CN114480535B - Preparation method of microcystin-LW - Google Patents
Preparation method of microcystin-LW Download PDFInfo
- Publication number
- CN114480535B CN114480535B CN202210258021.8A CN202210258021A CN114480535B CN 114480535 B CN114480535 B CN 114480535B CN 202210258021 A CN202210258021 A CN 202210258021A CN 114480535 B CN114480535 B CN 114480535B
- Authority
- CN
- China
- Prior art keywords
- microcystin
- solution
- incubator
- algae
- eluent
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/50—Cyclic peptides containing at least one abnormal peptide link
- C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
- C07K7/56—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation not occurring through 2,4-diamino-butanoic acid
-
- 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
- C12N1/20—Bacteria; Culture media therefor
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Biophysics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a preparation method of microcystin-LW, relates to the technical field of biopharmaceuticals, and solves the problems that a plurality of separation and purification steps are needed for preparing microcystins, the preparation efficiency and the yield are low, the yields of different microcystin types are unstable, and the microcystin-LW cannot be prepared in the prior art. The method comprises adding nutrient substances into water to obtain culture solution; adding microcystis aeruginosa into the culture solution and placing the microcystis aeruginosa into a incubator; placing the incubator in an illumination incubator, culturing to a mature period, and extracting by suction filtration to obtain algae cells; freeze thawing and ultrasonic extracting are carried out on the algae cells to obtain an extracting solution; pouring the extracting solution into an extraction column for eluting for a plurality of times, and collecting the eluting solution; separating the eluent by preparative chromatography, rotary evaporating, and collecting solid powder; in step 3, the pH value is detected, and when the pH value reaches a threshold value, phosphorus is added. The method can be used for preparing microcystin-LW.
Description
Technical Field
The invention relates to the technical field of biological pharmacy, in particular to a preparation method of microcystin-LW.
Background
The algae toxins are pollutants which have highest occurrence frequency and are most serious in hazard in the explosion of the harmful cyanobacteria bloom, and the algae toxins produced by the cyanobacteria are currently becoming global environmental problems. Among toxic blue algae, microcystis aeruginosa is often expressed as a dominant species, is a main algae species for producing blue algae bloom in the world today, belongs to the most common toxic blue algae widely distributed in lakes, ponds and reservoirs in different warm areas of the world, and can produce hepatotoxin microcystis toxins (MCs) with strong toxicity. The microcystin is a ring-shaped heptapeptide compound with biological activity, is an intracellular toxin generated by water bloom blue algae, is synthesized in cells, is released into water after the cells are broken, has a strong cancer promotion effect, and is a potential hazard to the safety of aquatic organisms and human drinking water and human health.
At present, the microcystins are found to be more than 90, but the microcystins are most studied as microcystins-LR, microcystins-RR, microcystins-YR, microcystins-LA, microcystins-LY, microcystins-LW, microcystins-LF and the like. The preparation of microcystins is difficult due to the limitations of preparation methods and techniques, and the price of pure MCs is up to $500,000 per gram.
In the prior art, the microcystins are prepared mainly by harvesting outdoor bloom blue algae or indoor cultured microcystins and freeze-drying to prepare algae powder, and then separating and purifying to prepare microcystins, for example, chinese patent applications CN101974080A, CN01145121.1 and CN1687114 respectively disclose the preparation of microcystins-LR and microcystins-RR, and Chinese patent application CN101168561 discloses the preparation of MC-LR, microcystins-YR and microcystins-RR microcystins.
However, there are some disadvantages to the above: the outdoor bloom-forming cyanobacteria or the indoor cultured copper green microcapsule is not subjected to purification culture, a plurality of algae species are mixed, so that more impurities are caused, a plurality of separation and purification steps are needed, the preparation efficiency is low, and the yield is low; due to the variation of the population and community of the collected samples, the yields of different microcystins are unstable, even algae degradation occurs, and no microcystins are produced; microcystin-LW could not be prepared.
Disclosure of Invention
In view of the above analysis, the present invention aims to provide an efficient, green, simple and convenient method for preparing microcystin-LW, which solves the problems that the preparation of microcystin in the prior art requires a plurality of separation and purification steps, the preparation efficiency and yield are low, the yield of different microcystin types is unstable, and the microcystin-LW cannot be prepared.
The aim of the invention is mainly realized by the following technical scheme:
the invention provides a preparation method of microcystin-LW, which comprises the following steps:
step 1: adding nutrient substances into water, wherein the composition of the nutrient substances comprises the following components in percentage by mass: 5.4 to 6.6mg/L of citric acid and 5.4 to 6.6mg/L, naNO of ferric citrate amine 3 540～660mg/L、 MgSO 4 ·7H 2 O 27～33mg/L、K 2 HPO 4 14.0～17.2mg/L、CaCl 2 ·2H 2 O 34.2～41.8mg/L、Na 2 CO 3 18～22mg/L、H 3 BO 3 2.57～3.15mg/L、MnCl 2 ·4H 2 O 1.63～1.99mg/L、ZnSO 4 ·7H 2 O 0.202～0.242mg/L、Na 2 MnO 4 ·2H 2 O 0.351～0.431mg/L、CuSO 4 ·5H 2 O0.071-0.087 mg/L and Co (NO) 3 ) 2 ·6H 2 O0.044-0.054 mg/L, cesium nitrate (CsNO) 3 ) The addition amount is 8.75-10.75 mg/L, and the culture solution is obtained;
step 2: adding microcystis aeruginosa into the culture solution, and placing the culture solution containing microcystis aeruginosa into a incubator;
step 3: placing the incubator in an illumination incubator, culturing to a mature period, and obtaining algae cells through centrifugal suction filtration;
step 4: freeze thawing is carried out on algae cells, and then methanol aqueous solution is added for ultrasonic extraction to obtain extract;
step 5: pouring the extract into activated C 18 The solid phase extraction column is eluted for a plurality of times by adopting methanol eluent until pigment is removed, and eluent is collected;
step 6: separating the eluent by preparative chromatography, rotationally evaporating, and collecting solid powder to obtain microcystin-LW;
in the process of the step 3, the pH value is detected, and when the pH value reaches a threshold value, phosphorus element is added.
Further, in the step 1, the raw material of the phosphorus element is yellow phosphorus, and the adding amount of the yellow phosphorus is 10-15 mg/L.
Further, in the step 2, the volume ratio of the microcystis aeruginosa to the culture solution is 1:49 to 59.
Further, the method comprises the steps of,in the step 3, the temperature of the illumination incubator is 25-30 ℃, and the illumination is 30-35 mu mol/m 2 /s。
Further, in step 4, the freeze thawing includes the steps of:
step 41: freezing the algae cells in a refrigerator at the temperature of minus 25 ℃ to minus 30 ℃;
step 42: thawing the frozen algal cells;
step 43: repeating steps 41 to 42 at least 2 to 3 times.
Further, in step 4, the aqueous methanol solution is required to cover the algal cells, so that the algal cells are immersed in the aqueous methanol solution.
Further, in step 4, the ultrasonic extraction further comprises the following steps:
and centrifuging the extract and filtering the extract with a filter membrane.
Further, in step 4, the volume ratio of methanol to water in the aqueous methanol solution is 1:4 to 4.5.
Further, in the step 5, the addition amount of the methanol eluent is 8-10 mL of eluent is added to each 1mL of the extracting solution.
Further, in the step 6, the temperature of the rotary evaporation is 40-45 ℃ and the air pressure is 337-370 mbar.
Compared with the prior art, the invention has at least one of the following beneficial effects:
a) The preparation method of microcystin-LW provided by the invention takes purified microcystin (FACHB-1752) as a preparation algae seed, the algae seed is utilized to have the capability of synthesizing microcystin-LW, microcystin-LW can be prepared, cs element is increased through the improvement of culture solution formula and step optimization, the growth and toxigenic efficiency of microcystis aeruginosa can be improved, and the degradation phenomenon of microcystis aeruginosa is basically avoided.
B) The preparation method of microcystin-LW provided by the invention has the advantages of low concentration of CsNO 3 Can promote the growth and photosynthesis of algae, strengthen photosynthesis, promote the increase of C element in algae cells,so that more C element participates in calvin circulation or glycolysis, and the carbon skeleton of the microcystin is increased to promote the synthesis of the microcystin; when the increase in element C, i.e., the relative amount of N decreases, exacerbating the N limitation situation, such that these carbon scaffolds in the algal cells are more applied to the synthesis of other nitrogen-depleted microcystins MC-LW (C: N ratio=6.8), the nitrogen-enriched MC-LR (C: N ratio=4.9) is relatively decreased, thereby enabling increased production of MC-LW.
C) According to the preparation method of microcystin-LW, provided by the invention, through purifying and culturing microcystin aeruginosa, impurity interference caused by other algae metabolites can be reduced, the types and the yields of microcystins are stabilized, and the types and the yields of microcystins are prevented from being changed due to population and community changes.
D) According to the preparation method of microcystin-LW, yellow phosphorus is added as a storage element of a nutrient element phosphorus element, so that chemical reaction can be automatically carried out to generate phosphate in the process of growing a large amount of algae, the phosphorus element required by growth is supplemented, the growth of the algae is promoted, and the stable period is prolonged.
Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objectives and other advantages of the invention may be realized and attained by the structure particularly pointed out in the written description and drawings.
Drawings
The drawings are only for the purpose of illustrating the invention and are not to be construed as limiting the invention, like reference numerals referring to like parts throughout the several views.
FIG. 1 is a schematic structural diagram of a preparation device used in the preparation method of microcystin-LW provided by the invention, wherein nutrient solution is manually supplemented;
FIG. 2 is a schematic structural diagram of a preparation device used in the preparation method of microcystin-LW provided by the invention, wherein the nutrient solution is automatically supplemented;
FIG. 3a is a graph showing the periodic variation of algal density and microcystin-LW content according to the first embodiment and the first comparative embodiment of the present invention;
FIG. 3b is a bar graph showing the periodic variation of algae density and microcystin-LW content for example I and comparative example I of the present invention;
FIG. 4 is a mass spectrum of microcystin-LW according to the first embodiment of the present invention.
Reference numerals:
1-a incubator; 2-a nutrient solution reservoir; 3-sampling tube; 4-sampling valve; 5-a fluid replacement pipe; 6-a fluid supplementing valve; 7-barrel setting; 8-a rotary drum; 9-a liquid inlet hole; 10-a liquid outlet hole; 11-a first connection tube; 12-a second connecting tube; 13-a reservoir; 14-a breathable layer; 15-temperature sensor.
Detailed Description
For the purposes of making the objects, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions of the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is apparent that the described embodiments are some embodiments of the present application, but not all embodiments. All other embodiments, which can be made by one of ordinary skill in the art without undue burden from the present disclosure, are within the scope of the present disclosure.
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. The term "and/or" as used in this specification includes any and all combinations of one or more of the associated listed items. In addition, the technical features of the different embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.
The invention provides a preparation method of microcystin-LW (microcystin-LW), which is shown in figures 1 to 4 and comprises the following steps:
step 1: adding nutrient substances into pure water, wherein the composition of the nutrient substances comprises the following components in percentage by mass: 5.4 to 6.6mg/L of citric acid and 5.4 to 6.6mg/L, naNO of ferric citrate amine 3 540～660mg/L、 MgSO 4 ·7H 2 O 27～33mg/L、K 2 HPO 4 14.0～17.2mg/L、CaCl 2 ·2H 2 O 34.2～41.8mg/L、Na 2 CO 3 18～22mg/L、H 3 BO 3 2.57～3.15mg/L、MnCl 2 ·4H 2 O 1.63～1.99mg/L、ZnSO 4 ·7H 2 O 0.202～0.242mg/L、Na 2 MnO 4 ·2H 2 O 0.351～0.431mg/L、CuSO 4 ·5H 2 O0.071-0.087 mg/L and Co (NO) 3 ) 2 ·6H 2 O0.044-0.054 mg/L, cesium nitrate (CsNO) 3 ) Sterilizing at 120-130 deg.c in high pressure steam kettle in the amount of 8.75-10.75 mg/L for 30-40 min to obtain culture liquid;
step 2: adding microcystis aeruginosa (FACHB-1752) to the culture broth, and placing the culture broth containing microcystis aeruginosa into a incubator (e.g., a petri dish);
step 3: placing the incubator in an illumination incubator, culturing for 2-3 weeks until the mature period, and obtaining algae cells through centrifugal suction filtration;
step 4: freeze thawing the algae cells, and adding a methanol aqueous solution for ultrasonic extraction for 10-15 min to obtain an extracting solution, wherein the methanol aqueous solution is prepared by regulating the polarity of methanol and water so as to better extract microcystin in the algae cells;
step 5: pouring the extract into activated C 18 A solid phase extraction column (1 g/6 mL), methanol eluent containing 0.1% -0.5% formic acid is adopted for eluting for multiple times until pigment is removed, and eluent is collected;
step 6: separating the eluent by preparative chromatography, rotationally evaporating, and collecting solid powder to obtain microcystin-LW;
during the above step 3, the pH is detected, and when the pH reaches a threshold value (for example, ph=6 to 7), phosphorus is added.
Compared with the prior art, the preparation method of microcystin-LW provided by the invention takes purified microcystin aeruginosa (FACHB-1752) as a preparation algae seed, and the algae seed is utilized to have the capability of synthesizing microcystin-LW, so that microcystin-LW can be prepared, and through improvement of culture solution formula and step optimization, cs element is increased, the growth and toxigenic efficiency of microcystis aeruginosa can be improved, and basically no degradation phenomenon of microcystis aeruginosa occurs.
Furthermore, low concentrations of CsNO 3 The growth and photosynthesis of algae can be promoted, the photosynthesis is enhanced, the increase of C element in algae cells can be promoted, more C element participates in calvin circulation or glycolysis, and the carbon skeleton of microcystin is increased along with the increase of C element so as to promote the synthesis of microcystin; when the increase in element C, i.e., the relative amount of N decreases, exacerbating the N limitation situation, such that these carbon scaffolds in the algal cells are more applied to the synthesis of other nitrogen-depleted microcystins MC-LW (C: N ratio=6.8), the nitrogen-enriched MC-LR (C: N ratio=4.9) is relatively decreased, thereby enabling increased production of MC-LW.
Meanwhile, through purifying and culturing microcystis aeruginosa, impurity interference caused by other algae metabolites can be reduced, the types and the yields of microcystin are stabilized, and the types and the yields of microcystin caused by population and community changes are prevented from being changed.
Specifically, the following steps in the preparation method are used:
in step 1, sterilization can prevent contamination of bacteria or microorganisms remaining in various reagents, affecting algae growth. In the step 4, the ultrasonic waves cause the cell wall of the algae cells to be broken, so that microcystins (intracellular toxins) in the cells are thoroughly released; in step 4, the extraction is to extract the algae toxin from the cells into the extractant by using a solvent of similar polarity as the algae toxin and using a similar compatibility principle. In step 5, the elution belongs to a purification process, and impurities (such as pigments) in the extract liquid are removed; in step 5, the extracted microcystins are not only microcystins-LW but also microcystins-LR, and pass through C 18 The microcystin-LR with short retention time is removed by the solid phase extraction column, and microcystin-LW with long retention time is collected.
Illustratively, in the step 1, the raw material of the phosphorus element is yellow phosphorus, and the adding amount of the yellow phosphorus is 10-15 mg/L. This is because, in practical applications, the nutrient elements of microcystis aeruginosa gradually decrease during mass propagation, especially the loss of phosphorus element which is the main element of genetic material, and at the same time, the pH value in the culture solution gradually increases during the growth of algae, and yellow phosphorus is added as a reserve element of phosphorus element which is the nutrient element, so that phosphate can be generated by yellow phosphorus under the alkaline condition of increasing the pH value during mass growth of algae to supplement the loss of phosphorus element, thereby promoting the growth and stationary phase of algae, preventing the premature arrival of decay phase caused by the reduction of phosphorus element, and simultaneously, phosphate is not generated under the neutral condition of the initial stage of cultivation, thereby reducing the phenomenon that excessive phosphorus element inhibits growth.
Wherein, yellow phosphorus reaction equation:
P 4 +3OH - +3H 2 O＝PH 3 +3H 2 PO 2 - 。
in order to ensure the sterilization effect and reduce the influence on the subsequent microcystin-LW preparation process, in the step 1, an autoclave is used for sterilization, the sterilization temperature is 120-130 ℃, and the sterilization time is 30-40 min.
In order to promote the rapid growth of algae cells, the growth cycle of algae cells is made to enter the stationary phase as soon as possible, and meanwhile, the time of the stationary phase is prolonged, and the premature arrival of the decay phase caused by the overlarge algae density is prevented, wherein in the step 2, the volume ratio of microcystis aeruginosa to the culture solution is 1:49 to 59. It should be noted that, too large a volume difference between the microcystis aeruginosa (i.e., inoculating solution) and the culture solution may result in too long a culture period, and the stationary phase cannot be reached quickly. When the volume difference is too small, the stable period is promoted to be too short, and the algae cells are rapidly grown, and insufficient growth space is provided, so that the algae density is too high, and the algae cells enter the decay period in advance.
In order to make the microcystis aeruginosa-1752 reach the optimal growth condition, in the step 3, the temperature of the illumination incubator is 25-30 ℃ and the illumination is 30-35 mu mol/m 2 /s。
In order to ensure that the algal cells can break the wall to release microcystin in the cells, in the step 4, the freeze thawing comprises the following steps:
step 41: freezing the algae cells in a refrigerator at the temperature of minus 25 ℃ to minus 30 ℃;
step 42: thawing the frozen algal cells;
step 43: repeating steps 41 to 42 at least 2 to 3 times.
In order to increase the extraction efficiency of microcystin in the algae cells, the polarity of the extraction solution is adjusted to be similar to that of microcystin-LW by using a mixed solution of methanol and water, and in the step 4, the algae cells need to be covered by the aqueous solution of methanol so that the algae cells are soaked in the aqueous solution of methanol.
In order to remove the algae cell residues, the ultrasonic extraction step 4 further comprises the following steps:
and centrifuging the extract and filtering the extract with a filter membrane.
In the above step 4, the volume ratio of methanol to water in the aqueous methanol solution is 1:4 to 4.5. This is because the polarity of the extract in this range is similar to that of microcystin-LW, and the extraction efficiency thereof is improved according to the similar principle of compatibility.
In practical application, in the step 5, the adding amount of the methanol eluent is 8-10 mL of the eluent in every 1mL of the extracting solution. This is because too little target may remain in the column and not completely eluted, resulting in a decrease in the yield of the target, and too much addition wastes reagent, increases the solvent removal time, and is time-consuming and laborious.
In order to accelerate the solvent removal and prevent degradation of microcystin-LW due to excessive temperature or pressure, in the step 6, the rotary evaporation temperature is 40-45 ℃ and the air pressure is 337-370 mbar.
Illustratively, the above-described method of preparing microcystin-LW employs a preparation apparatus including an incubator 1 and a nutrient solution reservoir 2 (e.g., a liquid bottle) in communication with the incubator 1, see fig. 1.
In the actual culture process, multiple sampling is needed, therefore, the preparation device further comprises a sampling tube 3, a sampling port for sampling is formed in the incubator 1, the sampling tube 3 is connected with the sampling port, and a sampling valve 4 is arranged on the sampling tube 3. When sampling is needed, the sampling valve 4 can be opened, and samples flow out through the sampling port and the sampling tube 3 in sequence, so that sampling is realized; the sampling valve 4 may be closed when sampling is not required.
Similarly, in the actual culture process, the nutrient solution is continuously consumed, so that the nutrient solution needs to be supplemented for many times on time, and the supplement of the nutrient solution can adopt two modes of manual supplement and automatic supplement.
For manual replenishment, the nutrient solution reservoir 2 is communicated with the incubator 1 through a fluid replacement pipe 5, and a fluid replacement valve 6 is arranged on the fluid replacement pipe 5. When fluid replacement is required, the sampling valve 4 may be opened, and when fluid replacement is not required, the fluid replacement valve 6 may be closed.
For automatic replenishment, the preparation apparatus further comprises an automatic replenishment module through which the nutrient solution reservoir 2 communicates with the incubator 1. To the structure of automatic fluid infusion subassembly, specifically, including fixed cylinder 7, locate fixed cylinder 7 in and with fixed cylinder 7 sealing rotation rotary drum 8 and be used for driving rotary drum 8 rotatory driving piece, feed liquor hole 9 is seted up to fixed cylinder 7's up end, feed liquor hole 10 is seted up to fixed cylinder 7's lower terminal surface, feed liquor hole 9 is connected with nutrient solution reserve ware 2 through first connecting pipe 11, feed liquor hole 10 is connected with incubator 1 through second connecting pipe 12, set up a plurality of reservoir 13 along the axial on the rotary drum 8, make reservoir 13 and feed liquor hole 9 or the position correspondence of play liquid hole 10 through the rotation. Taking one of the liquid storage tanks 13 as an example, a driving piece is started, so that relative rotation occurs between the rotary drum 8 and the fixed drum 7, the liquid storage tank 13 corresponds to the liquid inlet hole 9 in position, the nutrient solution reservoir 2 is communicated with the liquid storage tank 13 and supplies nutrient solution into the liquid outlet tank, the rotary drum 8 is continuously rotated, sealing of the liquid storage tank 13 is realized through the side wall of the fixed drum 7, the liquid storage tank 13 filled with the nutrient solution rotates to the liquid outlet hole 10, and the nutrient solution flows into the incubator 1 through the liquid outlet hole 10. Thus, by adjusting the rotational speed of the drum 8, multiple fluid replenishment can be performed on time.
By adopting the structure of sampling and replenishing the culture solution, the incubator 1 does not need to be opened, so that other microbial contamination caused by sampling and replenishing the culture solution for a plurality of times can be avoided.
To reduce evaporation of the culture medium on the basis of ensuring sufficient oxygen in the incubator 1, the top opening of the above-described incubator 1 may be, for example, a conical flask, and the opening may be covered with a gas-permeable layer 14 (e.g., kraft paper).
It is noted that the temperature in the incubator 1 also affects the preparation of microcystin-LW, and therefore, the above preparation apparatus further includes a temperature sensor 15 inserted into the culture liquid in the incubator 1 to monitor the temperature of the culture liquid in real time, preventing the productivity from being lowered due to temperature errors.
Example 1
Microcystis: microcystis aeruginosa FACHB-1752 is used as experimental algae species and purchased from a fresh water algae species library of China academy of sciences.
The preparation method of the microcystin-LW comprises the following steps:
step 1: adding nutrient substances into pure water, wherein the composition of the nutrient substances comprises the following components in percentage by mass: 6.0mg/L of citric acid and 6.0mg/L, naNO of ferric citrate amine 3 610mg/L、MgSO 4 ·7H 2 O 31mg/L、K 2 HPO 4 15.5mg/L、CaCl 2 ·2H 2 O 38.5mg/L、Na 2 CO 3 20mg/L、H 3 BO 3 2.85mg/L、MnCl 2 ·4H 2 O 1.95mg/L、ZnSO 4 ·7H 2 O 0.22mg/L、Na 2 MnO 4 ·2H 2 O 0.375mg/L、CuSO 4 ·5H 2 O0.076 mg/L and Co (NO) 3 ) 2 ·6H 2 O0.049 mg/L, cesium nitrate (CsNO) 3 ) Sterilizing at 130deg.C for 40min to obtain culture solution with addition amount of 8.80 mg/L;
step 2: adding algae seed solution, wherein the adding amount of the algae seed solution and the volume ratio of the culture solution are 1:52.
Step 3: the culture device is placed in an illumination incubator with the temperature of 26 ℃ and the illumination of 33 mu mol/m 2 Culturing for 2 weeks to obtain algae cells, centrifuging and suction filtering, detecting pH value during culturing, and collecting the algae cells when pH value is equal toWhen the concentration reaches 6, yellow phosphorus is added, and the addition amount of the yellow phosphorus is 11mg/L.
Step 4: freezing the algae cells in a refrigerator at-30deg.C, thawing, repeating for 2 times, adding methanol water solution (methanol and water volume ratio is 1:4.3), and ultrasonically extracting with ultrasonic instrument for about 12 min. After the ultrasonic treatment, the extract is centrifuged and filtered by a filter membrane to remove the residues of algae cells.
Step 5: pouring the extract into activated C 18 The solid phase extraction column (1 g/6 mL) was eluted by pouring methanol eluent containing 0.3% formic acid, eluting with 9mL eluent for each 1mL extract, collecting the eluent, and repeating the purification operation until the pigment was removed.
Step 6: the eluate was subjected to preparative chromatography and, after rotary evaporation at 42℃and 350mbar, the solid powder was collected.
The experimental results are as follows:
the effect of the culture medium on algae density and microcystin-LW content is shown in FIGS. 3 a-3 b.
After microcystin-LW is separated by preparative chromatography, the target fraction is collected, methanol is removed by rotary evaporation, and pure solid powder is obtained, and a pure quality spectrum is shown in figure 4.
Example two
Microcystis: microcystis aeruginosa FACHB-1752 is used as experimental algae species and purchased from a fresh water algae species library of China academy of sciences.
The preparation method of the microcystin-LW comprises the following steps:
step 1: adding nutrient substances into pure water, wherein the composition of the nutrient substances comprises the following components in percentage by mass: 5.6mg/L of citric acid and 5.5mg/L, naNO of ferric citrate amine 3 550mg/L、MgSO 4 ·7H 2 O 28mg/L、K 2 HPO 4 14.0mg/L、CaCl 2 ·2H 2 O 36.0mg/L、Na 2 CO 3 18mg/L、H 3 BO 3 2.80mg/L、MnCl 2 ·4H 2 O 1.63mg/L、ZnSO 4 ·7H 2 O 0.21mg/L、Na 2 MnO 4 ·2H 2 O 0.36mg/L、CuSO 4 ·5H 2 O 0.075mg/L and Co (NO) 3 ) 2 ·6H 2 O0.044 mg/L, cesium nitrate (CsNO) 3 ) Sterilizing at 120deg.C for 40min to obtain culture solution with the addition amount of 10 mg/L;
step 2: adding algae seed solution, wherein the adding amount of the algae seed solution and the volume ratio of the culture solution are 1:50.
Step 3: placing the culture device in an illumination incubator at 25deg.C with illumination of 35 μmol/m 2 And/s, culturing to a mature period, wherein the mature period is 2 weeks, extracting algae cells by centrifugation and suction filtration, detecting the pH value in the culture process, and adding yellow phosphorus when the pH value reaches 6, wherein the adding amount of the yellow phosphorus is 10mg/L.
Step 4: freezing the algae cells in a refrigerator at-25deg.C, thawing, repeating for 2 times, adding methanol water solution (methanol and water volume ratio of 1:4.5), and ultrasonic extracting with ultrasonic instrument for about 10 min. After the ultrasonic treatment, the extract is centrifuged and filtered by a filter membrane to remove the residues of algae cells.
Step 5: pouring the extract into activated C 18 The solid phase extraction column (1 g/6 mL) was eluted by pouring methanol eluent containing 0.5% formic acid, 10mL eluent was required for each 1mL extract to elute, the eluent was collected, and the purification operation was repeated until the pigment was removed.
Step 6: the eluate was subjected to preparative chromatography and, after rotary evaporation at 40℃and 370mbar, the solid powder was collected.
Example III
Microcystis: microcystis aeruginosa FACHB-1752 is used as experimental algae species and purchased from a fresh water algae species library of China academy of sciences.
The preparation method of the microcystin-LW comprises the following steps:
step 1: adding nutrient substances into pure water, wherein the composition of the nutrient substances comprises the following components in percentage by mass: 6.5mg/L of citric acid and 6.2mg/L, naNO of ferric citrate amine 3 650mg/L、MgSO 4 ·7H 2 O 33mg/L、K 2 HPO 4 16.0mg/L、CaCl 2 ·2H 2 O 41.0mg/L、Na 2 CO 3 22mg/L、H 3 BO 3 3.10mg/L、MnCl 2 ·4H 2 O 1.80mg/L、ZnSO 4 ·7H 2 O 0.24mg/L、Na 2 MnO 4 ·2H 2 O 0.42mg/L、CuSO 4 ·5H 2 O0.085 mg/L and Co (NO) 3 ) 2 ·6H 2 O0.054 mg/L, cesium nitrate (CsNO) 3 ) Sterilizing at 125 deg.C for 30min to obtain culture solution with the addition amount of 9.0 mg/L;
step 2: adding algae seed solution, wherein the adding amount of the algae seed solution and the volume ratio of the culture solution are 1:55.
Step 3: placing the culture device in an illumination incubator at 30deg.C with illumination of 30 μmol/m 2 And/s, culturing to a mature period, wherein the mature period is 3 weeks, extracting algae cells by centrifugation and suction filtration, detecting the pH value in the culture process, and adding yellow phosphorus when the pH value reaches 7, wherein the adding amount of the yellow phosphorus is 15mg/L.
Step 4: freezing the algae cells in a refrigerator at-28deg.C, thawing, repeating for 3 times, adding methanol water solution (methanol and water volume ratio of 1:4), and ultrasonic extracting with ultrasonic instrument for 15 min. After the ultrasonic treatment, the extract is centrifuged and filtered by a filter membrane to remove the residues of algae cells.
Step 5: pouring the extract into activated C 18 The solid phase extraction column (1 g/6 mL) was eluted by pouring methanol eluent containing 0.2% formic acid, eluting with 8mL eluent per 1mL extract, collecting the eluent, and repeating the purification operation until the pigment was removed.
Step 6: the eluate was subjected to preparative chromatography and, after rotary evaporation at 42℃and 350mbar, the solid powder was collected.
Comparative example one
Microcystis: microcystis aeruginosa FACHB-1752 is used as experimental algae species and purchased from a fresh water algae species library of China academy of sciences.
The present comparative example is for the purpose of explaining the addition of cesium nitrate (CsNO 3 ) And the effect of yellow phosphorus, the other steps of this comparative example are not considered prior art.
The preparation method of the microcystin-LW provided in the comparative example comprises the following steps:
step 1: adding nutrient substances into pure water, wherein the composition of the nutrient substances comprises the following components in percentage by mass: 5.4 to 6.6mg/L of citric acid and 5.4 to 6.6mg/L, naNO of ferric citrate amine 3 540～660mg/L、 MgSO 4 ·7H 2 O 27～33mg/L、K 2 HPO 4 14.0～17.2mg/L、CaCl 2 ·2H 2 O 34.2～41.8mg/L、Na 2 CO 3 18～22mg/L、H 3 BO 3 2.57～3.15mg/L、MnCl 2 ·4H 2 O 1.63～1.99mg/L、ZnSO 4 ·7H 2 O 0.202mg/L～0.242mg/L、Na 2 MnO 4 ·2H 2 O 0.351～0.431mg/L、CuSO 4 ·5H 2 O0.071-0.087 mg/L and Co (NO) 3 ) 2 ·6H 2 0.044-0.054 mg/L O, sterilizing for 30-40 min at 120-130 ℃ in a high pressure steam pot to obtain a culture solution;
step 2: adding microcystis aeruginosa (FACHB-1752) to the culture broth, and placing the culture broth containing microcystis aeruginosa into a incubator (e.g., a petri dish);
step 3: placing the incubator in an illumination incubator, culturing for 2-3 weeks until the mature period, and obtaining algae cells through centrifugal suction filtration;
step 4: freeze thawing is carried out on the algae cells, and then methanol aqueous solution is added for ultrasonic extraction for 10-15 min to obtain extract;
step 5: pouring the extract into activated C 18 A solid phase extraction column (1 g/6 mL), methanol eluent containing 0.1% -0.5% formic acid is adopted for eluting for multiple times until pigment is removed, and eluent is collected;
step 6: separating the eluent by preparative chromatography, rotary evaporating, and collecting solid powder to obtain microcystin-LW.
The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions easily contemplated by those skilled in the art within the technical scope of the present invention should be included in the scope of the present invention.
Claims (4)
1. The preparation method of microcystin-LW is characterized by comprising the following steps:
step 1: adding nutrient substances into water, wherein the composition of the nutrient substances comprises the following components in percentage by mass: 5.4-6.6 mg/L of citric acid and 5.4-6.6 mg/L, naNO of ferric citrate amine 3 540~660mg/L、MgSO 4 •7H 2 O 27~33mg/L、K 2 HPO 4 14.0~17.2mg/L、CaCl 2 •2H 2 O 34.2~41.8mg/L、Na 2 CO 3 18~22mg/L、H 3 BO 3 2.57~3.15mg/L、MnCl 2 •4H 2 O 1.63~1.99mg/L、ZnSO 4 •7H 2 O 0.202~0.242mg/L、Na 2 MnO 4 •2H 2 O 0.351~0.431mg/L、CuSO 4 •5H 2 O0.071-0.087 mg/L and Co (NO) 3 ） 2 •6H 2 O0.044-0.054 mg/L, cesium nitrate is added, the adding amount is 8.75-10.75 mg/L, the culture solution is obtained, and the culture solution is sterilized by adopting a high-pressure steam pot, wherein the sterilization temperature is 120-130 ℃ and the sterilization time is 30-40 min;
step 2: adding microcystis aeruginosa into the culture solution, and placing the culture solution containing microcystis aeruginosa FACHB-1752 into a incubator;
step 3: placing the incubator in an illumination incubator, culturing to a mature period, and obtaining algae cells through centrifugal suction filtration;
step 4: freeze thawing is carried out on algae cells, and then methanol aqueous solution is added for ultrasonic extraction to obtain extract;
step 5: pouring the extract into activated C 18 The solid phase extraction column is eluted for a plurality of times by adopting methanol eluent until pigment is removed, and eluent is collected;
step 6: separating the eluent by preparative chromatography, rotationally evaporating, and collecting solid powder to obtain microcystin-LW;
in the step 3, detecting the pH value, and adding phosphorus element when the pH value reaches 6-7, wherein the raw material of the phosphorus element is yellow phosphorus, and the adding amount of the yellow phosphorus is 10-15 mg/L;
in the step 2, the volume ratio of the microcystis aeruginosa to the culture solution is 1: 49-59;
in the step 4, the freeze thawing includes the following steps:
step 41: freezing the algae cells in a refrigerator at-25 ℃ to-30 ℃;
step 42: thawing the frozen algal cells;
step 43: repeating steps 41 to 42 at least 2-3 times;
in the step 4, the aqueous methanol solution needs to cover the algae cells, so that the algae cells are soaked in the aqueous methanol solution;
in the step 4, the volume ratio of methanol to water in the aqueous methanol solution is 1: 4-4.5;
in the step 5, the addition amount of the methanol eluent is 8-10 mL of the eluent in every 1mL of the extracting solution.
2. The method for preparing microcystin-LW according to claim 1, wherein in the step 3, the temperature of the illumination incubator is 25-30 ℃ and the illumination is 30-35 μmol/m 2 /s。
3. The method for preparing microcystin-LW according to claim 1, wherein in step 4, the following steps are further included after the ultrasonic extraction:
and centrifuging the extract and filtering the extract with a filter membrane.
4. The method for preparing microcystin-LW according to any one of claims 1 to 3, wherein in step 6, the rotary evaporation temperature is 40 ℃ to 45 ℃ and the air pressure is 337 to 370mbar.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210258021.8A CN114480535B (en) | 2022-03-16 | 2022-03-16 | Preparation method of microcystin-LW |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210258021.8A CN114480535B (en) | 2022-03-16 | 2022-03-16 | Preparation method of microcystin-LW |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114480535A CN114480535A (en) | 2022-05-13 |
| CN114480535B true CN114480535B (en) | 2023-07-25 |
Family
ID=81485495
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210258021.8A Active CN114480535B (en) | 2022-03-16 | 2022-03-16 | Preparation method of microcystin-LW |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114480535B (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110606876A (en) * | 2019-08-01 | 2019-12-24 | 青岛普瑞邦生物工程有限公司 | N-15 labeled microcystin and production method thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10809259B2 (en) * | 2016-10-21 | 2020-10-20 | The University Of Toledo | Protocol for preconcentration and quantification of microcystins using LC-MS |
-
2022
- 2022-03-16 CN CN202210258021.8A patent/CN114480535B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110606876A (en) * | 2019-08-01 | 2019-12-24 | 青岛普瑞邦生物工程有限公司 | N-15 labeled microcystin and production method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 微囊藻毒素的提取与分析研究进展;邓琳, 张维昊, 邓南圣, 宋立荣;重庆环境科学(第11期);全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114480535A (en) | 2022-05-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Linhardt et al. | Microbially produced rhamnolipid as a source of rhamnose | |
| CN104745655A (en) | Methods For The Production Of Ansamitocins | |
| JP2002542778A (en) | Culture method of basidiomycetes in liquid medium | |
| KR20170031727A (en) | Active fermentation and fermented drinks and products | |
| JPH0678757A (en) | Ray fungus capable of producing antibacterial cl-1577 | |
| SU786914A3 (en) | Method of preparing antibiotic complex | |
| CN101748173A (en) | Corn silk polysaccharide separation and decoloring method | |
| CN105906650B (en) | Macrolide new compounds and preparation method and application | |
| CN114480535B (en) | Preparation method of microcystin-LW | |
| SU576965A3 (en) | Method of preparing antibiotic possessing b-lactamase inhibiting activity | |
| CN105087419B (en) | A kind of application of bacillus subtilis in handling leather-making waste water total nitrogen | |
| CN104962491A (en) | Degradation strain of herbicide 2, 4-D, produced inoculum and application thereof | |
| CN105802872B (en) | Pseudomonas fluorescens, method for producing phenazine amide and application thereof | |
| JPH05504469A (en) | A83543 collection method | |
| JPS6010718B2 (en) | Process for producing maytansinol, maytanacin and maytansinol propionate | |
| CN111808115A (en) | Fermented organic matter with anti-pathogenic activity of potato endophyte, preparation process and application thereof, and fermented product | |
| CN109180635B (en) | Compound E1011, preparation method and application thereof, fermentation product of potato endophytic fungi and ethyl acetate extract liquid of fermentation product | |
| CN112342146A (en) | Ash-fragrant strain for synthesizing 2-phenethyl alcohol by utilizing L-phenylalanine biological fermentation | |
| CN111100823A (en) | Polymyxin B sulfate production strain, preparation method and application of polymyxin B sulfate | |
| RU2199582C2 (en) | Method of preparing selenium-enriched spirulina biomass (spirulina platensis) | |
| CN1068049C (en) | Antibiotic producing method utilizing hydropic streptomyce | |
| US20060105443A1 (en) | Process for obtaining biosynthesized lycopene from bacterial cells and the purified lycopene of the same | |
| CN110272486A (en) | The extracting method of phycocyanin in a kind of cyanobacteria | |
| JPS62195276A (en) | Cell cultivation | |
| CN116606753B (en) | Streptomyces 0728-27 and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |