CN110241047B - Method for screening linear spirulina filaments by using triclosan - Google Patents
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- 240000002900 Arthrospira platensis Species 0.000 title claims abstract description 58
- 235000016425 Arthrospira platensis Nutrition 0.000 title claims abstract description 57
- XEFQLINVKFYRCS-UHFFFAOYSA-N Triclosan Chemical compound OC1=CC(Cl)=CC=C1OC1=CC=C(Cl)C=C1Cl XEFQLINVKFYRCS-UHFFFAOYSA-N 0.000 title claims abstract description 52
- 229940082787 spirulina Drugs 0.000 title claims abstract description 51
- 229960003500 triclosan Drugs 0.000 title claims abstract description 51
- 238000000034 method Methods 0.000 title claims abstract description 24
- 238000012216 screening Methods 0.000 title claims abstract description 14
- 241000195493 Cryptophyta Species 0.000 claims abstract description 60
- 239000007788 liquid Substances 0.000 claims abstract description 24
- 238000007865 diluting Methods 0.000 claims abstract description 7
- 241000192700 Cyanobacteria Species 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 58
- 238000005286 illumination Methods 0.000 claims description 11
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- 239000012452 mother liquor Substances 0.000 claims description 4
- 238000005303 weighing Methods 0.000 claims description 3
- 239000002028 Biomass Substances 0.000 claims description 2
- 238000010790 dilution Methods 0.000 claims description 2
- 239000012895 dilution Substances 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 230000003068 static effect Effects 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 10
- 230000007246 mechanism Effects 0.000 abstract description 4
- 239000000178 monomer Substances 0.000 abstract description 4
- 238000012258 culturing Methods 0.000 abstract description 3
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 238000012136 culture method Methods 0.000 abstract description 2
- 238000005516 engineering process Methods 0.000 abstract description 2
- 238000013537 high throughput screening Methods 0.000 abstract description 2
- 238000000926 separation method Methods 0.000 abstract description 2
- 238000011160 research Methods 0.000 description 8
- 238000001514 detection method Methods 0.000 description 3
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 3
- 241000620196 Arthrospira maxima Species 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 230000000877 morphologic effect Effects 0.000 description 2
- 239000010413 mother solution Substances 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- HIXDQWDOVZUNNA-UHFFFAOYSA-N 2-(3,4-dimethoxyphenyl)-5-hydroxy-7-methoxychromen-4-one Chemical compound C=1C(OC)=CC(O)=C(C(C=2)=O)C=1OC=2C1=CC=C(OC)C(OC)=C1 HIXDQWDOVZUNNA-UHFFFAOYSA-N 0.000 description 1
- 241000813872 Oscillatoriaceae Species 0.000 description 1
- 241000192494 Oscillatoriales Species 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 150000001491 aromatic compounds Chemical class 0.000 description 1
- 229940011019 arthrospira platensis Drugs 0.000 description 1
- 239000003899 bactericide agent Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 210000002777 columnar cell Anatomy 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- RTZKZFJDLAIYFH-UHFFFAOYSA-N ether Substances CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012847 fine chemical Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000009182 swimming Effects 0.000 description 1
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- 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
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Abstract
The invention relates to a production technology of spirulina, and aims to provide a method for screening linear spirulina filaments by using triclosan. The method comprises the following steps: taking a proper amount of spirulina liquid with spiral and linear algae filaments, and diluting the turbidity T of the spirulina liquid to 0.2 by using Zarrouk's culture solution; equally dividing the diluted algae solution into multiple parts, respectively adding Zarrouk culture solution containing triclosan with gradient concentration, and diluting the turbidity T of the algae solution to 0.1; and after standing and culturing for 70 days, selecting blue-green algae solution, and taking a group with the highest triclosan concentration, wherein all algae filaments in the group of algae solution are linear algae filaments. The method screens the linear algae filaments based on the sensitivity difference of the algae filaments with different forms to the triclosan, compared with the traditional algae filament monomer separation culture method, the method is simple, convenient and efficient, has low cost, and can realize batch, automatic and high-throughput screening. Can provide the required linear spirulina filaments for further researching the internal mechanism and influencing factors of the spirulina filament straightening.
Description
Technical Field
The invention belongs to a production technology of spirulina, and particularly relates to a method for screening linear spirulina filaments by using triclosan.
Background
The Spirulina (Spirulina) belongs to the phylum of cyanophyta, order Oscillatoriales and family Oscillatoriaceae, at least 38 species of Spirulina (Spirulina), wherein, Arthrospira platensis (A. platensis) and Arthrospira maxima (A. maxima) and other minority species, because of the characteristics of edible safety, high nutritional health care and medical value, good production performance and the like, the Spirulina has realized large-scale industrial culture and is widely applied to a plurality of industries such as food, feed, aquatic product bait, medical health care, fine chemical industry and the like, and becomes the economic microalgae which has the largest scale and the widest application field in the current scientific research and industrialization at home and abroad.
The typical morphological feature of spirulina filaments in taxonomy is that a plurality of columnar cells are connected in series to form a filamentous, non-branched, regular, loose or compact spiral shape, but under certain conditions, polymorphic variation occurs and even complete straightening occurs. A plurality of experimental researches and production practices show that the spiral form of the spirulina is an important economic character in the industrial culture production. The spiral shape is not only beneficial to the floating and swimming of the algae, and obviously improves the utilization rate of light energy and nutrients, but also can be collected with low cost by using a simple and convenient bolting silk filtering mode. Once the algae filaments are straightened and lose the spiral form, the algae filaments are difficult to recover to the spiral form, serious consequences such as rapid reduction of yield and quality, difficulty in harvesting, susceptibility to other biological pollution and the like are brought to production, and the irreversible mutation in production is the degradation of algae seeds. In order to solve the serious problem of production, research and elucidation of the intrinsic mechanism and influencing factors of spirulina filament straightening are necessary.
The establishment of a method for simply, efficiently and massively screening out linear phycofilaments from spiral and linear mixed phycofilament groups of spirulina is a necessary premise for the development of the research. The traditional method for screening the linear spirulina filaments still continues to be used at home and abroad, namely the linear spirulina filament monomer is selected under a microscope → the single spirulina filament is cultured for about 1 month → the microscopic detection → the linear spirulina filament monomer is selected again to be cultured → … … → the linear spirulina filament group is obtained if the linear spirulina filaments are contained, and the traditional method for screening the linear spirulina filaments has the advantages of complicated process, long time consumption and low success rate. Therefore, there is a need to establish a new method for simply, efficiently and massively screening out linear phycofilaments from a spirulina spiral and linear mixed phycofilament population for research needs of spirulina morphological mutation mechanisms and the like.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defects in the prior art and provides a method for screening linear spirulina filaments by using triclosan.
In order to solve the technical problem, the solution of the invention is as follows:
the method for screening the linear spirulina filaments by using the triclosan comprises the following steps:
(1) taking a proper amount of spirulina liquid with spiral and linear algae filaments, and diluting the spirulina liquid to 0.2 by using Zarrouk's culture solution to obtain turbidity T (called T for short);
(2) equally dividing the diluted algae solution into multiple parts, respectively adding Zarrouk culture solution containing triclosan with gradient concentration, and diluting the turbidity T of the algae solution to 0.1; and after standing and culturing for 70 days, selecting blue-green algae solution, and taking a group with the highest triclosan concentration, wherein all algae filaments in the group of algae solution are linear algae filaments.
In the invention, the Zarrouk culture solution containing triclosan with gradient concentration refers to: the Zarrouk culture solution contains triclosan in multiple portions, and the concentration of the triclosan contained in the Zarrouk culture solution is increased by a gradient of 0.25 mg/L.
In the invention, the preparation method of the Zarrouk culture solution containing triclosan with gradient concentration in the step (2) comprises the following steps:
weighing 100mg of triclosan, dissolving the triclosan in 100ml of 0.5mol/L NaOH solution, and preparing triclosan mother liquor with the content of 1 g/L; then 0.50mL, 1.00mL, 1.50mL, 2.00mL, 2.50mL, 3.00mL, 3.50mL, 4.00mL of mother liquor are taken in turn, respectively added into 8 1L volumetric flasks containing 950mL of Zarrouk culture solution, and the Zarrouk culture solution is used for constant volume to 1L to prepare the Zarrouk culture solution with the triclosan content of 0.50mg/L, 1.00mg/L, 1.50mg/L, 2.00mg/L, 2.50mg/L, 3.00mg/L, 3.50mg/L, 4.00mg/L in turn.
In the present invention, the static culture conditions in the step (2) are such that a 40W fluorescent lamp is used as a light source and the light intensity of the culture liquid surface is 54. mu. mol photons.m-2·d-1(ii) a The illumination condition is controlled according to the alternative mode of illumination for 12h and darkness for 12h, and the temperature is 28 ℃ during illumination and 20 ℃ during darkness.
In the invention, the turbidity T of the algae liquid is measured by a spectrophotometer to characterize the biomass of the spirulina, the wavelength of the spectrophotometer is set to be 560nm, a 10mm cuvette is used, and Zarrouk's culture solution is used as a blank control.
In the present invention, the volumes of the taken algal solution and Zarrouk's culture solution were estimated as follows: let the initial turbidity of the spirulina solution be T1Volume is V1(ii) a The turbidity of the algae liquid to be prepared is T2Volume is V2(ii) a The volume of the culture solution to be added with Zarrouk's is V2-V1(ii) a According to the principle of linear dilution, from T1×V1＝T2×V2Obtaining V1＝(T2×V2)/T1Then is composed of (V)2-V1) The amount of the Zarrouk's culture solution added was determined.
Description of the inventive principles:
triclosan (Triclosan) is known as Triclosan and is known as 2,4,4 '-trichloro-2' -hydroxy-diphenyl ether and has the molecular formula C12H7Cl13O2And the molecular weight of 289.6 is an artificially synthesized chlorinated aromatic compound with ether and phenol functional groups. At present, the intrinsic cause of straightening spirulina filaments is not clear, and there is no method for completely inhibiting the straightening. Through years of research, the sensitivity of the spirulina spiral algae filaments to the broad-spectrum bactericide triclosan is obviously higher than that of the straightened algae filaments. That is, the spiral algae filament of one spiral algae strain can tolerate higher concentration of triclosan than the spiral algae filament after being straightened. Based on the discovery, the invention establishes a method for screening the linear spirulina filaments.
Compared with the prior art, the invention has the following remarkable advantages:
1. the method screens the linear algae filaments based on the sensitivity difference of the algae filaments with different forms to the triclosan, compared with the traditional algae filament monomer separation culture method, the method is simple, convenient and efficient, has low cost, and can realize batch, automatic and high-throughput screening.
2. The invention can provide the required linear spirulina filaments for further researching the internal mechanism and influencing factors of the spirulina filament straightening.
Detailed Description
The technical solution of the present invention will be described in detail with reference to specific embodiments.
1. Selecting sample materials: the known 9 strains of spirulina platensis strains CHM-5, CHM-6, CHM-7, CHM-8, CHM-9, CHM-10, CHM-11, ZJU0112 and ZJU0137 which are widely applied to scientific research and industrialized production and cultivation are also preserved by the nuclear agricultural science research institute of Zhejiang university, which belongs to the applicant. The applicant ensures that samples can be provided to the public at will during the period of patent validity.
2. Reagents and instrumentation: the triclosan used in the invention is a product of chemical Limited of Icuro, Jiangsu; other reagents are analytically pure; the optical density T of the algal solution was measured with an Ultrospec 2000 UV-visible spectrophotometer, Pharmacia, Sweden.
3. The method for screening the linear spirulina filaments comprises the following steps:
(1) weighing 100mg of triclosan purchased from Ika chemical Co., Ltd, Jiangsu, dissolving the triclosan in 100mL of 0.5mol/L NaOH solution to prepare a triclosan mother solution with the content of 1g/L, sequentially taking 0.50mL, 1.00mL, 1.50mL, 2.00mL, 2.50mL, 3.00mL, 3.50mL and 4.00mL of the mother solution in a 1L volumetric flask containing 950mL of Zarrouk culture solution, and fixing the volume to 1L by using the Zarrouk culture solution to prepare a Zarrouk culture solution with the triclosan content of 0.50mg/L, 1.00mg/L, 1.50mg/L, 2.00mg/L, 2.50mg/L, 3.00mg/L, 3.50mg/L and 4.00 mg/L;
(2) detecting the form of the spirulina platensis strain CHM-5 algae filament cultivated in a laboratory by a microscope, finding that the algae filament is straightened from a spiral shape, and measuring the turbidity T of the algae filament by an Ultrospec 2000 ultraviolet-visible spectrophotometer1＝0.423；
(3) Intended to be diluted with Zarrouk's medium to a volume of V1(units are L) and T1CHM-5 algae liquid of 0.423 to prepare target turbidity T20.2% algal solution V21L, according to V1＝(T2×V2)/T1＝(0.2×1)/0.423＝472.8(mL)、V2-V1When the concentration is 1000-472.8-527.2 (mL), 472.8mL of CHM-5 algal solution is added to 527.2mL of Zarrouk's culture solution to prepare 1L of algal solution with turbidity of 0.2, and 100mL of each solution is dispensed into 9 triangular flasks of 500 mL;
(4) adding the Zarrouk's culture solution and the Zarrouk culture solution containing 0.50mg/L, 1.00mg/L, 1.50mg/L, 2.00mg/L, 2.50mg/L, 3.00mg/L, 3.50mg/L and 4.00mg/L of the prepared triclosan in the step (1) into 9 500mL triangular flasks in sequence in the step (3), shaking up the mixture, diluting the turbidity T of the algae solution to 0.1, and sequentially adding the triclosan concentration to 0.00mg/L, 0.25mg/L, 0.50mg/L, 0.75mg/L, 1.00mg/L, 1.25mg/L, 1.50mg/L, 1.75mg/L and 2.00 mg/L;
(5) and (5) statically culturing the groups of algae liquid in the step (4) for 70d under the culture conditions that: at 40WThe fluorescent lamp is used as a light source, and the illumination intensity of the culture liquid surface is 54 mu mol phosns.m-2·d-1(ii) a The illumination condition is controlled according to the alternative mode of illumination for 12h and darkness for 12h, and the temperature is 28 ℃ during illumination and 20 ℃ during darkness.
Through detection, in a triangular flask with the triclosan concentration of 1.25mg/L, the algae filaments grow well, the algae liquid is blue-green, and all the algae filaments are linear algae filaments. In a triangular flask with the triclosan concentration higher than 1.25mg/L, all the algae filaments die; in a triangular flask with the triclosan concentration lower than 1.25mg/L, spiral and linear algae filaments are combined.
4. Results and analysis
The result shows that the spirulina platensis strain CHM-5 which is cultivated in the laboratory and has the spiral shape and the straight shape is detected by a microscope to be totally straight shape by taking 472.8mL of the algae solution with the turbidity of 0.423, adding 527.2mL of the Zarrouk's culture solution to prepare 1L of the algae solution with the turbidity of 0.2, subpackaging, carrying out the treatment and cultivation of different concentrations of triclosan for 70d, and then carrying out the treatment and the cultivation of the different concentrations of triclosan in a triangular flask.
As a verification example 1, an applicant uses a microscope to respectively detect the forms of spirulina platensis strains CHM-6, CHM-7, CHM-8, CHM-9, CHM-10 and CHM-11 which are cultivated in a laboratory, and finds out spirulina filaments with both spiral and linear forms, and uses an Ultrospec 2000 ultraviolet-visible spectrophotometer to measure the turbidities of the spirulina filaments to be 0.511, 0.365, 0.464, 0.682, 0.718 and 0.297 in sequence, and takes corresponding spirulina liquid 391.4mL, 547.9mL, 431.0mL, 293.3mL, 278.6mL and 673.4mL in sequence, and further adds Zarrouk's culture solution 608.6mL, 432.1mL, 569.0mL, 706.7mL, 721.4mL and 326.6mL in sequence to prepare 1L of spirulina liquid with the turbidities of 0.2, then subpackages and carries out triclosan treatment and cultivation for 70d by different concentrations, and CHM-6, CHM-7, CHM-8, CHM-9-10 and CHM-11 respectively show the highest triclosan concentrations of the spirulina liquid are CHM/CHM in sequence, 1.75mg/L, 2.00mg/L, 0.75mg/L, 1.50mg/L and 1.25mg/L, and the corresponding algal filaments in the triangular flask are all linear through microscope detection.
As a verification example 2, an applicant uses a microscope to respectively detect the forms of spirulina platensis strains ZJU0112 and ZJU0137 algae filaments cultivated in a factory production pool, finds out the algae filaments in both spiral forms and linear forms, uses an Ultrospec 2000 ultraviolet-visible spectrophotometer to measure that the turbidity of the algae filaments is 0.473 and 0.381 in sequence, sequentially takes 422.8mL and 524.9mL of corresponding algae liquid, further sequentially adds 577.2mL and 475.1mL of Zarrouk's culture liquid to prepare 1L of algae liquid with the turbidity of 0.2, subpackages the algae liquid and cultures the algae liquid for 70d after different concentrations of triclosan treatment, the highest triclosan concentration corresponding to the blue-green color of the ZJU0112 and ZJU0137 algae liquid is 1.50mg/L and 1.00mg/L in sequence, and detects the algae filaments in corresponding triangular flasks are all linear forms through the microscope.
The above examples further illustrate that the present invention provides a simple, batch-wise, high throughput method for screening linear spirulina filaments.
Claims (4)
1. A method for screening linear spirulina filaments by using triclosan is characterized by comprising the following steps:
(1) taking a proper amount of spirulina liquid with spiral and linear algae filaments, and diluting the spirulina liquid to 0.2 by using Zarrouk culture solution;
(2) equally dividing the diluted algae solution into multiple parts, respectively adding Zarrouk culture solution containing triclosan with gradient concentration, and diluting the turbidity T of the algae solution to 0.1; standing for 70 days, selecting blue-green algae solution, and selecting one group with highest triclosan concentration, wherein all algae filaments in the group of algae solution are linear algae filaments;
the Zarrouk culture solution containing triclosan with gradient concentration refers to the following components: the Zarrouk culture solution contains a plurality of parts of triclosan, and the concentration of the triclosan contained in the Zarrouk culture solution is increased by 0.25 mg/L; the preparation method comprises the following steps:
weighing 100mg of triclosan, dissolving the triclosan in 100ml of 0.5mol/L NaOH solution, and preparing triclosan mother liquor with the content of 1 g/L; then 0.50mL, 1.00mL, 1.50mL, 2.00mL, 2.50mL, 3.00mL, 3.50mL, 4.00mL of mother liquor are taken in turn, respectively added into 8 1L volumetric flasks containing 950mL of Zarrouk culture solution, and the Zarrouk culture solution is used for constant volume to 1L to prepare the Zarrouk culture solution with the triclosan content of 0.50mg/L, 1.00mg/L, 1.50mg/L, 2.00mg/L, 2.50mg/L, 3.00mg/L, 3.50mg/L, 4.00mg/L in turn.
2. The method according to claim 1, wherein the static culture conditions in the step (2) are: the illumination intensity of the culture liquid surface is 54 mu mol photons.m by using a 40W fluorescent lamp as a light source-2 · d-1(ii) a The illumination condition is controlled according to the alternative mode of illumination for 12h and darkness for 12h, and the temperature is 28 ℃ during illumination and 20 ℃ during darkness.
3. The method of claim 1, wherein the turbidity T of the algae broth is measured spectrophotometrically for characterizing the biomass of Spirulina, and the wavelength of the spectrophotometer is set at 560nm, a 10mm cuvette is used, and a blank is prepared with Zarrouk culture solution.
4. The method of claim 1, wherein the volumes of the algal solution and the Zarrouk culture solution taken are estimated as follows: let the initial turbidity of the spirulina solution be T1Volume is V1(ii) a The turbidity of the algae liquid to be prepared is T2Volume is V2(ii) a Volume of culture solution to be added with Zarrouk is V2-V1(ii) a According to the principle of linear dilution, from T1×V1=T2×V2Obtaining V1= (T2×V2)/T1Then is composed of (V)2-V1) The amount of the Zarrouk culture solution added was determined.
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| Title |
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