NOTE
Study of thermodegradation of phycocyanin from Spirulina platensis
C. Couteau1, S. Baudry1, C. Roussakis2, L. J.M. Coiffard1*
RÉSUMÉ
Étude de la thermodégradation de la phycocyanine issue de Spirulina platensis
L’objectif de ce travail est l’évaluation de la stabilité de la phycocyanine issue de Spirulina platensis. La thermodégradation de solutions de ce colo- rant a été étudiée à différentes températures (25, 35, 45 °C). Cette dégrada- tion semble suivre une cinétique d’ordre 1, quelle que soit la température et être pH-dépendante. La demi-vie est de 38, 41 et 29 jours, respectivement à pH 2,0, 6,5 et 8,0. On peut conclure que cette protéine est plus stable à pH inférieur à 7.
Mots clés
thermodégradation, phycocyanine, Spirulina platensis, pH.
SUMMARY
The objective of this present study was to evaluate the stability of phycocy- anin from Spirulina platensis. Thermodegradation of solutions of this color- ant at various temperatures (25, 35, 45 °C) was studied. This degradation appeared to follow first-order kinetics at whatever temperature and was found to be pH dependent. The experiments revealed an half-life of 38, 41 and 29 days at pH 2.0, 6.5 and 8.0 respectively. So it can be concluded that this protein is more stable at pH < 7.
Key words
thermodegradation, phycocyanin, Spirulina platensis, pH.
1. L.P.I.C. - School of Pharmacy - University of Nantes - 1, rue Gaston-Veil - 44035 Nantes cedex, France.
Tél. : + 33-240-412873/Fax + 33-240-412987.
1 – INTRODUCTION
Spirulina platensis is characterized by a spiral shape (ROSS et al., 1994). This cyanobacteria is rich in proteins (60 at 70%) and in minerals (5 at 12%). So, the nutritional and therapeutic application possibilities of Spirulina are very well documented (OMSTEDT et al., 1973 ; NARASIMHA et al., 1982 ; HAYASHI et al., 1998 ; BATH et al., 2000). The blue soluble pigment can be interesting to study its stability conditions at various pH values. We have studied degradation of phycocyanin at three temperatures 25, 35 and 45 °C and we have determined the half-life (t50%) at 20°C.
2 – MATERIALS AND METHODS
2.1 Materials
Phycocyanin (Linablue-A, Dainippon Ink & Chemicals) is a blue powder. It is easily soluble in water. All chemicals were of analytical reagent grade. Distilled H2O was obtained from an Autostill 4000X (Jencons).
2.2 Thermodegradation study
Aqueous solutions (about 400 mg.L–1) of phycocyanin at variable pH were packaged in small glass bottles (Duran, Schott, 100 ml) hermeticaly closed by a polypropylen cork. The pH of these solutions was adjusted to the desired value with HCl 10–2M, Na2B4O7 10–4M, H3BO3 1 M or NaOH 10–2M. The pH of these solutions were determined with a Metrohm Herisau pH-meter, model E300B, equipped with a Refill 9811 Ingold I 3556 (pH = 0 – 14, T = 0 – 80°C) electrode and standardized with Panreac solutions respectively at pH = 4 and pH = 10.
These measures were carried out at 20°C. The various solutions were stored in thermostatically controlled ovens (Memmert, type UE-200), at 25, 35 and 45 °C.
Samples were withdrawn at specific time intervals, throughout 40 days, cooled to room temperature and phycocyanin concentrations were determined.
2.3 Assay
Phycocyanin concentrations, initially and at various times, were determined by a spectrophotometric method (Hitachi UV-visible, double beam spectropho- tometer, model U-2000). The concentration of phycocyanin was calculated from Beer' plot of concentration versus absorbance. The analyses were carried out on triplicate sample and the difference between the triplicates was less than 1%.
2.4 Data analyses
The chemical kinetics of the thermodegradation reaction of phycocyanin was described by the least squares method with linear adjustment and by cal- culation of correlation coefficients. These methods were used in order to choose the order of the kinetic. The degradation rate constants (k) were deter- mined from the slope of the line of absorbance versus time. The degradation rate constants and the halfe-lives at 25, 35 and 45°C were calculated in accord- ance with the postulated order of the reaction. These thermal treatments or accelerated stability studies were based on the Arrhenius relationship where degradation rate constant of the substance is a function of the temperature, according to the equation:
dLn k/dT = Ea/RT2 Eq.1
where k is the degradation rate constant (s–1), A a constant for a given reac- tion, Ea the energy of activation (J.mol–1), R universal gas constant (J.mol–1.K–1) and T the absolute temperature (K). The statistical analysis of the results have been conducted using the Student t test (P < 0.05, ddl = 2) (GARETT, 1962; COLE
and LEADBEATER, 1966; BENTLEY, 1970).
3 – RESULTS AND DISCUSSION
3.1 Phycocyanin thermodegradation kinetic study
Optimum sensitivity of phycocyanin determination in aqueous solution was obtained at 617 nm. Under the experimental conditions, linear calibration (cor- relation coefficient, r > 0.99), was obtained over phycocyanin concentrations ranging from 70 to 500 mg.L–1. The thermodegradation of phycocyanin in aqueous solution (pH = 6.5) was expressed as the rate of change of absorbance at 617 nm.
We have observed a gradual decrease in absorbance at 617 nm during the thermal treatment. The degradation rate constant is calculated from the slope of the line of absorbance versus time. The percentage of substance remaining is calculated from Beer’s plot of concentration versus absorbance (table 1).
The thermodegradation of phycocyanin in aqueous solution follows apparent first order kinetics (figure 1) and is described by the following equation:
C/C0 = e–kt Eq. 2
where C and C0 were the phycocyanin concentrations at time t and initially respectively and k is the apparent first order degradation rate constant. The degradation rate constants and the half-lives concerning phycocyanin in aqueous solution (400 mg.l–1) at pH 6.5 can be found in table 2.
At 20°C, it can be deduced t50% (half-life or time necessary to obtain a
Table 1
C/C0 ratio for various temperatures at specific time intervals for phycocyanin aqueous solution (pH = 6.5).
Tableau 1
Rapports C/C0 à différentes températures et à intervalles de temps réguliers pour des solutions aqueuses de phycocyanine (pH = 6,5).
45°C 35°C 25°C
Time
(days) C/C0 C/C0 C/C0
0 1.000 1.000 1.000
1 0.738
2 0.718 0.827
3 0.657
4 0.602 0.716
5 0.551 0.888
6 0.505 0.621
7 0.462
8 0.538
10 0.465 0.798
20 0.643
30 0.520
40 0.420
– 1 – 0,9 – 0,8 – 0,7 – 0,6 – 0,5 – 0,4 – 0,3 – 0,2 – 0,1 0
0 10 20 30 40 50
Temps (d)
Ln C/C0
45°C 35°C 25°C
Figure 1
Thermodegradation of phycocyanin in aqueous solution at pH 6.5 at 25, 35 and 45°C.
3.2 Effect of pH
Then, the thermodegradation of phycocyanin (400 mg.L–1) at various pH has been studied. There was a gradual decrease at 624 nm (bathochrom effect) and 617 nm respectively at pH 8 and at pH 2. Whatever the pH, the thermodegrada- tion of phycocyanin follows apparent first order kinetic and is described by the following equation:
C/C0 = e–kt Eq. 3
where C and C0 are the phycocyanin concentrations at time t and initially respectively and k the apparent first order degradation rate 138) constant at a given pH.
No results can be obtained at pH 4 and at pH 10. At pH 4 we have observed a precipitation of the colorant. It is known that the phycobiliproteins precipitate at pH 4 (BOUSSIBA and RIECHMOND, 1980). At pH 10, we have noted a total loss of colouration.
The values of k and the values of t50% according to the pH at various tem- peratures can be found in table 3.
0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1
0,0031 0,0032 0,0033 0,0034 0,0035
1/T (K-1)
log k
Figure 2
Arrhenius relationship log k = f(/T) concerning phycocyanin (pH = 6.5).
Table 2
Degradation rate constants (confidence interval 95%) for phycocyanin (pH = 6.5) and half-time at various temperatures.
Tableau 2
Constantes de vitesse de dégradation (intervalle de confiance de 95%) pour la phycocyanine (pH = 6,5) et demi-vies à différentes températures.
Table 3
Degradation rate constants and halfe-lives values for phycocyanin at various pH at 20°C.
Tableau 3
Constantes de vitesse de dégradation et demi-vies pour la phycocyanine à différents pH, à 20 °C.
4 – CONCLUSION
The spiruline being more stable in acid medium, it is advisable to the formu- lator to adapt to this constraint. In prospects, it would be interesting to evaluate the interest of various technological additives and excipients, while exploiting, for example the potential redox of the medium. It appears also judicious to sup- plement this study by tests of photostability, in order to know the requirements as regards conditioning.
Temperatures (°C)
Degradation rate constants (10–2) (d–1) (confidence interval 95%)
Half-time (days)
25 35 45
2.15 +/– 0.11 7.18 +/– 0.43 8.80 +/– 0.54
31.8 9.0 3.8
pH
Degradation rate constants (confidence interval 95%)
(10–2) (d–1)
Half-lives (d)
2.0 6.5 8.0
1.82 +/– 0.02 1.62 +/– 0.01 2.63 +/– 0.09
38 41 29
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