A method for extracting microcystin from Aphanizomenon flos-aquae

Abstract

The present invention discloses a method for extracting microcystin from Anabaena flos-aquae, comprising the following steps: (1) obtaining the Anabaena flos-aquae strain that produces microcystin; (2) crushing the Anabaena flos-aquae strain, adding a methanol solution, extracting microcystin under stirring, then performing centrifugation, collecting the supernatant, and evaporating the supernatant to obtain a crude extract; (3) preliminarily purifying the crude extract using a solid-phase extraction column to obtain a refined extract; (4) further purifying the refined extract using semi-preparative liquid chromatography to obtain a solution containing microcystin, and then evaporating and drying to obtain the microcystin. The method for extracting microcystin from Anabaena flos-aquae of the present invention has the advantages of stable properties of the toxin-producing strain, single toxin-producing component, high toxin content, good repeatability of extraction and purification, etc., and can obtain a microcystin standard product with high purity and high yield.

Description

Technical Field

[0001] The present invention belongs to the field of biochemical separation and sustainable utilization of resources, and particularly relates to a method for extracting anatoxin-a. Background Art

[0002] The frequent outbreak of cyanobacterial blooms in eutrophic water bodies has become a major environmental problem faced globally. Some cyanobacterial blooms can produce harmful cyanotoxins, posing a serious threat to freshwater ecosystems and human health. Among them, anatoxin-a is a type of small molecule alkaloid cyanobacterial neurotoxin, mainly produced by filamentous cyanobacteria such as Aphanizomenon, Dolichospermum, Oscillatoria, and Pseudanabaena. Long-term exposure or direct drinking of water sources contaminated with toxic algae can cause animal neurotoxicity and even death.

[0003] The anatoxin-a standard sample is an important basic chemical reagent required for the scientific research and water body detection of anatoxin-a. With the extensive and in-depth research on anatoxin-a, the demand for anatoxin-a standard samples by scientific research personnel is increasing day by day. Due to the difficulty in obtaining anatoxin-a-producing samples and the limitations of extraction process technology, there are no reports on the extraction, purification, and preparation of pure anatoxin-a at home and abroad. The anatoxin-a standard sample also depends on imports and is very expensive, resulting in high research funding costs, and it is difficult for general small and medium-sized research units to carry out relevant research work.

[0004] At present, the development of extraction and purification processes for anatoxin-a standard samples has become an urgent problem to be solved. At present, in the extraction and purification processes of anatoxin-a standard samples, it is difficult to obtain anatoxin-a-producing samples in the wild, and they have complex components, low toxin content, and poor repeatability of preparation results. Moreover, due to the special molecular structure of anatoxin-a, the conventional extraction and purification processes have poor adaptability, and the extracted anatoxin-a has defects such as low purity and small quantity. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the above-mentioned deficiencies and defects in the background art, and provide a method for extracting anatoxin-a from Aphanizomenon with a large extraction amount and high purity. To solve the above technical problem, the technical solution proposed by the present invention is as follows:

[0006] A method for extracting anatoxin-a from Aphanizomenon, comprising the following steps:

[0007] (1) Obtaining an anatoxin-a-producing strain of Aphanizomenon;

[0008] (2) Crushing the Aphanizomenon strain obtained in step (1), adding a methanol solution, extracting anatoxin-a under stirring, then performing centrifugation, collecting the supernatant, and evaporating the supernatant to obtain a crude extract;

[0009] (3) The crude extract obtained in step (2) is preliminarily purified using a solid-phase extraction column to obtain a refined extract;

[0010] (4) The refined extract obtained in step (3) is further purified using semi-preparative liquid chromatography to obtain a solution containing microcystin, and then evaporated and dried to obtain the microcystin.

[0011] In the above method for extracting microcystin from Aphanizomenon flos-aquae, preferably, the toxin-producing strain of microcystin is obtained by culturing Aphanizomenon flos-aquae strains indoors. During the culture, aerated culture is carried out using BG11 medium, and sodium nitrate, the nitrogen source in BG11 medium, is replaced with 0.1 - 0.3 g / L of alanine.

[0012] In the above method for extracting microcystin from Aphanizomenon flos-aquae, preferably, the culture temperature is controlled at 20 - 25 °C, the light intensity is 25 - 35 μE, the light-dark ratio is 14 h:10 h, and the aeration rate is 0.1 - 0.3 L / min.

[0013] In the above method for extracting microcystin from Aphanizomenon flos-aquae, preferably, during the aerated culture, the gas introduced is controlled to contain 0.1 - 0.3% (by volume) of CO2 gas.

[0014] The selection of raw materials for cyanotoxin extraction generally follows the following approach: When a cyanotoxin-producing algal bloom breaks out, floating algal masses form on the water surface, and a large amount of cyanotoxin-producing algal raw materials can be obtained by fishing and concentration. However, the frequency of cyanotoxin-producing Aphanizomenon flos-aquae algal blooms is much lower than that of microcystin-producing algal blooms. In addition, the impurity components in wild cyanobacteria are complex, the toxin content is low and unstable, and it is difficult to obtain reproducible results for batch toxin purification. Relatively speaking, the toxin-producing strains cultured indoors have a single composition, a stable toxin production content under specific conditions, and good reproducibility. Based on the characteristics of Aphanizomenon flos-aquae strains, the present invention can obtain toxin-producing strains with higher toxin production and more stable toxin production through indoor culture. The above-obtained toxin-producing strains are centrifuged to collect algal cells in the late logarithmic growth phase, and after low-temperature vacuum freeze-drying, they are stored at low temperature as raw materials for toxin extraction for future use.

[0015] The present invention makes the following key optimizations to the culture medium and culture process conditions adopted: When aerobically culturing using BG11 medium, alanine is used instead of sodium nitrate as the modified nitrogen source, and the gas introduced is controlled to contain CO2 gas with a volume content of 0.1 - 0.3%. More preferably, 0.2 g / L of alanine is used in the BG11 medium instead of 1.5 g / L of sodium nitrate, and the gas introduced is controlled to contain CO2 gas with a volume content of 0.2%. For obtaining extraction materials by culturing Anabaena strains indoors, the present invention improves the toxin content of toxin-producing strains and the toxin production efficiency by optimizing the culture conditions. Specifically, when aerobically culturing BG11 with alanine as the nitrogen source, 0.1 - 0.3% of CO2 is also introduced. By adopting this technical solution, the biomass of algal cells in the pure culture system can be increased (the biomass on the 15th day is increased by 133%), and the toxin content in algal cells per unit dry weight can be increased (the toxin content on the 15th day is increased by 151%). For some cyanobacteria that can directly use CO2 as the inorganic carbon source for photosynthesis, an increase in the CO2 concentration can significantly promote their photosynthesis efficiency and the growth of algal strains. Especially when using flowing CO2, the promotion effect is better. However, the content of CO2 gas needs to be controlled during aerobic culture. If the content is too low, both the biomass and the toxin content will decrease. If the content is too high, the effect is not significant, and the culture cost will increase. In addition, anatoxin is rich in nitrogen elements. Our research shows that alanine, as a small molecule, can be efficiently absorbed and utilized by Anabaena. Alanine can significantly promote the growth of Anabaena, and the matching relationship between alanine and Anabaena is good. Moreover, adding alanine to the culture medium is beneficial to improving the adaptability of anatoxin to alanine and is beneficial to protecting the structural stability of anatoxin through alanine subsequently. In addition, when nitrogen is sufficient, an increase in carbon content will also cause an increase in toxin content.

[0016] In the above method for extracting anatoxin from Anabaena, preferably, the toxin-producing strain is broken by repeatedly freezing and thawing with liquid nitrogen (such as three times). The anatoxin produced by normally growing algal cells mainly exists in the intracellular form, and a small amount is secreted extracellularly. The first key step in extracting anatoxin is to break the algal cells to obtain a crude extract of algal toxin. Different cell-breaking methods have different effects on the extraction of toxins. Our research shows that compared with conventional breaking methods such as glass bead grinding, the repeated freezing and thawing treatment with liquid nitrogen adopted in the present invention results in a higher content of anatoxin finally extracted.

[0017] In the above method for extracting microcystin from Aphanizomenon flos-aquae, preferably, when adding the methanol solution in step (2), the mass concentration of the methanol solution is controlled to be 70-80%, and the addition amount of the methanol solution is controlled to be 50-70 ml of the methanol solution per gram of the toxin-producing strain. More preferably, when adding the methanol solution in step (2), the mass concentration of the methanol solution is controlled to be 70%, and the addition amount of the methanol solution is controlled to be 60 ml of the methanol solution per gram of the toxin-producing strain. Different extraction reagents have obvious effects on the extraction efficiency of the toxin. Our research shows that when the methanol concentration increases from 30% to 70%, the toxin extraction concentration also increases. However, when the methanol concentration increases to 100%, the toxin extraction concentration decreases, and at this time, the impurity content in the eluent is also relatively high. Therefore, a methanol extraction concentration of 70-80% is a better choice.

[0018] In the above method for extracting microcystin from Aphanizomenon flos-aquae, preferably, after centrifugation in step (2), a methanol solution with a mass concentration of 70-80% is added to the centrifugal precipitate for repeated extraction, and then centrifuged to collect the supernatant, which is combined with the supernatant collected from the previous centrifugation. Repeated extraction of the centrifugal precipitate after centrifugation can recycle the residual microcystin in the centrifugal precipitate, with a higher recovery rate.

[0019] In the above method for extracting microcystin from Aphanizomenon flos-aquae, preferably, glacial acetic acid is also added synchronously when adding the methanol solution in step (2), and the volume content of glacial acetic acid in the methanol solution is controlled to be 0.05-0.15%. More preferably, alanine is added when adding glacial acetic acid, and the dosage of alanine is controlled to be 0.5-1.5 times the mass of glacial acetic acid. Considering the specific molecular structure and molecular properties of microcystin in the present invention, our research shows that adding a certain amount of glacial acetic acid during the extraction process of microcystin can greatly improve the stability of microcystin, which is beneficial to obtaining microcystin with higher purity and higher yield in the later stage. However, the dosage of glacial acetic acid should not be too high. In a more preferred scheme, alanine consistent with the components in the culture medium is also added. Microcystin has good compatibility with alanine. Adding alanine and glacial acetic acid together can maintain the extraction environment of the extract, which is more beneficial to improving the stability of microcystin.

[0020] In addition, considering the sensitivity of microcystin to light and heat, the extraction process in step (2) should be carried out under low-temperature and light-proof environmental conditions as much as possible.

[0021] In the above method for extracting microcystin from Anabaena flos-aquae, preferably, in step (3), the solid-phase extraction column is a BUCHI FlashPure EcoFlex C18 packed column. First, the C18 packed column is rinsed with 5% methanol, then microcystin is eluted with 20% methanol. The 20% methanol elution fractions are collected in segments. Finally, 80% methanol is used to remove other impurities on the C18 packed column. The above C18 packed column uses high-quality silica gel and spherical particles with a smaller particle size to ensure a high separation degree for the mixed sample, improve the purity of the fractions, and its sample loading capacity can be extended from milligrams to grams, which is suitable for batch purification and preparation. Since the microcystin molecule has a strong polarity, first, the C18 packed column is rinsed with 5% methanol, then microcystin is eluted with 20% methanol. The 20% methanol elution fractions are collected in segments. The purity and concentration of microcystin in each collected fraction are detected by high-performance liquid chromatography. Finally, 80% methanol is used to remove other impurities on the C18 packed column. The fraction containing microcystin with high purity and concentration is transferred to a rotary evaporation flask, rotary evaporated to nearly dry at 35°C, centrifuged at 12,000 rpm for 15 min to obtain a refined extract, which is dissolved in 10% methanol (containing 0.05% glacial acetic acid by volume concentration and alanine with a mass 1 time that of glacial acetic acid) after freeze-drying.

[0022] In the above method for extracting microcystin from Aphanizomenon flos-aquae, preferably, in step (4), a Waters Atlantis T3 C18 column is used for semi-preparative separation. During separation, the column temperature is controlled at 30 °C, mobile phase A is acetonitrile, mobile phase B is ultrapure water containing 0.05% formic acid by volume concentration, the flow rate is 3.5 mL / min, the detector is a Waters diode array detector, the detection wavelength is 228 nm, and the injection volume is 50 μL. The chromatographic separation gradient conditions are as follows: at 1 min, mobile phase A is 10% and mobile phase B is 90%; at 12 min, mobile phase A is 10% and mobile phase B is 90%; at 13 min, mobile phase A is 90% and mobile phase B is 10%; at 14 min, mobile phase A is 10% and mobile phase B is 90%; at 15 min, mobile phase A is 10% and mobile phase B is 90%. The collection time of the microcystin ATX chromatographic peak is 10.1 - 10.45 min. Since microcystin has a small molecular weight, a relatively large molecular polarity, and strong hydrophilicity, the present invention uses a Waters Atlantis T3 C18 column for semi-preparative separation. This chromatographic column uses a triple-bonded C18 alkyl bonding with a low ligand density and also has a more effective end-capping. The greatest advantage is that it can improve the retention ability of polar small molecule compounds and is compatible with a 100% aqueous solution mobile phase. When optimizing the elution conditions using the Waters Atlantis T3 C18 chromatographic column, the target compound can be eluted with a low proportion of the organic phase. The present invention sets 10% acetonitrile to separate the target compound, and then the proportion is increased to 90% to elute the impurities in the sample. The chromatographic peak of ATX is collected on the semi-preparative chromatograph, and the collected solutions from multiple injections are combined, transferred to a rotary evaporation flask, and after rotary evaporation, an appropriate amount of MilliQ H2O is added for dissolution and freeze-drying respectively.

[0023] In the above method for extracting microcystin from Aphanizomenon flos-aquae, preferably, the microcystin obtained by evaporation and drying is measured for its quality and purity by HPLC-UV. The results show that the purity is above 95%, meeting the requirements of scientific research work for the toxin purity.

[0024] Some strains of Aphanizomenon flos-aquae can produce microcystins. Currently, the research and utilization of Aphanizomenon flos-aquae are almost blank. Extracting and utilizing microcystins from Aphanizomenon flos-aquae is a brand-new research and exploration direction. Based on the characteristics of Aphanizomenon flos-aquae, this invention adopts a special culture medium and culture method to obtain a toxin-producing strain with higher toxin production and more stable toxin production. Then, the toxin-producing strain is broken and extracted. Based on the molecular structure and molecular characteristics of microcystins, in a more preferred scheme, glacial acetic acid and alanine are added to methanol, which is beneficial to improving the structural stability of microcystins and is conducive to obtaining microcystins with higher purity and higher extraction yield in the later stage. Finally, solid-phase extraction columns and semi-preparative liquid chromatography are used to extract and purify microcystins. By optimizing the extraction conditions of solid-phase extraction columns and semi-preparative liquid chromatography, microcystin standards with high purity and high extraction yield can be obtained. This invention uses laboratory pure culture of toxin-producing algal strains to extract microcystins, and establishes a batch purification method capable of obtaining high-purity microcystin reference substances, providing a basis for subsequent research on microcystins, and also providing a reference for the preparation of other different isomeric microcystins.

[0025] Compared with the prior art, the advantages of this invention are as follows:

[0026] 1. The method for extracting microcystins from Aphanizomenon flos-aquae in this invention uses laboratory pure culture of the toxin-producing algal strain Aphanizomenon flos-aquae, then extracts by breaking the toxin-producing strain, and finally uses solid-phase extraction columns and semi-preparative liquid chromatography for two purifications. The toxin-producing algal strain has stable properties, single toxin-producing components, high toxin content, and good repeatability of extraction and purification, and can obtain microcystin standards with high purity (the purity can be as high as over 98%) and high yield.

[0027] 2. The method for extracting and purifying microcystins in this invention is simple to operate and low in cost, which is conducive to realizing the small-batch (mg level) production of microcystins. Description of the Drawings

[0028] In order to more clearly illustrate the technical solutions in the embodiments of this invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of this invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0029] Figure 1 Growth curves of Aphanizomenon flos-aquae strains producing microcystins under the culture conditions of alanine and 0.2% CO2 for Example 1 and Comparative Example 1.

[0030] Figure 2The content of microcystin in Microcystis aeruginosa strain under the culture conditions of alanine and 0.2% CO2 in Example 1 and Comparative Example 1.

[0031] Figure 3 Comparison of the effects of extracting microcystin by different cell disruption methods in Example 1 and Comparative Example 2.

[0032] Figure 4 Comparison of the effects of extracting microcystin with different extraction reagents in Example 1 and Comparative Example 3.

[0033] Figure 5 Chromatogram (A) before purification and chromatogram (B) after purification by semi-preparative liquid chromatography in Example 1. Detailed implementation manners

[0034] To facilitate the understanding of the present invention, the present invention will be described more comprehensively and meticulously below in conjunction with the accompanying drawings of the specification and preferred embodiments, but the protection scope of the present invention is not limited to the following specific embodiments.

[0035] Unless otherwise defined, all the technical terms used hereinafter have the same meanings as those commonly understood by those skilled in the art. The technical terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the protection scope of the present invention.

[0036] Unless otherwise specifically stated, all kinds of raw materials, reagents, instruments and equipment used in the present invention can be obtained through the market or can be prepared by existing methods.

[0037] Example 1:

[0038] A method for extracting microcystin from Microcystis aeruginosa, comprising the following steps:

[0039] (1) Raw material acquisition: Select the Anabaena circinalis strain that produces anatoxin (select the toxin-producing strain), culture the strain using the modified BG11 medium, and simultaneously culture it by introducing CO2 gas with a volume concentration of 0.2%. The modified BG11 medium uses alanine as the N source, and the specific formula is: alanine (Ala) 0.2 g / L, K2HPO4·3H2O 0.04 g / L, MgSO4·7H2O 0.075 g / L, CaCl2·2H2O 0.036 g / L, citric acid 0.006 g / L, ammonium ferric citrate 0.006 g / L, EDTA 0.001 g / L, Na2CO3 0.02 g / L, A5+Co stock solution 1 mL, distilled water 1000 mL. The composition of the A5+Co stock solution is: H3BO3 2.86 g, MnCl2·2H2O 1.81 g, ZnSO4·7H2O 0.222 g, CuSO4·5H2O 0.079 g, NaMoO4·2H2O 0.390 g, Co(NO3)2·6H2O 0.049 g, distilled water 1000 mL. The culture temperature is set at 20°C, the light intensity is 30 μE, the light-dark ratio is 14 h:10 h, and the ventilation volume is 0.2 L / min. A large number of Anabaena circinalis cells can be obtained in a relatively short time. Centrifuge at 6000 rpm for 8 min to collect the algal cells in the logarithmic growth phase (cultured for 15 - 18 days). The endotoxin content in the algal cells is the highest at this growth stage. Vacuum freeze-dry the collected algal cells to obtain dry algal powder.

[0040] (2) Crude extraction of anatoxin ATX: Weigh 6.82 g of algal powder, break the algal cells by repeatedly freezing and thawing with liquid nitrogen three times, add 70% methanol (containing 0.1% v / v glacial acetic acid) at a ratio of 60 ml / g to dissolve, add a magnetic stir bar, and stir at 200 rpm on a magnetic stirrer at room temperature for 1 h to promote the precipitation of anatoxin. Centrifuge at 8000 rpm for 15 min at room temperature to collect supernatant I. Add 70% methanol (containing 0.1% v / v glacial acetic acid) to the precipitate, repeat the above extraction step once to obtain supernatant II, and combine the extracted supernatants I and II. Use a rotary evaporator to vacuum-rotate and concentrate the supernatant at 35°C. Centrifuge the preliminarily concentrated extract at 12000 rpm for 20 min at 10°C to remove impurities to obtain the crude extract of anatoxin.

[0041] (3) Preliminary purification of anatoxin

[0042] Using a BUCHI FlashPure EcoFlex C18 packed column, the crude extract was injected into the pre-equilibrated C18 column. The extraction column was rinsed with 50 ml of 5% methanol, and then 150 ml of 20% methanol was used to elute the microcystin. Each 30 ml of the eluate was collected as a fraction. The purity and concentration of microcystin in each collected fraction were detected by high performance liquid chromatography. Finally, 100 ml of 80% methanol was used to remove other impurities on the C18 column. The purity and content of microcystin in the 20% methanol elution fractions collected in segments were detected. The liquid chromatography detection conditions were as follows: mobile phase A: acetonitrile (ACN); mobile phase B: ultrapure water (0.05% v / v formic acid, FA), column temperature: 30 °C, flow rate: 1.0 mL / min, detection wavelength: 228 nm, detector: ultraviolet spectrophotometric detector. The optimal elution conditions were as follows: 0 - 2 min, 5% mobile phase A; 2 - 15 min, 5 - 15% mobile phase A; 15 - 18 min, 15 - 90% mobile phase A; 18 - 20 min, 5% mobile phase A. The fraction containing high purity and concentration of microcystin was transferred to a rotary evaporation flask, rotary evaporated to nearly dry at 35 °C, centrifuged at 12000 rpm for 15 min to obtain the refined extract, and dissolved in 10% methanol (containing 0.05% v / v glacial acetic acid) after freeze-drying.

[0043] (4) Semi-preparative liquid chromatography separation of microcystin ATX

[0044] The ATX standard was purchased from Novakits, France. All chromatographic reagents used in the experiment were of HPLC grade. The specific chromatographic conditions were as follows:

[0045] Chromatographic column: Waters Atlantis T3 C18 column (250 mm * 8 mm, 5 μm);

[0046] Column temperature: 30 °C;

[0047] Mobile phase A: acetonitrile (ACN); mobile phase B: ultrapure water (0.05% v / v formic acid);

[0048] Flow rate: 3.5 mL / min;

[0049] Detector: Waters diode array detector (PDA), detection wavelength: 228 nm, injection volume: 50 μL;

[0050] The chromatographic separation gradient conditions are shown in Table 1 below:

[0051] Table 1: Chromatographic separation gradient conditions

[0052] Time (min) Flow rate (ml / min) Mobile phase A Mobile phase B 1 3.5 10 90 12 3.5 10 90 13 3.5 90 10 14 3.5 10 90 15 3.5 10 90

[0053] The chromatographic peak collection time is ATX (10.1 - 10.45 min). To improve the purity of the collected fractions, when collecting the target compound, the front and back segments of the chromatographic peak are preferably discarded as this part may contain a small amount of impurity components. The collected liquids from multiple injections are combined, transferred to a rotary evaporation flask, rotary evaporated, and then transferred to a sample vial for freeze-drying. The mass of the product is weighed as 3.01 mg. From the chromatogram Figure 5 It can be seen that after purification by semi-preparative chromatography, the chromatographic peak of microcystin is single and no obvious impurity peak is observed.

[0054] (5) HPLC-UV purity analysis of microcystin

[0055] HPLC-UV was used to determine the mass and purity of the extracted microcystin. When separating using a Waters Symmetry C18 column, 5 - 15% acetonitrile was set to separate the target compound, and then the proportion of acetonitrile was increased to 90% to elute the impurities in the sample.

[0056] Chromatographic column: Waters Symmetry C18 column (250 mm * 4.6 mm, 5 μm);

[0057] Column temperature: 30 °C;

[0058] Mobile phase A: acetonitrile (CAN); Mobile phase B: ultrapure water (0.05% v / v formic acid, FA);

[0059] Flow rate: 1.0 mL / min;

[0060] Detector: Waters diode array detector (PDA), detection wavelength: 228 nm, injection volume: 50 μL;

[0061] The chromatographic separation gradient conditions are shown in Table 2 below:

[0062] Table 2: Chromatographic separation gradient conditions

[0063] Time (min) Flow rate (ml / min) Mobile phase A Mobile phase B 0 1.0 5 95 2 1.0 5 95 15 1.0 15 85 20 1.0 90 0 21 1.0 90 0 23 1.0 5 95

[0064] The chromatographic purity of microcystin ATX calculated by the chromatographic peak area normalization method is 98.6%, meeting the requirements of scientific research work for the purity of the toxin.

[0065] Comparative Example 1:

[0066] This comparative example is different from Example 1 in that when culturing the toxin-producing strain using BG11 medium in step (1), air is directly introduced for culturing.

[0067] As Figure 1 、 Figure 2As shown in the figure, compared with the direct air culture in this comparative example and the culture with 0.2% CO2 gas introduced using the improved BG11 medium in Example 1, when using alanine as the nitrogen source and introducing 0.2% CO2 gas for culture simultaneously, it can increase the biomass of algal cells in the pure culture system (the biomass increased by 133% on the 15th day), and can increase the content of endotoxin in algal cells per unit dry weight (the toxin content increased by 151% on the 15th day). Therefore, using the alanine-modified medium and introducing 0.2% CO2 gas for culture to obtain the toxin-producing algal strain is a better condition.

[0068] Comparative Example 2:

[0069] Compared with Example 1, this comparative example is different in that the crushing methods in step (2) are different, and no treatment, ultrasonic ice bath for 15 min, and glass bead grinding are respectively used.

[0070] As Figure 3 shown, the content of toxin extraction was the highest when using liquid nitrogen freeze-thaw three times in Example 1, the extraction effect of glass bead grinding was the second, and there was no obvious difference in the effect of ultrasonic ice bath crushing and the untreated group.

[0071] Comparative Example 3:

[0072] Compared with Example 1, this comparative example is different in that the extraction reagents used to extract anatoxin in step (2) are different, and water (0.1% glacial acetic acid), 30% methanol (0.1% glacial acetic acid), 50% methanol (0.1% glacial acetic acid), 70% methanol (0.1% glacial acetic acid), and 100% methanol (0.1% glacial acetic acid) are respectively used as extraction reagents.

[0073] As Figure 4 shown, when the methanol ratio increases from 30% to 70%, the toxin extraction concentration also increases accordingly. When it increases to 100% methanol, the toxin extraction concentration decreases, and at this time, the impurity content in the eluent is also relatively high. Therefore, the reagent of 70% methanol (0.1% glacial acetic acid) used in Example 1 has the best effect.

[0074] Example 2:

[0075] A method for extracting anatoxin from Aphanizomenon flos-aquae, comprising the following steps:

[0076] (1) Raw material acquisition: Select the Anabaena circinalis strain that produces anatoxin-a, and use the modified BG11 medium for culturing the strain. At the same time, culture it by introducing CO2 gas with a volume concentration of 0.2%. The modified BG11 medium uses alanine as the N source, and the specific formula is as follows: alanine (Ala) 0.2 g / L, K2HPO4·3H2O 0.04 g / L, MgSO4·7H2O 0.075 g / L, CaCl2·2H2O 0.036 g / L, citric acid 0.006 g / L, ammonium ferric citrate 0.006 g / L, EDTA 0.001 g / L, Na2CO3 0.02 g / L, A5+Co stock solution 1 mL, distilled water 1000 mL. The components of the A5+Co stock solution are: H3BO3 2.86 g, MnCl2·2H2O 1.81 g, ZnSO4·7H2O 0.222 g, CuSO4·5H2O 0.079 g, NaMoO4·2H2O 0.390 g, Co(NO3)2·6H2O 0.049 g, distilled water 1000 mL. The culture temperature is set at 20 °C, the light intensity is 30 μE, the light-dark ratio is 14 h:10 h, and the aeration rate is 0.2 L / min. A large number of Anabaena circinalis cells can be obtained in a relatively short time. Centrifuge at 6000 rpm for 8 min to collect the algal cells in the logarithmic growth phase (cultured for 15 - 18 days). The endotoxin content in the algal cells is the highest at this growth stage. Vacuum freeze-dry the collected algal cells to form dry algal powder.

[0077] (2) Crude extraction of anatoxin-a: Weigh 6.82 g of algal powder, break the algal cells by repeatedly freezing and thawing with liquid nitrogen three times, add 70% methanol (containing 0.1% acetic acid by volume and alanine with a mass 1 time that of acetic acid) at a ratio of 60 ml / g for dissolution, add a magnetic stirrer, and stir at 200 rpm on a magnetic stirrer at room temperature for 1 h to promote the precipitation of anatoxin-a. Centrifuge at 8000 rpm for 15 min at room temperature to collect the supernatant I. Add 70% methanol (containing 0.1% acetic acid by volume and alanine with a mass 1 time that of acetic acid) to the precipitate, repeat the above extraction step once to obtain supernatant II, and combine the extracted supernatants I and II. Use a rotary evaporator to vacuum rotary evaporate and concentrate the supernatant at 35 °C. Centrifuge the preliminarily concentrated extract at 10 °C and 12000 rpm for 20 min to remove impurities, and obtain the crude extract of anatoxin-a.

[0078] Steps (3) - (4): The same as steps (3) - (4) in Example 1.

[0079] Adopt the same method for testing the quality and purity of the anatoxin-a product as in Example 1. After measurement, in this example, the quality of the obtained product is 3.12 mg, and the purity is 98.8%.

[0080] Example 3:

[0081] A method for extracting microcystin from Aphanizomenon flos-aquae, comprising the following steps:

[0082] (1) Raw material acquisition: Select an Aphanizomenon flos-aquae strain that produces microcystin, culture the strain using a modified BG11 medium, and simultaneously introduce CO2 gas with a volume concentration of 0.2% for culturing. The modified BG11 medium uses alanine as the N source, and the specific formula is: alanine (Ala) 0.2 g / L, K2HPO4·3H2O 0.04 g / L, MgSO4·7H2O 0.075 g / L, CaCl2·2H2O 0.036 g / L, citric acid 0.006 g / L, ammonium ferric citrate 0.006 g / L, EDTA 0.001 g / L, Na2CO3 0.02 g / L, A5+Co stock solution 1 mL, distilled water 1000 mL. The composition of the A5+Co stock solution is: H3BO3 2.86 g, MnCl2·2H2O 1.81 g, ZnSO4·7H2O 0.222 g, CuSO4·5H2O 0.079 g, NaMoO4·2H2O 0.390 g, Co(NO3)2·6H2O 0.049 g, distilled water 1000 mL. The culture temperature is set at 20 °C, the light intensity is 30 μE, the light-dark ratio is 14 h:10 h, and the ventilation volume is 0.2 L / min. A large number of Aphanizomenon flos-aquae cells can be obtained in a relatively short time. Centrifuge at 6000 rpm for 8 min to collect the algal cells in the logarithmic growth phase (cultured for 15 - 18 days). The endotoxin content in the algal cells is the highest at this growth stage. Vacuum freeze-dry the collected algal cells to form dry algal powder.

[0083] (2) Crude extraction of microcystin ATX: Weigh 6.82 g of algal powder, break the algal cells by repeatedly freezing and thawing with liquid nitrogen three times, add 70% methanol (containing 0.05% v / v glacial acetic acid and 1.5 times the mass of glacial acetic acid of alanine) at a ratio of 60 ml / g for dissolution, add a magnetic stirrer, and stir at 200 rpm on a magnetic stirrer at room temperature for 1 h to promote the precipitation of microcystin. Centrifuge at 8000 rpm for 15 min at room temperature to collect the supernatant I. Add 70% methanol (containing 0.05% v / v glacial acetic acid and 1.5 times the mass of glacial acetic acid of alanine) to the precipitate, repeat the above extraction step once to obtain the supernatant II, and combine the extracted supernatants I and II. Use a rotary evaporator to vacuum rotary evaporate and concentrate the supernatant at 35 °C. Centrifuge the preliminarily concentrated extract at 10 °C and 12000 rpm for 20 min to remove impurities to obtain the crude microcystin extract.

[0084] Steps (3) - (4): The same as steps (3) - (4) in Example 1.

[0085] Using the same method for testing the quality and purity of the microcystin product as in Example 1, it was determined that in this example, the product quality obtained was 3.18 mg and the purity was 98.8%.

[0086] Example 4:

[0087] A method for extracting microcystin from Aphanizomenon flos-aquae, comprising the following steps:

[0088] (1) Raw material acquisition: Selecting an Aphanizomenon flos-aquae strain that produces microcystin, culturing the strain using the traditional BG11 medium, and simultaneously introducing a CO2 gas with a volume concentration of 0.2% for culturing. The formula of the traditional BG11 medium is: NaNO3 1.5 g / L, K2HPO4·3H2O 0.04 g / L, MgSO4·7H2O 0.075 g / L, CaCl2·2H2O 0.036 g / L, citric acid 0.006 g / L, ammonium ferric citrate 0.006 g / L, EDTA 0.001 g / L, Na2CO3 0.02 g / L, A5+Co stock solution 1 mL, distilled water 1000 mL. The components of the A5+Co stock solution are: H3BO3 2.86 g, MnCl2·2H2O 1.81 g, ZnSO4·7H2O 0.222 g, CuSO4·5H2O 0.079 g, NaMoO4·2H2O 0.390 g, Co(NO3)2·6H2O 0.049 g, distilled water 1000 mL. The culture temperature is set at 20 °C, the light intensity is 30 μE, the light-dark ratio is 14 h:10 h, and the ventilation rate is 0.2 L / min. A large number of Aphanizomenon flos-aquae cells can be obtained in a relatively short time. Centrifuge at 6000 rpm for 8 min to collect the algal cells in the logarithmic growth phase (cultured for 15 - 18 days). The endotoxin content in the algal cells is the highest at this growth stage. Vacuum freeze-dry the collected algal cells into dry algal powder.

[0089] Steps (2) - (4): The same as steps (2) - (4) in Example 1.

[0090] Using the same method for testing the quality and purity of the microcystin product as in Example 1, it was determined that in this example, the product quality obtained was 2.92 mg and the purity was 98.5%.

Claims

1. A method for extracting microcystin from Aphanizomenon flos-aquae, characterized in that, It includes the following steps: (1) Obtain the toxigenic strain of Anabaena circinalis, a strain of Aphanizomenon flos-aquae; (2) Crush the Aphanizomenon flos-aquae strain obtained in step (1), add a methanol solution, extract anatoxin under stirring, then perform centrifugation, collect the supernatant, and evaporate the supernatant to obtain a crude extract; (3) Use a solid-phase extraction column to preliminarily purify the crude extract obtained in step (2) to obtain a refined extract; (4) Further purify the refined extract obtained in step (3) using semi-preparative liquid chromatography to obtain a solution containing anatoxin, and then evaporate and dry it to obtain the anatoxin; The toxigenic strain of anatoxin is obtained by culturing the Aphanizomenon flos-aquae strain indoors. During the culture, it is aerobically cultured using BG11 medium, and 0.1 - 0.3 g / L of alanine is used to replace sodium nitrate, the nitrogen source in BG11 medium; When adding the methanol solution in step (2), glacial acetic acid is also added simultaneously, and the volume content of glacial acetic acid in the methanol solution is controlled to be 0.05 - 0.15%; when adding glacial acetic acid, alanine is also added, and the dosage of alanine is controlled to be 0.5 - 1.5 times the mass of glacial acetic acid.

2. The method for extracting microcystin from Aphanizomenon flos-aquae according to claim 1, characterized in that, Control the culture temperature to be 20 - 25 °C, the light intensity to be 25 - 35 μE, the light-dark ratio to be 14 h:10 h, and the aeration rate to be 0.1 - 0.3 L / min.

3. The method for extracting microcystin from Aphanizomenon flos-aquae according to claim 1, characterized in that, During the aerobic culture, control the gas introduced to contain 0.1 - 0.3% (by volume) of CO2 gas.

4. The method for extracting microcystin from Arthrospira platensis according to any one of claims 1-3, characterized in that, When crushing the toxigenic strain, use the method of repeated freezing and thawing with liquid nitrogen for crushing.

5. The method for extracting microcystin from Aphanizomenon flos-aquae according to any one of claims 1-3, characterized in that, When adding the methanol solution in step (2), control the mass concentration of the methanol solution to be 70 - 80%, and the addition amount of the methanol solution is controlled to be 50 - 70 ml of methanol solution added per gram of the toxigenic strain.

6. The method for extracting microcystin from Aphanizomenon flos-aquae according to any one of claims 1-3, characterized in that, In step (3), the solid-phase extraction column is a BUCHI FlashPure EcoFlex C18 packed column. First, wash the C18 packed column with 5% methanol, then elute anatoxin with 20% methanol, fractionally collect the 20% methanol elution fractions, and finally use 80% methanol to remove other impurities on the C18 packed column.

7. The method for extracting microcystin from Aphanizomenon flos-aquae according to any one of claims 1-3, characterized in that, In step (4), use a Waters Atlantis T3 C18 column for semi-preparative separation. During the separation, control the column temperature to be 30 °C, mobile phase A to be acetonitrile, mobile phase B to be ultrapure water containing 0.05% (by volume) of formic acid, the flow rate to be 3.5 mL / min, the detector to be a Waters diode array detector, the detection wavelength to be 228 nm, and the injection volume to be 50 μL; the chromatographic separation gradient conditions are as follows: at 1 min, mobile phase A is 10%, mobile phase B is 90%; at 12 min, mobile phase A is 10%, mobile phase B is 90%; at 13 min, mobile phase A is 90%, mobile phase B is 10%; at 14 min, mobile phase A is 10%, mobile phase B is 90%; at 15 min, mobile phase A is 10%, mobile phase B is 90%; the collection time for the anatoxin ATX chromatographic peak is 10.1 - 10.45 min.

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