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MD4492C1 - Process for cultivation of Spirulina platensis cyanobacterium - Google Patents

Process for cultivation of Spirulina platensis cyanobacterium Download PDF

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MD4492C1
MD4492C1 MDA20160091A MD20160091A MD4492C1 MD 4492 C1 MD4492 C1 MD 4492C1 MD A20160091 A MDA20160091 A MD A20160091A MD 20160091 A MD20160091 A MD 20160091A MD 4492 C1 MD4492 C1 MD 4492C1
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cultivation
exopolysaccharides
spirulina
cyanobacterium
cuso4
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MDA20160091A
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MD4492B1 (en
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Валентина БУЛЬМАГА
Мария ПИСОВ
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Государственный Университет Молд0
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Abstract

Invenţia se referă la ficobiotehnologie, şi anume la un procedeu de cultivare a cianobacteriei Spirulina platensis, pentru utilizare în medicină şi industria alimentară.Procedeul, conform invenţiei, includeinocularea cianobacterieipe mediul nutritiv Zarrouk, cultivarea în decurs de 7 zile la iluminarea de 3500 lx şi temperatura de 35 °C, apoi la a 8-a zi mediul se suplimentează cu 2…4 mg/l CuSO4•5H2O şi cultivarea se efectuează încă 3 zile la iluminarea de 3500…5500 lx.Rezultatul constă în majorarea productivităţii cianobacteriei şi a conţinutului de exopolizaharide acide.The invention relates to phytobiotechnology, namely to a process for cultivation of the cyanobacterium Spirulina platensis, for use in medicine and the food industry. at 35 ° C, then on the 8th day the medium is supplemented with 2… 4 mg / l CuSO4 • 5H2O and cultivation is carried out for another 3 days at 3500… 5500 lx illumination. The result is increased productivity of cyanobacteria and the acidic exopolysaccharides.

Description

Invenţia se referă la ficobiotehnologie, şi anume la un procedeu de cultivare a cianobacteriei Spirulina platensis pentru utilizare în medicină şi industria alimentară. The invention relates to phycobiotechnology, namely to a method of cultivating the cyanobacterium Spirulina platensis for use in medicine and the food industry.

În ultimele decenii, o atenţie deosebită din partea cercetătorilor este orientată asupra proprietăţilor biologice şi chimice ale polizaharidelor, în special a polizaharidelor sulfatate. Polizaharidele sulfatate posedă o gamă largă de bioactivităţi importante: antioxidante, antivirale, anticoagulante şi trombocitare, precum şi de inhibare a unor tumori. Este deja bine cunoscută polizaharida sulfatată Ca-spirulina, obţinută din biomasa de spirulină, care posedă acţiune anticanceroasă. În acest context prezintă interes elaborarea procedeelor de inducere a sintezei polizaharidelor acide şi sulfatate la cianobacteria Spirulina platensis. In the last decades, special attention from researchers is focused on the biological and chemical properties of polysaccharides, especially sulfated polysaccharides. Sulfated polysaccharides possess a wide range of important bioactivities: antioxidant, antiviral, anticoagulant and thrombocyte, as well as tumor inhibition. The sulfated polysaccharide Ca-spirulina, obtained from spirulina biomass, is already well known and possesses anticancer action. In this context, the elaboration of procedures for inducing the synthesis of acidic and sulfated polysaccharides in the cyanobacterium Spirulina platensis is of interest.

Este cunoscut un procedeu de cultivare a cianobacteriei Spirulina subsalsa care utilizează stresul pentru sporirea producerii de exopolizaharide prin crearea deficienţei de fosfat sau nitrat, prin adaos de un surplus de NaCl (0,9M), precum şi nitrat de (10 mM) la mediul de cultivare [1]. A procedure for cultivating the cyanobacterium Spirulina subsalsa is known which uses stress to increase the production of exopolysaccharides by creating a phosphate or nitrate deficiency, by adding a surplus of NaCl (0.9M), as well as nitrate (10 mM) to the medium cultivation [1].

Cu toate că a fost stabilit efectul pozitiv al factorilor menţionaţi asupra acumulării exopolizaharidelor la cianobacteria Spirulina subsalsa, dezavantajul acestui procedeu constă în diminuarea productivităţii şi a conţinutului de proteină în biomasă, cantitatea de exopolizaharide nu este considerabil de înaltă, fiind influenţată de durata cultivării, care în prezenţa excesului de 0,9 M NaCl atinge valoarea maximă în a 28-a zi de cultivare, ceea ce nu este rentabil din punct de vedere economic. Although the positive effect of the mentioned factors on the accumulation of exopolysaccharides in the cyanobacterium Spirulina subsalsa has been established, the disadvantage of this process is the reduction of productivity and protein content in the biomass, the amount of exopolysaccharides is not considerably high, being influenced by the duration of cultivation, which in the presence of the excess of 0.9 M NaCl it reaches the maximum value on the 28th day of cultivation, which is not profitable from an economic point of view.

Mai este cunoscut un procedeu de cultivare a cianobacteriei Spirulina platensis cu conţinut bogat de polizaharide sulfatate, care prevede cultivarea în baloane Erlenmeyer de 4 litri, conţinând 3 litri de mediul Zarrouk autoclavat. Mediul a fost îmbogăţit cu azot (sub formă de nitrat de sodiu) în concentraţii de 45, 128, 293, 412 şi 622 ppm. pH-ul mediului a fost ajustat la 10,5 şi mediul a fost autoclavat. Algele au fost supuse barbotării cu 0,3% CO2 în aer şi au fost cultivate la 25 ± 3°C, la o iluminare continuă cu lămpi fluorescente. În biomasa de spirulină a fost determinat conţinutul de polizaharide sulfatate, care a constituit 5,02% şi 4,13% la cultivare pe mediu cu 412 ppm de azot (nivel optim) şi 45 ppm de azot (nivel limitat) [2]. There is also a method for cultivating the cyanobacterium Spirulina platensis with a rich content of sulfated polysaccharides, which provides for cultivation in 4-liter Erlenmeyer flasks, containing 3 liters of autoclaved Zarrouk medium. The medium was enriched with nitrogen (in the form of sodium nitrate) in concentrations of 45, 128, 293, 412 and 622 ppm. The pH of the medium was adjusted to 10.5 and the medium was autoclaved. Algae were bubbled with 0.3% CO2 in air and cultivated at 25 ± 3°C under continuous fluorescent light. The content of sulfated polysaccharides was determined in the spirulina biomass, which was 5.02% and 4.13% when grown on medium with 412 ppm nitrogen (optimal level) and 45 ppm nitrogen (limited level) [2].

Dezavantajul acestui procedeu constă în aceea că la cultivarea spirulinei pe medii ce conţin 412 ppm (nivel optim de N2) şi 45 ppm (nivel limitat de N2) se atinge un conţinut de doar 5,02% şi 4,13% polizaharide sulfatate sau 50,2 şi 41,3 g/kg de biomasă, respectiv. The disadvantage of this process is that when growing spirulina on media containing 412 ppm (optimal level of N2) and 45 ppm (limited level of N2) a content of only 5.02% and 4.13% sulfated polysaccharides is reached or 50 .2 and 41.3 g/kg of biomass, respectively.

Mai este cunoscut un procedeu de cultivare a cianobacteriei Spirulina platensis care include etapele: cultivarea spirulinei la intensitatea luminii de 96 µmol photons m(-2) s(-1) sau 7104 lx şi temperatura de 28°C şi acumularea biomasei, după care se majorează intensitatea luminii până la 192 µmol photons m(-2) s(-1) sau 14208 lx la temperatura de 38°C sau, ca factor stresant se utilizează 0,75 M NaCl şi cultivarea este continuată încă 2…3 zile în condiţii de stres [3]. A procedure for cultivating the cyanobacterium Spirulina platensis is also known which includes the steps: cultivating spirulina at a light intensity of 96 µmol photons m(-2) s(-1) or 7104 lx and a temperature of 28°C and biomass accumulation, after which increase the light intensity up to 192 µmol photons m(-2) s(-1) or 14208 lx at the temperature of 38°C or, as a stress factor, 0.75 M NaCl is used and the cultivation is continued for another 2...3 days under the same conditions of stress [3].

Dezavantajul acestui procedeu constă în consumul sporit de energie electrică în cazul cultivării spirulinei în regim de iluminare continuă cu utilizarea lămpilor fluorescente (7104 şi 14208 lx), precum şi diminuarea productivităţii cianobacteriei Spirulina platensis şi a conţinutului de exopolizaharide acide la cultivare în prezenţa 0,50…0,75 M NaCl. The disadvantage of this process is the increased consumption of electricity in the case of spirulina cultivation under continuous lighting with the use of fluorescent lamps (7104 and 14208 lx), as well as the decrease in the productivity of the cyanobacterium Spirulina platensis and the content of acidic exopolysaccharides when cultivated in the presence of 0.50 …0.75 M NaCl.

Problema pe care a rezolvă invenţia constă în asigurarea unei productivităţi mai înalte, stabile a spirulinei şi majorarea conţinutului de exopolizaharide acide produse de cianobacteria Spirulina platensis. The problem to be solved by the invention consists in ensuring a higher, stable productivity of spirulina and increasing the content of acidic exopolysaccharides produced by the cyanobacterium Spirulina platensis.

Procedeul de cultivare a cianobacteriei Spirulina platensis include inocularea cianobacteriei pe mediul nutritiv Zarrouk, cultivarea în decurs de 7 zile la iluminarea de 3500 lx şi temperatura de 35 °C, apoi la a 8-a zi mediul se suplimentează cu 2...4 mg/l CuSO4•5H2O şi cultivarea se efectuează încă 3 zile la iluminarea de 3500…5500 lx. The process of cultivating the cyanobacterium Spirulina platensis includes the inoculation of the cyanobacterium on the Zarrouk nutrient medium, the cultivation during 7 days at the illumination of 3500 lx and the temperature of 35 °C, then on the 8th day the medium is supplemented with 2...4 mg /l CuSO4•5H2O and the cultivation is carried out for another 3 days at the illumination of 3500...5500 lx.

Rezultatul constă în majorarea productivităţii cianobacteriei şi a conţinutului de exopolizaharide acide, şi anume majorarea randamentului de exopolizaharide acide de 2…4 ori şi a productivităţii spirulinei de 1,27 ori. The result consists in increasing the productivity of cyanobacteria and the content of acidic exopolysaccharides, namely increasing the yield of acidic exopolysaccharides by 2...4 times and the productivity of spirulina by 1.27 times.

Rezultatul tehnic obţinut se datorează utilizării în ziua a 8-a de cultivare a cianobacteriei Spirulina platensis a 2 factori de stres: suplimentarea cu CuSO4*5H2O şi majorarea intensităţii de iluminare, care induc producerea de exopolizaharide acide (inclusiv şi polizaharide sulfatate) pe suprafaţa pereţilor celulari şi eliminarea lor în lichidul de cultură, pentru anihilarea ionilor toxici de Cu2+, precum şi protejarea celulelor de efectul iluminării excesive la majorarea intensităţii de iluminare. The technical result obtained is due to the use on the 8th day of cultivation of the cyanobacterium Spirulina platensis of 2 stress factors: the addition of CuSO4*5H2O and the increase in lighting intensity, which induce the production of acidic exopolysaccharides (including sulfated polysaccharides) on the surface of the walls cells and their elimination in the culture liquid, for the annihilation of toxic Cu2+ ions, as well as protecting the cells from the effect of excessive illumination when the illumination intensity is increased.

Exemple de realizare a invenţiei Examples of realization of the invention

Exemplul 1 Example 1

150 ml suspensie de spirulină (0,4 mg/ml) se cultivă în mediul Zarrouk în decurs de 7 zile la iluminarea de 3500 lx, iar la a 8-a zi se suplimentează cu 2mg/l CuSO4•5H2O şi cultivarea este continuată încă 3 zile la 3500 lx. La a 11-a zi spirulina se separă de lichidul de cultură prin filtrare, lichidul cultural (fracţia 1 de exopolizaharide) se recoltează, iar biomasa se suspendă în 40 ml de apă distilată pentru extragerea exopolizaharidelor ataşate pe suprafaţa peretelui celular, se agită timp de 4…5 min, se filtrează cu pompa de vid, iar biomasa se spală cu 10 ml de apă bidistilată, extractele se colectează împreună (fracţia 2 de exopolizaharide). Fracţia 1 de exopolizaharide este concentrată de 10 ori la evaporatorul cu vacuum şi supusă dializei. Din fracţiile obţinute sunt luate probe pentru determinarea concentraţiei polizaharidelor acide cu reactivul alcian blue. 150 ml of spirulina suspension (0.4 mg/ml) is cultivated in the Zarrouk medium during 7 days at 3500 lx illumination, and on the 8th day it is supplemented with 2 mg/l CuSO4•5H2O and the cultivation is continued for a further 3 days at 3500 lx. On the 11th day, the spirulina is separated from the culture liquid by filtration, the culture liquid (fraction 1 of exopolysaccharides) is harvested, and the biomass is suspended in 40 ml of distilled water to extract the exopolysaccharides attached to the surface of the cell wall, it is stirred for 4...5 min, filter with a vacuum pump, and the biomass is washed with 10 ml of double-distilled water, the extracts are collected together (fraction 2 of exopolysaccharides). Fraction 1 of exopolysaccharides is concentrated 10 times in the vacuum evaporator and subjected to dialysis. From the fractions obtained, samples are taken to determine the concentration of acidic polysaccharides with the alcian blue reagent.

Exemplul 2 Example 2

150 ml suspensie de spirulină (0,4mg/ml) se cultivă în mediul Zarrouk în decurs de 7 zile la iluminarea de 3500 lx, iar la a 8-a zi se suplimentează cu 2 mg/l CuSO4•5H2O şi cultivarea este continuată încă 3 zile la temperatura de 35 °C la 5500 lx. La a 10-a zi spirulina se separă de lichidul cultural prin filtrare, lichidul de cultură (fracţia 1 de exopolizaharide) se recoltează, iar biomasa se suspendă în 40 ml de apă distilată pentru extragerea exopolizaharidelor ataşate pe suprafaţa peretelui celular, se agită timp de 4…5 min, se filtrează, iar biomasa se spală cu 10 ml de apă bidistilată, extractele se colectează împreună (fracţia 2 de exopolizaharide). Fracţia 1 de exopolizaharide este concentrată de 10 ori la evaporatorul cu vacuum şi supusă dializei. Din fracţiile obţinute sunt luate probe pentru determinarea concentraţiei polizaharidelor acide cu reactivul alcian blue. 150 ml of spirulina suspension (0.4 mg/ml) is cultivated in the Zarrouk medium during 7 days at 3500 lx illumination, and on the 8th day it is supplemented with 2 mg/l CuSO4•5H2O and the cultivation is continued for 3 days at a temperature of 35 °C at 5500 lx. On the 10th day, the spirulina is separated from the culture liquid by filtration, the culture liquid (fraction 1 of exopolysaccharides) is harvested, and the biomass is suspended in 40 ml of distilled water to extract the exopolysaccharides attached to the surface of the cell wall, it is stirred for 4...5 min, filter, and the biomass is washed with 10 ml of double-distilled water, the extracts are collected together (fraction 2 of exopolysaccharides). Fraction 1 of exopolysaccharides is concentrated 10 times in the vacuum evaporator and subjected to dialysis. From the fractions obtained, samples are taken to determine the concentration of acidic polysaccharides with the alcian blue reagent.

Analogic s-a procedat şi la cultivarea cianobacteriei Spirulina platensis, mediul fiind suplimentat cu 4 mg/l CuSO4*5H2O, la ambele intensităţi de iluminare. Rezultatele conform soluţiei propuse în invenţie sunt prezentate în tabel. Ele confirmă că utilizarea în ziua a 8-a de cultivare a cianobacteriei Spirulina platensis a 2 factori de stres: suplimentarea cu CuSO4*5H2O şi majorarea intensităţii de iluminare, induc o sporire de 2…4 ori a producţiei de exopolizaharide acide (inclusiv şi polizaharide sulfatate), faţă de soluţia de referinţă şi obţinerea unei cantităţi maxime de exopolizaharide acide de 238,3 g/kg la suplimentarea cu 2 mg/l CuSO4*5H2O la 5500 lx. Analogously, the cyanobacterium Spirulina platensis was cultivated, the medium being supplemented with 4 mg/l CuSO4*5H2O, at both light intensities. The results according to the solution proposed in the invention are presented in the table. They confirm that the use on the 8th day of cultivation of the cyanobacterium Spirulina platensis of 2 stress factors: the addition of CuSO4*5H2O and the increase of the lighting intensity, induce a 2...4-fold increase in the production of acidic exopolysaccharides (including polysaccharides sulfated), compared to the reference solution and obtaining a maximum amount of acidic exopolysaccharides of 238.3 g/kg when supplemented with 2 mg/l CuSO4*5H2O at 5500 lx.

Tabel Table

N Spirulina cultivată cu adaos de săruri în a 8-a zi Produc-tivitatea la a 11-a zi, g/l Biomasa totală, mg Fracţia 1 de exopolizaharide în lichidul cultural după concentrare (96,15 ml) Fracţia 2 de exopolizaharide extract exopolizaharide (50 ml) Exopoli- zaharide acide totale, mg/g sau g/kg ΔE600 mg/g biomasă ΔE600 mg/g biomasă Conform soluţiei de referinţă 1 2 3 4 3500 lx 0 0,25 M/l NaCl 0,50 M/l NaCl 0,75 M/l NaCl 1,03 0,96 0,87 0,77 309,60 286,50 260,40 230,47 0,059 0,032 0,013 0,010 45,98 26,95 12,05 9,61 0,031 0,039 0,031 0,025 12,62 17,20 15,00 12,10 58,60 44,15 27,05 21,71 Conform soluţiei propuse în invenţie 3500 lx 0 1 mg/l CuSO4*5H2O 2 mg/l CuSO4*5H2O 4 mg/l CuSO4*5H2O 1,03 1,01 1,01 1,03 309,60 309,00 302,70 303,00 0,059 0,148 0,167 0,171 45,98 115,57 133,13 136,18 0,031 0,044 0,054 0,045 12,62 18,30 22,50 18,40 58,60 133,87 155,63 154,58 5500 lx 0 1mg/l CuSO4*5H2O 2 mg/l CuSO4*5H2O 4 mg/l CuSO4*5H2O 1,15 1,10 1,27 1,12 345,30 330,90 382,20 334,50 0,144 0,128 0,331 0,245 100,63 93,34 208,98 176,74 0,066 0,097 0,089 0,064 24,10 36,90 29,30 24,1 124,73 130,24 238,30 200,84 N Spirulina cultured with the addition of salts on the 8th day Productivity on the 11th day, g/l Total biomass, mg Fraction 1 of exopolysaccharides in the culture liquid after concentration (96.15 ml) Fraction 2 of exopolysaccharides extract exopolysaccharides (50 ml) Total acidic exopolysaccharides, mg/g or g/kg ΔE600 mg/g biomass ΔE600 mg/g biomass According to reference solution 1 2 3 4 3500 lx 0 0.25 M/l NaCl 0.50 M /l NaCl 0.75 M/l NaCl 1.03 0.96 0.87 0.77 309.60 286.50 260.40 230.47 0.059 0.032 0.013 0.010 45.98 26.95 12.05 9.61 0.031 0.039 0.031 0.025 12.62 17.20 15.00 12.10 58.60 44.15 27.05 21.71 According to the solution proposed in the invention 3500 lx 0 1 mg/l CuSO4*5H2O 2 mg/l CuSO4*5H2O 4 mg/l CuSO4*5H2O 1.03 1.01 1.01 1.03 309.60 309.00 302.70 303.00 0.059 0.148 0.167 0.171 45.98 115.57 133.13 136.18 0.031 0.044 0.054 0.045 12.62 18.30 22.50 18.40 58.60 133.87 155.63 154.58 5500 lx 0 1mg/l CuSO4*5H2O 2 mg/l CuSO4*5H2O 4 mg/l CuSO4*5H2O 1, 15 1.10 1.27 1.12 345.30 330.90 382.20 334.50 0.144 0.128 0.331 0.245 100.63 93.34 208.98 176.74 0.066 0.097 0.089 0.064 24 ,10 36.90 29, 30 24.1 124.73 130.24 238.30 200.84

1. Chakraborty Tania, A. K. Sen and R. Pal. Stress induced extra cellular polysaccharide of Spirulina subsalsa and its chemical characterization. Journal of Algal Biomass Utilization. 2015, 6(3), p. 24-38 1. Tania Chakraborty, A. K. Sen and R. Pal. Stress induced extra cellular polysaccharide of Spirulina subsalsa and its chemical characterization. Journal of Algal Biomass Utilization. 2015, 6(3), pp. 24-38

2. Abd El Baky H, Hanaa El Baz KF and El-Latife S A. Induction of Sulfated Polysaccharides in Spirulina platensis as Response to Nitrogen Concentration and its Biological Evaluation. Journal Aquaculture Research Development 2013, vol. 5 no 1, p. 1-8 2. Abd El Baky H, Hanaa El Baz KF and El-Latife S A. Induction of Sulfated Polysaccharides in Spirulina platensis as Response to Nitrogen Concentration and its Biological Evaluation. Journal Aquaculture Research Development 2013, vol. 5 no. 1, pp. 1-8

3. Meng-Chou LEE. Yean-Chang CHEN. Tzu-Chien PENG. Two-stage culture method for optimized polysaccharide production in Spirulina platensis. Jurnal of the Science of Food and Agriculture, vol. 92, 2012, p.1562-1569. 3. Meng-Chou LEE. Yean-Chang CHEN. Tzu-Chien PENG. Two-stage culture method for optimized polysaccharide production in Spirulina platensis. Journal of the Science of Food and Agriculture, vol. 92, 2012, p.1562-1569.

Claims (1)

Procedeu de cultivare a cianobacteriei Spirulina platensis, care include inocularea acesteia pe mediul nutritiv Zarrouk, cultivarea în decurs de 7 zile la iluminarea de 3500 lx şi temperatura de 35 °C, apoi la a 8-a zi mediul se suplimentează cu 2…4 mg/l CuSO4 ▪5H2O şi cultivarea se efectuează încă 3 zile la iluminarea de 3500…5500 lx.Cultivation process of the cyanobacterium Spirulina platensis, which includes its inoculation on Zarrouk nutrient medium, cultivation during 7 days at 3500 lx illumination and 35 °C temperature, then on the 8th day the medium is supplemented with 2...4 mg /l CuSO4 ▪5H2O and the cultivation is carried out for another 3 days at the illumination of 3500...5500 lx.
MDA20160091A 2016-07-28 2016-07-28 Process for cultivation of Spirulina platensis cyanobacterium MD4492C1 (en)

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CN108265014B (en) * 2017-12-28 2019-06-25 中国科学院南海海洋研究所 One plant of high-quality seawater spirulina and application thereof obtained by space breeding

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MD167F1 (en) * 1993-03-18 1995-02-28 Iurie Potapov Heat generator and device for heating liquids
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