Phycocyanin from Arthrospira platensis: Recent advances in production, extraction, purification, stability enhancement, and industrial applications

Introduction

The continuously increasing global population poses a significant challenge to food security by intensifying the demand for safe, nutritious, and sustainable food resources. According to the United Nations, the global population is projected to reach approximately 9.8 billion by 2050, thereby exerting substantial pressure on existing agricultural systems and food production capacity (United Nations, 2017). Conventional agricultural practices alone may not be sufficient to satisfy future nutritional requirements; therefore, the exploration of alternative and sustainable biological resources has become an important research priority. Among the various alternative food resources, microalgae have emerged as one of the most promising candidates due to their rapid growth rate, high photosynthetic efficiency, minimal land requirement, and exceptional nutritional composition. Microalgae are rich sources of high-quality proteins, essential amino acids, polyunsaturated fatty acids, vitamins, minerals, carbohydrates, pigments, and numerous bioactive compounds. These valuable metabolites have attracted considerable attention for their potential applications in the food, nutraceutical, pharmaceutical, cosmetic, and biotechnology sectors (Lafarga, 2020; Lim et al., 2021) [1,2] . In addition to their nutritional benefits, microalgae contribute significantly to environmental sustainability. They efficiently convert solar energy and atmospheric carbon dioxide into valuable biomass through photosynthesis and can be cultivated using non-arable land, saline water, or wastewater, thereby reducing competition with conventional agricultural production. Their ability to support carbon sequestration and facilitate resource recycling further strengthens their role in the development of a sustainable circular bioeconomy (Lim et al., 2021; AlFadhly et al., 2022) [2,3] . Consequently, the commercial demand for algal biomass and algae-derived products has increased substantially, with the global algae products market expected to exhibit steady growth in the coming years. Among commercially cultivated microalgae, Arthrospira platensis (commonly known as Spirulina) is one of the most extensively investigated cyanobacteria because of its outstanding nutritional profile, ease of cultivation, rapid biomass productivity, and economic feasibility. Spirulina thrives under highly alkaline conditions, which naturally reduce microbial contamination and facilitate large-scale cultivation. Its dry biomass typically contains 55-70% protein, along with essential fatty acids, vitamins, minerals, polysaccharides, and several 203 biologically active pigments, making it an important functional food and dietary supplement (Paniagua-Michel, 2015; Sorrenti et al., 2021) [4,5] . Among the various bioactive compounds present in Spirulina, C-phycocyanin has attracted considerable scientific and industrial interest. C-phycocyanin is a water-soluble blue phycobiliprotein that functions as an accessory photosynthetic pigment by absorbing light energy and transferring it to chlorophyll during photosynthesis. Owing to its intense natural blue colour, non-toxic nature, excellent biocompatibility, and biodegradability, phycocyanin has emerged as a promising natural alternative to synthetic food colourants and is increasingly utilized in the food, pharmaceutical, cosmetic, and biomedical industries (de Morais et al., 2018) [6] . Beyond its colouring properties, C-phycocyanin exhibits a broad spectrum of biological activities, including antioxidant, anti-inflammatory, antimicrobial, immunomodulatory, hepatoprotective, neuroprotective, antidiabetic, anticancer, and wound-healing effects. These multifunctional biological properties have significantly expanded its applications as a functional ingredient, nutraceutical, and potential therapeutic agent for the prevention and management of various chronic diseases (Yuan et al., 2022) [7] . Despite its enormous commercial potential, the large-scale utilization of phycocyanin remains challenging due to its intracellular localization and its susceptibility to degradation during processing and storage. Efficient recovery of phycocyanin requires effective cell disruption and extraction techniques, while its stability is strongly influenced by environmental factors such as temperature, pH, light intensity, oxygen exposure, solvent composition, and storage conditions. These factors can adversely affect pigment yield, purity, colour stability, and biological activity, thereby limiting its industrial applications. Consequently, considerable research has focused on optimizing cultivation practices, extraction methods, purification techniques, stabilization strategies, and downstream processing to improve the recovery efficiency and functional stability of phycocyanin (Jaeschke et al., 2021; de Morais et al., 2018) [8,6] . In recent years, increasing consumer preference for natural colourants and clean-label food products has accelerated research on phycocyanin as a sustainable natural blue pigment. Advances in extraction technologies, purification processes, encapsulation methods, and stabilization approaches have further enhanced its commercial prospects across multiple industries. Nevertheless, additional research is required to develop cost-effective, scalable, and environmentally sustainable production technologies that can facilitate its wider industrial application. Therefore, this review comprehensively discusses the current advances in the production, extraction, purification, stabilization, and industrial applications of C-phycocyanin obtained from Arthrospira platensis. Furthermore, the review summarizes the critical factors influencing extraction efficiency and pigment stability, highlights recent technological developments, and outlines future research directions aimed at improving the commercial exploitation of this valuable natural blue pigment.

Structure of Phycocyanin

C-phycocyanin is a water-soluble blue phycobiliprotein predominantly present in cyanobacteria, particularly Arthrospira platensis (Spirulina), and certain species of red algae. It is an integral component of the phycobilisome, a supramolecular light-harvesting complex attached to the thylakoid membrane, where it functions as an accessory photosynthetic pigment. The primary role of C-phycocyanin is to absorb light energy within the orange-red region of the visible spectrum (approximately 615-620 nm) and efficiently transfer the absorbed energy to chlorophyll a, thereby enhancing photosynthetic efficiency under low-light conditions. This unique light-harvesting capability contributes significantly to the high photosynthetic productivity of cyanobacteria. (Glazer, 1994; Eriksen, 2008) [9,10] Structurally, C-phycocyanin is composed of two homologous protein subunits, designated as α (alpha) and β (beta), which assemble to form an αβ heterodimer. The α-subunit possesses a molecular weight of approximately 17-18 kDa, whereas the βsubunit ranges between 18 and 20 kDa. Three heterodimers further associate through non-covalent interactions to form a trimer ((aβ)₃) , and two trimers subsequently assemble into a hexameric complex ((αβ)₆) , representing the biologically active form of the pigment. The quaternary structure provides structural stability and facilitates efficient transfer of excitation energy during photosynthesis. (Glazer, 1994) [9] The characteristic blue colour of C-phycocyanin originates from phycocyanobilin, a linear tetrapyrrole chromophore that is covalently attached to conserved cysteine residues of the protein through thioether linkages. Typically, one chromophore is attached to the α-subunit and two chromophores are attached to the β-subunit, enabling efficient absorption and fluorescence emission. The pigment exhibits a maximum absorption wavelength of 620 nm and fluorescence emission at approximately 640-650 nm. Owing to its high fluorescence quantum yield, excellent photophysical properties, and low toxicity, C-phycocyanin is extensively employed as a fluorescent probe in biomedical diagnostics, immunoassays, flow cytometry, and fluorescence microscopy. (Sekar & Chandramohan, 2008) [11] Extraction Techniques of Phycocyanin Efficient extraction of phycocyanin is a critical step in maximizing its yield, purity, and functional properties. Because phycocyanin is an intracellular, water-soluble phycobiliprotein located within the thylakoid membranes of Arthrospira platensis (Spirulina), effective disruption of the rigid cell wall is essential for its release. The efficiency of extraction depends on several factors, including the cell disruption method, biomass condition (fresh or dried), extraction solvent, pH, temperature, biomass-to-solvent ratio, and processing time. Various physical, mechanical, chemical, and emerging nonthermal techniques have been developed to improve phycocyanin recovery while preserving its structural integrity and biological activity (Jaeschke et al., 2021; de Morais et al., 2018) [8,6] .

Freeze-Thaw Extraction

The freeze-thaw technique is one of the most widely employed laboratory-scale methods for extracting phycocyanin due to its simplicity and minimal equipment requirements. This method involves repeated cycles of freezing and thawing, during which intracellular ice crystal formation mechanically disrupts the cell membrane, facilitating the release of phycocyanin into the extraction medium. Sodium phosphate buffer (pH 6.8-7.0) is commonly used as the extraction solvent because it enhances pigment stability and improves extraction efficiency. Several studies have reported that three to four freeze-thaw cycles provide the optimum balance between extraction yield and purity, whereas excessive cycles may increase the release of contaminating proteins and reduce pigment quality. Although the method provides high-quality extracts, it is time-consuming, energy-intensive, and therefore more suitable for laboratoryscale applications than industrial production (Silveira et al., 2007; Tavanandi et al., 2018; Chentir et al., 2018) [12,13,14] .

Homogenization

Homogenization is a mechanical cell disruption technique that employs high shear forces to rupture the cell wall and release intracellular phycocyanin. Compared with simple mixing, homogenization provides more effective disruption due to its higher mechanical intensity, resulting in improved extraction efficiency. The method is relatively rapid, inexpensive, and easily adaptable to laboratory and pilot-scale operations. However, prolonged homogenization generates heat, which can accelerate phycocyanin denaturation and decrease pigment purity. In addition, mechanical disruption often releases significant quantities of cellular debris, necessitating subsequent clarification and purification steps. Therefore, careful optimization of homogenization speed, extraction time, and temperature is essential to maximize pigment recovery while minimizing degradation (Ilter et [15,16,12] .

Bead Milling

Bead milling is an efficient mechanical extraction technique in which microalgal cells are disrupted through collisions with rapidly moving beads within a milling chamber. The repeated impact between the beads and biomass effectively breaks the cell wall, allowing the release of intracellular phycocyanin. Extraction efficiency is influenced by bead material, bead size, filling ratio, agitation speed, and processing duration. Glass beads with high filling densities (approximately 80-85%) have been reported to provide efficient cell disruption while maintaining relatively low energy consumption. Bead milling is capable of processing biomass at high concentrations and is considered suitable for large-scale industrial applications. Nevertheless, the crude extract generally contains considerable cellular debris, resulting in relatively low purity and requiring additional purification procedures to obtain food-or pharmaceutical-grade phycocyanin (Günerken et [17,18,19] .

Ultrasonication

Ultrasonication is an emerging non-thermal extraction technology that utilizes high-frequency ultrasonic waves to generate cavitation bubbles within the extraction medium. The collapse of these bubbles produces localized shear forces and microjets that disrupt the microalgal cell wall, thereby enhancing the diffusion of phycocyanin into the solvent. [20,21,22,13] .

Factors Affecting Extraction and Stability of Phycocyanin

The extraction efficiency and stability of phycocyanin are strongly influenced by several physicochemical and processing parameters. Since phycocyanin is a water-soluble and thermolabile phycobiliprotein, its structural integrity can be readily affected by extraction conditions. Optimizing factors such as biomass form, temperature, light exposure, pH, extraction solvent, biomass-tosolvent ratio, and the use of stabilizing agents is essential for maximizing pigment yield, purity, and functional properties. Proper control of these parameters minimizes protein denaturation and pigment degradation while improving the commercial feasibility of phycocyanin production (Jaeschke et al., 2021; Yuan et al., 2022) [8,7] .

Biomass Form

The physical state of Arthrospira platensis biomass significantly influences the extraction efficiency of phycocyanin. Both fresh and dried biomass have been employed for pigment recovery; however, dried biomass is generally preferred because of its longer storage stability and ease of handling. Prior rehydration of dried biomass before extraction has been shown to improve cell permeability, thereby enhancing phycocyanin release. Nevertheless, conventional drying at temperatures above 60 °C can cause irreversible protein denaturation and substantial pigment degradation. Therefore, mild drying techniques such as freeze-drying or vacuum drying are recommended to preserve pigment quality and maximize extraction efficiency (Jaeschke et al., 2019; Tavanandi & Raghavarao, 2020) [23,20] . [24,25,26] .

Temperature

Light

Light intensity and spectral quality play a significant role in both phycocyanin biosynthesis during cultivation and pigment stability after extraction. Red light has been reported to enhance photosynthetic activity and stimulate higher phycocyanin production in Arthrospira platensis compared with blue light. Conversely, prolonged exposure of extracted phycocyanin to visible light, particularly at high intensities, accelerates photodegradation by promoting oxidative damage to the chromophore-protein complex. Therefore, phycocyanin extracts should be processed and stored under dark or low-light conditions to minimize pigment degradation and preserve antioxidant properties ( [27,21,28,29] .

pH

The stability of phycocyanin is highly dependent on the pH of the extraction medium because pH influences protein conformation and chromophore integrity. Acidic conditions, particularly within the pH range of 3.0-4.0, promote protein precipitation and accelerate pigment degradation, resulting in substantial losses in color intensity and functional activity. In contrast, phycocyanin exhibits maximum stability under slightly acidic to neutral conditions, with an optimum pH range of approximately 5.5-7.0. Maintaining the extraction buffer within this range is essential for preserving the native protein structure and minimizing denaturation during processing and storage (Chaiklahan et [30,31,24] .

Extraction Solvent

The choice of extraction solvent has a direct influence on the solubility, recovery, and stability of phycocyanin. Buffer systems provide better control of pH and ionic strength than distilled water, thereby improving protein stability throughout extraction. Sodium phosphate buffer (pH [8,16,32,23] .

Biomass-to-Solvent Ratio

The biomass-to-solvent ratio is an important processing parameter that determines extraction yield, pigment concentration, and process economics. Increasing the solvent volume generally enhances mass transfer and improves phycocyanin recovery; however, excessive solvent dilution may simultaneously increase the extraction of contaminating proteins, thereby reducing pigment purity. Several studies have reported that biomass-to-solvent ratios ranging from 1:6 to 1:10 provide an appropriate balance between extraction efficiency and purity, although the optimal ratio varies according to biomass characteristics and extraction technique. Consequently, optimization of the biomass-to-solvent ratio is essential for achieving maximum pigment recovery while minimizing processing costs and downstream purification requirements (Tavanandi & Raghavarao, 2020; Jaeschke et al., 2021; Pan-Utai & Iamtham, 2019) [20,8,16,23] .

Purification Techniques of Phycocyanin

Purification is an essential downstream processing step following the extraction of phycocyanin, as the crude extract contains various impurities, including contaminating proteins, carbohydrates, lipids, nucleic acids, and cell debris. The primary objective of purification is to enhance pigment purity while preserving its structural integrity, biological activity, and characteristic blue color. The required purity level depends on the intended application. Food-grade phycocyanin generally requires a purity ratio (A 620 /A 280 ) of ≥0.7 , reagent-grade preparations require a purity of ≥3.9, and analytical-grade phycocyanin typically exceeds a purity ratio of 4.0. The selection of an appropriate purification strategy is therefore crucial to obtain high-quality phycocyanin suitable for food, pharmaceutical, cosmetic, or biomedical applications (de Morais et al., 2018; Ashaolu et al., 2021; Jaeschke et al., 2021) [6, 33, 8] .

Centrifugation

Centrifugation is the initial clarification step employed after extraction to separate insoluble cellular debris from the phycocyanin-containing supernatant.

High-speed centrifugation effectively removes disrupted cell fragments and suspended particles, thereby reducing turbidity and facilitating subsequent purification processes. Although centrifugation does not significantly increase phycocyanin purity, it is an indispensable pretreatment that improves the efficiency of downstream purification techniques by minimizing particulate contaminants (de Morais et al., 2018; Jaeschke et al., 2021) [6,8] .

Ammonium Sulphate Precipitation

Ammonium sulphate precipitation is one of the most widely adopted methods for the partial purification and concentration of phycocyanin. This technique utilizes the principle of selective protein precipitation based on differences in protein solubility under increasing salt concentrations. Typically, sequential precipitation using 20-30% ammonium sulphate removes undesirable proteins, whereas phycocyanin is selectively precipitated at approximately 50-65% saturation. The recovered precipitate is subsequently dissolved in phosphate buffer for further purification. This method is simple, economical, and suitable for laboratory and pilot-scale production; however, complete removal of residual salt requires subsequent dialysis (Patil & Raghavarao, 2007; Pan-Utai & Iamtham, 2019) [31,16] .

Dialysis

Dialysis is commonly performed after salt precipitation to eliminate ammonium sulphate, lowmolecular-weight impurities, and other dissolved contaminants from the phycocyanin solution. In this technique, the protein solution is enclosed within a semi-permeable membrane that allows diffusion of salts and other small molecules into an external buffer while retaining the larger phycocyanin molecules. Dialysis improves protein purity and restores suitable buffer conditions for subsequent purification or analytical procedures. Although highly effective, the process is relatively slow and generally requires several hours to days at low temperatures to prevent pigment degradation (de Morais et al., 2018; Pan-Utai & Iamtham, 2019) [6,16] .

Membrane Filtration

Membrane-based separation techniques, including ultrafiltration and microfiltration, are increasingly employed for phycocyanin purification due to their operational simplicity and scalability. These pressure-driven processes separate biomolecules according to molecular size, allowing concentration of phycocyanin while simultaneously removing low-molecular-weight impurities and residual cellular components. Membrane filtration offers several advantages, including reduced processing time, minimal thermal exposure, lower chemical consumption, and improved retention of biological activity. Consequently, it is considered a promising alternative for industrial-scale purification of phycocyanin (Ashaolu et al., 2021; Jaeschke et al., 2021) [33,8] .

Chromatographic Purification

Chromatographic techniques represent the most effective purification methods for obtaining highpurity phycocyanin suitable for pharmaceutical, analytical, and diagnostic applications. Ionexchange chromatography is the most commonly employed approach because phycocyanin possesses charged amino acid residues that facilitate selective binding to ion-exchange resins. Gel filtration chromatography further separates proteins based on molecular size, whereas hydrophobic interaction chromatography exploits differences in protein surface hydrophobicity. These chromatographic methods significantly enhance pigment purity while preserving its native conformation and fluorescence properties. However, their relatively high operational cost and longer processing time often limit their application to high-value products (Patil & Raghavarao, 2007; Ashaolu et al., 2021; de Morais et al., 2018) [31, 33, 6] .

Applications of Phycocyanin

Phycocyanin is a naturally occurring blue phycobiliprotein predominantly isolated from Arthrospira platensis (Spirulina). Owing to its intense blue color, excellent water solubility, high nutritional value, antioxidant potential, and non-toxic nature, phycocyanin has gained considerable commercial and scientific attention. Beyond its application as a natural colorant, phycocyanin exhibits multiple biological activities, including antioxidant, anti-inflammatory, immunomodulatory, anticancer, hepatoprotective, and neuroprotective effects. These multifunctional properties have led to its widespread utilization in the food, nutraceutical, pharmaceutical, cosmetic, and biomedical sectors. The increasing consumer preference for natural ingredients and clean-label products has further accelerated the demand for phycocyanin in various industrial applications ( [6,33,7] .

Food Industry

The food industry represents the largest commercial application of phycocyanin due to its vibrant blue color and excellent safety profile. It is widely used as a natural food colorant to replace synthetic dyes in beverages, dairy products, confectionery, bakery products, ice cream, yogurt, candies, chewing gum, desserts, and functional foods. Unlike synthetic colorants, phycocyanin is biodegradable, non-toxic, and environmentally sustainable, making it highly attractive for cleanlabel food formulations. In addition to its coloring properties, phycocyanin enhances the nutritional value of foods by contributing antioxidant activity, thereby improving product functionality. However, its application is limited in thermally processed foods because of its sensitivity to high temperature, acidic pH, and prolonged light exposure. Consequently, stabilization techniques such as encapsulation, controlled pH, and lowtemperature processing are commonly employed to improve its industrial applicability (de Morais et al., 2018; Eriksen, 2008; Ashaolu et al., 2021) [6,10,33] .

Nutraceuticals

Phycocyanin has emerged as an important nutraceutical ingredient owing to its diverse healthpromoting properties. Numerous experimental and clinical studies have demonstrated that phycocyanin exhibits potent antioxidant, antiinflammatory, immunomodulatory, and free radicalscavenging activities, thereby reducing oxidative stress and protecting cellular components against damage. Regular consumption of phycocyanin-containing dietary supplements has been associated with improved immune function, enhanced liver protection, cardiovascular health, and metabolic regulation. Furthermore, its ability to inhibit lipid peroxidation and reduce inflammatory mediators makes it a promising functional ingredient for preventing chronic diseases associated with oxidative stress. Consequently, phycocyanin is increasingly incorporated into health supplements, functional beverages, protein powders, and fortified foods intended to promote overall well-being (Belay et al., 1993; Ashaolu et al., 2021; Yuan et al., 2022) [34,33,7] .

Pharmaceuticals

The pharmaceutical potential of phycocyanin has attracted significant research interest because of its broad spectrum of biological activities. Scientific investigations have demonstrated that phycocyanin possesses antioxidant, antiinflammatory, antimicrobial, hepatoprotective, neuroprotective, and anticancer properties. It has been reported to inhibit the proliferation of various cancer cell lines by inducing apoptosis and regulating cell-cycle progression. Additionally, phycocyanin suppresses the production of inflammatory cytokines and reactive oxygen species, thereby reducing tissue damage associated with inflammatory disorders. Preclinical studies have also indicated its therapeutic potential in liver diseases, diabetes, cardiovascular disorders, neurodegenerative diseases, and acute lung injury. Although these findings are promising, further clinical trials are necessary to establish standardized dosage, efficacy, and long-term safety for pharmaceutical applications (Cherng et al., 2007 [35,36,37,6] .

Cosmetics

The cosmetic industry has increasingly adopted phycocyanin as a natural bioactive ingredient because of its antioxidant, antiaging, anti-inflammatory, and skin-protective properties. Phycocyanin effectively scavenges reactive oxygen species generated by ultraviolet radiation and environmental pollutants, thereby minimizing oxidative damage to skin cells. These properties contribute to improved skin hydration, enhanced collagen protection, delayed wrinkle formation, and reduced pigmentation. Moreover, its intense natural blue color enables its use as a coloring agent in cosmetic formulations, including facial masks, creams, lotions, shampoos, soaps, and personal care products. As consumer demand for plant-based and naturally derived cosmetic ingredients continues to increase, phycocyanin has become an attractive alternative to synthetic pigments and antioxidants (de Morais et al., 2018; Ashaolu et al., 2021) [6,33] .

Conclusion

Arthrospira platensis is a promising sustainable source of nutrients and the natural blue pigment Cphycocyanin with broad industrial applications. Advances in cultivation, extraction, and purification technologies have improved its commercial potential; however, challenges related to pigment stability and cost-effective large-scale production remain. Future research should focus on sustainable processing, enhanced stabilization techniques, and industrial-scale applications to maximize the utilization of phycocyanin in clean-label food products and health-related industries.

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