CN102697806A - Biological antibacterial agent, as well as preparation method and application thereof - Google Patents

Biological antibacterial agent, as well as preparation method and application thereof Download PDF

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CN102697806A
CN102697806A CN2012101541250A CN201210154125A CN102697806A CN 102697806 A CN102697806 A CN 102697806A CN 2012101541250 A CN2012101541250 A CN 2012101541250A CN 201210154125 A CN201210154125 A CN 201210154125A CN 102697806 A CN102697806 A CN 102697806A
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composite nanometer
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nanometer particle
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CN102697806B (en
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杨秀荣
王小磊
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Changzhou Institute Of Energy Storage Materials & Devices
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Changchun Institute of Applied Chemistry of CAS
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Abstract

The invention belongs to the field of biological materials, and discloses a biological antibacterial agent, as well as a preparation method and application thereof. The biological antibacterial agent consists of lactic acid bacteria, Ag nano-particles and magnetic Fe3O4 nano-particles, wherein the Ag nano-particles wrap the surfaces of the magnetic Fe3O4 nano-particles to form Ag and Fe3O4 composite nano-particles; and the Ag/Fe3O4 composite nano-particles are loaded on the surface of the lactic acid bacteria through hydrogen bonds. The antibacterial activity can be promoted by wrapping the Ag nano-particles on the surfaces of the magnetic Fe3O4 composite particles, the magnetic Ag/Fe3O4 composite nano-particles loaded on lactic acid bacteria can float in pathogenic bacteria-containing solution for a long time without extra dispersing treatment by virtue of low density of the lactic acid bacteria, so that the magnetic Ag/Fe3O4 composite nano-particles can be sufficiently contacted with the pathogenic bacteria floating in the solution, and can be accurately immobilized in a designated area and kept in a dispersing state for a long time in the body, so that the bacteriostasis and antibiosis capabilities of the biological antibacterial agent can be sufficiently played.

Description

A kind of biological antibiotic agent and preparation method thereof and application
Technical field
The invention belongs to technical field of biological material, relate in particular to a kind of biological antibiotic agent and preparation method thereof and application.
Background technology
Antibiotic is to be had antipathogen or other active one a type of secondary metabolite by what microorganism or high animals and plants produced in the life metabolism, is the one type of chemical substance that under low concentration very, can kill, suppress or disturb other microorganisms.Simultaneously, antibiotic also is one type of particular chemical preparation, since nineteen twenty-nine is found first kind of antibiotic penicillin, has found about 10,000 kinds of antibiotics so far.China is that antibiotic uses big country, shows that according to the 2006-2007 year Ministry of Public Health whole nation bacterial resistance monitoring result China hospital antibacterials year utilization rate is up to 74%.And in developed countries such as the U.S. and Britains, the antibiotic utilization rate of hospital is merely 22%-25%.Further research shows; China's infectious disease accounts for whole diseases 49% of number of always falling ill, and wherein bacterial infection disease only accounts for about 20%, yet; 70% patient used at least a antibiotic in the while in hospital now; Wherein almost everybody uses antibiotic to surgical patients, and ratio that is to say that up to 97% all there is the problem of abuse of antibiotics in most cases.Thisly be close to odd-shaped antibiotic utilization rate and make various drug resistance strains emerge in an endless stream, many bacterial strains for the drug resistance of penicillin near 100%.It is the most general that this makes China become abuse of antibiotics problem in the world, and one of also serious country surpasses 80,000 people and directly or indirectly die from abuse of antibiotics every year.
In addition; Though traditional antibiotherapy is selective for specific bacterial strain, do not have targeting property for intravital concrete active position, every through an antibiotic regimen; All might change the bacterium colony system of all organs in the body, and cause various unpredicted serious consequences thus.To this problem, a feasible solution route is to utilize composite nano materials to carry out the location antibacterial therapy of specific region in the body.The material of some hypotoxic micro-/ nano yardsticks, like zinc oxide, silver etc.; Can be used as the complementary therapy of existing antibiotherapy; The antagonism various bacteria infects, thereby reduces even avoid a series of side effect that caused by antibiotherapy, as fever, dizzy, feel sick and anaphylaxis etc.Yet,, seldom have report to further investigate the service efficiency that how to improve antibacterial in these bodies although have the report that the various nano-particle of utilization of certain length carry out locating therapy in the body at present.Though many nano antibacterial agents can accurately be fixed on the appointed area, can not effectively be disperseed, thereby be difficult to fully contact with pathogenic bacterium, in this case,, just can only increase the input amount of nano antibacterial agent in order to guarantee antimicrobial efficiency.Thus, not only having increased patient's misery and expense, also is a extra burden to its metabolic system.Therefore, be necessary to develop and a kind ofly promptly can accurately be fixed on the appointed area, can keep the high-performance bio antibacterial of dispersity in vivo for a long time again.
Summary of the invention
In view of this, the objective of the invention is to the defective to prior art, a kind of high-performance bio antibacterial and preparation method thereof and application that not only can accurately be fixed on the appointed area but also can keep dispersity in vivo for a long time is provided.
For realizing the object of the invention, the present invention adopts following technical scheme:
A kind of biological antibiotic agent is by lactobacillus, Ag nano-particle and magnetic Fe 3O 4Nano-particle is formed, and wherein, the Ag nano-particle is coated on magnetic Fe 3O 4Nano grain surface is formed Ag/Fe 3O 4Composite nanometer particle, Ag/Fe 3O 4Composite nanometer particle loads on the lactobacillus surface through hydrogen bond.
Biological antibiotic agent according to the invention is by lactobacillus, Ag nano-particle and magnetic Fe 3O 4Nano-particle is formed.
Wherein, lactobacillus is lower as the density of carrier own, Ag/Fe 3O 4After combining with lactobacillus, composite nanometer particle makes the higher magnetic Ag/Fe of density originally 3O 4The composite nanometer particle average density reduces, and is suitable with the density of the solution that contains pathogenic bacterium, makes the magnetic Ag/Fe of lactobacillus institute load 3O 4Composite nanometer particle can be handled under the condition of (like vibrations, stirring, ultrasonic etc.) and swim in for a long time in the solution that contains pathogenic bacterium need not additional dispersion; Thereby fully contact with buoyant pathogenic bacterium in solution; Give full play to its antibacterial, antibacterial ability, improve convenience, economy and the universality used in the practical operation of biological antibiotic agent to the solution-treated that contains pathogenic bacterium greatly.
Lactobacillus area load according to the invention has Ag/Fe 3O 4Composite nanometer particle, magnetic Fe wherein 3O 4Therefore nano-particle can move freely under magnetic action, and having improved lactobacillus, to adsorb antibacterial system be that load according to the invention has Ag/Fe 3O 4The lactobacillus suspension phase shift ability of nano-particle can concentrate on selection area fast and effectively, helps urgent, the efficient processing of pathogenic bacterium in the body.Undersized silver-colored particulate load makes silver-colored granule and extraneous contact area be able to amplify on larger-size ferroferric oxide magnetic nanoparticle surface.Compared to the fine silver nano-particle of similar size, this Ag/Fe 3O 4The arrangement mode of composite nanometer particle when guaranteeing anti-microbial property, has significantly reduced silver-colored consumption, has not only reduced the preparation cost of system, and has reduced the probability of occurrence of the possible side effect that is caused by silver.In addition, magnetic Fe 3O 4Interspersing of the Ag nano-particle of nano grain surface not only helps composite nanometer particle in surperficial the fixing of thalline, significantly promoted the antibacterial activity of this system, makes it in containing the solution of pathogenic bacterium, to win struggle for existence, gives full play to its antibiotic, bacteriostasis.
The present invention also provides the method for preparing of said biological antibiotic agent for utilizing the reversed micelle method in magnetic Fe 3O 4Nano grain surface coats one deck silver nano-grain, forms the Ag/Fe of black 3O 4Composite nanometer particle utilizes the 3-aminopropyltriethoxywerene werene at Ag/Fe 3O 4Composite nanometer particle surface coverage amination group fully contacts with lactic acid bacteria fermentation solution then, makes lactobacillus area load Ag/Fe 3O 4Composite nanometer particle.
Method for preparing according to the invention is with magnetic Fe 3O 4Nano-particle is that matrix prepares composite nanometer particle, said magnetic Fe 3O 4Nano-particle can obtain or adopt methods such as the disclosed coprecipitation of prior art, deposition oxidizing process, microemulsion method, hydro-thermal method, mechanical milling method, coacervation, sol method to prepare through the commercial channel.
In a specific embodiments, magnetic Fe according to the invention 3O 4Nano-particle is for adopting coprecipitation, with Fe 2+, Fe 3+With ammonia be that primary raw material makes.
In a specific embodiments, said coprecipitation prepares magnetic Fe 3O 4Nano-particle comprises the steps:
A, under nitrogen protection, ammonia is joined Fe 2+With Fe 3+Mixed solution in, make solution pH value ≧ 10, stir down fast at 85 ℃;
B, question response solution colour deepen continued and stirred 100 minutes;
C, outside magnetic field effect lower magnetic force are collected the black Fe in the solution 3O 4Deposition, the redistilled water washing is drying to obtain to neutral.
Coprecipitation prepares magnetic Fe 3O 4During nano-particle, Fe 2+With Fe 3+Mol ratio directly influence the crystal structure of product.The theoretical molar of reaction is than being Fe 2+: Fe 3+=1:2, but because ferrous ion is oxidized to ferric ion easily, and also have the intermediate reaction and the by-product of many complicacies, so ferrous ion should be suitably excessive, therefore, said Fe 2+With Fe 3+Mixed solution in Fe 2+With Fe 3+Mol ratio be preferably 1:1.75 ~ 2.
Fe according to the invention 2+And Fe 3+Can derive from chloride, nitrate, acetate or the sulfate of ferrum.In a specific embodiments, Fe 2+And Fe 3+FeCl originates respectively 24H 2O and FeCl 36H 2O.
Method for preparing according to the invention utilizes the reversed micelle method in magnetic Fe 3O 4Nano grain surface coated with silver nano-particle.In specific embodiments, the reversed micelle method of utilizing according to the invention is in magnetic Fe 3O 4Nano grain surface coats one deck silver nano-grain and comprises the steps:
I, with Fe 3O 4Nano-particle mixes with the water-in-oil microemulsion liquid phase that contains Triton X-100, hexanol and cyclohexane extraction, mixes silver nitrate solution again, reacts 30 minutes;
II, adding sodium borohydride solution, and at room temperature stirred 3-8 hour, add excessive propanone then and make the product deposition in the solution;
III, collection step 2 gained deposition, ethanol and water washing, external magnetic field is collected black precipitate and is promptly got.
Wherein, the water-in-oil microemulsion of the said Triton of containing X-100, hexanol and cyclohexane extraction is meant and contains Triton X-100, hexanol and three kinds of materials of cyclohexane extraction in the water-in-oil microemulsion.Said Fe 3O 4The mol ratio of nano-particle and Triton X-100, hexanol and cyclohexane extraction is preferably 1:14:14:75, said Fe 3O 4The mol ratio of nano-particle and silver nitrate is preferably 1:12.5.
The use of water-in-oil microemulsion according to the invention can be controlled the size of end product.In a specific embodiments, said in magnetic Fe 3O 4The method that nano grain surface coats one deck silver nano-grain is specially: through churned mechanically mode; The ferroferric oxide nano granules of 100 μ L, 40mM is mixed with the water-in-oil microemulsion liquid phase that contains 1.4mL Triton X-100,1.4mL hexanol and 7.5mL cyclohexane extraction; After 20 minutes; The silver nitrate solution that adds the 0.1M of 200 μ L continues to stir 30 minutes.The sodium borohydride that adds 250 μ L 0.20M then at room temperature stirred 3-8 hour, added excessive propanone then and made the product deposition in the solution, and gained precipitates through second alcohol and water cyclic washing, collects through external magnetic field and promptly gets Ag/Fe 3O 4Composite nanometer particle.
Method for preparing according to the invention utilizes the 3-aminopropyltriethoxywerene werene at Ag/Fe 3O 4Composite nanometer particle surface coverage amination group.In specific embodiments, the 3-aminopropyltriethoxywerene werene of utilizing according to the invention is at Ag/Fe 3O 4The method of composite nanometer particle surface coverage amination group is specially Ag/Fe 3O 4Composite nanometer particle mixes with alcoholic solution, water and washing with alcohol behind the stirring 2-12h, and external magnetic field is collected and is promptly got, and wherein, said alcoholic solution contains 5v/v%3-aminopropyltriethoxywerene werene and 5v/v% water.
Wherein, by g/mL, said Ag/Fe 3O 4The w/v of composite nanometer particle and alcoholic solution is preferably 1:10.The addition that is the alcoholic solution of the said 5v/v%3-of containing aminopropyltriethoxywerene werene and 5v/v% water is every 1gAg/Fe 3O 4Add 10mL.
Method for preparing according to the invention is with amido modified Ag/Fe 3O 4Composite nanometer particle fully contacts with lactic acid bacteria fermentation solution, amido modified Ag/Fe 3O 4The amino on composite nanometer particle surface is with the carboxylate group on the lactobacillus surface formation hydrogen bond that combines, through Ag/Fe 3O 4Form hydrogen bond between composite nanometer particle and the lactobacillus and can make lactobacillus area load Ag/Fe 3O 4Composite nanometer particle.On the other hand, the lactobacillus surface is the electronegative semipermeable membrane of one deck, easily and the Ag/Fe of positively charged 3O 4Composite nanometer particle combines.Therefore, as amido modified Ag/Fe 3O 4Composite nanometer particle with fully contact with lactic acid bacteria fermentation solution, just can make lactobacillus area load Ag/Fe 3O 4Composite nanometer particle, obtaining area load has Ag/Fe 3O 4The lactobacillus of composite nanometer particle.
In a specific embodiments, adding 2mL concentration in the lactic acid bacteria fermentation solution of every 25mL is the amido modified Ag/Fe of 0.01g/mL 3O 4The aqueous solution of composite nanometer particle.For avoiding amido modified Ag/Fe 3O 4Composite nanometer particle deposits in fermentation liquid, be preferably in speed is the shaking table of 200rpm to carry out, and the response time is no less than half an hour.
The biological antibiotic agent that the present invention makes through scanning electron microscopic observation method for preparing according to the invention; It is thus clear that the area load of lactobacillus has many diameters to be about the granule of 150nm; The average diameter of particles of the area load through the transmission electron microscope observing lactobacillus is about 150nm, and the dimensional homogeneity between the granule is better.In addition; All there is the membranaceous material of a circle lighter color each granule outside, and its density is starkly lower than the metal inner core at granule center, and high resolution electron microscopy detects the lattice fringe that includes Fe and two kinds of elements of Ag in the granular metal kernel; In conjunction with the experimental program of describing before, this kernel of deducibility is Ag/Fe 3O 4Composite nanometer particle.Further carry out corresponding XRD analysis,, can know that the silver element in the kernel mainly exists with non-valent simple substance silver form according to the position and the intensity at Fe in the XRD figure spectrum and the corresponding peak of Ag element, and Fe 3O 4Then the content in kernel is occupied an leading position.The granule of decidable lactobacillus surface institute load is Ag/Fe thus 3O 4Composite nanometer particle.
The present invention also provides the biological antibiotic agent of above-mentioned method for preparing preparation.
Biological antibiotic agent according to the invention is a kind of multi-functional system that is integrated with antibacterial activity and magnetic force targeting ability.The Ag nano-particle intersperse the antibacterial activity that can promote this system; Lactobacillus combines magnetic Ag/Fe as low can the making of the density of carrier own 3O 4The composite nanometer particle average density reduces, and makes the magnetic Ag/Fe of lactobacillus institute load 3O 4Composite nanometer particle can swim in the solution that contains pathogenic bacterium under the condition that need not the additional dispersion processing for a long time; Thereby fully contact with buoyant pathogenic bacterium in solution; Can accurately be fixed on the appointed area, can keep dispersity in vivo for a long time again, give full play to its antibacterial, antibacterial ability.Experiment shows, biological antibiotic agent according to the invention can be suspended in the simulation human body body that contains pathogenic bacterium of higher density in complicacy and the heavy-gravity mixed solution for a long time, and fully contacts with pathogenic bacterium in the solution, and antibacterial activity will obviously be better than matched group.
Therefore, the invention provides the application of said biological antibiotic agent in suppressing pathogenic bacterium.Preferably, said pathogenic bacterium are escherichia coli, staphylococcus aureus, helicobacter pylori or bacillus subtilis.
Description of drawings
Fig. 1 shows amido modified Ag/Fe according to the invention 3O 4The transmission electron microscope picture of composite nanometer particle and X-ray diffractogram and biological antibiotic agent sem photograph according to the invention wherein, (a) are amido modified Ag/Fe according to the invention 3O 4The sem photograph of composite nanometer particle, illustration is the high-resolution-ration transmission electric-lens lattice sign of kernel portion in it; (b) be the according to the invention amido modified Ag/Fe shown in (a) figure 3O 4The X-ray diffractogram of composite nanometer particle, abscissa are 2 θ, and vertical coordinate is the intensity of X ray; (c) be the sem photograph of the biological antibiotic agent according to the invention shown in (b) figure;
Fig. 2 shows Ag/Fe 3O 4Composite nanometer particle (No. 1 beaker) and the dispersibility comparison diagram of biological antibiotic agent according to the invention (No. 2 beakers) in containing the mixed solution of staphylococcus aureus, wherein, a～d is the deployment conditions of different time, e and f are under the effect of external magnetic field, Ag/Fe 3O 4The deployment conditions of composite nanometer particle and biological antibiotic agent according to the invention;
Fig. 3 shows and uses Ag/Fe 3O 4The transmission electron microscope picture of staphylococcus aureus is handled in composite nanometer particle and biological antibiotic agent according to the invention, and wherein (a) is for using Ag/Fe 3O 4Composite nanometer particle is handled the transmission electron microscope picture of the staphylococcus aureus (30mL) after 4 hours; (b) use the biological antibiotic agent according to the invention of half amount to handle the transmission electron microscope picture of the staphylococcus aureus (30mL) after 2 hours; Red arrow is oriented to damaged staphylococcus aureus, and interior illustration is selected enlarged drawing;
Fig. 4 shows through Ag/Fe 3O 4Escherichia coli (E.coli) after composite nanometer particle or biological antibiotic agent according to the invention are handled; Staphylococcus aureus (S.aureus); The survival rate cartogram of helicobacter pylori (H.pylori) and bacillus subtilis (B.subtilis), wherein, matched group is for using Ag/Fe 3O 4Composite nanometer particle is handled survival rate cartogram after 4 hours, and test group is to use the biological antibiotic agent according to the invention of half amount to handle survival rate cartogram after 2 hours, tests triplicate at least for every, and chart data is expressed as meansigma methods and relative deviation thereof.
The specific embodiment
The embodiment of the invention discloses a kind of biological antibiotic agent and preparation method thereof and application.Those skilled in the art can use for reference this paper content, suitably improve technological parameter and realize.Special needs to be pointed out is that all similarly replace and change apparent to those skilled in the art, they all are regarded as and are included in the present invention.The present invention is described through preferred embodiment, and the related personnel obviously can change or suitably change and combination product as herein described, methods and applications in not breaking away from content of the present invention, spirit and scope, realizes and use technology of the present invention.
For realizing the object of the invention, the present invention adopts following technical scheme:
A kind of biological antibiotic agent is by lactobacillus, Ag nano-particle and magnetic Fe 3O 4Nano-particle is formed, and wherein, the Ag nano-particle is coated on magnetic Fe 3O 4Nano grain surface is formed Ag/Fe 3O 4Composite nanometer particle, Ag/Fe 3O 4Composite nanometer particle loads on the lactobacillus surface through hydrogen bond.
The method for preparing of biological antibiotic agent according to the invention comprises the steps:
1, under nitrogen protection, ammonia is joined Fe 2+With Fe 3+Mixed solution in, make solution pH value ≧ 10, stir down fast at 85 ℃, the question response solution colour deepens continued and stirred 100 minutes, outside magnetic field effect lower magnetic force is collected the black Fe in the solution then 3O 4Deposition, the redistilled water washing is to neutral, and drying makes magnetic Fe 3O 4Nano-particle;
2, with the magnetic Fe of step 1 gained 3O 4Nano-particle with contain Triton X-100, the Water-In-Oil of hexanol and cyclohexane extraction (W/O) microemulsion liquid-phase mixing added silver nitrate solution reaction 30 minutes again; Add sodium borohydride solution and at room temperature stirred 3-8 hour, add excessive propanone subsequently and make the product deposition in the solution, after centrifugalize; Wash repeatedly with ethanol and redistilled water; Last outside magnetic field effect lower magnetic force is collected the black precipitate in the solution, the redistilled water washing, and drying makes Ag/Fe 3O 4Composite nanometer particle;
3, with the Ag/Fe of step 2 gained 3O 4Composite nanometer particle mixes after 6 hours with the alcoholic solution that contains 5v/v%3-aminopropyltriethoxywerene werene and 5v/v% water and washs respectively with secondary water and ethanol, and outside magnetic field effect lower magnetic force is collected amido modified Ag/Fe 3O 4Composite nanometer particle;
4, the amido modified Ag/Fe that in lactic acid bacteria fermentation solution, adds step 3 gained 3O 4Composite nanometer particle is cultivated in the shaking table of 200rpm and is promptly got more than half an hour.
In order further to understand the present invention, the present invention is elaborated below in conjunction with embodiment.
Embodiment 1: the preparation of biological antibiotic agent according to the invention
Get 43.10mL1.00mol/LFeCl 24H 2O solution and 43.10mL 1.75mol/LFeCl 36H 2O solution mixes, and under nitrogen protection, adds 25% ammonia of 25mL, stirs fast down at 85 ℃, and reaction solution darkens, and has brown particle to generate, and continues to stir 100 minutes.Come sedimented particle to separate the supernatant with strong magnet, with distilled water cyclic washing precipitate, extremely the pH value of solution of washing is about 7.The collecting precipitation thing places vacuum drying oven, gets magnetic Fe at 75 ℃ of vacuum drying 5h 3O 4Nano-particle.
With 1mL concentration is the magnetic Fe of 40mM 3O 4Nano-particle with contain 14mL Triton X-100, Water-In-Oil (W/O) the microemulsion liquid-phase mixing of 14mL hexanol and 75mL cyclohexane extraction adds 5mL concentration and is the silver nitrate solution reaction 30 minutes of 0.1M again.Adding 2mL concentration is the sodium borohydride solution of 0.2M, at room temperature stirs 3 ~ 8 hours.Add excessive propanone subsequently and make the product deposition in the solution; After centrifugalize; With ethanol and each washed twice of redistilled water, last outside magnetic field effect lower magnetic force is collected the black precipitate in the solution, redistilled water washing 2 times; The collecting precipitation thing places vacuum drying oven, makes Ag/Fe at 75 ℃ of vacuum drying 5h 3O 4Composite nanometer particle;
Get the Ag/Fe that 1g makes 3O 4Composite nanometer particle joins 10mL and contains in the alcoholic solution of 5v/v%APTES and 5v/v% water; Fully stirred 6 hours; With ethanol and each washed twice of redistilled water, outside magnetic field effect lower magnetic force is collected the black precipitate in the solution then, redistilled water washing 2 times; The collecting precipitation thing places vacuum drying oven, makes amido modified Ag/Fe at 75 ℃ of vacuum drying 5h 3O 4Composite nanometer particle;
The amido modified Ag/Fe that will make with syringe 3O 4Composite nanometer particle disperses to join in the lactic acid bacteria fermentation solution, in the shaking table of 200rpm, cultivates 40min and promptly gets.
To the amido modified Ag/Fe that makes 3O 4Composite nanometer particle carries out the transmission electron microscope detection and X-ray diffraction detects, and sem photograph is carried out in the biological antibiotic agent that makes, and the result sees Fig. 1.Visible by Fig. 1, amido modified Ag/Fe 3O 4The equal diameter of composite nanometer particle is about 150nm, and the dimensional homogeneity between the granule is better.In addition, all there is the membranaceous material of a circle lighter color each granule outside, and its density is starkly lower than the metal inner core at granule center, and in conjunction with the preparation flow of narrating before, the deducibility skin is Ag-Fe 3O 4The amination organic polymer layer that nano grain surface covers.The granular metal kernel is carried out Electronic Speculum detect, include the lattice fringe of Fe and two kinds of elements of Ag in the visible kernel of result, analysis kernel is Ag/Fe 3O 4Composite nanometer particle.According to the position and the intensity at Fe in the X ray diffracting spectrum and the corresponding peak of Ag element, think that the silver element in the kernel mainly exists with non-valent simple substance silver form, and Fe 3O 4Then the content in kernel is occupied an leading position.Sem photograph by biological antibiotic agent shown in Fig. 1 c of the present invention is visible, amido modified Ag/Fe 3O 4Composite nanometer particle loads on the lactobacillus surface equably, and simultaneously owing to outer amino protective effect, the silver-colored granule in the kernel does not have too big destruction to the lactobacillus surface as carrier at short notice.
Embodiment 2: the dispersibility of biological antibiotic agent according to the invention and antibiotic property experiment
Carry out the antimicrobial potential superiority of targeting property in vivo in order to prove biological antibiotic agent according to the invention.Select for use and contain beef peptone (5g/L); Glucose (5g/L), complicacy and heavy-gravity liquid environment in the mixed aqueous solution of agar (20g/L) and compound vitamin (10mg/L) (pH=7.1,37 ℃) the simulation human body body; Investigate with this understanding; Biological antibiotic agent according to the invention is for the disposal ability of four kinds of common pathogens (escherichia coli, staphylococcus aureus, helicobacter pylori and bacillus subtilis).Contain in the bacterium solution biological antibiotic agent that the embodiment 1 of interpolation 5.4mg makes (wherein, effective antimicrobial component Ag/Fe at every 30mL 3O 4The content of composite nanometer particle is about 0.6mg), and examine or check its antibacterial activity.Simultaneously, select 2 times of effective antimicrobial component content (Ag/Fe 3O 4The content 1.2mg of composite nanometer particle) Ag/Fe that embodiment 1 makes 3O 4Composite nanometer particle is as matched group.Test triplicate at least for every group, Fig. 2～is 4. seen in statistical result
Visible by Fig. 2 result, because Ag/Fe 3O 4The density of composite nanometer particle own is higher, even if Ag/Fe 3O 4Composite nanometer particle is dispersed in advance and contains in the bacterium liquid; Also will deposit (No. 1 beaker of Fig. 2 c) at short notice; Its antimicrobial component of transmission electron microscope observing be difficult to solution in pathogenic bacterium fully contact (Fig. 3 a), therefore, although reach 4 hours action time; But its corresponding antibacterial activity is also not obvious, adopts Ag/Fe 3O 4The survival rate of the pathogenic bacterium that composite nanometer particle is handled is about the twice (Fig. 4 matched group) that adopts biological antibiotic agent according to the invention to handle the survival rate of pathogenic bacterium.Biological antibiotic agent according to the invention then can be suspended in contain (No. 2 beakers of Fig. 2 d) in the bacterium mixed solution of higher density for a long time; Its effective antimicrobial component of transmission electron microscope observing can fully contact (Fig. 3 b) with the pathogenic bacterium in the solution; Therefore; Although have only 2 hours action time, and antimicrobial component only is the half the of matched group, and its corresponding antibacterial activity will obviously be better than matched group (Fig. 4 test group).And visible under the control of external magnetic field by Fig. 2 f, biological antibiotic agent according to the invention can be collected easily equally and separated (No. 2 beakers of Fig. 2 d), therefore, and compared to Ag/Fe 3O 4Composite nanometer particle, it is antibiotic that biological antibiotic agent according to the invention can be used for intravital targeting property.
The explanation of above embodiment just is used for helping to understand method of the present invention and core concept thereof.Should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention, can also carry out some improvement and modification to the present invention, these improvement and modification also fall in the protection domain of claim of the present invention.

Claims (11)

1. a biological antibiotic agent is characterized in that, by lactobacillus, Ag nano-particle and magnetic Fe 3O 4Nano-particle is formed, and wherein, the Ag nano-particle is coated on magnetic Fe 3O 4Nano grain surface is formed Ag/Fe 3O 4Composite nanometer particle, Ag/Fe 3O 4Composite nanometer particle loads on the lactobacillus surface through hydrogen bond.
2. the method for preparing of a biological antibiotic agent is characterized in that, utilizes the reversed micelle method in magnetic Fe 3O 4Nano grain surface coats one deck silver nano-grain, forms the Ag/Fe of black 3O 4Composite nanometer particle utilizes the 3-aminopropyltriethoxywerene werene at Ag/Fe 3O 4Composite nanometer particle surface coverage amination group fully contacts with lactic acid bacteria fermentation solution then, makes lactobacillus area load Ag/Fe 3O 4Composite nanometer particle.
3. according to the said method for preparing of claim 2, it is characterized in that said magnetic Fe 3O 4Nano-particle is for adopting coprecipitation with Fe 2+, Fe 3+With ammonia be that primary raw material makes.
4. according to the said method for preparing of claim 2, it is characterized in that the said reversed micelle method of utilizing is in magnetic Fe 3O 4Nano grain surface coats one deck silver nano-grain and comprises the steps:
I, with Fe 3O 4Nano-particle mixes with the water-in-oil microemulsion liquid phase that contains Triton X-100, hexanol and cyclohexane extraction, mixes silver nitrate solution again, reacts 30 minutes;
II, adding sodium borohydride solution, and at room temperature stirred 3-8 hour, add excessive propanone then and make the product deposition in the solution;
III, collection step 2 gained deposition, ethanol and water washing, external magnetic field is collected black precipitate and is promptly got.
5. according to the said method for preparing of claim 4, it is characterized in that said Fe 3O 4The mol ratio of nano-particle and Triton X-100, hexanol and cyclohexane extraction is 1:14:14:75.
6. according to the said method for preparing of claim 4, it is characterized in that said Fe 3O 4The mol ratio of nano-particle and silver nitrate is 1:12.5.
7. according to the said method for preparing of claim 2, it is characterized in that the said 3-aminopropyltriethoxywerene werene of utilizing is at Ag/Fe 3O 4The method of composite nanometer particle surface coverage amination group is specially Ag/Fe 3O 4Composite nanometer particle mixes with alcoholic solution, water and washing with alcohol behind the stirring 2-12h, and external magnetic field is collected and is promptly got, and wherein, said alcoholic solution contains 5v/v%3-aminopropyltriethoxywerene werene and 5v/v% water.
8. according to the said method for preparing of claim 7, it is characterized in that, by g/mL, said Ag/Fe 3O 4The w/v of composite nanometer particle and alcoholic solution is 1:10.
9. the biological antibiotic agent of any said method for preparing preparation of claim 2 ~ 8.
10. claim 1 or the 9 said biological antibiotic agent application in suppressing pathogenic bacterium.
11., it is characterized in that said pathogenic bacterium are escherichia coli, staphylococcus aureus, helicobacter pylori or bacillus subtilis according to the said application of claim 10.
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CN109221136B (en) * 2018-10-15 2020-10-20 安徽绩溪县徽煌化工有限公司 Preparation method of low-residue nitenpyram combined pesticide for cucumbers
CN109331179A (en) * 2018-10-25 2019-02-15 中国人民解放军总医院 A kind of application of composite magnetic nano particle
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CN116267989A (en) * 2022-09-09 2023-06-23 兰州大学 Nanometer iron antibacterial agent for fusarium trilineum
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