CN103484394B - Toxic microcystis bacterial strain and purification method of toxins produced by toxic microcystis bacterial strain - Google Patents
Toxic microcystis bacterial strain and purification method of toxins produced by toxic microcystis bacterial strain Download PDFInfo
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- CN103484394B CN103484394B CN201310278386.8A CN201310278386A CN103484394B CN 103484394 B CN103484394 B CN 103484394B CN 201310278386 A CN201310278386 A CN 201310278386A CN 103484394 B CN103484394 B CN 103484394B
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Abstract
The invention relates to a microcystis bacterial strain with high toxin production rate and a purification method of toxins produced by the toxic microcystis bacterial strain, and belongs to the field of biotechnology. The toxic microcystis bacterial strain is Microcystisaeruginosa, and is preserved at China Center for Type Culture Collection (CCTCC) on 25th, April, 2013, and the preservation number is CCTCCM2013146. Biological characteristics of the toxic microcystis bacterial strain are that: growth speed is relatively high; successive transfer culturing and large-scaled culturing are easy; a major toxin produced by the toxic microcystis bacterial strain is MC-LR, and toxin content and toxicity are high; long-term large-scaled culturing of the toxic microcystis bacterial strain can be realized without change of morphology and inheritable characters; decrease or loss of toxicity will not happen in long-term culturing; MA medium can be replaced by cheap medium without change of toxin yield or toxicity; and the toxic microcystis bacterial strain can be used for large-scaled extraction and preparation of MC crude products and purified products, and commercialization.
Description
technical field
The present invention relates to separation and Culture and the toxin purification process thereof of a kind of high yield poison Microcystis aeruginosa bacterial strain, microbial technology field.
Background technology
Cause the frequent generation of blue-green alga bloom (cyanobacterial bloom) to become the water environment pollution problem of domestic and international common concern in recent years due to body eutrophication.Lake, the reservoir of China current more than 66% are in eutrophic state, wherein heavy eutrophy and superly eutrophicly account for 22 %; 70 more than % rivers are subject to the different pollution of degree, and eutrophication and blue-green alga bloom are the maximum now water environmental problems of China.Chinese large-sized lake blue algae wawter bloom frequently occurs, especially Dian Chi, Taihu Lake, Chaohu etc., as Taihu Lake blue-green alga bloom in 2007 once caused the crisis of Residents In Wuxi tap water, causes showing great attention to of news media and government.Blue-green alga bloom not only polluted-water, destroy drinking water source, cause fishery loss, and most blue-green alga bloom due to produce cyanophycean toxin (cyanotoxin) also affect and endanger the hydrocoles even health of people.The modal blue-green algae of blue-green alga bloom be microcystic aeruginosa (
microcystis aeruginosa), and mostly produce cyanophycean toxin (cyanotoxin) thus impact and harm the hydrocoles even health of people.Microcystin (microcystins, MCs) be a modal class hepatotoxin in cyanophycean toxin, tool broad-spectrum biological toxicity, long-term chronic low dosage exposes (as by tap water approach), and MC can cause Susceptible population suffer from hepatitis and inspire liver cancer.
MC generally by 7 by the little peptide of the ring-type of the Amino acid profile of modified, common have MC-LR, MC-RR and MC-YR 3 kinds, the wherein toxicity of MC-LR the highest (being generally 10 times of MC-RR), and be the kind that the World Health Organization (WHO) formulates drinking water standard.MC steady chemical structure is not easily degraded, anti-pH and high temperature, and heated and boiled can only small portion degraded (being not more than 20 %).Regular tap water treatment process, as chlorination, filtration etc. all can not thoroughly eliminate MC in water.Therefore, in the river of generation microcystis waterbloom, lake, reservoir even resident's tap water, all MC may be there is.It is 1mg/L that WHO has recommended tap water Microcystin (MC-LR) limit standard for 1998, and the Ministry of Health of China have employed this standard in 2002.
MC is mainly used in toxicology, biology and hepatopathy Related Experimental Study, and domestic at present only have 1-2 company to sell.Separation, the poisonous Microcystis aeruginosa bacterial strain of purifying, make it reach this type of bacterial strain of high yield; Meanwhile, the method and technology of optimized Separation and purified toxins, reduces costs and reduces environmental pollution, and the research for Microcystin is significant.
Summary of the invention
The object of the invention is the poisonous Microcystis aeruginosa bacterial strain providing the high producing microcystic toxins of a strain energy; Another object is to provide its purification process, reduces costs and reduces environmental pollution.
For realizing the object of the invention, Henan Province's reservoir by gather water sample, carry out separation and Culture and obtain monoculture thing, through be accredited as microcystic aeruginosa (
microcystis aeruginosa MN001); China typical culture collection center (CCTCC) preservation (Luo Jia Shan, wuchang, wuhan road, address, Wuhan University) is sent, preserving number CCTCC M 2013146 in 2013-4-15.
This algae strain has following biological property:
1. the speed of growth is very fast, easy subculture and mass propgation;
2. carry out toxicity appraisal after Batch Culture, be found to be poisonous Microcystis aeruginosa, the primary toxins of generation is MC-LR;
3. a toxin producing content high (HPLC detected result); Strong toxicity (produce MC be MC-LR); Can long-term mass propgation and form and inherited character not malleable; Long-term cultivation toxicity does not weaken or loses (acute toxicity test result); Cheap substrate can be used, as BG-11 substitutes MA substratum and toxicity does not change.
4. the toxin that current domestic Microcystis aeruginosa strain produces mostly is MC-RR, and MC-RR toxicity is low, price is also low; Advantage is that this algae strain generation toxin is MC-LR and has some characteristics and advantages comparatively given prominence to.
The method of the separation and Culture of poisonous Microcystis aeruginosa strain and toxin preparation and preliminary purification is as follows: 1) first gather water sample at Henan Province's reservoir, microscopic examination kind;
2) micro pipette method is adopted to carry out the separation of object Microcystis aeruginosa under the microscope;
3) the single colony Microcystis aeruginosa be separated is proceeded in the inherent illumination box of culturing bottle carry out illumination cultivation, culture condition and parameter: substratum is MA substratum, temperature 25 DEG C, intensity of illumination 2 000 lux, nutrient solution pH 8.6; Quiescent culture, stuffiness.
4) cultivate after 1 month, after single Microcystis aeruginosa culture well-grown (culturing bottle presents denseer blue-greenish colour), single Microcystis aeruginosa culture is carried out enlarged culturing, namely with this single Microcystis aeruginosa culture as algae kind, carry out aerated culture with 1 L culturing bottle, culture condition and parameter the same.
5) carry out batch/mass propgation after enlarged culturing, namely with this single Microcystis aeruginosa culture as algae kind, carry out aerated culture with 10 L culturing bottles, culture condition and parameter the same.
6), after obtaining mass propgation thing, carry out acute toxicity test with small white mouse, detect the toxicity of this algae strain;
7) laboratory is preserved this single Microcystis aeruginosa culture and is sent China typical culture collection center (CCTCC) preservation.
8) this single Microcystis aeruginosa culture of mass propgation, collects the centrifugal rear acquisition algae slurry of culture;
9) adopt boiling method to extract microcystis extract, boil by algae slurry, to leave standstill after cooling 12 000 ×
gcentrifugal, get supernatant liquor, namely obtain microcystis extract;
10) with extracting at the methyl alcohol 4 DEG C of mass percent 70%, 12 000 ×
gcentrifugal, get supernatant liquor;
11) supernatant liquor of acquisition is rotated evaporation drying at 60 DEG C;
12) PBS dissolves rear C excessively again
18oDS pillar (Sep-Pak
òplus, waters);
13) then mass percent 100% methanol-eluted fractions is used again with after mass percent 20% methanol-eluted fractions;
14) elutriant of collection is rotated evaporation drying at 60 DEG C;
15)-20 DEG C of preservations after the Raw toxin that PBS dissolving is dry;
16) high pressure liquid chromatography (HPLC) detects kind and the content of MC in Raw toxin.
This process can be reduced to Fig. 1.
The invention has the advantages that:
1) adopt boiling method to extract microcystis extract and replace acetic acid, methyl alcohol extraction process, do not need to carry out lyophilize process, save the energy, and decrease environmental pollution;
2) the poisonous Microcystis aeruginosa bacterial strain adopting the method to obtain, detect through HPLC, its toxin producing is mainly MC-LR, and content is higher.In Raw toxin, MC total content is 214.26 μ g/mL PBS solution, wherein MC-LR and MC-YR distribution account for 65.98% and 34.02%, MC-RR do not detect (see accompanying drawing 2).Can be used for mass propgation extraction and preparation MC crude product and sterling and commercialization.
Accompanying drawing explanation
Fig. 1 is purifying process route map of the present invention;
The HPLC spectrogram of the Raw toxin that Fig. 2 obtains for purifying of the present invention.
Embodiment
Embodiment 1
Its purification process is as follows:
1) reservoir gathers water sample, microscopic examination kind;
2) micro pipette method is adopted to carry out the separation of object Microcystis aeruginosa under the microscope;
3) the single colony Microcystis aeruginosa be separated is proceeded in the inherent illumination box of culturing bottle carry out illumination cultivation, culture condition and parameter: substratum is MA substratum, temperature 25 DEG C, intensity of illumination 2 000 lux, nutrient solution pH 8.6; Quiescent culture, stuffiness.
4) cultivate after 1 month, after single Microcystis aeruginosa culture well-grown (culturing bottle presents denseer blue-greenish colour), single Microcystis aeruginosa culture is carried out enlarged culturing, namely with this single Microcystis aeruginosa culture as algae kind, carry out aerated culture with 1 L culturing bottle, culture condition and parameter the same.
5) carry out batch/mass propgation after enlarged culturing, namely with this single Microcystis aeruginosa culture as algae kind, carry out aerated culture with 10 L culturing bottles, culture condition and parameter the same.
6), after obtaining mass propgation thing, carry out acute toxicity test with small white mouse, detect the toxicity of this algae strain;
7) laboratory is preserved this single Microcystis aeruginosa culture and is sent China typical culture collection center (CCTCC) preservation.
8) this single Microcystis aeruginosa culture of mass propgation, collects the centrifugal rear acquisition algae slurry of culture;
9) adopt boiling method to extract microcystis extract, boil 10-15 minute by algae slurry, to leave standstill after cooling 12 000 ×
gcentrifugal 10 minutes, get supernatant liquor, namely obtain microcystis extract;
10) with extracting at the methyl alcohol 4 DEG C of mass percent 70% 12 hours, 12 000 ×
gcentrifugal 15 minutes, get supernatant liquor;
11) supernatant liquor of acquisition is rotated evaporation drying at 60 DEG C;
12) PBS dissolves rear C excessively again
18oDS pillar (Sep-Pak
òplus, waters);
13) mass percent 100% methanol-eluted fractions is used again after mass percent 20% methanol-eluted fractions;
14) elutriant rotates evaporation drying at 60 DEG C;
15)-20 DEG C of preservations after the Raw toxin that PBS dissolving is dry;
16) high pressure liquid chromatography (HPLC) detects kind and the content of MC in Raw toxin.
Detect through HPLC, as accompanying drawing 2,1 is the peak at MC-LR place; 2 is the peak at MC-YR place; 3 is the absorption peak at MC-RR place.Can find out: this Microcystis aeruginosa strain institute toxin producing is mainly MC-LR, and content is high.In Raw toxin, MC total content is 214.26 μ g/mL PBS solution, wherein MC-LR and MC-YR distribution account for 65.98% and 34.02%, MC-RR do not detect.
Application examples 1
Acute toxicity test result:
(1) acute toxicity test in mice result shows, microcystis extract is to the LD of mouse
50be 35 μ g extract dry weight/kg body weight;
(2) Raw toxin is to the LD of mouse
50for 40-60 μ g Raw toxin dry weight/kg body weight;
Application examples 2
After substituting MA substratum mass propgation with cheap substrate BG-11, the growth of this algae strain, output and toxicity thereof are without noticeable change.
Claims (2)
1. a poisonous Microcystis aeruginosa bacterial strain, is characterized in that, its be microcystic aeruginosa (
microcystis aeruginosa), preserving number is CCTCC NO:M2013146.
2. the preliminary purification method of poisonous Microcystis aeruginosa bacterial strain toxin as claimed in claim 1, it is characterized in that, step is as follows:
(1) this single Microcystis aeruginosa culture of mass propgation, collects the centrifugal rear acquisition algae slurry of culture;
(2) adopt boiling method to extract microcystis extract, boil by algae slurry, to leave standstill after cooling 12 000 ×
gcentrifugal, get supernatant liquor, namely obtain microcystis extract;
(3) with extracting at the methyl alcohol 4 DEG C of mass percent 70%, 12 000 ×
gcentrifugal, get supernatant liquor;
(4) supernatant liquor of acquisition is rotated evaporation drying at 60 DEG C;
(5) PBS dissolves rear C excessively again
18oDS pillar;
(6) mass percent 100% methanol-eluted fractions is used again after mass percent 20% methanol-eluted fractions;
(7) elutriant of collection is rotated evaporation drying at 60 DEG C;
(8) PBS dissolves dry Raw toxin-20 DEG C preservation.
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| CN101451979A (en) * | 2008-12-31 | 2009-06-10 | 哈尔滨工业大学 | Method for detecting microcystis aeruginosa toxin |
| CN101974080A (en) * | 2010-11-22 | 2011-02-16 | 中国科学院水生生物研究所 | Method for separating and purifying microcystin by utilizing series-connected solid phase extraction columns |
| CN102516367A (en) * | 2011-12-30 | 2012-06-27 | 山东建筑大学 | Method for extracting high-purity microcystins |
| WO2013068654A1 (en) * | 2011-11-09 | 2013-05-16 | Turun Yliopisto | Method and primers for the detection of microcystin-producing toxic cyanobacteria |
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2013
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Patent Citations (4)
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| CN101451979A (en) * | 2008-12-31 | 2009-06-10 | 哈尔滨工业大学 | Method for detecting microcystis aeruginosa toxin |
| CN101974080A (en) * | 2010-11-22 | 2011-02-16 | 中国科学院水生生物研究所 | Method for separating and purifying microcystin by utilizing series-connected solid phase extraction columns |
| WO2013068654A1 (en) * | 2011-11-09 | 2013-05-16 | Turun Yliopisto | Method and primers for the detection of microcystin-producing toxic cyanobacteria |
| CN102516367A (en) * | 2011-12-30 | 2012-06-27 | 山东建筑大学 | Method for extracting high-purity microcystins |
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| Microcystins cause embryonic toxicity in mice;Yan-Zhen Bu et al;《Toxicon》;20060817;第48卷;966-972 * |
| Microwave oven and boiling waterbath extraction of hepatotoxins from cyanobacterial cells;James S. Metcalf et al;《FEMS Microbiology Letters》;20001231;材料和方法部分 * |
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| 微囊藻毒素MC-LR纯品的制备工艺研究;陈永芳;《中国优秀硕士学位论文全文数据库》;20111015;第2.1节、第3.1.2、3.3、4.1.2节 * |
| 微囊藻细胞抽提液对小鼠血液的亚慢性毒性;李效宇 等;《水生生物学报》;20081130;第32卷(第6期);811-817 * |
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