CN112646770A - Preparation method of extracting solution with ultraviolet protection effect on bacillus thuringiensis - Google Patents
Preparation method of extracting solution with ultraviolet protection effect on bacillus thuringiensis Download PDFInfo
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- CN112646770A CN112646770A CN202110102535.XA CN202110102535A CN112646770A CN 112646770 A CN112646770 A CN 112646770A CN 202110102535 A CN202110102535 A CN 202110102535A CN 112646770 A CN112646770 A CN 112646770A
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- bacillus thuringiensis
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- 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
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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
- C12N1/04—Preserving or maintaining viable microorganisms
Abstract
A method for preparing an extracting solution with ultraviolet protection effect on Bacillus thuringiensis comprises drying mushroom bran, pulverizing mushroom bran, extracting mushroom bran water extract, and evaluating ultraviolet resistance effect. The invention takes culture medium mushroom residue after picking lucid ganoderma as a main raw material, utilizes waste, and obtains mushroom residue water extract after crushing and adding water for high-temperature treatment. The aqueous extract of the mushroom bran is directly added into a bacillus thuringiensis preparation, the production steps are simple, the ultraviolet resistance effect of the bacillus thuringiensis is improved, and the application range of the mushroom bran is widened. The result shows that the fungus chaff leaching liquor can obviously improve the ultraviolet stability of the bacillus thuringiensis preparation, and the spores, crystal proteins and biological activity of the bacillus thuringiensis are well protected. The ultraviolet-resistant additive obtained by the invention has good ultraviolet-resistant effect, and the ultraviolet stability of the bacillus thuringiensis preparation is improved.
Description
Technical Field
The invention relates to a preparation method of an extracting solution with an ultraviolet protection effect on bacillus thuringiensis, in particular to a fungus chaff extracting solution for protecting bioactive components of bacillus thuringiensis from being damaged by ultraviolet rays, and belongs to the technical field of biological pesticides.
Background
Bacillus thuringiensis (B.thuringiensis) (B.thuringiensis)Bacillus thuringiensisBt) is a gram-positive bacterium that produces a variety of Crystal Proteins (ICPs) when it forms spores, and has a specific Insecticidal action against a variety of pests such as lepidoptera, diptera, coleoptera, homoptera, and nematodes. Therefore, the bacillus thuringiensis preparation is widely applied to preventing and controlling pests in the fields of agriculture, forestry and sanitation, and becomes a biopesticide preparation with the widest application range and the largest application amount in the world. However, the bacillus thuringiensis preparation has weak ultraviolet stability, and easily loses biological activity due to the irradiation of sunlight, so that the use of the bacillus thuringiensis is limited to a certain extent. The added ultraviolet antagonists currently studied are mainly some expensive and environmentally unfriendly preparations, and therefore, the use of these additives is also limited.
China is the biggest fungus producing country in the world, annual output accounts for 60% of the world, and annual output is rising year by year. The mushroom bran is agricultural waste left after mushroom production, contains rich organic matter components, and is widely applied to the fields of animal feed, microbial fermentation, soil improvement, high-value enzyme extraction and the like. However, the use amount of the existing fungus chaff cannot keep up with the output amount, and the optionally discarded fungus chaff causes harm to the environment. Therefore, if the fungus chaff is used for improving the ultraviolet stability of the bacillus thuringiensis, the fungus chaff can be changed into valuable, the cost of the bacillus thuringiensis is reduced, the environmental pollution can be reduced, and the ecological effect and the economic effect are obvious.
Disclosure of Invention
In view of the problems existing at present, the invention aims to prepare a mushroom bran water extract, determine the ultraviolet absorption and oxidation resistance of the mushroom bran water extract and evaluate the ultraviolet protection effect of the mushroom bran water extract on a bacillus thuringiensis preparation. The fungus chaff is applied to the ultraviolet protection of the bacillus thuringiensis through simple treatment, so that the utilization rate of the fungus chaff can be improved, the environmental pollution is reduced, and the stability of the bacillus thuringiensis can be improved. The mushroom bran refers to culture waste left after harvesting of the lucid ganoderma.
In order to achieve the above object, the technical solution of the present invention is: a preparation method of an extracting solution with ultraviolet protection effect on Bacillus thuringiensis is characterized by comprising the following operation steps:
removing polyethylene outer bags from harvested ganoderma lucidum fungus chaff (the fungus chaff comprises 2wt% of gypsum powder, 30 wt% of dicranopteris pedata, 48 wt% of miscanthus floridulus and 20 wt% of film), drying at 60 ℃ until the water content is lower than 10%, crushing the dried ganoderma lucidum fungus chaff by a universal high-speed crusher, and sieving by a standard sieve of 60 meshes; weighing a certain amount of sieved fungus chaff, adding single distilled water according to the mass ratio of 20 times, and shaking up for 2 hours in a shaking table with 150 rpm; treating the mixture in a 15-pound per inch pressure autoclave at 121 deg.C for 60 min; and cooling the mixed solution, centrifuging and collecting supernatant, and performing suction filtration by using a suction filter to remove filter residues to obtain the ganoderma lucidum bran water extract.
The rotating speed of the universal high-speed pulverizer is 30000 rpm; centrifuging by the centrifuge, wherein the centrifuging speed is not lower than 900 rpm, and the centrifuging time is not less than 5 min; the suction filter has suction pressure not greater than 0.45 MPa.
Further, the method for improving the ultraviolet stability of the bacillus thuringiensis by using the extracting solution prepared by the method comprises the following steps:
an ultraviolet irradiation box is adopted to evaluate the anti-ultraviolet activity of the fungus chaff water extract, the fungus chaff extract and the Bacillus thuringiensis fermentation liquor are mixed according to the proportion of 1:1 in a 25 mL glass small beaker, and are placed in the ultraviolet irradiation box to be irradiated for a certain time, and samples are respectively taken at 0 hour, 12 hour, 24 hour, 48 hour, 72 hour and 96 hour. Finally, single distilled water is used for fixing the volume, an LB plate is used for evaluating the survival condition of spores, SDS-PAGE protein gel is used for verifying the retention condition of crystal protein, and the biological activity protection effect is evaluated by cotton bollworm biological assay.
The technical principle of the invention is as follows: the ganoderma lucidum mycelia can secrete various enzymes in the growth process, the enzymes can decompose part of lignocellulose to form soluble polysaccharide and lignin, and then part of polysaccharide and lignin with reduction activity is dissolved in water under the action of hot water, so that the black brown fungus chaff water extract is obtained. The water extract of the mushroom bran can absorb ultraviolet rays and eliminate active oxygen generated by the ultraviolet rays, thereby achieving the purpose of protecting the active ingredients of the bacillus thuringiensis.
The invention has the following advantages:
1) solves the problem of fungus chaff treatment and application, prevents the waste of substances and the pollution to the environment, comprehensively utilizes the substances and has the function of changing waste into valuable.
2) The leaching liquor can be obtained only by soaking in hot water without a complex extraction process, the preparation threshold is low, the operation is easy, and the extraction cost is low.
3) The mushroom bran has wide source, low price and low use cost.
Drawings
FIG. 1 shows the protective effect of the aqueous extract of mushroom bran on the spores of Bacillus thuringiensis;
FIG. 2 shows the protective effect of the aqueous extract of mushroom bran on the crystal protein of Bacillus thuringiensis;
FIG. 3 shows the protective effect of the aqueous extract of mushroom bran on the biological activity of Bacillus thuringiensis.
Detailed Description
The specific implementation mode of the invention is as follows:
embodiment 1a method for preparing an extracting solution with ultraviolet protection effect on bacillus thuringiensis and application thereof:
(1) removing polyethylene outer bag from Ganoderma bran, oven drying at 60 deg.C until water content is less than 10%, pulverizing with universal high-speed pulverizer, and sieving with 60 mesh standard sieve;
(2) weighing a certain amount of sieved fungus chaff, adding single distilled water according to the mass ratio of 20 times, and shaking up for 2 hours in a shaking table with 150 rpm;
(3) treating the mixture in a 15-pound per inch pressure autoclave at 121 deg.C for 60 min; and cooling the mixed solution, centrifuging, collecting supernatant, performing suction filtration by using a suction filter to remove filter residues, drying the mushroom bran water extract to be absolutely dry by using a drying method at 50 ℃, weighing the mass difference before and after, and calculating the mass ratio of dry substances contained in the water extract according to the mass difference. Then, according to the proportion, the fungus chaff leaching solution is respectively diluted by water to obtain the ganoderma lucidum fungus chaff leaching solution with the mass fractions of 0.15%, 0.3% and 0.6%.
(4) Mixing the fungus chaff leaching liquor with a fermented 72h bacillus thuringiensis bacterial liquid according to a volume ratio of 1:1, wherein the bacterial liquid is mainly a spore mixture and contains 2 x 106Number of spores CFU/mL. Fully and uniformly shaking, and placing in a 25 mL glass beaker;
(5) placing the mixed sample in an ultraviolet irradiation box, and irradiating for 0, 12, 24, 48, 72 and 96 hours;
(6) adding water to a constant volume sample, coating a plate to calculate the survival rate of spores, measuring the protection condition of protein strips, and measuring the retention condition of biological activity of each treatment after irradiating for 96 hours. The experimental results are shown in fig. 1, fig. 2 and fig. 3.
As can be seen from fig. 1, the aqueous extract of mushroom bran can significantly improve the ultraviolet resistance of spores, and the higher the concentration of the aqueous extract of mushroom bran, the better the protection effect.
As can be seen from fig. 2, the aqueous extract of the mushroom bran can significantly improve the ultraviolet resistance of Cry1Ac crystal protein (130 kDa), and the higher the concentration of the aqueous extract of the mushroom bran, the better the protection effect.
As can be seen from fig. 3, the aqueous extract of mushroom bran can significantly improve the ultraviolet stability of the insecticidal activity of bacillus thuringiensis, and the higher the concentration of the aqueous extract of mushroom bran, the better the protection effect.
Note: the UV light is generated by UVA lamp (wavelength 352 nm) and UVB lamp (wavelength 306 nm), both at an intensity of 15W and at a distance of 12.5 cm.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various changes and modifications can be made without departing from the overall concept of the present invention, and these should also be considered as the protection scope of the present invention.
Claims (3)
1. A preparation method of an extracting solution with ultraviolet protection effect on Bacillus thuringiensis is characterized by comprising the following steps:
removing polyethylene outer bag from Ganoderma bran, oven drying at 60 deg.C until water content is less than 10%, pulverizing with universal high-speed pulverizer, and sieving with 60 mesh standard sieve; weighing sieved fungus chaff, adding single distilled water according to the mass ratio of 20 times, and shaking up for 2 hours in a shaking table at 150 rpm; treating the mixture in a 15-pound per inch pressure autoclave at 121 deg.C for 60 min; and cooling the mixed solution, centrifuging and collecting supernatant, and performing suction filtration by using a suction filter to remove filter residues to obtain the ganoderma lucidum bran water extract.
2. The method of claim 1 wherein said universal high speed pulverizer rotates at 30000 rpm; centrifuging by the centrifuge, wherein the centrifuging speed is not lower than 900 rpm, and the centrifuging time is not less than 5 min; the suction filter has suction pressure not greater than 0.45 MPa.
3. The application of the extracting solution prepared by the method of claim 1 in improving the ultraviolet stability of the bacillus thuringiensis is characterized by comprising the following steps: an ultraviolet irradiation box is adopted to evaluate the anti-ultraviolet activity of the fungus chaff water extract, the fungus chaff extract and the Bacillus thuringiensis fermentation liquor are mixed according to the volume ratio of 1:1 in a 25 mL glass small beaker, the mixture is placed in the ultraviolet irradiation box to be irradiated for 0-96h, finally, the volume is fixed by using single distilled water, the survival condition of spores is evaluated by using an LB (Langerhans) plate, the retention condition of crystal protein is verified by using SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) protein gel, and the biological activity protection effect is evaluated by using cotton bollworm biological assay.
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Citations (7)
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| US5427787A (en) * | 1993-11-15 | 1995-06-27 | Industrial Technology Research Institute | Anti-ultraviolet biocidal composition |
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2021
- 2021-01-26 CN CN202110102535.XA patent/CN112646770B/en active Active
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|---|---|---|---|---|
| US5427787A (en) * | 1993-11-15 | 1995-06-27 | Industrial Technology Research Institute | Anti-ultraviolet biocidal composition |
| CN1948463A (en) * | 2006-11-03 | 2007-04-18 | 华中农业大学 | Application of poly gamma glutamic acid as bacillus thuringiensis agent protector |
| CN102286413A (en) * | 2011-09-03 | 2011-12-21 | 福建农林大学 | Preparation method of liquid fermentation medium for bacillus thuringiensis |
| CN102757926A (en) * | 2012-08-07 | 2012-10-31 | 福建农林大学 | Bacillus thuringiensis emulsification fermentation culture medium |
| CN103232961A (en) * | 2013-04-21 | 2013-08-07 | 中国农业科学院植物保护研究所 | Uvioresistant bacillus thuringiensis strain and building method and application thereof |
| CN104472882A (en) * | 2014-12-29 | 2015-04-01 | 邳州市小河科技发展有限公司 | Liquid biological feed additive |
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