Introduction

Microalgae Arthrospira (A.) platensis (spirulina) has been considered as one of the important targets of biotechno-logical research because of its economic, ecological, and nutritional significance. A. platensis algae is of particular importance in the food industry (Beheshtipour et al., 2012, 2013; Hoseini et al., 2013a; Massoud et al., 2015; Mazinani et al., 2016), medical sector (anti- inflammatory and anticancer with several kidney and liver protective properties; Hoseini et al., 2013b; Soheili et al., 2011), and aquaculture industry because of its digestibility and high nutritional value (50–70% w/w protein) and also having essential amino acids, vitamins, mineral elements, and essential fatty acids (Fernández-Rojas et al., 2014). A. platensis produces high amounts of phycobili proteins (Antelo et al., 2008) and significant amounts of natural pigments chlorophyll, carotenoid, and phycocyanin (Banayan et al., 2020; Ghaeni et al., 2014; Santiago-Morales et al., 2018).

Phycocyanin is a blue pigment and light receptor with antioxidant and fluorescent properties in cyanobacteria. This pigment is a water-soluble compound with high antioxidant properties. The blue powder of phycocyanin is nontoxic, odorless, and slightly sweet. Phycocyanin and other phycobiliproteins (PBPs) are used in chewing gum, chocolate, jellies, beverages, cosmetic health industries, and disease diagnosis (Ansarifard et al., 2017).

The photoautotrophic production of phycocyanin by A. platensis is carried out in photobioreactors (PBRs; Panutai et al., 2020) and/or open large ponds or pools in tropical or subtropical areas at the edges of oceans. Biomass production efficiency is modified by supply of light. A. platensis grows in mixotrophic cultures; therefore, the conditions for growth and production of phycocyanin are optimized by providing suitable level of light and organic carbon sources (Banayan et al., 2020; Soheili et al., 2013).

Many researchers have optimized cultivation to increase production efficiency and reduce production costs of this pigment. The effect of carbon and nitrogen sources as well as exposure period and intensity of light on the production of phycocyanin is investigated (Banayan et al., 2020; Chen and Zhang, 1997). The extraction efficiency, purity, and concentration of phycocyanin depend on the process of cell disruption. Various methods of extraction, such as the use of lysozyme, ethylenediaminetetraacetic acid (EDTA), and phosphate buffer, are used to extract phycocyanin. Lysozyme affects the cell wall more, and EDTA and phosphate buffer release phycocyanin by chelating magnesium ions and destroying the cytoplasmic membrane. Ultrasound, homogenization, freezing, and thawing techniques, as well as organic and inorganic solvents, are applied for the extraction of phycocyanin (Safari et al., 2018).

The degradation of phycocyanin depends on its protein structure, which is affected by many factors, such as light, pH, temperature, and protein concentration. Hence, stabilizing substances are used to maintain the protein structure (Sarada et al., 1999). Research has shown that phycocyanin is more stable in cold temperature and acidic pH 5–5.4. Increase in temperature to more than 70°C leads to fast denaturation of phycocyanin (Safari et al., 2018). The proteinaceous nature of phycocyanin protects this pigment to be less affected by contamination and microbial decomposition at low temperatures and to denature at high temperatures. Phycocyanin extract is more stable at 50–55°C (Doke, 2005).

Many researchers have focused on thermal stability and prediction of the degradation rate of natural pigments used in food matrices. Thermal analysis of sweet potato purple anthocyanins at 90°C and pH of 3–7 showed that content of anthocyanin decreased with increase in heating time at all pH values and especially in neutral conditions (pH 7; Li et al., 2013). The anthocyanins obtained from red radish extract are much more resistant at pH 5 than at pH 3 (Wang et al., 2017). Modeling of thermal degradation constant (Dc) and half-life (t_½) of Monascos poroporeus was successfully carried out to predict its degradation rate and survival in food processes (Abdollahi et al., 2021). To the best of our knowledge, there is no report about modeling and predicting the behavior of phycocyanin stability against osmotic, temperature, and acid stress conditions in food matrices.

This study was designed to investigate the effect of temperature, pH, and salt on the stability of phycocyanin in simulated food conditions. Modeling of the effect of temperature (50–98°C), pH (4–7), and salt (0–2% w/w) on Dc and t_½ of phycocyanin was carried out by the response surface method (RSM). By controlling pigment degradation in thermal processes, similar conditions are predicted and the desired pigment content in food is calculated.

Material and Methods

Microalgae source and culture condition

The microalgae strain (A. platensis, APP1) was provided by the Microalgae Culture Collection of Tarbiat Modares University, Tehran, Iran. A. platensis was cultivated using Zarrouk media (pH 9.8) in 250-mL glass flasks containing 150 mL of cell suspension under sterile conditions. Growth and maintenance of the culture were carried out using illuminated (150 µmol m² s^-1) phytotron at 30±1°C under a 12–12-h light–dark cycle with mild agitation (100 rpm) for 12 days (Banayan et al., 2020; Gami et al., 2011; Zeng et al. 2012).

Phycocyanin extraction

To extract and measure phycocyanin pigment, freezed biomass and 0.15-M potassium phosphate buffer (pH = 7) were mixed at a ratio of 1:50 v/v. Then, the cell suspension was centrifuged (Universal 320R made in Iran) at 1800 g for 10 min at 25°C. Pellets were removed and the supernatant was collected. Absorbance of the extract was measured with an ultraviolet-visible (UV-VIS) spectrophotometer (2100 model; UNICO, China) at 615 nm and 652 nm, and the concentration of phycocyanin was calculated according to Equation (1) (Banayan et al., 2020; Ferreira-Santos et al., 2020):

Calculation of reaction kinetics

Effects of temperature (50–98°C), NaCl (0–2% w/w), and pH (4.3–7.7) on Dc and t_½ of phycocyanin extracted from A. platensis were assessed using a water bath. Phycocyanin solutions were heated for 30 min in a water bath (50–98°C), and samples were collected after 5 min. Degradation constant obtained in the first-order kinetic model was expressed according to Equation (2). Regression lines were obtained by plotting changes in the degradation of phycocyanin logarithmically as a function of heat treatment time (Vendruscolo et al., 2013):

where A is the amount of phycocyanin (g/L), A₀ is the amount of phycocyanin at t = 0, t is the time (minute), and Dc is the color instability constant because of heat (min^-1). Equation (2) had a logarithmic graph in Equation (3). To convert it into a linear graph, boundary conditions must be used for each parameter. The t_½ was obtained from the Dc parameter given in Equation (4), where t_½ means the time when the amount of phycocyanin was twice the amount of initial phycocyanin:

Statistical analysis

In this study, temperature (50–98°C), pH (4–7), and NaCl content (0–2% w/w) were analyzed as independent variables using central composite design (CCD) of response surface methodology (RSM) and the Expert Design Software v.7.0.0. Levels of real variables in CCD are shown in Table 1. Effects of significant independent variables were assessed in terms of D_C (Y1) and t_½ (Y2) of phycocyanin. The research was designed by applying RSM with α = 1.7. Data were analyzed by the Design 7.0.0 Expert software at 95% confidence level (95% CI).

Results and Discussion

Table 1 shows the effect of independent variables of temperature (50–98°C), pH (4–7), and NaCl (0–2% w/w) on the constant variables of phycocyanin pigment instability, Dc and t_½. Figure 1 shows changes in phycocyanin color degradation after exposure to 17 treatment combinations. Since the graph is logarithmic, ln(A/A₀) was used to linearize the same. The weakening of phycocyanin pigment during the heating period of 5 min is shown in Figure 1, where the slope of the line is the Dc of the pigment. As shown in Figure 1, placed for 30 min, the highest degradation was related to treatment 10 at a temperature of 98°C, pH = 5.5, and salt concentration of 1% w/w, and the lowest degradation was related to treatment 3, which was at a temperature of 59.7°C, pH = 6.4, and salt concentration of 0.44% w/w.

Figure 1. Degradation constant (Dc) of the phycocyanin produced by Arthrospira platensis (spirulina) in aqueous solution versus various conditions (temperature: 50–98°C; pH 4–7, and salt content: 0–2%).

The results of the final analysis of variance presented in Table 2 show that the model was significant (p < 0.0006) and the lack of fit was not significant (p = 0.99). The distribution of statistical data in samples was suitable (CV = 8.22). The value of regression coefficient, R², was 0.9060 and showed significant relationships between experimental and predicted values according to Equation (5):

Dc = 0.0126 + 0.0003 A – 0.0005 B + 0.0001 C – 0.0019 AC + 0.0021 BC + 0.0021 A² + 0.0018 B² (5)

In which A, B, and C are Temperature (ºC), NaCl (%), and pH. Also, AC and BC are interaction effects and A² & B² are quadratic effect of main factors A, B, C.

The t_½ indicates the stability of phycocyanin under processing conditions. In other words, the t_½ is the time taken by the pigment to reach half of its initial amount. The t_½ of a pigment has an inverse relationship with its thermal instability (Dc) (Abdollahi et al., 2021). As the results of the analysis of variance showed, the model was significant (p < 0.0013), error in the lack of fit was not significant (p = 0.6183), and the value of regression coefficient, R², was 0.8869.

The experimentally obtained data for t_½ and coefficient of variables led to an objective function as shown in Equation (6):

t_½ = 55.05 + (0.625 A)+(1.24 B) – (0.106 C) + (5.34 AC) – (5.9 BC) – (6.09 A²) – (5.51 B²) (6)

The linear effects of temperature, pH, and NaCl content and interaction of temperature–NaCl and pH–NaCl were significant on Dc and t_½ of phycocyanin (Table 2). Furthermore, the square effect of temperature and pH was significant (p < 0.05). With increasing temperature, stability of phycocyanin decreased to a minimum level of about 5.5. Figures 2B and 2D show that the interaction of salt and pH was significantly effective on both Dc and t_½ of phycocyanin (p ≤ 0.05). At pH = 6.4, Dc increased with increasing salt, while at pH = 4.6, changes in salt had no effect on it.

Figure 2. The 3D response surface plot demonstrating effects of: (A) temperature and pH and (B) NaCl and pH on the thermal degradation constant (Dc); and (C) temperature and pH and (D) NaCl and pH on half-life (t_½) of phycocyanin.

The results of the present study showed that at pH > 4.6, Dc of phycocyanin increased with increase in salt content. At low temperatures, addition of salt maintained the stability of the pigment. Also, at a temperature of 59.7°C, pH = 6.4, and a salt content of 0.44% w/w, the lowest Dc (0.011) and maximum t_½ (58.74 min) were observed. The Dc of phycocyanin increased and t_½ of phycocyanin decreased with increasing temperature (Figures 2A and 2C). The t_½ of phycocyanin increased if preservatives were used (Martelli et al., 2014).

Phycocyanin is a heat-sensitive protein. Increase in temperature destroys the protein structure of phycocyanin because at high temperatures, structure of the protein is affected by heating, breaking down of bonds between and within the chain. Therefore, the stability of phycocyanin decreases (Chaiklahan et al., 2012). This change is irreversible (Doke, 2005). Decreased color stability because of high temperatures is expected in most natural colors (Priatni, 2015).

Under thermal processing, minimum changes in phycocyanin pigment occur at a temperature of 59.7°C, and maximum degradation occurs at a temperature of more than 88.3°C. Degradation of the pigment is slow at a temperature of 26–43°C, but the rate of degradation is faster at a temperature of 47–64°C and the pigment is destroyed at a temperature of more than 64°C (Chaiklahan et al., 2012; Doke, 2005).

Salt stabilizes phycocyanin by covering the surface of phycocyanin and changing its structure (Chaiklahan et al., 2012; Hadiyanto et al., 2019; Martelli et al., 2014). Hence, the addition of NaCl at a concentration of more than 1% w/w prevents phycocyanin degradation by more than 50–70% (Chaiklahan et al., 2012). Use of preservatives decreases Dc but increases t_½ of phycocyanin (Figure 2).

One of the effective parameters in the degradation of phycocyanin pigment under different thermal processing conditions is pH. The results showed that the lowest degradation was at pH = 5.5, hence under these conditions, the lowest Dc and the highest t_½ of phycocyanin were observed (Figures 2B and 2D). Phycocyanin is much more stable at pH = 5 than 6 but shows more instability at pH = 7 (Antelo et al., 2008). Because of the protein-aceous nature of phycocyanin, pH depends on temperature and other environmental conditions (Chaiklahan et al., 2012; Priatni, 2015). By investigating the addition of preservatives to food formulations, degradation of the protein part of the pigment is delayed. Therefore, the Dc and t_½ of phycocyanin pigment decreases and increases, respectively. The use of different preservatives, such as NaCl, amino acids, and sorbitol, increases the stability of phycocyanin at different temperatures and pH conditions (Barbiroli et al. 2017; Hadiyanto et al., 2019).

Conclusions

The results of the current study demonstrated the stability of phycocyanin produced by A. platensis under the stress conditions of temperature, NaCl, and pH shocks. Kinetic model for the thermal degradation of aqueous phycocyanin produced by A. platensis was validated as the first-order model. In general, Dc increased and t_½ of the pigment decreased with increase in temperature. Instability of the pigment decreased with increase in pH from 4 to 5.5 at high temperatures. In addition, salt stress played a protective role regarding stability of the pigment. Modeling and predicting the degradation of phycocyanin in food products by the response surface method provided the possibility of calculating and designing the exact amount of this pigment to create a desired color in a food product. Data showed that the stability of this pigment was suitable during thermal processing, pasteurization, baking, and in food matrix at a near neutral pH of 6.6 and a low NaCl content of 0.40% w/w.