CN107354175B - Silver nano material and biological preparation method and application thereof - Google Patents
Silver nano material and biological preparation method and application thereof Download PDFInfo
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- CN107354175B CN107354175B CN201710512718.2A CN201710512718A CN107354175B CN 107354175 B CN107354175 B CN 107354175B CN 201710512718 A CN201710512718 A CN 201710512718A CN 107354175 B CN107354175 B CN 107354175B
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Abstract
The invention discloses a silver nano material, a biological preparation method and application thereof. The method adopts endophytic fungi separated from the root of Dendrobium officinaleFusarium solaniThe silver nano material is synthesized by D07 (with the preservation number of CCTCC NO. M2017145) in an extracellular mode, the reaction condition is mild, the control is easy, toxic or radioactive reagents in chemical and physical methods are avoided, and the method has the advantages of reliability and greenness. The silver nano material is a round or oval nano particle material, has good dispersibility and narrow particle size distribution, has the particle size of 16-46 nm, and has good antibacterial effect. The silver nano material has good antibacterial activity, and has good application prospect when being applied to the preparation of antibacterial materials.
Description
Technical Field
The invention relates to the field of preparation and application of silver nano materials, in particular to a biological preparation method and application of the silver nano materials.
Background
Metal nanomaterials have become one of the important directions of research in recent decades due to their potential applications in the fields of optoelectronics, catalysis, sensors, biology, and the like. Among them, nano silver, as a new functional material, has a unique physicochemical property, and is drawing attention in the research fields of nano photoelectric devices, biomarkers, surface enhanced raman, and biomedical composite materials.
Bacteriostatic materials can be divided into three major types, namely natural, organic and inorganic bacteriostatic materials. The natural biological bacteriostatic material is derived from natural extracts, is nontoxic or low-toxic, but has poor durability, is limited by processing conditions, and cannot be the first choice of a bacteriostatic agent. The organic bacteriostatic material is mainly divided into organic acids, phenols, quaternary ammonium salts and the like, and can inhibit the growth and reproduction of harmful bacteria and mould by acting on cell walls and cell membranes of the bacteria, biochemical reaction enzyme, genetic substances and the like. The organic bacteriostatic material has the defects of poor stability, easy hydrolysis, secondary pollution, toxicity and difficult control of slow release. In order to avoid the disadvantages of organic bacteriostatic materials, researchers are gradually looking at inorganic bacteriostatic materials.
The inorganic bacteriostatic material is prepared by selecting porous materials such as zeolite, silica gel and the like as a matrix by virtue of bacteriostatic performance of metal, loading the metal on the surface or in pore channels of the matrix by physical adsorption or ion exchange and the like, and introducing the metal into a bacteriostatic product. The inorganic antibacterial material has the advantages of low toxicity, good heat resistance, long duration and wide antibacterial spectrum, and is an antibacterial agent variety with good commercial prospect. Inorganic bacteriostatic agents can be classified into photocatalytic materials, metal ion-containing materials, rare earth materials, natural ores, and shells. Currently, Ag is commonly used+、Cu2+And Zn2+As the metal particle component of the metal inorganic bacteriostatic material. Trace amount of Ag+、Cu2+And Zn2+Is beneficial to human bodies, but has destructive effect on microorganisms, and the silver ion has the strongest bacteriostatic property and the weakest zinc ion, so the silver-series inorganic bacteriostatic agent has the most extensive application. Silver is a long-standing bacteriostatic agent, people begin to use silver instruments to preserve food in ancient times, and a silver nitrate solution is used by Halstead, one of surgical creators, for treating wounds and scalds by the beginning of the 20 th century.
The preparation of inorganic bacteriostatic materials currently has physical, chemical and biological methods, and because the chemical and physical methods relate to toxic or radioactive reagents, the biological method has the characteristics of reliability and greenness, and becomes a trend of silver nanotechnology development in recent years.
Disclosure of Invention
The invention aims to provide a silver nano material which is a round or oval nano particle material, has good dispersibility and narrow particle size distribution, has particle size of 16-46 nm, and has good antibacterial effect.
The invention also aims to provide a biological preparation method of the silver nano material, the method adopts endophytic fungus Fusarium solani D07 (with the collection number of CCTCC NO. M2017145) separated from the root of the dendrobium officinale to biologically synthesize the silver nano material in an extracellular mode, and the reaction condition is mild and easy to control.
The invention also aims to provide the application of the silver nano material in preparing the antibacterial material, and the silver nano material has good antibacterial activity, so the silver nano material has good application prospect when being applied to the preparation of the antibacterial material.
The purpose of the invention is realized by the following technical scheme.
A biological preparation method of silver nano material comprises the following steps:
fusarium solani D07 strain activation → seed culture solution → fermentation culture → suction filtration to obtain mycelium → mycelium culture in sterile deionized water → suction filtration to obtain filtrate → addition of AgNO3Reaction → material acquisition.
A biological preparation method of a silver nano material specifically comprises the following steps:
(1) activating the dendrobium officinale endophytic fungi strain, then performing seed liquid culture, and performing fermentation culture on the obtained seed liquid;
(2) carrying out suction filtration on the culture solution finally obtained in the step (1), washing with sterile deionized water, culturing the obtained hyphae in the sterile deionized water, carrying out suction filtration again, and adding AgNO into the obtained filtrate3Carrying out reaction; and after the reaction is finished, centrifuging and collecting the precipitate to obtain the silver nano material.
Further, in the step (1), the dendrobium officinale endophytic fungus strain is separated from the root of dendrobium officinale, is named as Fusarium solani D07, and is preserved in a Chinese typical culture collection center (Wuhan university, Lo Huan mountain Lo, Lo Jia mountain, Wuhan, Wuchang, Hubei province, 3 months and 27 days in 2017, and the preservation number is CCTCC NO. M2017145.
Further, in the step (1), the activation is carried out for 48-72 hours at 28 +/-1 ℃ by adopting a test tube slant culture method under the aseptic condition, and the culture medium is Potato Dextrose Agar (PDA).
Further, in the step (1), the seed liquid culture is performed by shaking culture in potato dextrose aqueous medium (PDB) at 28 +/-1 ℃ and 120rpm for 48-72 hours under aseptic conditions.
Further, in the step (1), the fermentation culture is carried out under aseptic conditions, the obtained seed solution is inoculated into a liquid culture medium according to the inoculum size of 10v%, and the culture is carried out for 3-4 days at the temperature of 28 +/-1 ℃ and the rotating speed of 120 rpm.
Further, in the step (2), the sterile deionized water is Milli-Q deionized water.
Further, in the step (2), the cultivation in sterile deionized water is carried out for 24-48 h under the conditions of the temperature of 28 +/-1 ℃ and the rotating speed of 120rpm under the aseptic condition.
Further, in the step (2), AgNO is added3Keeping AgNO added during the reaction3The concentration of (2) is 1 mmol/L.
Further, in the step (2), the reaction is carried out for 24-48 h at room temperature and at a rotation speed of 120 rpm.
Further, in the step (2), the centrifugation is carried out at 12000g for 20min at a temperature of 4 ℃.
The processes of activation, seed solution culture, fermentation culture, suction filtration and culture in sterile deionized water are all carried out under sterile conditions.
The preparation principle of the silver nano material of the invention is as follows: during the growth culture of Fusarium solani D07 strain, Fusarium solani D07 strain produced reductase which promoted Ag+The ions are reduced to Ag to produce the silver nano material.
The silver nano material prepared by any one of the preparation methods is a round or oval nano particle material, and the particle size of the particles is 16-46 nm.
The silver nano material has good antibacterial activity and has good inhibition effect on test bacteria including candida tropicalis, bacillus cereus, bacillus subtilis, staphylococcus aureus, escherichia coli, alcaligenes and salmonella; the silver nano material is applied to the preparation of antibacterial materials, including the development and preparation of antibacterial film coatings.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) the invention provides a biological preparation method of silver nano-material, which has simple and easy technical process, mild reaction condition and easy control, and adopts the biological method to prepare the silver nano-material, thereby avoiding toxic or radioactive reagents in chemical and physical methods and having the advantages of reliability and environmental protection;
(2) the silver nano material is a round or oval nano particle material, has good dispersibility, narrow particle size distribution and good bacteriostatic effect, and has good application prospect when being applied to the preparation of bacteriostatic materials.
Drawings
FIG. 1 shows the addition of AgNO in example 13The 440nm characteristic absorption curve graph of the cell filtrate under an ultraviolet-visible spectrophotometer is obtained;
fig. 2 is a transmission electron microscope image of the silver nanomaterial prepared in example 1.
Detailed Description
The technical solution of the present invention is further described below with reference to the following specific examples and drawings, but the present invention is not limited thereto.
The experimental procedures for which specific conditions are not noted in the following examples are generally performed according to conventional conditions, as described in laboratory manuals, or according to conditions recommended by the manufacturer.
The dendrobium candidum endophytic fungus strain Fusarium solani D07 adopted in the specific embodiment of the invention is preserved in a China typical culture collection center (Wuhan university, Lonchaa Loopjia mountain of Wuchang city, Wuhan, Hubei province, and the preservation number is CCTCC NO. M2017145 in 3 months and 27 days in 2017; the ribose in vivo transcribed spacer region (ITS) and the 5.8S ribosomal RNA coding gene (5.8SrDNA) of the strain are shown as SEQ ID No. 1.
The bacteriostatic activity of the silver nano material in the specific embodiment of the invention is tested and observed by the following method, and the specific flow is as follows:
pathogenic bacteria strain activation → seed liquid culture → plate coating inoculation → inoculation of silver-containing nanomaterial filter paper → culture → observation of bacteriostatic effect.
Example 1
The biological preparation of the silver nano material comprises the following preparation processes:
fusarium solani D07 strain activation → seed culture solution → fermentation culture → suction filtration to obtain mycelium → mycelium culture in sterile deionized water → suction filtration to obtain filtrate → addition of AgNO3Reaction → material acquisition.
The preparation process comprises the following steps:
(1) taking a dendrobium officinale endophytic fungus strain Fusarium solani D07, picking a small amount of hypha by using an inoculating needle under an aseptic condition, inoculating the hypha into a sterilized solid PDA culture medium test tube, and performing activated culture at the temperature of 28 +/-1 ℃ for 72 hours;
(2) taking the activated and cultured strains, transferring the strains into a sterilized liquid PDB seed culture medium under the aseptic condition, and carrying out shake culture at the temperature of 28 +/-1 ℃ and the rpm of 120 for 72 hours to obtain a seed solution;
(3) inoculating into 500ml liquid culture medium according to 10v% (volume percentage) under aseptic condition, and performing shake fermentation culture at 28 + -1 deg.C and 120rpm for 3 days;
(4) vacuum filtering the fermentation liquid under aseptic condition, washing mycelium with sterile water, and removing residual culture medium components;
(5) the harvested mycelia (wet weight 20g) were re-inoculated into 500ml Erlenmeyer flasks containing 200ml Milli-Q deionized water and incubated at 28. + -. 1 ℃ for 24 hours at 120 rpm;
(6) filtering to remove mycelium, adding AgNO into the collected culture solution3And ensure AgNO3The final concentration is 1mmol/l, and the reaction solution reacts for 24 hours at 25 ℃ and 120 rpm; scanning the reaction solution in an ultraviolet visible spectrophotometer at the wavelength of 200-800nm to determine that the silver nano material is generated;
adding AgNO3The 440nm characteristic absorption curve of the cell filtrate under an ultraviolet-visible spectrophotometer is shown in figure 1, and as can be seen from figure 1, AgNO is added into the cell filtrate3And in the cell bodyAddition of AgNO3The absorption peak of the later samples at 440nm is completely the same as the characteristic resonance absorption peak of the nano-silver, which proves that the two samples are formed by the silver nano-material; and as a control, cell filtrate alone and AgNO3There was no absorption at 440nm, indicating no nanosilver formation.
(7) After the reaction, the reaction solution was centrifuged at 12000g at 4 ℃ for 20 minutes to collect precipitates, which were dried and then characterized by structure by transmission electron microscopy.
The transmission electron microscope image of the prepared silver nano material is shown in fig. 2, and as can be seen from fig. 2, the prepared silver nano material is a round or oval particle material, and the particle size is 16-46 nm.
Comparative example 1
Comparative example blank control the procedure for preparing nanomaterials was the same as in example 1, except that the blank control was made without the addition of AgNO3The reaction is carried out.
Example 2
The nano materials obtained in the example 1 and the comparative example 1 are tested and observed for bacteriostatic activity, a filter paper sheet method is adopted for bacteriostatic activity evaluation, and the flow is as follows:
pathogenic bacteria strain activation → seed liquid culture → plate coating inoculation → inoculation of silver-containing nano filter paper → culture → observation of bacteriostatic effect.
The method for observing the antibacterial activity specifically comprises the following steps:
(1) selecting 7 common pathogenic bacteria: selecting a small amount of bacterial colonies by using an inoculating needle under the aseptic condition, inoculating the bacterial colonies into a sterilized beef extract peptone culture medium (solid state) test tube, and activating for 24 hours at the temperature of 37 +/-1 ℃;
(2) taking the activated and cultured strain, transferring the strain into a sterilized liquid beef extract peptone liquid culture medium (liquid state) under the aseptic condition, and carrying out shake cultivation for 24 hours at 37 +/-1 ℃ and 160rpm to obtain a seed solution;
(3) inoculating into 10ml liquid LB plate culture medium according to the inoculation amount of 2 v% (volume percentage) under aseptic condition, shaking the plate to uniformly mix the seed liquid and the LB liquid culture medium, and waiting for solidification in an ultra-clean workbench;
(4) a filter paper sheet (phi 0.6cm, sterilized) soaked in the silver nano material colloid solution extracellularly synthesized by the dendrobium officinale endophytic fungus D07 in example 1 was soaked in the colloid solution of comparative example 1 by the same method; after aseptic air drying, inoculating the mixture to a beef extract peptone plate culture medium coated with pathogenic bacteria, and carrying out inverted culture at 37 ℃ for 48 hours;
(5) and (3) placing the prepared test bacterium culture dish in an incubator at 37 ℃ for constant-temperature culture for 24h, observing, and measuring the size of the inhibition zone.
The results of the observation of the zone of inhibition are shown in table 1.
Table 1 results of antibacterial activity of nanomaterial of example 1 and comparative example 1
As can be seen from table 1, the inhibition zones of the silver nanomaterial synthesized in example 1 to the selected 7 common pathogenic bacteria are much larger than the inhibition zones of the nanomaterial of comparative example 1, which indicates that the silver nanomaterial synthesized in example 1 has obvious resistance to the selected 7 common pathogenic bacteria and has an obvious effect of inhibiting the common pathogenic bacteria.
It should be understood that the above specific embodiments are only illustrative of the technical solutions of the present invention, and are not intended to limit the scope of the present invention. Furthermore, various changes, modifications and equivalents of the embodiments of the invention that may occur to those skilled in the art after reading the teachings herein will also fall within the scope of the claims appended hereto.
SEQUENCE LISTING
<110> university of southern China's science
<120> silver nano material, biological preparation method and application thereof
<130>
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 572
<212> DNA
<213> Fusarium solani D07
<400> 1
tttcctccgg cctttgatat gcttaagttc agcgggtatt cctacctgat ccgaggtcaa 60
cattcagaag ttggggttta acggcgtggc cgcgacgatt accagtaacg agggttttac 120
tactacgcta tggaagctcg acgtgaccgc caatcaattt ggggaacgcg aattaacgcg 180
agtcccaaca ccaagctgtg cttgagggtt gaaatgacgc tcgaacaggc atgcccgcca 240
gaatactggc gggcgcaatg tgcgttcaaa gattcgatga ttcactgaat tctgcaattc 300
acattactta tcgcattttg ctgcgttctt catcgatgcc agaaccaaga gatccgttgt 360
tgaaagtttt gatttattta tggttttact cagaagttac atatagaaac agagtttagg 420
ggtcctctgg cgggccgtcc cgttttaccg ggagcgggct gatccgccga ggcaacaagt 480
ggtatgttca caggggtttg ggagttgtaa actcggtaat gatcctccgc tggttcacca 540
acggagacct tgttacgatt tttttacttc ca 572
Claims (6)
1. The biological preparation method of the silver nano material is characterized by comprising the following steps:
(1) activating the dendrobium officinale endophytic fungi strain, then performing seed liquid culture, and performing fermentation culture on the obtained seed liquid; the dendrobium officinale endophytic fungus strain is separated from the root of dendrobium officinale, is named as Fusarium solani DO7 by latin and has the preservation number of CCTCC NO. M2017145; the activation is carried out by adopting a test tube slant culture method at 28 +/-1 ℃ for 48-72 hours under the aseptic condition, wherein the culture medium is a potato glucose agar culture medium; the seed liquid culture is characterized in that under the aseptic condition, a potato glucose water culture medium is adopted to carry out shake culture for 48-72 hours at the temperature of 28 +/-1 ℃ and the rotating speed of 120 rpm;
(2) the step (1) is finally carried outFiltering the obtained culture solution, washing with sterile deionized water, culturing the obtained mycelium in sterile deionized water, filtering again, adding AgNO into the obtained filtrate3Carrying out reaction; after the reaction is finished, centrifuging and collecting the precipitate to obtain the silver nano material;
said addition of AgNO3Keeping AgNO added during the reaction3The concentration of (1) is 1 mmol/L; the reaction is carried out for 24-48 h at the temperature of 25 ℃ and the rotating speed of 120 rpm.
2. The biological preparation method of silver nano-material according to claim 1, wherein in the step (1), the fermentation culture is carried out under aseptic conditions by inoculating the obtained seed solution into a liquid culture medium according to an inoculum size of 10v%, and culturing for 3-4 days at 28 ± 1 ℃ and 120 rpm.
3. The biological preparation method of silver nano-material according to claim 1, wherein in the step (2), the sterile deionized water is Milli-Q deionized water; the culture in the sterile deionized water is carried out for 24-48 h under the conditions of the temperature of 28 +/-1 ℃ and the rotating speed of 120rpm under the sterile condition.
4. The biological preparation method of silver nano-material according to claim 1, wherein in the step (2), the centrifugation is performed at 12000g for 20min at 4 ℃.
5. The silver nanomaterial prepared by the preparation method of any one of claims 1 to 4, wherein the silver nanomaterial is a round or oval nanoparticle material, and the particle size of the particles is 16 to 46 nm.
6. The silver nano material of claim 5 is applied to preparation of antibacterial materials.
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| CN112159765B (en) * | 2020-10-22 | 2022-06-07 | 华侨大学 | Ageratum endophytic fungus Letenadraea sp.WZ07 and application thereof in nano silver synthesis |
| CN114806887B (en) * | 2022-03-28 | 2023-09-05 | 云南大学 | Fungus strain for promoting germination of Jinsha Jiang Danhu and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102888428A (en) * | 2011-07-21 | 2013-01-23 | 中国科学院过程工程研究所 | Method for synthesizing nano silver by utilizing Bacillus amyloliquefaciensBacillus amyloliquefaciens LSSE-62 |
| CN104762329A (en) * | 2015-02-06 | 2015-07-08 | 东南大学 | Method using fusarium venenatum 1281-2 to synthesize nano-silver |
| CN105935781A (en) * | 2016-06-06 | 2016-09-14 | 江苏省农业科学院 | Biological method for preparing nano-silver |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102888428A (en) * | 2011-07-21 | 2013-01-23 | 中国科学院过程工程研究所 | Method for synthesizing nano silver by utilizing Bacillus amyloliquefaciensBacillus amyloliquefaciens LSSE-62 |
| CN104762329A (en) * | 2015-02-06 | 2015-07-08 | 东南大学 | Method using fusarium venenatum 1281-2 to synthesize nano-silver |
| CN105935781A (en) * | 2016-06-06 | 2016-09-14 | 江苏省农业科学院 | Biological method for preparing nano-silver |
Non-Patent Citations (1)
| Title |
|---|
| Screening of Different Fusarium Species to Select Potential Species for the Synthesis of Silver Nanoparticles;Swapnil C. Gaikwad等;《J. Braz. Chem. Soc.》;20131231;第24卷(第12期);第1975-1976前言部分和第1980页结论部分 * |
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