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Phycocyanin Extraction from Spirulina platensis and Extract Stability under Various pH and Temperature


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Three extraction methods: sonication, repeated freezing and thawing (RFT) and enzymolysis were carried out on two strains of Spirulina platensis, IFRPD1183 (Sp1183) and IFRPD1213 (Sp1213). The sonication method using an ultrasonic processor was conducted for 5, 12.5 and 20 s at 70, 85 and 100% amplitude. Results showed that only the extraction time significantly affected (p
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As. J. Food Ag-Ind. 2009, 2(04), 819-826
Asian Journal of
Food and Agro-Industry
ISSN 1906-3040
Available online at
Research Article
Phycocyanin extraction from Spirulina platensis and extract
stability under various pH and temperature
Rachen Duangsee 1, Natapas Phoopat 2 and Suwayd Ningsanond 1*
1 School of Food Technology, Institute of Agricultural Technology, Suranaree University of
Technology, Nakhon Ratchasima 30000 Thailand.
2 Institute of Food Research and Product Development, Kasetsart University, Bangkok 10900
*Author to whom correspondence should be addressed, email:
This paper was originally presented at the Food Innovation Asia Conference 2009, Bangkok, Thailand.
Received 18 September 2009, Revised 7 October 2009, Accepted 8 October 2009.
Three extraction methods: sonication, repeated freezing and thawing (RFT) and enzymolysis
were carried out on two strains of Spirulina platensis, IFRPD1183 (Sp1183) and IFRPD1213
(Sp1213). The sonication method using an ultrasonic processor was conducted for 5, 12.5 and
20 s at 70, 85 and 100% amplitude. Results showed that only the extraction time significantly
affected (p<0.05) cell disruption. The RFT method was tested at -20oC for 1-3 h and 1-3
thawing cycles. Results showed that freezing time and thawing cycles significantly affected
(p<0.05) cell disruption. Cell disruption from sonication and RFT of Sp1183 was higher than
that of Sp1213. Sonication was more effective in breaking the cell envelope compared with
RFT. Further extraction of phycocyanin (PC) showed that temperature greatly contributed to
extraction efficiency (EE). The enzymolysis method using lysozyme was carried out under
various enzyme concentrations, extraction time and temperature. Results showed that %EE
was significantly affected (p<0.05) by extraction time and temperature, but not enzyme
concentration. Without lysozyme, the maximum EE was obtained at 44oC which was due to
ionic strength of buffer solution. Effects of pH and heat on crude PC solution (0.123 µg/ml)
were determined in citrate-phosphate buffer with an ionic strength of 0.1 M at pH 3.0, 4.0 4.5
5.0 and 6.0. Effects of temperature under continuous heating from 30oC to 68oC in 240 s at
each pH showed that PC exhibited the highest stability at pH 5.0. At pH 3, PC was very
sensitive to heat and underwent rapid changes.
As. J. Food Ag-Ind. 2009, 2(04), 819-826 820
Keywords: prokaryotic bacteria, algae, sonication, repeated freezing and thawing, RFT,
enzymolysis, lysozyme, Thailand
Phycocyanin (PC) is used for food colourant, nutraceutical and immuno diagnostic
applications and is mainly extracted from Spirulina [1, 2, 3]. Cell structure of Spirulina is
grouped into prokaryotic bacteria. In Spirulina cells, carotenoids, chlorophyll, and
phycocyanin (PC) are major pigments amounting to 0.4, 1.0 and 14% dry wt, respectively [4].
PC is (blue) pigment protein (biliprotein) located in the thylakoid system or photosynthetic
lamellas in the cytoplasmic membrane [1]. When the cell envelope is broken, thylakoid
membrane together with PC are released. Typically, cytoplasmic membrane of gram-negative
bacteria is enveloped with 4 layers of longitudinal cell wall: Layers I, II, III and IV [5]. Outer
membrane (layer IV) is composed of lipopolysaccharides (LPS). Each LPS molecule is linked
together with a calcium and magnesium ion. Layer III is composed of protein fibril. Layer II
is the strongest layer and is composed of peptidoglycan molecules. There are three main
methods for cell envelope disruption: 1) mechanical means such as bead mill, sonication and
high-pressure homogenization, 2) physical rupture such as heat, repeated freezing and
thawing (RFT), atomization and decompression and 3) disruption with lytic agent such as
chemical lysis and enzymatic lysis [6].
PC is a metal free tetrapyrrole, phycocyanobilin (PCB), which is a chromophore, attached to
apoprotein by thioether bond. The basic structure of PC consists of two helix subunits, alpha-
and beta subunits. These two subunits form a heterodimer. Near the neutral pH, PC usually
forms thrimer [7], but the hexameric form may be the basic functional unit of this protein [8].
It was found that pH and concentration of PC in solution are main factors of PC aggregation
and dissociation to form monomer, trimer, hexamer and other oligomers [9]. The absorption
spectra of PC, monomer and all aggregates, exhibit a strong first excited state band at ~ 615
nm and a much weaker second excited state band at ~360 nm [8]. Changes of PC and PCB
conformation relate to the absorptivity of PC at both wavelengths.
This work was conducted to study extraction of PC from Spirulina platensis and PC stability
under various pH and temperature.
Materials and Methods
Two strains of Spirulina platensis, IFRPD1183 (Sp1183) and IFRPD1213 (Sp1213) were
studied. The algae were obtained from the algae laboratory of the Institute of Food Research
and Product Development, Kasetsart University, Bangkok, Thailand. They were cultivated in
a 380 L outdoor raceway pond in Zarrouk medium [10] at pH 10.0 for 10 d before harvesting
for this study.
The optimized extraction of PC production from Spirulina platensis was studied with 3
extraction methods; sonication, freezing and thawing (RFT) and enzymolysis. The sonication
method was conducted with an Ultrasonic processor, model GE-100 equipped with 1/8 in.
stepped microtip, at 100% amplitude giving 20 KHz ultrasonic wave frequency, for 5, 12.5
and 20 s at 70, 85 and 100% amplitude. The RFT method was designed for 1, 2 and 3 h
freezing time (Ft) at -20oC and 1, 2 and 3 thawing cycles (FTC). After cell disruption, further
extraction of PC was carried out in a shaker at 25 and 37 oC for 4 h. The enzymolysis method
with chicken egg white lysozyme, L6876 obtained from Sigma-Aldrich, Co., Singapore) was
As. J. Food Ag-Ind. 2009, 2(04), 819-826 821
carried out at various enzyme concentration (EC), extraction time (Et) and extraction
temperature. Extraction efficiency (EE) was calculated from [A620 - 0.474 A652]/ 5.34 [11]
using a Biocrom UV/VIS Spectrophotometer, model Libra S22.
PC extraction after cell disruption was conducted in 0.1 M phosphate buffer (pH 7.0) solution.
Response surface methodology (RSM) with face centered design was used and the data were
analyzed with the Design-Expert version 7.1.6 program.
The effect of pH on PC was determined in sodium citrate-phosphate buffer at pH 3.0, 4.0, 4.5,
5.0 and 6.0 with ionic strength of 0.1 M in a quartz cell (1 cm path length) at room
temperature. The UV/Vis spectra of PC were recorded between 250 – 700 nm. The
absorbance ratio of A620/A370 was used for determining the conformation change of PC
[12]. The folding dynamics of PC structure affected by pH was determined from the emission
spectra of PC at an excitation lambda 290 nm [13] and 590 nm [12] using a Shimadzu
spectrofluorometer, model RF-1501.
The effect of heat on PC was carried out at pH 3.0 to 6.0 in the same buffer as above in a 1 cm
path length quartz cell. The sample cell was warmed to 30oC in a water bath prior to heating
in the cell holder of the spectrophotometer to 68oC under a circulating water bath.
Temperature changes and absorbance readings at 370 nm and 620 nm were recorded every 10
s during 240 s heating.
Results and Discussion
Effects of sonication and RFT on cell disruption
Cell disruption has been monitored via PC released and expressed as %EE. Figure 1 shows
the response surface plot (RSP) of EE affected by sonication time (St) and % amplitude of
Sp1183. EE of Sp1183 is higher than that of Sp1213 (Table 1) indicating more cell disruption.
In addition, RFT has greater cell rupture on Sp1183 than that on Sp1213. RSP for RFT of Sp
1183 is presented in Figure 2, showing significant effects of FTC and Ft. From our previous
microscopic study (not published), Sp1213 and Sp1183 cell wall thicknesses were 64.6 and
35.4 nm, respectively. This may explain why Sp1213 resists cell disruption higher than
Sp1183. Cell rupture comparison between sonication and RFT shows that sonication is more
effective. Vibration and bubbles generated by ultrasonic wave power inside algae cells are
rapid and effective to explode the cells [4] and therefore cell disruption is higher than that
treated with RFT.
PC extraction after sonication
After cell disruption with sonication, further extraction of PC was carried out on Sp1183 and
Sp1213. The extraction temperature significantly affects (p<0.05) EE. The maximum EE from
equation model from RSM of Sp1183 at 20 s sonication and 100% amplitude is 73.8% at
25oC and 8 h whereas 96.8% and 92.1% are obtained for Sp1183 and Sp 1213, respectively at
37oC and 3 h (Table 2.).
As. J. Food Ag-Ind. 2009, 2(04), 819-826 822
Figure 1. Response surface plot of EE of PC from Sp1183 affected by amplitude and
extraction time using sonication.
Design-Expert® Softw are
Transfor med Scale
Sqrt(EE (%))
Design points above predicted v alue
Design points below pr edicted value
X1 = B: Ft
X2 = A : FTC
Sqrt(EE (%))
B: Ft A: FT C
Figure 2. Response surface plot of EE of PC from Sp1183 affected by freeze-thaw cycle
and freezing time using RFT.
Table 1. Cell disruption as %EE of Spirulina platensis using sonication and RFT.
IFRPD No. Sonication RFT
St (sec) Amp (%) EE (%) FTC (cycle) Ft (h) EE (%)
1183 20 91 22.1 3 3 15.6
1213 20 100 14.2 3 3 2.0
Table 2. EE of PC extraction after cell disruption.
IFRPD No. St (sec) Amp (%) Et (h) Extraction Temp (oC) EE (%)
1183 20 100 8 25 73.8
1183 20 99 3 37 96.8
1213 20 100 3 37 92.1
Design-Expert® Software
EE (% )
Design points above pr edicted value
Design po ints below pr edicted v alue
X1 = B: Amp (%)
X2 = A: St (sec)
EE (%)
Amp (%) A: St (sec)
As. J. Food Ag-Ind. 2009, 2(04), 819-826 823
PC extraction with lysozyme
Cell wall hydrolysis was carried out using lysozyme. EE is significantly affected (p<0.05) by
extraction time and extraction temperature (Fig.3), but not enzyme concentration (RSP is not
shown). From response surface model, the maximum extraction efficiency is shown in Table
3. Without lysozyme, the maximum EE of Sp1183 at 44 is 87.5% at 2 h extraction while
Sp1213 is 103.4% at 2 h extraction. This indicates that extraction at 44oC in buffer solution
does not require lysozyme.
Figure 3. Response surface plot of EE of PC from Sp1183 affected by temperature and
extraction time using lysozyme.
Table 3. Maximum extraction efficiency of PC with lysozyme.
IFRPD No. EC (mg/g dry weight) Extraction Temp (oC) Et (h) EE (%)
1183 0 44 2 87.5
18.17 44 3 94.5
1213 0 44 2 103.4
16.12 44 2 108.2
In addition, the effect of ionic concentration was conducted on Sp1213 using only RO water
without lysozyme at 37oC. It was found that %EE of PC extracted in buffer is much higher
than that in RO water (Table 4.). These results confirm that ionic strength has a greater effect
on the EE.
Table 4. Extraction of PC from Sp 1213 without lysozyme at 37oC.
Extraction solution EE (%)
Et (h) 1 2 3 4
RO H2O 0.6 2.4 4.4 5.5
phosphate buffer 46.0 67.0 69.5 71.9
Effects of pH and heat on PC
This study has been conducted under low pH range and continuous heating. Spectra readings
are inevitably interfered by protein precipitates. Therefore, interpretation is based on relative
spectra readings.
Design-Expert® Softw are
Transformed Scale
Sq r t ( EE)
Design points above pr edicted value
Design points below predicted value
X1 = B: T (oC)
X2 = C: Et (h)
Actual Factor
A: EC (mg/g dw) = 9.0 9
B: T ( oC) C: Et (h)
As. J. Food Ag-Ind. 2009, 2(04), 819-826 824
At pH4.5, PC structure is unfolded, causing protein precipitates which were removed by
centrifugation. As a result, emission intensity and absorbance readings at the same lambda
peak are reduced (Fig.4). The lower pH, the higher precipitates occur resulting in inversely
reduced absorbance readings of centrifuged samples.
At pH 3, the spectra of centrifuged PC solution noticeably shows a shift in maximum
emission lambda and maximum absorbance lambda. This indicates that soluble PC molecules
underwent conformation changes due to breaking of salt bridges and hydrogen bonding at low
pH [9, 10].
Figure 4. Absorption and emission spectra of PC at pH 3.0-6.0.
During heating, the absorbance readings of PC solutions at 370 nm increase at all pH levels
(Fig.5). Thus, A620/A370 ratios are decreased with increased temperature. However, the
effect of heat, from the absorbance readings at 370 and 620 nm, at pH 5.0 is the least among
the other pHs. This suggests that at pH 5 PC has more stable conformation. PC at pH 4 and
4.5 are more stable when compared with those at pH 3.0 and 6.0, indicating that drastic
changes of PC conformation, perhaps protein unfolding, has already occurred and reached
more stable conformation before measurement. At pH 3, changes rapidly take place at early
heating, about 50 s, due to unstable state of PC which is sensitive to temperature changes at
very low pH.
Figure 5. Relative profile changes of A620, A370 and A617/A370 ratio affected by heat
and pH.
As. J. Food Ag-Ind. 2009, 2(04), 819-826 825
From A620/A370 ratios at pH 5-6, PC exhibits continuous changes throughout increasing
temperature profile indicating that PC is in its native form and continuously changes during
The sonication method is very effective in cell wall disruption of Spirulina sp., more so than
RFT. Sp-1213 has higher resistance to cell disruption by sonication and RFT than Sp-1183.
Further extraction is highly affected by temperature.
The effect of ionic strength of an extraction solvent is very high on EE. PC extraction in
buffer solution at 44oC for 2 h can be accomplished without lysozyme.
The conformation of PC structure is affected by pH. PC could well retain its native structure
at pH>5 and PC forms partial protein unfolding at pH<5.0. Heat strongly exhibits detrimental
effect on colour of PC solution at pH>5.0 and pH<3.
The results suggest that in applying sonication for a larger scale, this continuous and short
process would be favourable. In the case of RFT method, energy cost is a limiting constraint.
Although enzymolysis with lysozyme gives the highest EE, the enzyme is quite expensive and
difficult to handle. In addition, the extraction process needs pH and temperature control to
obtain a good yield with more stable form of PC.
The authors would like to thank the Thailand Research Fund (TRF) for financial support for
this project.
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... They observed that the PC was more stable between 50 and 55 °C at pH 6 and between 57 and 65 °C at pH 5. Furthermore, the addition of sorbitol between 10 and 50% (w/w) at 62 °C for 30 min increased the half-life values of the PC. Duangsee et al. (2009) evaluated the thermal and pH stability of PC from S. platensis at different pHs (3-6) and temperatures (30-68 °C). They found that the PC was stable at pH 5 while at pH 3, it was very sensitive to heat undergoing rapid changes. ...
... Eight papers evaluated the pH and thermal stability of the PCs (Antelo et al., 2008;Duangsee et al., 2009;Jespersen et al., 2005;Leu et al., 2013;Liang et al., 2018;Patel et al., 2004;Rahman et al., 2017;Wu et al., 2016). S. platensis is the most prolific cyanobacterial biomass for these PCs (Patel et al., 2004;Antelo et al., 2008;Duangsee et al., 2009;Wu et al., 2016, Rahman et al., 2017. ...
... Eight papers evaluated the pH and thermal stability of the PCs (Antelo et al., 2008;Duangsee et al., 2009;Jespersen et al., 2005;Leu et al., 2013;Liang et al., 2018;Patel et al., 2004;Rahman et al., 2017;Wu et al., 2016). S. platensis is the most prolific cyanobacterial biomass for these PCs (Patel et al., 2004;Antelo et al., 2008;Duangsee et al., 2009;Wu et al., 2016, Rahman et al., 2017. The other cyanobacterial biomass are C. merolae (Rahman et al., 2017), T. elongatus (Leu et al., 2013), and S. lividus sp. ...
Full-text available
The research in the field of food storage and preservation intensified in recent years to develop alternative preservatives and colorants to chemical ones. The algal preservatives and colorants have emerged as a viable green alternative in this context. The primary food preservatives from the algae are phycobiliproteins (PBPs) and their primary constituents: allophycocyanin (APC), phycocyanin (PC), and phycoerythrin (PE). As the foods have been subjected to the adverse environmental conditions during the production, storage, and utilization stages, it is important to research the determinants of the thermal, acidic, light, and color stability of the PBPs in these adverse environmental conditions such as light and acid exposure as well as elevated and sub-zero temperatures relevant to the food storage. Although there have been around 60 papers published in this research field, there has been no review of the research on the stability of the PBPs in these adverse environmental conditions relevant to the food storage. Hence, this paper reviews the research in this field as a first-ever population study. These studies reveal that these PBPs are stable at a wide range of acid and light exposure, and temperatures, making them perfect for the foods and beverages as a green alternative to chemical preservatives. Furthermore, a wide range of preservatives improve the stability of these PBPs through their close interaction with the PBPs and encapsulation of the PBPs is an efficient way to improve their stability, potentially using the nanotechnology. A couple of recommendations were made for the further research.
... The results of the effect of incubation time on the extraction of C-PC presented in Fig. 1(d) indicate that after an incubation time of 16 h, the maximum yield of 69.73 mg/g (Gupta & Sainis, 2010). Duangsee, Phoopat, and Ningsanond (2009) in their work inferred that extraction efficiency was significantly affected by incubation time during lysozyme-assisted extraction on two strains of A. platensis, IFRPD1183 (Sp1183) and IFRPD1213 (Sp1213) ( Duangsee et al., 2009). ...
... The results of the effect of incubation time on the extraction of C-PC presented in Fig. 1(d) indicate that after an incubation time of 16 h, the maximum yield of 69.73 mg/g (Gupta & Sainis, 2010). Duangsee, Phoopat, and Ningsanond (2009) in their work inferred that extraction efficiency was significantly affected by incubation time during lysozyme-assisted extraction on two strains of A. platensis, IFRPD1183 (Sp1183) and IFRPD1213 (Sp1213) ( Duangsee et al., 2009). ...
... Statistically significant (p < 0.05) effect of S/L ratio, enzyme concentration and their interaction was observed on the purity of C-PC. However, contradictory results were observed by Duangsee et al. (2009), in their work they inferred that extraction efficiency was not significantly affected by enzyme concentration during lysozyme-assisted extraction on two strains of A. platensis, IFRPD1183 (Sp1183) and IFRPD1213 (Sp1213) ( Duangsee et al., 2009). ...
... Additionally, direct scalability is possible in ultrasonication due to its ability of high intensity cavitation zones and therefore suitable for scaling up of the process (Peshkovsky et al. 2013). Further, most of the publications on ultrasound-assisted and enzyme-assisted extractions of microalgae are for wet biomass (Duangsee et al. 2009;Antonio-Marcos et al. 2016). ...
... Lower values of yield and purity at extreme pH can be attributed to the degradation of phycobiliproteins or lysozyme or both. Duangsee et al. (2009) reported that at lower pH, soluble phycocyanin molecules underwent conformation changes due to breakage of salt bridges and hydrogen bonding. Similarly, it was reported that extreme buffer pH causes internal electrostatic attraction by changing the charge on protein giving net positive charge and at which stage protein opens up leading to loss of bound solvent in turn resulting in denaturation of protein (Roe 2001). ...
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Spirulina maxima is reported to have the maximum protein content among different species of Cyanobacteria. In the present work, extraction of C-phycocyanin (C-PC) and Allophycocyanin (A-PC) from dry biomass of S. maxima was carried out for the first time using ultrasound-assisted extraction (UAE), enzyme-assisted extraction (EAE) and their combination. The total extractable C-PC and A-PC were estimated to be 47.89 and 39.95 mg/g dry biomass, respectively from serial extraction. UAE resulted in better yield and purity compared to EAE. At standardized conditions of UAE (1:15 solid-liquid ratio, 20% amplitude and 4 minutes ultrasonication time), the highest yield, purity and extraction efficiency achieved were 44.5 mg/g.d.b, 2.0 and 93% for C-PC and 29.93 mg/g.d.b, 0.89 and 75% for A-PC, respectively. The purity observed is the highest reported in primary extraction till date. Combination of methods did not result in significant improvement with respect to yield and purity. Considering both yield and purity, ultrasound assisted extraction alone was inferred to be the most suitable method for extraction of phycocyanins from the dry biomass of Spirulina maxima.
... They are formed of four layers: fibrils, peptidoglycans, proteins, and oligosaccharides, similar to gramnegative bacteria [53]. It has been reported that several methods rupture the cell wall, such as homogenization, sonication, microwave, supercritical fluid process, and disintegration of lysozyme [54][55][56]. The process must be efficient in terms of high extraction performance and must be respectful of the environment and further use for the food industry. ...
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Phycocyanin is a blue natural food colorant with multiple health benefits. Here we propose an efficient phycocyanin extraction method from Arthrospira platensis from Mexico. Three extraction methods were applied to optimize the extraction process, using water and buffer as solvents, with three pH values at two agitation times. The highest phycocyanin, 54.65 mg/g, was extracted from dry biomass with water as a solvent using an ultrasonication bar. The optimum condition of extraction was determined to be 1:50 biomass/solvent ratio for dry biomass, with the freeze/thaw method for 20 min repeated twice, and then agitated at 120 rpm for 24 h. The phycocyanin content was 48.88 mg/g biomass, with a purity of 0.47. For scalable phycocyanin productivity, the sonication method is recommended as there is no statistical difference. The phycocyanin stability was best at − 20 °C storage temperature at pH 7 for 35 days. Partial purification with ammonium sulfate was found to be suitable as a fractional precipitation method, first at 0–20% and then 20–65%, to get purity nearly 1. Total protein was found to be 55.52%, and total amino acids after phycocyanin extraction was 33%. The maximum phycocyanin yield using water as a solvent was the most interesting result regardless of the method used for extraction.
... This condition causes phycocyanin to become more unstable. According to Duangsee et al. (2009), the chemical structure of phycocyanin will open at pH < 4.5 due to damage to hydrogen bonds. Phycocyanin which is stored at a low pH makes the structure of phycocyanin expand caused by denatured protein. ...
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Spirulina maxima is a blue-green microalga that rich of pigment. The pigments in S. maxima grouped into primary pigment (Chlorophyll a) and accessory pigments (carotenoid & phycobiliprotein). Phycocyanin is an accessory pigment belongs to phycobiliprotein, blue colored, and can be used as natural food coloring and drugs. Phycocyanin has activities as antibacterial, antioxidant, anti-inflammatory, antihyperalgesic and many more. Because of that, phycocyanin usually used in the pharmaceutical industry. However, phycocyanin is a protein that unstable under lights, high temperature, and pH in the storage. The aim of this research is to obtain information about the effect of pH on the stability of phycocyanin extracted from S. maxima that cultivated in indoor and semi-outdoor. The steps are cultivation, extraction using different solutions to get blue pigment phycocyanin, and stability test. Phycocyanin was dissolved in buffer solution at pH 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, and 7.0 for 7 days. Color observations and concentration measurements are carried out every day to see changes in phycocyanin. Growth in indoor cultivation with 24 hours light is faster than semi-outdoor a that uses only sunlight. Cultivation gets the optical density value 0,6 at day 20 with indoor cultivation and day 34 with semi-outdoor cultivation. Results show that phycocyanin can be extracted using buffer phosphate solution. The stability of the pigments can be seen from the color changes and relative concentration using a spectrophotometer. Phycocyanin shows stable in the storage of pH 4 until pH 5.5. The highest relative concentration (CR) was shown in pH 5.
... Pre-treatment is needed in most cases to rupture the cell wall to make the algae more prone to extraction [15][16][17]. Conventional disintegration techniques like freezing and thawing, microwave, bead milling, ultrasound, homogenization and lysozyme treatments of the cell wall have been assayed [6,[18][19][20]. However, the main disadvantage of these methods is a lack of selectivity that cause the release of other internal compounds and cell debris that could negatively affect the quality of the extract. ...
Free access for 50 days: The study investigated the potential of pulsed electric fields (PEF) technology application for the improvement of cell disintegration and subsequent extraction/fractionation of cyanobacteria Arthrospira maxima into valuable components (phycocyanin, proteins, oils and carbohydrates). Pulsed electric fields were applied to fresh A. maxima suspension, dried powder and dried sticks in combination with water as extraction solvent and freeze-thawing. Pulsed electric fields application for cell disruption reached approximately 90% increase of C-phyco-cyanin extraction compared to bead milling. Obtained fractions of phycocyanin and bulk proteins were also of higher purity and had twice lower environmental impact than similar fractions obtained without Pulsed electric fields treatment. Extracts with improved purity can be directly applied in pharma and food industry without any further processing and purification steps.
... So far, extraction of C-Phycocyanin from A. platensis has been conducted using diverse cell disruption methods, such as sonication, bead milling, high pressure homogenization (HPH), or freeze-thawing (Patel et al., 2005;Duangsee et al., 2009). In addition to the energy demand of these cell disruption methods, purification of C-Phycocyanin from a mixture of cell debris is also counted as a major drawback. ...
Pulsed electric field (PEF) was conducted for the extraction of proteins/C-Phycocyanins from Arthrospira platensis. The cyanobacterial suspension was treated with 1 µs long pulses at an electric field strength of 40 kV·cm⁻¹ and a treatment energy of 114 kJ·kgsus⁻¹ and 56 kJ·kgsus⁻¹. For benchmarking, additional biomass was processed by high pressure homogenization. Homogeneity of the suspension prior to PEF-treatment influenced the protein/C-phycocyanin extraction efficiency. Stability of C-Phycocyanin during post-PEF incubation time was affected by incubation temperature and pH of the external medium. Biomass concentration severely affect proteins/C-Phycocyanins extraction yield via PEF-treatment. The optimum conditions for extraction of proteins/C-Phycocyanin was obtained at 23 °C while incubating in pH 8-buffer. The energy demand for PEF-treatment amounts to 0.56 MJ·kgdw⁻¹ when processing biomass at 100 gdw·kgsus⁻¹. PEF treatment enhances the protein/C-Phycocyanin extraction yield, thus, it can be suggested as preferential downstream processing method for the production of C-Phycocyanin from A. platensis biomass.
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Phyco-remediation of dyestuffs in textile wastewaters is of economic, industrial, and environmental importance. We evaluated the remediation of the textile dye, Direct Green 6 (DG6), by Spirulina platensis, and investigated the novel possibility that DG6 treatment enhances production of the biopolymer, polyhydroxybutyrate (PHB). We showed that both live and dead cells of Spirulina were capable of DG6 remediation, but live cells could be re-used with no loss of remediation efficiency. Furthermore, DG6 remediation by live cells resulted in increased algal biomass and trichome lengths, and stimulated production of valuable metabolites, including PHB, antioxidants, carbohydrates and pigments (phycobilins and astaxanthin). We determined the optimal conditions for DG6 remediation and an artificial neural network (ANN) accurately modeled the experimental data and predicted the concentration of dye as the most and algal turbidity as the least important parameters for DG6 removal efficiency. A DG6 concentration of 60 mg L-1 resulted in the highest simultaneous co-production of PHB (12.7±1.7% DW) and increase of astaxanthin (194%), carotenoids (50%), phenol (51%), carbohydrates (27%) total phycobilin (43%), together with the enhancement of biomass and trichome lengths (95%). Oxidative stress indices and enzyme activities such as peroxidases and laccase (involved in dye removal/antioxidant functions) were also increased by dye dosage. On the basis of our results, we propose that S. platensis may use DG6 dye as a nitrogen/carbon source for co-accumulation of valuable bioplastic and metabolites.
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امروزه همه مردم جهان براي داشتن زندگي سالم به داروها، فرآورده¬هاي گياهي و فرآورده¬هاي مشتق شده از گياهان وابسته¬اند که استفاده از آن‌ها بجاي منابع مصنوعي مضر مقبوليت عمومي يافته است. لازمه دانستن دانش فني روش کشت اسپيرولينا به‌منظور استخراج فيکوسيانين و انتخاب شرايط رشد از اهميت ويژه¬اي برخوردار است. بدين منظور اين پژوهش مروري بر نحوه کشت ريز جلبک تا استخراج رنگ‌دانه داشته است. در اين مطالعه مروري مقالات معتبر علمي نمايه شده در پايگاه¬هاي اطلاعاتي مختلف مانند Science Direct، Springer، Scopus و ديگر بانک¬ها داراي يکي از کليدواژه‌هاي اسپيرولينا، پلاتنسيس، رنگ‌دانه فيکوسيانين، کشت و روش استخراج بررسي شدند. اسپيرولينا پلاتنسيس ريزجلبک ميکروسکوپي رشته¬اي غني از پروتئين، ويتامين¬هاي گروه B، اسيدهاي آمينه ضروري و مواد معدني مهم است. اين جلبک اساساً سيانو باکتري غيرهتروسيست اوتوتروف و فتوسنتز کننده است. مطابق با نتايج مطالعات، فيکوسيانين به‌عنوان يکي از رنگ‌دانه‌هاي اين جلبک، کاربردهاي متنوعي از جمله در تهيه قرص دارويي به‌منظور درمان سرطان، ضدالتهاب، درمان ايدز و افزودني غذايي به‌عنوان مکمل و يا رنگ‌دانه طبيعي چهار مزيت اصلي است که بر ضرورت استخراج اين ريز جلبک تأکيد مي¬کند. توليد جلبک با مقادير بالا فيکوسيانين به عوامل متعددي ازجمله شرايط رشد جلبک، قابليت تجمع رنگيزه‌اي، فنّاوري توليد و کارايي فرآيند پايين‌دستي بستگي دارد. براي کشت اين ريزجلبک از محيط کشت¬هاي متعددي از جمله زاروک، زاروک اصلاح شده، کانوي، جردن، اف 2 و شوسلر به کار گرفته مي¬شود اما بهترين محيط کشت براي رشد اين ريزجلبک زاروک در شرايط آزمايشگاهي ͦ C 35-28، شدت نوردهي 4500-1500 لوکس، 5/9-9 =pH و هوادهي کافي معرفي شده است.
The equilibrium unfolding pathway of Aplysia apomyoglobin has been studied under various solvent conditions. The protein exhibits a single unfolding transition in acid in contrast to the two transitions observed for the mammalian apomyoglobins with which it shares a common fold but a low level of sequence identity (24%). This acid-unfolded species has considerable residual structure as evidenced by both tryptophan fluorescence and far-UV CD spectroscopy. It remains 40% α-helical under low salt conditions (2 mM citrate, 4°C) the folded form is 65% helical. A similar species is observed for the mammalian globins in mild acid conditions. Titration with GdnHCl at pH 7 reveals two unfolding transitions, the first having common features with that observed in acid and the second resulting in a completely unfolded state. Under the same conditions, urea unfolds the protein completely in an apparently single cooperative transition. Assuming a simple three-state model (F ↔ I ↔ U), data from GdnHCl and urea titrations over a range of pH conditions were used to derive values for the apparent stability (ΔGw(app)) and solvent accessibility (n(app)) of the folded (F) and intermediate (I) forms of the protein. Urea titrations were then repeated over a range of KCl concentrations in order to understand the contribution of Cl− to the different unfolding activity of GdnHCl. A three-state scheme is justified when changes in ΔGw(app) occur without changes in n(app). The change in free energy of folding of I ↔ F (ΔGw(F/I)) decreases to 0 at pH 4 as expected from the acid unfolding curve. ΔGw(I/U) reaches its maximum at pH 4.5, the isoelectric point of the protein. Variations of this value with pH and chloride are as much as 3 kcal mol−1 and correlate closely with changes in n(app) although there is no change in the α-helical content of I across the pH range. This observation is interpreted here as a deviation of the unfolding of the I state of Aplysia apomyoglobin from a cooperative behaviour.
C-Phycocyanin from an acido-thermophilic eukaryotic alga, Cyanidium caldarium, was characterized with respect to subunit structure, absorption spectrum and fluorescence properties and was found to be similar to C-phycocyanins from mesophilic sources. The pH-dependence of fluorescence polarization and the changes in sedimentation velocity as a function of pH, concentration and temperature indicate the presence of extremely large amounts of unusually stable 19S aggregates. It was not possible to disaggregate this phycocyanin completely to monomer under normal conditions. The amino acid composition is similar to that of phycocyanins from other thermophilic and halophilic sources. The isoelectric point of this C-phycocyanin was 5.11, an unusually high value. The properties of this C-phycocyanin suggest an increase in protein stability as its mode of adaptation to the environmental stress of high temperature.
The cell wall of the blue-green alga Spirulina platensis was studied with the electron microscope using ultra-thin sectioning, shadowing, carbon-replication or freeze-etching techniques for specimen preparation. The cell wall could be resolved into four layers, L-I through L-IV. The L-I and L-III layers contain fibrillar material. The septum is a three-layered wall: an L-II layer sandwiched between L-I layers. The shape in vitro of isolated septa might be an artifact due to the preparation technique used. Certain structural properties of the septum seem to allow tangential stretching; they might be reflected in the flexible gliding mobility of Spirulina species. The outer, L-IV layer contains material longitudinally arranged along the trichome axis.
Cyanobacterial phycobilisomes harvest light and cause energy migration usually toward photosystem II reaction centers. Energy transfer from phycobilisomes directly to photosystem I may occur under certain light conditions. The phycobilisomes are highly organized complexes of various biliproteins and linker polypeptides. Phycobilisomes are composed of rods and a core. The biliproteins have their bilins (chromophores) arranged to produce rapid and directional energy migration through the phycobilisomes and to chlorophyll a in the thylakoid membrane. The modulation of the energy levels of the four chemically different bilins by a variety of influences produces more efficient light harvesting and energy migration. Acclimation of cyanobacterial phycobilisomes to growth light by complementary chromatic adaptation is a complex process that changes the ratio of phycocyanin to phycoerythrin in rods of certain phycobilisomes to improve light harvesting in changing habitats. The linkers govern the assembly of the biliproteins into phycobilisomes, and, even if colorless, in certain cases they have been shown to improve the energy migration process. The Lcm polypeptide has several functions, including the linker function of determining the organization of the phycobilisome cores. Details of how linkers perform their tasks are still topics of interest. The transfer of excitation energy from bilin to bilin is considered, particularly for monomers and trimers of C-phycocyanin, phycoerythrocyanin, and allophycocyanin. Phycobilisomes are one of the ways cyanobacteria thrive in varying and sometimes extreme habitats. Various biliprotein properties perhaps not related to photosynthesis are considered: the photoreversibility of phycoviolobilin, biophysical studies, and biliproteins in evolution. Copyright 1998 Academic Press.
Aphanizomenon flos-aquae (AFA) is a fresh water unicellular blue-green alga (cyanophyta) rich in phycocyanin (PC), a photosynthetic pigment with antioxidant and anti-inflammatory properties. The purpose of this study was to evaluate the ability of a novel natural extract from AFA enriched with PC to protect normal human erythrocytes and plasma samples against oxidative damage in vitro. In red blood cells, oxidative hemolysis and lipid peroxidation induced by the aqueous peroxyl radical generator [2,2'-Azobis (2-amidinopropane) dihydrochloride, AAPH] were significantly lowered by the AFA extract in a time- and dose-dependent manner; at the same time, the depletion of cytosolic glutathione was delayed. In plasma samples, the natural extract inhibited the extent of lipid oxidation induced by the pro-oxidant agent cupric chloride (CuCl2); a concomitant increase of plasma resistance to oxidation was observed as evaluated by conjugated diene formation. The involvement of PC in the antioxidant protection of the AFA extract against the oxidative damage was demonstrated by investigating the spectral changes of PC induced by AAPH or CuCl2. The incubation of the extract with the oxidizing agents led to a significant decrease in the absorption of PC at 620 nm accompanied with disappearance of its blue color, thus indicating a rapid oxidation of the protein. In the light of these in vitro results, the potential clinical applications of this natural compound are under investigation.