CN103484394A

CN103484394A - Toxic microcystis bacterial strain and purification method of toxins produced by toxic microcystis bacterial strain

Info

Publication number: CN103484394A
Application number: CN201310278386.8A
Authority: CN
Inventors: 李效宇; 周春娥; 段红英; 刘洋; 张榜军; 马军国; 方倩
Original assignee: Henan Normal University
Current assignee: Henan Normal University
Priority date: 2013-07-04
Filing date: 2013-07-04
Publication date: 2014-01-01
Anticipated expiration: 2033-07-04
Also published as: CN103484394B

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

Poisonous Microcystis aeruginosa bacterial strain and toxin purification process thereof

technical field

The present invention relates to a kind of separation and Culture and toxin purification process thereof of high yield poison Microcystis aeruginosa bacterial strain, microbial technology field.

Background technology

Because causing the frequent generation of blue-green alga bloom (cyanobacterial bloom), body eutrophication become the water environment pollution problem of domestic and international common concern in recent years.The current lake more than 66% of China, reservoir be in eutrophic state, wherein heavy eutrophy and super eutrophic 22 % that account for; The 70 above rivers of % are subject to the pollution that degree is different, and eutrophication and blue-green alga bloom are the current maximum water environmental problems of China.Chinese large-sized lake blue algae wawter bloom frequently occurs, and 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, cause 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 also affects and endangers even people's health of hydrocoles owing to producing cyanophycean toxin (cyanotoxin).The modal blue-green algae of blue-green alga bloom be microcystic aeruginosa ( microcystis aeruginosa), and mostly produce cyanophycean toxin (cyanotoxin) thereby impact and harm hydrocoles people's health even.Microcystin (microcystins, MCs) is a modal class hepatotoxin in cyanophycean toxin, tool broad-spectrum biological toxicity, and the long-term chronic low dosage exposes (as by the tap water approach), and MC can cause Susceptible population's trouble hepatitis and inspire liver cancer.

MC is generally by 7 little peptides of the ring-type by the Amino acid profile of modified, common have 3 kinds of MC-LR, MC-RR and MC-YR, the toxicity of MC-LR the highest (be generally MC-RR 10 times) wherein, and be the kind that the World Health Organization (WHO) formulates drinking water standard.The MC steady chemical structure is difficult for degraded, anti-pH and high temperature, and heated and boiled can only small portion degraded (being not more than 20 %).Conventional water treatment technique, as chlorination, filtration etc. all can not thoroughly be eliminated MC in water.Therefore, even in resident's tap water, all may there is MC at the river that microcystis waterbloom occurs, lake, reservoir.It is 1mg/L that WHO has recommended tap water Microcystin (MC-LR) limit standard in 1998, and China Ministry of Health has adopted this standard in 2002.

MC is mainly used in toxicology, biology and hepatopathy Related Experimental Study, and at present domestic 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; Simultaneously, the method and technology of optimized Separation and purified toxins, reduce costs and environmental contamination reduction, significant for the research of Microcystin.

Summary of the invention

The object of the invention is to provide the poisonous Microcystis aeruginosa bacterial strain of the high producing microcystic toxins of a strain energy; Another purpose is to provide its purification process, reduces costs and environmental contamination reduction.

For realizing the object of the invention,, carry out separation and Culture and obtain the monoculture thing by gathering water sample at Henan Province's reservoir, through be accredited as microcystic aeruginosa ( microcystis aeruginosa MN001); Send Chinese Typical Representative culture collection center (CCTCC) preservation (wuchang, wuhan, address Lu Luojia Shan， Wuhan University), preserving number CCTCC M 2013146 in 2013-4-15.

This algae strain has following biological property:

1. the speed of growth is very fast, easily subculture and a large amount of the cultivation;

2. carry out the toxicity evaluation after Batch Culture, be found to be poisonous Microcystis aeruginosa, the main toxin of generation is MC-LR;

3. institute's toxin producing content high (HPLC detected result); Strong toxicity (MC that produces is MC-LR); Can be for a long time cultivation and form and inherited character malleable not in a large number; Long-term cultivation toxicity does not weaken or loses (acute toxicity test result); Can use cheap substrate, as BG-11 substitutes the 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 more outstanding characteristics and advantages.

The method of the separation and Culture of poisonous Microcystis aeruginosa strain and toxin preparation and preliminary purification is as follows: 1) at first at Henan Province's reservoir, gather water sample, microscopic examination kind;

2) adopt the micro pipette method to carry out under the microscope the separation of purpose Microcystis aeruginosa;

3) the single colony Microcystis aeruginosa separated is proceeded in the inherent illumination box of culturing bottle and carry out illumination cultivation, culture condition and parameter: substratum is MA substratum, 25 ℃ of temperature, intensity of illumination 2 000 lux, nutrient solution pH 8.6; Standing cultivation, stuffiness.

4) after cultivating 1 month, after list is planted Microcystis aeruginosa culture well-grown (culturing bottle presents denseer blue-greenish colour), will singly plant the Microcystis aeruginosa culture and carry out enlarged culturing, with this list, plant Microcystis aeruginosa culture as the algae kind, carry out aerated culture with 1 L culturing bottle, culture condition and parameter are the same.

5) carry out batch/a large amount of cultivation after enlarged culturing, with this list, plant Microcystis aeruginosa culture as the algae kind, with 10 L culturing bottles, carry out aerated culture, culture condition and parameter are the same.

6) after obtaining a large amount of cultures, with small white mouse, carry out acute toxicity test, detect the toxicity of this algae strain;

7) laboratory is preserved this list kind Microcystis aeruginosa culture and is sent Chinese Typical Representative culture collection center (CCTCC) preservation.

8) cultivate in a large number this list and plant the Microcystis aeruginosa culture, collect the centrifugal rear acquisition algae slurry of culture;

9) adopt boiling method to extract the microcystis extract, be about to the algae slurry and boil, standing cooling after 12 000 * gcentrifugal, get supernatant liquor, obtain the microcystis extract;

10) with extracting under 4 ° of C of methyl alcohol of mass percent 70%, 12 000 * gcentrifugal, get supernatant liquor;

11) supernatant liquor of acquisition is rotated to evaporation drying under 60 ° of C;

12) PBS dissolves the rear C of mistake again ₁₈oDS pillar (Sep-Pak ^òplus, waters);

13) then with using again mass percent 100% methanol-eluted fractions after mass percent 20% methanol-eluted fractions;

14) elutriant of collection is rotated to evaporation drying under 60 ° of C;

15) after PBS dissolves dry thick toxin ,-20 ° of C preserve;

16) high pressure liquid chromatography (HPLC) detects kind and the content of MC in thick toxin.

This process can be reduced to Fig. 1.

The invention has the advantages that:

1) adopt boiling method to extract the microcystis extract and replace acetic acid, methyl alcohol extraction process, do not need to carry out the lyophilize processing, saved the energy, and reduced environmental pollution;

2) the poisonous Microcystis aeruginosa bacterial strain that adopts the method to obtain, detect through HPLC, and its toxin producing is mainly MC-LR, and content is higher.In thick toxin, the MC total content is 214.26 μ g/mL PBS solution, wherein MC-LR and MC-YR distribute account for 65.98% and 34.02%, MC-RR do not detect (seeing accompanying drawing 2).Can be used for a large amount of the cultivation extracts and preparation MC crude product and sterling commercialization.

The accompanying drawing explanation

Fig. 1 is purifying process route map of the present invention;

The HPLC spectrogram that Fig. 2 is the thick toxin that obtains of purifying of the present invention.

Embodiment

Embodiment 1

Its purification process is as follows:

1) reservoir gathers water sample, microscopic examination kind;

9) adopt boiling method to extract the microcystis extract, be about to the algae slurry and boil 10-15 minute, standing cooling after 12 000 * gcentrifugal 10 minutes, get supernatant liquor, obtain the microcystis extract;

10) with extracting under 4 ° of C of methyl alcohol of mass percent 70% 12 hours, 12 000 * gcentrifugal 15 minutes, get supernatant liquor;

13) use again mass percent 100% methanol-eluted fractions after mass percent 20% methanol-eluted fractions;

14) elutriant rotates evaporation drying under 60 ° of C;

15) after PBS dissolves dry thick toxin ,-20 ° of C preserve;

Through HPLC, detect, as accompanying

drawing

2,1 peak that is the MC-LR place; 2 peaks that are the MC-YR place; 3 absorption peaks that are the MC-RR place.Can find out: this Microcystis aeruginosa strain institute toxin producing is mainly MC-LR, and content is high.In thick toxin, the MC total content is 214.26 μ g/mL PBS solution, wherein MC-LR and MC-YR distribute account for 65.98% and 34.02%, MC-RR do not detect.

Application examples 1

The acute toxicity test result:

(1) the acute toxicity test in mice result shows, the LD of microcystis extract to mouse ₅₀be 35 μ g extract dry weight/kg body weight;

(2) LD of thick toxin to mouse ₅₀for the thick toxin dry weight/kg body weight of 40-60 μ g;

Application examples 2

After substituting a large amount of cultivations of MA substratum with cheap substrate BG-11, the growth of this algae strain, output and toxicity thereof are without noticeable change.

Claims

1. a poisonous Microcystis aeruginosa bacterial strain, for microcystic aeruginosa ( microcystis aeruginosa), preserving number is CCTCC M 2013146.

2. the purification process of poisonous Microcystis aeruginosa bacterial strain toxin as claimed in claim 1.

3. step is as follows:

(1) cultivate in a large number this list and plant the Microcystis aeruginosa culture, collect the centrifugal rear acquisition algae slurry of culture;

(2) adopt boiling method to extract the microcystis extract, be about to the algae slurry and boil, standing cooling after 12 000 * gcentrifugal, get supernatant liquor, obtain the microcystis extract;

(3) with extracting under 4 ° of C of methyl alcohol of mass percent 70%, 12 000 * gcentrifugal, get supernatant liquor;

(4) supernatant liquor of acquisition is rotated to evaporation drying under 60 ° of C;

(5) PBS dissolves the rear C of mistake again ₁₈the ODS pillar;

(6) use again mass percent 100% methanol-eluted fractions after mass percent 20% methanol-eluted fractions;

(7) elutriant of collection is rotated to evaporation drying under 60 ° of C;

(8) PBS dissolves ° C preservation of dry thick toxin-20.