MD167Y - Process for the cultivation of cyanobacterium Spirulina platensis - Google Patents

Process for the cultivation of cyanobacterium Spirulina platensis Download PDF

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MD167Y
MD167Y MDS20090211A MDS20090211A MD167Y MD 167 Y MD167 Y MD 167Y MD S20090211 A MDS20090211 A MD S20090211A MD S20090211 A MDS20090211 A MD S20090211A MD 167 Y MD167 Y MD 167Y
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Moldova
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cultivation
days
spirulina platensis
phycobiliproteins
cyanobacterium spirulina
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MDS20090211A
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Romanian (ro)
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Liudmila Batir
Valeriu Rudic
Valentina Bulimaga
Liliana Zosim
Daniela ELENCIUC
Aurelian Gulea
Natalia Bejenari
Victor Tapcov
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Universitatea De Stat Din Moldova
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Abstract

Inventia se refera la ficobiotehnologie, in particular la un procedeu de cultivare a cianobacteriei Spirulina platensis cu un continut sporit de ficobiliproteine, care pot fi utilizate in industria farmaceutica si medicina. Procedeul de cultivare a cianobacteriei Spirulina platensis include cultivarea pe un mediu nutritiv, continand, g/L: NaHCO3 - 16,8; K2HPO4·3H2O - 1,0; NaNO3 - 2,5; NaCl - 1,0; K2SO4 - 1,0; CaCl2·6H2O - 0,04; MgSO4·7H2O - 0,20; H3BO3 - 0,00286; MnCl2·4H2O - 0,00181; ZnSO4·7H2O - 0,00022; CuSO4·5H2O - 0,00008; MoO3 - 0,000015; 1mL de solutie FeHEDTA de 0,09 M, apa distilata pana la 1 L. La a 2-a zi de cultivare in mediu se adauga 3...4 mg/L de hidrat al nitratului de 5-nitrosalicilidentiosemicarbazido(1-)aquacupru(II). Cultivarea se efectueaza timp de 6 zile, la temperatura de 30 ... 35°C, la iluminarea de 2000...3000 lx in primele 2 zile si de 3000...4000 lx in urmatoarele zile.The invention relates to phyobiotechnology, in particular to a process for cultivating cyanobacterium Spirulina platensis with a higher content of phycobiliproteins, which can be used in the pharmaceutical and medical industries. The process of cultivation of the cyanobacterium Spirulina platensis includes cultivation on a nutrient medium, containing, g / L: NaHCO3 - 16.8; K2HPO4 · 3H2O - 1.0; NaNO3 - 2.5; NaCl - 1.0; K2SO4 - 1.0; CaCl2 · 6H2O - 0.04; MgSO4 · 7H2O - 0.20; H3BO3 - 0.00286; MnCl2 · 4H2O - 0.00181; ZnSO4 · 7H2O - 0.00022; CuSO4 · 5H2O - 0.00008; MoO3 - 0.000015; 1mL of 0.09 M FeHEDTA solution, distilled water up to 1 L. On the second day of cultivation in the medium add 3 ... 4 mg / L of hydrate of 5-nitrosalicylidentosemicarbazido (1-) aquacupure nitrate. (II). The cultivation is carried out for 6 days, at a temperature of 30 ... 35 ° C, at the lighting of 2000 ... 3000 lx in the first 2 days and 3000 ... 4000 lx in the following days.

Description

Invenţia se referă la ficobiotehnologie, în particular la un procedeu de cultivare a cianobacteriei Spirulina platensis cu un conţinut sporit de ficobiliproteine, care pot fi utilizate în industria farmaceutică şi medicină. The invention relates to phycobiotechnology, in particular to a process for cultivating the cyanobacterium Spirulina platensis with an increased content of phycobiliproteins, which can be used in the pharmaceutical and medical industries.

Ficobiliproteinele sunt utilizate pe scară largă în industria cosmetică şi farmaceutică, au proprietăţi antiinflamatoare, neuro- şi hepatoprotectoare, dar şi antioxidante. Phycobiliproteins are widely used in the cosmetic and pharmaceutical industries, they have anti-inflammatory, neuro- and hepatoprotective, as well as antioxidant properties.

Este cunoscut un procedeu de cultivare a algelor şi cianobacteriilor, unde dintre 18 tulpini studiate, cianobacteria Spirulina platensis NCCU-S5 are un conţinut de ficobiliproteine de 62,5 mg/g din masa celulară uscată. Acest procedeu constă în creşterea culturii la pH 8,0, la temperatura de 30±1°C, intensitatea luminii 25 µmol fotoni/m2/s, în regim de noapte/zi timp de 27 zile [1]. A method for cultivating algae and cyanobacteria is known, where among 18 strains studied, the cyanobacterium Spirulina platensis NCCU-S5 has a phycobiliprotein content of 62.5 mg/g of dry cell mass. This method consists of growing the culture at pH 8.0, at a temperature of 30±1°C, light intensity 25 µmol photons/m2/s, in a night/day regime for 27 days [1].

Dezavantajul acestui procedeu constă în durata îndelungată de cultivare, care are loc 27 zile şi nu duce la un conţinut sporit de ficobiliproteine. The disadvantage of this process is the long cultivation time, which takes 27 days and does not lead to an increased content of phycobiliproteins.

Mai este cunoscut un procedeu de cultivare a cianobacteriei Spirulina platensis cu un conţinut sporit de ficobiliproteine, care include inocularea şi cultivarea spirulinei pe un mediu nutritiv cu următorul conţinut al ingredientelor (g/L): NaHCO3 - 16,8; K2HPO4·3H2O - 1,0; NaNO3 - 2,5; NaCl - 1,0; K2SO4 - 1,0; CaCl2·6H2O - 0,04; MgSO4·7H2O - 0,20; H3BO3 - 0,00286; MnCl2·4H2O - 0,00181; ZnSO4·7H2O - 0,00022; CuSO4·5H2O - 0,00008; MoO3 - 0,000015. În a treia zi de cultivare în acest mediu se adaugă unul dintre compuşii coordinativi: azotat de hexa-µ-glicinato(O,O')-µ3-oxotriacvotrifier(III)trihidrat-[Fe3O(Gly)6 (H2O)3]NO3·3H2O, de hexa-µ-treoninato(O,O')-µ3-oxotriacvotrifier(III)-[Fe3O (Tre)6 (H2O)3]NO3 sau de hexa-µ-alaninato(O,O')-µ3-oxotriacvotrifier(III)trihidrat-[Fe3 O(Ala)6(H2O)3]NO3·3H2O în cantitate de 5 … 10 mg/L, cultivarea se efectuează timp de 6 zile, la o temperatură de 30…35°C şi iluminarea de 3000…4000 lx [2]. There is also a known process for cultivating the cyanobacterium Spirulina platensis with an increased content of phycobiliproteins, which includes inoculating and cultivating spirulina on a nutrient medium with the following content of ingredients (g/L): NaHCO3 - 16.8; K2HPO4·3H2O - 1.0; NaNO3 - 2.5; NaCl - 1.0; K2SO4 - 1.0; CaCl2·6H2O - 0.04; MgSO4·7H2O - 0.20; H3BO3 - 0.00286; MnCl2·4H2O - 0.00181; ZnSO4·7H2O - 0.00022; CuSO4·5H2O - 0.00008; MoO3 - 0.000015. On the third day of cultivation, one of the coordinating compounds is added to this medium: hexa-µ-glycinato(O,O')-µ3-oxotriquattrifier(III) trihydrate nitrate [Fe3O(Gly)6 (H2O)3]NO3·3H2O, hexa-µ-threoninato(O,O')-µ3-oxotriquattrifier(III) trihydrate [Fe3O(Tre)6 (H2O)3]NO3·3H2O or hexa-µ-alaninato(O,O')-µ3-oxotriquattrifier(III) trihydrate [Fe3 O(Ala)6(H2O)3]NO3·3H2O in an amount of 5...10 mg/L, cultivation is carried out for 6 days, at a temperature of 30...35°C and illumination of 3000...4000 lx [2].

Dezavantajul acestui procedeu constă în faptul că cultivarea spirulinei conform procedeului asigură obţinerea unei biomase cu un conţinut doar de 21,68% de ficobiliproteine ca parte componentă a biomasei. The disadvantage of this process is that cultivating spirulina according to the process ensures the production of biomass with a content of only 21.68% of phycobiliproteins as a component of the biomass.

Problema pe care o rezolvă invenţia propusă constă în elaborarea unui procedeu de cultivare a cianobacteriei Spirulina platensis cu un conţinut sporit de ficobiliproteine - sursă de substanţe bioactive utilizate în industria farmaceutică, medicina clinică şi experimentală. The problem solved by the proposed invention consists in developing a process for cultivating the cyanobacterium Spirulina platensis with an increased content of phycobiliproteins - a source of bioactive substances used in the pharmaceutical industry, clinical and experimental medicine.

Procedeul de cultivare a cianobacteriei Spirulina platensis include cultivarea pe un mediu nutritiv, conţinând, g/L: NaHCO3 - 16,8; K2HPO4·3H2O - 1,0; NaNO3 - 2,5; NaCl - 1,0; K2SO4 - 1,0; CaCl2·6H2O - 0,04; MgSO4·7H2O - 0,20; H3BO3 - 0,00286; MnCl2·4H2O - 0,00181; ZnSO4·7H2O - 0,00022; CuSO4·5H2O - 0,00008; MoO3 - 0,000015; 1mL de soluţie FeHEDTA de 0,09 M, apă distilată până la 1L. La a 2-a zi de cultivare în mediu se adaugă 3...4 mg/L de hidrat al nitratului de 5-nitrosalicilidentiosemicarbazido(1-)aquacupru(II). Cultivarea se efectuează timp de 6 zile, la temperatura de 30 ... 35°C, la iluminarea de 2000...3000 lx în primele 2 zile şi de 3000...4000 lx în următoarele zile. The process of cultivating the cyanobacterium Spirulina platensis includes cultivation on a nutrient medium, containing, g/L: NaHCO3 - 16.8; K2HPO4·3H2O - 1.0; NaNO3 - 2.5; NaCl - 1.0; K2SO4 - 1.0; CaCl2·6H2O - 0.04; MgSO4·7H2O - 0.20; H3BO3 - 0.00286; MnCl2·4H2O - 0.00181; ZnSO4·7H2O - 0.00022; CuSO4·5H2O - 0.00008; MoO3 - 0.000015; 1mL of 0.09 M FeHEDTA solution, distilled water up to 1L. On the 2nd day of cultivation, 3...4 mg/L of 5-nitrosalicylidentiosemicarbazido(1-)aquacopper(II) nitrate hydrate is added to the medium. Cultivation is carried out for 6 days, at a temperature of 30...35°C, with illumination of 2000...3000 lx in the first 2 days and 3000...4000 lx in the following days.

Compusul coordinativ al Cu(II), hidratul nitratului de 5-nitrosalicilidentiosemicarbazido(1-)aquacupru(II) este descris în MD 3890 G2 2009.04.30 şi are următoarea formulă de structură: The coordination compound of Cu(II), 5-nitrosalicylidentiosemicarbazido(1-)aquacopper(II) nitrate hydrate is described in MD 3890 G2 2009.04.30 and has the following structural formula:

Rezultatul invenţiei constă în asigurarea unei majorări a conţinutului de ficobiliproteine în biomasa de spirulină de 1,27 … 1,40 ori sau 27,50 … 30,33 % din biomasa absolut uscată (BAU). The result of the invention consists in ensuring an increase in the content of phycobiliproteins in spirulina biomass by 1.27 ... 1.40 times or 27.50 ... 30.33% of the absolute dry biomass (BAU).

Rezultatul obţinut se datorează faptului că compuşii coordinativi ai Cu(II) cauzează stresul oxidativ în celulele spirulinei şi ca rezultat are loc sinteza sporită a ficobiliproteinelor, pigmenţi ce fac parte din fotosistemul II. The result obtained is due to the fact that Cu(II) coordination compounds cause oxidative stress in spirulina cells and as a result, increased synthesis of phycobiliproteins, pigments that are part of photosystem II, occurs.

Exemplu de realizare a invenţiei Example of embodiment of the invention

Exemplul 1 Example 1

Se prepară mediul nutritiv mineral SP-1 cu următoarea componenţă (g/L): NaNO3 2,5; NaHCO3 16,8; NaCl 1,0; K2SO4 1,0; K2HPO4·3H2O 1,0; MgSO4·7H2O 0,20; CaCl2·6H2O 0,04; H3BO3 0,00286; MnCl2·4H2O 0,00181; ZnSO4·7H2O 0,00022; CuSO4·5H2O 0,00008; MoO3 0,000015; 1mL soluţie FeHEDTA de 0,09M şi apă distilată până la 1L. Prepare the SP-1 mineral nutrient medium with the following composition (g/L): NaNO3 2.5; NaHCO3 16.8; NaCl 1.0; K2SO4 1.0; K2HPO4·3H2O 1.0; MgSO4·7H2O 0.20; CaCl2·6H2O 0.04; H3BO3 0.00286; MnCl2·4H2O 0.00181; ZnSO4·7H2O 0.00022; CuSO4·5H2O 0.00008; MoO3 0.000015; 1mL of 0.09M FeHEDTA solution and distilled water up to 1L.

La mediul preparat se adaugă suspensia de Spirulina platensis în cantitate de 0,40 g/L. În a doua zi de cultivare suspensia de spirulină se suplimentează cu 3 mg/L de hidrat al nitratului de 5-nitrosalicilidentiosemicarbazido(1-)aquacupru(II). The Spirulina platensis suspension is added to the prepared medium in an amount of 0.40 g/L. On the second day of cultivation, the spirulina suspension is supplemented with 3 mg/L of 5-nitrosalicylidentiosemicarbazido(1-)aquacopper(II) nitrate hydrate.

Cultivarea se efectuează în baloane Erlenmeyer a câte 250 mL cu 100 mL suspensie în următoarele condiţii: iluminarea de 2000 … 3000 lx în primele 2 zile şi de 3000 … 4000 lx în următoarele zile ale cultivării, la pH 9,5 … 10,0 şi la o temperatură de 30° C. Cultivation is carried out in 250 mL Erlenmeyer flasks with 100 mL of suspension under the following conditions: illumination of 2000 ... 3000 lx in the first 2 days and 3000 ... 4000 lx in the following days of cultivation, at pH 9.5 ... 10.0 and at a temperature of 30° C.

La ziua a şaptea biomasa se separă de lichidul cultural, se supune demineralizării şi se determină conţinutul de ficobiliproteine. On the seventh day, the biomass is separated from the culture liquid, subjected to demineralization and the phycobiliprotein content is determined.

Biomasa obţinută conţine 27,50% de ficobiliproteine din BAU faţă de 21,68% în cazul soluţiei celei mai apropiate. The obtained biomass contains 27.50% phycobiliproteins from BAU compared to 21.68% in the case of the closest solution.

Exemplul 2 Example 2

Se prepară mediul nutritiv mineral SP-1 cu următoarea componenţă (g/L): NaNO3 2,5; NaHCO3 16,8; NaCl 1,0; K2SO4 1,0; K2HPO4·3H2O 1,0; MgSO4·7H2O 0,20; CaCl2·6H2O 0,04; H3BO3 0,00286; MnCl2·4H2O 0,00181; ZnSO4·7H2O 0,00022; CuSO4·5H2O 0,00008; MoO3 0,000015; 1 mL soluţie FeHEDTA de 0,09M şi apă distilată până la 1L. Prepare the SP-1 mineral nutrient medium with the following composition (g/L): NaNO3 2.5; NaHCO3 16.8; NaCl 1.0; K2SO4 1.0; K2HPO4·3H2O 1.0; MgSO4·7H2O 0.20; CaCl2·6H2O 0.04; H3BO3 0.00286; MnCl2·4H2O 0.00181; ZnSO4·7H2O 0.00022; CuSO4·5H2O 0.00008; MoO3 0.000015; 1 mL of 0.09M FeHEDTA solution and distilled water up to 1L.

La mediul preparat se adaugă suspensia de Spirulina platensis în cantitate de 0,40 g/L. În a doua zi de cultivare suspensia de spirulină se suplimentează cu 4 mg/L de hidrat al nitratului de 5-nitrosalicilidentiosemicarbazido(1-)aquacupru(II). The Spirulina platensis suspension is added to the prepared medium in an amount of 0.40 g/L. On the second day of cultivation, the spirulina suspension is supplemented with 4 mg/L of 5-nitrosalicylidentiosemicarbazido(1-)aquacopper(II) nitrate hydrate.

Cultivarea se efectuează în baloane Erlenmeyer a câte 250mL cu 100mL suspensie în următoarele condiţii: iluminarea de 2000 … 3000 lx în primele 2 zile şi de 3000 … 4000 lx în următoarele zile ale cultivării, la pH 9,5…10,0 şi la o temperatură de 32° C. Cultivation is carried out in 250mL Erlenmeyer flasks with 100mL suspension under the following conditions: illumination of 2000...3000 lx in the first 2 days and 3000...4000 lx in the following days of cultivation, at pH 9.5...10.0 and at a temperature of 32° C.

La ziua a şaptea biomasa se separă de lichidul cultural, se supune demineralizării şi se determină conţinutul de ficobiliproteine. On the seventh day, the biomass is separated from the culture liquid, subjected to demineralization and the phycobiliprotein content is determined.

Biomasa obţinută conţine 30,33% ficobiliproteine din BAU faţă de 21,68% în cazul soluţiei celei mai apropiate. The obtained biomass contains 30.33% phycobiliproteins from BAU compared to 21.68% in the case of the closest solution.

Conţinutul ficobiliproteinelor în biomasa cianobacteriei Spirulina platensis Phycobiliprotein content in the biomass of the cyanobacterium Spirulina platensis

la cultivare conform procedeului propus în invenţie when cultivated according to the method proposed in the invention

şi soluţiei cele mai apropiate and the closest solution

Procedeul utilizat Compusul Concentraţia compusului, mg/L Conţinutul ficobiliproteinelor, % din BAU Conform soluţiei celei mai apropiate [Fe3O(Gly)6(H2O)3]NO3x3H2O 5 21,68±0,580 Conform invenţiei Hidratul nitratului de 5-nitrosalicilidentiosemicarbazido (1-)aquacupru(II) 3 4 27,50±0,110 30,33±0,130Process used Compound Compound concentration, mg/L Phycobiliprotein content, % of BAU According to the closest solution [Fe3O(Gly)6(H2O)3]NO3x3H2O 5 21.68±0.580 According to the invention 5-nitrosalicylidentiosemicarbazido (1-)aquacopper(II) nitrate hydrate 3 4 27.50±0.110 30.33±0.130

Datele tabelului demonstrează majorarea de 1,27 … 1,4 ori a conţinutului de ficobiliproteine în biomasa de spirulină în procedeul propus în invenţie faţă de procedeul cel mai apropiat. The data in the table demonstrate a 1.27...1.4-fold increase in the content of phycobiliproteins in the spirulina biomass in the process proposed in the invention compared to the closest process.

1. Hemlata Tasneem Fatma. Screening of cyanobacteria for phycobiliproteins and effect of different environmental stress on its yield. Bulletin of environmental contamination and toxicology, 2009, Vol. 83, № 4, 509-515 1. Hemlata Tasneem Fatma. Screening of cyanobacteria for phycobiliproteins and effect of different environmental stress on its yield. Bulletin of environmental contamination and toxicology, 2009, Vol. 83, No. 4, 509-515

2. MD 2386 C2 2003.02.28 2. MD 2386 C2 2003.02.28

Claims (1)

Procedeu de cultivare a cianobacteriei Spirulina platensis, care include cultivarea timp de 6 zile pe un mediu nutritiv, conţinând, g/L: NaHCO3 - 16,8; K2HPO4·3H2O - 1,0; NaNO3 - 2,5; NaCl - 1,0; K2SO4 - 1,0; CaCl2·6H2O - 0,04; MgSO4·7H2O - 0,20; H3BO3 - 0,00286; MnCl2·4H2O - 0,00181; ZnSO4·7H2O - 0,00022; CuSO4·5H2O - 0,00008; MoO3 - 0,000015 şi apă distilată până la 1 L, la care se adaugă un compus coordinativ, totodată cultivarea se efectuează la iluminarea de 3000 ... 4000 lx şi temperatura de 30 ... 35°C, caracterizat prin aceea că mediul nutritiv conţine suplimentar 1mL/L de soluţie FeHEDTA de 0,09 M, iar în calitate de compus coordinativ se utilizează hidratul nitratului de 5-nitrosalicilidentiosemicarbazido(1-)aquacupru(II), care se adaugă în mediu la a 2-a zi de cultivare în cantitate de 3...4 mg/L, totodată în primele 2 zile cultivarea se efectuează la iluminarea de 2000...3000 lx.Process for cultivating the cyanobacterium Spirulina platensis, which includes cultivation for 6 days on a nutrient medium, containing, g/L: NaHCO3 - 16.8; K2HPO4·3H2O - 1.0; NaNO3 - 2.5; NaCl - 1.0; K2SO4 - 1.0; CaCl2·6H2O - 0.04; MgSO4·7H2O - 0.20; H3BO3 - 0.00286; MnCl2·4H2O - 0.00181; ZnSO4·7H2O - 0.00022; CuSO4·5H2O - 0.00008; MoO3 - 0.000015 and distilled water up to 1 L, to which a coordinating compound is added, at the same time the cultivation is carried out at an illumination of 3000 ... 4000 lx and a temperature of 30 ... 35°C, characterized in that the nutrient medium additionally contains 1mL/L of 0.09 M FeHEDTA solution, and as a coordinating compound the hydrate of 5-nitrosalicylidentiosemicarbazido(1-)aquacopper(II) nitrate is used, which is added to the medium on the 2nd day of cultivation in an amount of 3...4 mg/L, at the same time in the first 2 days the cultivation is carried out at an illumination of 2000...3000 lx.
MDS20090211A 2009-11-13 2009-11-13 Process for the cultivation of cyanobacterium Spirulina platensis MD167Z (en)

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MD163C2 (en) * 1989-12-22 1995-06-30 Валериу РУДИК Method for Spirulina microalga cultivation
MD240G2 (en) * 1994-08-22 1995-12-31 Vladimir Bacicov The sail with variable profile
MD614G2 (en) * 1994-09-07 1997-06-30 Государственный Университет Молд0 Process for spirulina biomass production
MD2386C2 (en) * 2001-05-15 2004-10-31 Институт Микробиологии И Биотехнологии Академии Наук Молдовы Process for cultivation of cyanobacterium Spirulina platensis
MD3101G2 (en) * 2005-12-26 2007-02-28 Государственный Университет Молд0 Process for Spirulina platensis biomass obtaining
MD3129G2 (en) * 2006-01-31 2007-03-31 Государственный Университет Молд0 Process for Spirulina platensis cyanobacterium biomass obtaining
MD3128G2 (en) * 2006-01-31 2007-03-31 Государственный Университет Молд0 Process for Spirulina platensis biomass obtaining
MD3171G2 (en) * 2006-02-17 2007-05-31 Государственный Университет Молд0 Process for Spirulina platensis cyanobacterium biomass obtaining
MD3417G2 (en) * 2007-03-16 2008-05-31 Государственный Университет Молд0 Nutrient medium for cultivation of Spirulina platensis cyanobacyterium
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