Biochemical Engineering Journal 41 (2008) 43–47

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Biochemical Engineering Journal

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Thermal degradation kinetics of the phycocyanin from Spirulina platensis

Francine S. Antelo, Jorge A.V. Costa, Susana J. Kalil ∗
Department of Chemistry, Fundação Universidade Federal do Rio Grande, Rua Alfredo Huch 475, CEP Rio Grande, RS, Brazil

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

Article history: The cyanobacterium Spirulina platensis is a source of pigments, such as phycocyanin, which is used in the
Received 15 May 2007 food, cosmetic and pharmaceutical industries. The thermal degradation kinetics of the liquid extract at
Received in revised form 22 February 2008 pH values of 5, 6 and 7 was studied, evaluating its stability between 50 and 65 ◦ C. The kinetic model was
Accepted 13 March 2008
assumed and validated as being of the first order. Between 50 and 55 ◦ C the extract was more stable at pH
6 and between 57 and 65 ◦ C at pH 5, but was shown to be increasingly unstable at pH 7 as the temperature
Keywords:
of the treatment increased. The addition of sorbitol between 10 and 50% (w/w) in the treatment at 62 ◦ C
Phycocyanin
for 30 min increased the half-life values of the phycocyanin extract, proving that its de-colorization was
Sorbitol
Protein
related to degradation of the protein chain.
Microalgae © 2008 Elsevier B.V. All rights reserved.
Biokinetics
Kinetic parameters

1. Introduction
The cyanobacterium Spirulina platensis has been considered to
be an important subject for biotechnological research due to its eco-
nomical, ecological and nutritional importance [1]. S. platensis is a
blue-green microalga which can produce large quantities of high
value products such as phycobiliproteins [2]. Phycobiliproteins are
a brilliantly colored family of water-soluble proteins bearing cova-
lently attached, open-chain tetrapyrroles known as phycobilins
[3]. On the basis of their visible absorption properties, the phy-
cobiliproteins have been assigned to four spectroscopic classes:
phycoerythrocyanin, phycoerythrins, phycocyanins and allophyco-
cyanin [4].
Phycocyanin is the main pigment produced by the cyanobac-
terium S. platensis and may reach 20% in dry weight of the cell
protein [5]. This phycobiliprotein is not only used as a natural, nutri-
tious and coloring ingredient for foods and cosmetics, but also as a
potential therapeutic agent in the treatment of oxidative diseases
and as fluorescent marker in biomedical research [6–8].
The current tendency to use natural pigments has turned phy-
cocyanin into an attractive bioproduct. In Europe, the search for
natural dyes is growing since the artificial dyes are generally
considered to be toxic or somehow dangerous [9]. Color is an
important attribute related to the visual appeal and to the quality
of nutritious products [10]. Thus excessive de-colorization dur-
ing thermal processing compromises commercialization, and the
∗ Corresponding author. Tel.: +55 53 3233 8754; fax: +55 53 3233 8720.
E-mail address: susana.kalil@vetorial.net (S.J. Kalil).
kinetic parameters such as the reaction order, reaction constant and
activation energy can supply profitable information on the change
in quality of a food occurring during thermal processing [11].
The kinetics of color degradation have been considered to follow
a first order reaction as shown by Ahmed et al. [12], Ahmed et al.
[13], Gunawan and Barringer [14], Weemaes et al. [15] and Steet and
Tong [16]. Due to the possible denaturation of the protein fraction of
the phycocyanin and consequent loss of color, it is of interest to use
stabilizing agents to maximize the shelf life of the protein solutions
of greater biotechnical and pharmaceutical interest [17,18]. Sugars
and polyhydric alcohols have been used to stabilize proteins, and
are now being used widely as stabilizing agents in the food indus-
try as well as in pharmaceutical formulations, in concentrations
inoffensive to the ingestion [18].
The objective of the present work was to study the thermal
degradation kinetics of the aqueous extract of phycocynin from S.
platensis, in the temperature range from 50 to 65 ◦ C at pH values of
5, 6 and 7, and with the addition of a stabilizing agent under the
conditions showing greater instability of the phycobiliprotein.
2. Material and methods
2.1. Culture conditions of Spirulina platensis
S. platensis LEB 52 [19] was cultivated in a 450 L open
outdoor photo-bioreactor under uncontrolled conditions in the
south of Brazil. During these cultivations, the water was supple-
mented with 20% Zarrouk [20] synthetic medium, with an initial
biomass concentration of 0.30 (g dm−3 ). Samples were taken every
24 h to determine the biomass concentration via optical density

1369-703X/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.bej.2008.03.012
44 F.S. Antelo et al. / Biochemical Engineering Journal 41 (2008) 43–47

Table 1
Degradation rate constants (Kd), and the respective correlation coefficients (r2 ) and half-life values (t1/2 ) of the aqueous phycocyanin crude extract at pH values 5, 6 and 7,
for each temperature studied

pH 5 pH 6 pH 7
◦ −1 −1
T ( C) Kd (s ) r2
t1/2 (s) Kd (s ) r2
t1/2 (s) Kd (s−1 ) r2 t1/2 (s)
−5 −6 −5
50 1.0 × 10 0.95 69,315 8.0 × 10 0.96 86,643 2.0 × 10 0.97 34,657
53 3.0 × 10−5 0.93 23,105 2.0 × 10−5 0.94 34,657 3.0 × 10−5 0.97 23,105
55 2.0 × 10−4 0.97 3,466 5.0 × 10−5 0.95 13,863 1.0 × 10−4 0.96 6,931
57 4.0 × 10−4 0.94 1,733 6.0 × 10−4 0.96 1,155 1.4 × 10−4 0.93 495
60 1.1 × 10−3 0.97 630 3.8 × 10−3 0.92 182 1.8 × 10−4 0.95 385
62 2.5 × 10−3 0.94 277 7.8 × 10−3 0.95 89
65 2.9 × 10−3 0.93 239

measurements at 670 nm in a spectrophotometer (FEMTO Espec- phycocyanin), and OD652 is the optical density of the sample at
trofotômetro 700 Plus) according to Costa et al. [19] At the end of 652 nm (maximum absorption of phycoerythrin).
cultivation, the biomass was recovered by filtration, pressing and
extrusion, dried at 50 ◦ C for 6 h, frozen at −18 ◦ C, ground in a ball
mill and sieved (the perforations of the sieve being 150 mesh). 2.6. Degradation rate constant

2.2. Extraction of phycocyanin The Kd (s−1 ) of phycocyanin was estimated by regression of the
experimental data for time, protein concentration with time and
Phycocyanin was extracted according to Silveira et. al [21]. After initial protein concentration. Assuming first order reaction kinetics,
extraction, the suspension was centrifuged and vacuum filtered and Kd was determined according to Eq. (2) [22]:
the supernatant collected.
dCF
= −KdC (2)
dt
2.3. Determination of the phycocyanin degradation kinetics
where CF is the phycocyanin concentration (mg mL−1 ), t is the time
The phycocyanin degradation kinetics was studied in duplicate, (s) and Kd is the degradation rate constant (s−1 ).
in covered flasks at constant temperature using the crude phy-
cocyanin extract. The temperatures of 50, 53, 55, 57, 60, 62 and
65 ◦ C were studied at three pH values: 5, 6 and 7, so as to obtain 2.7. Half-life values
the degradation rate constant (Kd) for each proposed condition.
Samples were removed periodically until half the initial concentra- The half-life values t1/2 (s) for the first-order degradation kinetic
tion of the phycocyanin was reached. The values for Kd obtained at model were given by Eq. (3) [23]:
each temperature for each pH value allowed one to determine the
half-life of the phycobiliprotein in each situation. The correlation ln 2
t1/2 = (3)
between the kinetic constants supplied the kinetic model for the Kd
thermal degradation of the crude phycocyanin extract at each pH
value studied.
2.8. Arrhenius equation

2.4. Determination of the phycocyanin degradation kinetics with

The Arrhenius equation relates the temperature to the constant
the addition of a stabilizing agent
for the speed of elementary reactions, and allows for the determina-
tion of the activation energy and the frequency factor of for protein
To prevent the de-colorization of the aqueous crude phyco-
degradation reactions, as expressed by Eq. (4) [24]:
cyanin extract due to degradation of the protein fraction, the
addition of a stabilizing agent was studied. Sorbitol was added at
Kd = Ae−Ed/RT (4)
concentrations of 10, 20, 30, 40 and 50% (w/w) to the extract incu-
bated at 62 ◦ C, triplicate samples being removed periodically for
where Kd is a degradation rate constant (s−1 ), A is the frequency
up to 30 min. The degradation rate constants and half-lives of the
factor (s−1 ), Ed is the activation energy degradation reaction
crude phycocyanin extract were calculated for each sorbitol con-
(cal gmol−1 ), T is the temperature (K) and R is the gas constant
centration added to the crude extract, under each of the established
(cal gmol−1 K−1 ).
conditions. Using Tukey’s test with the statistical means, the signif-
icant differences amongst the sorbitol concentrations added to the
phycocyanin extract at each pH value were determined at a 95% 2.9. Relative concentration of phycocyanin
confidence level.
The relative concentration of phycocyanin (%) relates the
2.5. Concentration of the phycocyanin remaining and initial concentrations of this phycobiliprotein to the
time, for each different sorbitol concentration (%) studied, as shown
The phycocyanin concentration (PC), according to Abalde et al. in Eq. (5):
[4], was defined as:
CF
[OD615 − 0.474 × OD652 ] %CRF = × 100 (5)
PC = (1) CFO
5.34
where PC is the phycocyanin concentration (mg cm−3 ), OD615 is the where CF and CFO are the remaining and initial concentrations
optical density of the sample at 615 nm (maximum absorption of of phycocyanin, respectively.
 F.S. Antelo et al. / Biochemical Engineering Journal 41 (2008) 43–47 45

3. Results and discussion Table 2

Frequence factor (A), activation energy (Ed) and the respective correlation coeffi-
cients (r2 ) to each pH values studied, 5, 6 and 7
3.1. Determination of the phycocyanin degradation kinetics
pH A (s−1 ) Ed (kcal gmol−1 ) r2
Phycocyanin samples at the three pH values studied, were sub- 5 1.86 × 10 54
87.36 0.94
mitted to temperatures of 50, 53, 55, 57, 60, 62 and 65 ◦ C, so as to 6 2.45 × 1086 135.57 0.96
obtain the Kd and the values for the phycobiliprotein half-lives (t1/2 ) 7 2.03 × 1070 111.14 0.90

under each condition proposed. At 65 ◦ C and pH values of 6 and

7, the aqueous crude phycocyanin extract was quickly denatured,
making the practical procedure unfeasible. According to Àvila and
Silva [25] and Corzo et al. [26], numerous researchers have studied
the application of the first order model to describe the degradation
of color in nutritious products. Using Eqs. (2) and (3), the Kd and t1/2
values were obtained for the different T and pH values, as presented
in Table 1.
It was verified that phycocyanin degradation or the protein frac-
tion degradation, followed a first order kinetic model, as shown
by the high correlations of between 0.92 and 0.97 obtained in
the kinetic constant determination. It was also verified that the
phycocyanin was denatured more quickly at higher temperatures,
since the half-life values (the time taken for the initial phycocyanin
concentration to be reduced by half) decreased as the working tem-
perature increased. The same was observed by Koca et al. [27] for
the chlorophyll of pea grains, where the half-life of the pigment was
reduced as the working temperature increased.
Between 50 and 55 ◦ C, the phycocyanin was more stable at pH
6 and between 57 and 65 ◦ C at pH 5. Thus the higher the work-
ing temperature the lower the pH at which the aqueous extract of
phycocyanin was more stable, showing that the degradation tem-
perature and pH were inversely proportional with respect to the
degradation of phycobiliprotein. In the present study it was shown
that between 50 and 65 ◦ C the aqueous crude phycocyanin extract
was less stable at pH 7. In addition to the temperature dependence
of phycocyanin degradation and consequently of color loss, it was
observed that the inverse of the temperature was correlated with
the logarithm of the kinetic constants for protein heat degradation
at each pH value.
Using Eq. (4), the values for A and Ed were determined for each
pH value, as shown in Table 2.
3.2. Determination of the phycocyanin degradation kinetics with
the addition of a stabilizing agent
As the color maintenance of phycocyanin is related to the non-
degradation of the protein fraction, as affirmed by Fukui et al. [28],
it is important to use a stabilizing agent to maintain the high struc-
tural order of the protein-chain, and thus maintain the phycocyanin
color. Sorbitol is a polyol that can be found in a vast range of nutri-
tious products as mentioned by Dierckx and Huyghebaert [29], and
for this reason was chosen as the stabilizing agent for the crude phy-
cocyanin extract. Its effect on protein degradation is well known,
favoring its use in the stabilization of the phycocyanin color, since
the development of the color is tied up with the conformation of
the protein fraction.

Fig. 1. Relative phycocyanin concentration (%) for treatment times of 30–2400 s with sorbitol concentrations between 10 and 50% (w/w), for aqueous crude extract at pH 5
(a), 6 (b) and 7 (c).
46 F.S. Antelo et al. / Biochemical Engineering Journal 41 (2008) 43–47

Table 3
Degradation rate constants (Kd), correlation coefficients (r2 ) and half-life values (t1/2 ), for the aqueous phycocyanin crude extract, at pH values 5, 6 and 7, for each sorbitol
concentration (%, w/w) added

pH 5 pH 6 pH 7
−1 −1
Sorbitol concentration (%, w/w) Kd (s ) r2
t1/2 (s) Kd (s ) r2 1/2
t (s) Kd (s−1 ) r2 t1/2 (s)
−4 −4 −4
10 3.9 × 10 0.94 1,732 7.7 × 10 0.96 866 6.7 × 10 0.92 990
20 2.3 × 10−4 0.88 3,465 5.0 × 10−4 0.95 1,386 4.0 × 10−4 0.90 1,733
30 2.9 × 10−4 0.95 2,310 1.2 × 10−4 0.98 6,931 3.0 × 10−4 0.92 2,310
40 2.2 × 10−4 0.93 3,465 1.1 × 10−4 0.98 6,931 2.1 × 10−4 0.94 3,466
50 6.0 × 10−5 0.94 11,552 6.0 × 10−5 0.95 11,552 6.0 × 10−5 0.92 11,552

Fig. 1 shows the relative concentrations of phycocyanin (%) after
treatment of the aqueous crude extracts in an immersion water
bath at 62 ◦ C for pH values of 5, 6 and 7 without sorbitol, and with
the addition of 10, 20, 30, 40 and 50% (w/w) of the stabilizing agent,
for treatment times of 30–1800 s.
As shown by the results obtained, sorbitol tended to maintain
the conformation of the protein molecule of the phycocyanin with
the addition of any of the concentrations studied (%, w/w), caus-
ing an increment in the relative phycocyanin concentration at all
the times analyzed between 30 and 1800 s. As the treatment time
increased, higher percentages of added sorbitol resulted in larger
relative phycocyanin concentrations.
Table 3 presents the values for the Kd and the half-life times for
phycocyanin at the three pH values studied (5, 6 and 7) at 62 ◦ C,
for each of the sorbitol concentrations added to the aqueous crude
extract.
An analysis of the results obtained for the aqueous crude phyco-
cyanin extract at pH values of 5, 6 and 7, showed that the half-life
values increased proportionally with the sorbitol concentration
(%, w/w) added. At pH 5, the half-life value of the phycocyanin
crude extract at 62 ◦ C was 5 min without the addition of a sta-
bilizer, whilst the addition of 10% (w/w) sorbitol provided an
increase of 29 min. This fact confirmed that sorbitol was an effi-
cient protein-stabilizing agent, delaying the degradation process
of the phycocyanin molecule at concentrations starting from 10%
(w/w).
Using Tukey’s test, significant differences were found between
the different sorbitol concentrations added to the aqueous crude

Fig. 2. Analysis of the effect of the different sorbitol concentrations (%, w/w) on the relative phycocyanin concentration (%) of the extracts at pH 5 (a) and 6 (b), after 30 min
immersed in a water bath and pH 7 (c) after 10 min of treatment.
 F.S. Antelo et al. / Biochemical Engineering Journal 41 (2008) 43–47
phycocyanin extracts at each pH value and for each treatment time,
at a 95% level of confidence.
Fig. 2 shows the effect of the sorbitol concentrations (%, w/w) on
the relative phycocyanin concentrations (%) of the extracts at pH 5
and 6 at the end of 30 min, and of those at pH 7 after 10 min, where
different letters represent statistical difference at p ≤ 0.05.
For the extract at pH 5 the addition of 30, 40 and 50% (w/w)
sorbitol to the aqueous crude phycocyanin extract showed no
significant differences in the mean relative phycocyanin concen-
trations (%) at the end of 30 min of heat treatment, at a 95% level of
confidence. Thus the crude phycocyanin extract at pH 5 maitained a
relative concentration of about 80% with the addition of 30% (w/w)
of stabilizing agent. For the extract at pH 6, the addition of 30, 40
and 50% (w/w) of sorbitol to the aqueous phycobiliprotein extract
showed no significant differences at the 95% confidence level at the
end of 30 min of heat treatment. Thus with 30% (w/w) of stabiliz-
ing agent, the phycocyanin extract at pH 6 maintained a relative
concentration of about 79%.
For the extract at pH 7, the addition of 40 and 50% (w/w) of sor-
bitol to the aqueous phycobiliprotein extract showed no significant
differences at the 95% confidence level after 3 min of heat treat-
ment. However, as from 10 min of heat treatment, all the sorbitol
concentrations (10, 20, 30, 40 and 50% (w/w)) presented statis-
tically significant differences with respect to the relative mean
phycocyanin concentrations. The addition of 50% (w/w) of sorbitol
maintained 92% of the relative phycocyanin concentration at the
end of 10 min and 85% at the end of 30 min.
4. Conclusions
The kinetic model for the thermal degradation of the aqueous
crude phycocyanin extract was validated as being of the first order
according to the correlations obtained in the determination of the
degradation rate kinetic constants for each of the temperatures
studied. At temperatures between 50 and 55 ◦ C, the aqueous crude
phycocyanin extract was more stable at pH 6 and between 57 and
65 ◦ C at pH 5. It was shown to be least stable between 50 and 65 ◦ C
at pH 7.
The half-life values decreased as the temperature of the heat
treatment increased, while the addition of 10 to 50% stabilizing
agent increased the half-life of the aqueous phycocyanin crude
extracts from 277 s to between 1732 and 11,552 s at pH 5, from
89 s to between 866 and 11,552 s at pH 6 and to between 990 and
11,552 s at pH 7, showing that phycocyanin de-colorization is prob-
ably related to protein-chain degradation, since sorbitol stabilizes
proteins.
At the end of 30 min of heat treatment, the phycocyanin extract
at pH 5 maintained about 80% of its mean relative concentration
with the addition of 30% (w/w) sorbitol. For the extract at pH 6,
about 79% of the relative phycocyanin concentration was main-
tained with the addition of 30% (w/w) of stabilizing agent, and
at pH 7, statistically significant differences in the mean relative
phycocyanin concentrations obtained were found between all the
concentrations of sorbitol added, 50% (w/w) of sorbitol maintain-
ing 85% of the protein concentration at the end of 30 min of heat
treatment.
Acknowledgement
The authors gratefully acknowledge the financial support of
the Coordenação de Aperfeiçoamento de Pessoal de Nı́vel Superior
(CAPES).
47
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