MD4543C1 - Process for cultivation of Spirulina platensis cyanobacterium - Google Patents
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Abstract
Invenţia se referă la biotehnologie, şi anume la un procedeu de cultivare a cianobacteriei Spirulina platensis.Procedeul de cultivare a cianobacteriei Spirulina platensis prevede cultivarea culturii pe un mediu nutritiv mineral ce conţine, g/L: NaNO3 - 2,25, NaHCO3 - 8,0, NaCl - 1,0, K2SO4 - 0,3, Na2HPO4 - 0,2, MgSO4·7H2O - 0,2, CaCl2 - 0,024, FeSO4 - 0,01, EDTA - 0,08, H3BO3 - 0,00286, MnCl2·4H2O - 0,00181, ZnSO4·7H2O - 0,00022, CuSO4·5H2O - 0,00008, MoO3 - 0,000015, nanoparticule hidrosolubile de aur cu dimensiunea de 5 nm în concentraţie de 0,0088…0,0091 g/L şi apă distilată până la 1L, la temperatura de 25…28°C, pH 8,0…10,0, iluminarea de 3000…4000 lx în regim continuu în decurs de 5 zile.Rezultatul invenţiei constă în stimularea producerii de biomasă de spirulină şi a conţinutului de lipide în biomasă cu scopul obţinerii materiei prime pentru elaborarea şi fabricarea remediilor cu acţiune anticanceroasă, imunostimulatoare şi antioxidantă.The invention relates to biotechnology, namely a process for cultivating cyanobacterium Spirulina platensis. The process for cultivating cyanobacterium Spirulina platensis provides for cultivation of the culture on a mineral nutrient medium containing, g / L: NaNO3-2.25, NaHCO3-8, 0, NaCl - 1.0, K2SO4 - 0.3, Na2HPO4 - 0.2, MgSO4 · 7H2O - 0.2, CaCl2 - 0.024, FeSO4 - 0.01, EDTA - 0.08, H3BO3 - 0.00286, MnCl2 · 4H2O - 0.00181, ZnSO4 · 7H2O - 0.00022, CuSO4 · 5H2O - 0.00008, MoO3 - 0.000015, water-soluble gold nanoparticles with a size of 5 nm at a concentration of 0.0088 ... 0.0091 g / L and distilled water up to 1L, at 25 ... 28 ° C, pH 8.0 ... 10.0, illumination of 3000 ... 4000 lx continuously during 5 days. The result of the invention consists in stimulating the production of biomass. of spirulina and lipid content in biomass for the purpose of obtaining the raw material for the elaboration and manufacture of anti-cancer, immunostimulatory and antioxidant remedies.
Description
Invenţia se referă la biotehnologie, şi anume la un procedeu de cultivare a cianobacteriei Spirulina platensis. The invention relates to biotechnology, namely to a process for cultivating the cyanobacterium Spirulina platensis.
Nanotehnologiile au imigrat rapid din domeniul tehnic în medicină, dar şi biotehnologie, creând un nou domeniu ştiinţifico-practic - bionanotehnologia. A fost extinsă aria aplicării nanoparticulelor. A fost demonstrat efectul pozitiv al nanoparticulelor asupra creşterii şi a metabolismului celular. Dimensiunile mici ale nanoparticulelor favorizează interacţiunea lor cu suprafaţa celulară şi pătrunderea în citosol. Nanoparticulele pot fi o sursă alternativă favorabilă oligoelementelor care au funcţia de stimulatori ai activităţii biosintetice. Procedeul propus se include în noua direcţie de utilizare a nanoparticulelor în domeniul biotehnologiei. Nanotechnologies have rapidly migrated from the technical field to medicine, but also biotechnology, creating a new scientific-practical field - bionanotechnology. The area of application of nanoparticles has been expanded. The positive effect of nanoparticles on cell growth and metabolism was demonstrated. The small size of the nanoparticles favors their interaction with the cell surface and penetration into the cytosol. Nanoparticles can be a favorable alternative source of trace elements that act as stimulators of biosynthetic activity. The proposed procedure is included in the new direction of using nanoparticles in the field of biotechnology.
Este cunoscut procedeul de cultivare a cianobacteriei Spirulina platensis pe mediul mineral nutritiv ce conţine, g/L: NaHCO3 - 4,5, K2HPO4 - 0,5, NaNO3 - 1,5, K2SO4 - 1,0, NaCl - 1,0, MgSO4·7H2O - 1,2; CaCl2·2H2O - 0,04, FeSO4 - 0,01 şi pH 9,0. Durata cultivării este de 10 zile. În compoziţia mediului de cultivare fosfatul de potasiu a fost înlocuit cu 0,09 g/L Ca5[OH|(PO4)3] sub forma de nanoparticule cu dimensiunea de 200 nm. Conform acestui procedeu producerea de biomasă creşte cu 21% [1]. The process of cultivating the cyanobacterium Spirulina platensis on the nutritious mineral medium containing, g/L: NaHCO3 - 4.5, K2HPO4 - 0.5, NaNO3 - 1.5, K2SO4 - 1.0, NaCl - 1.0, MgSO4·7H2O - 1.2; CaCl2·2H2O - 0.04, FeSO4 - 0.01 and pH 9.0. The duration of cultivation is 10 days. In the composition of the growing medium, potassium phosphate was replaced by 0.09 g/L Ca5[OH|(PO4)3] in the form of nanoparticles with a size of 200 nm. According to this process, biomass production increases by 21% [1].
Dezavantajul acestui procedeu constă lipsa unui efect vizibil de stimulare a activităţii biosintetice şi în durata ciclului de cultivare. The disadvantage of this procedure is the lack of a visible effect of stimulating the biosynthetic activity during the cultivation cycle.
Cea mai apropiată soluţie este procedeul de cultivare a cianobacteriei Arthrospira (Spirulina) maxima pe mediul mineral Zehnder varianta Z8 cu componenţa în g/l: NaNO3-0,467, Na2CO3 - 0,02, Ca(NO3)2·4H2O-0,059, NH4Cl-0,031, H3BO3-0,0031, MnSO4·4H2O- 0,00223, ZnSO4·7H2O - 0,00022, (NH4)6Mo7O24·4H2O-0,000088, Co(NO3)2·6H2O-0,000146, VOSO4·6H2O-0,000054, Al2(SO4)3K2SO4·2H2O-0,000474, NiSO4(NH4)2SO4·6H2O-0,000198, Cd(NO3)2·4H2O-0,000154, Cr(NO3)3·7H2O-0,000037, Na2W4·2H2O- 0,000033, KBr - 0,000119, KI-0,000083. Mediul de cultivare este suplimentat cu 2% CO2. Fierul din mediul nutritiv a fost înlocuit cu Fe nanoparticule în concentraţia 0,0051 g/L. Durata cultivării a fost de 9 zile. Cultivarea a fost efectuată în godeuri în condiţii axenice. Conform procedeului, producerea de biomasă creşte cu 16%, iar conţinutul lipidelor creşte cu 21% [2]. The closest solution is the process of cultivating the cyanobacterium Arthrospira (Spirulina) maxima on the mineral medium Zehnder version Z8 with the composition in g/l: NaNO3-0.467, Na2CO3 - 0.02, Ca(NO3)2·4H2O-0.059, NH4Cl- 0.031, H3BO3-0.0031, MnSO4·4H2O- 0.00223, ZnSO4·7H2O - 0.00022, (NH4)6Mo7O24·4H2O-0.000088, Co(NO3)2·6H2O-0.000146, VOSO4·6H2O -0.000054, Al2(SO4)3K2SO4·2H2O-0.000474, NiSO4(NH4)2SO4·6H2O-0.000198, Cd(NO3)2·4H2O-0.000154, Cr(NO3)3·7H2O-0 .000037, Na2W4·2H2O- 0.000033, KBr - 0.000119, KI-0.000083. The cultivation medium is supplemented with 2% CO2. The iron in the nutrient medium was replaced by Fe nanoparticles in a concentration of 0.0051 g/L. The duration of cultivation was 9 days. Cultivation was carried out in wells under axenic conditions. According to the procedure, biomass production increases by 16%, and lipid content increases by 21% [2].
Dezavantajul acestui procedeu constă în durata ciclului de cultivare şi lipsa unui spor de biomasă de spirulină. The disadvantage of this process is the duration of the cultivation cycle and the lack of an increase in spirulina biomass.
Problema pe care o rezolvă prezenta invenţie constă în elaborarea unui procedeu eficient şi reproductibil de sporire a producerii de biomasă şi a conţinutului de lipide în biomasa cianobacteriei Spirulina platensis. The problem that the present invention solves consists in the development of an efficient and reproducible process for increasing the production of biomass and the content of lipids in the biomass of the cyanobacterium Spirulina platensis.
Procedeul de cultivare a cianobacteriei Spirulina platensis conform invenţiei, înlătură dezavantajele menţionate mai sus prin aceea că prevede cultivarea culturii pe un mediu nutritiv mineral ce conţine, g/L: NaNO3 - 2,25, NaHCO3 - 8,0, NaCl - 1,0, K2SO4 - 0,3, Na2HPO4 - 0,2, MgSO4·7H2O - 0,2, CaCl2 - 0,024, FeSO4 - 0,01, EDTA - 0,08, H3BO3 - 0,00286, MnCl2·4H2O - 0,00181, ZnSO4·7H2O - 0,00022, CuSO4·5H2O - 0,00008, MoO3 - 0,000015, nanoparticule hidrosolubile de aur cu dimensiunea de 5 nm în concentraţie de 0,0088…0,0091 g/L şi apă distilată până la 1L, la temperatura de 25…28°C, pH 8,0…10,0, iluminarea de 3000…4000 lx în regim continuu în decurs de 5 zile. The process of cultivating the cyanobacterium Spirulina platensis according to the invention removes the disadvantages mentioned above in that it provides for the cultivation of the culture on a mineral nutrient medium containing, g/L: NaNO3 - 2.25, NaHCO3 - 8.0, NaCl - 1.0 , K2SO4 - 0.3, Na2HPO4 - 0.2, MgSO4·7H2O - 0.2, CaCl2 - 0.024, FeSO4 - 0.01, EDTA - 0.08, H3BO3 - 0.00286, MnCl2·4H2O - 0.00181 , ZnSO4·7H2O - 0.00022, CuSO4·5H2O - 0.00008, MoO3 - 0.000015, gold nanoparticles with a size of 5 nm in a concentration of 0.0088...0.0091 g/L and distilled water up to 1L, at a temperature of 25...28°C, pH 8.0...10.0, illumination of 3000...4000 lx in continuous mode within 5 days.
Rezultatul invenţiei constă în asigurarea unei majorări de producere de biomasă cu 40-44% şi a conţinutului de lipide în biomasă cu 28-33%. The result of the invention consists in ensuring an increase in biomass production by 40-44% and the lipid content in biomass by 28-33%.
Rezultatul obţinut este condiţionat de efectul nanoparticulelor de Au care, datorită dimensiunilor mici (5 nm) pătrund rapid în celule şi stimulează reproducerea celulară, drept urmare durata ciclului de cultivare se reduce până la 5 zile. Contactul nanoparticulelor cu membrana celulară induce stimularea producerii lipidelor. Totodată, în biomasa cianobacteriană nu se diminuează conţinutul de proteine, glucide şi pigmenţi. The result obtained is conditioned by the effect of Au nanoparticles which, due to their small size (5 nm) quickly penetrate the cells and stimulate cell reproduction, as a result the duration of the cultivation cycle is reduced to 5 days. The contact of nanoparticles with the cell membrane induces the stimulation of lipid production. At the same time, the content of proteins, carbohydrates and pigments does not decrease in the cyanobacterial biomass.
Exemple de realizare a invenţiei. Examples of realization of the invention.
Exemplul 1 Example 1
Se prepară mediul mineral nutritiv cu următoarea componenţă (g/L): NaNO3 - 2,25, NaHCO3 - 8,0, NaCl - 1,0; K2SO4 - 0,3, Na2HPO4 - 0,2; MgSO4·7H2O - 0,2, CaCl2 - 0,024, FeSO4 - 0,01, EDTA - 0,08, H3BO3 - 0,00286, MnCl2·4H2O - 0,00181, ZnSO4·7H2O - 0,00022, CuSO4·5H2O - 0,00008, MoO3 - 0,000015 şi apă distilată până la 1L. La mediul preparat se adaugă 0,0088 g/L nanoparticule de Au. Cultura start este suspensia de Spirulina platensis CNMN-CB-11 în cantitate de 0,3 g/L. Cultivarea se efectuează în baloane Erlenmeyer cu volumul de 500 ml şi volumul de lucru de 250 ml la temperatura de 25°C, pH-ul 8,0-10,0 şi iluminarea de 3000-4000 lx în regim continuu. Prepare the nutritious mineral medium with the following composition (g/L): NaNO3 - 2.25, NaHCO3 - 8.0, NaCl - 1.0; K2SO4 - 0.3, Na2HPO4 - 0.2; MgSO4·7H2O - 0.2, CaCl2 - 0.024, FeSO4 - 0.01, EDTA - 0.08, H3BO3 - 0.00286, MnCl2·4H2O - 0.00181, ZnSO4·7H2O - 0.00022, CuSO4·5H2O - 0.00008, MoO3 - 0.000015 and distilled water up to 1L. 0.0088 g/L Au nanoparticles are added to the prepared medium. The starting culture is the suspension of Spirulina platensis CNMN-CB-11 in an amount of 0.3 g/L. Cultivation is carried out in Erlenmeyer flasks with a volume of 500 ml and a working volume of 250 ml at a temperature of 25°C, pH 8.0-10.0 and illumination of 3000-4000 lx in continuous mode.
La a 5-a zi se colectează biomasa de spirulină şi se determină conţinutul de lipide. A fost obţinut 1,30 g/L biomasă. Conţinutul lipidelor în biomasă este de 6,27%. On the 5th day, the spirulina biomass is collected and the lipid content is determined. 1.30 g/L biomass was obtained. The lipid content in the biomass is 6.27%.
Exemplul 2 Example 2
Se prepară mediul mineral nutritiv cu următoarea componenţă (g/L): NaNO3 - 2,25, NaHCO3 - 8,0, NaCl - 1,0, K2SO4 - 0,3, Na2HPO4 - 0,2, MgSO4·7H2O - 0,2, CaCl2 - 0,024, FeSO4 - 0,01, EDTA - 0,08, H3BO3 - 0,00286, MnCl2·4H2O - 0,00181, ZnSO4·7H2O - 0,00022, CuSO4·5H2O - 0,00008, MoO3 - 0,000015 şi apă distilată până la 1L. La mediul preparat se adaugă 0,0091 g/L nanoparticule de Au. Cultura start este suspensia de Spirulina platensis CNMN-CB-01 în cantitate de 0,3 g/L. Cultivarea se efectuează în baloane Erlenmeyer cu volumul de 500 ml şi volumul de lucru de 250 ml la temperatura de 28°C, la pH-ul 8,0-10,0 şi iluminarea de 3000-4000 lx în regim continuu. Prepare the nutrient mineral medium with the following composition (g/L): NaNO3 - 2.25, NaHCO3 - 8.0, NaCl - 1.0, K2SO4 - 0.3, Na2HPO4 - 0.2, MgSO4·7H2O - 0, 2, CaCl2 - 0.024, FeSO4 - 0.01, EDTA - 0.08, H3BO3 - 0.00286, MnCl2·4H2O - 0.00181, ZnSO4·7H2O - 0.00022, CuSO4·5H2O - 0.00008, MoO3 - 0.000015 and distilled water up to 1L. 0.0091 g/L Au nanoparticles are added to the prepared medium. The starting culture is the suspension of Spirulina platensis CNMN-CB-01 in an amount of 0.3 g/L. Cultivation is carried out in Erlenmeyer flasks with a volume of 500 ml and a working volume of 250 ml at a temperature of 28°C, at a pH of 8.0-10.0 and illumination of 3000-4000 lx in continuous mode.
La a 5-a zi se colectează biomasa de spirulină şi se determină conţinutul de lipide. A fost obţinut 1,38 g/L biomasă. Conţinutul lipidelor în biomasă este de 6,52%. On the 5th day, the spirulina biomass is collected and the lipid content is determined. 1.38 g/L biomass was obtained. The lipid content in the biomass is 6.52%.
Tabel Table
Cantitatea de biomasă de Spirulina platensis şi conţinutul lipidelor în biomasă la cultivare conform procedeului propus în invenţie şi soluţiei celei mai apropiate The quantity of Spirulina platensis biomass and the lipid content in the biomass during cultivation according to the method proposed in the invention and the closest solution
Procedeul utilizat Compusul (nanoparticule), concentraţia g/L Durata ciclului de cultivare, zile Sporul de biomasă de spirulină, % (biomasa, g/L) Sporul conţinutului de lipide în biomasă, % (conţinutul, % biomasă) Conform soluţiei [1] Ca5[OH|(PO4)3], 0,09 g/L 10 21% - Conform celei mai apropiate soluţii [2] FeNP, 0,0051 g/L 9 16% 21% Conform soluţiei revendicate AuNP, 0,0088 g/L 5 40% (1,30±0,06) 28% (6,27±0,10) AuNP, 0,0091 g/L 5 44% (1,38±0,03) 33% (6,52±0,07) The procedure used Compound (nanoparticles), concentration g/L Cultivation cycle duration, days Increase in spirulina biomass, % (biomass, g/L) Increase in lipid content in biomass, % (content, % biomass) According to the solution [1] Ca5[OH|(PO4)3], 0.09 g/L 10 21% - According to the closest solution [2] FeNP, 0.0051 g/L 9 16% 21% According to the claimed solution AuNP, 0.0088 g /L 5 40% (1.30±0.06) 28% (6.27±0.10) AuNP, 0.0091 g/L 5 44% (1.38±0.03) 33% (6, 52±0.07)
Astfel, datele din tabel demonstrează majorarea de 1,9-2,0 ori a producerii de biomasă şi de 1,3-1,57 ori a conţinutului de lipide în biomasa spirulinei în procedeul propus în invenţie faţă de procedeele cele mai apropiate. Thus, the data in the table demonstrate the increase of 1.9-2.0 times the biomass production and 1.3-1.57 times the lipid content in the spirulina biomass in the process proposed in the invention compared to the closest processes.
1. J. A. Lone, A. Kumar, S. Kundu, F. A. Lone, M. R. Suseela. Characterization of Tolerance Limit inSpirulina platensis in Relation to Nanoparticles. Water Air Soil Pollut, 2013, 224:1670. 1. J.A. Lone, A. Kumar, S. Kundu, F.A. Lone, M.R. Suseela. Characterization of Tolerance Limit in Spirulina platensis in Relation to Nanoparticles. Water Air Soil Pollut, 2013, 224:1670.
2. Karolína Pádrová, Jaromír Lukavský, Linda Nedbalová, Alena Čejková, Tomáš Cajthaml, Karel Sigler, Milada Vítová, Tomáš Řezanka. Trace concentrations of iron nanoparticles cause overproduction of biomass and lipids during cultivation of cyanobacteria and microalgae. Journal of Applied Phycology <http://link.springer.com/journal/10811>, 2015, 27(4), p. 1443-1451 2. Karolína Pádrová, Jaromír Lukavský, Linda Nedbalová, Alena Čejková, Tomáš Cajthaml, Karel Sigler, Milada Vítová, Tomáš Řezanka. Trace concentrations of iron nanoparticles cause overproduction of biomass and lipids during cultivation of cyanobacteria and microalgae. Journal of Applied Phycology <http://link.springer.com/journal/10811>, 2015, 27(4), pp. 1443-1451
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