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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 www.ajofai.info
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
Thailand.
*Author to whom correspondence should be addressed, email: suwayd@hotmail.com
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.
Abstract
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
Introduction
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
4.07981
0.463003
X1 = B: Ft
X2 = A : FTC
1.00
1.50
2.00
2.50
3.00
1.00
1.50
2.00
2.50
3.00
0.4
1.325
2.25
3.175
4.1
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
26.393
3.03376
X1 = B: Amp (%)
X2 = A: St (sec)
70.00
77.50
85.00
92.50
100.00
5
8.75
12.5
16.25
20
3.0
9.0
15.0
21.0
27.0
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
9.53696
2.98045
X1 = B: T (oC)
X2 = C: Et (h)
Actual Factor
A: EC (mg/g dw) = 9.0 9
30
34
37
41
44
1
2
2
3
3
3.30
4.83
6.35
7.88
9.40
Sqrt(EE)
B: T ( oC) C: Et (h)
As. J. Food Ag-Ind. 2009, 2(04), 819-826 824
At pH≤4.5, 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
heating.
Conclusions
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.
Acknowledgement
The authors would like to thank the Thailand Research Fund (TRF) for financial support for
this project.
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