CN113717917A - Combined antibiotic application method for pure culture of haematococcus pluvialis - Google Patents
Combined antibiotic application method for pure culture of haematococcus pluvialis Download PDFInfo
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- 241000168517 Haematococcus lacustris Species 0.000 title claims abstract description 45
- 230000003115 biocidal effect Effects 0.000 title claims abstract description 36
- 238000000034 method Methods 0.000 title claims abstract description 27
- 239000002904 solvent Substances 0.000 claims abstract description 53
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 claims abstract description 36
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 claims abstract description 36
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims abstract description 26
- 229960000723 ampicillin Drugs 0.000 claims abstract description 24
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 claims abstract description 24
- 229930027917 kanamycin Natural products 0.000 claims abstract description 24
- 229960000318 kanamycin Drugs 0.000 claims abstract description 24
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 claims abstract description 24
- 229930182823 kanamycin A Natural products 0.000 claims abstract description 24
- 229930182566 Gentamicin Natural products 0.000 claims abstract description 21
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 claims abstract description 21
- 229960002518 gentamicin Drugs 0.000 claims abstract description 21
- 235000010333 potassium nitrate Nutrition 0.000 claims abstract description 18
- 239000004323 potassium nitrate Substances 0.000 claims abstract description 18
- 229960005322 streptomycin Drugs 0.000 claims abstract description 18
- QHFQAJHNDKBRBO-UHFFFAOYSA-L calcium chloride hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].[Cl-].[Ca+2] QHFQAJHNDKBRBO-UHFFFAOYSA-L 0.000 claims abstract description 16
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 claims abstract description 16
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 claims abstract description 16
- ISPYRSDWRDQNSW-UHFFFAOYSA-L manganese(II) sulfate monohydrate Chemical compound O.[Mn+2].[O-]S([O-])(=O)=O ISPYRSDWRDQNSW-UHFFFAOYSA-L 0.000 claims abstract description 16
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims abstract description 16
- 229910000368 zinc sulfate Inorganic materials 0.000 claims abstract description 16
- 229960001763 zinc sulfate Drugs 0.000 claims abstract description 16
- 239000001963 growth medium Substances 0.000 claims abstract description 14
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims abstract description 13
- JZCCFEFSEZPSOG-UHFFFAOYSA-L copper(II) sulfate pentahydrate Chemical compound O.O.O.O.O.[Cu+2].[O-]S([O-])(=O)=O JZCCFEFSEZPSOG-UHFFFAOYSA-L 0.000 claims abstract description 13
- RWVGQQGBQSJDQV-UHFFFAOYSA-M sodium;3-[[4-[(e)-[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfonatophenyl)methyl]azaniumylidene]-2-methylcyclohexa-2,5-dien-1-ylidene]methyl]-n-ethyl-3-methylanilino]methyl]benzenesulfonate Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C(=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C)C=2C(=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C)C=C1 RWVGQQGBQSJDQV-UHFFFAOYSA-M 0.000 claims abstract description 13
- ARPLKSKOWFTTTQ-UHFFFAOYSA-L cobalt(2+);dichloride;dihydrate Chemical compound O.O.Cl[Co]Cl ARPLKSKOWFTTTQ-UHFFFAOYSA-L 0.000 claims abstract description 11
- 239000003242 anti bacterial agent Substances 0.000 claims abstract description 8
- 239000007787 solid Substances 0.000 claims abstract description 8
- 229940088710 antibiotic agent Drugs 0.000 claims abstract description 7
- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 claims abstract description 7
- 238000012258 culturing Methods 0.000 claims abstract description 5
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 83
- 238000005303 weighing Methods 0.000 claims description 50
- 239000012452 mother liquor Substances 0.000 claims description 41
- 239000003109 Disodium ethylene diamine tetraacetate Substances 0.000 claims description 15
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 claims description 15
- 235000019301 disodium ethylene diamine tetraacetate Nutrition 0.000 claims description 15
- 239000010413 mother solution Substances 0.000 claims description 15
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 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
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 12
- 239000012498 ultrapure water Substances 0.000 claims description 12
- 238000002156 mixing Methods 0.000 claims description 9
- 229920001817 Agar Polymers 0.000 claims description 7
- 239000001888 Peptone Substances 0.000 claims description 7
- 108010080698 Peptones Proteins 0.000 claims description 7
- 239000008272 agar Substances 0.000 claims description 7
- 229940041514 candida albicans extract Drugs 0.000 claims description 7
- 235000019319 peptone Nutrition 0.000 claims description 7
- 239000011780 sodium chloride Substances 0.000 claims description 7
- 239000012138 yeast extract Substances 0.000 claims description 7
- -1 heptahydrate ferric sulfate Chemical class 0.000 claims description 6
- 229910000360 iron(III) sulfate Inorganic materials 0.000 claims description 6
- 239000002609 medium Substances 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 5
- 238000005286 illumination Methods 0.000 claims description 4
- 239000012153 distilled water Substances 0.000 claims description 3
- XNCMOUSLNOHBKY-UHFFFAOYSA-H iron(3+);trisulfate;heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+3].[Fe+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O XNCMOUSLNOHBKY-UHFFFAOYSA-H 0.000 claims description 3
- 238000000967 suction filtration Methods 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 238000000746 purification Methods 0.000 claims 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 abstract 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 abstract 1
- 235000019797 dipotassium phosphate Nutrition 0.000 abstract 1
- 229960001484 edetic acid Drugs 0.000 abstract 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 abstract 1
- 239000008399 tap water Substances 0.000 abstract 1
- 235000020679 tap water Nutrition 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 15
- JEBFVOLFMLUKLF-IFPLVEIFSA-N Astaxanthin Natural products CC(=C/C=C/C(=C/C=C/C1=C(C)C(=O)C(O)CC1(C)C)/C)C=CC=C(/C)C=CC=C(/C)C=CC2=C(C)C(=O)C(O)CC2(C)C JEBFVOLFMLUKLF-IFPLVEIFSA-N 0.000 description 12
- 235000013793 astaxanthin Nutrition 0.000 description 12
- MQZIGYBFDRPAKN-ZWAPEEGVSA-N astaxanthin Chemical compound C([C@H](O)C(=O)C=1C)C(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)C(=O)[C@@H](O)CC1(C)C MQZIGYBFDRPAKN-ZWAPEEGVSA-N 0.000 description 12
- 229940022405 astaxanthin Drugs 0.000 description 12
- 239000001168 astaxanthin Substances 0.000 description 12
- 241000195493 Cryptophyta Species 0.000 description 6
- 230000009105 vegetative growth Effects 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 239000003963 antioxidant agent Substances 0.000 description 3
- 235000006708 antioxidants Nutrition 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- RCUTWANGTCNSSX-UHFFFAOYSA-L O.O.[Co+2].[O-]S([O-])(=O)=O Chemical compound O.O.[Co+2].[O-]S([O-])(=O)=O RCUTWANGTCNSSX-UHFFFAOYSA-L 0.000 description 2
- 230000003078 antioxidant effect Effects 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 210000000947 motile cell Anatomy 0.000 description 2
- 230000009182 swimming Effects 0.000 description 2
- 241000251468 Actinopterygii Species 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- 241000195627 Chlamydomonadales Species 0.000 description 1
- 241000196319 Chlorophyceae Species 0.000 description 1
- 241000195628 Chlorophyta Species 0.000 description 1
- 241000238557 Decapoda Species 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000316848 Rhodococcus <scale insect> Species 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 239000003674 animal food additive Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 150000001749 carotenones Chemical class 0.000 description 1
- 235000005472 carotenones Nutrition 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000013505 freshwater Substances 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 125000000468 ketone group Chemical group 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000013028 medium composition Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 230000004792 oxidative damage Effects 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 230000029553 photosynthesis Effects 0.000 description 1
- 238000010672 photosynthesis Methods 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 230000003334 potential effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 235000015170 shellfish Nutrition 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
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Classifications
-
- 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
- C12N1/38—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
-
- 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
Abstract
The invention relates to a combined antibiotic application method for pure culture of haematococcus pluvialis. The experimental haematococcus pluvialis culture medium MCM comprises: the formula of the solution A comprises 500mg/L potassium nitrate, 20mg/L dipotassium phosphate and 100mg/L magnesium sulfate heptahydrate; the formula of the solution B is 80mg/L calcium chloride hexahydrate; the formula of the solution C is 372 mu g/L of ethylene diamine tetraacetic acid and 417 mu g/L of iron sulfate heptahydrate; the formula of the solution D comprises 7.189 mu g/L zinc sulfate, 1.237 mu g/L phosphoric acid, 0.476 mu g/L cobalt chloride dihydrate, 6.242 mu g/L copper sulfate pentahydrate, 8.451 mu g/L manganese sulfate monohydrate and 0.726 mu g/L sodium molybdate dihydrate; the formula solvent is tap water, and after autoclaving, the solution A, the solution B, the solution C and the solution D are dissolved in the solvent in sequence; using 4 antibiotics of streptomycin, kanamycin, gentamicin and ampicillin in combination, adjusting pH to 6.99-7.03, respectively controlling the concentration of each antibiotic to be 600 mug/ml, culturing for 3d on a shaking table in a light box, and purifying and checking by combining a solid culture medium to obtain pure haematococcus pluvialis.
Description
Technical Field
The invention relates to an obtaining method suitable for pure microalgae, in particular to a combined antibiotic application method suitable for pure culture of haematococcus pluvialis, and particularly relates to a pure culture obtaining and culturing method suitable for a green cell vegetative growth stage of haematococcus pluvialis.
Background
Haematococcus pluvialis (Haematococcus pluvialis)Haematococcus pluvialis) Is a unicellular algae suitable for growth in fresh water, belonging to Chlorophyta, Chlorophyceae, Volvocales, Rhodococcus, and its genus. Haematococcus pluvialis produces chlamydospores when the environment is poor, and a large amount of astaxanthin is accumulated. Haematococcus pluvialis contains a large amount of astaxanthin (up to 7% of the dry weight of cells), is an important molecular biological test object and a raw material for producing natural astaxanthin, is generally recognized as the safest source of astaxanthin, has a very wide market, has the largest industry of Haematococcus pluvialis culture and a global stable production base in Hubei and Yunnan of China, has a very large industrial scale and has a solid industrial foundation.
Natural astaxanthin is a red keto-carotenoid, is a fat-soluble unsaturated macromolecular organic compound, widely exists in the biological world, particularly in aquatic organisms such as fish, shrimp, shellfish and algae, is one of the strongest antioxidants worldwide, and has been subjected to more than 10 international certifications such as the U.S. FDA and the european union. The molecular structure of astaxanthin contains long conjugated double-bond chain, and the terminal contains unsaturated keto group and hydroxyl group, thus providing important antioxidant capacity for the molecule. In the photosynthesis process of haematococcus pluvialis, astaxanthin can be used as an auxiliary pigment to absorb light energy, and can also be used as an antioxidant to remove excessive active oxygen in cells and protect the cells from oxidative damage. In addition, the astaxanthin also has the functions of resisting aging, resisting inflammation, improving immunity, repairing ultraviolet injury, inhibiting tumor growth and the like, can be used as a feed additive for aquatic animals and livestock breeding, can also be used as a therapeutic drug to be applied to industries such as medicines, skin care products and the like, and has higher economic value.
There are two distinct cell types in the Haematococcus pluvialis life cycle, namely, the green motile cells which undergo vegetative growth and the red immobile cells which accumulate astaxanthin. Under favorable growth conditions, the cells grow vigorously and exist as green motile cells, and the cells contain a very small amount of astaxanthin; under adverse environment, cells are stressed, grow slowly, the tails of swimming cells fall off, the kinetic energy loss gradually becomes a static state from a swimming state, the cell volume is increased, the cells are converted into chlamydospores, simultaneously a large amount of astaxanthin is accumulated, and the cells are turned from green to red. Haematococcus pluvialis begins to accumulate astaxanthin primarily at the red cell stage.
The haematococcus pluvialis strain obtained by the traditional method is not a true pure strain, the culture medium contains other intersophyta bacteria, and the intersophyta bacteria and the haematococcus pluvialis have a complex mutual relationship and have a potential effect on improving or reducing the productivity of the haematococcus pluvialis. The invention can provide a large amount of pure haematococcus pluvialis strains as experimental materials for relevant follow-up research.
Disclosure of Invention
The invention aims to solve the technical problem of providing a combined antibiotic application method suitable for pure culture of haematococcus pluvialis, aiming at the defects in the prior art, so that pure haematococcus pluvialis can be obtained, and a large amount of experimental materials are provided for correlation research. The method for applying the combined antibiotics is suitable for the vegetative growth stage of the haematococcus pluvialis green cells.
In order to achieve the purpose, the invention adopts the following technical scheme:
a combined antibiotic application method suitable for pure culture of haematococcus pluvialis comprises the following steps: culturing Haematococcus pluvialis with MCM culture medium, performing shake culture treatment on Haematococcus pluvialis liquid in an illumination incubator for 3d by jointly using 4 antibiotics with the concentrations of streptomycin, kanamycin, gentamicin and ampicillin respectively of 600 mu g/ml, and purifying by combining with a solid culture medium to obtain pure Haematococcus pluvialis.
Further, the preparation method of the MCM medium specifically comprises the following steps:
(1) preparing a solution A mother solution: weighing 50g of potassium nitrate, and dissolving in a small amount of solvent to completely dissolve the potassium nitrate; weighing 2g of potassium dihydrogen phosphate, and dissolving in a small amount of solvent to completely dissolve the potassium dihydrogen phosphate; weighing 10g of magnesium sulfate heptahydrate, and dissolving in a small amount of solvent to completely dissolve the magnesium sulfate heptahydrate; mixing the dissolved potassium nitrate solution, the potassium dihydrogen phosphate solution and the magnesium sulfate heptahydrate solution together, and preparing a solution A mother liquor by using a solvent to fix the volume to 1L, wherein the concentration of potassium nitrate is 50g/L, the concentration of potassium dihydrogen phosphate is 2g/L, and the concentration of magnesium sulfate heptahydrate is 10 g/L;
(2) preparing a solution B mother solution: weighing 8g of calcium chloride hexahydrate, dissolving the calcium chloride hexahydrate in a small amount of solvent to completely dissolve the calcium chloride hexahydrate, and preparing a B liquid mother liquor by using the solvent to fix the volume to 1L to ensure that the concentration of the calcium chloride hexahydrate is 8 g/L;
(3) preparing a solution C mother solution: weighing 3.72g of disodium ethylene diamine tetraacetate, and dissolving in a small amount of solvent to completely dissolve the disodium ethylene diamine tetraacetate; weighing 4.17g of iron sulfate heptahydrate, and dissolving in a small amount of solvent to completely dissolve the iron sulfate heptahydrate; mixing the dissolved disodium ethylene diamine tetraacetate solution and the heptahydrate ferric sulfate solution together, and preparing a C solution mother solution by using a solvent to fix the volume to 1L, wherein the concentration of the disodium ethylene diamine tetraacetate is 3.72g/L, and the concentration of the heptahydrate ferric sulfate is 4.17 g/L;
(4) preparing solution D mother liquor: weighing 71.89mg of zinc sulfate, and dissolving in a small amount of solvent to completely dissolve the zinc sulfate; weighing 12.37mg of phosphoric acid, and dissolving in a small amount of solvent to completely dissolve; weighing 4.76mg/L g cobalt chloride dihydrate to dissolve in a small amount of solvent completely; weighing 62.42mg/L copper sulfate pentahydrate, and dissolving in a small amount of solvent to completely dissolve; weighing 84.51mg of manganese sulfate monohydrate, and dissolving in a small amount of solvent to completely dissolve the manganese sulfate monohydrate; weighing 7.26mg/L sodium molybdate dihydrate to be dissolved in a small amount of solvent to be completely dissolved; mixing the dissolved zinc sulfate solution, the phosphoric acid solution, the cobalt chloride dihydrate solution, the copper sulfate pentahydrate solution, the manganese sulfate monohydrate solution and the sodium molybdate dihydrate solution together, and fixing the volume to 1L by using a solvent to prepare a D solution mother solution, wherein the concentration of zinc sulfate is 71.89mg/L, the concentration of phosphoric acid is 12.37mg/L, the concentration of cobalt chloride dihydrate is 4.76mg/L, the concentration of copper sulfate pentahydrate is 62.42mg/L, the concentration of manganese sulfate monohydrate is 84.51mg/L, and the concentration of sodium molybdate dihydrate is 7.26 mg/L;
(5) when the potassium nitrate-containing aqueous solution is used, 10mL of A solution mother liquor, 10mL of B solution mother liquor, 100 mu L C solution mother liquor and 100 mu L D solution mother liquor are sequentially dissolved in 900mL of solvent, so that the concentration of potassium nitrate in the A solution is 500mg/L, the concentration of dipotassium hydrogen phosphate is 20mg/L, the concentration of magnesium sulfate heptahydrate is 100mg/L, the concentration of calcium chloride hexahydrate in the B solution is 80mg/L, the concentration of disodium ethylene diamine tetraacetate in the C solution is 372 mu g/L, the concentration of ferric sulfate heptahydrate is 417 mu g/L, the concentration of zinc sulfate in the D solution is 7.189 mu g/L, the concentration of phosphoric acid is 1.237 mu g/L, the concentration of cobalt sulfate dihydrate is 0.476 mu g/L, the concentration of copper sulfate pentahydrate is 6.242 mu g/L, the concentration of manganese sulfate monohydrate is 8.451 mu g/L, and the concentration of sodium molybdate dihydrate is 0.726 mu g/L; adjusting pH to 6.9-7.1 with pH meter, and inoculating Haematococcus pluvialis strain to be cultured.
Further, the preparation method of the 4 antibiotics with the concentrations of 600 mug/ml streptomycin, kanamycin, gentamicin and ampicillin specifically comprises the following steps:
(1) preparing 60g/L of antibiotic mother liquor: weighing 6g of streptomycin, dissolving in 100mL of ultrapure water, and completely dissolving; weighing 6g of kanamycin, and dissolving the kanamycin in 100mL of ultrapure water to completely dissolve the kanamycin; weighing 6g of gentamicin, and dissolving in 100mL of ultrapure water to completely dissolve the gentamicin; weighing 6g of ampicillin, and dissolving the ampicillin in 100mL of ultrapure water to completely dissolve the ampicillin; the streptomycin concentration is 60g/L, the kanamycin concentration is 60g/L, the gentamicin concentration is 60g/L, and the ampicillin concentration is 60 g/L; degerming by adopting a suction filtration mode;
(2) when in use, 1mL of streptomycin antibiotic mother liquor, 1mL of kanamycin antibiotic mother liquor, 1mL of gentamicin antibiotic mother liquor and 1mL of ampicillin antibiotic mother liquor are sequentially dissolved in 96mL of MCM medium, so that the streptomycin concentration is 600 mu g/mL, the kanamycin concentration is 600 mu g/mL, the gentamicin concentration is 600 mu g/mL and the ampicillin concentration is 600 mu g/mL.
Further, the preparation method of the solid culture medium specifically comprises the following steps:
5g of peptone, 1g of yeast extract and 9g of sodium chloride are weighed and dissolved in 1000mL of distilled water in sequence, so that the concentration of the peptone is 5g/L, the concentration of the yeast extract is 1g/L, the concentration of the sodium chloride is 9g/L, the pH is adjusted to 7.2 by using a pH meter, and then 20g of agar is weighed and dissolved in the secondary solution, so that the concentration of the agar is 20 g/L.
Further, the haematococcus pluvialis is purified by jointly using antibiotics, the temperature is 20 ℃, and the illumination intensity is 60 mu mol m in an illumination incubator-2·s-1The light-dark period is 14 h: 10h and the treatment time is 72 h.
Furthermore, the rotating speed of the shaking table is 180r/min, and the time is 72 h.
Further, the combined antibiotic application method is used for purifying haematococcus pluvialis algae.
The combined antibiotic application method is simple to operate and has obvious effect; the combined antibiotic is applied in the MCM culture medium, the growth rate and biomass accumulation of the cultured algae are high, the pure Haematococcus pluvialis is particularly beneficial to obtaining, and a large amount of experimental materials are provided for related scientific research.
Detailed Description
The method of administration of the combination antibiotic of the present invention and the pure species of Haematococcus pluvialis will be described in detail with reference to the following examples:
example 1:
an MCM medium formulation suitable for use in the administration of a combination antibiotic and the cultivation of pure haematococcus pluvialis comprising the steps of:
(1) preparing a solution A mother solution: weighing 50g of potassium nitrate, and dissolving in a small amount of solvent to completely dissolve the potassium nitrate; weighing 2g of potassium dihydrogen phosphate, and dissolving in a small amount of solvent to completely dissolve the potassium dihydrogen phosphate; weighing 10g of magnesium sulfate heptahydrate, and dissolving in a small amount of solvent to completely dissolve the magnesium sulfate heptahydrate; mixing the dissolved potassium nitrate solution, the potassium dihydrogen phosphate solution and the magnesium sulfate heptahydrate solution together, and preparing a solution A mother liquor by using a solvent to fix the volume to 1L, wherein the concentration of potassium nitrate is 50g/L, the concentration of potassium dihydrogen phosphate is 2g/L, and the concentration of magnesium sulfate heptahydrate is 10 g/L;
(2) preparing a solution B mother solution: weighing 8g of calcium chloride hexahydrate, dissolving the calcium chloride hexahydrate in a small amount of solvent to completely dissolve the calcium chloride hexahydrate, and preparing a B liquid mother liquor by using the solvent to fix the volume to 1L to ensure that the concentration of the calcium chloride hexahydrate is 8 g/L;
(3) preparing a solution C mother solution: weighing 3.72g of disodium ethylene diamine tetraacetate, and dissolving in a small amount of solvent to completely dissolve the disodium ethylene diamine tetraacetate; weighing 4.17g of iron sulfate heptahydrate, and dissolving in a small amount of solvent to completely dissolve the iron sulfate heptahydrate; mixing the dissolved disodium ethylene diamine tetraacetate solution and the heptahydrate ferric sulfate solution together, and preparing a C solution mother solution by using a solvent to fix the volume to 1L, wherein the concentration of the disodium ethylene diamine tetraacetate is 3.72g/L, and the concentration of the heptahydrate ferric sulfate is 4.17 g/L;
(4) preparing solution D mother liquor: weighing 71.89mg of zinc sulfate, and dissolving in a small amount of solvent to completely dissolve the zinc sulfate; weighing 12.37mg of phosphoric acid, and dissolving in a small amount of solvent to completely dissolve; weighing 4.76mg/L g cobalt chloride dihydrate to dissolve in a small amount of solvent completely; weighing 62.42mg/L copper sulfate pentahydrate, and dissolving in a small amount of solvent to completely dissolve; weighing 84.51mg of manganese sulfate monohydrate, and dissolving in a small amount of solvent to completely dissolve the manganese sulfate monohydrate; weighing 7.26mg/L sodium molybdate dihydrate to be dissolved in a small amount of solvent to be completely dissolved; mixing the dissolved zinc sulfate solution, the phosphoric acid solution, the cobalt chloride dihydrate solution, the copper sulfate pentahydrate solution, the manganese sulfate monohydrate solution and the sodium molybdate dihydrate solution together, and fixing the volume to 1L by using a solvent to prepare a D solution mother solution, wherein the concentration of zinc sulfate is 71.89mg/L, the concentration of phosphoric acid is 12.37mg/L, the concentration of cobalt chloride dihydrate is 4.76mg/L, the concentration of copper sulfate pentahydrate is 62.42mg/L, the concentration of manganese sulfate monohydrate is 84.51mg/L, and the concentration of sodium molybdate dihydrate is 7.26 mg/L;
(5) when the potassium nitrate-containing aqueous solution is used, 10mL of A solution mother liquor, 10mL of B solution mother liquor, 100 mu L C solution mother liquor and 100 mu L D solution mother liquor are sequentially dissolved in 900mL of solvent, so that the concentration of potassium nitrate in the A solution is 500mg/L, the concentration of dipotassium hydrogen phosphate is 20mg/L, the concentration of magnesium sulfate heptahydrate is 100mg/L, the concentration of calcium chloride hexahydrate in the B solution is 80mg/L, the concentration of disodium ethylene diamine tetraacetate in the C solution is 372 mu g/L, the concentration of ferric sulfate heptahydrate is 417 mu g/L, the concentration of zinc sulfate in the D solution is 7.189 mu g/L, the concentration of phosphoric acid is 1.237 mu g/L, the concentration of cobalt sulfate dihydrate is 0.476 mu g/L, the concentration of copper sulfate pentahydrate is 6.242 mu g/L, the concentration of manganese sulfate monohydrate is 8.451 mu g/L, and the concentration of sodium molybdate dihydrate is 0.726 mu g/L; adjusting pH to 6.9-7.1 with pH meter, and inoculating Haematococcus pluvialis strain to be cultured.
Table 1: the MCM culture medium formula of the invention contains a Chinese name and chemical formula comparison table:
MCM culture medium formula
MCM medium composition
Example 2: the preparation and application method of the combined antibiotic mother liquor comprises the following steps:
(1) preparing 60g/L of antibiotic mother liquor: weighing 6g of streptomycin, dissolving in 100mL of ultrapure water, and completely dissolving; weighing 6g of kanamycin, and dissolving the kanamycin in 100mL of ultrapure water to completely dissolve the kanamycin; weighing 6g of gentamicin, and dissolving in 100mL of ultrapure water to completely dissolve the gentamicin; weighing 6g of ampicillin, and dissolving the ampicillin in 100mL of ultrapure water to completely dissolve the ampicillin; the streptomycin concentration is 60g/L, the kanamycin concentration is 60g/L, the gentamicin concentration is 60g/L, and the ampicillin concentration is 60 g/L; degerming by adopting a suction filtration mode;
(2) when in use, 1mL of streptomycin antibiotic mother liquor, 1mL of kanamycin antibiotic mother liquor, 1mL of gentamicin antibiotic mother liquor and 1mL of ampicillin antibiotic mother liquor are sequentially dissolved in 96mL of MCM medium, so that the streptomycin concentration is 600 mu g/mL, the kanamycin concentration is 600 mu g/mL, the gentamicin concentration is 600 mu g/mL and the ampicillin concentration is 600 mu g/mL.
Example 3: the invention relates to a method for obtaining pure haematococcus pluvialis strains, which comprises the following steps:
(1) preparing: 6 100mL autoclaved transparent glass conical flasks, wherein 95mL of the MCM culture medium is added into the transparent glass conical flasks, and the 1mL of streptomycin antibiotic mother liquor, 1mL of kanamycin antibiotic mother liquor, 1mL of gentamicin antibiotic mother liquor and 1mL of ampicillin antibiotic mother liquor are sequentially added and uniformly mixed;
(2) inoculation: 1ml of haematococcus pluvialis solution is respectively inoculated into each conical flask on the 1 st day, and the algae solution contains algae seeds in the green vegetative growth stage of the haematococcus pluvialis;
(3) culturing: placing 6 conical bottles on a shaking table, wherein the rotation speed of the shaking table is 180 r/min; placing shaking table in light for culturingCulturing in a box with daylight as light source at 20 deg.C and dark period of 14:10 and light intensity of 60 μmol · m-2·s-1；
(4) Preparing a solid culture medium: weighing 5g of peptone, 1g of yeast extract and 9g of sodium chloride, sequentially dissolving the peptone, the yeast extract and the sodium chloride in 1000mL of distilled water to ensure that the concentration of the peptone is 5g/L, the concentration of the yeast extract is 1g/L and the concentration of the sodium chloride is 9g/L, adjusting the pH to 7.2 by using a pH meter, weighing 20g of agar, and dissolving the agar in the secondary solution to ensure that the concentration of the agar is 20 g/L;
(5) and (3) bacteria inspection, wherein after 3 days of culture, the sterilization effect is detected on a solid culture medium by adopting a plate marking method, and pure haematococcus pluvialis colonies are finally obtained.
Claims (7)
1. A combined antibiotic application method suitable for pure culture of haematococcus pluvialis is characterized by comprising the following steps: culturing Haematococcus pluvialis with MCM culture medium, performing shake culture treatment on Haematococcus pluvialis liquid in an illumination incubator for 3d by jointly using 4 antibiotics with the concentrations of streptomycin, kanamycin, gentamicin and ampicillin respectively of 600 mu g/ml, and purifying by combining with a solid culture medium to obtain pure Haematococcus pluvialis.
2. The method as claimed in claim 1, wherein the MCM medium is formulated by the method comprising the following steps:
(1) preparing a solution A mother solution: weighing 50g of potassium nitrate, and dissolving in a small amount of solvent to completely dissolve the potassium nitrate; weighing 2g of potassium dihydrogen phosphate, and dissolving in a small amount of solvent to completely dissolve the potassium dihydrogen phosphate; weighing 10g of magnesium sulfate heptahydrate, and dissolving in a small amount of solvent to completely dissolve the magnesium sulfate heptahydrate; mixing the dissolved potassium nitrate solution, the potassium dihydrogen phosphate solution and the magnesium sulfate heptahydrate solution together, and preparing a solution A mother liquor by using a solvent to fix the volume to 1L, wherein the concentration of potassium nitrate is 50g/L, the concentration of potassium dihydrogen phosphate is 2g/L, and the concentration of magnesium sulfate heptahydrate is 10 g/L;
(2) preparing a solution B mother solution: weighing 8g of calcium chloride hexahydrate, dissolving the calcium chloride hexahydrate in a small amount of solvent to completely dissolve the calcium chloride hexahydrate, and preparing a B liquid mother liquor by using the solvent to fix the volume to 1L to ensure that the concentration of the calcium chloride hexahydrate is 8 g/L;
(3) preparing a solution C mother solution: weighing 3.72g of disodium ethylene diamine tetraacetate, and dissolving in a small amount of solvent to completely dissolve the disodium ethylene diamine tetraacetate; weighing 4.17g of iron sulfate heptahydrate, and dissolving in a small amount of solvent to completely dissolve the iron sulfate heptahydrate; mixing the dissolved disodium ethylene diamine tetraacetate solution and the heptahydrate ferric sulfate solution together, and preparing a C solution mother solution by using a solvent to fix the volume to 1L, wherein the concentration of the disodium ethylene diamine tetraacetate is 3.72g/L, and the concentration of the heptahydrate ferric sulfate is 4.17 g/L;
(4) preparing solution D mother liquor: weighing 71.89mg of zinc sulfate, and dissolving in a small amount of solvent to completely dissolve the zinc sulfate; weighing 12.37mg of phosphoric acid, and dissolving in a small amount of solvent to completely dissolve; weighing 4.76mg/L g cobalt chloride dihydrate to dissolve in a small amount of solvent completely; weighing 62.42mg/L copper sulfate pentahydrate, and dissolving in a small amount of solvent to completely dissolve; weighing 84.51mg of manganese sulfate monohydrate, and dissolving in a small amount of solvent to completely dissolve the manganese sulfate monohydrate; weighing 7.26mg/L sodium molybdate dihydrate to be dissolved in a small amount of solvent to be completely dissolved; mixing the dissolved zinc sulfate solution, the phosphoric acid solution, the cobalt chloride dihydrate solution, the copper sulfate pentahydrate solution, the manganese sulfate monohydrate solution and the sodium molybdate dihydrate solution together, and fixing the volume to 1L by using a solvent to prepare a D solution mother solution, wherein the concentration of zinc sulfate is 71.89mg/L, the concentration of phosphoric acid is 12.37mg/L, the concentration of cobalt chloride dihydrate is 4.76mg/L, the concentration of copper sulfate pentahydrate is 62.42mg/L, the concentration of manganese sulfate monohydrate is 84.51mg/L, and the concentration of sodium molybdate dihydrate is 7.26 mg/L;
(5) when in use, 10mL of A solution mother liquor, 10mL of B solution mother liquor, 100 mu L C solution mother liquor and 100 mu L D solution mother liquor are sequentially dissolved in 900mL of solvent, so that the concentration of potassium nitrate in the A solution is 500mg/L, the concentration of dipotassium hydrogen phosphate is 20mg/L, the concentration of magnesium sulfate heptahydrate is 100mg/L, the concentration of calcium chloride hexahydrate in the B solution is 80mg/L, the concentration of disodium ethylene diamine tetraacetate in the C solution is 372 mu g/L, the concentration of ferric sulfate heptahydrate is 417 mu g/L, the concentration of zinc sulfate in the D solution is 7.189 mu g/L, the concentration of phosphoric acid is 1.237 mu g/L, the concentration of cobalt chloride dihydrate is 0.476 mu g/L, the concentration of copper sulfate pentahydrate is 6.242 mu g/L, the concentration of manganese sulfate monohydrate is 8.451 mu g/L, and the concentration of sodium molybdate dihydrate is 0.726 mu g/L; adjusting pH to 6.9-7.1 with pH meter, and inoculating Haematococcus pluvialis strain to be cultured.
3. The method as claimed in claim 1, wherein the preparation method of 4 antibiotics with the concentration of 600 μ g/ml streptomycin, kanamycin, gentamicin and ampicillin comprises the following steps: preparing 60g/L of antibiotic mother liquor: weighing 6g of streptomycin, dissolving in 100mL of ultrapure water, and completely dissolving; weighing 6g of kanamycin, and dissolving the kanamycin in 100mL of ultrapure water to completely dissolve the kanamycin; weighing 6g of gentamicin, and dissolving in 100mL of ultrapure water to completely dissolve the gentamicin; weighing 6g of ampicillin, and dissolving the ampicillin in 100mL of ultrapure water to completely dissolve the ampicillin; the streptomycin concentration is 60g/L, the kanamycin concentration is 60g/L, the gentamicin concentration is 60g/L, and the ampicillin concentration is 60 g/L; degerming by adopting a suction filtration mode; when in use, 1mL of streptomycin antibiotic mother liquor, 1mL of kanamycin antibiotic mother liquor, 1mL of gentamicin antibiotic mother liquor and 1mL of ampicillin antibiotic mother liquor are sequentially dissolved in 96mL of MCM medium, so that the streptomycin concentration is 600 mu g/mL, the kanamycin concentration is 600 mu g/mL, the gentamicin concentration is 600 mu g/mL and the ampicillin concentration is 600 mu g/mL.
4. The method according to claim 1, wherein the method for preparing the solid culture comprises the following steps: 5g of peptone, 1g of yeast extract and 9g of sodium chloride are weighed and dissolved in 1000mL of distilled water in sequence, so that the concentration of the peptone is 5g/L, the concentration of the yeast extract is 1g/L, the concentration of the sodium chloride is 9g/L, the pH is adjusted to 7.2 by using a pH meter, and then 20g of agar is weighed and dissolved in the secondary solution, so that the concentration of the agar is 20 g/L.
5. The method of claim 1, wherein the haematococcus pluvialis is purified using an antibiotic in combination in a light incubator at a temperature of 20 ℃ and a light intensity of 60 μmol m-2·s-1The light-dark period is 14 h: 10h and the treatment time is 72 h.
6. The method of claim 1, wherein the shaker is rotated at a speed of 180r/min for a period of 72 hours.
7. A method of combined antibiotic administration according to claims 1 to 6, wherein the method of combined antibiotic administration is used for Haematococcus pluvialis species purification.
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