**2. Phycobiliproteins**

*Spirulina* is filamentous, helical, photosynthetic cyanobacteria naturally inhabiting alkaline brackish and saline waters in tropical and subtropical regions. Biochemical analysis has revealed its exceptional nutritive properties, so it is referred in the literature as "super food" or "food of the future" [2]. *Spirulina* is one of the richest natural sources of proteins and essential amino acids, as well as an excellent source of vitamins (primarily A, K, and vitamin B complex), macro- and micro-elements (calcium, potassium, magnesium, iron, iodine, selenium, chromium, zinc, and manganese), essential fatty acids, including γ-linoleic acid (GLA), glycolipids, lipopolysaccharides, and sulfolipids [3]. *Spirulina* is especially rich in a variety of pigments, such as chlorophylls, β-carotene, xanthophylls, and phycobilins (phycobiliproteins) (**Table 1**). A huge number of *in vitro* and *in vivo* studies, published in the last few decades, have revealed potentially beneficial effects of *Spirulina* on human health. Health benefits mainly arise from the antioxidant effect of algae as a whole, or from its individual ingredients, such as phycobiliproteins (Section 2). Moreover, the presence of significant amounts of GLA, sulfated polysaccharide (calcium spirulin), and sulfolipids additionally contribute to health-promoting

Several dried biomass products of *Spirulina* have categorized as "generally recognized as safe" (GRAS) by the Food and Drug Administration (FDA) of USA. A recommended dosage for adults is usually in the range of 3–10 g of *Spirulina* per day, while maximum daily intake should not exceed 30 g [3]. Extensive safety studies of *Spirulina* did not show the presence of cyanobacterial toxins [1]. *Spirulina* production requires the use of high quality nutrients and accurate determination of heavy metals in the culture medium, as well as in the biomass. Heavy metal analysis of commercial *Spirulina* products did not found to exceed the regulatory levels [2]. Nevertheless, it should be paid much attention during *Spirulina* cultivation to prevent contamination with heavy metals or the other cyanobacteria, capable to produce toxins.

> **% DV\*\***

Total carbohydrates <1 g <1 Chromium 50 μg 41 Proteins 2 g 4 Sodium 35 mg <2

Vitamin K 75 μg 94 C-phycocyanin 240 mg – Vitamin B12 9 μg 150 GLA 32 mg – Iron 7 mg 39 Chlorophyla a 30 mg – Magnesium 15 mg 4 Total carotenoids 15 mg –

11,250 IU 230 Potassium 60 mg 2

dismutase

**Substance Quantity/activity per** 

**serving (3 g\***

**)**

2500 U –

**% DV\*\***

activities of *Spirulina* [3].

130 Microalgal Biotechnology

**Substance Quantity/activity per** 

Recommended daily value of *Spirulina* powder.

\*\*Percent daily values (DV) are based on a 2000 calories diet.

Vitamin A (as β-carotene)

**\***

**serving (3 g\***

**)**

Manganese 0.4 mg 20 Superoxid

**Table 1.** Nutritional profile of commercial Spirulina powder (Nutrex, Hawaii, USA).

Phycobiliproteins are photosynthetic antenna pigments in the cyanobacteria, red and cryptophyte algae, that efficiently harvest light energy, which is subsequently transferred to chlorophylls during photosynthesis. Therefore, phycobiliproteins significantly contribute to the global photosynthesis. Phycobiliproteins are deeply colored, highly fluorescent, and watersoluble proteins with high propensity to form oligomers (hexamers) that constitute the building blocks of the extra-membranous antenna complex, phycobilisomes. Its intensive color arises from covalently attached linear tetrapyrrole chromophores (phycobilins) *via* thioether bonds to the cysteine residues [4].

Phycobilins are produced by heme metabolism. Heme is synthesized from protoheme IX by ferrochelatase. Then, heme oxygenase cleaves heme and biliverdin IXα is obtained. Biliverdin IXα is reduced by ferredoxin-dependent bilin reductases to obtain phycobilins. Final step in phycobiliproteins biosynthesis is the covalent attachment of bilin chromophores to the apoproteins, catalyzed by phycobiliprotein lyases. Slow spontaneous *in vitro* attachment of tetrapyrrole chromophores to the apoproteins has low fidelity and mixture of oxidation products is obtained [5].

*Spirulina* produces two phycobiliproteins: C-phycocyanin (C-PC) as the major pigment and allophycocyanin (APC), which is present in much smaller quantities, approximately at an 10:1 ratio [3]. C-phycocyanin level varies based on growing conditions, and may constitute up to 20% of the dry weight of *Spirulina* [6]. C-phycocyanin and APC are homologous proteins and both bind phycocyanobilin (PCB) chromophore [7, 8]. The presence of the third phycobiliprotein, red phycoerythrin, in *Arthrospira platensis* is the subject of debate. While some studies have found that *Spirulina* produces small amounts of phycoerythrin, the other ones did not detect phycoerythrin in *Spirulina* [9].

#### **2.1. Structure and physicochemical properties of C-phycocyanin and phycocyanobilin**

C-phycocyanin (CAS registry number 11016-15-2) is water-soluble, intensive blue protein with strong fluorescence. It is the heterodimer consisting of α- (~18 kDa) and β-subunits (~19 kDa), which form αβ monomers, further aggregating to trimmers (αβ)<sup>3</sup> and hexamers (αβ)<sup>6</sup> . Hexamer form represents a functional unit of phycobilisomes. C-phycocyanin is α-helicoidal protein, with one well-defined domain (similar to the globins) observed within the 3D structure of both chains. Color and intensive fluorescence of C-PC arises from PCB, covalently attached to Cys-84 of α-subunits, while β-subunit binds two PCB molecules *via* Cys-82 and Cys-153 residues [8]. Allophycocyanin has similar structure and physicochemical properties as C-PC. Unlike C-PC, β-subunit of APC binds only one PCB molecule (at Cys-84) [7–8]. Amino acid variation of phycocyanins between cyanobacteria and red algae species are very minor [10].

The VIS absorption spectrum of the native C-PC has pronounced specific peak at 620 nm, arising from bound PCB. Phycocyanobilin has a molecular weight of 586.7 g/mol and characteristic fluorescence spectrum with an emission peak at 640 nm. Spectra of free PCB differ from spectra of native protein, in sense of intensity and shape of absorption and emission bands [11]. Bilin chromophore is a very sensitive indicator of the conformational state of the protein, enabling monitoring of C-PC denaturation/renaturation by standard spectroscopic methods. Thermal denaturation of C-PC induces shift of absorption maximum from 620 to 600 nm with significant decrease in protein absorbance (color intensity) and fluorescence [12]. Changes of PCB conformation upon denaturation induce these phenomena: chromophore in native protein has stretched conformation, while denaturation changes PCB conformation to the cyclic, similar to the free chromophore [13].

lasting up to 16 hours [10]. Performing ethanolysis in the sealed vessel at 120°C decreases reaction time to 30 minutes, and obtained PCB has higher purity in comparison to conventional reflux method [17]. Phycocyanobilin can be produced in mammalian cells by metabolic engineering, introducing genes for heme oxygenase-1 and PCB:ferrodoxin oxidoreductase, with simultaneous knock-down of biliverdin reductase A to prevent PCB reduction to phycocyanorubin [19].

Natural food colorants are often sensitive to heat, light, oxygen, acidic conditions, and exposure to oxidants, such as ascorbic acid and trace metal ions. Generally speaking, natural C-PC is not a particularly stable protein. It was found to be unstable to heat and light in aqueous solution. The presence of photosensitive PCB makes C-PC sensitive to light and prone to freeradical oxidation [20]. The optimum pH range for C-PC was found to be 5.0–6.0 [21] and it is insoluble in acidic solution (pH 3) [22]. The critical temperature for C-PC stability is 47°C, with a sharp drop in the protein half-life values above this temperature. At 50°C, the C-PC solution showed maximum stability at pH 6.0, while at 60°C the maximum protein stability

and 7 caused ~80% of its degradation [22]. Therefore, although C-PC has high potential for applications in food industry, biotechnology, and medicine, stability issue is one of the limit-

There are an increasing number of studies dealing with development of methods to increase C-PC/PCB stability and expand their application to different food systems. Addition of 20% glucose, 20% sucrose, or 2.5% sodium chloride was considered suitable for prolonging the stability of the C-PC extract [23]. The natural protein cross-linker methylglyoxal does not significantly stabilize C-PC, whereas addition of honey or high concentration of sugars greatly diminishes thermal degradation of protein. After sterilization (80 and 100°C) of fructose syrups with mixture of C-PC and yellow pigment of *Carthamus tinctorius*, the syrups remain clear, with only partial blue color degradation even after 2 months of storage [6]. The rate of C-PC thermal degradation was decreased in the presence of benzoic acid, followed by citric acid and sucrose, while calcium chloride and ascorbic acid supported the least protein stability in comparison to the other food preservatives studied [24]. After solubilization into reverse micelles, C-PC embedded into the structured interfacial water layer was protected from the bleaching processes, reflecting in stable protein spectral parameters as long as the microemulsion was stable [25]. Incorporation of C-PC into polyethylene oxide nanofibers, or addition of sorbitol (50%) and glucose (20%), increased protein thermostability, considering its almost twice extended half-life [26]. C-phycocyanin incorporated into polysaccharide beads such as alginate/chitosan microcapsules and alginate microspheres, showed greater antioxidant activity and thermal stability. These beads are resistant in simulated gastric fluid, while rapidly release C-PC in simulated intestinal fluid [27]. The addition of anionic and ferulated beet pectin enhanced the color stability of the C-PC extract upon heating (65°C) and slowed down its degradation and color lost by proteases, such as Alcalase 2.4 L, papain, and bromelain [28]. C-phycocyanin stabilized by cross-linking of its subunits with formaldehyde exhibited

lux for 24 hours in aqueous solution at pH 5

*Spirulina* Phycobiliproteins as Food Components and Complements

http://dx.doi.org/10.5772/intechopen.73791

133

**3. Food applications of C-phycocyanin and phycocyanobilin**

**3.1. Stability and technologies to improve stability**

was at pH 5.5 [23]. Exposure to light of 3 × 105

ing factors for its successful application.

#### *2.1.1. Production, isolation, and purification of C-phycocyanin and phycocyanobilin*

Thanks to the high protein (C-PC) content, as well as large availability, *Arthrospira platensis* is culture of choice for C-PC production. *Spirulina* growth requires dry, hot, and sunny climatic conditions [14]. Photoautotrophic *Spirulina* production is outdoor method, used for commercial production of C-PC at tropical and subtropicals regions, in open ponds and raceways. In the mixotrophic production, *Spirulina* cultivation is performed in an enclosed reactor with the addition of glucose, yielding a higher amount of C-PC. *Spirulina* can grow even heterotrophically, but in this case small yield of pigments is obtained [10]. Presence of covalently attached chromophore makes recombinant production of C-PC more complicated in comparison to other proteins. Complete synthesis of C-PC depends not only on co-expression of α- and β-chains, but also on parallel synthesis of PCB and its covalent attachment to protein [15].

Crucial parameters for C-PC production are lighting conditions (light spectrum, quality, intensity, and cycle), climatic conditions (pH and temperature), and media type. Their optimization strategies are reviewed in [16], with higher productivity in closed bioreactor systems than open ponds. Utilization of agricultural waste to replace the synthetic chemicals in algae cultivation media could also have enviro-economical impact.

Isolation of C-PC in high yield requires efficient extraction process. There are several effective approaches used for C-PC extraction: freezing and thawing, homogenization with mortar and pestle, sonication, high pressure homogenization, osmotic shock (using distilled water), acid treatment, enzymatic treatment (by lysozyme), organic solvent extraction, etc. [17]. Potential applications of C-PC in medicine or for research purposes (as fluorescent tag) require its high purity. The purity of C-PC is evaluated using ratio between absorbance at 620 and 280 nm (A620/A280). C-PC preparations with A620/A280 greater than 0.7 is considered as food grade, while preparations with A620/A280 more than 3.9 and 4 have reactive and analytical grade of purity, respectively [14]. C-phycocyanin price strongly depends on its purity, ranging from \$200 to \$2.2 million per kilogram. Numerous different procedures for C-PC purification (usually after protein precipitation with ammonium sulfate) use one or more chromatographic steps (ion-exchange chromatography, hydrophobic chromatography, gel filtration, hydroxyapatite chromatography, and expanded bed adsorption chromatography) or two-phase aqueous extraction [10]. Changing light conditions during cultivation of *Spirulina* (blue and red light *vs*. normal) could increase yield and purity of C-PC [18].

Phycocyanobilin (CAS 20298-86-6) isolation requires cleavage of thioether bond between apoprotein and bilin chromophore, by acid hydrolysis, enzymatic cleavage, or alcohol reflux. The most common procedure for the cleavage of PCB from C-PC is still conventional reflux in methanol, lasting up to 16 hours [10]. Performing ethanolysis in the sealed vessel at 120°C decreases reaction time to 30 minutes, and obtained PCB has higher purity in comparison to conventional reflux method [17]. Phycocyanobilin can be produced in mammalian cells by metabolic engineering, introducing genes for heme oxygenase-1 and PCB:ferrodoxin oxidoreductase, with simultaneous knock-down of biliverdin reductase A to prevent PCB reduction to phycocyanorubin [19].
