**4. Other applications of C-phycocyanin and phycocyanobilin**

As we have seen, C-PC and PCB have excellent antioxidant properties. Irradiation of these molecules with visible light produces reactive oxygen species, making them good candidates for application in photodynamic therapy (PTD). Indeed, it was shown that anticancer activities of C-PC against breast cancer MCF-7 cells increases upon exposure to He-Ne laser (632.8 nm wavelength) [90]. C-phycocyanin has specific affinity for tumor-associated macrophages (TAM), which have been proposed to be a "target for cancer therapy". Formation of non-covalent conjugate between Zn-phthalocyanine and C-PC resulted in an enhanced photodynamic effect with selective accumulation in the tumor site, probably through the specific binding of C-PC to TAMs [91].

In comparison to other fluorophores, phycobiliproteins have a high molar extinction coefficient and fluorescence quantum yield, as well as a large Stokes shift. Therefore, C-PC could be a good candidate for applications as fluorescent marker. When C-PC is extracted by low ionic strength buffers monomers will be the dominant form, inducing decrease of protein fluorescence. Therefore, in order to obtain stabilized highly fluorescent oligomers cross-linking of C-PC is needed. Chemically stabilized C-PC, fused to the biospecific domains such as streptavidin, is used as a biospecific fluorescent assay. Further, C-PC fluorescence can be also used for *in vivo* monitoring of cyanobacterial growth and detection of toxic cyanobacteria in drinking water [15]. Furthermore, strong quenching of C-PC fluorescence Hg2+ ions implies its potential application as biosensor for heavy metals in aquatic systems [92]. Interestingly, PCB synthesized in mammalian cells through metabolic engineering could be useful optogenetic tool for regulation of cell processes by light [19].

Artificial photosynthesis is currently a hot topic in science and technology. Consequently, there are growing demands for designing photoelectron-chemical (PEC) cells, capable to perform artificial photosynthesis. In PEC devices, light-harvesting proteins (such as C-PC) are used to "sensitize" metal and semiconductor surfaces. BioPEC solar hydrogen generator with a hematite-phycocyanin hybrid photo anode was designed. In order to obtain PEC cells with higher performances, the stability of immobilized C-PC needs to be improved [93].

Beside the application for food and drink coloring, C-PC is also used as a cosmetics colorant in lipsticks, eyeliners, and eye shadows preparations [94].

### **5. Conclusion**

In this moment, there are only few studies investigating the functional properties of *Spirulina* protein concentrate/isolate, with phycobilins being the main functional protein component. Proteins isolated from *Spirulina* are quite capable of reducing the interfacial tension at the aqueous-air interface at relatively lower bulk concentrations than common food proteins [79]. In comparison with soy protein isolates, *Spirulina* protein isolate (SPI) demonstrates lower water, but higher oil absorption capacity. SPI showed good emulsifying and foaming capacity, and ability to form protein films and gels [80]. This study demonstrated that emulsifying capacity, the emulsion aging stability, the emulsion microstructure and opacity as well as the foaming capacity and the foam stability were pH dependent. Also, emulsifying and foaming capacities have shown to be positively correlated to the protein solubility [80]. *Spirulina* protein isolate forms gels after heating (90°C) and cooling, showing fairly low minimum critical gelling concentrations (1.5 wt% in aqueous solution) compared to other food (soy) proteins [81]. *Spirulina* protein concentrate (SPC) have shown higher emulsifying and similar foaming capacity, when compared with soybean meal [82]. The rheological and textural parameters increased linearly with increased C-PC addition (0.25–1.25% w/w) in oil-in-water emulsions, suggesting C-PC emulsion stabilizing role [83]. From food technology point of view, these studies imply that C-PC or *Spirulina* proteins are promising food ingredients and additives, and that further studies are needed to fully exploit their most

In contrast to other natural food colors, but similar to other food-derived proteins, C-PC can be used to modify techno-functional properties of food matrices or as carrier of bioactive substances. As a biodegradable, biocompatible, and poor immunogenic protein molecule, C-PC is suitable as carrier for preparation of protein-based nanoparticles. Drug delivery *via* their loading into C-PC nanoparticles have shown to be more effective and safer [84, 85]. By analogy, C-PC-based nanoparticles can be used for food applications in the future as carrier for other active substances, acting together in synergistic manner and complementing mutual benefits. In order to fully utilize all benefits of valued bilin component, C-PC should be added

The natural food colorings are often associated with functional properties. C-Phycocyanin/ PCB with their extraordinary antioxidative activities could have role in maintaining of the lipid oxidative stability, especially in food products with high lipid contents. Addition of C-PC was found to inhibit linoleic acid peroxidation and decrease TBARS value in liposome-meat system [86]. Some studies demonstrated that C-PC exhibited antibacterial and antifungal potential [87, 88], suggesting that C-PC/PCB can also can serve as antimicrobial agent. Silver nanoparticles-based antimicrobial packaging is a promising form of active food packaging, and C-PC was used for synthesis of bio-silver nanoparticles [89]. Incorporation of *Spirulina* powder in strudels can significantly retard lipid oxidation and reduced the number of yeast and mold resulting in prolonged shelf life [68]. Therefore, C-PC/PCB in addition to their role as food colors can contribute to food preservation and improvement of food shelf life and/or to the reduce addition of

likely excellent functional properties.

140 Microalgal Biotechnology

only after all thermal pretreatments.

non-natural food preservatives.

The vast majority of studies regarding *Spirulina* bioactive components used whole algal (dried) mass or its aqueous extracts. Major *Spirulina* deep blue color protein C-phycocyanin and its bilin chromophore have remarkable potential for use in food technology, as safe food colorant, functional food additive, nutraceutical and/or dietary supplement, given their excellent healthrelated properties, and opportunity for sustainable and relatively inexpensive mass production.
