CN114606133A - Method for extracting exosome from schizochytrium limacinum waste residues - Google Patents
Method for extracting exosome from schizochytrium limacinum waste residues Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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Abstract
The invention discloses a method for extracting exosomes from schizochytrium limacinum waste residues. The method comprises the following steps: treating schizochytrium limacinum algae residue with weak alkaline water, filtering, collecting filtrate, and dropwise adding a neutralizing agent to neutrality; and separation and extraction: sequentially centrifuging for four times, wherein the rotating speed of the second centrifugation is higher than that of the first centrifugation; the third and fourth centrifugation are performed at higher speeds than the second centrifugation. The precipitate obtained by the fourth centrifugation is the exosome; the method realizes the purpose of changing waste into valuable, overcomes the problems of insufficient extraction technology of the exosomes from non-animal sources and the lack of channels for producing the exosomes in a large scale, and simultaneously has high content of the prepared exosomes.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for extracting exosomes from schizochytrium limacinum waste residues.
Background
The exosome is an extracellular vesicle, and is an extracellular vesicle-like corpuscle with a membrane structure actively taken up and secreted by cells. The diameter of the metal wire is about 30 to 300 nm. In 1983, Pan et al discovered that sheep immature erythrocytes can secrete vesicle structures. In 1987, Johnstone et al named Exosomes (Exosomes). Scientists subsequently found that a variety of cells can secrete exosomes. In 1996, Stoorvogel et al discovered that exosomes have important functions of changing extracellular microenvironment, participating in immune regulation, affecting body health and the like, and the exosomes begin to get attention. In 2013, scientists James e.rothman, Randy w.schekman and germany scientist Thomas c.sudhof from usa asked the nobel prize for revealing the regulation mechanism of transport of intracellular vesicles such as secreta exosomes.
The exosome has wide sources and various functions, almost all cells can secrete the exosome, and the exosome has various characteristics of source cells. The exosome carries a plurality of nucleic acids, proteins, lipids and other substances, can change the information transmission and physiological state of cells, achieves a plurality of achievements in the research of disease diagnosis and treatment at present, and shows that the exosome participates in the processes of immune response, cell migration, cell proliferation and differentiation, cell apoptosis, tumor invasion and the like of organisms. In recent years, exosomes have emerged as a drug carrier with low immunogenicity, high biocompatibility, and high administration efficiency. KIM and the like develop and optimize an exosome containing aminoethylethanolamine-polyethylene glycol (AA-PEG) modified carrier loaded with anticancer drug Paclitaxel (PTX), and the exosome has higher carrying capacity on the PTX, strong accumulation capacity in cancer cells after systemic administration and obviously improved treatment effect, and provides a strong and novel carrier for anticancer treatment.
Most of the current research efforts on exosomes have focused on exosomes of animal cell origin. However, the extraction process is complex, the cost is high, and the yield is low; if the exosome derived from animal cells is used as a drug carrier and contains lipids, nucleic acids, proteins and the like consistent with donor cells, the application process has a safety problem. And the plant-derived exosome is more reliable in safety as a drug carrier. Researches find that specific exosome is selected as a drug carrier to carry curcumin, so that the blood concentration and the drug stability can be increased, off-target effect is reduced, the curative effect of the drug is improved, no obvious side effect exists, and the method overcomes the defects of poor water solubility, low utilization rate and limited drug effect of the curcumin. Exosomes prepared from tomatoes, such as Wujulian and the like, are also successfully used as carrier-loaded model drugs. In addition, the plant exosome contains rich miRNA, which is an endogenous single-stranded non-coding RNA, plays an important role in organisms and is a high-efficiency gene expression regulatory factor. Many researches prove that miRNA from plant exosome as a new bioactive component can interact with a mammal system, realize cross-boundary regulation and have wide application prospect.
Docosahexaenoic acid (DHA) is a long-chain unsaturated fatty acid, is an important component substance of cerebral cortex and retina of a human body, has important significance for promoting the health of the human body, can prevent metabolic disorder, improve the immune function and reproductive health, effectively inhibit the formation of thrombus, and prevent the occurrence of cardiovascular diseases, and is widely used for foods, medicaments and the like. Previously, the main source from which people have access to DHA products was fish with high fat content. However, with the degeneration of marine fishery in China and the threat of water body environmental pollution, the traditional source DHA product is influenced by the problems of small catch amount, poor quality caused by water body pollution, poor taste caused by fishy smell and the like.
The schizochytrium limacinum is also called schizochytrium limacinum, belongs to Eumycota, Nemacystaceae, thraustochytriales, and is in the form of spherical single cells with a diameter of 7-15 μm. The schizochytrium limacinum is a marine alga with high DHA content, becomes a main alga species for the current industrial production of DHA, and DHA alga oil produced by using the schizochytrium limacinum has been approved by the Ministry of health of China to become a new resource food raw material. And the reutilization of a large amount of waste residues after the algae oil is extracted is worthy of exploration.
The algae residue generated after unsaturated fatty acid is extracted from schizochytrium limacinum is mostly treated as feed or fertilizer at present, and the algae residue resource is not fully developed and utilized with high value. The method takes schizochytrium residue after industrialized oil extraction as a raw material to extract novel and medicinal active ingredient exosomes. Plant-derived exosomes are currently available at a price of tens to hundreds of 1 mg. The patent provides a better process for preparing schizochytrium limacinum exosomes, and aims to provide a new channel and a new method for high-value utilization of algae residue waste.
Patent application CN111567798A utilizes fresh brown algae homogenate, obtains the brown algae juice, with the centrifugation of brown algae juice, obtains the exosome, because exosome content is extremely low, can produce a large amount of solid waste, not only leads to the huge waste of resource, and the brown algae sediment directly discharges easily to the ecological environment cause the pollution in the environment moreover.
The patent application CN112451488A preparation method of plant source type exosome product adopts a preparation process combining low-speed centrifugation and ultrafiltration, and the process has the defects that a filter membrane is easy to block, and the exosome loss is large and the purity is low.
Disclosure of Invention
The schizochytrium limacinum is a marine alga with high docosahexaenoic acid (DHA) content, and has become a main alga species for the current industrial production of DHA, however, the schizochytrium limacinum waste residue contains a small amount of DHA alga oil, and also contains more viscous impurities such as phospholipid, protein, sugar ester and the like, and the oily viscous substances are easy to adsorb exosomes, so that the extraction amount of the exosomes is seriously influenced. The invention aims to overcome the defects of the existing extraction technology of exosomes from non-animal sources and the channel for producing exosomes in a large scale, and provides a preparation method of exosomes from schizochytrium limacinum waste residues.
In order to achieve the aim, the invention provides a method for extracting exosomes from schizochytrium limacinum waste residues, which is characterized by comprising the following steps:
pretreatment: treating schizochytrium limacinum algae residue with weak alkaline water, filtering, collecting filtrate, and dropwise adding a neutralizing agent to neutrality;
separation and extraction: adding PBS, mixing, centrifuging for the first time, and centrifuging the supernatant for the second time; the rotating speed of the second centrifugation is higher than that of the first centrifugation; filtering with a filter membrane; carrying out heavy suspension precipitation after the third centrifugation, carrying out centrifugation for the fourth time, and taking the precipitation as an exosome; the third and fourth centrifugations have higher rotation speed than the second centrifugations.
Further, the schizochytrium limacinum algae residues are the schizochytrium limacinum algae residues after the oil is industrially extracted.
Further, the weak base water is a saturated sodium bicarbonate solution.
Further, the neutralizing agent is acetic acid.
Further, in the pretreatment, the amount of the weak base water added is that the solid-liquid volume ratio is 1: 10 to 30.
Further, the first centrifugation is performed for 10min to 100min under the conditions of 1 ℃ to 25 ℃ and 1000 Xg to 6000 Xg.
Further, the second centrifugation is carried out for 30min to 100min under the conditions of 1 ℃ to 25 ℃ and 10,000 Xg to 60,000 Xg.
Further, the third centrifugation is carried out for 30min to 300min under the conditions that the temperature is between 1 and 25 ℃ and is more than 100,000 Xg.
Further, the filter membrane is a microporous filter membrane.
Further, the fourth centrifugation is carried out for 30min to 300min under the conditions that the temperature is between 1 and 25 ℃ and is more than 100,000 Xg.
The existing exosome extraction is a little extraction, and is not industrialized, and waste materials are not used for extracting exosomes. And the common algae are processed into waste residues, so that exosomes cannot be extracted, and analysis may be damaged. The fresh algae has exosome and can be extracted by a certain method. The inventor has discovered that the residue left by the processed schizochytrium has exosomes by an accidental chance, and the exosomes are protected from being damaged by the high oil content which is analyzed to be high.
The raw material is derived from the schizochytrium limacinum waste residues, so that the method has the advantages of sufficient raw material source and low cost, and the prepared exosome has the advantages of high yield and stable quality, and a new thought is provided for widening plant source channels and recycling the high value of the microalgae waste residues. Compared with the traditional exosomes derived from animals, the exosome has the advantage of more reliable biological safety.
The invention has no special requirements on the schizochytrium limacinum waste residues, and the schizochytrium limacinum waste residues obtained by the normal production process can be obtained. Such as schizochytrium waste residues after the schizochytrium oil extraction process. The extraction process of schizochytrium algae oil is a conventional technology: firstly, squeezing out the water on the outer surface of the fresh schizochytrium limacinum, and then drying and dehydrating. Squeezing the dried schizochytrium limacinum, extracting the squeezed schizochytrium limacinum cake, sequentially extracting, draining and repeatedly circulating the schizochytrium limacinum cake through extracting agents with different concentrations in a negative pressure state to extract the algae oil in the schizochytrium limacinum to the maximum, and finally removing the algae oil residues through rotary liquid separation.
In the pretreatment of the schizochytrium limacinum waste residues, the invention discovers that if strong alkali such as sodium hydroxide or potassium hydroxide is added into the algae residues, the free fatty acid in the algae residues is easy to generate saponification reaction, the generated soapstock has strong adsorption capacity, so that a large amount of exosomes are adsorbed and lost, the strong alkali can damage the membrane structure of the exosomes, and the exosomes cannot be basically seen in the later electron microscope analysis. If medium-strength alkali such as saturated sodium carbonate is added into the algae residue, the membrane structure of exosome is damaged due to strong alkalinity and pH exceeding 10, and the yield is low. Finally, the inventor finds that the weak base, particularly the saturated sodium bicarbonate aqueous solution is added into the algae residues, so that the oily viscous property of the schizochytrium limacinum waste residues is improved to a great extent, the dispersibility of the schizochytrium limacinum waste residues in the aqueous solution is promoted, and the subsequent extraction of water-soluble exosomes is facilitated. The weak alkali water elution of schizochytrium limacinum waste residue is mainly favorable for separating lipid and water, so that solid impurities such as free fatty acid, mucus, protein, phospholipid and the like are flocculated and settled and separated from water-soluble exosome. The pretreatment method is simple to operate, the reagent is cheap, and the reaction is mild.
The invention can recover the original treatment way used as feed or fertilizer after the exosome is extracted from the algae residue generated after unsaturated fatty acid is extracted from schizochytrium limacinum, so that the algae residue is utilized at a high value.
The schizochytrium limacinum waste residue is treated by weak alkaline water, filtered and collected, and then a neutralizing agent is dripped into the schizochytrium limacinum waste residue until the schizochytrium limacinum waste residue is neutral. The exosome is a vesicle with a membrane structure, and in order not to damage the biological structure, the solution is adjusted to be neutral, and then PBS is added to keep the separation environment to have salt balance and proper pH value.
The preparation method is suitable for separating and extracting exosome with specific particle size, and the controllability of the particle size range is strong. The grain diameter of schizochytrium limacinum algae residue exosomes is approximately 40-160nm, the invention adopts the combination of low-speed centrifugation and high-speed centrifugation and the change of the revolution of the ultra-high-speed centrifugation to obtain exosomes with the grain diameter of about 70nm, and the exosomes fall into the highest abundance area of 60-80nm of schizochytrium limacinum algae residue exosomes, and are shown in a grain diameter figure 3 of the attached figure.
Drawings
FIG. 1 is a transmission electron micrograph of the exosomes obtained in example 1. Wherein A is a 100nm field view and B is a 200nm field view.
Figure 2 is a standard graph of BCA assay exosome protein concentration.
FIG. 3 is a graph of the particle size abundance of Schizochytrium sp.
Detailed Description
Reference will now be made in detail to embodiments of the invention, examples of which are illustrated in the accompanying drawings, wherein like or similar abbreviations refer to the same or similar reagents or products having the same or similar functions throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1: exosome is extracted from schizochytrium limacinum waste residue
1. Pretreatment:
1) 1g of schizochytrium limacinum waste residue is put into a 50ml flat-bottomed flask, 20ml of saturated sodium bicarbonate aqueous solution is added, and the mixture is placed on a magnetic stirrer and stirred for 1 hour at room temperature.
2) Pouring the obtained solution into a funnel with qualitative filter paper, filtering off solid impurities, collecting filtrate, and dropwise adding dilute acetic acid into the filtrate until phenolphthalein does not change color.
2. Separation and extraction:
1) measuring about 10mL of schizochytrium limacinum filtrate obtained by pretreatment, adding PBS to 50mL, and mixing uniformly.
2) Centrifugation was carried out at 2000 Xg for 10min at 4 ℃.
3) The supernatant was transferred to a new centrifuge tube and centrifuged again at 10,000 Xg for 30min at 4 ℃.
4) Collecting supernatant, filtering with 0.45 μm filter membrane, and collecting filtrate.
5) The filtrate was transferred to a new centrifuge tube and centrifuged for 70min at 100,000 Xg and 4 ℃ using an ultracentrifuge rotor.
6) The supernatant was removed, resuspended in 10mL of pre-cooled 1 XPBS, and the ultrarotor selected, and ultracentrifuged again at 4 ℃ and 100,000 Xg for 70 min.
7) The supernatant was removed, resuspended in 70. mu.L of pre-cooled 1 XPBS and the pellet collected, i.e.the exosomes, were stored at-80 ℃.
Transmission electron microscope observation of exosome samples of schizochytrium limacinum waste residues:
sucking 10 μ L of the obtained exosome, dripping the exosome on a copper net for precipitation for 1min, and sucking the floating liquid by filter paper. 10 mu L of uranyl acetate is dripped on a copper net for precipitation for 1min, and the floating liquid is absorbed by filter paper. Drying for several minutes at normal temperature. And (5) performing electron microscope detection imaging at 100 kv. Transmission electron microscopy imaging results were obtained, see figure 1. Panel A is a 100nm field view and panel B is a 200nm field view, from which it can be seen that vesicles are circular cakes, filled on the periphery and invaginated in the middle, which are typical exosome characteristics, indicating that it is feasible to extract exosomes using the present method.
Extracting protein of an exosome sample of schizochytrium limacinum waste residue and measuring the concentration:
(1) the resulting exosomes were thawed at 37 ℃ and 5 x RIPA lysate was added quickly.
(2) After mixing, the mixture was lysed on ice for 30min, during which time it was mixed.
(3) Preparing a standard sample for measuring the protein concentration by using the BCA method, taking 5 mu L of the sample, adding the sample into the BCA mixed solution, and uniformly mixing.
(4) Incubate 30min at 37 deg.C, detect the absorbance at OD562 nm on the microplate reader and record.
(5) The protein concentration of the sample to be tested was calculated from the standard curve (see FIG. 2: standard curve for BCA assay for exosome protein concentration).
The measured OD562 of the exosome protein was substituted into the above formula (formula y-10.706 x-0.7096, R, fig. 2) respectively20.9983), the resulting exosome protein concentrations were extracted as shown in table 1 below:
table 1: protein concentration profile for exosomes
It can be seen that if the exosome solution is approximately considered to be a pure solution, the protein concentration of the lysed sample represents the amount of exosomes, and the protein concentration of exosomes of schizochytrium waste residue obtained through a series of extractions is 2.24 μ g/μ L, then the protein concentration of exosomes of 1g of initial algal residue is approximately 313.6 μ g/g, which is significantly higher than the prevalent protein concentration (1-5 μ g/g) of exosomes of the existing plant source.
Particle size analysis of exosome samples:
1 exosomes were taken out 10. mu.L and diluted to 30. mu.L.
2, the sample of the exosome sample can be loaded after the instrument performance test is qualified by using the standard sample, and the sample is required to be diluted in a gradient manner to avoid the sample from blocking the sample injection needle.
And 3, obtaining the particle size of the exosome detected by the instrument after the sample is detected.
From FIG. 3, it can be calculated that the average particle size of the sample exosomes is 72.97 nm.
Example 2: exosome is extracted from schizochytrium limacinum waste residue
1. Pretreatment:
1) 1g of schizochytrium limacinum waste residue is put into a 50ml flat-bottomed flask, 30ml of saturated sodium bicarbonate aqueous solution is added, and the mixture is placed on a magnetic stirrer and stirred for 1 hour at room temperature.
2) Pouring the obtained solution into a funnel with qualitative filter paper, filtering off solid impurities, collecting filtrate, and dropwise adding dilute acetic acid into the filtrate until phenolphthalein does not change color.
2. Separation and extraction:
1) measuring about 10mL of schizochytrium limacinum filtrate obtained by pretreatment, adding PBS to 50mL, and mixing uniformly.
2) Centrifuge at 3000 Xg for 100min at 4 ℃.
3) The supernatant was transferred to a new centrifuge tube and centrifuged again at 60,000 Xg for 20min at 4 ℃.
4) Collecting supernatant, filtering with 0.45 μm filter membrane, and collecting filtrate.
5) The filtrate was transferred to a new centrifuge tube, an ultracentrifuge rotor was selected, and centrifuged at 200,000 Xg for 60min at 4 ℃.
6) The supernatant was removed and resuspended in 10mL of pre-cooled 1 XPBS, and the ultracentrifuge selected again at 4 ℃ and 200,000 Xg for 60 min.
7) The supernatant was removed, resuspended in 70. mu.L of pre-cooled 1 XPBS and the pellet collected, i.e.the exosomes, were stored at-80 ℃.
Transmission electron microscope observation of exosome samples of schizochytrium limacinum waste residues:
observed by a transmission electron microscope in the same way as in example 1, the vesicle is in a circular cake shape, is full at the periphery and is sunken in the middle, and is a typical exosome characteristic.
The protein concentration of the exosome of the separated and purified schizochytrium limacinum waste residue is 1.93 g/mu L by detecting the method of the embodiment 1.
The average particle size of the exosomes of the sample was calculated to be around 65nm by the same method as in example 1.
Example 3: exosome is extracted from schizochytrium limacinum waste residue
1. Pretreatment:
1) 1g of schizochytrium limacinum waste residue is put into a 50ml flat-bottomed flask, 10ml of saturated sodium bicarbonate aqueous solution is added, and the mixture is placed on a magnetic stirrer and stirred for 1 hour at room temperature.
2) Pouring the obtained solution into a funnel with qualitative filter paper, filtering off solid impurities, collecting filtrate, and dropwise adding dilute acetic acid into the filtrate until phenolphthalein does not change color.
2. Separation and extraction:
1) measuring about 10mL of schizochytrium limacinum filtrate obtained by pretreatment, adding PBS to 50mL, and mixing uniformly.
2) Centrifugation was carried out at 6000 Xg for 20min at 4 ℃.
3) The supernatant was transferred to a new centrifuge tube and centrifuged again at 30,000 Xg for 60min at 4 ℃.
4) Collecting supernatant, filtering with 0.45 μm filter membrane, and collecting filtrate.
5) The filtrate was transferred to a new centrifuge tube and centrifuged for 70min at 300,000 Xg and 4 ℃ using an ultracentrifuge rotor.
6) The supernatant was removed, resuspended in 10mL of pre-cooled 1 × PBS, and the ultracentrifuge selected, again at 4 deg.C, 300,000 × g, and ultracentrifuged for 70 min.
7) The supernatant was removed, resuspended in 70. mu.L of pre-cooled 1 XPBS and the pellet collected, i.e.the exosomes, were stored at-80 ℃.
Observed by a transmission electron microscope in the same way as in example 1, the vesicle is in a circular cake shape, is full at the periphery and is sunken in the middle, and is a typical exosome characteristic.
The protein concentration of the exosome of the separated and purified schizochytrium limacinum waste residue is 2.56 mu g/mu L by detecting the method of the embodiment 1.
The average particle size of the exosomes of the sample was calculated to be around 60nm by the same method as in example 1.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made in the above embodiments by those of ordinary skill in the art without departing from the principle and spirit of the present invention.
Claims (10)
1. A method for extracting exosomes from schizochytrium limacinum waste residues is characterized by comprising the following steps:
pretreatment: treating schizochytrium limacinum algae residue with weak alkaline water, filtering, collecting filtrate, and dropwise adding a neutralizing agent to neutrality;
separation and extraction: adding PBS, mixing, centrifuging for the first time, and centrifuging the supernatant for the second time; the rotating speed of the second centrifugation is higher than that of the first centrifugation; filtering with a filter membrane; carrying out heavy suspension precipitation after the third centrifugation, carrying out centrifugation for the fourth time, and taking the precipitation as an exosome; the third and fourth centrifugation are performed at higher speeds than the second centrifugation.
2. The method for extracting exosomes from schizochytrium limacinum waste residues as claimed in claim 1, wherein the schizochytrium limacinum algae residues are the algae residues of schizochytrium limacinum after the oil is industrially extracted.
3. The method of extracting exosomes from schizochytrium limacinum waste residues as claimed in claim 1, wherein the weak alkaline water is saturated sodium bicarbonate solution.
4. The method of extracting exosomes from schizochytrium limacinum waste residues as claimed in claim 1, wherein the neutralizing agent is acetic acid.
5. The method for extracting exosomes from schizochytrium limacinum waste residues as claimed in claim 1, wherein in the pretreatment, the amount of weak alkaline water added is that the solid-liquid volume ratio is 1: 10 to 30.
6. The method for extracting exosomes from schizochytrium limacinum waste residues according to claim 1, wherein the conditions of the first centrifugation are 1-25 ℃, 1000 Xg-6000 Xg, and 10-100 min of centrifugation.
7. The method for extracting exosomes from schizochytrium limacinum waste residues according to claim 1, wherein the conditions of the second centrifugation are 1-25 ℃, 10,000-60,000 Xg, and 30-100 min of centrifugation.
8. The method for extracting exosomes from schizochytrium limacinum waste residues as claimed in claim 1, wherein the conditions of the third centrifugation are 1-25 ℃, more than 100,000 Xg, and the centrifugation is carried out for 30-300 min.
9. The method of extracting exosomes from schizochytrium limacinum waste residues as claimed in claim 1, wherein said filter is a microporous filter.
10. The method for extracting exosomes from schizochytrium limacinum waste residues as claimed in claim 1, wherein the conditions of the fourth centrifugation are 1-25 ℃, more than 100,000 Xg, and 30-300 min of centrifugation.
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