CN104894206A - Method for enriching huperzine A through huperzia serrata cell and endophytic fungi coculture - Google Patents
Method for enriching huperzine A through huperzia serrata cell and endophytic fungi coculture Download PDFInfo
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Abstract
The invention relates to a technology for producing lycopodium alkaloid through culturing huperzia serrata cells. The method includes the following steps: (1), artificial habituated culture of huperzia serrata for producing huperzine A; (2) , acquisition of huperzia serrata single cells; (3), establishment of a huperzia serrata-endophytic fungi co-culture system; (4), observation and counting of living cells; (5), extraction of huperzine A and ergosterol; (6) detection of huperzine A and ergosterol, namely: adopting HPLC to detect the contents of huperzine A and ergosterol in the system respectively, and adopting the following mobile phases: methyl alcohol: 0.08 mol/L ammonium acetate (25:75, v/v), and methyl alcohol to water (95:5, v/v) respectively. Through observing the content change of huperzine A and ergosterol, the facts that the endophytic fungi inside the system does not obviously improve the content of huperzine A, and the content of huperzine A reaches a peak value increased by 33.05% compared with the original content are obtained, and at the time, acquisition or subculturing can be performed; according to the invention, under the premise that the huperzia serrata is not completely sterilized, huperzine A in the system is enriched, and the process is quick and economic.
Description
Technical field:
The present invention relates to a kind of culture plant cell and produce lycopodium alkaloid technology, be specifically means with plant cell culture technology, utilize Herba Lycopodii serrati cell and endophyte Dual culture enrichment lycopodium alkaloid selagine, and be testing tool with high performance liquid chromatograph, selagine and ergosterol are detected, to determine best harvest time and the succeeding transfer culture time point of selagine.
Background technology:
Selagine (huperzine A) is a kind of alkaloid extracted in Huperziaceae plants of Huperzia Herba Lycopodii serrati, there is the inhibiting activity of acetylcholinesterase, of highly selective, can be used for treatment myasthenia gravis, improve adolescents with learning ability and improve memory of elderly person function etc., have the great market requirement.But Herba Lycopodii serrati wild resource is limited, and selagine content in herb is very micro-, excavates the exhaustion that wild Herba Lycopodii serrati will cause resource for a long time, and in addition, selagine synthesis cost is comparatively high, is difficult to realize industrialization.Herba Lycopodii serrati and endogenetic fungus coexist for a long time, based on biosimulation effect, endogenetic fungus often can produce with host same or analogous bioactive ingredients, and ergosterol (ergosterol) can as the criterion of fungal biomass.
Utilize plant cell culture technology to produce selagine to be day by day subject to people's attention because it has the advantages such as environmental friendliness, with short production cycle, controllability is strong.
Present invention obviates current callus induction method and obtain the unicellular middle extremely difficult difficult point removing endogenetic fungus completely of Herba Lycopodii serrati, enrichment selagine is carried out by Herba Lycopodii serrati cell and endogenetic fungus Dual culture, recycling high performance liquid chromatography detects the content of Herba Lycopodii serrati and ergosterol in nutrient solution, by the content determination Subculture of the two and the Best Times of selagine results.
Summary of the invention:
The object of this invention is to provide a kind of method that co-culture system by Herba Lycopodii serrati cell and endogenetic fungus carrys out enrichment selagine, the harvest time of cell is determined by the relative content of selagine and ergosterol.This is to the protection of Herba Lycopodii serrati natural resources and the exploitation of selagine and realize suitability for industrialized production and have very important theory significance and practical value.
For achieving the above object, the technical solution used in the present invention is:
(1) produce effective alkaloidal Herba Lycopodii serrati domestication to cultivate: be orchid soil for cultivating the soil constitution of Herba Lycopodii serrati: river sand=2: 1, soil pH is about 7; Keep the living environment humidity of Herba Lycopodii serrati more than 95%; Keep the living environment illumination of Herba Lycopodii serrati at about 100Lx; Be used for taming in cultivation by liver moss first, liver moss is laid on soil surface, advantage is that liver moss plays moisture-keeping function, and maintains a good ecotope.
(2) the unicellular acquisition of Herba Lycopodii serrati: get Herba Lycopodii serrati blade, carries out surface sterilization with following methods: 70% alcohol 50s, 0.5mg/L malachite green 8min, 3% hydrogen peroxide 10min, sterilized water 3 times successively; By means of scotch tape stripping blade surface wax, be affixed on by blade on the adhesive plaster of ultraviolet sterilization, scrape off leaf surface waxes with pocket knife, blend compounds band pastes 2 ~ 3 times repeatedly to peel off wax; It is unicellular that mechanical crushing method obtains plant mesophyll, the blade peeling off wax is divided into minimum fritter, scrape to be placed in and grind alms bowl, add 5 ~ 10mL liquid nutrient medium, mill after 1 ~ 3min, suspension will be mixed by double-deck 200 order nylon net filters, and with a small amount of nutrient solution drip washing filter cloth, by filtrate centrifugal 2min under the rotating speed of 2000rpm, get precipitation nutrient solution settling flux.
(3) foundation of Herba Lycopodii serrati-endogenetic fungus co-culture system: Herba Lycopodii serrati single-cell suspension liquid is placed in 24 DEG C of constant incubator light culture, within about about 10 days, change a liquid nutrient medium, liquid nutrient medium main component used comprises MS minimum medium, 5.0mg/L2,4-D, 1.0mg/LKT, 20.0g/L sucrose, regulates pH to be 5.8.
(4) the observation counting of viable cell: Herba Lycopodii serrati suspension cell and toluylene red dye liquor is dyed blended in the ratio of 4: 1, and after leaving standstill 15 ~ 20min, observation counts.
(5) extraction of selagine and ergosterol: get nutrient solution 5.0mL, be placed in freezer compartment of refrigerator, repeatedly freeze-thaw three times; After nutrient solution and cell thawing, rotary evaporation removing moisture, uses 10mL dissolve with methanol, supersound process 2 times at 30 DEG C, each 20min, therebetween interval 10min, filter, obtain filtrate and be placed in Erlenmeyer flask, add 10mL methyl alcohol after filter paper shreds, again extract as stated above, filter, merge twice filtrate, rotary evaporation removing methyl alcohol; Fully dissolve the medicinal extract revolving and steam and obtain with chromatogram methyl alcohol, be transferred to cillin bottle, methyl alcohol volatilizees, and obtains medicinal extract; The medicinal extract 1mL chromatogram methyl alcohol obtained after methyl alcohol volatilization fully dissolves, with the filtering with microporous membrane of 0.22 μm.
(6) detection of selagine and ergosterol: the reverse-phase chromatographic column of employing is Agilent TC C
18post, produced by Agilent company of the U.S., specification 4.6 × 150mm, filler granularity 5 μm, the moving phase detecting selagine is methyl alcohol: 0.08mol/L ammonium acetate (25: 75, v/v), wavelength is 310nm, and the moving phase detecting ergosterol is methyl alcohol: water (95: 5, v/v), wavelength is 282nm, and both are 1.000mL/min by flow velocity used, and temperature is 25 DEG C.Wherein the retention time of selagine is 9.191min, and the retention time of ergosterol is 10.716min.
(7) analyze the variation tendency of selagine content with incubation time: utilize high performance liquid chromatography to detect the content of selagine and ergosterol in culture system rapidly, draw best results and succeeding transfer culture time, with enrichment selagine.
Tool of the present invention has the following advantages:
1, enrichment selagine is carried out by the method for Dual culture, utilize the feature of the long-term and endogenetic fungus symbiosis of Herba Lycopodii serrati, sterilizing is not thoroughly carried out to explant, make endogenetic fungus and vegetable cell balanced growth, endogenetic fungus also can produce selagine on the one hand, is enriched product; Endogenetic fungus can impel vegetable cell to generate selagine as elicitor on the other hand.
2, by means of scotch tape stripping blade surface wax, thus make vegetable cell easier from parent come off and the single vegetable cell that dissociates to carry out suspension culture.
3, directly obtain Herba Lycopodii serrati with mechanical crushing method unicellular, overcome two shortcomings: 1) complete sterilizing is extremely difficult with endophyte symbiosis because of long-term; 2) callus is formed because of poor growth from explant consuming time extremely long.
4, adopt intravital stain, recognize viable cell easily, determine the growth conditions of suspension cell.
5, after sampling at once by ultrasonic for nutrient solution 20min, and put into refrigerator freezing, be conducive to the fragmentation of cell and prevent the growth of cell and bacterium, ensure that the accuracy of experiment.
Accompanying drawing illustrates:
Fig. 1 is that selagine standard substance contrast figure with co-culture system extracting solution high performance liquid chromatography;
Fig. 2 is that ergosterol standard substance contrast figure with co-culture system extracting solution high performance liquid chromatography;
Fig. 3 is the content curve of selagine and ergosterol in 24 days culture cycle;
Fig. 4 is selagine viable cell number change curve in 24 days culture cycle;
Fig. 5 is co-culture system first 7 days selagine content curves.
Embodiment:
Below in conjunction with color atlas, most the present invention is further described.
Laboratory apparatus and reagent: Agilent 1100 series of high efficiency liquid chromatograph is produced by Agilent company of the U.S., and reference substance selagine and ergosterol are all purchased from Wei Keqi bio tech ltd of Sichuan Province; Selagine-endogenetic fungus culture is provided by applicant; HPLC analyzes chromatographically pure reagent: methyl alcohol, pure water and 0.08mol/L Spirit of Mindererus; Extraction analytical reagent: methyl alcohol.
Chromatographic condition: chromatographic column is Agilent TC C
18post, produced by Agilent company of the U.S., specification 4.6 × 150mm, filler granularity 5 μm, flow velocity is 1.000mL/min, ultraviolet detection wavelength selagine is 310nm, and ergosterol is 282nm, and column temperature is 25 DEG C, selagine moving phase is methyl alcohol: 0.08mol/L ammonium acetate (25: 75, v/v), ergosterol moving phase is methyl alcohol: water (95: 5, v/v).
Formulate typical curve: according to the standard content of two kinds of composition reference substances, adopt external standard method, the content of selagine and ergosterol in accurate quantitative analysis selagine-endophyte culture system.The standardized solution that precision takes selagine, ergosterol 5mg makes 100 μ g/mL, this mark liquid getting different volumes is respectively made into 5,10,15,20,25,30,35,40, the standardized solution of 45 μ g/mL series concentration gradients, gets the reference substance solution 10 μ L sample introduction of above each concentration, measures respectively by above-mentioned chromatographic condition, take peak area as ordinate zou, concentration is X-coordinate, draws the typical curve of selagine and ergosterol, obtains respective regression equation and relation conefficient.
Tolerance range is tested: the selagine reference substance solution of getting 5 μ g/mL repeats sample introduction 5 times, calculates RSD;
Replica test: get nutrient solution extract replication 5 times, calculates the RSD of peak area;
Stability test: get nutrient solution extract respectively at 1,2,4,8,12h sample introduction, calculates the RSD of peak area;
Average recovery is tested: joined by 0.5mL standard substance in 1.0mL nutrient solution extract, repeats sample introduction 5 times, calculates average recovery experiment and RSD thereof.
Methodological study result is as follows:
Embodiment one
1. the optimization of Herba Lycopodii serrati domestication culture condition: Herba Lycopodii serrati plant source-Fujian Province.To divide at random with a collection of Herba Lycopodii serrati plant is planted in four flowerpots, each flowerpot three strain plant.Probe into soil ratio respectively and with or without paving liver moss on the impact of its upgrowth situation, and measure the humidity of its soil, pH and intensity of illumination respectively with wet bulb thermometer.Experimental results is as shown in the table:
Concluded by form, determine that Herba Lycopodii serrati domestication culture condition is: for cultivate the soil constitution of Herba Lycopodii serrati be orchid soil: river sand=2: 1, soil pH is about 7; Keep the living environment humidity of Herba Lycopodii serrati more than 95%; Keep the living environment illumination of Herba Lycopodii serrati at about 100Lx.As can be seen from the experiment, liver moss is very important for the upgrowth situation of Herba Lycopodii serrati, and reason may be liver moss to be laid on soil surface, and liver moss plays moisture-keeping function, and maintains a good ecotope.
2. the foundation of Herba Lycopodii serrati-endogenetic fungus co-culture system: get 80, Herba Lycopodii serrati blade at random, by banister brush and washing composition clean surface, then Bechtop is brought into, surface sterilization is carried out successively: 70% alcohol 50s with following methods, 0.5mg/L malachite green 8min, 3% hydrogen peroxide 10min, sterilized water 3 times.Blade is affixed on the adhesive plaster of ultraviolet sterilization, leaf surface waxes is scraped off with pocket knife, blend compounds band pastes 3 times repeatedly to peel off wax, the blade peeling off wax is divided into minimum fritter, scrapes and be placed in sterilized mortar, add 10mL liquid nutrient medium, mill after 3min, suspension will be mixed by double-deck 200 order nylon net filters, and with a small amount of nutrient solution drip washing filter cloth, by filtrate centrifugal 2min under the rotating speed of 2000rpm, get precipitation nutrient solution settling flux.Liquid nutrient medium main component used comprises MS minimum medium, 5.0mg/L2,4-D, 1.0mg/LKT, 20.0g/L sucrose, regulates pH to be 5.8.
Herba Lycopodii serrati single-cell suspension liquid is placed in 24 DEG C of constant incubator light culture, within 10th day, change a liquid nutrient medium, within every 2 days, carry out observation counting to viable cell, method is: Herba Lycopodii serrati suspension cell and toluylene red dye liquor is dyed blended in the ratio of 4: 1, observes after standing 20min.Find after observing, the starting point concentration of Herba Lycopodii serrati cell is 5.0 × 10
4/ mL, reached peak concentration 8.0 × 10 at the 8th day
4/ mL, after this cell quantity sharply reduces, and within the 16th day, rises substantially dead.
3. the preparation of selagine-endogenetic fungus nutrient solution extract: get nutrient solution 5.0mL, repeatedly freeze-thaw three times, rotary evaporation removing moisture, use 10mL dissolve with methanol, supersound process 2 times at 30 DEG C, each 20min, interval 10min therebetween, filter, obtain filtrate and be placed in Erlenmeyer flask, after filter paper shreds, add 10mL methyl alcohol, again extract as stated above, filter, merge twice filtrate, rotary evaporation removing methyl alcohol.Fully dissolved by medicinal extract 1mL chromatogram methyl alcohol, with the filtering with microporous membrane of 0.22 μm, filtrate measures for HPLC.
4. the compartment analysis of selagine and ergosterol: selagine and each sample introduction of ergosterol are 10 μ L, temperature is 25 DEG C, flow velocity is 1.000mL/min, detecting selagine moving phase used is methyl alcohol: 0.08mol/L ammonium acetate (25: 75, v/v), determined wavelength 310nm; Ergosterol moving phase is methyl alcohol: water (95: 5, v/v), determined wavelength 282nm.In Herba Lycopodii serrati-endophyte co-culture system, the content of selagine reached peak value at the 4th day, and ergosterol reached peak value at the 12nd day.
Embodiment two
1. according to soil constitution be orchid soil: river sand=2: 1, soil pH is about 7; Keep the living environment humidity of Herba Lycopodii serrati more than 95%; The living environment illumination of Herba Lycopodii serrati is kept to carry out domestication at about 100Lx to Herba Lycopodii serrati plant.
2. the foundation of Herba Lycopodii serrati-endophyte co-culture system: get 80, Herba Lycopodii serrati blade at random, by banister brush and washing composition clean surface, then Bechtop is brought into, surface sterilization is carried out successively: 70% alcohol 50s with following methods, 0.5mg/L malachite green 8min, 3% hydrogen peroxide 10min, sterilized water 3 times.Blade is affixed on the adhesive plaster of ultraviolet sterilization, leaf surface waxes is scraped off with pocket knife, blend compounds band pastes 3 times repeatedly to peel off wax, the blade peeling off wax is divided into minimum fritter, scrapes and be placed in sterilized mortar, add 10mL liquid nutrient medium, mill after 3min, suspension will be mixed by double-deck 200 order nylon net filters, and with a small amount of nutrient solution drip washing filter cloth, by filtrate centrifugal 2min under the rotating speed of 2000rpm, get precipitation nutrient solution settling flux.Herba Lycopodii serrati single-cell suspension liquid is placed in 24 DEG C of constant incubator light culture, within 10th day, change a liquid nutrient medium, within every 2 days, carry out observation counting to viable cell, method is: Herba Lycopodii serrati suspension cell and toluylene red dye liquor is dyed blended in the ratio of 4: 1, observes after standing 20min.
This time Experimental Research impact of different basic medium on the vegetable cell in vegetable cell-endogenetic fungus co-culture system.Liquid nutrient medium main component used, except minimum medium, includes 5.0mg/L2,4-D, 1.0mg/LKT, 20.0g/L sucrose, regulates pH to be 5.8.Investigation result is as shown in the table:
For Herba Lycopodii serrati explant, MS substratum and 1/2MS substratum are on its impact also not quite; And in co-culture system, MS is better than 1/2MS, it can thus be appreciated that because co-culture system consumes nutritive substance sooner, 1/2MS provides nutrient not enough, therefore MS substratum is more suitable for co-culture system.
3. the preparation of selagine-endogenetic fungus nutrient solution extract: get nutrient solution 5.0mL, repeatedly freeze-thaw three times, rotary evaporation removing moisture, use 10mL dissolve with methanol, supersound process 2 times at 30 DEG C, each 20min, interval 10min therebetween, filter, obtain filtrate and be placed in Erlenmeyer flask, after filter paper shreds, add 10mL methyl alcohol, again extract as stated above, filter, merge twice filtrate, rotary evaporation removing methyl alcohol.Fully dissolved by medicinal extract 1mL chromatogram methyl alcohol, with the filtering with microporous membrane of 0.22 μm, filtrate measures for HPLC.
4. the compartment analysis of selagine and ergosterol: selagine and each sample introduction of ergosterol are 10 μ L, temperature is 25 DEG C, flow velocity is 1.000mL/min, detecting selagine moving phase used is methyl alcohol: 0.08mol/L ammonium acetate (25: 75, v/v), determined wavelength 310nm; Ergosterol moving phase is methyl alcohol: water (95: 5, v/v), determined wavelength 282nm.In Herba Lycopodii serrati-endophyte co-culture system, the content of selagine reached peak value at the 4th day, and ergosterol reached peak value at the 12nd day, improve 33.05% than starting point concentration, and ergosterol to the increase of selagine content in system without obvious effect.
Claims (10)
1. utilize the method for Herba Lycopodii serrati cell and endophyte Dual culture enrichment selagine, it is characterized in that the method comprises the following steps:
(1) the Herba Lycopodii serrati domestication producing selagine cultivates;
(2) the unicellular acquisition of Herba Lycopodii serrati;
(3) foundation of Herba Lycopodii serrati-endophyte co-culture system;
(4) the observation counting of viable cell;
(5) extraction of selagine and ergosterol;
(6) detection of selagine and ergosterol;
(7) analyze the selagine content variation tendency with incubation time, show that best harvest time is with enrichment selagine or carry out succeeding transfer culture.
2. according to method described in claim 1, it is characterized in that: with vegetable cell vital stain, Herba Lycopodii serrati suspension cell in co-culture system is dyeed and counted.
3. method according to claim 1, is characterized in that: the growing state weighing vegetable cell and endophyte in co-culture system with selagine and ergosterol.
4. method according to claim 1, is characterized in that: the Herba Lycopodii serrati domestication cultivation that described step (1) produces selagine refers to: simulation Herba Lycopodii serrati Natural Survival environment.Domestication's culture condition is as follows:
(1) be sandy soil for cultivating the soil constitution of Herba Lycopodii serrati: river sand=2: 1, soil pH is about 7.
(2) keep the living environment humidity of Herba Lycopodii serrati more than 95%.
(3) keep the living environment illumination of Herba Lycopodii serrati at about 100Lx.
(4) be used for taming in cultivation by liver moss first, liver moss is laid on soil surface, advantage is that liver moss plays moisture-keeping function, maintains a good ecotope.
5. method according to claim 1, it is characterized in that: the unicellular acquisition of described step (2) Herba Lycopodii serrati refers to: carry out surface sterilization to Herba Lycopodii serrati blade, to remove fungi and the bacterium of blade surface, obtain mesophyll cell with mechanical crushing method, carry out as follows:
(1) Herba Lycopodii serrati blade carries out surface sterilization, gets Herba Lycopodii serrati blade, successively with fungi and the bacterium of following methods removing blade surface: 70% alcohol 50s, 0.5mg/L malachite green 8min, 3% hydrogen peroxide 10min, sterilized water 3 times.
(2) by means of scotch tape stripping blade surface wax, be affixed on by blade on the adhesive plaster of ultraviolet sterilization, scrape off leaf surface waxes with pocket knife, blend compounds band pastes 2 ~ 3 times repeatedly to peel off wax.
(3) mechanical crushing method acquisition plant mesophyll is unicellular, the blade peeling off wax is divided into minimum fritter, scrape to be placed in and grind alms bowl, add 5 ~ 10mL liquid nutrient medium, mill after 1 ~ 3min, suspension will be mixed by double-deck 200 order nylon net filters, and with a small amount of nutrient solution drip washing filter cloth, by filtrate centrifugal 2min under the rotating speed of 2000rpm, get precipitation nutrient solution settling flux.
6. method according to claim 1, it is characterized in that: the foundation of described step (3) Herba Lycopodii serrati-endogenetic fungus co-culture system refers to: Herba Lycopodii serrati single-cell suspension liquid is placed in 24 DEG C of constant incubator light culture, within about about 10 days, change a liquid nutrient medium, liquid nutrient medium main component used comprises MS minimum medium, 5.0mg/L2,4-D, 1.0mg/LKT, 20.0g/L sucrose, regulates pH to be 5.8.
7. according to method described in claim 1, it is characterized in that: the observation of described step (4) viable cell counting refers to: by dyed blended in the ratio of 4: 1 to Herba Lycopodii serrati suspension cell and toluylene red dye liquor, after leaving standstill 15 ~ 20min, observation counts.
8. method according to claim 1, is characterized in that: the extraction of described step (5) selagine and ergosterol is carried out as follows:
(1) sample: get nutrient solution 5.0mL, be placed in freezer compartment of refrigerator, repeatedly freeze-thaw three times;
(2) by after nutrient solution and cell thawing, rotary evaporation removing moisture, uses 10mL dissolve with methanol, supersound process 2 times at 30 DEG C, each 20min, therebetween interval 10min, filter, obtain filtrate and be placed in Erlenmeyer flask, add 10mL methyl alcohol after filter paper shreds, again extract as stated above, filter, merge twice filtrate, rotary evaporation removing methyl alcohol.
(3) fully dissolve the medicinal extract revolving and steam and obtain with chromatogram methyl alcohol, be transferred to cillin bottle, methyl alcohol volatilizees, and obtains medicinal extract.
(4) the medicinal extract 1mL chromatogram methyl alcohol obtained after methyl alcohol volatilization fully dissolves, with the filtering with microporous membrane of 0.22 μm.
9. according to method described in claim 1, it is characterized in that: use high performance liquid chromatograph, with Agilent TC C
18post is analyzed selagine and ergosterol.The detection of described step (6) selagine and ergosterol refers to:
(1) with methyl alcohol: 0.08mol/L ammonium acetate (25: 75, v/v) be moving phase, flow velocity is 1.000mL/min, and temperature is 25 DEG C, selagine typical curve is set up, the content of selagine in Detection and Extraction thing under similarity condition under wavelength 310nm;
(2) with methyl alcohol: water (95: 5, v/v) is moving phase, flow velocity is 1.000mL/min, and temperature is 25 DEG C, sets up ergosterol typical curve under wavelength 282nm, the content of ergosterol in Detection and Extraction thing under similarity condition;
(3) according to the experimental result detected, the content curve of selagine and ergosterol in culture system is made.
10. according to method described in claim 1, it is characterized in that: make change curve by the detection level of selagine in system and ergosterol, determine the time point carrying out best results and succeeding transfer culture, with enrichment selagine.
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| CN109258148A (en) * | 2018-09-29 | 2019-01-25 | 西北大学 | A kind of method of Huperzia serrata rooting of cuttings |
| CN109258425B (en) * | 2018-09-29 | 2023-03-03 | 西北大学 | Method for increasing content of huperzine A in huperzia serrata |
| CN110468055A (en) * | 2019-07-29 | 2019-11-19 | 西北大学 | Colletotrichum gloeosporioides Penz and its application in a kind of serrate clubmoss herb |
| CN110468055B (en) * | 2019-07-29 | 2021-09-14 | 西北大学 | Huperzia serrata colletotrichum and application thereof |
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