CN109810934B - Application of N-acetylglucosamine in promoting cordyceps sinensis bud spore development to form hypha - Google Patents
Application of N-acetylglucosamine in promoting cordyceps sinensis bud spore development to form hypha Download PDFInfo
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Abstract
The invention discloses application of N-acetylglucosamine in promoting cordyceps sinensis bud spores to develop to form hypha. Aiming at the problems of long mycelium formation time, long artificial culture period, low efficiency and the like of cordyceps sinensis spores during the development of the cordyceps sinensis spores, N-acetylglucosamine is added into a cordyceps sinensis culture medium to perform hypha induction culture on the cordyceps sinensis spores, hypha formation of the spores is found after the germination of the spores on the 2 nd day of culture, 62% of the spores on the 8 th day of culture germinate to form hypha, and the hypha formation rate is remarkably improved compared with that of the spores without induction substances. The invention promotes the growth of the blastospores in a hypha forming mode, and shortens the artificial cultivation time of the cordyceps sinensis.
Description
The technical field is as follows:
the invention belongs to the technical field of fungus culture, and particularly relates to application of N-acetylglucosamine in promoting cordyceps sinensis spore growth to form hypha.
Background art:
the Chinese caterpillar fungus Ophiocerceps sinensis is a traditional famous and precious Chinese medicinal material, and is known as Chinese medical science with ginseng and pilose antler. Cordyceps sinensis is a muscardine and fungal stroma complex formed by Cordyceps sinensis (also known as hirsutella sinensis, hirsutella sinensis) infecting hepialdae larvae. The cordyceps sinensis has a special habitat (only naturally propagates in a high-altitude alpine meadow environment), is slow in growth speed and long in period, and has extremely limited natural resources. Although Cordyceps sinensis is listed in the second-level protective species of national famous records of important protective wild plants, excessive mining of Cordyceps sinensis is not effectively inhibited. Under the comprehensive influence of various factors such as habitat degradation and global climate warming, especially over-development and utilization of cordyceps sinensis, the natural cordyceps sinensis resource storage amount is sharply reduced.
In recent years, the cordyceps sinensis is successfully cultured by simulating the ecological environment of the Qinghai-Tibet plateau and infecting hepialus larvae with cordyceps sinensis, but in the process of artificially culturing cordyceps sinensis, hemolymph of the hepialus larvae mainly exists in the form of geminispores, the geminispores are proliferated in a way of producing new geminispores through budding and germination, the geminispores need more than 5 months to form mycelia, and some hepialus larvae carry the geminispores to normally grow for more than 1 year. The hypha formed by the spore development of the gemma is a key link for killing larva and growing sporocarp of cordyceps sinensis. Therefore, how to promote the spore of the bud to develop and form hypha so as to shorten the culture time of the stroma is an important problem facing the artificial cultivation industry of the cordyceps sinensis at present.
N-acetylglucosamine is the basic building block of many important polysaccharides within biological cells, especially in the highest content in the exoskeleton of crustaceans. Since N-acetylglucosamine is a precursor of glycosaminoglycan, which is a main component of articular cartilage, N-acetylglucosamine is frequently used clinically as an adjuvant therapy for rheumatic and rheumatoid arthritis. In terms of the effect of this substance on fungal growth, there are reports that Neurospora crassa cannot form hyphae in a medium containing N-acetylglucosamine (Gaderer et al, N-acetylglucosamine, the building block of chitin, inhibition growth of Neurospora crassa. fungal Genetics and Biology,2017,107: 1-11).
The invention content is as follows:
based on the problems, the invention aims to provide the application of the N-acetylglucosamine in promoting the growth of the spores of the cordyceps sinensis buds to form hyphae, so that the problems of long time for the spores of the cordyceps sinensis buds to form the hyphae and low efficiency of artificially culturing the cordyceps sinensis are solved.
Experiments show that the addition of N-acetylglucosamine into a culture medium of cordyceps sinensis can promote the spore development of cordyceps sinensis buds to form hypha.
Therefore, the invention can apply the N-acetylglucosamine to the promotion of the cordyceps sinensis bud spore development to form hypha. For example, the preparation can be applied to the preparation of the preparation for promoting the spore development of the cordyceps sinensis buds to form hypha.
The second object of the present invention is to provide a method for promoting the development of mycelia from the blastospores of Cordyceps sinensis, which comprises adding N-acetylglucosamine to a medium (e.g., a liquid medium) containing the blastospores of Cordyceps sinensis, and culturing the mixture.
The concentration of the N-acetylglucosamine in the liquid medium is preferably 0.5mg/L to 30 g/L.
The culture medium containing Cordyceps sinensis spore is prepared by inoculating Cordyceps sinensis spore in liquid culture medium, wherein the final inoculation concentration of spore is not higher than 107one/mL, more preferably 105-107one/mL.
The liquid culture medium can be conventional culture medium of Cordyceps sinensis, preferably containing per 150ml filtrate of cooked potato 30g, peptone 1.5g, maltose or glucose 3g, magnesium sulfate 0.08g, potassium dihydrogen phosphate 0.25g, and vitamin B3 mg1And the balance being water.
The culture conditions are as follows: culturing at 12 + -2 deg.C and 100 rpm in dark.
Aiming at the problems of long mycelium formation time, long artificial culture period, low efficiency and the like of cordyceps sinensis spores during the development of the cordyceps sinensis spores, N-acetylglucosamine is added into a cordyceps sinensis culture medium to perform hypha induction culture on the cordyceps sinensis spores, hypha formation of the spores is found after the germination of the spores on the 2 nd day of culture, 62% of the spores on the 8 th day of culture germinate to form hypha, and the hypha formation rate is remarkably improved compared with that of the spores without induction substances. The invention promotes the growth of the blastospores in a hypha forming mode, and shortens the artificial cultivation time of the cordyceps sinensis.
Description of the drawings:
FIG. 1 shows spores of Cordyceps sinensis buds inoculated with the culture medium of the present invention (stained with 28 fluorescent dyes, 400X).
FIG. 2 shows the induction of hyphae (fluorescence 28 staining, 400X) formed by the spore growth of Cordyceps sinensis buds.
The specific implementation mode is as follows:
the following examples are further illustrative of the present invention and are not intended to be limiting thereof.
The blastospores used in the examples below were of the species Cordyceps sinensis.
The blastospore is prepared by the following method:
filtering the bacterial liquid containing the cordyceps sinensis blastospores through three layers of lens wiping paper or filter screens, centrifuging for 15 minutes at 10 ℃ under the condition of 5000 r/min, discarding part of supernatant, counting the number of the blastospores through a blood counting plate to obtain concentrated blastospore inoculation mother liquid, and inoculating by using the concentrated blastospore inoculation mother liquid.
Example 1:
preparing Cordyceps liquid culture medium by decocting 30g of cooked potato filtrate (30 g of potato is decocted in water, and then filtering, and collecting filtrate to obtain 30g of cooked potato filtrate, the same below), 1.5g of peptone, 3g of maltose, 0.08g of magnesium sulfate, 0.25g of potassium dihydrogen phosphate, and 3mg of vitamin B1Placing into a triangular flask, and adding water to a constant volume of 150 ml. Sterilizing under high pressure, placing in culture room at 12 + -2 deg.C for precooling, inoculating blastospore (figure 1) into 150mL liquid culture medium to final concentration of 105Adding N-acetylglucosamine (with the contrast of not adding N-acetylglucosamine) to make the final concentration 5mg/l, placing on a shaking table with rotation speed of 100 r/min, culturing at 12 + -2 deg.C in the dark, culturing for 2 days, taking out 1mL of culture medium, observing by microscope to find that geminispores start to germinate, culturing for 8 days, taking out 1mL of culture medium, observing by microscope to obtain mycelium formation rate of geminispores (figure 2) reaching 62%, and improving the mycelium formation rate by 151.32% compared with the contrast of not adding inducing substance N-acetylglucosamine.
Example 2:
preparing Cordyceps liquid culture medium by mixing cooked potato filtrate 30g, peptone 1.5g, maltose 3g, magnesium sulfate 0.08g, potassium dihydrogen phosphate 0.25g, and vitamin B3 mg1Placing into a triangular flask, and adding water to a constant volume of 150 ml. Sterilizing under high pressure, pre-cooling in 12 + -2 deg.C culture chamber, inoculating spore to 150mL liquid culture medium to obtain final concentration of 107Per mL, and adding N-acetyl grapeAnd (3) adding glucosamine (taking the glucosamine without adding N-acetylglucosamine as a control) to ensure that the final concentration is 5mg/l, placing the glucosamine on a shaking table with the rotating speed of 100 r/min, culturing at 12 ℃ under the dark condition, culturing for 2 days, taking out 1mL of culture bacterial liquid, observing by microscopy to find that the blastospores start to germinate, culturing for 8 days, taking out 1mL of culture bacterial liquid, observing by microscopy under a microscope to ensure that the hypha formation rate of the blastospores reaches 54 percent, and the hypha formation rate is increased by 127.94 percent compared with the control without adding the inducing substance N-acetylglucosamine.
Example 3:
preparing Cordyceps liquid culture medium by mixing cooked potato filtrate 30g, peptone 1.5g, maltose 3g, magnesium sulfate 0.08g, potassium dihydrogen phosphate 0.25g, and vitamin B3 mg1Placing into a triangular flask, and adding water to a constant volume of 150 ml. Sterilizing under high pressure, pre-cooling in 12 + -2 deg.C culture chamber, inoculating spore to 150mL liquid culture medium to obtain final concentration of 105Adding N-acetylglucosamine (with the condition that the N-acetylglucosamine is not added as a control) to enable the final concentration to be 50mg/l, placing the mixture on a shaking table with the rotating speed of 100 revolutions per minute, culturing at 12 +/-2 ℃ in the dark, culturing for 2 days, taking out 1mL of culture medium liquid, observing by a microscope to find that the blastospores start to germinate, culturing for 8 days, taking out 1mL of culture medium liquid, observing by a microscope to enable the hypha formation rate of the blastospores to reach 51.11%, and improving the hypha formation rate by 107.17% compared with that without adding an inducing substance.
Example 4:
preparing Cordyceps liquid culture medium by mixing cooked potato filtrate 30g, peptone 1.5g, maltose 3g, magnesium sulfate 0.08g, potassium dihydrogen phosphate 0.25g, and vitamin B3 mg1Placing into a triangular flask, and adding water to a constant volume of 150 ml. Sterilizing under high pressure, pre-cooling in 12 + -2 deg.C culture chamber, inoculating spore to 150mL liquid culture medium to obtain final concentration of 107Adding N-acetylglucosamine (with N-acetylglucosamine not added as control) to make its final concentration be 50mg/l, placing in a shaking table with rotation speed of 100 rpm, culturing at 12 + -2 deg.C in dark, culturing for 2 days, taking out 1mL of culture medium, observing by microscope to find that geminispores start to germinate, culturing for 8 days, taking out 1mL of culture medium, and observing by microscope to obtain mycelium formation rate of geminisporesTo 45.33%, the hyphal formation rate was increased 83.75% over the control without the addition of the inducer N-acetylglucosamine.
Example 5:
preparing Cordyceps liquid culture medium by mixing cooked potato filtrate 30g, peptone 1.5g, maltose 3g, magnesium sulfate 0.08g, potassium dihydrogen phosphate 0.25g, and vitamin B3 mg1Placing into a triangular flask, and adding water to a constant volume of 150 ml. Sterilizing under high pressure, pre-cooling in 12 + -2 deg.C culture chamber, inoculating spore to 150mL liquid culture medium to obtain final concentration of 105Adding N-acetylglucosamine (with the N-acetylglucosamine not added as a control) to make the final concentration 10g/l, placing the mixture on a shaking table with the rotation speed of 100 r/min, culturing at 12 +/-2 ℃ in the dark, culturing for 2 days, taking out 1mL of culture medium liquid, observing by microscope to find that the blastospores start to germinate, culturing for 8 days, taking out 1mL of culture medium liquid, observing by microscope to obtain the blastospores with the mycelium formation rate of 44%, and increasing the mycelium formation rate by 78.35% compared with the control without adding the inducing substance N-acetylglucosamine.
Example 6:
preparing Cordyceps liquid culture medium by mixing cooked potato filtrate 30g, peptone 1.5g, maltose 3g, magnesium sulfate 0.08g, potassium dihydrogen phosphate 0.25g, and vitamin B3 mg1Placing into a triangular flask, and adding water to a constant volume of 150 ml. Sterilizing under high pressure, pre-cooling in 12 + -2 deg.C culture chamber, inoculating spore to 150mL liquid culture medium to obtain final concentration of 107Adding N-acetylglucosamine (with the contrast of not adding N-acetylglucosamine) to make the final concentration 10g/l, placing on a shaking table with the rotation speed of 100 r/min, culturing at 12 + -2 deg.C in the dark, culturing for 2 days, taking out 1mL of culture medium, observing by microscope to find that the blastospores start to germinate, culturing for 8 days, taking out 1mL of culture medium, observing by microscope to obtain the mycelium formation rate of the blastospores reaching 41.01%, and increasing the mycelium formation rate by 73.11% compared with the contrast of not adding inducing substance N-acetylglucosamine.
Example 7:
preparing Cordyceps liquid culture medium by mixing cooked potato filtrate 30g, peptone 1.5g, maltose 3g, magnesium sulfate 0.08g,0.25g of monopotassium phosphate and 3mg of vitamin B1Placing into a triangular flask, and adding water to a constant volume of 150 ml. Sterilizing under high pressure, pre-cooling in 12 + -2 deg.C culture chamber, inoculating spore to 150mL liquid culture medium to obtain final concentration of 105Adding N-acetylglucosamine (with the contrast of not adding N-acetylglucosamine) to make the final concentration 30g/l, placing on a shaking table with the rotation speed of 100 r/min, culturing at 12 + -2 deg.C in the dark, culturing for 2 days, taking out 1mL of culture medium, observing by microscope to find that the blastospores start to germinate, culturing for 8 days, taking out 1mL of culture medium, observing by microscope to obtain the mycelium formation rate of the blastospores reaching 38%, and improving the mycelium formation rate by 54.03% compared with the contrast of not adding inducing substance N-acetylglucosamine.
Example 8:
preparing Cordyceps liquid culture medium by mixing cooked potato filtrate 30g, peptone 1.5g, maltose 3g, magnesium sulfate 0.08g, potassium dihydrogen phosphate 0.25g, and vitamin B3 mg1Placing into a triangular flask, and adding water to a constant volume of 150 ml. Sterilizing under high pressure, pre-cooling in 12 + -2 deg.C culture chamber, inoculating spore to 150mL liquid culture medium to obtain final concentration of 105Adding N-acetylglucosamine (with N-acetylglucosamine not added as a control) to make the final concentration 0.5mg/l, placing on a shaking table with the rotation speed of 100 r/min, culturing at 12 + -2 deg.C in the dark, culturing for 2 days, taking out 1mL of culture medium, observing by microscope that blastospores begin to germinate, culturing for 8 days, taking out 1mL of culture medium, observing by microscope that the hypha formation rate of the blastospores reaches 32%, and the hypha formation rate is increased by 29.71% compared with the control without adding inducing substance N-acetylglucosamine.
Comparative example 1:
preparing Cordyceps liquid culture medium by mixing cooked potato filtrate 30g, peptone 1.5g, maltose 3g, magnesium sulfate 0.08g, potassium dihydrogen phosphate 0.25g, and vitamin B3 mg1Placing into a triangular flask, and adding water to a constant volume of 150 ml. Sterilizing under high pressure, pre-cooling in 12 + -2 deg.C culture chamber, inoculating spore to 150mL liquid culture medium to obtain final concentration of 105Placing the strain/mL strain in a shaking table at a rotation speed of 100 rpm in a condition of 12 +/-L without adding hypha inducing substancesCulturing at 2 ℃ in the dark, taking out 1mL of culture medium liquid after culturing for 2 days, observing by microscope to find that the blastospores start to germinate, and taking out 1mL of culture medium liquid after culturing for 8 days, and observing by microscope to obtain the mycelium formation rate of the blastospores reaching 24.67%.
Comparative example 2:
preparing Cordyceps liquid culture medium by mixing cooked potato filtrate 30g, peptone 1.5g, maltose 3g, magnesium sulfate 0.08g, potassium dihydrogen phosphate 0.25g, and vitamin B3 mg1Placing into a triangular flask, and adding water to a constant volume of 150 ml. Sterilizing under high pressure, pre-cooling in 12 + -2 deg.C culture chamber, inoculating spore to 150mL liquid culture medium to obtain final concentration of 107And (2) culturing the germinated spores in a shaking table at the rotating speed of 100 rpm at 12 +/-2 ℃ in the dark condition without adding hypha inducing substances in each mL, taking out 1mL of culture bacterial liquid after the 2 nd day of culture, observing by a microscope to find that the germinated spores start to germinate, and taking out 1mL of culture bacterial liquid after the 8 th day of culture, and observing by a microscope to ensure that the hypha formation rate of the germinated spores reaches 23.69%.
Example 9:
preparing Cordyceps liquid culture medium by mixing cooked potato filtrate 30g, peptone 1.5g, glucose 3g, magnesium sulfate 0.08g, potassium dihydrogen phosphate 0.25g, and vitamin B3 mg1Placing into a triangular flask, and adding water to a constant volume of 150 ml. Sterilizing under high pressure, pre-cooling in 12 + -2 deg.C culture chamber, inoculating spore to 150mL liquid culture medium to obtain final concentration of 105Adding N-acetylglucosamine (with the contrast of not adding N-acetylglucosamine) to make the final concentration be 5mg/l, placing on a shaking table with rotation speed of 100 r/min, culturing at 12 + -2 deg.C in dark, culturing for 2 days, taking out 1mL of culture medium, observing by microscope to find that geminispores start to germinate, culturing for 8 days, taking out 1mL of culture medium, observing by microscope to obtain mycelium formation rate of geminispores reaching 42.04%, and increasing mycelium formation rate by 159.35% compared with the contrast of not adding inducing substance N-acetylglucosamine.
Example 10:
preparing Cordyceps liquid culture medium by mixing cooked potato filtrate 30g, peptone 1.5g, glucose 3g, magnesium sulfate 0.08g, potassium dihydrogen phosphate 0.25g, and vitamin 3mgElement B1Placing into a triangular flask, and adding water to a constant volume of 150 ml. Sterilizing under high pressure, pre-cooling in 12 + -2 deg.C culture chamber, inoculating spore to 150mL liquid culture medium to obtain final concentration of 107Adding N-acetylglucosamine (with the contrast of not adding N-acetylglucosamine) to make the final concentration be 5mg/l, placing on a shaking table with rotation speed of 100 r/min, culturing at 12 + -2 deg.C in the dark, culturing for 2 days, taking out 1mL of culture medium, observing by microscope to find that the blastospores start to germinate, culturing for 8 days, taking out 1mL of culture medium, observing by microscope to obtain the blastospores with mycelium formation rate of 34.55%, and improving the mycelium formation rate by 154.22% compared with the contrast of not adding inducing substance N-acetylglucosamine.
Example 11:
preparing Cordyceps liquid culture medium by mixing cooked potato filtrate 30g, peptone 1.5g, glucose 3g, magnesium sulfate 0.08g, potassium dihydrogen phosphate 0.25g, and vitamin B3 mg1Placing into a triangular flask, and adding water to a constant volume of 150 ml. Sterilizing under high pressure, pre-cooling in 12 + -2 deg.C culture chamber, inoculating spore to 150mL liquid culture medium to obtain final concentration of 105Adding N-acetylglucosamine (with the contrast of not adding N-acetylglucosamine) to make the final concentration be 50mg/l, placing on a shaking table with rotation speed of 100 r/min, culturing at 12 + -2 deg.C in the dark, culturing for 2 days, taking out 1mL of culture medium, observing by microscope to find that the blastospores start to germinate, culturing for 8 days, taking out 1mL of culture medium, observing by microscope to obtain the mycelium formation rate of the blastospores reaching 41.50%, and increasing the mycelium formation rate by 156.01% compared with the contrast of not adding inducing substance N-acetylglucosamine.
Example 12:
preparing Cordyceps liquid culture medium by mixing cooked potato filtrate 30g, peptone 1.5g, glucose 3g, magnesium sulfate 0.08g, potassium dihydrogen phosphate 0.25g, and vitamin B3 mg1Placing into a triangular flask, and adding water to a constant volume of 150 ml. Sterilizing under high pressure, pre-cooling in 12 + -2 deg.C culture chamber, inoculating spore to 150mL liquid culture medium to obtain final concentration of 105one/mL, and N-acetylglucosamine was added (with no N-acetylglucosamine added as a control) to a final concentration of 0.5 mg-And l, placing the mixture on a shaking table with the rotating speed of 100 revolutions per minute, culturing the mixture at 12 +/-2 ℃ in the dark, taking out 1mL of culture bacterial liquid after culturing for 2 days, observing by a microscope to find that the blastospores start to germinate, and culturing the mixture for 8 days, taking out 1mL of culture bacterial liquid, observing by a microscope to ensure that the hypha formation rate of the blastospores reaches 42.96 percent, wherein the hypha formation rate is improved by 165.02 percent compared with that of the control without adding the inducing substance N-acetylglucosamine.
Comparative example 3:
preparing Cordyceps liquid culture medium by mixing cooked potato filtrate 30g, peptone 1.5g, glucose 3g, magnesium sulfate 0.08g, potassium dihydrogen phosphate 0.25g, and vitamin B3 mg1Placing into a triangular flask, and adding water to a constant volume of 150 ml. Sterilizing under high pressure, pre-cooling in 12 + -2 deg.C culture chamber, inoculating spore to 150mL liquid culture medium to obtain final concentration of 105And (2) culturing the germinated spores in a shaking table at the rotating speed of 100 rpm at 12 +/-2 ℃ in the dark condition without adding hypha inducing substances in each mL, taking out 1mL of culture bacterial liquid after the 2 nd day of culture, finding that the germinated spores start to germinate by microscopic examination, taking out 1mL of culture bacterial liquid after the 8 th day of culture, and carrying out microscopic examination on the germinated spores to ensure that the hypha formation rate reaches 16.21%.
Comparative example 4:
preparing Cordyceps liquid culture medium by mixing cooked potato filtrate 30g, peptone 1.5g, glucose 3g, magnesium sulfate 0.08g, potassium dihydrogen phosphate 0.25g, and vitamin B3 mg1Placing into a triangular flask, and adding water to a constant volume of 150 ml. Sterilizing under high pressure, pre-cooling in 12 + -2 deg.C culture chamber, inoculating spore to 150mL liquid culture medium to obtain final concentration of 107And (2) culturing the germinated spores in a shaking table at the rotating speed of 100 rpm at 12 +/-2 ℃ in the dark condition without adding hypha inducing substances in each mL, taking out 1mL of culture bacterial liquid after the 2 nd day of culture, observing by a microscope to find that the germinated spores start to germinate, and taking out 1mL of culture bacterial liquid after the 8 th day of culture, and observing by a microscope to ensure that the hypha formation rate of the germinated spores reaches 13.39%.
Claims (3)
1. A method for promoting germination and development of cordyceps sinensis spore to form hypha is characterized in that N-acetylglucosamine is added into a culture medium containing the cordyceps sinensis spore, and then the culture is carried out;
the culture medium is a liquid culture medium;
the concentration of the N-acetylglucosamine in a liquid culture medium is 5mg/L-50 mg/L;
the culture medium containing the cordyceps sinensis spore is obtained by inoculating the cordyceps sinensis spore in a liquid culture medium, and the inoculation concentration of the spore is 105-107one/mL.
2. The method as claimed in claim 1, wherein the liquid medium contains 30g of boiled potato filtrate, 1.5g of peptone, 3g of maltose or glucose, 0.08g of magnesium sulfate, 0.25g of potassium dihydrogen phosphate, 3mg of vitamin B per 150ml1And the balance being water.
3. The method of claim 1, wherein said culturing is carried out under conditions selected from the group consisting of: culturing at 12 + -2 deg.C and 100 rpm in dark.
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| CN104130992A (en) * | 2014-06-30 | 2014-11-05 | 浙江工业大学 | Chitinase A coming from cordyceps sinensis hirsutella sinensis, encoding gene and application of two |
| CN106810366A (en) * | 2017-01-24 | 2017-06-09 | 北京新创青龙湖种植专业合作社 | A kind of cultural method for improving cordycepin content in Cordyceps militaris mycelia and silkworm chrysalis Cordyceps sinensis |
| CN108739050A (en) * | 2018-05-25 | 2018-11-06 | 广东省生物资源应用研究所 | A kind of aweto fluid nutrient medium and the efficient method for obtaining host of Cordyceps sinensis insect infection blastopore |
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