CN106011204B - Method for synthesizing echinocandin B by fermentation method - Google Patents

Abstract

The invention discloses a method for synthesizing echinocandin B by a fermentation method, which comprises the following steps: inoculating aspergillus nidulans serving as a production strain into a fermentation medium, culturing in two stages, culturing for 4-8 days at 30-37 ℃ in the first stage, culturing for 4-8 days at 23-27 ℃ in the second stage, and separating and purifying fermentation liquor to obtain echinocandin B; the invention provides a method for improving the yield of an anidulafungin precursor compound echinocandin B through two stages of temperature or the synergy with a feed phase, and the yield of the anidulafungin precursor compound ECB is improved through the two stages of temperature and the synergy with the feed phase. The result shows that the highest yield in the fermentation culture system reaches 2.05g/L, which is about 1.7 times of the original traditional culture scheme and is improved by 10 percent compared with the single two stages. The invention has important significance for producing ECB by fermenting the production strain with the aspergillus nidulans.

Description

A kind of method for synthesizing echinocandin B by fermentation method

(1) Technical field

The present invention relates to a preparation method of echinocandin B, in particular to a method for fermenting and producing anidungin precursor compound echinocandin B (ECB) by controlling environmental factors (temperature) and feeding in stages .

(2) Background technology

In recent years, organ transplantation has led to the extensive use of immunosuppressants, chemotherapy and the application of more invasive medical methods, etc., resulting in an increase in the number of immunocompromised patients, and a significant increase in the incidence of fungal infections, especially deep fungal infections. The rate and case fatality rate are increasing year by year. According to the statistics of the World Health Organization (WHO) in 2004, the global annual number of systemic fungal infections is 335,000. The most common of these is systemic candidiasis, which accounts for around 70% of all such infections, and causes mortality rates as high as around 40%. With the continuous progress of medicine, after the 1990s, various antifungal drugs have been continuously developed, bringing revolutionary progress to the treatment of various fungal diseases. Due to the increasing demand for antifungal drugs year by year, its market size has become more and more prosperous.

Echinocandin antibiotics are a group of natural products discovered in the 1970s. They have similar structural features of a cyclic polypeptide core and different fatty acid side chains, and can non-competitively inhibit fungal cell wall β-1, 3-glucan synthase activity, so as to achieve the purpose of antifungal. Anidulafungin is the third generation of systemic antifungal echinocandin derivatives after Caspofungin and Micafungin. On February 21, 2006, by Anidulafungin produced by Pfizer has passed the US FDA certification, mainly for the treatment of candidemia and other forms of Candida infections (abdominal abscess, peritonitis) and esophageal candida. Anidungin is a precursor compound echinocandin B (ECB) that removes the side chain linoleyl group by the action of acylase produced by Actinomyces utah, and then reacts with the active intermediate 4"-pentane in DMF. Oxy-[1,1',4',1"]triphenyl-4-carboxylic acid-2,4,5-trichloro-phenyl ester was obtained by reaction.

According to literature and patent reports, ECB is currently prepared by microbial fermentation at home and abroad, and the main one is the fermentation of Aspergillus nidulans. Maria Papagianni et al. reviewed the effects of inoculum size, types and concentrations of carbon and nitrogen sources, pH, temperature and stirring rate on the growth of Aspergillus nidulans during the fermentation process. ECB is the main precursor compound for the synthesis of anidulafungin, and the level of its fermentation unit will directly affect the market prospect of anidulafungin. Scholars such as FritzBenz determined the molecular structure of ECB by infrared, nuclear magnetic resonance and other methods, and found that its hydrolyzed components included linoleic acid, 4-hydroxy-L-proline, 4-carbonyl-L-proline, L - Threonine, (2S,3S,4S)-4-methyl-3-hydroxyproline, etc., and the addition of carbonate, phosphate, sulfate and nitrate to the fermentation medium has an effect on ECB production increase has a stimulative effect.

The secondary metabolite fermentation process is divided into two stages: the cell growth stage and the product synthesis stage. Generally speaking, the optimum temperature of the two stages is different. The two-stage temperature control strategy is an easy-to-operate and effective fermentation strategy, which can meet the temperature requirements of bacteria in different stages. The feeding strategy can avoid substrate inhibition during the fermentation process and ensure that the fermentation process is carried out at an appropriate substrate concentration. In secondary metabolite fermentation, two-stage temperature control strategy and feeding strategy are of great significance. Since the 1980s, Eli Lilly and Company in the United States has been optimizing the fermentation conditions of echinocandin B for many years, but there has been no major breakthrough in fermentation yield. At present, the main problem is that the level of domestic ECB fermentation preparation is still low. Therefore, it is extremely important for the industrial production of anidulafungin to optimize the fermentation process by means of fermentation metabolism regulation and increase the production of ECB.

(3) Contents of the invention

The purpose of the present invention is to provide a technology for improving the fermentation level of ECB in view of the defect that the existing ECB fermentation yield is low.

The technical scheme adopted in the present invention is:

The present invention provides a method for synthesizing echinocandin B by fermentation. Cultivated at ℃ for 4-8 days, and then cultured at 23-27 ℃ for 4-8 days in the second stage, and separated and purified the fermentation broth to obtain echinocandin B (ECB); the final concentration of the fermentation medium was composed of: glycerol 5 ～50g/L, peptone 5～50g/L, L-proline 1～5g/L, mannitol 10～100g/L, soybean flour 10～100g/L, ornithine 1～5g/L, peanut oil 5～50g/L, K ₂ HPO ₄ ·3H ₂ O 2～20g/L, the solvent is distilled water, the pH value is natural, and the final concentration of the fermentation medium is preferably composed of: glycerol 1%, peptone 0.9%, L-proline 0.5%, mannitol 9.7%, soybean flour 4%, L-ornithine 0.5%, peanut oil 2%, K ₂ HPO ₄ ·3H ₂ O 0.84%, the solvent is distilled water, the pH value is 6.8-7.0. In the present invention, the composition of the medium is expressed in terms of mass-volume (w/v) percentage, and a concentration of 1% of a certain component means that 100 mL of medium contains 1 g of the component.

Further, the Aspergillus nidulans is Aspergillus nidulans ZJB09223, which is preserved in the China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, zip code 430072, preservation number is CCTCC NO: M2012300, preservation date is 2012 On July 25th, it was published in "Mutagenesis breeding of highechinocandin B producing strain and further titer improvement with culturemedium optimization".

Further, the first-stage culture for 6 days was transferred to the second-stage culture.

Further, the first stage was cultured at 30°C for 6 days, and then switched to the second stage at 25°C for 6 days on the 7th day.

Further, mannitol is added to the culture solution during the first and second stages of fermentation and culture at the same time, and the total amount of mannitol added is 30-50 g/L of the culture solution. Preferably, the method for adding mannitol is: in the first stage. On the 3rd to 9th days of the first and second stage fermentation, mannitol is added 1 to 3 times, more preferably on the 3rd, 6th and 9th days of the fermentation culture, respectively, add mannitol to the culture medium once , the amount of each addition is 15g/L culture medium.

Further, the Aspergillus nidulans is subjected to slant culture and seed culture before fermentation culture: (1) slant culture: inoculate Aspergillus nidulans into a slant medium, and culture at 25°C for 12 days to obtain slant cells; the slant The final concentration of the medium is: potato 200g/L, sucrose 20g/L, agar powder 20g/L, the solvent is water, and the pH value is natural; (2) seed culture: inoculate step (1) slant cells into the seed medium, Incubate at 25-30°C for 24-48h to obtain seed liquid; the final concentration of seed medium is composed of cottonseed meal 25g/L, glucose 10g/L, glycerol 10g/L, pH 6.8-7.0, and water as solvent.

Further, the method for synthesizing echinocandin B by the fermentation method of the present invention is as follows: inoculating Aspergillus nidulans CCTCC NO: M2012300 into the fermentation medium, and culturing in two stages, the first stage is cultured at 30°C for 6 days, Then the second stage was cultured at 25°C for 6 days, and mannitol was added to the culture solution on the 3rd, 6th and 9th days of the first and second stages of fermentation and culture, respectively, and the amount of each addition was 15g/L culture broth, the fermentation broth was separated and purified to obtain echinocandin B.

The invention aims to realize the increase of ECB output through the synergy of temperature two-stage and feeding, so as to provide technical support for industrialized production of ECB, and to lay a foundation for the production of anidulafungin.

Compared with the prior art, the beneficial effects of the present invention are mainly reflected in: the present invention provides a method for increasing the yield of anidulafungin precursor compound echinocandin B by two-stage temperature or synergistically with feeding. Two-stage synergy with feeding increases the production of anidulafungin precursor compound ECB. The results showed that the highest yield in the fermentation culture system reached 2.05 g/L, which was about 1.7 times that of the original traditional culture scheme, and was 10% higher than that of the two-stage alone. The invention has important significance for using Aspergillus nidulans as a production strain to ferment and produce ECB.

(4) Description of drawings

Figure 1 The effect of different culture temperatures on the dry weight of bacteria;

Fig. 2 The effect of different culture temperature on ECB yield;

Fig. 3 The effect of temperature change on ECB fermentation on day 3;

Fig. 4 The effect of temperature change on ECB fermentation on the 9th day;

Figure 5 The effect of temperature change on ECB fermentation on the 6th day;

Fig. 6 Effects of different feed concentrations on ECB fermentation;

Figure 7 Effect of feeding on ECB fermentation on day 3;

Figure 8 Effect of feeding on ECB fermentation on day 6;

Fig. 9 The effect of feeding on ECB fermentation on day 9;

Figure 10 Effects of feeding on ECB fermentation on days 3, 6, and 9;

Fig. 11 The effect of synergistic effect of two-stage temperature control strategy and feeding strategy on ECB fermentation.

(5) Specific implementation methods

The present invention is further described below in conjunction with specific embodiment, but the protection scope of the present invention is not limited to this:

Example 1 The effect of temperature on the growth of Aspergillus nidulans ZJB09223 and synthesis of ECB

(1) Inoculate Aspergillus nidulans ZJB09223 into slant medium, cultivate at 25° C. for 12 days to obtain slant cells; the final concentration of the slant medium is: potato 200g/L (boil supernatant), sucrose 20g/L , Agar powder 20g/L, solvent is water, pH value is natural, after 121 ℃, 20min high temperature sterilization and cooling before use.

(2) Inoculate the slanted cells in step (1) into the seed medium, and cultivate at 25°C for 48 hours to obtain seed liquid; the final concentration of the seed medium is composed of: cottonseed meal 25g/L, glucose 10g/L, glycerol 10g/L, pH6.8～7.0, the solvent is water, sterilized and cooled at 115℃ for 30min and used after high temperature sterilization.

(3) Fermentation culture: inoculate the seed liquid in step (2) with an inoculation amount of 10% by volume to 1 L fermentation medium, cultivate at 20° C. and 220 rpm for 12 days, and sample and detect the biomass (dry weight) in the fermentation liquid every day. and echinocandin B concentration; the final concentration of the fermentation medium is composed as follows: glycerol 10g/L, peptone 8.6g/L, L-proline 5g/L, mannitol 97g/L, soybean meal powder 40g/L , ornithine 5g/L, peanut oil 20g/L, K ₂ HPO ₄ ·3H ₂ O 8.4g/L, the solvent is distilled water, and the pH value is natural.

The content of ECB in the fermentation broth was detected by HPLC. Sample pretreatment: take 5ml of fermentation broth and place it in a 10ml centrifuge tube, centrifuge at 5000rpm for 5min, discard the precipitate and collect the supernatant; mix the supernatant with absolute ethanol in a volume ratio of 1:4, centrifuge at 10000rpm for 10min, and collect the supernatant It was filtered through a 0.45 μm microfiltration membrane, and the filtrate was analyzed by Shimadzu HPLC. HPLC mobile phase preparation: accurately weigh 0.30g KH ₂ PO ₃ and 0.35g Na ₂ HPO ₃ , dissolve in ultrapure water and dilute to 500ml, filter with 0.45μm microfiltration membrane; the filtrate and chromatographically pure acetonitrile are in a volume ratio of 3: 7 Mix and use ultrasonic to remove gas. HPLC analysis conditions: Shimadzu LC-20AT pump, Shimadzu SPD-20A UV-Vis detector, chromatographic column is C18 column (Dalian Elite 4.6mm × 250mm), and the mobile phase ratio is acetonitrile: methanol: water = 2:7 : 1, the flow rate is 1.0ml/min, the UV detection wavelength is 222nm, the injection volume is 20μL, and the column temperature is 40℃.

When the fermentation temperature was 20°C, the maximum biomass (dry weight) during the fermentation process was 56.8 g/L, and the maximum concentration of ECB was 0.92 g/L; when the fermentation temperature was 25°C, the maximum biomass (dry weight) during the fermentation process was 0.92 g/L. When the fermentation temperature is 30°C, the maximum biomass (dry weight) during the fermentation process is 72.8g/L, and the maximum concentration of ECB is 1.22g/L. L; When the fermentation temperature was 35°C, the maximum biomass (dry weight) during the fermentation process was 67.6 g/L, and the maximum concentration of ECB was 0.98 g/L. The test results are shown in Figures 1 and 2.

Fermentation of secondary metabolites is generally divided into bacterial growth phase and product synthesis phase. The optimum fermentation temperature for the two processes is different. It can be seen from the test results that when the culture temperature is 30 °C, the bacterial growth is the fastest in the early stage of fermentation. , the maximum dry weight was obtained, indicating that the optimum growth temperature of the bacteria was 30 °C. When the culture temperature was 25°C, the final ECB yield was the largest, indicating that the optimum temperature for ECB formation was 25°C.

The influence that embodiment 2 fermentation adopts temperature two-stage produces on ECB synthesis

The slant and seed culture are the same as in Example 1, and the fermentation culture in step (3) of Example 1 is carried out in two stages. , from the first stage to the second stage, using the method of Example 1 to detect the concentration of product ECB in the fermentation broth.

On the third day of the liquid submerged fermentation, the first stage was changed to the second stage, and the concentration of the product ECB was 1.44 g/L. The test results are shown in Figure 3.

Example 3 Effect on ECB synthesis when fermentation adopts two stages of temperature

The slant and seed culture are the same as in Example 1, and the fermentation culture in step (3) of Example 1 is carried out in two stages. The culture temperature of the first stage is 30 ° C, and the cultivation temperature of the second stage is 25 ° C. On the 9th day of fermentation , from the first stage to the second stage, using the method of Example 1 to detect the concentration of product ECB in the fermentation broth.

On the 9th day of the liquid submerged fermentation, the first stage was transferred to the second stage, and the concentration of the product ECB was 0.98g/L; the test results are shown in Figure 4.

Example 4 Effect on ECB synthesis when fermentation adopts two stages of temperature

The slant and seed cultivation are the same as in Example 1, and the fermentation culture in step (3) of Example 1 is carried out in two stages. The cultivation temperature of the first stage is 30° C., and the cultivation temperature of the second stage is 25° C. On the sixth day of fermentation , from the first stage to the second stage, using the method of Example 1 to detect the concentration of product ECB in the fermentation broth.

On the 6th day of the liquid submerged fermentation, the concentration of the product ECB was 1.72 g/L from the first stage to the second stage; the test results are shown in Figure 5.

It can be seen from the test results that the ECB yield is the highest when the temperature transition time point is the 6th day. sky.

The influence that embodiment 5 produces on ECB synthesis when adopting temperature two-stage adding feeding mannitol

Slope and seed culture are with embodiment 1, with embodiment 1 step (3) fermentation culture (mannitol content is 85g/L mannitol in fermentation medium), adopts temperature two-stage control and feeding collaboration when carrying out liquid submerged fermentation Strategic fermentation, 30°C fermentation was used for the first 6 days, and the fermentation temperature was changed to 25°C on the 7th day. During the fermentation process, mannitol was added, and 15g mannitol was added per liter of fermentation broth. The other culture conditions were exactly the same. The concentration of ECB It is 1.75g/L, and the test results are shown in Figure 6.

The influence that embodiment 6 produces on ECB synthesis when adopting temperature two-stage adding feeding mannitol

Slope and seed culture are the same as in Example 1, with the fermentation culture of Example 1 step (3) (the mannitol content in the fermentation medium is 70g/L mannitol), and the temperature two-stage control and feeding collaboration are adopted when carrying out liquid submerged fermentation Strategic fermentation, 30°C fermentation was used for the first 6 days, and the fermentation temperature was changed to 25°C on the 7th day. During the fermentation process, mannitol was added, and 30g mannitol was added per liter of fermentation broth. The other culture conditions were exactly the same, and the concentration of ECB It is 1.81g/L, and the test results are shown in Figure 6.

The influence that embodiment 7 produces on ECB synthesis when adopting temperature two-stage adding feeding mannitol

Slope and seed culture are with embodiment 1, with embodiment 1 step (3) fermentation culture (mannitol content is 40g/L mannitol in fermentation medium), adopt temperature two-stage control and feeding collaboration when carrying out liquid submerged fermentation Strategic fermentation, 30 °C fermentation was used for the first 6 days, and the fermentation temperature was changed to 25 °C on the 7th day. During the fermentation process, mannitol was added, and 60 g of mannitol was added per liter of fermentation broth. Other culture conditions were exactly the same. The concentration of ECB It is 1.85g/L, and the test results are shown in Figure 6.

Example 8 The effect on ECB synthesis when two stages of temperature are added with feeding mannitol

Slope and seed culture are with embodiment 1, with embodiment 1 step (3) fermentation culture (mannitol content is 55g/L mannitol in fermentation medium), adopt temperature two-stage control and feeding collaboration when carrying out liquid submerged fermentation Strategic fermentation, 30 °C fermentation was used for the first 6 days, and the fermentation temperature was changed to 25 °C on the 7th day. During the fermentation process, mannitol was added, and 45g mannitol was added per liter of fermentation broth. Other culture conditions were exactly the same, and the concentration of ECB It is 1.87g/L, and the test results are shown in Figure 6.

It can be seen from the test results that when the feeding concentration is 45g/L and 60g/L, the output of ECB is higher. Considering economic factors, 45g/L is the optimal feeding concentration. The collaborative strategy controlled the fermentation, the ECB yield was increased by 30.7% and the optimal feed concentration for ECB fermentation was 45 g/L.

Example 9 Effects of different ways of supplementing mannitol on the synthesis of ECB when producing ECB by temperature two-stage fermentation

Slope and seed culture are with embodiment 1, with embodiment 1 step (3) fermentation culture (mannitol content is 55g/L mannitol in fermentation medium), adopt temperature two-stage control and feeding collaboration when carrying out liquid submerged fermentation Strategic fermentation, 30°C fermentation was used for the first 6 days, the fermentation temperature was changed to 25°C on the 7th day, mannitol was added during the fermentation process, and 45g mannitol was added per liter of fermentation broth on the third day of fermentation, and other culture conditions were complete Similarly, the concentration of ECB was 1.68 g/L, and the test results are shown in Figure 7.

Example 10 Effects of different ways of supplementing mannitol on the synthesis of ECB when producing ECB by temperature two-stage fermentation

Slope and seed culture are with embodiment 1, with embodiment 1 step (3) fermentation culture (mannitol content is 55g/L mannitol in fermentation medium), adopt temperature two-stage control and feeding collaboration when carrying out liquid submerged fermentation Strategic fermentation, 30 °C fermentation was used for the first 6 days, the fermentation temperature was changed to 25 °C on the 7th day, mannitol was added during the fermentation process, and 45g mannitol was added per liter of fermentation broth on the sixth day of fermentation, and other culture conditions were complete Similarly, the concentration of ECB is 1.87g/L, and the test results are shown in Figure 8.

Example 11 Effects of different ways of supplementing mannitol on the synthesis of ECB when producing ECB by temperature two-stage fermentation

Slope and seed culture are with embodiment 1, with embodiment 1 step (3) fermentation culture (mannitol content is 55g/L mannitol in fermentation medium), adopt temperature two-stage control and feeding collaboration when carrying out liquid submerged fermentation Strategic fermentation, 30°C fermentation was used for the first 6 days, the fermentation temperature was changed to 25°C on the 7th day, mannitol was added during the fermentation process, and 45g mannitol was added per liter of fermentation broth on the 9th day of fermentation, and other culture conditions were complete Similarly, the concentration of ECB was 0.9 g/L, and the test results are shown in Figure 9.

Example 12 Effects of different ways of supplementing mannitol on the synthesis of ECB when producing ECB by two-stage fermentation at temperature

Slope and seed culture are with embodiment 1, with embodiment 1 step (3) fermentation culture (mannitol content is 55g/L mannitol in fermentation medium), adopt temperature two-stage control and feeding collaboration when carrying out liquid submerged fermentation For strategic fermentation, 30°C was used for the first 6 days, the fermentation temperature was changed to 25°C on the 7th day, mannitol was added during the fermentation process, and 15g mannitol was added per liter of fermentation broth on the 3rd, 6th, and 9th days of fermentation. , the other culture conditions are exactly the same, the concentration of ECB is 2.05g/L, and the test results are shown in Figure 10.

As can be seen from Table 1, when the number of feedings was 3 times, and 45g/L of mannitol was added for 3, 6, and 9 days respectively, the ECB yield was the highest, which was 2.05g/L. When the cooperative feeding strategy was used to control the fermentation, the most effective feeding strategy was to add 15g/L mannitol at 3, 6 and 9 days, respectively.

Table 1 Effects of different feed concentrations on ECB fermentation

Example 13 Effects of two-stage temperature control strategy and feeding strategy synergistically on ECB synthesis.

The slant and seed culture are the same as in Example 1, and the fermentation culture in step (3) of Example 1 adopts two groups of parallel experiments, wherein the first group is the control group, and the original culture strategy is adopted, and the culture is maintained at a constant temperature of 30 ° C until the fermentation is completed. , the second group was the experimental group. During the liquid submerged fermentation, two-stage temperature control and feeding cooperation strategy were adopted for fermentation. The first 6 days were fermented at 30 °C (the mannitol content in the fermentation medium was 55 g/L mannitol). The seventh The fermentation temperature was changed to 25 °C on the 3rd day, and 15g/L mannitol was added on the 3rd, 6th, and 9th days respectively, for a total of 45g/L three times. The test results are shown in Figure 11.

It can be seen from the test results that when the fermentation is controlled by the temperature two-stage and feeding cooperative strategy, the production of ECB is significantly increased, which is about 1.7 times of the control group. It has obvious promoting effect and has broad industrial application prospects.

Claims (3)

1. a method for synthesizing echinocandin B by fermentation method, it is characterized in that described method is: take Aspergillus nidulans ( Aspergillus nidulans ) CCTCC NO:M 2012300 as production strain, inoculate in fermentation medium, divide into two The first stage was cultured at 30°C for 6 days, followed by the second stage at 25°C for 4 to 8 days. On the 3rd, 6th and 9th days of the fermentation culture, 1 was added to the culture medium. Submannitol, each addition amount is 15g/L culture solution, and the total addition amount of mannitol is 45g/L culture solution, and the fermentation solution is separated and purified to obtain echinocandin B; the final concentration of the fermentation medium is composed of: Glycerin 5～50g/L, Peptone 5～50 g/L, L-Proline 1～5 g/L, Mannitol 10～100 g/L, Soybean Cake Powder 10～100 g/L, Ornithine 1 ～5 g/L, peanut oil 5～50 g/L, K ₂ HPO ₄ ·3H ₂ O 2～20 g/L , the solvent is distilled water, and the pH value is natural. 2. the method for synthesizing echinocandin B by fermentation method as claimed in claim 1, it is characterized in that in described Aspergillus nidulans CCTCCNO:M 2012300 before fermentation culture, carry out slant culture and seed culture first: (1) slant culture: Inoculate Aspergillus nidulans into a slant medium, and cultivate at 25°C for 12 days to obtain slant cells; the final concentration of the slant medium is: potato 200g/L, sucrose 20g/L, agar powder 20g/L, and the solvent is water , the pH value is natural; (2) Seed culture: inoculate the slanted bacteria in step (1) into the seed medium, and cultivate at 25 to 30 °C for 24 to 48 hours to obtain seed liquid; the final concentration of the seed medium is: cottonseed powder 25g/ L, glucose 10g/L, glycerol 10g/L, pH 6.8～7.0, solvent is water. 3. the method for synthesizing echinocandin B by fermentation method as claimed in claim 1, it is characterized in that in described method: inoculate Aspergillus nidulans CCTCC NO:M 2012300 in fermentation medium, cultivate in two stages , the first stage was cultured at 30 °C for 6 days, and then the second stage was cultured at 25 °C for 6 days, and the culture medium was added to the culture medium on the 3rd, 6th and 9th days of the first and second stages of the fermentation process, respectively. Mannitol was added to the medium, and the amount of each addition was 15 g/L of the culture broth, and the fermentation broth was separated and purified to obtain echinocandin B.

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