N-15 labeled microcystin and production method thereof
阅读说明：本技术 一种n-15标记的微囊藻毒素及其生产方法 (N-15 labeled microcystin and production method thereof ) 是由 吴振兴 高春蕾 刘金丽 刘红兵 于 2019-08-01 设计创作，主要内容包括：本发明公开了一种N-15标记的微囊藻毒素及其生产方法,其生产方法包括以下步骤：微囊藻的清洗：选用处于对数生长期的微囊藻作为藻种,采用去离子水对藻种进行悬浮清洗,离心后收集藻体,清洗次数至少2次；微囊藻的培养：将清洗干净的微囊藻接种到带有N-15的专用培养基中,并置于光下培养,光强约2500-5000LUX,温度为25-28℃,每天摇匀培养瓶1-5次,培养10-30天,得到培养液；微囊藻毒素的提取：对培养液中的微囊藻细胞进行裂解处理,离心收集上清液,得到微囊藻毒素粗提液。本发明的生产方法简单易操作,制备的微囊藻毒素的N-15标记率高,作为标准品使用降低基质效应,定量更准确,相比现有非标微囊藻毒素,具有更好的应用前景。(The invention discloses an N-15 marked microcystin and a production method thereof, wherein the production method comprises the following steps: cleaning the microcystis: selecting microcystis in logarithmic growth phase as algae seeds, performing suspension cleaning on the algae seeds by using deionized water, centrifuging, and collecting algae for at least 2 times; culturing microcystis: inoculating the cleaned microcystis into a special culture medium with N-15, culturing under light with light intensity of 2500-; extracting microcystin: and (3) cracking the microcystis cells in the culture solution, centrifuging and collecting supernatant to obtain crude microcystin extract. The production method is simple and easy to operate, the prepared microcystins have high N-15 labeling rate, the matrix effect is reduced when the microcystins are used as standard substances, the quantification is more accurate, and the method has better application prospect compared with the existing non-standard microcystins.)
1. A method for producing N-15 labeled microcystins, comprising the steps of:
cleaning the microcystis: selecting microcystis in logarithmic growth phase as algae seeds, performing suspension cleaning on the algae seeds by using deionized water, and collecting algae after centrifugation; washing for at least 2 times;
culturing microcystis: inoculating the cleaned microcystis into a special culture medium with N-15, culturing under light with light intensity of 2500-;
extracting microcystin: and (3) cracking the microcystis cells in the culture solution, centrifuging and collecting supernatant to obtain crude microcystin extract.
2. The production method according to claim 1, wherein the dedicated medium comprises macroelement components and microelement components at the following concentrations:
the macroelement component comprises: n-15 labeled nitrogen source 0.1-1.8g/L, KH2PO4
20-50mg/L、KCl20-40mg/L、MgSO4·7H2O50-100mg/L、CaCl2·2H2O20-50mg/L, citric acid 4-8mg/L, FeCl3 4-6mg/L、Na2CO3 10-30mg/L；
The trace element component comprises: h3BO3 1.5-3.0mg/L、MnCl2·4H2O1.5-2.5mg/L、ZnSO4·7H2O0.1-0.3mg/L、Na2MoO4·2H2O0.2-0.5mg/L、CuSO4·5H2O0.05-0.1mg/L、CoCl2·6H2O0.03-0.06mg/L；
The balance being solvent ddH2O。
3. The production method according to claim 2, wherein the N-15 labeled nitrogen source is one or a combination of several of the following N-15 labeled components: CH (CH)4N2O100-250mg/L、NaNO3 1.2-1.6g/L、KNO3 1.4-1.8g/L。
4. The production method according to claim 3, wherein the exclusive medium further comprises an N-15 labeled additional nitrogen-containing trace element component; preferably, the N-15 marked other nitrogen-containing trace element component is 5-8mg/L, EDTANa of ferric ammonium citrate2 0.8-1.2mg/L、Co(NO3)2·5H2One or more of O0.04-0.08 mg/L.
5. The method of claim 2, wherein the ratio of the micro-capsules to the special culture medium is 1:10 to 1: 30.
6. The method according to claim 2, wherein the algal solution obtained from the first transfer is continuously transferred 3-5 times as algal species during the cultivation of the microcystis to increase the microcystin concentration and the labeling rate of N-15 in the cultivation product.
7. The production method according to claim 1, wherein the pyrolysis method is a high temperature treatment or an ultrasonic treatment, wherein the conditions of the high temperature treatment are as follows: treating at 100-; the conditions of the ultrasonic treatment were: centrifuging the culture solution, collecting algae cell precipitate, adding appropriate amount of water into the precipitate, and performing ultrasonic treatment at 50-70 deg.C for 15-30 min.
8. The method of claim 1, further comprising a step of purifying the microcystin:
performing vacuum rotary evaporation on the crude microcystin extract, and performing primary purification through an adsorption column to obtain primary pure liquid;
concentrating the primary pure solution by vacuum rotary evaporation, purifying the concentrated toxin primary pure solution again by HPLC, concentrating and freeze-drying the product to obtain high-purity N-15 labeled microcystin powder.
9. An N-15 labeled microcystin, characterized in that it is prepared by the production method of claims 1-8.
Technical Field
The invention relates to the technical field of production methods of microcystins, in particular to an N-15 labeled microcystin and a production method thereof.
Background
Microcystis (Microcystis aeruginosa) is the dominant species for the formation of blue algae "fresh water bloom". The method has strong adaptability and high propagation speed, the produced microcystins (Microcystis) are mainly LR, RR, YR and the like, are monocyclic heptapeptides with biological activity, can be enriched in aquatic zooplankton and fish, when algae cells are cracked and die, the toxins can be directly released into a water body, and human beings can induce liver injury, even liver cancer and other diseases by drinking polluted water sources or eating aquatic animals, so that the microcystins content in the water sources and food needs to be quickly, efficiently and accurately extracted and detected to ensure the dietary health. Therefore, the production of high-purity and high-stability microcystin standard products is not only a technical problem which needs to be overcome urgently, but also has great economic significance.
In recent years, the microcystin standard products in domestic markets have few varieties, low purity and low stability, and are difficult to meet the requirement of accurate detection, but the imported standard products have high price and unbalanced supply and demand, and isotope-labeled microcystin internal standard products do not exist. In the mass spectrum detection process, the isotope internal standard can reduce the matrix effect, so that the quantification is more accurate, and meanwhile, the isotope internal standard can be used as a reference standard substance to evaluate the efficiency of the extraction and processing process and the detection process, and can not be replaced by a common standard substance. Therefore, there is a need to develop a simple, efficient and low-cost method for producing isotope-labeled microcystins, which promotes the rapid development of the industrialization of isotope internal standard products and creates higher technical and economic values.
At present, the synthesis route of microcystin molecules in microcystis is not clear, the source of C, H, O in the molecules is complex, if the isotope of the three elements is used for marking microcystin, the cost is higher, the process is uncontrollable, and the nitrogen source in a culture medium is relatively single. At present, no report about isotope-labeled microcystin internal standard products and production thereof is found.
Disclosure of Invention
Aiming at the technical problems, the embodiment of the invention provides the N-15 marked microcystin and the production method thereof, the production method of the microcystin has simple steps and controllable isotope marking, and the yield and the marking rate of the prepared N-15 marked microcystin are high.
The invention provides the following technical scheme:
a method for producing N-15 labeled microcystins, comprising the steps of:
s1, cleaning microcystis: selecting microcystis in logarithmic growth phase as algae seeds, performing suspension cleaning on the algae seeds by using deionized water, and collecting algae after centrifugation; the number of washing times is 3-5;
s2, culturing microcystis: inoculating the cleaned microcystis into a special culture medium with N-15, culturing under light with the light intensity of 2500-;
s3, extracting microcystin: and (3) cracking the microcystis cells in the culture solution, centrifuging and collecting supernatant to obtain crude microcystin extract.
Preferably, in step S2, the special culture medium includes macroelement components and trace element components at the following concentrations:
the macroelement component comprises: n-15 labeled nitrogen source 0.1-1.8g/L, KH2PO4 20-50mg/L、KCl20-40mg/L、MgSO4·7H2O 50-100mg/L、CaCl2·2H2O20-50mg/L, citric acid 4-8mg/L, FeCl3 4-6mg/L、Na2CO3 10-30mg/L；
The trace element component comprises: h3BO3 1.5-3.0mg/L、MnCl2·4H2O 1.5-2.5mg/L、ZnSO4·7H2O0.1-0.3mg/L、Na2MoO4·2H2O 0.2-0.5mg/L、CuSO4·5H2O 0.05-0.1mg/L、CoCl2·6H2O0.03-0.06mg/L；
The balance being solvent ddH2O。
The special culture medium not only provides macronutrients such as N, P, K, Mg, Ca, Fe and the like necessary for the growth and the propagation of the microcystis and a plurality of trace elements, but also removes CH marked by N-15 in all the macroelements and the trace elements4N2O, no N element, and the only nitrogen source is marked by N-15, which is convenient for producing N-15 marked microcystin, and the isotope marking method of the nitrogen source is simpler and controllable, the culture cost is low, and the isotope marking rate of the microcystin is high.
Such as the use of C-13 for labeling C-containing compounds in the medium and C-13-labeled CO2As a carbon source of microcystis in the culture process, C-13 labeled microcystin can be cultured, but the culture process needs to strictly prevent air (or CO2 in the air) from entering a culture system, and the process is difficult to control; in addition, the culture medium contains a plurality of carbon-containing compounds, and the carbon-containing compounds are completely replaced by the C-13 marker, so that the culture cost is greatly increased; if only the macroelements are replaced by isotopes (Na)2CO3，CO2) The growth of algae cells will be affected without adding other compounds with low C content. Similar problems can occur when other elements H and O are isotopically labeled.
Preferably, the N-15 labeled nitrogen source is one or a combination of several of the following N-15 labeled components: CH (CH)4N2O 100-250μg/L、NaNO3 1.2-1.6g/L、KNO3 1.4-1.8g/L。
Further, the special culture medium also comprises other nitrogen-containing trace element components marked by N-15, preferably, the other nitrogen-containing trace element components marked by N-15 are ferric ammonium citrate 5-8mg/L, EDTANa20.8-1.2mg/L、Co(NO3)2·5H2One of O0.04-0.08mg/LOne or more of them. Therefore, other trace elements can be supplemented, the special culture medium can be further optimized, and N-15 isotopes can be further introduced, so that the specific gravity of the N-15 isotopes in the culture medium is improved, and the labeling rate of the microcystins is improved.
Wherein CH is adopted4N2O100-250 mg/L is most preferred as the nitrogen source because of the N-15 labeled CH4N2O and N in the urea are high in percentage content and are easily absorbed and utilized by plants, so that the production cost of the isotope-labeled microcystins is effectively reduced.
More preferably, the specialized medium comprises the following concentrations of macroelement components and micronutrient components:
the macroelement component comprises: n-15 labeled Nitrogen Source CH4N2O 180-220mg/L、KH2PO4 40-50mg/L、KCl 30-40mg/L、MgSO4·7H2O 60-90mg/L、CaCl2·2H235-50mg/L of O and 5-7mg/L, FeCl of citric acid3 5-6mg/L、Na2CO3 15-25mg/L；
The trace element component comprises: h3BO3 2.0-3.0mg/L、MnCl2·4H2O 1.8-2.3mg/L、ZnSO4·7H2O0.2-0.3mg/L、Na2MoO4·2H2O 0.3-0.5mg/L、CuSO4·5H2O 0.06-0.1mg/L、CoCl2·6H2O0.04-0.06 mg/L. The balance being solvent ddH2O。
Preferably, the inoculation ratio of the microcystis to the special culture medium is 1:10-1: 30.
Preferably, during the culture process of the microcystis, the algae liquid obtained by the first transfer is continuously transferred for 3-5 times as the algae seed so as to continuously improve the concentration of microcystin in the culture product and the N-15 marking rate of the microcystin.
Preferably, the cracking method is high temperature treatment or ultrasonic treatment, wherein the conditions of the high temperature treatment are as follows: treating at 100-; the conditions of the ultrasonic treatment were: centrifuging the culture solution, collecting algae cell precipitate, adding appropriate amount of water into the precipitate, and performing ultrasonic treatment at 50-70 deg.C for 15-30 min.
Preferably, the method also comprises a purification step of the microcystins:
performing vacuum rotary evaporation on the crude microcystin extract, and performing primary purification through an adsorption column to obtain primary pure liquid;
concentrating the primary pure solution by vacuum rotary evaporation, purifying the concentrated toxin primary pure solution again by HPLC, concentrating and freeze-drying the product to obtain high-purity N-15 labeled microcystin powder.
The invention also provides N-15 marked microcystin which is prepared by the production method.
The invention has the following beneficial effects:
1. the production method is simple and easy to operate, has strong practicability, and is suitable for industrial production of products.
2. The microcystin prepared by the invention has high N-15 labeling rate and high purity, can be used as a standard substance to reduce matrix effect, has more accurate quantification, and has better application prospect compared with the existing non-standard microcystin.
3. The invention selects the nitrogen source as the isotope labeled element, and has the advantages of easy operation, easy quantification, low culture cost and no inhibition of the growth of algae cells compared with other nutrient elements.
Drawings
FIG. 1 is a HPLC-UV chromatogram after purification of microcystin-LR;
FIG. 2 is a HPLC-UV chromatogram after purification of microcystin-RR.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items. In addition, the technical features involved in the different embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
Experimental materials:
1. micro-capsule algae: adopting microcystis aeruginosa FACHB-315 mainly producing MC-LR and microcystis aeruginosa FACHB-912 mainly producing MC-RR as experimental algae species, which are purchased from freshwater algae seed bank of Chinese academy of sciences; the microcystin standard substance adopts the following components: the specification of the microcystin LR and the microcystin RR are both 10 mug/ml, and the microcystin LR and the microcystin RR are purchased from Beijing Wanjiayi biological science and technology limited company.
2. Culture medium:
(1) macroelement mother liquor: n-15 labeled CH4N2O 100-250g/L；KH2PO4 20-50g/L；KCl20-40g/L；MgSO4·7H2O 50-100g/L；CaCl2·2H2O 20-50g/L；Citric acid 4-8g/L；FeCl34-6g/L；Na2CO310-30 g/L; the above mother solutions are prepared separately. Sterilizing and cooling the macroelement mother liquor, and storing at 4 ℃, wherein the dosage of each macroelement mother liquor is 1 ml/L.
(2) The formula of the microelement mother solution is as follows: h3BO3 1.5-3.0g/L；MnCl2·4H2O 1.5-2.5g/L；ZnSO4·7H2O 0.1-0.3g/L；Na2MoO4·2H2O 0.2-0.5g/L；CuSO4·5H2O 0.05-0.1g/L；CoCl2·6H2O0.03-0.06 g/L. After the above microelement mother liquor is prepared, sterilizing, cooling and storing at 4 deg.C, each dosage is 1 ml/L.