CN108315381A - Chlorella pyrenoidosa cell utilizes the method that manioc waste is that primary raw material prepares grease - Google Patents
Chlorella pyrenoidosa cell utilizes the method that manioc waste is that primary raw material prepares grease Download PDFInfo
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- CN108315381A CN108315381A CN201810423474.5A CN201810423474A CN108315381A CN 108315381 A CN108315381 A CN 108315381A CN 201810423474 A CN201810423474 A CN 201810423474A CN 108315381 A CN108315381 A CN 108315381A
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- medium
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- grease
- seed
- chlorella pyrenoidosa
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- 239000004519 grease Substances 0.000 title claims abstract description 51
- 244000249214 Chlorella pyrenoidosa Species 0.000 title claims abstract description 33
- 235000007091 Chlorella pyrenoidosa Nutrition 0.000 title claims abstract description 33
- 238000000034 method Methods 0.000 title claims abstract description 26
- 240000003183 Manihot esculenta Species 0.000 title claims abstract description 22
- 235000016735 Manihot esculenta subsp esculenta Nutrition 0.000 title claims abstract description 22
- 239000002699 waste material Substances 0.000 title claims abstract description 18
- 239000002994 raw material Substances 0.000 title claims abstract description 15
- 241000228245 Aspergillus niger Species 0.000 claims abstract description 29
- 239000007788 liquid Substances 0.000 claims abstract description 25
- 241000499912 Trichoderma reesei Species 0.000 claims abstract description 24
- 235000003239 Guizotia abyssinica Nutrition 0.000 claims abstract description 11
- 239000002609 medium Substances 0.000 claims description 36
- 239000001963 growth medium Substances 0.000 claims description 22
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 21
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 20
- 241000894006 Bacteria Species 0.000 claims description 17
- 239000000843 powder Substances 0.000 claims description 17
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 16
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 16
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 16
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 claims description 16
- 230000005684 electric field Effects 0.000 claims description 14
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 12
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 12
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 12
- 239000011259 mixed solution Substances 0.000 claims description 10
- 229910052757 nitrogen Inorganic materials 0.000 claims description 10
- 238000012545 processing Methods 0.000 claims description 10
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 8
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 8
- 229930006000 Sucrose Natural products 0.000 claims description 8
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 8
- 239000001110 calcium chloride Substances 0.000 claims description 8
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 8
- WORJEOGGNQDSOE-UHFFFAOYSA-N chloroform;methanol Chemical compound OC.ClC(Cl)Cl WORJEOGGNQDSOE-UHFFFAOYSA-N 0.000 claims description 8
- 238000000605 extraction Methods 0.000 claims description 8
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 8
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 8
- 239000011780 sodium chloride Substances 0.000 claims description 8
- 239000004317 sodium nitrate Substances 0.000 claims description 8
- 235000010344 sodium nitrate Nutrition 0.000 claims description 8
- 239000005720 sucrose Substances 0.000 claims description 8
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims description 8
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 8
- 239000011686 zinc sulphate Substances 0.000 claims description 8
- 235000009529 zinc sulphate Nutrition 0.000 claims description 8
- 238000000855 fermentation Methods 0.000 claims description 7
- 230000004151 fermentation Effects 0.000 claims description 7
- 229960002413 ferric citrate Drugs 0.000 claims description 7
- 238000011081 inoculation Methods 0.000 claims description 7
- NPFOYSMITVOQOS-UHFFFAOYSA-K iron(III) citrate Chemical compound [Fe+3].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NPFOYSMITVOQOS-UHFFFAOYSA-K 0.000 claims description 7
- 238000002137 ultrasound extraction Methods 0.000 claims description 7
- 244000061456 Solanum tuberosum Species 0.000 claims description 5
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- 229920001817 Agar Polymers 0.000 claims description 4
- 240000008042 Zea mays Species 0.000 claims description 4
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 4
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 4
- 239000000654 additive Substances 0.000 claims description 4
- 230000000996 additive effect Effects 0.000 claims description 4
- 239000008272 agar Substances 0.000 claims description 4
- 235000019270 ammonium chloride Nutrition 0.000 claims description 4
- 238000005119 centrifugation Methods 0.000 claims description 4
- 235000005822 corn Nutrition 0.000 claims description 4
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 4
- 235000013312 flour Nutrition 0.000 claims description 4
- 238000004108 freeze drying Methods 0.000 claims description 4
- 229940099596 manganese sulfate Drugs 0.000 claims description 4
- 239000011702 manganese sulphate Substances 0.000 claims description 4
- 235000007079 manganese sulphate Nutrition 0.000 claims description 4
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 4
- 238000005406 washing Methods 0.000 claims description 4
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 3
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 3
- 239000002893 slag Substances 0.000 claims description 3
- 230000003203 everyday effect Effects 0.000 claims description 2
- 241000223259 Trichoderma Species 0.000 claims 2
- 235000009754 Vitis X bourquina Nutrition 0.000 claims 1
- 235000012333 Vitis X labruscana Nutrition 0.000 claims 1
- 240000006365 Vitis vinifera Species 0.000 claims 1
- 235000014787 Vitis vinifera Nutrition 0.000 claims 1
- 238000011218 seed culture Methods 0.000 claims 1
- 239000012879 subculture medium Substances 0.000 claims 1
- 239000002023 wood Substances 0.000 claims 1
- 238000005516 engineering process Methods 0.000 abstract description 4
- 241000195493 Cryptophyta Species 0.000 description 21
- 239000002028 Biomass Substances 0.000 description 12
- 230000000052 comparative effect Effects 0.000 description 11
- 238000006243 chemical reaction Methods 0.000 description 10
- 239000003225 biodiesel Substances 0.000 description 9
- 239000003921 oil Substances 0.000 description 9
- 235000019198 oils Nutrition 0.000 description 9
- 210000004027 cell Anatomy 0.000 description 8
- 230000008901 benefit Effects 0.000 description 6
- 239000002054 inoculum Substances 0.000 description 6
- 230000003834 intracellular effect Effects 0.000 description 6
- 235000000346 sugar Nutrition 0.000 description 6
- 241000195649 Chlorella <Chlorellales> Species 0.000 description 5
- 241000196324 Embryophyta Species 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 5
- 239000000306 component Substances 0.000 description 5
- 150000002632 lipids Chemical class 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 4
- 229910052799 carbon Inorganic materials 0.000 description 4
- 210000000170 cell membrane Anatomy 0.000 description 4
- 235000013339 cereals Nutrition 0.000 description 4
- 235000015097 nutrients Nutrition 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 238000012549 training Methods 0.000 description 4
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- 229920002472 Starch Polymers 0.000 description 3
- 239000002283 diesel fuel Substances 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 235000016709 nutrition Nutrition 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 235000019698 starch Nutrition 0.000 description 3
- 239000008107 starch Substances 0.000 description 3
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical group OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 229910002092 carbon dioxide Inorganic materials 0.000 description 2
- 229960004424 carbon dioxide Drugs 0.000 description 2
- 210000002421 cell wall Anatomy 0.000 description 2
- 239000000460 chlorine Substances 0.000 description 2
- 229910052801 chlorine Inorganic materials 0.000 description 2
- 230000009514 concussion Effects 0.000 description 2
- 230000002354 daily effect Effects 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005265 energy consumption Methods 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 235000019441 ethanol Nutrition 0.000 description 2
- 239000003925 fat Substances 0.000 description 2
- 235000019197 fats Nutrition 0.000 description 2
- 238000005755 formation reaction Methods 0.000 description 2
- 238000005286 illumination Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 239000012533 medium component Substances 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000035764 nutrition Effects 0.000 description 2
- 239000005416 organic matter Substances 0.000 description 2
- 230000035699 permeability Effects 0.000 description 2
- 238000011020 pilot scale process Methods 0.000 description 2
- 239000013535 sea water Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 230000002195 synergetic effect Effects 0.000 description 2
- 231100000033 toxigenic Toxicity 0.000 description 2
- 230000001551 toxigenic effect Effects 0.000 description 2
- 235000015112 vegetable and seed oil Nutrition 0.000 description 2
- 239000008158 vegetable oil Substances 0.000 description 2
- 235000017060 Arachis glabrata Nutrition 0.000 description 1
- 244000105624 Arachis hypogaea Species 0.000 description 1
- 235000010777 Arachis hypogaea Nutrition 0.000 description 1
- 235000018262 Arachis monticola Nutrition 0.000 description 1
- 240000002791 Brassica napus Species 0.000 description 1
- 235000004977 Brassica sinapistrum Nutrition 0.000 description 1
- 239000004215 Carbon black (E152) Substances 0.000 description 1
- 241000195628 Chlorophyta Species 0.000 description 1
- 241000192699 Chroococcales Species 0.000 description 1
- 241000206751 Chrysophyceae Species 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- NIPNSKYNPDTRPC-UHFFFAOYSA-N N-[2-oxo-2-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 NIPNSKYNPDTRPC-UHFFFAOYSA-N 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 241001529246 Platymiscium Species 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000012271 agricultural production Methods 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
- BIGPRXCJEDHCLP-UHFFFAOYSA-N ammonium bisulfate Chemical compound [NH4+].OS([O-])(=O)=O BIGPRXCJEDHCLP-UHFFFAOYSA-N 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 235000014121 butter Nutrition 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002090 carbon oxide Inorganic materials 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000000875 corresponding effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 235000013325 dietary fiber Nutrition 0.000 description 1
- 239000008157 edible vegetable oil Substances 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 239000010685 fatty oil Substances 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000002803 fossil fuel Substances 0.000 description 1
- 239000013505 freshwater Substances 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 235000013402 health food Nutrition 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
- 150000002430 hydrocarbons Chemical class 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- -1 long chain fatty acids Ester Chemical class 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 150000004767 nitrides Chemical class 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 235000014593 oils and fats Nutrition 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 235000020232 peanut Nutrition 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 230000029553 photosynthesis Effects 0.000 description 1
- 238000010672 photosynthesis Methods 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 210000001082 somatic cell Anatomy 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 238000011282 treatment Methods 0.000 description 1
- YWYZEGXAUVWDED-UHFFFAOYSA-N triammonium citrate Chemical compound [NH4+].[NH4+].[NH4+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O YWYZEGXAUVWDED-UHFFFAOYSA-N 0.000 description 1
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 1
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- 239000002351 wastewater Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6436—Fatty acid esters
- C12P7/6445—Glycerides
- C12P7/6463—Glycerides obtained from glyceride producing microorganisms, e.g. single cell oil
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biotechnology (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Cell Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to Alga technology fields, disclose the method that chlorella pyrenoidosa cell prepares grease using manioc waste for primary raw material comprising following steps：Step 1）Prepare trichoderma reesei seed liquor, step 2）Prepare chlorella pyrenoidosa liquid, step 3）Prepare aspergillus niger seed liquor, step 4）Mixed culture, step 5）Extract grease.The method of the present invention reduces cost, and grease yield improves.
Description
Technical field
The invention belongs to Alga technology fields, and in particular to chlorella pyrenoidosa cell is primary raw material system using manioc waste
The method of standby grease.
Background technology
Biodiesel is a kind of long chain fatty acids Ester, is by short-chain alcohols substance (methanol or ethyl alcohol) and certain
A little fatty oil substances product obtained by the reaction.The source of grease is the emphasis studied at present, and main source includes：Plant origin,
Animal origin and alga-derived.Plant origin is the oil extracted using rapeseed, soybean, peanut and various oil crops
Fat is raw material；Animal origin is to utilize animal tallows or the used edible oils such as lard, butter and sheep oil；It is alga-derived, it is micro-
Algae can synthesize a large amount of greases in growth course, and microalgae grease belongs to Unicell Oils and Fats, and key component is glycerine and aliphatic acid, is
Under certain conditions by microalgae, it using carbohydrate, hydrocarbon and common grease as carbon source, is synthesized in frond
, mainly as biofilm components, metabolin and energy source.Due to plant origin, animal origin, raw material life cycle is long,
Total resources is insufficient, and economic benefit is low and its influence to agricultural product price, cultivated land resource, grain security, constrains biology
The development of diesel oil.Relative to traditional plant origin and animal origin biodiesel raw material, microalgae has widely distributed, growth
Period is short, biomass is big, strong environmental adaptability, fat content are high, with grain does not strive ground, does not strive the huge advantages such as grain with people, by
To pursuing energetically for scientific research personnel, it is considered to be solve one of insufficient important channel of current biodiesel raw material.Currently, algae
The grease in source is the hot spot of research.As biodiesel raw material of new generation, microalgae possesses many advantages.Algal kind is various,
It is distributed widely in fresh water and seawater.The identified microalgae in the whole world has tens of thousands of kinds, and its quantity is also being continuously increased.Relatively
In traditional oil crops, microalgae is big with biomass, growth cycle is short.The growth rate of microalgae is significantly larger than terrestrial crop,
General microalgae can be doubled in its interior biomass for 24 hours, and 3-5h is generally in the biomass doubling time of exponential phase of growth.Microalgae
The ingredient of oil is similar to vegetable oil, is the substitute of vegetable oil, directly the prior art can be used to produce biodiesel.Normal culture
Under the conditions of, the oil content of general microalgae is all up 20-50%, and microalgae can use cultivation in sea water, is resistant to desert, punja, half-dried
The extreme environments such as nonirrigated farmland are not take up arable land, therefore will not constitute a threat to the production of cereal crops.Microalgae can absorb and utilize work
The a large amount of C02 and nitride or acquirement nitrogen, phosphorus etc. from waste water given off in agricultural production, are conducive to improve environment.
The biochemical composition of microalgae can be adjusted by the change of environmental condition, to improve oil content.Zhejiang
Regional algae species are abundant, have the natural advantage for carrying out algae production biodiesel research.It is produced currently with algae
The problem of diesel oil industrialization maximum is how to reduce toxigenic capacity and raising oil productivity.Due to the grease of variety classes microalgae
Content and growth ability have differences, and need to screen microalgae type before carrying out microdisk electrode.The microalgae bacterium of selection
Strain must have high productivity and high fat content, have stronger stain resistance, and can adapt to the variation of environment, can be with
Large-scale culture.Currently, most common can have chlorella, Du Shi algaes, chrysophyceae etc. with the algae of pilot scale culture, because itself compared with
High fat content and growth rate is commonly used for the production of microalgae biodiesel.But also there are many researchers by changing microalgae
Growing environment or the fat content and growth rate of microalgae are improved using genetic engineering.
Chlorella pyrenoidosa belongs to Chlorophyta, Chroococcales, Chlorella, is uniquely to have pyrenoids in the platymiscium
Type.Contain abundant protein, polysaccharide, unsaturated fatty acid, dietary fiber, vitamin and micro- in chlorella pyrenoidosa cell
Secondary element etc. has very high nutritive value, is the excellent health food of the mankind and aquaculture bait.It is total to by the Chinese people
It is approved as new resource food with the Ministry of Public Health of state.Chlorella pyrenoidosa can be in light autotrophy as a kind of renewable energy source biomass
Grease is accumulated under the condition of culture of heterotrophism, solar energy can be efficiently used and has function to carry out fast-growth and accumulation grease, egg
The grease of white nucleus chlorella can be used as the renewable sources of energy, and for making biodiesel, biodiesel is in substitute fossil fuels diesel oil
It is upper that there are very big potentiality.But current chlorella pyrenoidosa pilot scale culture, there is slow-growing, biomass is low, grease
The low problem of content, is unfavorable for large-scale culture.CN106754383A discloses chlorella and oleaginous yeast co-incubation to carry
The method of high grease yield, this method are by the two while to be inoculated into culture medium, and the mixed culture total fat of microbial dry powder contains
Amount can reach 40.55%, and total fatty acids yield reaches 175.64mg/l/d, the microalgae cell noticeably greater than individually cultivated.But
There are still fat content is to be improved and the higher defect of fermentation costs.Manioc waste is the by-product after cassava extraction starch
Object, leading indicator include crude fibre, coarse ash, moisture, and nutritional cost is relatively low, are typically used as feed or discarded.In In Hangzhou Region of Zhe Jiang Province
There is a large amount of starch factory, will produce many manioc wastes when processing starch with cassava, how manioc waste is carried out effective
Using being also the technical issues that need to address.
Invention content
It is low and the defects of toxigenic capacity is high present invention aim to address prior art algae Lipid-producing efficiency, provide albumen
Core chlorella cells utilize the method that manioc waste is that primary raw material prepares grease.
The present invention is achieved by the following technical solution：
Chlorella pyrenoidosa cell utilizes the method that manioc waste is that primary raw material prepares grease comprising following steps：Step 1）
Prepare trichoderma reesei seed liquor, step 2）Prepare chlorella pyrenoidosa liquid, step 3）Prepare aspergillus niger seed liquor, step 4）Mixing
Culture, step 5）Extract grease.
Specifically, described method includes following steps：Step 1）Prepare trichoderma reesei seed liquor：Trichoderma reesei scribing line is connect
Kind is cultivated in PDA culture medium, obtains single bacterium colony；Picking single bacterium colony is inoculated into primary-seed medium and is cultivated, then into
Row secondary seed medium culture obtains trichoderma reesei seed liquor；
Step 2）Prepare chlorella pyrenoidosa liquid：Picking chlorella pyrenoidosa is inoculated into the container containing growth medium, light
According to intensity 6000lux, 28 DEG C of cultures are shaken every day container 2-3 times, to be grown to exponential phase, obtain chlorella pyrenoidosa
Liquid；
Step 3）Prepare aspergillus niger seed liquor：Aspergillus niger streak inoculation is cultivated on slant medium, obtains single bacterium colony；Picking
Single bacterium colony is inoculated into primary-seed medium and is cultivated, and then carries out secondary seed medium culture, obtains aspergillus niger seed
Liquid；
Step 4）Mixed culture：By in exponential phase chlorella pyrenoidosa liquid and trichoderma reesei seed liquor be inoculated into containing
In the reaction tank of the culture medium of manioc waste, the inoculum density of chlorella pyrenoidosa and trichoderma reesei is respectively 1 × 106A/ml and 1
×107Cfu/ml, temperature are 28 DEG C, 24 hours continuous lights, intensity 6000-8000Lux, rotating speed 100rpm, when fermentation
Between be 2-3 days, then access aspergillus niger seed liquor, inoculum density be 5 × 107Cfu/ml continues culture 3-4 days, and culture terminates
Afterwards, to get to powder after centrifugation, washing and freeze-drying；
Step 5）Extract grease：Powder is handled using impulse electric field, then powder is added in chloroform methanol mixed solution,
Additive amount is 1g powder：2ml chloroform methanol mixed solutions, ultrasonic extraction are then centrifuged for, and are collected chloroform phase, are placed in nitrogen and blow
It is dry, and be dried in vacuo, obtain grease.
Preferably, the step 1）In, primary-seed medium and secondary seed medium are PDA liquid medium.
Preferably, the step 2）In, the group of growth medium is divided into：Glucose 10g/L, ammonium chloride 2g/L, sodium nitrate
1g/L, potassium dihydrogen phosphate 0.5g/L, sodium chloride 0.1g/L, epsom salt 100mg/L, calcium chloride 30mg/L, ironic citrate
Ammonium 20mg/L, white vitriol 10mg/L, manganese sulfate 10mg/L.
Preferably, the step 3）In, the slant medium group is divided into：Potato 150g/L, sucrose 20g/L, agar
15g/L；The component of the primary-seed medium and secondary seed medium is：Corn flour 50g/L, sucrose 10g/L, sulfuric acid
Ammonium 5g/L, potassium dihydrogen phosphate 1g/L, dipotassium hydrogen phosphate 1g/L.
Preferably, the step 4）In, the concrete composition of the culture medium is as follows：Manioc waste 50-80g/L, sodium nitrate 1-
2g/L, potassium dihydrogen phosphate 0.5-1g/L, sodium chloride 0.1-0.2g/L, epsom salt 100-200mg/L, calcium chloride 50-
70mg/L, ferric citrate 20-30mg/L, white vitriol 10-15mg/L.
Preferably, the impulse electric field, which is handled, is：Electric field strength 20kV/cm, pulse width 4us, processing time 200us.
Preferably, the ultrasonic extraction is：Extracting temperature is 60-65 DEG C, ultrasonic power 100-200W, and extraction time is
60-90min。
Preferably, the chloroform methanol mixed solution is 2 according to volume ratio by chloroform and methanol:1 is made.
Compared with prior art, the advantageous effect that the present invention obtains includes but is not limited to mainly several aspects：
The increment and grease yield of algae are not fully positively correlated ratio, by adjusting extraneous factor so that increment is in
In reasonable range and grease yield maximizes.Trichoderma reesei generates reduced sugar using fermentation cassava slag, and reduced sugar can promote
Into the growth rate of algae, increase biomass, after algae reaches certain growth amount, inoculated aspergillus niger, aspergillus niger can be very fast
Utilization nitrogen source and partial reduction sugar, to generate competition with algae so that algae is in nitrogen limitation and the stress deprived of nutrient
Under the conditions of, higher fat content is obtained by nitrogen limitation or nutrition limitation.And aspergillus niger can also generate a large amount of two
The inorganic carbon source of carbonoxide, to promote the grease yield of algae.Trichoderma reesei and aspergillus niger can also generate oil substances, and
And used manioc waste as primary raw material in culture of the present invention, and it is cheap, reduce entreprise cost.Microalgae in mixed culture
The oxygen of photosynthesis release can be utilized by somatic cells, so that co-culture system is in an equilibrium state.Properly
The highfield of processing time can have an impact intracellular polar molecule, and concussion is generated on cell membrane, occur irrecoverable
Destruction so that permeability of cell membrane enhances, and may finally accelerate the exchange efficiency of the outer grease of intracellular.Work as ul-trasonic irradiation
When liquid reaction system, due to the cavitation of ultrasonic wave, a large amount of small bubble formations, and these gas are had in liquid
The generation of bubble and vanish very rapid, reaction system will produce local high temperature and high pressure, can reach the work for destroying cell wall
With the touch opportunity of solvent and intracellular organic matter can be increased.The present invention is one using impulse electric field synergistic supersonic wave extraction grease
A efficient method combines the advantage of the two, not only shortens the time of reaction, can also reduce energy consumption of reaction.
Description of the drawings
Fig. 1：Influence of the different impulse electric field times to grease yield；
Fig. 2：The influence of ultrasonic power and ultrasonic time to grease yield.
Specific implementation mode
Those skilled in the art can use for reference present disclosure, be suitably modified technological parameter realization.In particular, it should be pointed out that
All similar substitutions and modifications are apparent to those skilled in the art, they are considered as being included in this hair
It is bright.The product and method of the present invention is described by preferred embodiment, and related personnel can obviously not depart from this hair
Product as described herein and method are modified or are suitably changed and combined in bright content, spirit and scope, to realize and answer
Use the technology of the present invention.For a further understanding of the present invention, the following describes the present invention in detail with reference to examples.
Embodiment 1
Chlorella pyrenoidosa cell utilizes the method that manioc waste is that primary raw material prepares grease comprising following steps：
Trichoderma reesei streak inoculation is cultivated in PDA culture medium, obtains single bacterium colony；Picking single bacterium colony is inoculated into first order seed training
Foster base is cultivated, and secondary seed medium culture is then carried out, and obtains trichoderma reesei seed liquor；The primary-seed medium
It is PDA liquid medium with secondary seed medium；
Picking chlorella pyrenoidosa is planted into the container containing growth medium, intensity of illumination 6000lux, 28 DEG C of cultures, daily
Shake container 2-3 times, it is to be grown to exponential phase, obtain chlorella pyrenoidosa liquid；The group of the growth medium is divided into：Portugal
Grape sugar 10g/L, ammonium chloride 2g/L, sodium nitrate 1g/L, potassium dihydrogen phosphate 0.5g/L, sodium chloride 0.1g/L, epsom salt
100mg/L, calcium chloride 30mg/L, ferric citrate 20mg/L, white vitriol 10mg/L, manganese sulfate 10mg/L；
Aspergillus niger streak inoculation is cultivated on slant medium, obtains single bacterium colony；Picking single bacterium colony is inoculated into first order seed training
Foster base is cultivated, and secondary seed medium culture is then carried out, and obtains aspergillus niger seed liquor；The slant medium component
For：Potato 150g/L, sucrose 20g/L, agar 15g/L；The component of the primary-seed medium and secondary seed medium
It is：Corn flour 50g/L, sucrose 10g/L, ammonium sulfate 5g/L, potassium dihydrogen phosphate 1g/L, dipotassium hydrogen phosphate 1g/L.
By in exponential phase chlorella pyrenoidosa liquid and trichoderma reesei seed liquor be inoculated into containing the anti-of culture medium
The inoculum density of Ying Chizhong, chlorella pyrenoidosa and trichoderma reesei is respectively 1 × 106A/ml and 1 × 107Cfu/ml, temperature are
28 DEG C, 24 hours continuous lights, intensity 6000Lux, rotating speed 100rpm, fermentation time is 3 days, then accesses aspergillus niger kind
Sub- liquid, inoculum density are 5 × 107Cfu/ml continues culture 3 days, after culture, after centrifugation, washing and freeze-drying,
Obtain powder；The concrete composition of the culture medium is as follows：Manioc waste 50g/L, sodium nitrate 1g/L, potassium dihydrogen phosphate 0.5g/
L, sodium chloride 0.1g/L, epsom salt 100mg/L, calcium chloride 50mg/L, ferric citrate 20mg/L, white vitriol
10mg/L。
Powder is handled using impulse electric field, electric field strength 20kV/cm, pulse width 4us, processing time 200us, then
Powder is added to chloroform methanol mixed solution（The volume ratio of chloroform and methanol is 2:1）In, additive amount is 1g powder：2ml chlorine
Imitative methanol mixed solution, ultrasonic extraction, Extracting temperature are 60 DEG C, ultrasonic power 200W, extraction time 60min, then from
The heart collects chloroform phase, is placed in nitrogen and dries up, and be dried in vacuo, obtain grease.
Embodiment 2
Chlorella pyrenoidosa cell utilizes the method that manioc waste is that primary raw material prepares grease comprising following steps：
Trichoderma reesei streak inoculation is cultivated in PDA culture medium, obtains single bacterium colony；Picking single bacterium colony is inoculated into first order seed training
Foster base is cultivated, and secondary seed medium culture is then carried out, and obtains trichoderma reesei seed liquor；The primary-seed medium
It is PDA liquid medium with secondary seed medium；
Picking chlorella pyrenoidosa is planted into the container containing growth medium, intensity of illumination 6000lux, 28 DEG C of cultures, daily
Shake container 2-3 times, it is to be grown to exponential phase, obtain chlorella pyrenoidosa liquid；The group of the growth medium is divided into：Portugal
Grape sugar 10g/L, ammonium chloride 2g/L, sodium nitrate 1g/L, potassium dihydrogen phosphate 0.5g/L, sodium chloride 0.1g/L, epsom salt
100mg/L, calcium chloride 30mg/L, ferric citrate 20mg/L, white vitriol 10mg/L, manganese sulfate 10mg/L；
Aspergillus niger streak inoculation is cultivated on slant medium, obtains single bacterium colony；Picking single bacterium colony is inoculated into first order seed training
Foster base is cultivated, and secondary seed medium culture is then carried out, and obtains aspergillus niger seed liquor；The slant medium component
For：Potato 150g/L, sucrose 20g/L, agar 15g/L；The component of the primary-seed medium and secondary seed medium
It is：Corn flour 50g/L, sucrose 10g/L, ammonium sulfate 5g/L, potassium dihydrogen phosphate 1g/L, dipotassium hydrogen phosphate 1g/L.
By in exponential phase chlorella pyrenoidosa liquid and trichoderma reesei seed liquor be inoculated into containing the anti-of culture medium
The inoculum density of Ying Chizhong, chlorella pyrenoidosa and trichoderma reesei is respectively 1 × 106A/ml and 1 × 107Cfu/ml, temperature are
28 DEG C, 24 hours continuous lights, intensity 7000Lux, rotating speed 100rpm, fermentation time is 3 days, then accesses aspergillus niger kind
Sub- liquid, inoculum density are 5 × 107Cfu/ml continues culture 4 days, after culture, after centrifugation, washing and freeze-drying,
Obtain powder；The concrete composition of the culture medium is as follows：Manioc waste 80g/L, sodium nitrate 2g/L, potassium dihydrogen phosphate 1g/L,
Sodium chloride 0.2g/L, epsom salt 200mg/L, calcium chloride 70mg/L, ferric citrate 30mg/L, white vitriol
15mg/L。
Powder is handled using impulse electric field, electric field strength 20kV/cm, pulse width 4us, processing time 200us, then
Powder is added to chloroform methanol mixed solution（The volume ratio of chloroform and methanol is 2:1）In, additive amount is 1g powder：2ml chlorine
Imitative methanol mixed solution, ultrasonic extraction, Extracting temperature are 65 DEG C, ultrasonic power 100W, extraction time 90min, then from
The heart collects chloroform phase, is placed in nitrogen and dries up, and be dried in vacuo, obtain grease.
Comparative example 1
Trichoderma reesei and aspergillus niger are not added, remaining is the same as embodiment 1.
Comparative example 2
Aspergillus niger is not added, remaining is the same as embodiment 1.
Comparative example 3
Chlorella pyrenoidosa, trichoderma reesei and aspergillus niger add simultaneously, remaining is the same as embodiment 1.
Embodiment 3
Biomass dry weight content, total lipid content in the embodiment of the present invention and comparative example（Account for the percentage of biomass dry weight）And
Grease yield detects.Specific testing result is shown in Table 1：
Table 1
| Group | Incubation time d | Biomass dry weight content g/L | Total lipid content % | Grease yield g/L |
| Embodiment 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 |
Conclusion：Strain type is demonstrated by table 1, addition opportunity produces biomass dry weight content, total lipid content and grease
The influence of amount, it is found that 1 substance dry weight content of embodiment, total lipid content and each index of grease yield are above comparative example 1-
3；Comparative example 1 does not add bacterial strain, and only with algae culture, various aspects index is minimum, and grease yield reduces 40% than embodiment 1
Left and right；Comparative example 2 is only with trichoderma reesei, wherein trichoderma reesei generates reduced sugar using fermentation cassava slag, and reduced sugar can promote
Into the growth rate of algae, increase biomass, but compared with Example 1, continue sufficient nutrition although can maintain higher
Microbial quality, but be reduction of fat content；Comparative example 3 while inoculated aspergillus niger cause aspergillus niger transition to compete carbon source,
It causes algae nutrient insufficient, causes algal grown slow；Embodiment 1 is inoculated after algae basically reaches compared with height increament
Aspergillus niger, aspergillus niger can utilize nitrogen source and partial reduction sugar faster, to generate competition with algae so that algae is in nitrogen
Under the stress conditions that limitation and nutrient are deprived, higher fat content is obtained by nutrient limitation, and aspergillus niger can also
The inorganic carbon source of a large amount of carbon dioxide is generated, to promote the grease yield of algae.
Embodiment 4
Influence of the different impulse electric field times to grease yield is respectively set 0,100,200,400 by taking embodiment 1 as an example,
Influences of the 600us to grease yield, as shown in Figure 1, with the increase of processing time, grease yield amplification is apparent, after 200us,
Increase processing time can't bring to grease yield to be significantly affected, therefore selects 200us the most suitable.The suitable treatments time
Highfield can have an impact intracellular polar molecule, and concussion is generated on cell membrane, expendable destruction occurs,
So that permeability of cell membrane enhancing, may finally accelerate the exchange efficiency of the outer grease of intracellular.
Embodiment 5
The influence of ultrasonic power and ultrasonic time to grease yield：
Setting ultrasonic power is 50w, 100w, 200w, 400w, 800w；The ultrasonic extraction time is 15,30,60,90,120min, such as
Shown in Fig. 2, with the increase of ultrasonic power and ultrasonic time, grease yield is stepped up, and final ultrasonic power selects 100-
When 200W, processing time are 60-90min, the extraction effect of grease is best.When ul-trasonic irradiation is in liquid reaction system, by
In having a large amount of small bubble formations in the cavitation of ultrasonic wave, liquid, and the generation of these bubbles and vanish very
Rapidly, reaction system will produce local high temperature and high pressure, can have the function that destroy plant cell wall, can increase solvent
With the touch opportunity of intracellular organic matter.The present invention is an efficient method using impulse electric field synergistic supersonic wave extraction grease, knot
The advantage of the two has been closed, the time of reaction is not only shortened, can also reduce energy consumption of reaction.
Finally it should be noted that：The above embodiments are merely illustrative of the technical solutions of the present invention, rather than its limitations；Although reference
Invention is explained in detail for previous embodiment, it will be understood by those of ordinary skill in the art that：It still can be right
Technical solution recorded in previous embodiment is modified or equivalent replacement of some of the technical features；And these
Modification or replacement, the spirit and scope for technical solution of the embodiment of the present invention that it does not separate the essence of the corresponding technical solution.
Claims (9)
1. chlorella pyrenoidosa cell utilizes the method that manioc waste is that primary raw material prepares grease comprising following steps：Step
1）Prepare trichoderma reesei seed liquor, step 2）Prepare chlorella pyrenoidosa liquid, step 3）Prepare aspergillus niger seed liquor, step 4）It is mixed
Close culture, step 5）Extract grease.
2. according to the method described in claim 1, it is characterized in that, described method includes following steps：Step 1）Prepare Richter scale
Trichoderma seed liquor：Trichoderma reesei streak inoculation is cultivated in PDA culture medium, obtains single bacterium colony；Picking single bacterium colony is inoculated into one
Grade seed culture medium is cultivated, and secondary seed medium culture is then accessed, and obtains trichoderma reesei seed liquor；
Step 2）Prepare chlorella pyrenoidosa liquid：Picking chlorella pyrenoidosa is inoculated into the container containing growth medium, light
According to intensity 6000lux, 28 DEG C of cultures are shaken every day container 2-3 times, to be grown to exponential phase, obtain chlorella pyrenoidosa
Liquid；
Step 3）Prepare aspergillus niger seed liquor：Aspergillus niger streak inoculation is cultivated on slant medium, obtains single bacterium colony；Picking
Single bacterium colony is inoculated into primary-seed medium and is cultivated, and then accesses secondary seed medium culture, obtains aspergillus niger seed
Liquid；
Step 4）Mixed culture：By step 2）Gained is in the chlorella pyrenoidosa liquid and step 1 of exponential phase）Gained Richter scale
Trichoderma seed liquor is inoculated into the culture medium containing manioc waste, and temperature is 28 DEG C, 24 hours continuous lights, intensity 6000-
8000Lux, rotating speed 100rpm, fermentation time are 2-3 days, then access step 3）Gained aspergillus niger seed liquor, continues to cultivate
3-4 days, after culture, to get to powder after centrifugation, washing and freeze-drying；
Step 5）Extract grease：Powder is handled using impulse electric field, then powder is added in chloroform methanol mixed solution,
Additive amount is 1g powder：2ml chloroform methanol mixed solutions, ultrasonic extraction are then centrifuged for, and are collected chloroform phase, are placed in nitrogen and blow
It is dry, and be dried in vacuo, obtain grease.
3. according to the method described in claim 2, it is characterized in that, the step 1）In, primary-seed medium and two level kind
Sub- culture medium is PDA liquid medium.
4. according to the method described in claim 2, it is characterized in that, the step 2）In, the group of growth medium is divided into：Grape
Sugared 10g/L, ammonium chloride 2g/L, sodium nitrate 1g/L, potassium dihydrogen phosphate 0.5g/L, sodium chloride 0.1g/L, epsom salt
0.1g/L, calcium chloride 30mg/L, ferric citrate 20mg/L, white vitriol 10mg/L, manganese sulfate 10mg/L.
5. according to the method described in claim 2, it is characterized in that, the step 3）In, the slant medium group is divided into：Horse
Bell potato 150g/L, sucrose 20g/L, agar 15g/L；The component of the primary-seed medium and secondary seed medium is：
Corn flour 50g/L, sucrose 10g/L, ammonium sulfate 5g/L, potassium dihydrogen phosphate 1g/L, dipotassium hydrogen phosphate 1g/L.
6. according to the method described in claim 2, it is characterized in that, the step 4）In, the composition of the culture medium is as follows：Wood
Potato slag 50-80g/L, sodium nitrate 1-2g/L, potassium dihydrogen phosphate 0.5-1g/L, sodium chloride 0.1-0.2g/L, epsom salt
0.1-0.2g/L, calcium chloride 50-70mg/L, ferric citrate 20-30mg/L, white vitriol 10-15mg/L.
7. according to the method described in claim 2, it is characterized in that, impulse electric field processing is：Electric field strength 20kV/cm,
Pulse width 4us, processing time 200us.
8. according to the method described in claim 2, it is characterized in that, the ultrasonic extraction is：Extracting temperature is 60-65 DEG C, is surpassed
Acoustical power is 100-200W, extraction time 60-90min.
9. according to the method described in claim 2, it is characterized in that, the chloroform methanol mixed solution by chloroform and methanol according to
Volume ratio is 2:1 is made.
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