Method for extracting and preparing exosome from euglena fermentation liquor

Abstract

The invention discloses a method for extracting and preparing an exosome from euglena gracilis fermentation liquor. According to the method, an euglena gracilis mutant algae species ZWang109 is used as a fermentation algae species for heterotrophic culture, fermentation and extraction preparation are performed under a specific process, a differential centrifugation method and a precipitation method are combined, and the exosome is extracted and prepared by changing the concentration of a precipitator and the size of centrifugal force. At present, most research results about exosomes are mostly focused on the exosomes from animal cells, and the safety problem exists in the application process of the exosomes. The plant-derived exosome as a drug carrier is more reliable in safety, the research on the extraction and preparation process of the euglena exosome is less at present, and the problems of difficulty in extraction and preparation of the plant exosome, low purity and the like exist. According to the method, the problem of difficulty in extraction and preparation is solved, and a foundation is laid for wider application of euglena added-value products.

Description

Technical Field

[0001] The invention belongs to the field of microalgae biotechnology, and particularly relates to a method for extracting and preparing exosomes from a fermentation broth of Euglena gracilis. Background Art

[0002] Euglena is a type of single-cell eukaryotic organism with both animal and plant attributes. It can be cultivated in photoautotrophic, mixotrophic and heterotrophic ways. During the cultivation process, it can synthesize a variety of active substances, including euglena polysaccharides, carotenoids, essential vitamins, amino acids, minerals and unsaturated fatty acids, etc. Therefore, it has high economic and application value and has broad application space in the fields of feed, food, nutrition, medicine, cosmetics and bioenergy.

[0003] Extracellular vesicles (EVs) are double-layer lipid-structured vesicles secreted by cells. They play a role in material transfer and information communication between various cells and tissues. Their subpopulations are mainly distinguished by diameter size. Exosomes are small membrane vesicles that are released into the extracellular environment after the fusion of multivesicular bodies (MVBs) formed by intracellular multivesicular bodies with cell membranes. Their diameter is 30 to 150 nm. Studies have shown that naked algae cells produce and secrete EVs into the culture environment, which has application potential in the fields of microalgae environmental adaptation, nutrient transport, viral infection, regulation of ecological balance, disease treatment, and drug delivery platform construction.

[0004] In recent years, exosomes have become a research hotspot in the fields of biology and medicine. At present, most of the research results on exosomes are concentrated on exosomes derived from animal cells. However, the extraction and preparation process is complicated, the cost is high, and the yield is low. If exosomes derived from animal cells are used as drug carriers, they contain lipids, nucleic acids and proteins consistent with donor cells, and there are safety issues in the application process. Plant-derived exosomes are more reliable as drug carriers. At present, there are few studies on the extraction and preparation process of euglena exosomes, and there are problems such as difficulty in extraction and low purity in the extraction and preparation of plant exosomes. Summary of the invention

[0005] Euglena cells are rich in high-value natural pigments, polyunsaturated fatty acids, polysaccharides and other nutrients and functional components. In the process of producing exosomes, Euglena sorts a variety of cell active components into exosomes, so that Euglena exosomes can act directly on receptor cells as a therapeutic agent, showing the effect of cell function regulation, and exercising therapeutic functions in the fields of medical treatment, cosmetics and health care products. Since there is no report on the research on the extraction and preparation method of Euglena exosomes, the present invention provides a method for extracting and preparing exosomes from Euglena fermentation broth, which uses a mutant strain of Euglena gracilis as a fermentation algae species for heterotrophic culture, and ferments and extracts and prepares under a unique process. The obtained Euglena exosomes have good prospects in the application fields of medical treatment, cosmetics and health care products.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A method for extracting and preparing exosomes from euglena fermentation broth comprises the following steps:

[0008] (1) Transferring Euglena Gracilis CGMCC No.40684 to solid EM culture medium to prepare algae strains;

[0009] (2) preparing a liquid EM liquid culture medium for washing the plate to prepare a seed solution;

[0010] (3) Transferring the EM seed solution to EG liquid medium for expansion;

[0011] (4) The expanded EG seed solution is transferred to the seed tank for secondary expansion;

[0012] (5) transferring the seed solution to a fermentation tank for fermentation;

[0013] (6) pretreatment of fermentation broth;

[0014] (7) Extraction and preparation of exosomes from Euglena fermentation broth;

[0015] The EM solid culture medium in step (1) above comprises the following ingredients: 5-20 g / L anhydrous ethanol, 0.03-0.08 g / L urea, 0.01-0.015 g / L calcium chloride, 0.2-0.7 g / L potassium dihydrogen phosphate, 1.2-2.0 g / L magnesium sulfate, 0.001-0.002 g / L manganese chloride, 0.001-0.002 g / L cobalt nitrate, 0.0004-0.0008 g / L zinc sulfate, 0.0002-0.0006 g / L copper sulfate, 0.002-0.008 g / L iron sulfate, 0.0005-0.0008 g / L disodium acetate, 0.0001-0.0004 g / L vitamin B1, 0.000005-0.000008 g / L vitamin B12 , 5-20 g / L agar powder; the composition of the EM liquid culture medium in the above step (2) is: prepared by not adding agar powder to the EM solid culture medium;

[0016] The EG liquid culture medium in step (3) above comprises the following ingredients: 10-30 g / L glucose, 0.03-0.08 g / L urea, 0.01-0.015 g / L calcium chloride, 0.2-0.7 g / L potassium dihydrogen phosphate, 1.2-2.0 g / L magnesium sulfate, 0.001-0.002 g / L manganese chloride, 0.001-0.002 g / L cobalt nitrate, 0.0004 -0.0008g / L zinc sulfate, 0.0002-0.0006g / L molybdic acid, 0.00002-0.00006g / L copper sulfate, 0.002-0.008g / L iron sulfate, 0.0005-0.0008g / L disodium acetate, 0.0001-0.0004g / L vitamin B1, 0.000005-0.000008g / L vitamin B 12 ;

[0017] Furthermore, the culture conditions in step (1) are:

[0018] The culture temperature is 25-30°C, the culture time is 7-10 days, the inoculation volume is 100-200 μl, and the culture is carried out under light.

[0019] Furthermore, the culture conditions of steps (2) and (3) are:

[0020] The culture temperature is 25-30°C, the culture time is 3-5 days, the inoculation amount is 5-10 v / v%, and the culture is carried out under light;

[0021] Furthermore, the culture conditions in step (4) are:

[0022] 0-72h: culture temperature 25-30℃, inoculation volume 5-10%, pH 3-5, tank pressure 0.05-0.1Mpa, rotation speed 50-150rpm, ventilation volume 0.3-1.0m 3 / h, light culture;

[0023] 72-120h: culture temperature 25-30℃, inoculation amount 5-10%, pH 3-5, tank pressure 0.05-0.1Mpa, rotation speed 50-150rpm, ventilation volume 0.3-1.0m 3 / h;

[0024] Furthermore, the culture conditions in step (5) are:

[0025] 0-72h: culture temperature 25-30℃, inoculation volume 5-10%, pH 3-5, tank pressure 0.05-0.1Mpa, rotation speed 50-150rpm, ventilation volume 0.3-1.0m 3 / h, light culture;

[0026] 72-120h: culture temperature 25-30℃, inoculation amount 5-10%, pH 3-5, tank pressure 0.05-0.1Mpa, rotation speed 50-150rpm, ventilation volume 0.3-1.0m 3 / h;

[0027] Preferably, during the culture process, when the glucose concentration is lower than 10 g / L, a one-time EG medium fed-batch fermentation is performed;

[0028] Furthermore, the pretreatment conditions in step (6) are:

[0029] Take 30mL of fresh algal liquid and centrifuge at 5000g for 30min to remove large pieces of debris in the supernatant; after centrifugation, transfer the upper liquid to a new container and discard the lower precipitate to obtain the crude exosome extract, which is frozen in a -80℃ refrigerator and thawed in a 4℃ refrigerator the night before when needed.

[0030] Furthermore, the preparation in step (7) involves membrane, precipitation, and centrifugation;

[0031] In the membrane filtering process, the filter membrane size is 0.22 um.

[0032] The precipitation process is specifically implemented as follows: the crude exosome extract is filtered through a 0.22um filter membrane to obtain a filtrate. The PEG6000 solution prepared in advance is mixed with the filtrate in a volume ratio of 1:1 to start a precipitation, and the solution is placed in a 4°C refrigerator overnight for precipitation.

[0033] The concentration of the precipitant is 18%-26%; preferably, the concentration of the precipitant is 24%;

[0034] The precipitation process is specifically implemented as follows: centrifugation at a centrifugal force of 8000g-12000g for 1 hour; preferably, the centrifugal force is 11000g.

[0035] Beneficial effects of the present invention:

[0036] 1. The present invention adopts a method combining centrifugation and precipitation. The method is simple to operate and the biological activity of the separated exosomes is not affected, laying a foundation for the extraction, preparation and application of exosomes from Euglena fermentation broth.

[0037] 2. The exosome content in the euglena fermentation filtrate of the present invention is the highest at the end of fermentation, and sampling is performed at the end of fermentation to reduce the risk of bacterial contamination during the fermentation process.

[0038] 3. The centrifugal speed used in the present invention is lower than that of other ultra-high-speed centrifugal extraction and preparation methods, and combined with the precipitation method, the precipitant is easy to obtain, the process is simple, and the feasibility is high. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is the standard curve of BCA determination of exosome protein concentration in Euglena.

[0040] Figure 2 This is a graph of the abundance of exosomes in Euglena. DETAILED DESCRIPTION

[0041] In order to better illustrate the technical solution, advantages and active efficacy of the present invention, the present invention will be further introduced in conjunction with specific implementation cases. Although the following embodiment describes the present invention in detail, it is only a part of the embodiment of the present invention, not all embodiments. People can also obtain other embodiments based on this embodiment without inventiveness, and these embodiments all belong to the protection scope of the present invention.

[0042] The Euglena gracilis strain used in this example is the Euglena gracilis mutant strain ZWang109, which was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on June 28, 2023. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is: CGMCC No. 40684.

[0043] Example 1 A method for extracting and preparing exosomes from euglena fermentation broth comprises the following steps:

[0044] (1) Transferring the algae seeds to a solid EM plate to prepare solid EM plate seeds, wherein the solid EM plate culture medium comprises: 12 g / L anhydrous ethanol, 0.05 g / L urea, 0.012 g / L calcium chloride, 0.5 g / L potassium dihydrogen phosphate, 1.5 g / L magnesium sulfate, 0.0015 g / L manganese chloride, 0.0010 g / L cobalt nitrate, 0.0005 g / L zinc sulfate, 0.0002 g / L molybdic acid, 0.00002 g / L copper sulfate, 0.002 g / L iron sulfate, 0.0005 g / L disodium acetate, 0.0001-0.0004 g / L vitamin B1, 0.000005-0.000008 g / L vitamin B 12 . Sterilization conditions: 121℃, 20min sterilization or 0.22μm filter filtration sterilization. Cultivation temperature 27℃, cultivation time 10d.

[0045] (2) Preparation of EM seed liquid, the detailed steps are: add 3 mL of newly configured, sterilized liquid EM culture medium to the solid seed plate to elute the algae species, obtain a bacterial suspension, transfer to 20 mL of EM liquid culture medium, culture temperature 27°C, inoculation amount 10%, culture time 4 days. All 20 mL of cultured fermentation liquid are transferred to 130 mL of EM culture medium, culture temperature 27°C, culture time 4 days. After that, all 150 mL of algae species are further transferred to 850 mL of EM liquid culture medium, culture temperature 27°C, culture time 3 days, and obtain 1 L of EM seed liquid. The culture medium sterilization conditions: 121°C, 20 min sterilization or 0.22 μm filter membrane filtration sterilization.

[0046] (3) Preparation of EG seed solution, the detailed steps are: transfer the EM seed solution to 900 mL of EG culture medium, the inoculation amount is 10%, the culture time is 3 days, and the culture temperature is 27° C. The culture medium sterilization conditions are: 121° C., 20 min sterilization or 0.22 μm filter membrane filtration sterilization.

[0047] (4) Seed tank culture, the detailed steps are: the cultured EG medium is transferred to the seed tank via the seed bottle for expansion culture, the culture time is 5 days, the culture temperature is 27°C, the inoculation amount is 10%, the tank pressure is 0.06Mpa, and the rotation speed is 60rpm. The seed tank fermentation medium is EG medium, and the sterilization conditions are 121°C, 20min sterilization.

[0048] (5) Fermentation tank culture, the detailed steps are: transferring the cultured seed tank fermentation liquid to a 1T fermentation tank through a sterile pipeline for culture, the culture time is 12 days, the culture temperature is 27°C, the inoculation amount is 20%, the tank pressure is 0.06Mpa, and the rotation speed is 60rpm. The seed tank fermentation medium is EG medium, and the sterilization conditions are 121°C, 20min sterilization.

[0049] (6) Fermentation liquid pretreatment, the detailed steps are as follows: take 30 mL of fresh algae liquid from 5, 6, 7, 8, 9, 10, and 11 days of fermentation, centrifuge at 5000 g for 30 min, and remove large pieces of debris in the supernatant; after the centrifugation, transfer the upper layer of liquid to a new container, discard the lower layer of sediment, and obtain the crude exosome extract, which is placed in a -80°C refrigerator for freezing and thawing in a 4°C refrigerator the night before when needed.

[0050] (7) Extraction and preparation of exosomes from naked algae fermentation broth, the detailed steps are as follows: filter the crude exosome extract through a 0.22um filter membrane to obtain a filtrate. Mix the 18%-26% PEG6000 solution prepared in advance with the filtrate in a volume ratio of 1:1, start a precipitation, and place it in a 4°C refrigerator overnight for precipitation. After 24 hours, take out the suspension, and you can see flocs suspended in the solution. Centrifuge it with a centrifugal force of 8000g-12000g for 1 hour; discard the supernatant. Add PBS buffer solution at a ratio of 2mL to 100mL of the supernatant to resuspend the obtained product. Detection of naked algae exosome content (BCA method for determining protein concentration)

[0051] Preparation of protein standards

[0052] a) Take 0.8mL of protein standard solution and add it to a tube of protein standard (20mg BSA) to fully dissolve it to prepare a 25mg / mL protein standard solution. It can be used immediately after preparation or stored at -20℃ for a long time.

[0053] b) Take 20mL of 25mg / mL protein standard and dilute it with PBS buffer solution to a final concentration of 0.5mg / mL. The diluted 0.5mg / mL protein standard can be stored at -20℃ for a long time.

[0054] Preparation of BCA working solution

[0055] Prepare an appropriate amount of BCA working solution by adding 1 volume of BCA reagent B to 50 volumes of BCA reagent A (50:1) and mix thoroughly. Take 20mL of BCA reagent A and add 0.4mL of BCA reagent B, mix well, and prepare BCA working solution. BCA working solution is stable at room temperature for 24 hours.

[0056] Protein concentration determination

[0057] a) Add 0, 10, 20, 40, 80, 120, 160, and 200 μL of the standard into a clean test tube, and add PBS solution to make up to 200 μL, which is equivalent to the standard concentration of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL respectively;

[0058] b) Add an appropriate volume of sample to the test tube. If the sample volume is less than 200uL, add PBS solution to make it up to 200uL. Record the sample volume;

[0059] c) Add 2 mL of BCA working solution to each well and place at 37°C for 30 min;

[0060] d) measuring the absorbance at wavelength A562 using a spectrophotometer;

[0061] The protein concentration of the samples was calculated based on the standard curve and the sample volume used.

[0062] The OD of exosome protein was measured 562 Substitute them into the above formula ( Figure 2 The resulting formula is Y = 0.7307X + 0.1415, R 2 =0.9968) was used for calculation, and the concentration of exosome protein extracted and prepared is shown in Table 1-3 below

[0063]

[0064] Table 1

[0065]

[0066] Table 2

[0067]

[0068] Table 3

[0069] Exosome sample particle size analysis:

[0070] 1 The exosome heavy suspension obtained in step (7) was graded diluted with PBS buffer, with the dilution multiples being 100 times, 1000 times, and 10000 times.

[0071] 2 Using nanoparticle tracking technology, the particle size and number of exosome particles are analyzed and counted, and finally the relevant detection values ​​of exosomes are obtained.

[0072] from Figure 1 It can be calculated that the number of exosome particles in the sample is 4.44×10 11 / mL, the median particle size is 183.9nm, and the optimal dilution factor is 1000 times.

Claims

1. A method for extracting and preparing exosomes from Euglena fermentation broth, comprising the following steps: (1) Transferring Euglena Gracilis CGMCC No.40684 to solid EM culture medium to prepare algae strains; (2) preparing a liquid EM liquid culture medium for washing the plate to prepare a seed solution; (3) Transferring the EM seed solution to EG liquid medium for expansion; (4) The expanded EG seed solution is transferred to the seed tank for secondary expansion; (5) transferring the seed solution to a fermentation tank for fermentation; (6) pretreatment of fermentation broth; (7) Extraction and preparation of exosomes from algae fermentation broth.

2. A method for extracting and preparing exosomes from Euglena fermentation broth as claimed in claim 1, characterized in that: The EM solid culture medium comprises the following components: 5-20 g / L anhydrous ethanol, 0.03-0.08 g / L urea, 0.01-0.015 g / L calcium chloride, 0.2-0.7 g / L potassium dihydrogen phosphate, 1.2-2.0 g / L magnesium sulfate, 0.001-0.002 g / L manganese chloride, 0.001-0.002 g / L cobalt nitrate, 0.0004-0.0008 g / L zinc sulfate, 0.0002-0.0006 g / L copper sulfate, 0.002-0.008 g / L iron sulfate, 0.0005-0.0008 g / L disodium acetate, 0.0001-0.0004 g / L vitamin B1, 0.000005-0.000008 g / L vitamin B 12 , 5-20g / L agar powder.

3. A method for extracting and preparing exosomes from Euglena fermentation broth as claimed in claim 1, characterized in that: The EM liquid culture medium is composed of: EM solid culture medium without adding agar powder.

4. A method for extracting and preparing exosomes from Euglena fermentation broth as claimed in claim 1, characterized in that: The EG liquid culture medium contains the following ingredients: 10-30 g / L glucose, 0.03-0.08 g / L urea, 0.01-0.015 g / L calcium chloride, 0.2-0.7 g / L potassium dihydrogen phosphate, 1.2-2.0 g / L magnesium sulfate, 0.001-0.002 g / L manganese chloride, 0.001-0.002 g / L cobalt nitrate, 0.0004-0.0 0.008g / L zinc sulfate, 0.0002-0.0006g / L molybdic acid, 0.00002-0.00006g / L copper sulfate, 0.002-0.008g / L ferrous sulfate, 0.0005-0.0008g / L disodium acetate, 0.0001-0.0004g / L vitamin B1, 0.000005-0.000008g / L vitamin B 12 .

5. A method for extracting and preparing exosomes from Euglena fermentation broth as claimed in claim 1, characterized in that: The culture conditions of step (1) are: culture temperature 25-30°C, culture time 7-10d, inoculation amount 100-200μl, light culture; further, the culture conditions of steps (2) and (3) are: culture temperature 25-30°C, culture time 3-5d, inoculation amount 5-10v / v%, light culture; further, the culture conditions of step (4) are: 0-72h: culture temperature 25-30°C, inoculation amount 5-10%, pH 3-5, tank pressure 0.05-0.1Mpa, rotation speed 50-150rpm, ventilation volume 0.3-1.0m 3 / h, light culture; 72-120h: culture temperature 25-30℃, inoculation amount 5-10%, pH 3-5, tank pressure 0.05-0.1Mpa, rotation speed 50-150rpm, ventilation volume 0.3-1.0m 3 / h; further, the culture conditions of step (5) are: 0-72h: culture temperature 25-30°C, inoculation amount 5-10%, pH 3-5, tank pressure 0.05-0.1Mpa, rotation speed 50-150rpm, ventilation volume 0.3-1.0m 3 / h, light culture; 72-120h: culture temperature 25-30℃, inoculation amount 5-10%, pH 3-5, tank pressure 0.05-0.1Mpa, rotation speed 50-150rpm, ventilation volume 0.3-1.0m 3 / h.

6. A method for extracting and preparing exosomes from Euglena fermentation broth as claimed in claim 1, characterized in that: The pretreatment is as follows: take 30 mL of fresh algae liquid and centrifuge at 5000 g for 30 minutes to remove large pieces of debris in the supernatant; after the centrifugation, transfer the upper liquid to a new container, discard the lower sediment, and obtain the exosome crude extract, which is frozen in a -80°C refrigerator and thawed in a 4°C refrigerator the night before when needed.

7. A method for extracting and preparing exosomes from Euglena fermentation broth as claimed in claim 1, characterized in that: The precipitation process is specifically implemented as follows: filtering the crude exosome extract through a 0.22um filter membrane to obtain a filtrate; mixing the pre-prepared PEG6000 solution and the filtrate in a volume ratio of 1:1, starting a precipitation, and placing in a 4°C refrigerator for overnight precipitation.

8. A method for extracting and preparing exosomes from Euglena fermentation broth as claimed in claim 1, characterized in that: The concentration of the precipitant is 18%-26%.

9. A method for extracting and preparing exosomes from Euglena fermentation broth as claimed in claim 1, characterized in that: The precipitation process is centrifuged at 8000g-12000g for 1 hour.

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