CN108315381B - Method for preparing grease by using chlorella pyrenoidosa cells with cassava residues as main raw materials - Google Patents
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Abstract
The invention belongs to the technical field of algae, and discloses a method for preparing grease by using chlorella pyrenoidosa cells with cassava dregs as a main raw material, which comprises the following steps: step 1) preparing a trichoderma reesei seed solution, step 2) preparing a protein core chlorella solution, step 3) preparing an aspergillus niger seed solution, step 4) performing mixed culture, and step 5) extracting grease. The method of the invention reduces the cost and improves the oil yield.
Description
Technical Field
The invention belongs to the technical field of algae, and particularly relates to a method for preparing grease by using chlorella pyrenoidosa cells with manioc waste as a main raw material.
Background
Biodiesel is a long-chain fatty acid ester substance, and is a product obtained by reacting a short-chain alcohol substance (methanol or ethanol) with certain fatty oil substances. The source of grease is the focus of current research, and the main sources include: plant origin, animal origin and algal origin. The plant source is that grease extracted from rapeseeds, soybeans, peanuts and various oil crops is used as a raw material; the animal source is animal fat such as lard, beef tallow, mutton fat and the like or waste catering grease; the microalgae is used as a source of algae, a large amount of grease can be synthesized by the microalgae in the growth process, the microalgae grease belongs to single-cell grease, the main components of the microalgae grease are glycerol and fatty acid, the microalgae is synthesized in an algae body by using carbohydrate, hydrocarbon and common grease as carbon sources under a certain condition and mainly used as components of biomembranes, metabolites and energy sources. The development of the biodiesel is restricted due to the plant source, the animal source, the long life cycle of the raw materials, the insufficient total amount of resources, the low economic benefit and the influence on the price of agricultural products, farmland resources and grain safety. Compared with the traditional biodiesel raw materials from plant sources and animal sources, the microalgae have the great advantages of wide distribution, short growth cycle, large biomass, strong environmental adaptability, high oil content, no land competition with grains, no competition with people and the like, are greatly pursued by researchers, and are considered to be one of important ways for solving the problem of insufficient biodiesel raw materials at present. Currently, grease of algae origin is the focus of research. Microalgae have many advantages as a new generation of biodiesel feedstock. Algae are widely distributed in fresh water and seawater. There are tens of thousands of species of microalgae that have been identified worldwide, and their number is increasing. Compared with traditional oil crops, the microalgae have large biomass and short growth period. The growth rate of the microalgae is far higher than that of terrestrial crops, the biomass of the microalgae can be doubled within 24 hours generally, and the biomass doubling time in the exponential growth phase is 3-5 hours generally. The microalgae oil has similar components to vegetable oil, is a substitute of vegetable oil, and can be directly used for producing biodiesel by the prior art. Under normal culture conditions, the oil content of general microalgae can reach 20-50%, the microalgae can be cultured by seawater, can tolerate extreme environments such as deserts, arid lands, semi-arid lands and the like, and does not occupy cultivated lands, so that the production of grain crops is not threatened. The microalgae can absorb and utilize a large amount of C02 and nitride discharged in industrial and agricultural production or extract nitrogen, phosphorus and the like from waste water, and is beneficial to improving the environment.
The biochemical composition of microalgae can be adjusted by changing environmental conditions, thereby increasing oil content. The algae species in Zhejiang are abundant, and the method has natural favorable conditions for researching the production of biodiesel by the algae. The biggest problem in the current industrialization of producing diesel oil by utilizing algae is how to reduce the culture cost and improve the oil yield. Because of the differences in oil content and growth capacity of different microalgae species, the microalgae species need to be screened before microalgae culture. The selected microalgae strain has high productivity, high oil content and strong anti-fouling capability, can adapt to environmental changes and can be cultured on a large scale. At present, the most common algae which can be cultured in a large scale include chlorella, dunaliella and chrysophyceae, and are often used for producing microalgae biodiesel due to higher oil content and growth rate. However, there are many researchers to improve the oil content and growth rate of microalgae by changing the growth environment of microalgae or using genetic engineering.
Chlorella pyrenoidosa belongs to Chlorophyta, Chroococcales and Chlorella, and is the only species with pyrenoidosa in the plants of the genus. The chlorella pyrenoidosa cells contain rich protein, polysaccharide, unsaturated fatty acid, dietary fiber, vitamins, trace elements and the like, have high nutritional value and are excellent health-care food and aquaculture bait for human beings. Has been approved as a new resource food by the Ministry of health of the people's republic of China. The chlorella pyrenoidosa serving as a renewable energy biomass can accumulate grease under photoautotrophic and heterotrophic culture conditions, can effectively utilize solar energy and organic energy to rapidly grow and accumulate the grease, can serve as renewable energy to be used for manufacturing biodiesel, and has great potential in replacing fossil fuel diesel. However, the existing large-scale culture of the chlorella pyrenoidosa has the problems of slow growth, low biomass and low oil content, and is not beneficial to large-scale culture. CN106754383A discloses a method for co-culturing chlorella and oleaginous yeast to improve oil yield, which comprises inoculating the chlorella and oleaginous yeast into a culture medium at the same time, wherein the total fat content of the mixed culture microorganism dry powder can reach 40.55%, the total fatty acid yield can reach 175.64mg/l/d, and is obviously larger than that of the microalgae cells cultured independently. But still has the disadvantages of increased fat content and higher fermentation costs. The cassava dregs are byproducts generated after starch is extracted from cassava, the main indexes comprise crude fiber, crude ash and water, the nutrition cost is low, and the cassava dregs are generally used as feed or waste. In Hangzhou areas, a large number of starch factories exist, a large number of cassava residues are generated when cassava is used for processing and producing starch, and the technical problem to be solved is how to effectively utilize the cassava residues.
Disclosure of Invention
The invention aims to overcome the defects of low oil and fat production efficiency, high culture cost and the like of algae in the prior art, and provides a method for preparing oil and fat by using cassava residues as main raw materials by using chlorella pyrenoidosa cells.
The invention is realized by the following technical scheme:
the method for preparing the grease by using the chlorella pyrenoidosa cells as the main raw material comprises the following steps: step 1) preparing a trichoderma reesei seed solution, step 2) preparing a protein core chlorella solution, step 3) preparing an aspergillus niger seed solution, step 4) performing mixed culture, and step 5) extracting grease.
Specifically, the method comprises the following steps: step 1) preparation of a trichoderma reesei seed solution: inoculating the trichoderma reesei on a PDA culture medium for culturing by streaking to obtain a single colony; selecting a single colony, inoculating the single colony to a primary seed culture medium for culture, and then performing secondary seed culture medium culture to obtain a trichoderma reesei seed solution;
step 2) preparing a protein core chlorella solution: selecting Chlorella pyrenoidosa, inoculating into a container containing growth medium, culturing at 28 deg.C under illumination intensity of 6000lux, shaking the container 2-3 times per day, and growing to logarithmic phase to obtain Chlorella pyrenoidosa solution;
step 3) preparation of an Aspergillus niger seed solution: inoculating Aspergillus niger on a slant culture medium for culturing by streaking to obtain a single colony; selecting a single colony, inoculating the single colony to a primary seed culture medium for culture, and then performing secondary seed culture medium culture to obtain an Aspergillus niger seed solution;
step 4), mixed culture: inoculating Chlorella pyrenoidosa solution and Trichoderma reesei seed solution in logarithmic growth phase into reaction tank containing culture medium containing cassava residue, wherein the inoculation density of Chlorella pyrenoidosa and Trichoderma reesei is 1 × 106Each/ml and 1X 107cfu/ml, temperature 28 deg.C, 24 hr continuous illumination intensity 6000-7cfu/ml, continuously culturing for 3-4 days, and after the culture is finished, centrifuging, washing and freeze-drying to obtain powder;
step 5), extracting grease: treating the powder by adopting a pulsed electric field, and then adding the powder into a chloroform-methanol mixed solution, wherein the addition amount is 1g of powder: 2ml of chloroform-methanol mixed solution, ultrasonically extracting, then centrifuging, collecting a chloroform phase, blow-drying in nitrogen, and vacuum-drying to obtain the grease.
Preferably, in the step 1), the primary seed culture medium and the secondary seed culture medium are both PDA liquid culture media.
Preferably, in step 2), the growth medium has the following composition: 10g/L of glucose, 2g/L of ammonium chloride, 1g/L of sodium nitrate, 0.5g/L of monopotassium phosphate, 0.1g/L of sodium chloride, 100mg/L of magnesium sulfate heptahydrate, 30mg/L of calcium chloride, 20mg/L of ferric ammonium citrate, 10mg/L of zinc sulfate heptahydrate and 10mg/L of manganese sulfate.
Preferably, in the step 3), the slant culture medium comprises: 150g/L of potato, 20g/L of cane sugar and 15g/L of agar; the first-stage seed culture medium and the second-stage seed culture medium comprise the following components: 50g/L of corn flour, 10g/L of cane sugar, 5g/L of ammonium sulfate, 1g/L of monopotassium phosphate and 1g/L of dipotassium phosphate.
Preferably, in the step 4), the specific composition of the culture medium is as follows: 50-80g/L of cassava dregs, 1-2g/L of sodium nitrate, 0.5-1g/L of potassium dihydrogen phosphate, 0.1-0.2g/L of sodium chloride, 200mg/L of magnesium sulfate heptahydrate, 50-70mg/L of calcium chloride, 20-30mg/L of ferric ammonium citrate and 10-15mg/L of zinc sulfate heptahydrate.
Preferably, the pulsed electric field treatment is: electric field intensity 20kV/cm, pulse width 4us, and processing time 200 us.
Preferably, the ultrasound extraction is: the extraction temperature is 60-65 ℃, the ultrasonic power is 100-.
Preferably, the chloroform-methanol mixed solution is prepared by mixing chloroform and methanol according to the volume ratio of 2: 1.
Compared with the prior art, the invention has the advantages that the invention mainly comprises but is not limited to the following aspects:
the growth amount of the algae and the oil yield are not in a positive correlation proportion completely, the growth amount is in a reasonable range by adjusting external factors, and the oil yield is maximized. The trichoderma reesei utilizes fermented cassava residues to generate reducing sugar, the reducing sugar can promote the growth rate of algae and increase biomass, aspergillus niger is inoculated after the algae reaches a certain growth amount, and the aspergillus niger can quickly utilize a nitrogen source and part of the reducing sugar so as to compete with the algae, so that the algae can obtain higher grease content through nitrogen limitation or nutrient limitation under the stress conditions of nitrogen limitation and nutrient deprivation. And Aspergillus niger can also generate a large amount of carbon dioxide inorganic carbon source, so that the grease yield of algae is improved. Trichoderma reesei and Aspergillus niger can also produce grease substances, and cassava residues are used as main raw materials in the culture process, so that the price is low, and the enterprise cost is reduced. Oxygen released by photosynthesis of the microalgae in the mixed culture can be utilized by somatic cells, so that the mixed culture system is in an equilibrium state. The strong electric field with proper treatment time can affect polar molecules in cells, generate oscillation on cell membranes, generate unrecoverable destructive action, enhance the permeability of the cell membranes and finally accelerate the exchange efficiency of intracellular and extracellular grease. When ultrasonic wave acts on a liquid reaction system, a large number of tiny bubbles are formed in the liquid due to the cavitation action of the ultrasonic wave, the bubbles are generated and destroyed very quickly, the reaction system can generate local high temperature and high pressure, the effect of destroying cell walls can be achieved, and the contact chance of a solvent and intracellular substances can be increased. The method for extracting the grease by using the pulsed electric field and the ultrasonic wave is an efficient method, combines the advantages of the pulsed electric field and the ultrasonic wave, shortens the reaction time, and can reduce the reaction energy consumption.
Drawings
FIG. 1: the influence of different pulse electric field time on the oil yield;
FIG. 2: the influence of the ultrasonic power and the ultrasonic time on the oil yield.
Detailed Description
Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the products and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations and modifications, or appropriate alterations and combinations, of the products and methods described herein may be made and utilized without departing from the spirit, scope, and spirit of the invention. For a further understanding of the present invention, reference will now be made in detail to the following examples.
Example 1
The method for preparing the grease by using the chlorella pyrenoidosa cells as the main raw material comprises the following steps:
inoculating the trichoderma reesei on a PDA culture medium for culturing by streaking to obtain a single colony; selecting a single colony, inoculating the single colony to a primary seed culture medium for culture, and then performing secondary seed culture medium culture to obtain a trichoderma reesei seed solution; the primary seed culture medium and the secondary seed culture medium are both PDA liquid culture media;
selecting Chlorella pyrenoidosa, inoculating into a container containing growth medium, culturing at 28 deg.C under illumination intensity of 6000lux, shaking the container 2-3 times per day, and growing to logarithmic phase to obtain Chlorella pyrenoidosa solution; the components of the growth medium are as follows: 10g/L of glucose, 2g/L of ammonium chloride, 1g/L of sodium nitrate, 0.5g/L of monopotassium phosphate, 0.1g/L of sodium chloride, 100mg/L of magnesium sulfate heptahydrate, 30mg/L of calcium chloride, 20mg/L of ferric ammonium citrate, 10mg/L of zinc sulfate heptahydrate and 10mg/L of manganese sulfate;
inoculating Aspergillus niger on a slant culture medium for culturing by streaking to obtain a single colony; selecting a single colony, inoculating the single colony to a primary seed culture medium for culture, and then performing secondary seed culture medium culture to obtain an Aspergillus niger seed solution; the slant culture medium comprises the following components: 150g/L of potato, 20g/L of cane sugar and 15g/L of agar; the first-stage seed culture medium and the second-stage seed culture medium comprise the following components: 50g/L of corn flour, 10g/L of cane sugar, 5g/L of ammonium sulfate, 1g/L of monopotassium phosphate and 1g/L of dipotassium phosphate.
Inoculating Chlorella pyrenoidosa solution and Trichoderma reesei seed solution in logarithmic growth phase into a reaction tank containing culture medium, wherein the inoculation density of Chlorella pyrenoidosa and Trichoderma reesei is 1 × 106Each/ml and 1X 107cfu/ml, temperature 28 deg.C, 24 hr continuous illumination, intensity 6000Lux, rotation speed 100rpm, fermentation time 3 days, inoculating Aspergillus niger seed solution with inoculation density of 5 × 107cfu/ml, continuously culturing for 3 days, and centrifuging after the culture is finishedWashing and freeze-drying to obtain powder; the specific composition of the culture medium is as follows: 50g/L of cassava dregs, 1g/L of sodium nitrate, 0.5g/L of monopotassium phosphate, 0.1g/L of sodium chloride, 100mg/L of magnesium sulfate heptahydrate, 50mg/L of calcium chloride, 20mg/L of ferric ammonium citrate and 10mg/L of zinc sulfate heptahydrate.
The powder was treated with a pulsed electric field having an electric field strength of 20kV/cm, a pulse width of 4us, and a treatment time of 200us, and then added to a chloroform-methanol mixed solution (the volume ratio of chloroform to methanol was 2: 1) in an amount of 1g of the powder: 2ml of chloroform-methanol mixed solution is subjected to ultrasonic extraction at the temperature of 60 ℃, the ultrasonic power of 200W and the extraction time of 60min, then the mixture is centrifuged, the chloroform phase is collected and dried in nitrogen, and the mixture is dried in vacuum to obtain the grease.
Example 2
The method for preparing the grease by using the chlorella pyrenoidosa cells as the main raw material comprises the following steps:
inoculating the trichoderma reesei on a PDA culture medium for culturing by streaking to obtain a single colony; selecting a single colony, inoculating the single colony to a primary seed culture medium for culture, and then performing secondary seed culture medium culture to obtain a trichoderma reesei seed solution; the primary seed culture medium and the secondary seed culture medium are both PDA liquid culture media;
selecting Chlorella pyrenoidosa, inoculating into a container containing growth medium, culturing at 28 deg.C under illumination intensity of 6000lux, shaking the container 2-3 times per day, and growing to logarithmic phase to obtain Chlorella pyrenoidosa solution; the components of the growth medium are as follows: 10g/L of glucose, 2g/L of ammonium chloride, 1g/L of sodium nitrate, 0.5g/L of monopotassium phosphate, 0.1g/L of sodium chloride, 100mg/L of magnesium sulfate heptahydrate, 30mg/L of calcium chloride, 20mg/L of ferric ammonium citrate, 10mg/L of zinc sulfate heptahydrate and 10mg/L of manganese sulfate;
inoculating Aspergillus niger on a slant culture medium for culturing by streaking to obtain a single colony; selecting a single colony, inoculating the single colony to a primary seed culture medium for culture, and then performing secondary seed culture medium culture to obtain an Aspergillus niger seed solution; the slant culture medium comprises the following components: 150g/L of potato, 20g/L of cane sugar and 15g/L of agar; the first-stage seed culture medium and the second-stage seed culture medium comprise the following components: 50g/L of corn flour, 10g/L of cane sugar, 5g/L of ammonium sulfate, 1g/L of monopotassium phosphate and 1g/L of dipotassium phosphate.
Inoculating Chlorella pyrenoidosa solution and Trichoderma reesei seed solution in logarithmic growth phase into a reaction tank containing culture medium, wherein the inoculation density of Chlorella pyrenoidosa and Trichoderma reesei is 1 × 106Each/ml and 1X 107cfu/ml, temperature 28 deg.C, 24 hr continuous illumination, intensity 7000Lux, rotation speed 100rpm, fermentation time 3 days, inoculating Aspergillus niger seed solution with inoculation density of 5 × 107cfu/ml, continuously culturing for 4 days, and after the culture is finished, centrifuging, washing and freeze-drying to obtain powder; the specific composition of the culture medium is as follows: 80g/L of cassava residue, 2g/L of sodium nitrate, 1g/L of monopotassium phosphate, 0.2g/L of sodium chloride, 200mg/L of magnesium sulfate heptahydrate, 70mg/L of calcium chloride, 30mg/L of ferric ammonium citrate and 15mg/L of zinc sulfate heptahydrate.
The powder was treated with a pulsed electric field having an electric field strength of 20kV/cm, a pulse width of 4us, and a treatment time of 200us, and then added to a chloroform-methanol mixed solution (the volume ratio of chloroform to methanol was 2: 1) in an amount of 1g of the powder: 2ml of chloroform-methanol mixed solution is subjected to ultrasonic extraction at 65 ℃ for 90min under the ultrasonic power of 100W, and then centrifuged, the chloroform phase is collected, dried in nitrogen, and vacuum-dried to obtain the grease.
Comparative example 1
The procedure of example 1 was repeated except that Trichoderma reesei and Aspergillus niger were not added.
Comparative example 2
The procedure of example 1 was followed without adding Aspergillus niger.
Comparative example 3
Chlorella pyrenoidosa, Trichoderma reesei, and Aspergillus niger were added simultaneously, as in example 1.
Example 3
The dry weight content of biomass, the total fat content (as a percentage of the dry weight of biomass) and the oil yield were measured in the examples and comparative examples of the invention. Specific detection results are shown in table 1:
TABLE 1
| Group of | Incubation time d | Dry weight content g/L of biomass | The total lipid content% | Oil yield g/L |
| Example 1 | 6 | 4.37 | 44.5 | 1.94 |
| Comparative example 1 | 6 | 3.02 | 35.2 | 1.06 |
| Comparative example 2 | 6 | 4.19 | 37.8 | 1.58 |
| Comparative example 3 | 6 | 3.74 | 41.6 | 1.56 |
And (4) conclusion: the influence of the strain type and the addition timing on the dry matter content, the total fat content and the oil and fat yield was verified by table 1, and it was found that each index of the dry matter content, the total fat content and the oil and fat yield of example 1 was higher than those of comparative examples 1 to 3; the comparative example 1 has the advantages that no strain is added, only algae culture is adopted, indexes in all aspects are lowest, and the oil yield is reduced by about 40% compared with that of the example 1; comparative example 2 only using trichoderma reesei, which produced reducing sugars that could promote the growth rate of algae and increase biomass by fermenting cassava residue, but reduced the oil content compared to example 1, while maintaining a higher microbial quality with continued adequate nutrition; comparative example 3 aspergillus niger was inoculated at the same time, resulting in the aspergillus niger competing for the carbon source transitionally, causing insufficient nutrients in the algae, resulting in slow growth of the algae; in example 1, after the algae substantially reaches a high growth amount, aspergillus niger is inoculated, wherein the aspergillus niger can rapidly utilize a nitrogen source and part of reducing sugar, so as to compete with the algae, so that the algae can obtain a higher oil content through nutrient limitation under the stress conditions of nitrogen limitation and nutrient deprivation, and the aspergillus niger can also generate a large amount of carbon dioxide inorganic carbon source, so as to improve the oil yield of the algae.
Example 4
Taking example 1 as an example, the influences of different pulse electric field times on the grease yield are respectively set to 0, 100, 200, 400 and 600us, as shown in fig. 1, the grease yield is obviously increased along with the increase of the processing time, and after 200us, the increase of the processing time does not obviously influence the grease yield, so that 200us is most suitable for selection. The strong electric field with proper treatment time can affect polar molecules in cells, generate oscillation on cell membranes, generate unrecoverable destructive action, enhance the permeability of the cell membranes and finally accelerate the exchange efficiency of intracellular and extracellular grease.
Example 5
Influence of ultrasonic power and ultrasonic time on grease yield:
setting the ultrasonic power to be 50w, 100w, 200w, 400w and 800 w; the ultrasonic extraction time is 15, 30, 60, 90 and 120min, as shown in fig. 2, the oil yield is gradually increased along with the increase of the ultrasonic power and the ultrasonic time, and finally, when the ultrasonic power is selected to be 100 and 200W and the treatment time is 60-90min, the oil extraction effect is optimal. When ultrasonic wave acts on a liquid reaction system, a large number of tiny bubbles are formed in the liquid due to the cavitation action of the ultrasonic wave, the bubbles are generated and destroyed very quickly, the reaction system can generate local high temperature and high pressure, the effect of destroying plant cell walls can be achieved, and the contact opportunity of a solvent and intracellular substances can be increased. The method for extracting the grease by using the pulsed electric field and the ultrasonic wave is an efficient method, combines the advantages of the pulsed electric field and the ultrasonic wave, shortens the reaction time, and can reduce the reaction energy consumption.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (6)
1. The method for preparing the grease by using the chlorella pyrenoidosa cells with the cassava dregs as the main raw material is characterized by comprising the following steps:
step 1) preparation of a trichoderma reesei seed solution: inoculating the trichoderma reesei on a PDA culture medium for culturing by streaking to obtain a single colony; selecting a single colony, inoculating the single colony to a primary seed culture medium for culture, and then inoculating to a secondary seed culture medium for culture to obtain a trichoderma reesei seed solution;
step 2) preparing a protein core chlorella solution: selecting Chlorella pyrenoidosa, inoculating into a container containing growth medium, culturing at 28 deg.C under illumination intensity of 6000lux, shaking the container 2-3 times per day, and growing to logarithmic phase to obtain Chlorella pyrenoidosa solution;
step 3) preparation of an Aspergillus niger seed solution: inoculating Aspergillus niger on a slant culture medium for culturing by streaking to obtain a single colony; selecting a single colony, inoculating the single colony to a primary seed culture medium for culture, and then inoculating the single colony to a secondary seed culture medium for culture to obtain an Aspergillus niger seed solution;
step 4), mixed culture: inoculating the protein core chlorella solution in the logarithmic phase obtained in the step 2) and the trichoderma reesei seed solution obtained in the step 1) into a culture medium containing cassava residues, wherein the temperature is 28 ℃, the illumination is continuously carried out for 24 hours, the intensity is 6000-plus 8000Lux, the rotating speed is 100rpm, the fermentation time is 2-3 days, then inoculating the aspergillus niger seed solution obtained in the step 3), continuously culturing for 3-4 days, and after the culture is finished, obtaining powder after centrifugation, washing and freeze drying;
step 5), extracting grease: treating the powder by adopting a pulsed electric field, and then adding the powder into a chloroform-methanol mixed solution, wherein the addition amount is 1g of powder: 2ml of chloroform-methanol mixed solution is subjected to ultrasonic extraction and then centrifugation, a chloroform phase is collected, the chloroform phase is dried in nitrogen, and vacuum drying is carried out to obtain grease;
the pulsed electric field treatment comprises the following steps: the electric field intensity is 20kV/cm, the pulse width is 4us, and the processing time is 200 us;
the ultrasonic extraction comprises the following steps: the extraction temperature is 60-65 ℃, the ultrasonic power is 100-.
2. The method as claimed in claim 1, wherein in step 1), the primary seed culture medium and the secondary seed culture medium are both PDA liquid culture medium.
3. The method according to claim 1, wherein in step 2), the growth medium has a composition of: 10g/L glucose, 2g/L ammonium chloride, 1g/L sodium nitrate, 0.5g/L potassium dihydrogen phosphate, 0.1g/L sodium chloride, 0.1g/L magnesium sulfate heptahydrate, 30mg/L calcium chloride, 20mg/L ferric ammonium citrate, 10mg/L zinc sulfate heptahydrate and 10mg/L manganese sulfate.
4. The method according to claim 1, wherein in the step 3), the slant culture medium comprises the following components: 150g/L of potato, 20g/L of cane sugar and 15g/L of agar; the first-stage seed culture medium and the second-stage seed culture medium comprise the following components: 50g/L of corn flour, 10g/L of cane sugar, 5g/L of ammonium sulfate, 1g/L of monopotassium phosphate and 1g/L of dipotassium phosphate.
5. The method according to claim 1, wherein in step 4), the composition of the culture medium is as follows: 50-80g/L of cassava dregs, 1-2g/L of sodium nitrate, 0.5-1g/L of monopotassium phosphate, 0.1-0.2g/L of sodium chloride, 0.1-0.2g/L of magnesium sulfate heptahydrate, 50-70mg/L of calcium chloride, 20-30mg/L of ferric ammonium citrate and 10-15mg/L of zinc sulfate heptahydrate.
6. The method as claimed in claim 1, wherein the chloroform-methanol mixed solution is prepared from chloroform and methanol at a volume ratio of 2: 1.
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