Process Biochemistry 47 (2012) 659664

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Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio

Stability of phycocyanin extracted from Spirulina sp.: Inuence of temperature,

pH and preservatives
Ratana Chaiklahan a, , Nattayaporn Chirasuwan a , Boosya Bunnag a,b
a
Pilot Plant Development and Training Institute, King Mongkuts University of Technology Thonburi, Bangkhuntien, Bangkok 10150, Thailand
b
School of Bioresources and Technology, King Mongkuts University of Technology Thonburi, Bangkhuntien, Bangkok 10150, Thailand

a r t i c l e i n f o a b s t r a c t

Article history: Temperature and pH play an important role in the stability of phycocyanin, a natural blue colorant.
Received 20 October 2011 Systematic investigations showed the maximum stability of phycocyanin was in the pH range 5.56.0.
Received in revised form Incubation at temperatures between 47 and 64 C caused the concentration (CR ) and half-life of phyco-
30 December 2011
cyanin in solution to decrease rapidly. The CR value remained at approximately 50% after incubating for
Accepted 9 January 2012
30 min at 59 C. After heating at 60 C for 15 min, the CR value of phycocyanin at pH 7.0 was maintained
Available online 16 January 2012
at around 6270% when 2040% glucose or sucrose was added, and the half-life increased from 19 min
to 3044 min. 2.5% sodium chloride was found to be an effective preservative for phycocyanin at pH 7.0
Keywords:
Phycocyanin
as a CR value of 76% was maintained and the half-life of 67 min was increased.
Spirulina 2012 Elsevier Ltd. All rights reserved.
Stability
Temperature
pH
Preservative

1. Introduction
Phycocyanin is an accessory photosynthetic pigment of the phy-
cobiliprotein family. In general, phycobiliproteins are made up of
chromophore-bearing polypeptides containing and subunits,
which have a molecular weight of around 20 kDa [1]. Phycobilisome
from the cyanobacterium Spirulina sp. consists of allophycocyanin
(APC) cores surrounded by c-phycocyanin (CPC) peripherally. CPC is
the major phycobiliprotein in Spirulina and constitutes up to 20% of
its dry weight [2,3]. Phycocyanin is a natural blue colorant, and has
been used as a colorant in health drinks, beverages, confectionary
and cosmetics. Small quantities are also used as biochemical tracers
in immunoassays due to its uorescent properties [4]. Furthermore,
phycocyanin has proven to posses therapeutic properties including
antioxidant, anti-inammatory and anti-cancer activities [5,6]. The
market value of phycocyanin was reported to be US$ 1050 million
per annum [7,8]. The price of food-grade phycocyanin is around
Australian $500 per kg [9,10].
In solution, phycocyanin is found as a complex mix of
monomers, trimers, hexamers and other oligomers, and its
Corresponding author at: Pilot Plant Development and Training Institute, King
Mongkuts University of Technology Thonburi (KMUTT), 49 Tientalay 25, Thakham,
Bangkhuntien, Bangkok 10150, Thailand. Tel.: +66 2470 7483; fax: +66 2452 3455.
E-mail addresses: ratana.cha@kmutt.ac.th, ratana@pdti.kmutt.ac.th
(R. Chaiklahan).
molecular weight, therefore, ranges from 44 to 260 kDa [11]. Phyco-
cyanin is water-soluble and can be easily extracted from Spirulina
sp. as a proteinpigment complex. Appropriate control of the pH
and ionic strength during extraction, separation and purication
processes are crucial for the stability of phycocyanin molecules.
The degradation of phycocyanin depends on the aggregation state
of the protein, which is inuenced by parameters such as light,
temperature, pH and protein concentration [1214].
The use of phycocyanin in food and other applications is limited
due to its sensitivity to heat treatment, which results in precipita-
tion and fading of the blue color. Sodium azide and dithiothreitol
are commonly used as preservatives for phycocyanin for analyt-
ical purposes, but they are toxic and thus cannot be used for
food-grade phycocyanin production [15]. Sugars and polyhydric
alcohols have been used to stabilize proteins, and are being used
widely at present as stabilizing agents in the food industry as well
as in pharmaceutical formulations since they are safe for con-
sumption [16]. Moreover, many studies have reported that the
modication of the protein conformation itself can improve the
stability of proteins. Fukui et al. [17] reported that DSP [dithio-
bis(succinimidyl propionate)]-modied phycocyanin was resistant
to bleaching by urea treatment because the amino groups of
modied lysine residues were cross-linked, thus maintaining the
proteins high-order structure. Another method used to improve
stability was reported by Li et al. [18]. Their study showed that the
measured photodamage rate constant of phycocyanin entrapped
in a silica matrix (immobilized biomolecules of protein with silica)

1359-5113/$ see front matter 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2012.01.010
660 R. Chaiklahan et al. / Process Biochemistry 47 (2012) 659664

was 25 times lower than that of phycocyanin in a buffer solution.

The aim of the current study is to investigate the effect of tem-
perature and pH on the stability of phycocyanin and to determine
which preservatives are suitable for maintaining the stability of
food-grade preparations.

2. Materials and methods

2.1. Materials and extraction of phycocyanin

Spirulina sp. was cultured in Zarrouks medium in 100 L open raceway ponds
under outdoor conditions, in a semi-continuous mode. The cell concentration was
kept constant at approximately OD560 0.40.5. Approximately 3040% of the total
volume of the culture was harvested every other day. Spirulina cells were then
separated by ltration, spread on aluminum trays and sun-dried.
Extraction, separation and purication of phycocyanin were carried out using
the procedures reported by Chaiklahan et al. [19]. Briey, extraction was done using
100 mM phosphate buffer (pH 7.0) at a ratio of 1:100 (w/v) with continuous stirring
at 300 rpm at room temperature for 4 h. The sample was then centrifuged at 4800 g
for 15 min to remove the cell residue. The crude extract was rst ltered through a
5 m membrane at a ow rate of 150 mL min1 and then through a 0.8/0.2 m mem-
brane at 100 mL min1 . The phycocyanin ltrate was then ltered again through a
membrane with a molecular weight cut-off of 50 kDa at 69 kPa and 75 mL min1 .
Food-grade phycocyanin powder was obtained after lyophilization. Fig. 1. The CR value of phycocyanin solution after 30 min incubation at each tem-
perature.
2.2. Effect of temperature, pH and preservatives on stability of phycocyanin

To evaluate the effect of temperature, 30 mg dried phycocyanin was dissolved in

30 mL citrate phosphate buffer at pH 5.0, 6.0 and 7.0. The solutions were incubated
in a temperature gradient box with twelve temperatures (26, 31, 35, 39, 43, 47, 51,
55, 59, 64, 69 and 74 1 C) for 30 min.
To study the effect of pH and temperature, phycocyanin solutions at ve pH
levels (pH 5.0, 5.5, 6.0, 6.5 and 7.0 0.1, citrate phosphate buffer) were incubated
at two different temperatures (50 C and 60 C) using the same concentration of
phycocyanin as above (1 mg mL1 ). Samples were taken for examination after 15,
30, 60, 90, 120, 180, and 240 min.
To determine the efcacy of preservatives, each of the following preservatives
was added to 30 mL of a 1 mg mL1 phycocyanin solution at pH 7.0 before incubation
at 60 C: glucose (Glu, 6.0 g), sucrose (Su, 6.0 g), d-sorbitol (Sor, 6.0 g), sodium chlo-
ride (NaCl, 6.0 g), ascorbic acid (Asc, 0.12 g), citric acid (CA, 0.12 g), sorbic acid (SA,
0.12 g) and sodium azide (So-azide, 0.015 g). The concentrations of these preser-
vatives were based on literature reviews [4,15,20]. Effective preservatives were
investigated further at various concentrations under the same conditions.
2.3. Analyses
The purity and the concentration of phycocyanin were measured using a UVvis
spectrophotometer (UVIKONXL) at the wavelengths of 280, 620 and 652 nm. The
purity ratio of the phycocyanin extract was determined spectrophotometrically by
the A620 /A280 ratio.
The phycocyanin concentration was taken as the total amount of CPC and APC (C;
CPC + APC). These were measured as absorbance at 620 and 652 nm and calculated
using the following equation [21,22]:
A620 0.474 (A652 )
CPC (mg mL1 ) = (1)
5.34
A652 0.208 (A620 )
APC (mg mL1 ) = (2)
5.09
The relative concentration of phycocyanin (CR , %) is the remaining concentration
of phycocyanin as a percentage of the initial concentration, as shown in Eq. (3) [20]:
C of the solutions at pH 5.0, 6.0 and 7.0 were approximately 83%, 94%
CR (%) = 100 (3)
C0
The thermal degradation of the phycocyanin solution was conrmed as being
of rst order kinetics according to the correlation obtained while determining the
degradation rate constant, which was calculated according to the equation [23,24]:
 
C
ln = kt (4)
C0
where C is the concentration at time t, C0 is the initial concentration, and k is the
rst-order rate constant (min1 ).
The half-life values (t1/2 , the time taken for the initial phycocyanin concentra-
tion to be reduced by half) of phycocyanin degradation were calculated using the
equation given below [20,24]:
t1/2 = ln 2k (5)
To assess statistical signicance, results from independent experiments con-
ducted in triplicate were analyzed using the analysis of variance (ANOVA) test with
a condence level of 95% (P < 0.05).
2.4. Microstructure examination
The powder of phycocyanin extract, denatured phycocyanin and phycocyanin
with preservatives were obtained after lyophilization. After coating these samples
with gold, they were investigated for characteristics of shape structure using the
scanning electron microscope (SEM; JEOL-JSM-6400).
3. Results and discussion
3.1. Effect of temperature and pH
After phycocyanin extract was ltered through microltra-
tion membrane of pore size 5 m and 0.8/0.2 m, followed by
ultraltration membrane with cut off 50 kDa, the purity ratio of
phycocyanin was increased from 0.75 to 1.43. This value is con-
sistent with the quality of food and cosmetic grade phycocyanin
[25,26]. When food grade phycocyanin powder was dissolved in
citrate phosphate buffer and incubated in a temperature gradient
box for 30 min, the CR value of the solutions at pH 5.0, 6.0 and 7.0
demonstrated 3 degradation phases. In the rst phase (2643 C),
phycocyanin solutions showed a slow rate of degradation. During
the second phase, the degradation rate increased dramatically as
the incubation temperature was raised from 47 to 64 C. Further
increases in the degradation rate were minimal during the third
phase, when the temperature was increased above 64 C (Fig. 1).
The half-life of phycocyanin also decreased rapidly as the temper-
ature was raised from 47 to 64 C (Table 1). At 47 C, the CR values
and 87% of the initial values, respectively, whereas at 59 C the CR
values decreased to approximately 50%. After incubating for 30 min
at 74 C, the CR values of solutions at pH 5.0, 6.0 and 7.0 were about
36%, 38% and 25% of the initial values, respectively. The half-lives
of phycocyanin solutions at pH 5.0, 6.0 and 7.0, were 116.5 min,
309.4 min and 128.6 min, respectively, at 47 C, but these values
were reduced to 9.4 min, 28.1 min and 8.9 min at 64 C. Between
43 and 59 C, the phycocyanin solution at pH 7.0 was more stable
than at pH 5.0 but above 59 C the solution was more stable at pH
5.0. The highest stability of the phycocyanin solution was observed
at pH 6.0. In addition, the phycocyanin precipitated out of solution
at pH 5.0 and 6.0 after incubation at 59 C for 15 min resulting in
fading of the blue color, while the phycocyanin solution at pH 7.0
showed precipitation after incubation at 64 C for 30 min. Jespersen
et al. [12] also reported that aqueous phycocyanin at pH 5.0 and 7.0
 R. Chaiklahan et al. / Process Biochemistry 47 (2012) 659664 661

Table 1
Degradation rate (k) and half-life values (t1/2 ) of phycocyanin at pH 5.0, 6.0 and 7.0.

Temperature ( C) pH 5.0 pH 6.0 pH 7.0

k (min1 ) t1/2 (min) k (min1 ) t1/2 (min) k (min1 ) t1/2 (min)

47 0.0060 0.0013 116.5 22.3 0.0022 0.0001 309.4 12.0 0.0054 0.0005 128.6 13.3
51 0.0112 0.0006 62.1 3.1 0.0041 0.0020 167.0 39.1 0.0064 0.0003 108.3 28.0
55 0.0202 0.0013 34.4 2.2 0.0075 0.0018 92.7 23.2 0.0146 0.0034 47.5 8.9
59 0.0320 0.0051 21.6 3.6 0.0150 0.0031 46.4 9.7 0.0309 0.0042 22.8 2.7
64 0.0735 0.0030 9.4 2.6 0.0247 0.0052 28.1 5.4 0.0776 0.0066 8.9 0.9
69 0.0928 0.0102 7.5 1.7 0.0481 0.0064 14.5 4.2 0.1156 0.0328 6.0 1.7
74 0.1068 0.0232 6.5 1.4 0.0707 0.0030 9.7 1.6 0.1361 0.0256 5.3 1.0

In this table and subsequent table, value are expressed as mean SD of n = 3.

Table 2
The CR value of phycocyanin solution at each temperature.

Temperature ( C) The CR value (%) at

pH 5.0 pH 6.0 pH 7.0

1 (min) 2.5 (min) 1 (min) 2.5 (min) 1 (min) 2.5 (min)

59 nda 89 10 nda 100 5 nda 94 8

64 98 3 87 6 101 4 99 3 101 8 88 7
69 97 1 83 8 100 4 97 3 101 7 86 14
74 96 4 78 11 98 3 91 6 101 7 76 15
a
Not determined.

precipitated after incubation at 45, 50 and 55 C for 24 h, indicating
protein degradation.
Experiments under commercial HTST (high temperatureshort
time) pasteurization conditions (72 C for 15 s; [27]), showed that
phycocyanin remained almost intact. The CR value of the solutions
at pH 5.0, 6.0 and 7.0 was in range 96100% after incubation at 74 C
for 1 min (Table 2).
Moreover, the results indicated that phycocyanin solution was
more stable at low temperature (4 C). The CR value of the solu-
tions at pH 6.0 and 7.0 remained more than 80%, whereas, pH 5.0
remained approximately 70% after incubation at 4 C for 120 days
(Table 3). Although, the CR value of the solutions at room temper-
ature remained more than 80% after incubation for 10 days, but
turbidity and odor of the solution was observed. This nding was
also observed by Doke [28]. Due to phycocyanin being a natural pro-
the working temperature the lower the pH at which the aqueous
extract of phycocyanin was more stable, showing that the degrada-
tion temperature and pH were inversely proportional with respect
to the degradation of phycobiliprotein. Although the trend found
by Antelo et al. [20] was similar to these ndings, they reported that
between 50 and 55 C, phycocyanin was more stable at pH 6.0 but
between 57 and 65 C it was more stable at pH 5.0. Some studies
have reported that a pH between 5.0 and 6.0 was most suitable for
A 120
50 oC
100
80
C R (%)

tein, microorganisms such as bacteria can easily utilize it at room 60

temperature.
When phycocyanin solutions were tested for stability at 5 dif- 40 pH 5.0
ferent pH values, the maximum stability at 50 C was observed pH 5.5
pH 6.0
at pH 6.0 and the lowest stability was at pH 5.0. At pH 5.5 and 20 pH 6.5
pH 7.0
6.5, the concentration of the phycocyanin solution decreased by
0
approximately 30% from the initial concentration after incubating
for 120 min at 50 C (Fig. 2A). At 60 C, the stability of the phyco- 0 30 60 90 120 150 180 210 240 270

cyanin solution at pH 5.5 was signicantly higher (P < 0.05) than Time (min)
the solutions at pH 6.0 and pH 6.5. The stability was much lower
at pH 5.0 and 7.0. After heating at 60 C for 15 min, the CR value B 120
of the solution at pH 5.5 sharply decreased from 100% to 70%, 60 oC
100
and then slightly decreased to approximately 55% after 180 min
(Fig. 2B), leading to fading of the color. This result is consistent 80
with the study by Antelo et al. [20], who reported that the higher
CR (%)

60
Table 3
The CR value of phycocyanin solution at low temperature. 40
pH 5.0
pH The CR value (%) at pH 5.5
20 pH 6.0
pH 6.5
Room temperature 4 C pH 7.0
(25 2 C) 0
2 days 10 days 30 days 60 days 90 days 120 days 0 30 60 90 120 150 180 210
Time (min)
5.0 90 5 88 4 99 3 86 5 77 11 73 17
6.0 91 3 84 8 98 3 92 5 90 5 88 6
Fig. 2. The CR value of phycocyanin solution with varied pH at 50 C (A) and 60 C
7.0 92 4 79 16 98 4 95 7 89 19 83 24
(B).
662 R. Chaiklahan et al. / Process Biochemistry 47 (2012) 659664

A 120
100 100 102 100 98 T = 0 min
100 91
88 87 85 T= 30 min
82
77
80

CR (%)
64 61
58 57
60
46 46
44
40

20

0
Cont. Glu Su Sor Asc CA SA NaCl So-azide

Preservative

B 8.0
7.1 7.1 7.1
6.8 6.9 6.9
7.0 6.6

6.0 5.7 5.7

5.0
pH value

4.0

3.0

2.0

1.0

0.0
Cont. Glu Su Sor Asc CA SA NaCl So-azide

Preservative

Fig. 3. The CR (A) and pH (B) value of the phycocyanin solution with various preservatives at 60 C.

maintaining phycocyanin properties [15]. However, the results
from this study indicated that the phycocyanin solution was highly
stable in the pH range 5.56.0 with low stability at pH 5.0. The
pH value and the phycocyanin concentration are the main fac-
tors controlling phycocyanin aggregation and dissociation to form
monomers, trimers, hexamers and other oligomers in solution.
While the hexameric structure is protected against denaturation,
they dissociate to trimers at higher pH values. Indeed, Kao et al.
[29] and Adams et al. [30] reported that at pH 6.0, the trimeric form
constituted 23% of the protein with the remainder being hexam-
eric, while at pH 7.0, 82% of the phycocyanin was trimeric. In this
study, the hexameric form may predominate in the phycocyanin
solution at pH 5.56.0, resulting in the higher stability observed.
Judging by color and clarity, the phycocyanin was highly soluble at
pH 7.0, but insoluble in acidic solutions. At pH 3.0, the phycocyanin
solution precipitated and immediately denatured to 25% of the ini-
tial phycocyanin concentration. The solution was turbid at pH 4.0
(data not shown).
3.2. Effect of preservative
Fig. 3A shows that at t = 0, the addition glucose, sucrose or sor-
bitol at a concentration of 20% (w/v, 6.0 g in 30 mL phycocyanin
solution) resulted in a 1215% reduction of the CR value when
compared with the control (without the addition of preservative).
Likewise, the addition of sodium chloride at the same concentra-
tion resulted in a reduction of the CR value of around 9% and the
pH also decreased from 7.1 to 5.7 (Fig. 3B). However, after 30 min
of heat treatment at 60 C, the CR values of samples with added
glucose, sucrose, sorbitol, ascorbic acid, citric acid or sodium chlo-
ride were signicantly higher (P < 0.05) than in the control, whereas
addition of sorbic acid or sodium azide did not result in a signicant
difference. The study conducted by Antelo et al. [20] also found that
the addition of sorbitol at 10% and 50% (w/w) increased the half-
life values of phycocyanin at 62 C. Mishra et al. [15] also found
that both calcium chloride and sucrose are effective in maintaining
the stability of phycocyanin solutions at low temperature (0 C),
and 4 mg mL1 citric acid was one of the best preservatives at pH
7.0 and 35 C. Results from this study showed that the pH value of
the phycocyanin solution decreased approximately 1.5 units after
the addition of 4 mg mL1 citric acid to the phycocyanin solution.
Specically, the phycocyanin solution at pH 7.1 decreased to 5.7
after the addition of 4 mg mL1 citric acid, which was close to the
best pH value for maintaining the stability of phycocyanin at 60 C.
In contrast, the pH value of the phycocyanin solution at pH 6.0
decreased to 4.5 after the addition of 4 mg mL1 citric acid, and
this resulted in low stability of phycocyanin (data not shown). Glu-
cose, sucrose and sodium chloride were selected for further study
to determine the most effective concentrations for stability of the
phycocyanin extract on the basis of the CR value and the price.
The CR value of samples with the addition of 20% (w/v) glucose,
20% (w/v) sucrose or 2.5% (w/v) sodium chloride was signicantly
higher (P < 0.05) than in the control (no preservative) (Fig. 4). After
15 min of heat treatment at 60 C, the highest CR value (82%)
was obtained with the addition of 5% sodium chloride, while the
CR values of the samples remained in the range 5470% when
supplemented with 1040% (w/v) glucose or sucrose, and was
approximately 47% in the control. The half-lives of phycocyanin
solutions with the addition of 1040% glucose or sucrose also
increased from 19 min to between 21.2 min and 44.2 min, while the
half-life of the phycocyanin solution with 2.5% sodium chloride was
67.7 min (Table 4). In addition, the scanning electron microscope
(SEM) images showed that sugar and salt were efcient protein-
stabilizing agents that coated the surface of phycocyanin, thereby
 R. Chaiklahan et al. / Process Biochemistry 47 (2012) 659664 663

Table 4
Degradation rate (k) and half-life values (t1/2 ) of phycocyanin at pH 7.0 which added each preservative concentration.

Conc. (%) Glucose Sucrose NaCl

k (min1 ) r2 t1/2 (min) k (min1 ) r2 t1/2 (min) k (min1 ) r2 t1/2 (min)

0 0.0362 0.99 19.1 6.1

2.5 0.0362 0.95 19.1 5.0 0.0352 0.99 19.7 2.8 0.0102 0.94 67.7 1.0
5 0.0365 0.98 19.0 2.8 0.0311 0.81 22.3 2.0 0.0079 0.93 87.8 6.6
10 0.0327 0.97 21.2 4.6 0.0296 0.93 23.4 3.2 0.0088 0.95 78.2 10.6
20 0.0206 0.93 33.6 5.0 0.0231 0.82 30.0 4.6 0.0101 0.95 68.5 14.6
40 0.0157 0.98 44.2 6.1 0.0173 0.96 40.0 5.9

100 maintaining and protecting its structure. This resulted in delay of

Glu Su NaCl
90 82 80 81 the degradation process, whereas the phycocyanin extract with-
80 76 out the addition of a stabilizer was easy to crack at 60 C (Fig. 5).
70
70 65
62
65 The stabilization of the protein structure by sugar was studied by
57 Arakawa and Timasheff [31]. They reported that the addition of
60
CR (%)

51 54
50 49
47 47 sugar results in a positive free-energy change, and this change is
50
40 presumed to increase with an increase in the surface tension of
30 water. The free-energy change is a very important factor governing
20 the preferential interaction of protein with solvent components in
10 aqueous sugar systems, and hence important for the stabilization
0 of proteins. Likewise the study conducted by Kaushik and Bhat [32]
0 2.5% 5% 10% 20% 40% found that surface tension measurements of aqueous solutions of
salts indicate an increase in the surface tension that very strongly
Concentration (w/v)
correlated with the increase in the thermal stability of proteins.
Fig. 4. The CR value of the phycocyanin solution with Glu, Su, and NaCl at each Moreover, Adams et al. [30] reported that at pH 7.0, phycocyanin
concentration after 15 min of heating at 60 C. was virtually completely converted to hexamer in the presence of
2 M sodium chloride.

Fig. 5. SEM images (500) of the phycocyanin extract (A), denatured phycocyanin (B), phycocyanin with 2.5% NaCl (C) and phycocyanin with 20% sucrose (D). The image at
3500 was inserted on the right corner of A, C and D.
664 R. Chaiklahan et al. / Process Biochemistry 47 (2012) 659664
Although 5% sodium chloride was a good preservative for main-
taining the stability of the phycocyanin solution at pH 7.0 and 60 C,
the solution became turbid. Therefore, using the CR value and the
half-life of phycocyanin as criteria, 20% glucose, 20% sucrose or
2.5% sodium chloride were considered suitable for prolonging the
stability of the phycocyanin extract.
4. Conclusions
This study demonstrated that 47 C is the critical temperature
for stability of phycocyanin solutions, with a sharp drop in the
CR and half-life values being detected at temperatures above this.
However, the phycocyanin solution was stable during HTST pas-
teurization. At 50 C the phycocyanin solution showed maximum
stability at pH 6.0, while at 60 C the maximum stability was at
pH 5.5. Glucose, sucrose and sodium chloride showed potential for
protection and delay of the degradation process.
Acknowledgment
This work was supported by King Mongkuts University of Tech-
nology Thonburi, Bangkok, Thailand.
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