulfite, preferably sodium dithionite.

According to a preferred embodiment of the present invention, the intercalated clay obtained in step 1 is mixed with a chemical reducing agent for reaction, wherein the weight ratio of the two is 1: (2-6), preferably 1: (3-5), more preferably 1: 4.

in the invention, before the reducing agent is mixed with the clay after intercalation modification, the clay after intercalation is prepared into suspension. Preferably, the intercalated clay is dissolved in CB (citric acid-sodium bicarbonate, sodium citrate) buffer solution, and ultrasonic stirring is carried out to prepare clay suspension.

In a further preferred embodiment, the mixing reaction is carried out under a water bath condition, and the temperature of the water bath is 90-100 ℃, preferably 100 ℃.

Preferably, the water bath time is 15-25 min.

In a still further preferred embodiment, the intercalated clay is reduced with a chemical reducing agent under inert gas or nitrogen atmosphere conditions.

Wherein, inert gas or nitrogen is firstly introduced under the condition of water bath, and after a certain time (15-20 min), a chemical reducing agent is added into the clay solution, so that the weight ratio of the clay in the solution to the chemical reducing agent is 1: (2-6), preferably 1: (3-5), more preferably 1: 4; then continuously introducing inert gas or nitrogen for 5min, and placing the mixture in a closed container to react for 24h at the constant temperature of 60 ℃.

Wherein, the reduced clay needs to be washed by deionized water under anaerobic condition, preferably by the deionized water with oxygen removed in an anaerobic box.

In a fourth aspect, the present invention provides a method for preparing the bactericidal composition according to the second aspect, the method comprising the following steps:

step I, cleaning the modified clay.

The cleaning is to clean the prepared modified clay by using deionized water, and the cleaning frequency is 2-4 times, preferably 3 times.

And II, adjusting the pH value of the modified clay.

In the invention, the undiluted modified clay after cleaning is mother liquor of the modified clay, and the pH of the mother liquor is adjusted to the pH required by the optimal sterilization effect, such as 6-8, by adopting acid and alkali.

Wherein the formulating is performed under anaerobic conditions to prevent oxidation of the reduced clay, which affects the ferrous content.

And III, mixing the modified clay adjusted in the step II with a buffering agent to prepare the bactericidal composition.

According to a preferred embodiment of the invention, the buffering agent and the modified clay are mixed according to a predetermined ratio, wherein the molar ratio of the modified clay to the buffering agent is (0.5-10): 5, preferably (2-6): 5, more preferably 4: 5.

wherein the pH of the buffer is adjusted to the same range as the modified clay prior to mixing.

In a further preferred embodiment, the divalent iron is used in the modified clay at a concentration of more than 0.08mM, preferably more than 0.1 mM;

the buffer is used in a concentration of more than 0.8mM, preferably more than 1 mM.

According to a preferred embodiment of the present invention, the modified clay and the buffering agent are mixed under anaerobic conditions with shaking, and the mixing time is 10 to 50min, preferably 20 to 40min, and more preferably 30 min.

In a further preferred embodiment, the mixed system is freeze-dried under vacuum to prepare the bactericidal composition.

In the present invention, the prepared bactericidal composition is preferably vacuum-sealed and packaged (filled with nitrogen or inert gas), and when in use, the bactericidal composition is added with water to form a paste, and then the paste is applied to the surface of a wound.

In a fifth aspect, the present invention provides a use of the modified clay of the first aspect for the preparation of a biocidal material.

Examples

The present invention is further described below by way of specific examples, which are merely exemplary and do not limit the scope of the present invention in any way.

Example 1

(1) Iron-rich nontronite (NAu-2) having a particle size of <2 μm was prepared according to the following procedure:

(1.1) grinding nontronite with an agate mortar to particles having a particle size of 200 mesh (<74 μm);

(1.2) soaking 30g of nontronite in 600ml of NaCl solution (concentration 500 mM; 29.22g/L) for 24h (ensuring that the clay concentration is 50 g/L);

(1.3) adding a rotor into a conical flask filled with clay and NaCl liquid, and rotationally stirring in a magnetic stirrer for 24 hours; for uniform mixing, ultrasonic and stirring methods (every 2h, stirring 1h) can be adopted at the same time until the clay and the NaCl solution are uniformly mixed;

(1.4) subpackaging the uniformly mixed clay solution into 50ml centrifuge tubes, centrifuging for 5min at 4000G, and pouring off the supernatant; after the clay is cleaned, the volume is regulated to 35ml by Milli-Q Water in a 50ml centrifuge tube, the centrifugation is carried out for 6min at 50G, and the supernatant is clay particles with the particle size less than 2 mu m;

(1.5) subpackaging the centrifuged supernatant into a dialysis bag, sealing, placing, adding a certain amount of Milli-Q water into a large beaker, and placing the subpackaged and sealed dialysis bag into the beaker for dialysis to remove residual Cl^-Dialyzing for 12-18h, and replacing Milli-Q Water in the beaker every 2-3 h;

(1.6) AgNO₃Solution detection of Cl^-Whether the removal is complete or not;

(1.7) filling the dialyzed clay into 50ml centrifuge tubes, freezing and storing at-80 ℃, removing covers, wrapping the centrifuge tube openings with sealing films and rubber bands, and freeze-drying by using a freeze dryer.

Meanwhile, 5g of chitosan is weighed and dissolved in 100ml of 2% acetic acid to prepare chitosan mother liquor.

(2) According to the weight ratio of 1:1 mixing the chitosan mother liquor and the iron-rich nontronite, fully shaking and mixing for 24 hours, and washing the obtained intercalated clay for 3 times by using deionized water.

(3) Dissolving 1g of intercalated clay in 100ml of CB (citric acid-sodium bicarbonate, sodium citrate) buffer solution for 3 times (ultrasonic + stirring, 15min each) to prepare 10g/L clay suspension, and placing the clay suspension in a reaction container; introducing nitrogen for 20min under the condition of water bath at 100 ℃, then adding 4g of sodium hydrosulfite, continuously introducing the nitrogen for 5min, sealing the reactor, placing the reactor in a 60 ℃ drying oven for reacting for 24h to obtain the intercalated and reduced modified clay, and washing the reduced clay in an anaerobic box with deoxidized deionized water for three times;

detecting the concentration of ferrous iron and total iron in the modified clay, and the steps are as follows:

(3.1) 200. mu.L of reduced clay (undiluted stock solution) was added to 480. mu.L of 3.6mol/L H₂SO₄Performing the following steps;

(3.2) adding 80 μ L of 10% 1, 10-o-phenanthroline solution (1g of 1, 10-o-phenanthroline solid is dissolved in 10ml of 95% ethanol) and 40 μ L of 48% HF solution into the system;

(3.3) carrying out water bath at 100 ℃ for 30 min;

(3.4) Cooling at room temperature for 15min and adding 400. mu.L of 5% H₃BO₃(5g boric acid solids dissolved in 100mL deionized water);

(3.5) after uniformly mixing, adding 0.1mL of the mixed solution of the system into 1mL of 1% sodium citrate, reacting for 5min, and reading an OD value at 510nm to obtain the concentration of ferrous iron; adding 0.1mL of the mixed solution of the system into 1mL of 10% hydroxylamine hydrochloride, reacting for 12h, and reading an OD value at 510nm to obtain the total iron concentration;

wherein, the reduction degree is bivalent iron concentration/total iron concentration is 75%;

among them, the general formula (K) based on nontronite_0.01Na_0.30Ca_0.15)(Al_0.55Fe_3.53Mg_0.12)(Si_7.57Al_0.15Fe_0.48)O₂₀(OH)₄1g/L of nontronite contained about 5mM of total iron, and the concentration of the above reduced clay was 10g/L, and the concentration of divalent iron in the modified clay was about 37.5mM in terms of the degree of reduction of 75%.

(4) Adjusting the pH of the reduced modified clay to 6, then shaking and mixing the modified clay (calculated by ferrous iron) and MES (the pH is adjusted to 6 in advance) in an anaerobic operation box according to a molar ratio of 3:5 for 30min, and carrying out freeze drying treatment under a vacuum condition to obtain the bactericidal composition powder.

Examples 2 to 3

Examples 2 to 3 were carried out in a similar manner to example 1 except that the buffer solutions in (4) were PIPES and HEPES, respectively, and the pH of the adjusted solutions were 7 and 8, respectively.

Examples 4 to 8

Examples 4 to 8 were carried out in a similar manner to example 1 except that (4) the modified clay (in terms of ferrous iron) and MES (pH adjusted to 6 beforehand) were mixed in a molar ratio of 1.5:5, 2:5, 4:5, 6:5 and 10:5, respectively.

Comparative example

Comparative example 1

This comparative example was carried out in a similar manner to example 1, except that the clay was directly subjected to a reduction treatment without intercalation modification.

Comparative examples 2 to 3

Comparative examples 2-3 used methods similar to comparative example 1 except that the buffer solutions were PIPES and HEPES, respectively, and the adjusted solutions had pH's of 7 and 8, respectively.

Comparative examples 4 to 8

Comparative examples 4 to 8 were similar to comparative example 1 except that (4) the non-intercalated, only reduced clay (in terms of ferrous iron) and MES (pH adjusted to 6 in advance) were mixed in molar ratios of 1.5:5, 2:5, 4:5, 6:5 and 10:5, respectively.

Comparative example 9

This comparative example is similar to example 1 except that the clay has been intercalated and not reduced.

Comparative examples 10 to 11

Comparative examples 10 to 11 were similar to comparative example 9 except that only the intercalated, unreduced clay (in terms of ferrous iron) and MES (pH adjusted to 6 in advance) were mixed in the bactericidal composition powder in (4) at a molar ratio of 1.5:5 and 2:5, respectively.

Comparative examples 12 to 14

The clay used in comparative examples 12-14 was nontronite that had not been intercalated and reductively modified.

Comparative example 15

This comparative example was carried out in a similar manner to example 1, except that the reducing agent used was a bioreductive agent, iron-reducing bacteria (CN-32), which was reduced by the action of the electron shuttle AQDS using sodium lactate as a carbon source.

Examples of the experiments

Experimental example 1

And (3) detection of Zeta potential:

the clays of example 1 and comparative examples 1, 9 and 12 were each diluted to the following concentrations at room temperature and pH 6: the concentration of divalent iron in the intercalated, reductively-modified clay (rC-NAu-2) in example 1 was 0.3mM, and the total iron concentration was 0.4 mM; the divalent iron concentration in the reduced only non-intercalated clay (rNAu-2) of comparative example 1 was 0.3mM, and the total iron concentration was 0.46 mM; the total iron concentration in the clay intercalated with only unreduced clay (C-NAu-2) in comparative example 9 was 0.4 mM; the non-intercalated, reductively-modified clay (NAu-2) of comparative example 12 had a total iron concentration of 0.46 mM; each system contained 5mM MES, and then each suspension and the clay solution in comparative example 15 were subjected to zeta potential analysis (ZetapALS; Brookheaven Instruments) to determine the potential value thereof and calculate the reduction degree of divalent iron, the results of which are shown in Table 1.

TABLE 1

Diluting a sample	Zeta potential	Degree of reduction
			Example 1	3.47	75％
Comparative example 1	-44.57	65％
			Comparative example 9	16.525	-
Comparative example 12	-33.98	-
			Comparative example 15	-9.23	23％

Wherein, the reduction degree is bivalent iron concentration/total iron concentration × 100%; "-" indicates that the degree of reduction was 0, i.e., no reduction occurred.

As can be seen from table 1, the surface charges of the intercalated and modified clays of example 1 and comparative example 9 are both positive, and the degree of reduction of the clays also increased (from 65% to 75%) with the intercalation of chitosan, indicating that the same divalent iron concentration can be achieved with less raw material (earth).

And as can be seen from table 1, the degree of reduction of divalent iron in the clay obtained by the biological reduction method is low, only 23%, which is significantly lower than that of the chemical reducing agent.

Further, XRD analysis was performed on the above diluted sample, and as a result, as shown in FIG. 1, it can be seen from FIG. 1 and Table 1 that the interlayer distance of sample C-NAu-2(1.81nm) in comparative example 9 and sample rC-NAu-2(1.46nm) in example 1 was relatively increased with respect to those of comparative example 12NAu-2(1.27nm) and sample rNAu-2(1.25nm) in comparative example 1. While the corresponding Zeta potential values were also increased from-33.98 (comparative example 12NAu-2) and-44.57 (comparative example 1rNAu-2) to 16.525 (comparative example 9C-NAu-2) and3.47 (example 1 rC-NAu-2).

Experimental example 2

The samples used in the experimental examples are the bactericidal composition powder samples described in examples 1-3 and comparative examples 1-3, and water and the concentration of the bactericidal composition powder sample are 10⁸CFU/ml of escherichia coli (e.coli) suspension so that the concentration of ferrous iron in the mixed system was 3mM and the concentrations of MES, PIPES, and HEPES were all 5 mM.

After mixing for 24h, the pH changes before and after the reaction were less than 0.1, the results of the sterilization of the samples under different pH conditions (6, 7, 8) were compared, and no sterilization sample was added to the control group, as shown in FIG. 2.

As can be seen from FIG. 2, the samples prepared in examples 1 to 3 had superior bactericidal effects to those of the samples in comparative examples 1 to 3 under the same pH condition.

The samples prepared in examples 1 to 3 and comparative examples 1 to 3 all had the best bactericidal effect under the condition of pH 6. The effective sterilization pH range of the samples prepared in the embodiments 1-3 is wide, and about 90% (1 order of magnitude) of escherichia coli can be killed even under the conditions that the pH is 7 and 8; whereas the samples prepared in comparative examples 1 to 3 could not be sterilized at pH 7 and 8.

Experimental example 3

The samples used in the present experimental examples were the samples prepared in examples 1,4 and 5 and comparative examples 1,4, 5 and 9 to 14, and water was added to the samples prepared in examples 1,4 and 5 and comparative examples 1,4, 5 and 9 to 14 to give a concentration of 10⁸CFU/ml Escherichia coli (E.coli) bacterial liquid (wherein MES buffer solution is added in comparative examples 12-14) is adopted, so that the concentration of ferrous iron in the mixed system is respectively 1.5mM (example 4, comparative examples 4, 10 and 12), 2mM (example 5, comparative examples 5, 11 and 13) and 3mM (example 1, comparative examples 1, 9 and 14), and the concentration of MES in each sample is 5 mM.

After mixing for 24h, the pH change before and after the reaction was less than 0.1, and the results of the sterilization of the samples at pH6 were compared, wherein the control group was not supplemented with the sterilization sample, and the results are shown in FIG. 3.

As can be seen from FIG. 3, the intercalated, unreduced clays prepared in comparative examples 9-11 had no bactericidal activity.

Under the same divalent iron condition, the bactericidal effect of the clays prepared in examples 1,4 and 5 is significantly better than that of the clays prepared in comparative examples 1,4 and 5 which are not intercalated and only reduced; wherein 3mM ferrous iron can be 10 in 24h⁸CFU/ml of E.coli was completely killed.

Experimental example 4

The samples used in this experimental example were the germicidal composition powders prepared in example 1 and comparative example 1, to which water was added in a concentration of 10%⁸CFU/ml of E.coli (E.coli) suspension, so that the concentration of ferrous iron in the mixed system was 3mM and the concentration of MES was 5 mM.

After mixing for 24h, the pH change before and after the reaction was less than 0.1, and the bactericidal activity of comparative example 1 and comparative example 1 was shown in fig. 4.

As can be seen from fig. 4, the sample prepared in example 1 has significantly higher bactericidal power and efficiency than the sample prepared in comparative example 1.

Experimental example 5

The samples used in the experimental examples are the bactericidal combinations prepared in examples 5-7 and comparative examples 5-7Adding water to the powder at a concentration of 10%⁸CFU/ml of a Stenotrophomonas maltophilia (S. maltophilia) solution so that the concentrations of divalent iron in the mixed system were 2mM (example 5 and comparative example 5), 4mM (example 6 and comparative example 6), 6mM (example 7 and comparative example 7), and 5mM of MES, respectively.

After mixing for 24h, the pH change before and after the reaction was less than 0.1, and the bactericidal effect of the comparative sample on killing stenotrophomonas maltophilia under the condition that the pH of the sample is 6 is shown in FIG. 5.

As can be seen from FIG. 5, the samples prepared in comparative examples 5 to 7 have no bactericidal activity against stenotrophomonas maltophilia, the samples prepared in examples 5 to 7 have bactericidal activity against stenotrophomonas maltophilia, and particularly the samples prepared in examples 6 and 7 can kill more than 90% (1 order of magnitude) of stenotrophomonas maltophilia. The samples prepared in the examples of the present invention were shown to have spectral antimicrobial properties.

Experimental example 6

The samples used in this experimental example were the germicidal composition powders prepared in example 8 and comparative example 8, to which water was added at a concentration of 10%⁸CFU/ml of E.coli (E.coli) suspension, so that the concentration of ferrous iron and the concentration of MES in the mixed system are 10mM and 5mM respectively.

Mixed for 24 hours and then re-injected 10⁸CFU/ml of Escherichia coli (E.coli) was reacted for 24h, and the pH change before and after 48h reaction was less than 0.1. The results of comparison of the bactericidal effect are shown in fig. 6.

As can be seen from FIG. 6, the sample prepared in comparative example 8 loses the bactericidal ability after 24 hours of oxidation sterilization, while the sample prepared in example 8 can kill Escherichia coli again by one order of magnitude after 24 hours of oxidation sterilization, which indicates that the sample prepared in example 8 of the present invention has a longer bacteriostatic time.

The invention has been described in detail with reference to specific embodiments and illustrative examples, but the description is not intended to be construed in a limiting sense. Those skilled in the art will appreciate that various equivalent substitutions, modifications or improvements may be made to the technical solution of the present invention and its embodiments without departing from the spirit and scope of the present invention, which fall within the scope of the present invention.

Claims

1. The use of a biocidal composition comprising a modified clay and a buffer in the preparation of a biocidal material,

the sterilization composition is prepared by the method comprising the following steps:

step I, cleaning modified clay, wherein the preparation method of the modified clay comprises the following steps:

step 2, mixing the intercalation modified clay prepared in the step 1 with a reducing agent to obtain the modified clay;

step III, mixing the modified clay mother liquor prepared in the step II with a buffering agent to prepare the bactericidal composition;

the modified clay is obtained by carrying out intercalation modification on the natural clay containing iron by using an intercalation agent and reducing by using a reducing agent, and the surface of the modified clay is positively charged; the iron-containing natural clay is nontronite,

the intercalation agent is chitosan, and the intercalation agent is chitosan,

the intercalation comprises the following steps:

step 1-1, preparing a solution of natural clay containing iron and an organic intercalation agent with a certain granularity;

step 1-2, mixing organic intercalation agent solution and natural clay containing iron according to a preset proportion;

step 1-3, oscillating and washing the mixed system in the step 1-2;

the weight ratio of the organic intercalation agent to the natural clay containing iron is 1: 1;

the reducing agent is sodium hydrosulfite;

the weight ratio of the iron-containing natural clay mixed with the intercalation agent to the reducing agent is 1: (3-5);

the pH value of the sterilization composition is maintained at 7-8 through the adjustment of a buffering agent;

the buffer is PIPES or HEPES,

in the bactericidal composition, the molar ratio of the modified clay to the buffering agent is (0.5-10) calculated by ferrous iron: 5;

the divalent iron in the modified clay is used at a concentration of more than 0.08mM, and the buffer is used at a concentration of more than 0.8 mM.

2. The use of the germicidal composition as claimed in claim 1, wherein in step III, the mixing is performed under anaerobic conditions, and the mixing time is 10-50 min.