KR20140077645A - Microbial agent formulations for improving water quality and sediment quality - Google Patents
Microbial agent formulations for improving water quality and sediment quality Download PDFInfo
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- KR20140077645A KR20140077645A KR1020120146678A KR20120146678A KR20140077645A KR 20140077645 A KR20140077645 A KR 20140077645A KR 1020120146678 A KR1020120146678 A KR 1020120146678A KR 20120146678 A KR20120146678 A KR 20120146678A KR 20140077645 A KR20140077645 A KR 20140077645A
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
- C02F3/341—Consortia of bacteria
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Abstract
More particularly, the present invention relates to a gel-type formulation for improving water quality and low quality, and more particularly, to a method for producing a gel-type formulation of the present invention by inoculating mixed cultivars into various culture materials and culturing them at an ultra-high temperature. And a method for producing the same. According to the present invention, it is possible to manufacture a microorganism preparation which can be safely used in a wide application environment without any variation, and it is possible to effectively improve water quality and low quality in a short time, It provides the environmental improvement effect of the circulation. It compensates the disadvantages of the liquid or powder form, and it is possible to maintain and manage for a long time. It is effective for long-term storage of microorganisms and is easy to store because of its small volume, Lt; / RTI >
Description
The present invention relates to a microbial agent gel-type formulation for improving the quality of poultry, lake water and the like and their preparation.
Although there is an increase in the use demand of the basin and the lake coast as a hydrophilic space, the pollution of water quality and low quality is increased to such an extent that the non-point pollutant flowing from the land is increased, have. In the case of fresh water reservoirs such as low-tide, low-polluted, and polluted water, polluted conditions are much more polluted than water quality. This is because not only the exchange of water within and outside the water body is not smooth, but also the bottom layer is severely decayed due to the continuous inflow of pollutants into the water. Water quality is also continuously contaminated by pollutants released from these low-level sediments. The decrease in the pH of the contaminated sediment layer, the decrease in dissolved oxygen (DO) in the bottom layer of the water, and the rapid increase in nutrients and hydrogen sulfide (H2S) accelerate the eutrophication of water quality in general, It is the cause of the occurrence.
Therefore, water quality improvement was not achieved due to the elution of nutrients such as nitrogen and phosphorus in sedimentary juni (low-level sediments) in the ecosystem and in the closed waters of the low tide area where the eutrophication proceeded, It is urgently required to develop a technique for improving the quality of the floor. As a remedy for degradation, sedimentation of sediments deposited in the lower reservoir has been carried out. However, it has not been possible to improve the quality of sediments.
In recent years, medicines for improvement of low quality have been aimed at removing water pollution by forming sediments by forming an insoluble compound with acidic water pollutants such as yellow loam, lime lime and quick lime. However, Causing the water quality to deteriorate.
Common microbial agents are mostly used in liquid or powder form. In the case of liquid microbial formulations, it is advantageous to be activated immediately upon administration, but it is bulky and has a heavy weight and has problems of carrying or carrying. On the other hand, in the case of powder, it has an advantage of being able to be semi-permanently preserved, while a large amount of non-freezing agent and other substances other than freeze-dried powders are added during manufacturing to adversely affect activity, The products have the disadvantage that they are pre-cultured when they are administered to the aeration tank. In addition, these products are applied to a large-scale treatment facility with a constant load, but it is required to develop a formulation suitable for application to a small-scale treatment facility such as an individual or a small farm with a low load.
Accordingly, the present inventors have developed a microbial agent having an activity of decomposing contaminants accumulated in water quality and low quality by activating water quality and low self-tacking force, and it has the advantage of having a microbial agent of liquid or powder form, The present invention has been completed by preparing a gel-type microorganism preparation in which a liquid microorganism preparation is solidified.
It is an object of the present invention to provide a water quality and low quality improvement agent which is applied to a lower layer of water bodies such as low tide, low water level, etc. to improve their low quality. In addition to not only decomposing contaminants deposited in the low tile, A microorganism preparation of a gel type formulation which exhibits an effect of improving water quality and low quality through sterilization of harmful microorganisms, which can be maintained and managed for a long time, has a long-term storage effect of microorganisms and is small in volume and easy to store, and a method for producing the microorganism preparation And the like.
Particularly, in the water quality and low quality improvement agent for improving water quality and low quality of low tide and lake according to the present invention, montmorillonite in powder form in which Bacillus subtilis is adsorbed is mixed, Etc. are adsorbed to montmorillonite in a dormant state and start to react when they come into contact with water in water.
Specifically, the object of the present invention is to provide a microorganism preparation having an activity of decomposing accumulated contaminants by activating self-tacking force without secondary contamination, and has the advantage of having a liquid or powdery microorganism, Based on the above-mentioned various problems, it is possible to make effective use of a microorganism preparation which is environmentally friendly and does not cause secondary pollution in the water body, It is a main object of the present invention to provide a low quality improvement agent capable of effectively improving the quality of the lower layer of the water body, and a field application technique of simply administering such a microorganism based low quality improvement agent at a contaminated site is also an object of the present invention It is one.
In order to achieve the above object,
1) Bacillus sonorensis, Bacillus sp. TUT1206, Bacillus sp. Q-12, and Bacillus sp., Which are composed of Bacillus subtilis and represented by Bacillus subtilis; At least one microorganism mixed seed selected from the group consisting of Bacillus thermoamylovorans, Leuconostoc paramesenteroides, and Pediococcus pentosaceis strain LM2632. %, 2 to 5% by weight of rice bran, 2 to 4% by weight of molasses, 2 to 4% by weight of sulfuric acid, nitric acid (2 to 5% by weight) Calcium carbonate (Ca (NO 3 ) 2 .4H 2 O) 20% (the highest concentration that can dissolve phosphorus from the low-level sediments and coagulate with agar), coagulant (agar) 1.2 wt% Since the water-soluble inhibits the rate at which mobile by pore), relaxes, continue to the inhibition rate of calcium carbonate (CaCO 3) 20 wt% (calcium nitrate), and dispersed or dissolved, such as water 38.8% by weight (in each reagent) To prepare a microorganism preparation;
2) 20 to 30% by weight of at least one culture raw material selected from the group consisting of rice bran, agricultural by-products, and food waste dry powder, and 70 to 80% by weight of the mixed bacterial liquid, 0.01 to 0.1% by weight of the mixed seed is inoculated on a weight basis;
3) culturing the inoculated mixed material at a temperature in the range of 80 to 90 ° C for 2 to 10 hours;
4) adding water to the culture, adding gelatin, agar and carboxymethyl cellulose to the microorganism preparation at a weight ratio of 10: 1: 1, and solidifying the formulation into a formulation. The present invention provides a method for producing a gel-type formulation.
The present invention also provides a microbial preparation for improving the water quality and low quality of a gel-type formulation prepared by the production method according to the present invention.
In addition, the present invention provides a method for improving water quality and low quality, comprising the step of administering the gel-type formulation of the present invention to a water quality and a low quality microorganism preparation for improvement of water quality and low quality.
Hereinafter, the present invention will be described in detail.
The present invention relates to a microbial agent having an activity of decomposing accumulated contaminants by activating self-tacky forces without secondary contamination, and has the advantage of having a liquid or powdery microbial agent, A microorganism preparation base capable of effectively improving the quality of the lower layer of the water body while making effective use of a microorganism preparation that does not cause secondary contamination in a water body such as low tide, And a method for producing the same.
The present invention is not only to decompose accumulated contaminants by activating self-tacking force without secondary contamination, but also to exhibit the effect of improving water quality and low quality through sterilization of harmful microorganisms, being able to maintain and manage for a long time, A microbial agent of a gel type which is small in volume and easy to store, and a method for producing the same.
Specifically, the present invention can be produced by a manufacturing method comprising the following steps.
1) a mixture of at least one microorganism selected from the group consisting of Bacillus subtilis (Bacillus subtilis) in an amount of 40 to 50% by weight 20% by weight of calcium nitrate (Ca (NO 3 ) 2 .4H 2 O) (the highest concentration which can be solidified with agar as a main ingredient for inhibiting the elution of phosphorus from low-layer sediments), coagulant (agar) % (it inhibits the rate at which mobile by pore because it is effective calcium nitrate is a water-soluble), (relaxes, continues to the inhibition rate of calcium nitrate), calcium carbonate (CaCO 3) 20% by weight, water 38.8% by weight (in each reagent such as To prepare a microorganism preparation;
2) 20 to 30% by weight of at least one culture raw material selected from the group consisting of rice bran, agricultural by-products, and food waste dry powder, and 70 to 80% by weight of the mixed bacterial liquid, 0.01 to 0.1% by weight of the microorganism mixed seeds is inoculated on a weight basis;
3) culturing the inoculated mixed material at a temperature in the range of 80 to 90 ° C for 2 to 10 hours;
4) adding gelatin, agar and carboxymethyl cellulose to the cultured microorganism preparation at a weight ratio of 10: 1: 1 to solidify into a formulation.
In the above production method, step 1) is carried out in such a manner that 40% by weight of at least one mixed microorganism selected from the group consisting of Bacillus genus (the ratio of the specific gravity of sediment to be sedimented to the lower sediments, ), 20% by weight of calcium nitrate (Ca (NO 3 ) 2 .4H 2 O) (the highest concentration that can coagulate with agar as the main ingredient for inhibiting the elution of phosphorus from low-layer sediments), 1.2% 20% by weight of calcium carbonate (alleviating / sustaining the phosphorus inhibition rate of calcium nitrate) and 38.8% by weight of water (dispersing and dissolving each reagent) To prepare a microorganism preparation;
2) 20 to 30% by weight of at least one culture raw material selected from the group consisting of rice bran, agricultural by-products, and food waste dry powder, and 70 to 80% by weight of the mixed bacterial liquid, 0.01 to 0.1% by weight of the microorganism mixed seeds is inoculated on a weight basis;
3) culturing the inoculated mixed material at a temperature in the range of 80 to 90 ° C for 2 to 10 hours;
4) adding gelatin, agar and carboxymethyl cellulose to the cultured microorganism preparation at a weight ratio of 10: 1: 1 to solidify into a formulation.
In the above production method, step 1) is carried out in such a manner that 40% by weight of at least one mixed microorganism selected from the group consisting of Bacillus genus (the ratio of the specific gravity of sediment to be sedimented to the lower sediments, ), 20% by weight of calcium nitrate (Ca (NO 3 ) 2 .4H 2 O) (the highest concentration that can coagulate with agar as the main ingredient for inhibiting the elution of phosphorus from low-layer sediments), 1.2% inhibits the rate at which mobile by pore because it is water soluble), it is dispersed, dissolved, such as calcium carbonate (CaCO 3) 20% by weight (thus continued, relieve the inhibition rate of calcium nitrate), water 38.8% by weight (in each reagent ) Is adjusted to 100%, air is kept at a temperature of 20 to 25 ° C, and air of 5 to 10 m 3 / h is aerated for 1 to 3 hours at intervals of 2 to 6 hours repeatedly for 2 to 6 times a day For 15 to 21 days It is preferable to prepare the microorganism preparation by adding the microorganism.
The Bacillus spp. Or Lactobacillus sp. Are all known to have the Bacillus spp. Or Lactobacillus spp. Listed above. For example, Bacillus subtilis, also known as Bacillus subtilis, is a type of aerobic bacterium belonging to the diatomaceae and is a bacterium distributed in nature. Gram positive bacterium that forms heat-resistant spores. The live cells move by the main flagella in the size of 0.7-0.8 ㎛ × 2.0-3.0 ㎛. Since the spores are resistant to boiling for several hours, the sterilization of the spores must be done by autoclaving, and the liquid culture forms a film with a slightly wrinkled surface. It is used to industrially produce these enzymes because they produce strong amylase proteases. Bacillus subtilis is used in genetic studies such as transformation, and recently it has also been used in research on genetic engineering.
The lactic acid bacteria belong to gram-positive bacteria and are taxonomically classified into Eubacteriales and classified into Lactobacilleae and Streptococcaceae. Lactobacillus is the former and Lactobacillus is the latter. Streptococcus, Pediococcus, Leucooxtoc, and the like. It is distinguished morphologically by lactic acid bacterium (Lactobacillus sp.) And lactic acid bacterium (Streptococcus sp., Pediococcus sp., Leuconostok sp.), But there are various kinds and 20 kinds of lactic acid bacteria are applied to the fermented milk production and lactic acid bacteria industry have. The lactic acid bacteria according to the present invention are preferably, but not limited to, Leuconostoc paramesenteroides or Pediococcus pentosaceis strain LM2632.
The mixed seed bacteria further include one or more strains selected from the group consisting of Enterobacter hormaechei, Weissella helenica, or Xanthomonas sp. EBHRS01-2. can do.
In the above production method, step 2) comprises preparing 20 to 30% by weight of one or more culture materials selected from the group consisting of rice bran, agricultural by-products and food waste dry powder, and the mixed bacterial liquid agent 70 to 80 % By weight of the mixed seed culture, and then inoculating the mixed seed culture with 0.01 to 0.1% by weight of the mixed seed culture.
The culture material may be used alone or in combination with the rice bran, agricultural by-products or food waste, preferably 35 to 45 wt% rice bran, 25 to 35 wt% agricultural by-products, 20 to 30 wt% . It is preferable that the culture material is pulverized to about 100 mesh in advance and used as a powder.
The mixed and pulverized raw materials are put into a flask and the water is controlled with a liquid mixed microbial agent to maintain an environment suitable for microbial fermentation, and then mixed seeds including three or more species of Bacillus sp. Or Lactobacillus are inoculated. The microorganism having the seasonal and environmental diversity is collected as it is without any artificial filtration and is subjected to environmental adaptation process for 6 months on the natural material medium to remove the harmfulness to prepare the powdery microorganism. In the production of the microorganism preparation according to the present invention, it is more preferable to inoculate 0.01 to 0.1% by weight of the mixed germs with respect to the total weight of the raw materials.
In the above production method, it is preferable that the step 3) is carried out after the inoculating step is followed by a high-temperature culturing step in which the inoculated mixed material is cultured at 80 to 85 ° C for 2 to 8 hours. Considering that ordinary microorganism cultivation is generally performed at a temperature in the range of 20 to 40 DEG C, the microorganism preparation of the present invention is carried out under ultrahigh temperature conditions. In particular, in one embodiment of the present invention, the cultured raw material inoculated with the mixed seed is incubated at 80 to 85 ° C for 2 to 8 hours at a rate of 30 to 80 rpm / min while being stirred at an ultra-high temperature. At this time, the incubation temperature is preferably 80 ° C. or higher to inhibit the growth of unnecessary microorganisms, but it is preferably carried out at 85 ° C. or lower to maintain the activity of the mixed bacteria according to the present invention. There are no particular restrictions on the composition of the medium that can be used in the composition of the culture medium used for the culture, and the composition can be suitably selected according to the kind of the microorganism. In particular, magnesium sulfate (MgSO 4), yeast extract, casein Casein, Glucose, ammonium sulfate (NH4) 2SO4), sodium carbonate (CaCO3), sodium acetate (CH3COONa), potassium monophosphate (KH2PO4), potassium phosphate dibasic (K2HPO4) edible defoamer and ammonium chloride NH4Cl) or the like.
In the microorganism preparation for improving water quality and low quality of the above-described low-runoff and lake, montmorillonite powder-form in which Bacillus subtilis is adsorbed is mixed and the Bacillus subtilis is adsorbed to montmorillonite in a dormant state When it comes into contact with water in water, it starts its activity.
The present invention also provides a microbial preparation for improving the water quality and low quality of a gel-type formulation prepared by the production method according to the present invention.
In addition, the present invention provides a method for improving water quality or low quality, comprising the step of administering the gel-type formulation of the present invention to a water quality or a low quality microorganism preparation for improvement of water quality and low quality.
The microbial formulation according to the present invention may contain various additives such as mineral (coagulant), alginic acid and its salt, organic acid, protective colloid thickening agent, or formulation used for molding if necessary.
The microbial formulation according to the present invention activates the self-tacking force without secondary contamination of water quality and low quality to decompose the contaminants deposited in the lower layer, and also shows the effect of improving water quality and low quality by sterilizing harmful microorganisms. In addition, the microbial formulation of the gel type formulation according to the present invention can simultaneously solve the problems of conventional microbial agents, that is, in the case of solid type, degradation of treatment efficiency due to low activation of microorganisms and storage problems of liquid type, It is possible to maintain and manage for a long time, has a long-term storage effect of microorganisms, is small in volume, and is easy to store.
Hereinafter, the present invention will be described in more detail by way of examples of the present invention. However, the following examples are intended to assist the understanding of the present invention, and the scope of the present invention is not limited to the following examples.
< Example 1> Preparation of liquid microorganism preparation
<1-1> Isolation of mixed seeds
The soil samples containing microorganisms (composted soil in the forests of Jecheon, Chungbuk) were heat-treated at 60 ℃ for 30 min and then finely ground in a mortar. Then, 1 g of the sample was suspended in 9 ml of 0.85% NaCl, 0 to 10 <" 7 & gt ;. 100 μl of each dilution suspension was plated on TSA, BL, and BBL medium (DIFCO), cultured at 28 ° C, and cultured using ANAEROGENTM COMPACT (OXOID) to identify anaerobic strains. The cultured strains were selected by colony morphology and cultured at least three times in TSA medium to separate into a single colony. The isolated strains were cultured in liquid, purified by genomic DNA, amplified with 16S RNA primer Next, the amplified region was used as a template and the sequence was sequenced using 519r and 785r primers. The primers used to identify bacteria with 16S RNA were named "Nucleic acid techniques in bacterial systematics (John Wiley and Sons, England)" and "nucleic acid research (2000) Bacillus subtilis, Bacillus sonorensis, Bacillus sp. TUT1206, Bacillus subtilis, Bacillus subtilis, and Bacillus spp. Bacillus thermoamylovorans, Leuconostoc paramesenteroides, Pediococcus pentosaceis strain LM2632, Enterococcus pentosaceus strain LM2632, Bacillus spp. Enterobacter hormaechei, Weissella hellenica, Xanthomonas sp. EBHRS01-2, Bordetella avium, Bacillus oryzae, Bacillus spp. Bacillus cereus, Bacillus cereus, Microbacterium esteraromaticum, Bordetella petrii, Streptomyces albus, Bacillus megaterium, Bacillus cereus, Bacillus badius, Bacillus licheniformis, Paenibacillus cineris, and Staphylococcus pasteuri.
≪ 1-2 > Liquid Produce
20% by weight of calcium nitrate (Ca (NO 3 ) 2 .4H 2 O) (the specific gravity for sedimentation to about 5 cm into the lower layer sediments (juniors) 1.2% by weight of coagulant (agar) (calcium nitrate is effective to inhibit the rate at which it moves to pore water because of its water solubility), calcium carbonate (CaCO 3 ) 20% by weight of calcium nitrate (the phosphorus inhibiting rate of calcium nitrate is lowered and maintained), and 38.8% by weight of water (dispersing and dissolving each reagent) are mixed and mixed at a weight of 100 kg, Aeration of 10 ㎥ / h of air for 2 hours at intervals of 4 hours was repeated four times a day for 21 days to prepare a mixed bacterial suspension. The mixed bacterial suspension had a total number of bacteria of 4,3 x 10 9 cfu / g. Then, pre-milled 65 kg of rice bran and 35 kg of agricultural by-products were placed in an incubator with mixing function and mixed for 30 minutes. 30 kg of the mixed bacterial suspension was added to the mixed raw materials to adjust the moisture concentration to 70%, and then the mixed seeds of 4.3 x 10 9 cfu / g were inoculated at 0.01% of the total weight of the culture materials. The inoculated culture material was agitated at an external temperature of 300 ° C and an internal temperature of the incubator of 80 to 85 ° C at 30 to 80 rpm / min for 4 hours. The cultured raw material after completion of cultivation was cooled to room temperature (20 to 25 캜).
< Example 2> Liquid microorganism preparation Gel type Manufacturing of formulations
The final volume of the liquid microorganism preparation prepared in Example 1 was adjusted to 100 L, and an appropriate amount was added to the lyophilized plate, followed by freezing in a deep freezer for 24 hours. After the freezing was completed, it was rapidly transferred to the freeze dryer and recovered 2 to 3 days later. In the method for preparing the gel-type composition of the liquid microorganism preparation, gelatin and other components were wet-sterilized in distilled water at 121 ° C for 15 minutes. The wet sterilized gelatin solution was maintained at about 38 to 43 캜, and then the gelatin solution was mixed with the lyophilized liquid microorganism bacteria in the agitator. The above mixed solution was poured into a cylindrical mold having a diameter of 8.5 cm and a height of 16 cm and solidified in a refrigerated room at 4 ° C for 12 hours. Finally, gel formulations of liquid microbial preparations were prepared by separating the mixture from the mold and the solidified gelatin mixture. The components of the gel-type formulation are shown in Table 1 below.
< Example 3> Microorganism preparation through vegetable cultivation Ineffective black
<3-1> Confirming the Effect of Growing on Vegetables
60 kg of the gel-type microorganism preparation prepared in Example 2 was treated at a paddy field of 300 pyeongs, and 21 days later, the alpine and alpine lettuce were inoculated and cultivated and cultivated at a ratio of 21: 17: 17 The validity of traits (leaf number, leaf length, rhizome, rootstock, weekday) was compared with one control. As a result, the vegetables of the experimental group treated with the microbial formulation of the gel type showed about 25 ~ 30% of the increase effect compared to the control without the treatment.
<3-2> Confirm reusability
When the gel composition was wrapped and stored for one week in a refrigerator and then re-treated, the effect of the gel composition on the growth of vegetables was measured. As a result, the same tendency as in the case of the immediately processed preparation was observed, Respectively.
Claims (7)
2) 20 to 30% by weight of at least one culture raw material selected from the group consisting of rice bran, agricultural by-products, and food waste dry powder, and 70 to 80% by weight of the mixed bacterial liquid, Inoculating 0.01 to 0.1% by weight of the soil microorganism mixed seeds on the basis of weight;
3) culturing the inoculated mixed material at a temperature in the range of 80 to 90 ° C for 2 to 10 hours;
4) adding gelatin, agar and carboxymethyl cellulose (CMC) to the cultured microorganism preparation at a weight ratio of 10: 1: 1 to solidify the mixture into a formulation; and A method for producing a gel-type formulation for microbial preparation for improving low quality.
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| KR1020120146678A KR20140077645A (en) | 2012-12-14 | 2012-12-14 | Microbial agent formulations for improving water quality and sediment quality |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105941197A (en) * | 2016-04-22 | 2016-09-21 | 苏州依科曼生物农业科技有限公司 | Micro nutrient solution and application thereof |
| KR102277780B1 (en) * | 2020-12-10 | 2021-07-15 | 세움 주식회사 | Method for manufacturing wastewater treatment agent by soil microbes |
| CN114836333A (en) * | 2021-02-01 | 2022-08-02 | 武汉渔庆家电子商务有限公司 | Blue algae purifying agent and preparation and application thereof |
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2012
- 2012-12-14 KR KR1020120146678A patent/KR20140077645A/en not_active Application Discontinuation
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105941197A (en) * | 2016-04-22 | 2016-09-21 | 苏州依科曼生物农业科技有限公司 | Micro nutrient solution and application thereof |
| KR102277780B1 (en) * | 2020-12-10 | 2021-07-15 | 세움 주식회사 | Method for manufacturing wastewater treatment agent by soil microbes |
| CN114836333A (en) * | 2021-02-01 | 2022-08-02 | 武汉渔庆家电子商务有限公司 | Blue algae purifying agent and preparation and application thereof |
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