CN108865894A - A kind of preparation method of sterile micro- quasi- ball algae - Google Patents
A kind of preparation method of sterile micro- quasi- ball algae Download PDFInfo
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- CN108865894A CN108865894A CN201811023262.4A CN201811023262A CN108865894A CN 108865894 A CN108865894 A CN 108865894A CN 201811023262 A CN201811023262 A CN 201811023262A CN 108865894 A CN108865894 A CN 108865894A
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- ball algae
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- 241000195493 Cryptophyta Species 0.000 title claims abstract description 128
- 238000002360 preparation method Methods 0.000 title claims abstract description 25
- 230000003115 biocidal effect Effects 0.000 claims abstract description 85
- 241000894006 Bacteria Species 0.000 claims abstract description 33
- 238000000034 method Methods 0.000 claims abstract description 29
- 229960005091 chloramphenicol Drugs 0.000 claims abstract description 25
- 239000004098 Tetracycline Substances 0.000 claims abstract description 24
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 claims abstract description 24
- 235000019364 tetracycline Nutrition 0.000 claims abstract description 24
- 150000003522 tetracyclines Chemical class 0.000 claims abstract description 24
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- 229930101283 tetracycline Natural products 0.000 claims abstract description 23
- 229960000479 ceftriaxone sodium Drugs 0.000 claims abstract description 22
- FDRNWTJTHBSPMW-GNXCPKRQSA-L disodium;(6r,7r)-7-[[(2e)-2-(2-amino-1,3-thiazol-4-yl)-2-methoxyiminoacetyl]amino]-3-[(2-methyl-6-oxido-5-oxo-1,2,4-triazin-3-yl)sulfanylmethyl]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate Chemical compound [Na+].[Na+].S([C@@H]1[C@@H](C(N1C=1C([O-])=O)=O)NC(=O)/C(=N/OC)C=2N=C(N)SC=2)CC=1CSC1=NC(=O)C([O-])=NN1C FDRNWTJTHBSPMW-GNXCPKRQSA-L 0.000 claims abstract description 22
- 230000012010 growth Effects 0.000 claims abstract description 18
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- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Inorganic materials [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 description 2
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- ULGZDMOVFRHVEP-RWJQBGPGSA-N Erythromycin Natural products O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)C(=O)[C@H](C)C[C@@](C)(O)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 ULGZDMOVFRHVEP-RWJQBGPGSA-N 0.000 description 1
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- 235000016496 Panda oleosa Nutrition 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 108010093965 Polymyxin B Proteins 0.000 description 1
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- 229960004261 cefotaxime Drugs 0.000 description 1
- 229960002588 cefradine Drugs 0.000 description 1
- 229940124587 cephalosporin Drugs 0.000 description 1
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- 230000001684 chronic effect Effects 0.000 description 1
- 229960003405 ciprofloxacin Drugs 0.000 description 1
- 229960002227 clindamycin Drugs 0.000 description 1
- KDLRVYVGXIQJDK-AWPVFWJPSA-N clindamycin Chemical compound CN1C[C@H](CCC)C[C@H]1C(=O)N[C@H]([C@H](C)Cl)[C@@H]1[C@H](O)[C@H](O)[C@@H](O)[C@@H](SC)O1 KDLRVYVGXIQJDK-AWPVFWJPSA-N 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
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- 238000010790 dilution Methods 0.000 description 1
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- 229960003722 doxycycline Drugs 0.000 description 1
- 229960003276 erythromycin Drugs 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 229940032958 ferric phosphate Drugs 0.000 description 1
- 230000001408 fungistatic effect Effects 0.000 description 1
- 229960001625 furazolidone Drugs 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- WBJZTOZJJYAKHQ-UHFFFAOYSA-K iron(3+) phosphate Chemical compound [Fe+3].[O-]P([O-])([O-])=O WBJZTOZJJYAKHQ-UHFFFAOYSA-K 0.000 description 1
- 229910000399 iron(III) phosphate Inorganic materials 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 229960004023 minocycline Drugs 0.000 description 1
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- 239000011574 phosphorus Substances 0.000 description 1
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- 230000001766 physiological effect Effects 0.000 description 1
- 229920000024 polymyxin B Polymers 0.000 description 1
- 229960005266 polymyxin b Drugs 0.000 description 1
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- 230000002441 reversible effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000010865 sewage Substances 0.000 description 1
- 229940082787 spirulina Drugs 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 1
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 1
- 229960003165 vancomycin Drugs 0.000 description 1
- MYPYJXKWCTUITO-LYRMYLQWSA-N vancomycin Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C2C=C3C=C1OC1=CC=C(C=C1Cl)[C@@H](O)[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@H]3C(=O)N[C@H]1C(=O)N[C@H](C(N[C@@H](C3=CC(O)=CC(O)=C3C=3C(O)=CC=C1C=3)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)O2)=O)NC(=O)[C@@H](CC(C)C)NC)[C@H]1C[C@](C)(N)[C@H](O)[C@H](C)O1 MYPYJXKWCTUITO-LYRMYLQWSA-N 0.000 description 1
- MYPYJXKWCTUITO-UHFFFAOYSA-N vancomycin Natural products O1C(C(=C2)Cl)=CC=C2C(O)C(C(NC(C2=CC(O)=CC(O)=C2C=2C(O)=CC=C3C=2)C(O)=O)=O)NC(=O)C3NC(=O)C2NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(CC(C)C)NC)C(O)C(C=C3Cl)=CC=C3OC3=CC2=CC1=C3OC1OC(CO)C(O)C(O)C1OC1CC(C)(N)C(O)C(C)O1 MYPYJXKWCTUITO-UHFFFAOYSA-N 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
- 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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- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Botany (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Microbiology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention discloses a kind of preparation method of sterile micro- quasi- ball algae (Nannochloropsis gaditana), based on antibacterial/bactericidal effect of antibiotic, there is Efficient antibacterial/bactericidal activity by screening, and it is small to algae toxicity, it is provided simultaneously with the antibiotic of different antibacterial/bactericidal mechanisms, in a manner of variety classes antibiotic synergism, achieve the purpose that bacterium in the micro- quasi- ball algae (Nannochloropsis gaditana) of removal；The antibiotic and its concentration are respectively the Ceftriaxone Sodium of the chloramphenicol of 25~100 μ g/mL, the tetracycline of 25~150 μ g/mL and 25~400 μ g/mL.This method simple process, convenient for operation, replicability is strong, still keeps good activity using axenic culture prepared by the present invention, can normal growth.
Description
Technical field
The invention belongs to the sterilization preparation technical fields of microalgae, and in particular to micro- using the method removal of antibiotic treatment
The method that bacterium prepares sterile micro- quasi- ball algae in quasi- ball algae algae solution.
Background technique
Microalgae is a kind of important living resources, have be distributed it is wide, full of nutrition, many kinds of, adaptable, can determine
To the features such as culture, it is widely used in fields such as feed, medicine research and development, sewage treatment and chemical industry.Micro- quasi- ball algae is that ocean is micro-
Main Populations in the big eye algae guiding principle of algae, energy high yield unsaturated fatty acid, and grow rapidly, it is that bait common in aquaculture is micro-
Algae.
There are a large amount of different types of miscellaneous bacterias in the routine culture of micro- quasi- ball algae, in algae solution, is deposited between these bacterium and algae
In many unknown and complicated relationships, and this is obviously unable to satisfy the side such as biochemical, molecular genetic and phycomycete relationship to algal physiology
The research in face, therefore sterile pure algae is obtained with particularly important meaning.Currently, the preparation without bacterium algae is still in algology research
A difficult point.Common preparation has coating method of scoring, centrifuge washing technology, irradiation technique, capillary syring without the technology of bacterium algae
Technology and antibiotic method etc., wherein coating method of scoring is the classical way for obtaining pure culture, but this method is not applicable in generally, is had
Researcher combines coating scribing line purifying spirulina using dilution but does not succeed, because the algae has gelatinous sheath, and moves energy
Power is strong；Centrifuge washing technology is exactly to be centrifuged repeatedly, and can eliminate bacterium to a certain extent, but can not for most of algae
A possibility that realizing sterilization, can only increasing pure separation, therefore, in sterilization research, it is sterile that centrifuge washing can be used as other
Change the ancillary measure of research method；The disadvantages of irradiation method is there are cumbersome, heavy workload, success rate is low, narrow application range, and irradiate
Frond physiological property susceptible, be easily mutated, lethal effect easily occur, it is difficult it cannot be guaranteed that screen the algae strain of nature
To obtain ideal result；Capillary syring technology can not usually go the miscellaneous bacteria that may adhere on frustule, capillary syring method
It removes, if the high requirements on the equipment, skilled operation degree is more demanding in conjunction with microtechnic.In contrast, antibiotic method is
A kind of strong operability, no bacterium algae technology of preparing applied widely.Antibiotic has suppression (killing) bacterium effect, pure using antibiotic
Change microalgae, usually in addition to considering to the toxicity of bacterium, must also consider antibiotic to microalgae form or (and) physiology, biochemical characteristic
It may influence, therefore selecting suitable antibiotic is key, ideal antibiotic should have two features, i.e. suppression (killing) bacterium activity
The characteristics of by force and to algae small toxicity or promotion algae growth.Lin Wei filters out 4 kinds in the sterilization culture of several marine microalgaes and resists
Raw element, i.e. chloramphenicol, penicillin, gentamicin and kanamycins, by experiments have shown that, brown born of the same parents algae only needs 2 kinds in 6 kinds of microalgaes
Sterilization can be realized in antibiotics penicillin and gentamicin, but cultivates 3 and be commissioned to train in nutrient solution and bacterium occur, but pass through addition
Kanamycins, and order of addition is prorocentrum minimum, anterior canal algae after penicillin, gentamicin and kanamycins (3 kinds of antibiotic)
And integral asepsis can be realized in brown 3 kinds of micro algae culturing liquids of born of the same parents algae；It is found by antibiotic mix-incubation experiment, by continuously locating
It manages 2 times, 4 kinds of microalgaes realization sterilizations, in remaining 2 kinds of microalgaes, prorocentrum minimum micro algae growth is heavily suppressed, cultivates 40 days
Death, bacterium exclusion still not yet in effect shares three kinds of antibiotic, not yet by adjusting antibiotic concentration in brown born of the same parents' algae culturing liquid
Eliminate bacterium in brown born of the same parents' algae culturing liquid, so, during algae sterilization Antibiotics, quantity and its concentration and effect when
Between and addition antibiotic method play a crucial role.Therefore, because microalgae type or living environment are different, miscellaneous bacteria
Group is also different, Antibiotics, quantity and its concentration and action time and the method for adding antibiotic, to the nothing to secure good health
Bacterium algae strain becomes difficult point, realizes that sterilization has not predictability to algae.It is sterile there is presently no being prepared using antibiotic method
The patent report of micro- quasi- ball algae.
Summary of the invention
In view of the above-mentioned problems, the object of the present invention is to provide a kind of sterilization cultural method of micro- quasi- ball algae, present invention choosing
Micro- quasi- ball algae (Nannochloropsis gaditana) is isolated from Qingdao brilliance ocean group breeding wastewater
, being preserved in China General Microbiological preservation administrative center on August 1st, 2018, (abbreviation CGMCC, address are court of Beijing
No. 3 Institute of Microorganism, Academia Sinica of institute of positive area's North Star West Road 1), deposit number is CGMCC No.16188.Needle of the present invention
To the growth characteristic of micro- quasi- ball algae, addition manner and the effect of suitable Antibiotics and its concentration and antibiont are provided
Time accurately studies micro- quasi- ball algae for the later period and lays the foundation for the sterile micro- quasi- ball algae providing method to secure good health.
The purpose of the present invention is achieved through the following technical solutions：
The present invention provides the antibiotic for preparing sterile micro- quasi- ball algae, and the antibiotic and its concentration are respectively 25~100 μ
The Ceftriaxone Sodium of the chloramphenicol of g/mL, the tetracycline of 25~150 μ g/mL and 25~400 μ g/mL.
Preferably, the antibiotic and its concentration are respectively：The chloramphenicol of 100 μ g/mL, 150 μ g/mL tetracycline and
The Ceftriaxone Sodium of 400 μ g/mL.
The present invention provides above-mentioned antibiotic to prepare the application in sterile micro- quasi- ball algae.
The present invention provides a kind of preparation method of sterile micro- quasi- ball algae, the specific steps are：
(1) culture of micro- quasi- ball algae
Micro- quasi- ball algae is forwarded to again in fresh f/2 culture medium and is cultivated, algae solution and culture solution are with 1~2:10~12 ratios
Carry out transferred species activation；
(2) sterile micro- quasi- ball algae preparation
Antibiotic treatment is sequentially added in batches into the activated micro- quasi- ball algae algae solution of above-mentioned steps (1)：Chloramphenicol (25~
100 μ g/mL) 1~7d of processing, 10,000rpm are centrifuged 10min, collect frond, wash 3~4 with the fresh f/2 culture medium of sterilizing
Time, it is transferred in fresh f/2 culture medium and cultivates 1d；
Tetracycline (25~150 μ g/mL) handles 3~7d, and 10,000rpm centrifugation 10min collect frond, new with sterilizing
Fresh f/2 culture medium washs 3~4 times, is transferred in fresh f/2 culture medium and cultivates 1d；
Ceftriaxone Sodium (25~400 μ g/mL) handles 3~7d, and 10,000rpm centrifugation 10min collect frond, with sterilizing
Fresh f/2 culture medium wash 3~4 times, be transferred in fresh f/2 culture medium and cultivate；
(3) bacteria-eliminating efficacy is examined
After taking step (2) antibiotic treatment, 0.2~0.5mL of algae solution coating in one week is grown in antibiotic-free culture medium
In 2216E solid medium, 3d is cultivated in 28 DEG C of biochemical cultivation cases, whether detect in algae solution has bacterium；
After taking step (2) antibiotic treatment, one week 1~1.5mL of algae solution is grown in antibiotic-free culture medium, is added
SYBR fluorescent dye is protected from light dyeing 15min, fluorescence microscope bacteria-eliminating efficacy after film-making；
After taking step (2) antibiotic treatment, one week 10~15mL of algae solution is grown in antibiotic-free culture medium and is carried out
PCR verifies PCR product with 1% agarose gel electrophoresis, and whether detect in algae solution has bacterium；
(4) micro- quasi- ball algae growth activity detection after antibiotic treatment
It after the completion of antibiotic treatment, periodically samples, with the fluorescence intensity of microplate reader measurement algae solution, whether can with detecting algae
Normal growth.
Preferably, the antibiotic and its concentration are respectively：The chloramphenicol of 100 μ g/mL, 150 μ g/mL tetracycline and
The Ceftriaxone Sodium of 400 μ g/mL.
Preferably, it is 24 ± 1 (DEG C), intensity of illumination 3000lux, Light To Dark Ratio that step (1) described condition of culture, which is temperature,
For 12L：12D, pH are 6.5~7.0.
Preferably, the antibiotic and its concentration described in (2) are respectively：The chloramphenicol of 100 μ g/mL, 150 μ g/mL
The Ceftriaxone Sodium of tetracycline and 400 μ g/mL.
Preferably, f/2 nutrient media components and its process for preparation described in step (1) and (2) are as follows：
A：NaNO375mg/L, NaH2PO4·H2O 5mg/L, Na2SiO3·9H2O 30mg/L；
B：F/2 microelement：Na2EDTA 4.36g/L, FeCl3·6H2O 3.16g/L, CuSO4·5H2O 0.01g/L,
ZnSO4·7H2O 0.023g/L, CoCl2·6H2O 0.012g/L, MnCl4H2O 0.18g/L, Na2MoO4·2H2O
0.07g/L；
C：F/2 vitamin solution：Vitamin B121.0mg/L, biotin 1.0mg/L, vitamin B1200.0mg/L, 0.2
μm filtering, it is spare；
Process for preparation：(1) in 121 DEG C after distributing above-mentioned A and B each group, sterilize 20min, spare；
(2) nature seawater 1L is taken, 121 DEG C after 0.2 μm of filtering, sterilize 20min；By A, B, component C point after seawater is cooling
It Jia Ru not be to get f/2 culture medium.
Preferably, 2216E solid medium process for preparation described in step (3) is as follows：Protein 5g, yeast extract 1g, phosphorus
Sour high-speed rail 0.001g, 0.2 μm of filtering Chen Haishui 1000mL adjust pH 7.6~7.8；
Then 1.5% agar is added based on the 20min that sterilizes under the conditions of 121 DEG C to get solid medium in above-mentioned culture.
Preferably, in step (3), the preparation method of the template of PCR is：By 10mL algae solution 12,000rpm is centrifuged 10min,
Supernatant is removed, 200 μ L TE buffers are added, after boiling water bath 10min after mixing, -20 DEG C of placements 30min, room temperature thawing, 12,
000rpm is centrifuged 10min and collects supernatant, using obtained supernatant as template.
Preferably, in step (3), the primer of PCR is bacterial 16 S rDNA universal primer, forward primer SEQ
IDNO.1, reverse primer are SEQ IDNO.2.
Preferably, in step (3), the 30 μ L reaction systems of PCR include：3 μ L 10 × Ex Taq Buffer, 2 μ L
2.5mMdNTPs, each each 1 μ L, 10 μM of primers of primer, 0.3 μ L 5U/ μ L Ex Taq, 1.5 μ L templates, 21.2 μ L ddH2O。
Preferably, in step (3), PCR amplification program is：
Preferably, fluorescence detection condition is in step (4)：Excitation wavelength 440nm, launch wavelength 690nm.
Screening and optimizing of the present invention goes out above-mentioned three kinds of antibiotic and its concentration, and wherein chloramphenicol, tetracycline are quick-acting bacteriostatic agents
(antimicrobial mechanism different), Ceftriaxone Sodium belong to multiplication stage bactericide, the present invention and by successively adding said medicine, i.e., first make
Inhibit most of bacterial growth with quick-acting bacteriostatic agents, fungicide is then selected to kill the bacterium of remaining, discovery has reached good
Bacteria-eliminating efficacy.In addition, antibiotic in conjunction with certain ingredients of sterilization object ball algae and can change its Physiology and biochemistry ingredient, thus
The growth of micro- quasi- ball algae is directly affected, or since antibiotic can inhibit nocuousness or be conducive to the fungal component of micro- quasi- ball algae growth
And the growth conditions of micro- quasi- ball algae are affected indirectly, from examples it can be seen that the micro- quasi- ball algae obtained is extensive through a period of time
It can normal growth after multiple.So screening and optimizing of the present invention Antibiotics and its concentration and antibiont addition manner and
Action time is best to sterile micro- quasi- ball algae effect is obtained.
Beneficial effects of the present invention
1, using sterile micro- quasi- ball algae preparation method of the invention, simple process, convenient for operation, repeatability is strong, is obtained
The micro- quasi- ball algae obtained can normal growth after restoring for a period of time.
2, there is no varied bacteria growing in micro- quasi- ball algae culture medium that the present invention obtains, study micro- quasi- ball algae for the later period and establish base
Plinth.
Detailed description of the invention
Fig. 1：Influence of the various concentration chloramphenicol to micro- quasi- ball algae.
Fig. 2：Influence of the various concentration tetracycline to micro- quasi- ball algae.
Fig. 3：Influence of the Ceftriaxone solutions of different concentration to micro- quasi- ball algae.
Fig. 4：Flat band method detects antibiotic bacteria-eliminating efficacy.
Fig. 5：Fluorescence microscope detects antibiotic bacteria-eliminating efficacy.
Fig. 6：16S PCR detects antibiotic bacteria-eliminating efficacy, A. blank control；B. antibiotic treatment group is not used；C. implement
Example 5 uses antibiotic group；D. embodiment 6 uses antibiotic group.
Fig. 7：Micro- quasi- ball algae Activity determination after antibiotic treatment.
Specific embodiment
Feature of present invention and other correlated characteristics are described in further detail by the following examples, in order to the same industry
The understanding of technical staff：
Drug sensitive test paper in the present invention is purchased from Hangzhou microorganism reagent Co., Ltd.
Used medium of the present invention and its be formulated as follows：
F/2 nutrient media components and its process for preparation of the present invention are as follows：
A：NaNO375mg/L, NaH2PO4·H2O 5mg/L, Na2SiO3·9H2O 30mg/L；
B：F/2 microelement：Na2EDTA 4.36g/L, FeCl3·6H2O 3.16g/L, CuSO4·5H2O 0.01g/L,
ZnSO4·7H2O 0.023g/L, CoCl2·6H2O 0.012g/L, MnCl4H2O 0.18g/L, Na2MoO4·2H2O
0.07g/L；
C：F/2 vitamin solution：Vitamin B121.0mg/L, biotin 1.0mg/L, vitamin B1200.0mg/L, 0.2
μm filtering, it is spare；
Process for preparation：(1) in 121 DEG C after distributing above-mentioned A and B each group, sterilize 20min, spare；
(2) nature seawater 1L is taken, 121 DEG C after 0.2 μm of filtering, sterilize 20min；A, B, component C are added after seawater is cooling
Enter.
2216E solid medium process for preparation of the present invention is as follows：Protein 5g, yeast extract 1g, high ferric phosphate
0.001g, 0.2 μm of filtering Chen Haishui 1000mL adjust pH 7.6~7.8；
Then 1.5% agar is added based on the 20min that sterilizes under the conditions of 121 DEG C to get solid medium in above-mentioned culture.
The culture of the micro- quasi- ball algae of embodiment 1
The micro- quasi- ball algae (Nannochloropsis gaditana) being separated to is forwarded to fresh f/2 culture medium again
In, algae solution and culture solution are with 1:10 ratios carry out transferred species activation.Cultivation temperature be 24 ± 1 (DEG C), intensity of illumination 3000lux,
Light To Dark Ratio is 12L：12D, pH are 6.5~7.0.
The sterile micro- quasi- ball algae preparation of embodiment 2
1, the screening of Antibiotics
The algae solution (3d is cultivated in fresh culture) for taking 1ml to activate is added in the 2216E culture medium of 30ml sterilizing, in 28
Shaken cultivation 18h in DEG C biochemical cultivation case, to be enriched with the bacterium in algae solution.0.2mL bacterium solution is taken to be coated on 2216E solid culture
Base, the drug sensitive test paper respectively containing variety classes antibiotic is smooth in the planar surface, it is cultivated in 28 DEG C of biochemical cultivation cases
3d detects antibacterial circle diameter.It selects antibacterial circle diameter >=20mm antibiotic for more sensitive type, is selected as degerming drug.Such as table 1
Shown, the present invention filters out 4 kinds of degerming drugs, chloramphenicol, tetracycline, Ceftriaxone Sodium and cefuroxime.
Inhibiting effect of the different antibiotic of table 1 to bacterium in algae solution
| Antibiotic | Inhibition zone (has "+", no "-") | Antibiotic | Inhibition zone (has "+", no "-") |
| AMP | - | Ofloxacin | + |
| Kana | - | Norfloxacin | - |
| Stre | Polymyxin B | - | |
| Chloramphenicol | ++ | Doxycycline | + |
| Penicillin | - | Cefradine | - |
| Gentamicin | - | Ciprofloxacin | - |
| Erythromycin | - | Cefuroxime | ++ |
| Tetracycline | ++ | Vancomycin | - |
| Furazolidone | - | Minocycline | - |
| Cefotaxime | + | Cefazolin | + |
| Clindamycin | - | Oxacillin | - |
| Ceftriaxone | ++ |
* it infuses："-" refers to no fungistatic effect；"+" refers to antibacterial circle diameter < 20mm；" ++ " refers to antibacterial circle diameter >=20mm.
Antibiotic can be divided into four major class by its interaction property：1. multiplication stage bactericide, 2. bactericide for rest period, 3. is quick-acting
Antibacterial class agent, 4. chronic antibacterial class agent；Chloramphenicol, tetracycline are quick-acting bacteriostatic agents (antimicrobial mechanisms different), Ceftriaxone Sodium and
Cefuroxime belongs to multiplication stage bactericide；Drug sensitive test discovery, the bactericidal effect of ceftriaxone is more more significant than cefuroxime, and two
Person belongs to cephalosporins, and bactericidal mechanism is identical, therefore selects Ceftriaxone Sodium.So the present invention is by above-mentioned
4 kinds of degerming drugs of screening, preferably chloramphenicol, tetracycline, Ceftriaxone Sodium out.
2, the screening of antibiotic concentration
It is separately added into various concentration antibiotic treatment into activated algae solution, periodically samples, measures algae solution with microplate reader
Fluorescence intensity, to detect algae whether can normal growth.Determine suitable antibiotic concentration.
(such as Fig. 1,2 and 3) as the result is shown, micro- quasi- ball algae growth is inhibited by different degrees of after antibiotic is added, micro-
Intend ball algae is respectively to the tolerable concentration range of three kinds of antibiotic：Chloramphenicol (0~100 μ g/mL), tetracycline (0~150 μ g/
ML), Ceftriaxone Sodium (0~400 μ g/mL).More than maximal tolerable concentration, micro- quasi- ball algae will receive irreversible damage, at one section
Between after it is dead；Antibiotic concentration is too low, and bacteria-eliminating efficacy is not achieved.
3, the preparation of sterile micro- quasi- ball algae
Antibiotic treatment is added portionwise into activated algae solution：Chloramphenicol (25~100 μ g/mL) handle 1~7d, 10,
000rpm is centrifuged 10min, collects frond, is washed 3 times with the fresh f/2 culture medium of sterilizing, be transferred to the fresh f/2 culture medium of 30ml
Middle culture 1d；
Tetracycline (25~150 μ g/mL) handles 3~7d, and 10,000rpm centrifugation 10min collect frond, new with sterilizing
Fresh f/2 culture medium washs 3 times, is transferred in the fresh f/2 culture medium of 30ml and cultivates 1d；
Ceftriaxone Sodium (25~400 μ g/mL) handles 3~7d, and 10,000rpm centrifugation 10min collect frond, with sterilizing
Fresh f/2 culture medium wash 3 times, be transferred in the fresh f/2 culture medium of 30ml and cultivate.
3 bacteria-eliminating efficacy of embodiment is examined
Detection method 1：After taking antibiotic treatment, one week algae solution 0.2mL is grown in antibiotic-free culture medium and is coated on
2216E solid medium cultivates 3d in 28 DEG C of biochemical cultivation cases, and whether detect in algae solution has bacterium；
Detection method 2：After taking antibiotic treatment, one week algae solution 1mL is grown in antibiotic-free culture medium, and SYBR is added
Fluorescent dye is protected from light dyeing 15min, fluorescence microscope bacteria-eliminating efficacy after film-making；
Detection method 3：After taking antibiotic treatment, one week algae solution 10mL is grown in antibiotic-free culture medium, 12,
000rpm is centrifuged 10min, removes supernatant, and 200 μ L TE buffers, boiling water bath 10min after mixing, -20 DEG C of placements are added
After 30min, room temperature is melted, and 12,000rpm centrifugation 10min collect supernatant.Using obtained supernatant as template, 16S universal primer
(27F:AGAGTT TGATCMTGGCTCAG/1492R:GGTTACCTTGTTACGACTT) it is primer, expands bacterial 16 S rDNA
Sequence.30 μ L reaction system of PCR includes：3 μ L 10 × Ex Taq Buffer, 2 μ L 2.5mM dNTPs, each each 1 μ L of primer
10 μM of primers, 0.3 μ L 5U/ μ L Ex Taq, 1.5 μ L templates, 21.2 μ L ddH2O.PCR amplification program is：95 DEG C of 5min, 30
A circulation (95 DEG C of 1min, 55 DEG C of 1min, 72 DEG C of 90s), 72 DEG C of extension 6min.It is produced with 1% agarose gel electrophoresis verifying PCR
Whether object, detecting in algae solution has bacterium.
Micro- quasi- ball algae growth activity detection after 4 antibiotic treatment of embodiment
It after the completion of antibiotic treatment, periodically samples, with the fluorescence intensity of microplate reader measurement algae solution, whether can with detecting algae
Normal growth.Fluorescence detection condition is：Excitation wavelength 440nm, launch wavelength 690nm.
Embodiment 5
Antibiotic treatment is added portionwise into activated algae solution：100 μ g/mL of chloramphenicol handles 1d, 10,000rpm centrifugations
10min collects frond, is washed 3 times with the fresh f/2 culture medium of sterilizing, be transferred in the fresh f/2 culture medium of 30ml and cultivate 1d；
150 μ g/mL of tetracycline handles 3d, and 10,000rpm centrifugation 10min collect frond, cultivated with the fresh f/2 of sterilizing
Base washs 3 times, is transferred in the fresh f/2 culture medium of 30ml and cultivates 1d；
400 μ g/mL of Ceftriaxone Sodium handles 7d, and 10,000rpm centrifugation 10min collect frond, with the fresh f/2 of sterilizing
Culture medium washs 3 times, is transferred in the fresh f/2 culture medium of 30ml and cultivates 7d.
After the completion of processing, by detection bacteria-eliminating efficacy and 4 micro- quasi- ball algae growth activity detection effects described in embodiment 3.
As a result as also shown in e.g. figs. 4-7, after adding antibiotic, good bacteria-eliminating efficacy has been reached, and the micro- quasi- ball algae obtained is through a period of time
It can normal growth after recovery.
Embodiment 6
Antibiotic treatment is added portionwise into activated algae solution：100 μ g/mL of chloramphenicol handles 3d, 10,000rpm centrifugations
10min collects frond, is washed 3 times with the fresh f/2 culture medium of sterilizing, be transferred in the fresh f/2 culture medium of 30ml and cultivate 1d；
100 μ g/mL of tetracycline handles 3d, and 10,000rpm centrifugation 10min collect frond, cultivated with the fresh f/2 of sterilizing
Base washs 3 times, is transferred in the fresh f/2 culture medium of 30ml and cultivates 1d；
200 μ g/mL of Ceftriaxone Sodium handles 3d, and 10,000rpm centrifugation 10min collect frond, with the fresh f/2 of sterilizing
Culture medium washs 3 times, is transferred in the fresh f/2 culture medium of 30ml and cultivates 7d.
After the completion of processing, by detection bacteria-eliminating efficacy and 4 micro- quasi- ball algae growth activity detection effects described in embodiment 3.
As a result as also shown in e.g. figs. 4-7, after adding antibiotic, good bacteria-eliminating efficacy has been reached, and the micro- quasi- ball algae obtained is through a period of time
It can normal growth after recovery.
Variety classes Antibiotic combination application can behave as collaboration, cumulative, unrelated, four kinds of effects of antagonism.The present invention is preferred
Chloramphenicol (25~100 μ g/mL), tetracycline (25~150 μ g/mL) and Ceftriaxone Sodium (25~400 μ g/mL) combination out, lead to
It crosses and successively adds said medicine, i.e., first inhibit most of bacterial growth using quick-acting bacteriostatic agents, then select fungicide to kill residual
The bacterium deposited, discovery can achieve good bacteria-eliminating efficacy.In addition, antibiotic can with the micro- quasi- ball algae of sterilization target it is certain at
Divide and combine and change its Physiology and biochemistry ingredient, to directly affect the growth of micro- quasi- ball algae, or since antibiotic can inhibit
Fungal component that is harmful or being conducive to micro- quasi- ball algae growth and the growth conditions for affecting indirectly microalgae, as can be seen from Figure 7 degerming
Micro- quasi- ball algae afterwards can normal growth after restoring for a period of time.So Antibiotics of screening and optimizing of the present invention and its dense
The addition manner and action time of degree and antibiont are best to sterile micro- quasi- ball algae effect is obtained
Comparative example 1：
Mixed antibiotic is added into activated algae solution：By chloramphenicol (100 μ g/mL), tetracycline (150 μ g/
ML it) and after Ceftriaxone Sodium (400 μ g/mL) mixing is added in activated algae solution.It is detected by detection method 1 in embodiment 4
Whether there is bacterium in algae solution.Difference from Example 5 is to add the method difference of antibiotic, remaining is all the same.
Comparative example 2：
Into activated algae solution, the sequence of addition antibiotic treatment is：Chloramphenicol (100 μ g/mL), Ceftriaxone Sodium
(400 μ g/mL), tetracycline (150 μ g/mL).Detect in algae solution whether have bacterium by detection method 1 in embodiment 4.With embodiment
5 except that the sequence of addition antibiotic is different, remaining is all the same.
Comparative example 3：
Mixed antibiotic is added into activated algae solution：By chloramphenicol (100 μ g/mL), tetracycline (150 μ g/
ML it) and after cefuroxime (400 μ g/mL) mixing is added in activated algae solution.Algae is detected by detection method 1 in embodiment 4
Whether there is bacterium in liquid.Difference from Example 5 is Ceftriaxone Sodium to be replaced with cefuroxime, remaining is all the same.
By the study found that the ball algae pollution in 1 culture medium of comparative example is serious, the ball algae ball in the culture medium of comparative example 2 and 3
Algae equally also has pollution, but without seriously polluted in comparative example 1, this is because generating after 1 three kinds of antibiotic mixing of comparative example
Antagonism completely cannot exclude the bacterium in culture medium.
Therefore, the type and order of addition of antibiotic of the present invention and processing time are optimal, are that the later period is sterile micro- quasi-
The research of ball algae provides basis.
Finally it should be noted that the foregoing is only a preferred embodiment of the present invention, it is not limited to this hair
It is bright, although the present invention is described in detail referring to the foregoing embodiments, for those skilled in the art, still
It can modify to technical solution documented by previous embodiment, or part is equivalently replaced.It is all in this hair
Within bright spirit and principle, any modification, equivalent replacement, improvement and so on should be included in protection scope of the present invention
Within.Above-mentioned, although the foregoing specific embodiments of the present invention is described with reference to the accompanying drawings, not to the scope of the present invention
Limitation, those skilled in the art should understand that, based on the technical solutions of the present invention, those skilled in the art are not required to
Make the creative labor the various modifications or changes that can be made still within protection scope of the present invention.
Claims (10)
1. preparing the antibiotic of sterile micro- quasi- ball algae (Nannochloropsis gaditana), which is characterized in that the antibiosis
Element and its concentration are respectively the cephalo of the chloramphenicol of 25~100 μ g/mL, the tetracycline of 25~150 μ g/mL and 25~400 μ g/mL
Qusong sodium；Micro- quasi- ball algae (Nannochloropsis gaditana) has been preserved on August 1st, 2018 Chinese common
Microbial preservation administrative center, deposit number are CGMCC No.16188.
2. antibiotic according to claim 1, which is characterized in that the antibiotic and its concentration are respectively：100μg/mL
Chloramphenicol, the tetracycline of 150 μ g/mL and the Ceftriaxone Sodium of 400 μ g/mL.
3. antibiotic of any of claims 1 or 2 is in preparing sterile micro- quasi- ball algae (Nannochloropsis gaditana)
Application.
4. a kind of preparation method of sterile micro- quasi- ball algae (Nannochloropsis gaditana), which is characterized in that including with
Lower step：
(1) culture of micro- quasi- ball algae (Nannochloropsis gaditana)：By micro- quasi- ball algae (Nannochloropsis
Gaditana it) is forwarded in fresh f/2 culture medium and cultivates again, algae solution and culture solution are with 1:10 ratios carry out transferred species activation；
(2) it is sequentially added in batches into activated micro- quasi- ball algae (Nannochloropsis gaditana) algae solution of step (1)
Following antibiotic treatment：
Firstly, 25~100 μ g/mL chloramphenicol, which are added, handles 1~7d, centrifugation collects frond, cultivates 1d after washing；Secondly, being added
25~150 μ g/mL tetracyclines handle 3~7d, and centrifugation collects frond, cultivates 1d after washing；Then, 25~400 μ g/mL are added
Ceftriaxone Sodium handles 3~7d, and centrifugation is collected frond, cultivated after washing.
5. the preparation side of the sterile micro- quasi- ball algae (Nannochloropsis gaditana) of one kind according to claim 4
Method, which is characterized in that further include that step (3) bacteria-eliminating efficacy is examined：
After taking step (2) antibiotic treatment, one week algae solution is grown in antibiotic-free culture medium and is coated on 2216E solid culture
Base, whether 3~4d of culture culture in 28~30 DEG C of biochemical cultivation cases, detecting in algae solution has bacterium；
After taking step (2) antibiotic treatment, one week algae solution is grown in antibiotic-free culture medium, and SYBR fluorescent dye is added and keeps away
Light dyes 15~20min, fluorescence microscope bacteria-eliminating efficacy after film-making；
After taking step (2) antibiotic treatment, one week algae solution is grown in antibiotic-free culture medium and carries out PCR, it is solidifying with agarose
Gel electrophoresis verifies PCR product, and whether detect in algae solution has bacterium.
6. the preparation side of the sterile micro- quasi- ball algae (Nannochloropsis gaditana) of one kind according to claim 4
Method, which is characterized in that further include micro- quasi- ball algae (Nannochloropsis gaditana) growth after step (4) antibiotic treatment
Activity determination：It after the completion of antibiotic treatment, periodically samples, measures the fluorescence intensity of algae solution, with microplate reader whether to detect algae
It can normal growth.
7. the preparation side of the sterile micro- quasi- ball algae (Nannochloropsis gaditana) of one kind according to claim 4
Method, which is characterized in that the antibiotic described in step (2) and its concentration are respectively：The chloramphenicol of 100 μ g/mL, 150 μ g/mL
Tetracycline and 400 μ g/mL Ceftriaxone Sodium.
8. the preparation method according to claim 4, which is characterized in that the condition of culture is that temperature is 24 ± 1 (DEG C), light
It is 3000lux, Light To Dark Ratio 12L according to intensity：12D, pH are 6.5~7.0.
9. the preparation method according to claim 4, which is characterized in that solution used in washing and culture is fresh f/2 training
Support base.
10. sterile micro- quasi- ball algae that any preparation method of claim 4~9 obtains.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110819535A (en) * | 2019-12-04 | 2020-02-21 | 曲阜师范大学 | Sterile culture method and sterile inspection method for red tide microalgae Karenia mikimotoi |
| CN114107140A (en) * | 2022-01-27 | 2022-03-01 | 中国科学院烟台海岸带研究所 | Method for in-situ sterile enrichment culture of synechococcus |
| CN114214201A (en) * | 2021-12-23 | 2022-03-22 | 日照职业技术学院 | Method for long-term stable storage of marine nannochloropsis oceanica |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010111707A1 (en) * | 2009-03-27 | 2010-09-30 | Sapphire Energy, Inc. | Variant isoprenoid producing enzymes and uses thereof |
| CN102660464A (en) * | 2012-05-17 | 2012-09-12 | 国家海洋局第一海洋研究所 | Oil-enriched marine microalga and application thereof |
| CN103849615A (en) * | 2013-12-31 | 2014-06-11 | 浙江省海洋开发研究院 | Immobilized algal-bacterial symbiotic system for processing petroleum pollutants and application thereof |
| CN104593263A (en) * | 2015-01-13 | 2015-05-06 | 新奥科技发展有限公司 | Method for treating protozoon pollution in microalgae culture |
| WO2015118457A1 (en) * | 2014-02-05 | 2015-08-13 | Reliance Industries Limited | A modified microorganism having enhanced biomass synthesis capacity and a method thereof |
| CN105779292A (en) * | 2016-03-23 | 2016-07-20 | 国家开发投资公司 | Ultralow-temperature preservation method for haematococcus pluvialis subjected to aseptic treatment |
-
2018
- 2018-09-03 CN CN201811023262.4A patent/CN108865894B/en not_active Expired - Fee Related
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010111707A1 (en) * | 2009-03-27 | 2010-09-30 | Sapphire Energy, Inc. | Variant isoprenoid producing enzymes and uses thereof |
| CN102660464A (en) * | 2012-05-17 | 2012-09-12 | 国家海洋局第一海洋研究所 | Oil-enriched marine microalga and application thereof |
| CN103849615A (en) * | 2013-12-31 | 2014-06-11 | 浙江省海洋开发研究院 | Immobilized algal-bacterial symbiotic system for processing petroleum pollutants and application thereof |
| WO2015118457A1 (en) * | 2014-02-05 | 2015-08-13 | Reliance Industries Limited | A modified microorganism having enhanced biomass synthesis capacity and a method thereof |
| CN104593263A (en) * | 2015-01-13 | 2015-05-06 | 新奥科技发展有限公司 | Method for treating protozoon pollution in microalgae culture |
| CN105779292A (en) * | 2016-03-23 | 2016-07-20 | 国家开发投资公司 | Ultralow-temperature preservation method for haematococcus pluvialis subjected to aseptic treatment |
Non-Patent Citations (5)
| Title |
|---|
| TEIXEIRA JACLYN R ET AL.: ""Effects of environmentally-relevant antibiotic mixtures on marine microalgal growth"", 《SCIENCE OF THE TOTAL ENVIRONMENT》 * |
| 刘晓娟等: ""眼点拟微绿球藻对抗生素的敏感性及其无菌藻株的培养"", 《生态科学》 * |
| 刘莹莹等: ""抗生素在微藻工程中的应用研究进展"", 《生命科学》 * |
| 李亚军等: ""不同抗生素和防腐剂对小球藻Chlorella sorokiniana细胞生长的影响"", 《南方农业学报》 * |
| 耿金峰等: ""眼点拟微绿球藻养殖过程中细菌污染的治理方法"", 《安徽农业科学》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110819535A (en) * | 2019-12-04 | 2020-02-21 | 曲阜师范大学 | Sterile culture method and sterile inspection method for red tide microalgae Karenia mikimotoi |
| CN110819535B (en) * | 2019-12-04 | 2023-06-30 | 曲阜师范大学 | Sterile culture method and sterile inspection method of red tide microalgae Karenia miq |
| CN114214201A (en) * | 2021-12-23 | 2022-03-22 | 日照职业技术学院 | Method for long-term stable storage of marine nannochloropsis oceanica |
| CN114214201B (en) * | 2021-12-23 | 2024-02-27 | 日照职业技术学院 | Method for long-term stable preservation of marine nannochloropsis |
| CN114107140A (en) * | 2022-01-27 | 2022-03-01 | 中国科学院烟台海岸带研究所 | Method for in-situ sterile enrichment culture of synechococcus |
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