CN106520657A - Method for increasing euglena biomass and fatty acid content - Google Patents
Method for increasing euglena biomass and fatty acid content Download PDFInfo
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- 241000195620 Euglena Species 0.000 title claims abstract description 46
- 239000002028 Biomass Substances 0.000 title claims abstract description 25
- 235000014113 dietary fatty acids Nutrition 0.000 title claims abstract description 25
- 229930195729 fatty acid Natural products 0.000 title claims abstract description 25
- 239000000194 fatty acid Substances 0.000 title claims abstract description 25
- 150000004665 fatty acids Chemical class 0.000 title claims abstract description 25
- 238000000034 method Methods 0.000 title claims abstract description 20
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims abstract description 32
- 229910052742 iron Inorganic materials 0.000 claims abstract description 15
- 239000002245 particle Substances 0.000 claims abstract description 14
- 239000006228 supernatant Substances 0.000 claims abstract description 14
- 238000005119 centrifugation Methods 0.000 claims abstract description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 6
- 238000005286 illumination Methods 0.000 claims abstract description 4
- 239000008367 deionised water Substances 0.000 claims abstract description 3
- 229910021641 deionized water Inorganic materials 0.000 claims abstract description 3
- 241000195493 Cryptophyta Species 0.000 claims description 18
- 239000012531 culture fluid Substances 0.000 claims description 6
- 239000008187 granular material Substances 0.000 claims description 3
- 238000009825 accumulation Methods 0.000 abstract description 12
- 239000001963 growth medium Substances 0.000 abstract description 10
- 230000012010 growth Effects 0.000 abstract description 9
- 239000000463 material Substances 0.000 abstract description 6
- 239000004519 grease Substances 0.000 abstract description 3
- 238000012136 culture method Methods 0.000 abstract description 2
- 239000007788 liquid Substances 0.000 abstract 2
- 239000000243 solution Substances 0.000 description 16
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 12
- 235000019387 fatty acid methyl ester Nutrition 0.000 description 12
- 230000035508 accumulation Effects 0.000 description 10
- 239000000843 powder Substances 0.000 description 8
- 238000004321 preservation Methods 0.000 description 8
- 238000007127 saponification reaction Methods 0.000 description 8
- 239000000047 product Substances 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 6
- 239000002904 solvent Substances 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 230000007774 longterm Effects 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- 235000020777 polyunsaturated fatty acids Nutrition 0.000 description 5
- 230000009514 concussion Effects 0.000 description 4
- 238000001816 cooling Methods 0.000 description 4
- 238000001035 drying Methods 0.000 description 4
- 230000032050 esterification Effects 0.000 description 4
- 238000005886 esterification reaction Methods 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 239000007789 gas Substances 0.000 description 4
- 239000011521 glass Substances 0.000 description 4
- FUKUFMFMCZIRNT-UHFFFAOYSA-N hydron;methanol;chloride Chemical compound Cl.OC FUKUFMFMCZIRNT-UHFFFAOYSA-N 0.000 description 4
- 150000002500 ions Chemical class 0.000 description 4
- 230000005499 meniscus Effects 0.000 description 4
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 4
- 235000014593 oils and fats Nutrition 0.000 description 4
- GRONZTPUWOOUFQ-UHFFFAOYSA-M sodium;methanol;hydroxide Chemical compound [OH-].[Na+].OC GRONZTPUWOOUFQ-UHFFFAOYSA-M 0.000 description 4
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 4
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 4
- 239000012224 working solution Substances 0.000 description 4
- 230000000975 bioactive effect Effects 0.000 description 3
- 235000019197 fats Nutrition 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000003643 water by type Substances 0.000 description 3
- GVJHHUAWPYXKBD-UHFFFAOYSA-N (±)-α-Tocopherol Chemical compound OC1=C(C)C(C)=C2OC(CCCC(C)CCCC(C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-UHFFFAOYSA-N 0.000 description 2
- 208000030507 AIDS Diseases 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000195649 Chlorella <Chlorellales> Species 0.000 description 2
- 229920002984 Paramylon Polymers 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 239000013543 active substance Substances 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 239000012159 carrier gas Substances 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- FDJOLVPMNUYSCM-WZHZPDAFSA-L cobalt(3+);[(2r,3s,4r,5s)-5-(5,6-dimethylbenzimidazol-1-yl)-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl] [(2r)-1-[3-[(1r,2r,3r,4z,7s,9z,12s,13s,14z,17s,18s,19r)-2,13,18-tris(2-amino-2-oxoethyl)-7,12,17-tris(3-amino-3-oxopropyl)-3,5,8,8,13,15,18,19-octamethyl-2 Chemical compound [Co+3].N#[C-].N([C@@H]([C@]1(C)[N-]\C([C@H]([C@@]1(CC(N)=O)C)CCC(N)=O)=C(\C)/C1=N/C([C@H]([C@@]1(CC(N)=O)C)CCC(N)=O)=C\C1=N\C([C@H](C1(C)C)CCC(N)=O)=C/1C)[C@@H]2CC(N)=O)=C\1[C@]2(C)CCC(=O)NC[C@@H](C)OP([O-])(=O)O[C@H]1[C@@H](O)[C@@H](N2C3=CC(C)=C(C)C=C3N=C2)O[C@@H]1CO FDJOLVPMNUYSCM-WZHZPDAFSA-L 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
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- 235000016709 nutrition Nutrition 0.000 description 2
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- 239000003921 oil Substances 0.000 description 2
- 229920001296 polysiloxane Polymers 0.000 description 2
- -1 polysiloxanes Polymers 0.000 description 2
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- 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 2
- 239000011573 trace mineral Substances 0.000 description 2
- 235000013619 trace mineral Nutrition 0.000 description 2
- 239000011691 vitamin B1 Substances 0.000 description 2
- 239000011715 vitamin B12 Substances 0.000 description 2
- 238000010792 warming Methods 0.000 description 2
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- 241000894006 Bacteria Species 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 1
- 229910016876 Fe(NH4)2(SO4)2 Inorganic materials 0.000 description 1
- 229920001503 Glucan Polymers 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 102000016611 Proteoglycans Human genes 0.000 description 1
- 108010067787 Proteoglycans Proteins 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- GWEVSGVZZGPLCZ-UHFFFAOYSA-N Titan oxide Chemical compound O=[Ti]=O GWEVSGVZZGPLCZ-UHFFFAOYSA-N 0.000 description 1
- 229930003268 Vitamin C Natural products 0.000 description 1
- 229930003427 Vitamin E Natural products 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- OENHQHLEOONYIE-UKMVMLAPSA-N all-trans beta-carotene Natural products CC=1CCCC(C)(C)C=1/C=C/C(/C)=C/C=C/C(/C)=C/C=C/C=C(C)C=CC=C(C)C=CC1=C(C)CCCC1(C)C OENHQHLEOONYIE-UKMVMLAPSA-N 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
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- 230000003078 antioxidant effect Effects 0.000 description 1
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- 239000011648 beta-carotene Substances 0.000 description 1
- TUPZEYHYWIEDIH-WAIFQNFQSA-N beta-carotene Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1=C(C)CCCC1(C)C)C=CC=C(/C)C=CC2=CCCCC2(C)C TUPZEYHYWIEDIH-WAIFQNFQSA-N 0.000 description 1
- 229960002747 betacarotene Drugs 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- AIUDWMLXCFRVDR-UHFFFAOYSA-N dimethyl 2-(3-ethyl-3-methylpentyl)propanedioate Chemical class CCC(C)(CC)CCC(C(=O)OC)C(=O)OC AIUDWMLXCFRVDR-UHFFFAOYSA-N 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- WIGCFUFOHFEKBI-UHFFFAOYSA-N gamma-tocopherol Natural products CC(C)CCCC(C)CCCC(C)CCCC1CCC2C(C)C(O)C(C)C(C)C2O1 WIGCFUFOHFEKBI-UHFFFAOYSA-N 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 230000003050 macronutrient Effects 0.000 description 1
- 235000021073 macronutrients Nutrition 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 150000002739 metals Chemical class 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 235000021281 monounsaturated fatty acids Nutrition 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 229940066779 peptones Drugs 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- OGIDPMRJRNCKJF-UHFFFAOYSA-N titanium oxide Inorganic materials [Ti]=O OGIDPMRJRNCKJF-UHFFFAOYSA-N 0.000 description 1
- 235000019154 vitamin C Nutrition 0.000 description 1
- 239000011718 vitamin C Substances 0.000 description 1
- 235000019165 vitamin E Nutrition 0.000 description 1
- 239000011709 vitamin E Substances 0.000 description 1
- 229940046009 vitamin E Drugs 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
- OENHQHLEOONYIE-JLTXGRSLSA-N β-Carotene Chemical compound CC=1CCCC(C)(C)C=1\C=C\C(\C)=C\C=C\C(\C)=C\C=C\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C OENHQHLEOONYIE-JLTXGRSLSA-N 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/12—Unicellular algae; Culture media therefor
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Abstract
The invention discloses a method for increasing euglena biomass and fatty acid content. The method comprises the steps that 1, euglena liquid in the logarithmic phase is taken, centrifugation is carried out, and supernatant is taken; 2, euglena cells are resuspended with a traditional culture medium, centrifugation is carried out, supernatant is taken, and the euglena cells are resuspended through with traditional culture medium; 3, nano iron particles are resuspended with deionized water and added into culture liquid; and 4, illumination culture is carried out at the temperature of 25 DEG C to 30 DEG C. Aiming at the contradiction between growth and grease accumulation during euglena culture, a trace amount of nano iron material is reasonably added, and the novel culture method simple in formula and low in cost is invented. Therefore, the ideal euglena large-scale culture aims of guaranteeing low cost and growth speed and increasing grease accumulation are achieved.
Description
Technical field
The present invention relates to Euglena culture technique field, more particularly to a kind of to improve Euglena Biomass and content of fatty acid culture
Base.
Background technology
Euglena is a kind of very potential food and nourishing additive agent, because no cell wall, the nutrition contained by it has
Very high utilizability.Abundant required polyunsaturated fatty acid PUFAs (the prevention of cardiovascular diseases that it is not only needed containing human body
Disease), protein (provide various essential amino acids) and antioxidant content, such as beta-carotene, vitamin C, Vitamin E, moreover it is possible to product
Tire out substantial amounts of Euglena distinctive Paramylon product (Pm) (enhancing immunity).Pm is a kind of β -1, and 3- glucosans can strengthen life
Thing immunity, Sulfated Pm also have opposing effect to acquired immune deficiency syndrome (AIDS) inhibition of HIV, after Pm includes diet, it is possible to decrease body's cholesterol.
Euglena is constituted and at a relatively high bioactive substance such as Paramylon with the fatty acid being approached with human body, with good city
Field prospect.On October 30th, 2013, national health State Family Planning Commission issue bulletin, and it is new food to ratify including 8 kinds of materials including Euglena
Product raw material.There are many natural algae kinds in microalgae with relatively large number of oils and fatss ability is accumulated under stress, be the current biological energy
One of best source.
Stress-inducing is most commonly used that various required macronutrient such as nitrogen, phosphorus, sulfur etc., oils and fatss under the conditions of great majority
Yield can have the increase of 20-50%.But such stress disadvantage is that replacing culture medium, increased production cost and
Labor cost.In addition, the positive correlation between Oxdative stress and microbial grease accumulation is in oleaginous yeast and a part
It is a small amount of in chlorella to find and report, such as add titanium oxide and can induce a certain amount of fatty acid accumulation (Kang of chlorella
NK,et al.,Korean J Chem Eng,2014,31:861-867), add nano iron particles and can improve oleaginous yeast
Biomass and content of fatty acid (Karolina et al., Folia Microbiol., 2015, doi:10.1007/s12223-
015-0442-7).This addition Oxdative stress factor is simple, cost is relatively low, therefore has good application prospect.
There is the optimization of culture medium and condition of culture in Euglena large-scale production, however, promoting cell using nano material
Growth and the accumulation of fatty acid, during Euglena large-scale culture and corresponding fatty acid are produced, related application has no invention
And report.
Under normal circumstances, the cultural method of Euglena has two kinds of autotrophy culture and Heterotrophic culture.The autotrophy culture speed of growth is delayed
Slowly, it is unfavorable for the accumulation of the raising bioactive substance of Biomass；Heterotrophic culture is more using rich in nutrition such as proteoglycans at present
The synthetic medium of material, wherein comprising organic substances such as yeast extract, Carnis Bovis seu Bubali cream and peptones, although the Euglena in the culture medium
The speed of growth is very fast, but cost of material is high, is highly prone to antibacterial, miscellaneous bacteria algae and primary during opening or large-scale culture
The microorganism pollutions such as animal so that Euglena yield is greatly reduced, may cause the failure of large-scale culture by the gross when serious,
It is difficult to industrialized production.How the Biomass of Euglena and the content of bioactive substance improved simultaneously, also just into Euglena
Realize the main bugbear of industrialized production.
The content of the invention
Contradiction of the present invention for growth and oil and fat accumulation in Euglena culture, using the nanometer iron material of rationally addition trace
Material, invented a kind of simple, the with low cost Novel culture method of formula, so as to reach ensure low cost, the speed of growth with
And improve the purpose of the preferable Euglena large-scale culture of oil and fat accumulation.
A kind of raising Euglena Biomass and the method for content of fatty acid that the present invention is provided, including step：1) take the logarithm life
Long-term algae solution, centrifuging and taking supernatant；2) the resuspended frustule of conventional medium, centrifugation is used to remove supernatant, then use conventional medium weight
Outstanding frustule；3) the resuspended nano iron particles of deionized water, are added in culture fluid；4) 25-30 DEG C of illumination cultivation.
Further, the present invention provide a kind of raising Euglena Biomass and content of fatty acid method the step of 3) in receive
The diameter range of the granule of rice iron particle is in 25-50 nanometers.
Further, the present invention provide a kind of raising Euglena Biomass and content of fatty acid method the step of 3) in
Suitable concn is 5-80mg/L.
The present invention applies business-like nano iron particles Nanofer 25, water it is mutually not coated, mean particle size exists
The iron granule of 25-50 rans, fresh particle directly can be applied.Effectively using concentration range in 5-80mg/
L.Euglena can grow into platform early stage (～1-5 × 10 under the conditions of 25-30 DEG C in heterotrophism or Euglena culture medium6cells/
ML), it is transferred in fresh culture fluid with 1/10 inoculation ratio as seed liquor, while adding respective concentration toward culture fluid
Nano iron particles, continue culture make cell growth to the platform later stage (～107Cells/mL), reach harvest time.
The process can improve the Biomass of 5-15%, and the fatty acid accumulation of 20-30% illustrates simply to add the nanometer of trace
Ferrum not only can increase the Biomass of Euglena and can stimulate more fat acid accumulations.
Compared with traditional, conventional technical method, the raising Euglena Biomass and content of fatty acid of present invention offer
Method accelerates can the Euglena speed of growth, more biomass accumulations, and the content of oils and fatss and unsaturated fatty acid has significantly
Property improve, play quality etc. the purpose that production cost and run time cycle is greatly lowered and active substance is improved.
Specific embodiment
Embodiment one：
The addition of microdisk electrode and trace element：The algae solution for growing to logarithmic (log) phase is taken, it is 0.5 to be diluted to OD750,
Using traditional Euglena culture medium culturing Euglena as control.
The nano iron particles (product type Nanofer 25, average particulate diameter are 25-50 nanometers) of fresh purchase, spend
Ionized water is resuspended to suitable concn (concentration well known within the skill of those ordinarily skilled), can ensure that 2 months steady at normal temperatures
Periodically.
Be separately added into toward culture fluid different volumes Nanoscale Iron suspension (final concentration is respectively 1,3,5,8,13,17,50,
120mg/L)；Inoculation grows to the frustule of logarithmic (log) phase, and cultivation temperature is 23 ± 1 DEG C, illumination cultivation.
The formula of the Euglena culture medium adopted in the present embodiment is as shown in table 1 below.
Table 1：Euglena culture medium prescription (according to the composition of weight portion).
Biomass measuring：10mL algae solutions were taken in the 15mL centrifuge tubes weighed in advance respectively at the 4th, 7,9 day same time,
8000rpm is collected by centrifugation frustule, abandons supernatant, is placed in -80 DEG C of preservations.After whole samples collection is finished, the mouth of pipe is sealed with preservative film
It is good, some apertures are pricked, -45 DEG C is placed in freezer dryer and is dried 24h.After drying is finished, weigh again, calculate Euglena biological
Amount.Dried algae powder is placed in -80 DEG C long-term and preserves.
Fatty acid and its component analyses：Claim the cryodesiccated algae powder of 5-10mg in glass centrifuge tube with cover, add 50 μ L
C19:0 internal standard working solution, 1mL 2M NaOH-CH3OH solution, places 1h on shaking table of the rotating speed for 100rpm.After 75 DEG C
Water-bath 15min carries out saponification, and saponification is cooled down completely afterwards, adds 1mL4M HCl-CH3OH solution, and pH ＜ 2 are adjusted with dense HCl,
Again in 75 DEG C of water-bath 15min so as to fully esterification, 1mL normal hexane after cooling, is added, be vortexed concussion 10s twice, make extraction
Completely.4000rpm centrifugation 2min promote layering, careful to draw the supernatant into chromatograph bottle, after the completion of be placed in fume hood.
After solvent volatilizees completely, prepare the fatty acid methyl ester for adding the dissolving of 500 μ L dichloromethane to obtain, and mark of ruling at concave meniscus
Note, -20 DEG C of preservations, goes up machine testing as early as possible.The same standard substance of instrument operational factor.
The analysis of fatty acid component and content：Using the GC-MS instruments of Agilent companies, by retention time and feature
Ion pair fatty acid methyl ester carry out it is qualitative, with internal standard method by calculate peak area each fatty acid methyl ester component is carried out quantitatively.
Gas chromatographic column：The gas chromatographic column for adopting is tested for capillary column, model VF-23ms (Part No.:
CP8827), it belongs to polysiloxanes gas-like phase capillary tube polarity chromatographic column, is especially suitable for the analysis of fatty acid methyl ester.The chromatograph
The maximum temperature of post tolerance is 260 DEG C, and the specification that this experiment is adopted is for 0.25 μm of 30.0m × 320 μ m.Operational factor：Sample introduction
Mouth temperature is 250 DEG C；Carrier gas is high-purity He (purity is higher than 99.999%), constant voltage mode；Sampling volume is 1 μ L, and split ratio is
10:1；Column oven intensification degree is as follows：70 DEG C of holding 4min, it is with 25 DEG C/min ramps to 195 DEG C then fast with 3 DEG C/min
Rate is warming up to 205 DEG C, then with 8 DEG C/min ramps to 230 DEG C, keeps 1min；Transmission line temperature is 250 DEG C；Solvent prolongs
It is 3min late；Mass Spectrometer Method pattern is full scan pattern, and mass number (m/z) detection range is 50-550；Ionization mode is EI
(70eV) ion source temperature is 230 DEG C, and quadrupole rod temperature is 150 DEG C.
The Collecting and dealing of sample：Respectively at the 3rd, 5,7 day same time take 10mL algae solutions in the 15mL for weighing in advance from
In heart pipe, 8000rpm is collected by centrifugation frustule, abandons supernatant, is placed in -80 DEG C of preservations.After whole samples collection is finished, the mouth of pipe is protected
Fresh film is sealed, and pricks some apertures, is placed in freezer dryer -45 DEG C and is dried 24h.After drying is finished, weigh again, calculate Euglena
Biomass.Dried algae powder is placed in -80 DEG C long-term and preserves.
The preparation of press proof product on fatty acid methyl ester：Claim the cryodesiccated algae powder of 5-10mg in glass centrifuge tube with cover, plus
Enter 50 μ L C19:0 internal standard working solution, 1mL2M NaOH-CH3OH solution, places 1h on shaking table of the rotating speed for 100rpm.Afterwards
Saponification is carried out in 75 DEG C of water-bath 15min, saponification is cooled down completely afterwards, add 1mL 4M HCl-CH3OH solution, and adjusted with dense HCl
PH ＜ 2, again in 75 DEG C of water-bath 15min so as to fully esterification, adds 1mL normal hexane after cooling, is vortexed concussion 10s twice,
Make extraction complete.4000rpm centrifugation 2min promote layering, careful to draw the supernatant into chromatograph bottle, after the completion of be placed in it is logical
In wind cupboard.After solvent volatilization completely, prepare the fatty acid methyl ester for adding the dissolving of 500 μ L dichloromethane to obtain, and in concave meniscus
Place's marking, -20 DEG C of preservations, goes up machine testing as early as possible.Qualitative and quantitative analysis are carried out as control according to standard sample subsequently various
The quality and quantity of fatty acid component.
Embodiment two：
The addition of microdisk electrode and trace element：The algae solution for growing to logarithmic (log) phase is taken, it is 0.5 to be diluted to OD750,
Using traditional Euglena culture medium culturing Euglena as control.
The nano iron particles (product type Nanofer 25, average particulate diameter are 25-50 nanometers) of fresh purchase, spend
Ionized water is resuspended to suitable concn (concentration well known within the skill of those ordinarily skilled), can ensure that 2 months steady at normal temperatures
Periodically.
Be separately added into toward culture fluid different volumes Nanoscale Iron suspension (final concentration is respectively 1,3,5,8,13,17,50,
120mg/L)；Inoculation grows to the frustule of logarithmic (log) phase, and cultivation temperature is 23 ± 1 DEG C.
Biomass measuring：10mL algae solutions were taken in the 15mL centrifuge tubes weighed in advance respectively at the 4th, 7,9 day same time,
8000rpm is collected by centrifugation frustule, abandons supernatant, is placed in -80 DEG C of preservations.After whole samples collection is finished, the mouth of pipe is sealed with preservative film
It is good, some apertures are pricked, -45 DEG C is placed in freezer dryer and is dried 24h.After drying is finished, weigh again, calculate Euglena biological
Amount.Dried algae powder is placed in -80 DEG C long-term and preserves.
Fatty acid and its component analyses：Claim the cryodesiccated algae powder of 5-10mg in glass centrifuge tube with cover, add 50 μ L
C19:0 internal standard working solution, 1mL 2M NaOH-CH3OH solution, places 1h on shaking table of the rotating speed for 100rpm.After 75 DEG C
Water-bath 15min carries out saponification, and saponification is cooled down completely afterwards, adds 1mL4M HCl-CH3OH solution, and pH ＜ 2 are adjusted with dense HCl,
Again in 75 DEG C of water-bath 15min so as to fully esterification, 1mL normal hexane after cooling, is added, be vortexed concussion 10s twice, make extraction
Completely.4000rpm centrifugation 2min promote layering, careful to draw the supernatant into chromatograph bottle, after the completion of be placed in fume hood.
After solvent volatilizees completely, prepare the fatty acid methyl ester for adding the dissolving of 500 μ L dichloromethane to obtain, and mark of ruling at concave meniscus
Note, -20 DEG C of preservations, goes up machine testing as early as possible.The same standard substance of instrument operational factor.
The analysis of fatty acid component and content：Using the GC-MS instruments of Agilent companies, by retention time and feature
Ion pair fatty acid methyl ester carry out it is qualitative, with internal standard method by calculate peak area each fatty acid methyl ester component is carried out quantitatively.
Gas chromatographic column：The gas chromatographic column for adopting is tested for capillary column, model VF-23ms (Part No.:
CP8827), it belongs to polysiloxanes gas-like phase capillary tube polarity chromatographic column, is especially suitable for the analysis of fatty acid methyl ester.The chromatograph
The maximum temperature of post tolerance is 260 DEG C, and the specification that this experiment is adopted is for 0.25 μm of 30.0m × 320 μ m.Operational factor：Sample introduction
Mouth temperature is 250 DEG C；Carrier gas is high-purity He (purity is higher than 99.999%), constant voltage mode；Sampling volume is 1 μ L, and split ratio is
10:1；Column oven intensification degree is as follows：70 DEG C of holding 4min, it is with 25 DEG C/min ramps to 195 DEG C then fast with 3 DEG C/min
Rate is warming up to 205 DEG C, then with 8 DEG C/min ramps to 230 DEG C, keeps 1min；Transmission line temperature is 250 DEG C；Solvent prolongs
It is 3min late；Mass Spectrometer Method pattern is full scan pattern, and mass number (m/z) detection range is 50-550；Ionization mode is EI
(70eV) ion source temperature is 230 DEG C, and quadrupole rod temperature is 150 DEG C.
The Collecting and dealing of sample：Respectively at the 3rd, 5,7 day same time take 10mL algae solutions in the 15mL for weighing in advance from
In heart pipe, 8000rpm is collected by centrifugation frustule, abandons supernatant, is placed in -80 DEG C of preservations.After whole samples collection is finished, the mouth of pipe is protected
Fresh film is sealed, and pricks some apertures, is placed in freezer dryer -45 DEG C and is dried 24h.After drying is finished, weigh again, calculate Euglena
Biomass.Dried algae powder is placed in -80 DEG C long-term and preserves.
The preparation of press proof product on fatty acid methyl ester：Claim the cryodesiccated algae powder of 5-10mg in glass centrifuge tube with cover, plus
Enter 50 μ L C19:0 internal standard working solution, 1mL2M NaOH-CH3OH solution, places 1h on shaking table of the rotating speed for 100rpm.Afterwards
Saponification is carried out in 75 DEG C of water-bath 15min, saponification is cooled down completely afterwards, add 1mL 4M HCl-CH3OH solution, and adjusted with dense HCl
PH ＜ 2, again in 75 DEG C of water-bath 15min so as to fully esterification, adds 1mL normal hexane after cooling, is vortexed concussion 10s twice,
Make extraction complete.4000rpm centrifugation 2min promote layering, careful to draw the supernatant into chromatograph bottle, after the completion of be placed in it is logical
In wind cupboard.After solvent volatilization completely, prepare the fatty acid methyl ester for adding the dissolving of 500 μ L dichloromethane to obtain, and in concave meniscus
Place's marking, -20 DEG C of preservations, goes up machine testing as early as possible.Qualitative and quantitative analysis are carried out as control according to standard sample subsequently various
The quality and quantity of fatty acid component.
As shown in table one and table two, 1 Biomass of embodiment is improved to some extent and (such as adds trace nanometer ferrum element
Biomass (10.53-23.1g/L of 3-7 days grown cultures) is than the control Biomass (9.12-19.38g/ of 3-7 days grown cultures
)), L the amount of satisfied fatty acid has been remarkably decreased (the 28.6-22.5ug/mg dry weights contrast of addition group 3-7 days grown cultures
70.3-108.5ug/mg), but, the amount of the unsaturated fatty acid with high added value but significantly has lifting.Such as, it is single
5.6-9.7ug/mg of the amount of unsaturated fatty acid from the 4.3-8.3ug/mg of 3-7 days grown cultures of matched group to addition group,
26.7-44.9ug/ of the polyunsaturated fatty acid from the 73.9-104.5ug/mg of 3-7 days grown cultures of matched group to addition group
Mg, DHA content 73.9-104.5ug/mg then from the 9.9-15.3ug/mg of 3-7 days grown cultures of matched group to addition group.
Therefore compared with traditional, conventional technical method, the invention provides unit is weighed in a kind of efficient, simple addition of culture Euglena
Element, the content such that it is able to make the quickening of the Euglena speed of growth, more biomass accumulations, oils and fatss and unsaturated fatty acid have aobvious
Work property is improved, and plays quality etc. the purpose that production cost and run time cycle is greatly lowered and active substance is improved.
Heterotrophic culture based formulas, according to the composition of weight portion：
A) precise 0.084g Fe (NH4)2(SO4)2·6H2O, is dissolved in 10mL deionized waters, matching while using.
B) " Metals 60A " formula, according to the composition of weight portion：
c)Vitamin B1：Precise 0.1g Vitamin B1, 100mL deionized waters are dissolved in, filtration sterilization is stored in 4
DEG C refrigerator.
d)Vitamin B12：Lucifuge precise 0.02g Vitamin B12, 1000mL deionized waters are dissolved in, acquirement is arrived
Solution 1mL be diluted to 100mL, filtration sterilization is stored in 4 DEG C of refrigerators.
Different time is cultivated in Heterotrophic culture base after addition nanogold particle of table 1 (8mg/L) and is spaced life in (3,5,7 days)
The comparison of thing amount, Nanoscale Iron-culture three days (nFe-3d), three days (CN-3d) after matched group-culture
Different time interval (3,5,7 days) is cultivated after addition nanogold particle of table 2 (8mg/L) in Heterotrophic culture base fatty
The comparison of sour key component, Nanoscale Iron-culture three days (nFe-3d), three days (CN-3d) after matched group-culture, SFA, MUFA,
PUFA is satisfied fatty acid, monounsaturated fatty acid and polyunsaturated fatty acid respectively；
Claims (3)
1. a kind of method for improving Euglena Biomass and content of fatty acid, including step：
1) take the logarithm the algae solution of trophophase, centrifuging and taking supernatant；
2) the resuspended frustule of conventional medium, centrifugation is used to remove supernatant, then with the resuspended frustule of conventional medium；
3) the resuspended nano iron particles of deionized water, are added in culture fluid；
4) 25-30 DEG C of illumination cultivation.
2. a kind of method for improving Euglena Biomass and content of fatty acid as claimed in claim 1, it is characterised in that：The side
The step of method 3) in nano iron particles granule diameter range in 25-50 nanometers.
3. a kind of method for improving Euglena Biomass and content of fatty acid as claimed in claim 1, it is characterised in that the side
The step of method 3) in suitable concn be 5-80mg/L.
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| CN111088211A (en) * | 2020-01-13 | 2020-05-01 | 武汉理工大学 | Culture method of microalgae |
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| WO2016038573A1 (en) * | 2014-09-11 | 2016-03-17 | Reliance Industries Limited | A process for preparing crude bio-oil from feedstock |
| KR20160053565A (en) * | 2014-11-05 | 2016-05-13 | 한국에너지기술연구원 | Method for harvesting microalgae and subsequent extracting lipid using cationic surfactant-functionalized magnetic nanoparticle composite |
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2016
- 2016-11-24 CN CN201611044256.8A patent/CN106520657A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016038573A1 (en) * | 2014-09-11 | 2016-03-17 | Reliance Industries Limited | A process for preparing crude bio-oil from feedstock |
| KR20160053565A (en) * | 2014-11-05 | 2016-05-13 | 한국에너지기술연구원 | Method for harvesting microalgae and subsequent extracting lipid using cationic surfactant-functionalized magnetic nanoparticle composite |
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| CN111088211A (en) * | 2020-01-13 | 2020-05-01 | 武汉理工大学 | Culture method of microalgae |
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