Nutritional profile of commercial
Spirulina has a documented history of use as a food for more than 1000 years, and has been in production as a dietary supplement for 40 years. Among many of Spirulina bioactive components, blue protein C-phycocyanin and its linear tetrapyrrole chromophore phycocyanobilin occupy a special place due to broad possibilities for application in various areas of food technology. The subject of this chapter is up-to-date food applications of these Spirulina components, with a focus on their use as food colorants, additives, nutriceuticals, and dietary supplements. Their other actual and future food application possibilities will also be briefly presented and discussed.
- food components
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Spirulinaas a superfood
The blue-green microalgae
|Substance||Quantity/activity per serving (3 g*)||% DV**||Substance||Quantity/activity per serving (3 g*)||% DV**|
|Total carbohydrates||<1 g||<1||Chromium||50 μg||41|
|Proteins||2 g||4||Sodium||35 mg||<2|
|Vitamin A (as β-carotene)||11,250 IU||230||Potassium||60 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||–|
|Manganese||0.4 mg||20||Superoxid dismutase||2500 U||–|
A huge number of
Several dried biomass products of
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 water-soluble 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)
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
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)3 and hexamers (αβ)6. 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
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 . 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 . 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 .
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,
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 , 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. . 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 . 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 . Changing light conditions during cultivation of
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 . 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 . 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 .
3. Food applications of C-phycocyanin and phycocyanobilin
3.1. Stability and technologies to improve stability
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 free-radical oxidation . The optimum pH range for C-PC was found to be 5.0–6.0  and it is insoluble in acidic solution (pH 3) . 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 was at pH 5.5 . Exposure to light of 3 × 105 lux for 24 hours in aqueous solution at pH 5 and 7 caused ~80% of its degradation . Therefore, although C-PC has high potential for applications in food industry, biotechnology, and medicine, stability issue is one of the limiting factors for its successful application.
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 . 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
3.2. Safety and bioavailability
Numerous toxicological studies, such as acute, sub-chronic, chronic, mutagenic, teratogenic/developmental toxicity, carcinogenic, and multiple generational/reproduction tests, have confirmed excellent safety profile of
Desert Lake Technologies, LLC got GRAS notification in 2012 for its CyaninPlus™ product, consistent with section 201(s) of the Federal Food, Drug and Cosmetic Act. It is a water extract of the
In animal models, C-PC possesses low toxicity and lack of adverse effects. For example, in acute oral toxicity study, the measured LD50 values were estimated to be greater than 3 g/kg for rats and mice, without mortality even at the highest dose of C-PC from
Bioavailability is the term used to describe how much of the nutrient are easily absorbed into the body and so is able to have an active effect.
3.3. Interactions with food matrix components
In addition to their sensitivity to light, heat, and oxidants, natural food colors are prone to interact with other food ingredients, especially if they are carrying proteinous component, such as C-PC. Several studies have found that both C-PC and PCB binds to food matrix components, such as proteins, lectins, saccharides, lipids, and polyphenols [40, 41, 42, 43, 44, 45, 46, 47].
C-phycocyanin non-covalently binds to bovine serum albumin (BSA), with binding constant 6.8 × 105 M−1 and n = 1.2, as determined by fluorescence quenching of BSA. FT-IR, and synchronous fluorescence spectroscopy confirmed the conformation of BSA has been affected the interaction with C-PC . In the recent study, we found that PCB also interact with BSA, showing high affinity binding (
C-phycocyanin also interacts with food-derived lectins. Jacalin, tumor-specific lectin from cempedak, binds C-PC specifically in a carbohydrate-independent manner, and with affinities better than that for porphyrins. The binding pattern involves both ionic and hydrophobic interactions and more than one contact site . Concanavalin A and peanut agglutinin can also interact with C-PC, although the nature of the interaction is distinctly different from that for jacalin. The legume lectins bind C-PC
Well-known cryoprotecting disaccharide trehalose interacts with C-PC and decreases the internal protein dynamics, slowing down molecular motions responsible for its unfolding and denaturation . Although it was found that C-PC interacts with lipids at the air-water interface, the oxidation of monogalactosyldiacylglycerol could not be prevented by the introduction of C-PC molecules at the lipid-water interface . Formation of complex between C-PC fragments and polyphenols, in order to obtain more stable blue color for application in food, feed, cosmetic, and pharmaceutical products, was recently patented .
3.4. Health-promoting effects
A good part of the bioactivity properties of
Phycocyanobilin is potent inhibitor of certain NADPH oxidase isoforms, likely because in mammalian cells it is rapidly reduced to phycocyanorubin, a close homolog of bilirubin. Over-activity of NADPH oxidase causes oxidative stress, and is known to mediate and/or exacerbate numerous pathological conditions .
The suitable clinical dose of PCB remains to be defined. Without mass-produced pigment derived from commercially available PCB-enriched
3.4.1. Antioxidant properties
Proteins bearing colored prosthetic groups, such as a highly conjugated linear tetrapyrrole chromophore in C-PC, can be both the source and target of reactive species in biological systems. An extremely high antioxidant capacity of C-PC was unambiguously established, based on experiments carried out both in
C-phycocyanin is a more efficient peroxynitrite scavenger than free PCB due to (additional) interactions with tyrosine and tryptophan residues of the apoprotein . Differences in the amino acid composition affect C-PC antioxidant capacity. Selenium-C-phycocyanin purified from Se-enriched
C-phycocyanin generates hydroxyl radicals in the light, while scavenging them in the dark. Radical generation ability disappears, but scavenging greatly increases in denaturated protein, confirming the role of phycobilin moiety in scavenging. Trypsin hydrolysis of C-PC demonstrated the apoprotein portion also made a significant contribution to the antioxidant activity . The heat denatured (spray-dried) C-PC shows the same level of activity as the intact protein, finding important for preparation and utilization of C-PC . C-phycocyanin can be cloned and expressed in
3.5. Food colorants
In the last decades, consumers are becoming more educated and aware of what they eat, demanding for clean labeling of the food/beverage products and making the pressure to food industry to switch from artificial to natural ingredients and additives. The main consumers of vividly colored food products are children. Due to their low weight, they are at constant risk to exceed recommended daily intake (mg/kg weight) of artificial colorants. Nowadays, the leading confectioners switched to natural colors to avoid obligatory label warning for acceptable daily intake levels of the colorings. Consequently, market of natural food colors is in prominent expansion, expecting to reach $2.5 billion by 2025.
Compared with other natural pigments, natural blue pigments are rare, because a complex combination of molecular features (such as π-bond conjugation, aromatic ring systems, heteroatoms, and ionic charges) is required to absorb red light (~600 nm region) . Anthocyanins are the primary source of blue color in plants, but their color is pH dependent. On the other hand, fungi and microorganisms produce many blue compounds in response to stress or predators and therefore their unpredictable biological activities make their safety for food use questionable. None of discovered natural blue pigments cannot reach shade, brilliance, vividness, molar absorptivity, and stability of Brilliant Blue FCF (Blue 1 or E133), the most used of approved synthetic blue food colorants, and concomitantly to be safe and cost-effective . In this moment, the only permitted natural blue food colorants are gardenia blue (in Japan), blue anthocyanins and
Demand for C-PC as a natural blue food colorant has experienced exponential growth in the past 5 years, especially after FDA approval of
Commercial powder formulations of C-PC, such as Linablue® (DIC Corporation, Japan), are declared as completely soluble in cold and warm water and <20% ethanol, making a homogeneous transparent solution, with stable color shade in the pH range 4.5–8.0 (except at C-PC pI value around pH 4.2), which can be improved by the presence of protein-containing ingredients; low thermal stability, which can be improved in high density sucrose solutions; with low light stability, which can be improved in the presence of antioxidant like ascorbate; and with no tongue dyeing effect. In combination of C-PC with red, yellow, and other natural colorants, it is possible to obtain vibrant green, purple, and other natural colors. As FDA is still considering the petition for copper chlorophyllin, natural green food color often involves C-PC or
Due to its refreshing ice cool color, C-PC is also increasingly promoted as natural color for alcoholic beverages, such as FIRKIN Blue gin.
In comparison to artificial colors, natural colorings are less vivid, and interactions with food matrix components can result in further decrease in their vibrancy, or unwanted change in color and flavor. For example, our research group observed an instant clear color change from blue to green when PCB interacts with BSA . Therefore, switching from artificial to natural colorings in existing food products can be challenging and complex.
3.6. Functional food additives, nutraceuticals, and dietary supplements
In last decade, there is an increase in chronic diseases and increasing costs of health care due to busy lifestyles and unhealthy nutrition. On the other hand, people are more health conscious and more interested in health-promoting products to improve their health quality. This imposed a demand for functional food ingredients and additives, nutraceuticals and dietary supplements of natural origin.
There are several studies dealing with incorporation of
Although protein component of C-PC added nutritive value to these products, bioactivity of sensitive PCB component cannot be exploited. The only way to take full advantage of health-promoting effects of bilin component, is addition of
Possible advantages of joint administration of flavanol-rich cocoa powder and
Besides being component of many dietary supplements, C-PC also becomes popular component of different wellness bioactive drinks, providing attractive blue color and nutraceutical properties [e.g. Ocean Mist by Allgalio Biotech, B Blue bioactive drink by B blue, Bloo tonic by Cidererie Nicol, Holy water by Juice Generation, Natura blue by Natura4Ever, Smart chimp by Smart chimp and many other drinks based on Blue Majik (C-PC-enriched organic extract of
Purified PCB is still not available as a nutraceutical supplement, but new research turned toward methods for efficient cleavage of PCB from the C-PC . Further stabilization will enable commercially available PCB as food colorant and dietary supplement.
3.7. Future carriers of bioactive substances and food additives with promising techno-functional and food-preserving properties
In recent years, the food industry is in increasing search for new sources of inexpensive food protein having nutritional and techno-functional characteristics similar to high-cost animal proteins. In addition to plant one, proteins extracted from microalgae are becoming favorable alternative due to availability and sustainability of their production on one hand, and due to their extraordinary nutritional and bioactive properties, as well as suitable functional properties on the other hand.
In this moment, there are only few studies investigating the functional properties of
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
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 . 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 . Incorporation of
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) . 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 .
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
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 .
Beside the application for food and drink coloring, C-PC is also used as a cosmetics colorant in lipsticks, eyeliners, and eye shadows preparations .
The vast majority of studies regarding
This study was supported by the grant No. 172024 obtained from the Ministry of Education and Science of the Republic of Serbia.