J Appl Phycol (2008) 20:113–136 DOI 10.1007/s10811-007-9188-1

Phycobiliproteins as a commodity: trends in applied research, patents and commercialization

Soundarapandian Sekar & Muruganandham Chandramohan

Received: 23 March 2007 /Revised and accepted: 25 May 2007 /Published online: 16 August 2007 # Springer Science + Business Media B.V. 2007

Abstract Phycobiliproteins are a group of colored proteins means for improvements in the application and production commonly present in cyanobacteria and red algae possess- of phycobiliproteins. ing a spectrum of applications. They are extensively commercialized for fluorescent applications in clinical and Keywords Colorant . Cyanobacteria . Fluorophore . Patent immunological analysis. They are also used as a colorant, analysis . Phycobiliprotein extraction . Red alga and their therapeutic value has also been categorically demonstrated. However, a comprehensive knowledge and technological base for augmenting their commercial utilities is lacking. Hence, this work is focused towards this Introduction objective by means of analyzing global patents and commercial activities with application oriented research. The phycobiliproteins (PBPs) are antennae-protein pigments Strategic mining of patents was performed from global involved in light harvesting in cyanobacteria (blue-green patent databases resulting in the identification of 297 algae, procaryotic), rhodophytes (red algae, eukaryotic), patents on phycobiliproteins. The majority of the patents cryptomonads (biflagellate unicellular eukaryotic algae) and are from USA, Japan and Europe. Patents are grouped into cyanelles (endosymbiotic plastid-like organelles) (Glazer fluorescent applications, general applications and produc- 1994). In cyanobacteria and red algae, the phycobiliproteins tion aspects of phycobiliproteins and the features of each are organized in supramolecular complexes, called phycobi- group are discussed. Commercial and applied research lisomes (PBSs), which are assembled in regular arrays on the activities are compared in parallel. It revealed that US outer surface of the thylakoid membranes. Phycobiliproteins patents are mostly related to fluorescent applications while are oligomeric proteins, built up from chromophore-bearing Japanese are on the production, purification and application polypeptides belonging to two families (α and β) probably for therapeutic and diagnostic purposes. Fluorescent appli- originating from a common ancestor (Glazer 1989). The cations are well represented in research, patents and colors of phycobiliproteins originate mainly from covalently commercial sectors. Biomedical properties documented in bound prosthetic groups that are open-chain tetrapyrrole research and patents are not ventured commercially. Several chromophores bearing A, B, C and D rings named novel applications are reported only in patents. The paper phycobilins. They are either blue colored phycocyanobilin further pinpoints the plethora of techniques used for cell (PCB), red colored phycoerythrobilin (PEB), the yellow breakage and for extraction and purification of phycobili- colored phycourobilin (PUB), or the purple colored phyco- proteins. The analysis identifies the lacuna and suggests biliviolin(PXB), also named cryptoviolin. These chromo- phores are generally bound to the polypeptide chain at conserved positions either by one cysteinyl thioester linkage through the vinyl substituent on the pyrrole ring A of the * : S. Sekar ( ) M. Chandramohan tetrapyrrole or occasionally by two cysteinyl thioester link- Department of Biotechnology, Bharathidasan University, Tiruchirappalli 620 024, India ages through the vinyl substituent on both A and D pyrrole e-mail: [email protected] rings (Glazer 1985). 114 J Appl Phycol (2008) 20:113–136

In cyanobacteria and red algae, four main classes of than trimers or hexamers, and (5) they contain several phycobiliproteins exist: allophycocyanin (APC, bluish unusual bilins, for example in the cryptophycean biliprotein green), phycocyanin (PC, blue), phycoerythrin (PE, purple), phycocyanin 645, three chemically different bilins such as and phycoerythrocyanin (PEC, orange) having lAmax of Cys-bilin 618, DiCys-bilin 584 and Cys-bilin 584 are 650–655 nm, 615–640 nm, 565–575 nm and 575 nm, present (Becker et al. 1998; Wedemayer et al. 1991). respectively and emit light at 660 nm, 637 nm, 577 nm and Cyanobacterial phycobiliproteins have gained impor- 607 nm, respectively (Bryant et al. 1979). Isolated intact tance in the commercial sector, as they have several appli- phycobilisomes exhibit a fluorescence emission maximum cations. The primary potential of these molecules are as of ∼680 nm (Gantt and Lipschuttz 1973). Light energy is natural dyes but a number of investigations have shown on absorbed mainly by the pheripheral rods, where the shortest their health-promoting properties and broad range of wavelength absorbing phycobiliproteins (PE or PEC) are pharmaceutical applications. Thus, one of the applications located. The light energy absorbed by PE or PEC is of phycocyanin is to use as food pigment replacing current transferred by radiationless dipole induced dipole resonance synthetic pigments. They are used as a colourant in energy transfer to C-phycocyanin and then to allophyco- chewing gum, ice sherberts, popsicles, candies, soft drinks, cyanin (APC) and finally transmitted to PS II (and partially) dairy products and cosmetics like lipstick and eyeliners. In to PS I reaction centers (Suter and Holzwarth 1987). addition, phycobiliproteins are widely used in clinical and The assembly of the phycobilisome is stabilized by a immunological research laboratories (Spolaore et al. 2006). group of polypeptides named ‘linker’ polypeptides (L). They serve as labels for antibodies, receptors and other They induce a face-to-face aggregation of phycoerythrin or biological molecules in a fluorescence-activated cell sorter, phycoerythrocyanin and phycocyanin trimers. They addi- and they are used in immunolabelling experiments, fluo- tionally cause the tail-to-tail joining of hexameric assem- rescence microscopy and diagnostics. The major organisms blies of these biliproteins to form larger aggregates such as exploited for production are the cyanobacterium Spirulina peripheral rods and core cylinders. Allophycocyanin as- for phycocyanin and the red alga Phorphyridium for semble into phycobilisome core with the assistance of three phycoerythrin (Roman et al. 2002). types of linker polypeptides: the large core-membrane The purpose of the review is to assess the wealth of linker polypeptide (LCM) involved in the interaction of knowledge on the applications of phycobiliproteins and the PBS core with the thylakoids, rod-core linker polypeptides technological backup available for commercial venturing.

(LRC) that mediate the attachment of the peripheral rods to This can be envisaged by analyzing application oriented the PBS core and the small core-linker polypeptides (LC) research, patents and commercial activities. A compilation which participate in the assembly of core substructure. The of this knowledge and its analysis will be of immense use peripheral rods of the phycobilisomes are composed of for attaining further improvements. phycoerythrin or phycoerythrocyanin and phycocyanin in association with the appropriate rod-linker polypeptides

(LR). Phycoerythrin hexamers of some marine cyanobac- Applied research teria and red algae contain a third type of subunit γPE in the central cavity of the hexamer. These subunits are bifunc- Utility of phycobiliproteins tional phycobiliproteins that act as light harvesting phyco- biliproteins and linker polypeptides (Sidler 1994). The As colorant morphology of phycobilisomes varies with the organisms. These particles may be ellipsoidal, hemidiscoidal, or There is an increasing demand for natural colors which are bundles of rod shaped elements. The differences in gross of use in food, pharmaceuticals, cosmetics, textiles and as morphology do not reflect fundamental differences in the printing dyes. However, their utility is limited to few of placement of the major phycobiliproteins or in the these since the natural dyes have low tinctorial values and fundamental properties of the particle (Glazer 1989). persistence. Due to the toxic effect of several synthetic In contrast to the proteins of the cyanobacteria and of the dyes, there is an increasing preference to use natural colors red algae, the cryptomonad phycobiliproteins are unusual in for various end uses. Phycobiliproteins are used as a natural several ways such as (1) phycobilisomes are lacking due to protein dye in the food industry (C-phycocyanin) and in the the formation of tetrameric complexes and therefore not cosmetic industry (C-phycocyanin and R-phycoerythrin). possible to form phycobilisomes, (2) each species of Phycocyanin derived from Spirulina platensis is used as a cryptomonad contains only one type of phycobiliproteins natural pigment in food such as chewing gum, dairy either phycoerythrin or phycocyanin, (3) the proteins are products and jellies (Santos et al. 2004). Despite its lower found on the lumenal rather than the stromal side of the stability to heat and light, phycocyanin is considered more thylakoid membrane, (4) the proteins form dimers, rather versatile than gardenia and indigo, showing a bright blue J Appl Phycol (2008) 20:113–136 115 color in jelly gum and coated soft candies (Lone et al. accessed as a fluorochrome for flow cytometric immuno- 2005). They are also used in coloring of many other food detection of surface antigens on immune cells (Telford et al. products such as fermented milk products, ice creams, soft 2001b). The lesser-known but potentially important series drinks, desserts, sweet cake decoration, milk shakes and of low-molecular weight cryptomonad-derived phycobili- cosmetics. The shade of blue color produced from the red proteins are evaluated for their applicability to flow microalga Phorphyridium aerugineum does not change with cytometry both in extracellular and intracellular labeling pH. The color was stable under light, but sensitive to heat. applications (Telford et al. 2001a). Within a pH range of 4 to 5, the blue color produced is Fluorescent labeling reagents are an essential component stable at 60°C for 40 min. This property was important for of a huge industry built on sensitive fluorescence detection. food uses, since many food items are acidic, particularly This powerful multi-chromophore protein-labeling reagent drink and confections. The blue color was added to has changed the flow cytometry industry and permitted beverages without heat application (Pepsi® and Bacardi sensitive two-color lymphocyte subset analysis with a Brezzer®) which did not lose their color for at least 1 month single argon ion laser. Phycoerythrin excites very strongly at room temperature. The color was very stable in dry at 488 nm but the emission is shifted to 580 nm by energy preparations. Sugar flowers for cake decoration maintained transfer events between chromophores within the protein. their colors for years of storage. Foods prepared with the Phycoerythrin as a second color works well with fluores- phycobiliproteins include gelatin and ice cream. cein-labeled antibodies. They can also be excited at 488 nm In addition to its coloring properties, phycoerythrin but are detected separately at 525 nm to provide the second possesses a yellow fluorescence. Opportunities for exploit- colour of detection. HIV monitoring and cancer diagnostics ing this property for special effects were studied. A range of were a strong driving force for the growth of this reagent. foods that fluoresce under natural light and UV light were Three- and four-color analyses with a single laser are now prepared and tested. These include transparent lollipops commonplace with phycobiliprotein-based energy transfer made from sugar solution, dry sugar-drop candies for cake reagents. Low molecular weight fluorescent labels such as decoration (that fluoresce under UV light), and soft drinks cyanine dyes Cy5 (Indodicarbocyanine) and Cy7 (Indotri- and alcoholic beverages that fluoresce at pH 5–6. Fluores- carbocyanine) are good acceptors from excited PE and shift cent color has also been added to alcoholic beverages emission farther to red and deep red wavelengths and 11- containing up to 30% alcohol but the shelf life for such color analyses are now possible (Waggoner 2006). PE is products is short (Dufosse et al. 2005). also a very important reagent in proteomics and genomics and form the basis of the detection system in Affymetrix As fluorescent agent chips (DNA microarrays). Phycoerythrin labeled streptavi- din is added after complete binding and produces a strong Phycobiliproteins play an important role in fluorescent signal from array elements containing the biotin-labeled based detection systems, particularly for flow cytometry. DNA or protein probes (De Rosa et al. 2003). The spectral properties, such as (1) excitation and emission at the red end of the spectrum, where interference from As pharmaceutical agent biological matrices tend to be less, (2) a large Stokes shift, so that interference from Rayleigh and Raman scatter and In the last decade, the screening of micro algae, especially other fluorescing components is less significant or nonex- the cyanobacteria, for antibiotics and pharmaceutically istent, (3) immunity from quenching by naturally occurring active compounds has received ever increasing interest biological substances, (4) high solubility in aqueous (Borowitzka 1995). The pharmacological property attribut- environment so that non specific binding effects are ed by phycocyanin includes antioxidant, anti-inflammatory, minimal, and (5) fluorescence quantum yield independent neuroprotective and hepatoprotective activity. When it was of pH of phycobiliproteins, particularly R-phycoerythrin evaluated as an antioxidant in vitro, it was able to scavenge (R-PE) and allophycocyanin (APC), have made them alkoxyl, hydroxyl and peroxyl radicals, and inhibits dominant reagents in this class of flurochromes (Kronick microsomal lipid peroxidation induced by Fe+2 -ascorbic and Grossman 1983). acid or the free radicals initiators 2, 2′ Azobis (2- Synthesis of conjugates of phycobiliproteins with mol- amidinopropane) dihydrochloride, (AAPH). They also ecules having biological specificity, like immunoglobulins, reduced edema, histamine (Hi) release, myeloperoxide protein A, biotin and avidin, were reported and showed that (MPO) activity and the levels of prostaglandin (PGE2) and phycobiliproteins conjugates are excellent reagents for two- leukotriene (LTB4) in the inflamed tissues. Phycocyanin color fluorescence analysis of single cells using fluores- also reduces the levels of tumor necrosis factor (TNF-α)in cence activated cell sorter (FACS) (Oi et al. 1982). The the blood serum of mice-treated with endotoxin and it stabilized phycobilisomes designated PBXL-3L was showed neuroprotective effects in the rat cerebellar granule 116 J Appl Phycol (2008) 20:113–136 cell cultures. Aphanizomenon flos-aquae (AFA) as a source they have to be extracted from phycobilisome and purified. of phycocyanin have been described as a strong antioxidant Extraction of phycobiliproteins from cyanobacteria is (Bhat and Madyastha 2000; Romay et al. 2003). The notoriously difficult because of the extremely resistant cell involvement of phycocyanin in the antioxidant protection of wall and the small size of the bacteria (Wyman 1992; the AFA extract against the oxidative damage was Stewart and Farmer 1984). Various methods can be demonstrated in vitro (Benedetti et al. 2004). In red blood employed for extraction and purification of phycobilipro- cells, oxidative hemolysis and lipid peroxidation induced teins, but no standard technique to quantitatively extract by aqueous peroxyl radical generator, AAPH were signif- pigments from micro algae exists (Jeffrey and Mantoura icantly lowered. In plasma samples, they inhibited the 1997; Wiltshire et al. 2000). A method that works well in extent of lipid oxidation induced by pro-oxidant agent cupric one organism may not be the method of choice for another chloride (CuCl2). Such findings about the phycocyanin organism (Ranjitha and Kaushik 2005). consider the potential benefits in the prevention of many Several different physical and chemical cell disruption pathological disorders associated with oxidative stress and and protein extraction methods exist. Sonication in an ultra inflammation. sound water bath is a very easy way to promote cell break- Allophycocyanin was found to inhibit enterovirus 71- age and has commonly been used with Phorphyridium induced apoptosis. At concentration nontoxic to the host cruentum and Synechococcus (Roman et al. 2002; Vernet et cells (0.045±0.012 μM), allophycocyanin was found to al. 1990). To further aid the disruption process, sonication inhibit enterovirus 71-induced cytopathic effects, viral with sand, mainly small-particle silica, can be advantageous plaque formation, and viral-induced apoptosis (Shih et al. (Wiltshire et al. 2000). Cell disruption by French press relies 2003). In addition, inhibitory effect of phycocyanin from on blunt force to treat the samples as they are squeezed Spirulina platensis on the growth of human leukemia K562 through a small orifice by the press, which disrupts the cells. cells in a dose- and time-dependent manner was reported Repeated freeze–thaw cycles of the samples in liquid (Liu et al. 2000). R-PE subunits were indicated as an nitrogen can aid the cell disruption process. Also, grinding attractive option for improving the selectivity of photody- the sample in a tissue grinder will result in cell breakage namic therapy (PDT). Mouse tumor cells S180 and human (Stewart and Farmer 1984). In some cases, it might be bene- liver carcinoma cells SMC 7721 cells was treated with R- ficial to freeze the sample in liquid nitrogen first and then PE subunits (Bei et al. 2002). grind it frozen. These techniques are classically used as part C-Phycocyanin derived from Spirulina platensis power- of extraction process for cyanobacteria. Nitrogen cavitation fully influenced serum cholesterol concentrations and is a gentle method of cell disruption that has not been used imparted a stronger hypocholesterolemic activity (Nagaoka as much as the other techniques for the extraction of et al. 2005). Phycocyanin also exerts hepatoprotective and phycobiliproteins (Viskari and Colyer 2003). Extraction of anti-inflammatory effects in a human hepatitis animal phycobiliproteins using dried biomass of Spirulina sp. by model. It reduced alanine amino transferase (ALT), aspar- incubating at 4°C for 24 h in phosphate buffer, pH 7.0, was tate amino transferase (AST) and malondialdehyde (MDA) reported (Doke 2005). By using dilute phosphate buffer for in the serum (Gonzalez et al. 2003). In addition, at a extraction, the resulting osmotic shock can cause the concentration of 0.25 mg/ml, influence of C-phycocyanin breakage of cell walls. Combination of EDTA/lysozyme on hepatocellular parameters related to liver oxidative stress was used by many researchers for phycobiliproteins and kupffer cell functioning indicated that C-phcocyanin extraction (Stewart and Farmer 1984; Vernet et al. 1990; elicite a concenteration-depedent inhibition of carbon Kilpatrick 1985). phagocytosis and carbon-induced O2 uptake by perfused Extraction of phycocyanin from the wet biomass of livers, with a 52% diminution in the carbon-induced Spirulina is possible using the following methods: extrac- sinusoidal release of lactate dehydrogenase. C-phycocyanin tion with distilled water, extraction by homogenization in a also suppressed the 3,3′,5-triiodothyronine (T3) induced mortar and pestle in the presence of acid-washed neutral increase in serum nitrite levels and in the activity of hepatic sand using 50 mM sodium phosphate buffer of pH 6.8, nitric oxide synthase (NOS) (Remirez et al. 2002). extraction by homogenization in a Virtimixer in 50 mM phosphate buffer of pH 6.8, or extraction with various Production of phycobiliproteins concentrations of hydrochloric acid (2 to 10 N) at room temperature (Sarada et al. 1999). Of the extraction methods Extraction and purification tested, freezing and thawing of cells, homogenization using a mortar and pestle in the presence of abrasive material and In light of the considerable commercial application partic- homogenization using a blender at 10,000 rpm yielded ularly as fluorescence tags, purity of the pigments plays a 19.4 mg phycocyanin per 100 mg (dwt) of Spirulina, while major role. In order to exploit these colored substances, water extraction was found to be a slow process. Acid J Appl Phycol (2008) 20:113–136 117 treatment also resulted in the leaching of phycocyanin. method for extracting phycobiliprotein from Synechococcus Phycocyanin was found to be stable over a pH range of 5.0 (CCMP 833) using a buffer composed of Chaps (3- to 7.5 at 9°C, whereas temperatures above 40°C led to [(3-cholamidoprophyl) dimethylammonio] propanesulfonic instability. acid and asolectin for cell wall disruption and protein Enzymatic disintegration of cell wall with lysozyme solubilization, combined with nitrogen cavitation was per- followed by fractionization in hydroxyapatite column was formed (Viskari and Colyer 2003). The analysis of the also reported (Boussiba and Richmond 1979). Purification extracted samples was carried out by capillary electrophore- of large amounts of phycocyanin and allophycocyanin from sis with laser-induced fluorescence detection (Viskari et al. the cyanbacterium Microcystis aeruginosa by repeated 2001). A method for recovery of phycocyanin from S. extraction of broken cells with distilled water followed by platensis by expanded bed adsorption chromatography using purification by chromatography on DEAE cellulose were streamline-DEAE anion exchanger was explained (Bermejo demonstrated (Padgett and Krogmann 1987). A method for et al. 2006). This reduced the processing time, costs and efficient separation and purification of C-phycocyanin and increased the yield of the product. Extraction employing allophycocyanin was reported (Zhang and Chen 1999). liquid nitrogen for cell disruption followed by enzymatic This involves fraction precipitation with ammonium sul- disintegration and purification by sepharose column yielded phate, ion exchange chromatography on a DEAE-Sepharose phycocyanin with an A620/A280 value of 4.98 (Bhaskar et al. CL-6b column and gel filtration chromatography on a 2005). sephadex G-100 column, which yielded pure C-phycocyanin In Calothrix sp, lysozyme (2 mg g−1 wet cells) was used and allophycocyanin with an A620/A280 value of 5.06 and to extract the pigment during 24-h incubation at 30°C. an A665/A280 value of 5.34. Selective use of polyethylene Following centrifugation to remove the debris, the blue glycol 6000 for the precipitation of phycobilisomes to supernatant was purified by anion chromatography using further obtain phycobiliproteins were tried (Rigbi et al. Q-Sepharose fast-flow and hydrophobic interaction chro- 1980). Extraction of C-Phycocyanin (C-PC) from S. matography with a methyl macro-prep column. A high fusiformis involves treatment of crude extract with rivanol proportion (93%) of the protein was recovered and the (10:1, v/v) followed by addition of ammonium sulphate to purity was good enough to allow its use in foods and 40% saturation. Rivanol was removed by gel filtration, cosmetics (Sajilata and Singhal 2006). giving a C-PC yield of approximately 46% (Minkova et al. Osmotic membrane distillation is a novel alternate 2003). The feasibility of using the red algae Porphyridium membrane process for the concentration of heat-sensitive cruentum for preparation of large quantities of homoge- natural colors, thereby preserving the desired nutritional neous B-PE and R-PC were described (Roman et al. 2002). and organoleptic properties. It employs a porous hydropho- Further, the yield in each step was evaluated in the initial bic membrane that separates two aqueous solutions (feed stage and showed that mainly phycobiliproteins were and osmotic solution), with different solute concentrations. released in the extraction steps and precipitation. Thus, in Water evaporates from the surface of the solution of higher the extraction process, only 60% of the phycobiliproteins vapor pressure (feed) and the vapors pass through the pores were recovered, whereas in the precipitation process the of the membrane and condense on the surface of the yield was 75%. The overall recovery at the end of the pre- solution at lower vapour pressure (osmotic agent). The mi- treatment was only 43%. Separation of APC and C-PC gration of water in the form of vapors concentrates the feed from S. platensis with low- and high-performance liquid and dilutes the osmotic agent, and the products are chromatographies using UV detection was described. concentrated to 70°Brix without damage. Osmotic mem- However, these separations required 25 and 310 min, re- brane distillation appears to be a feasible method for the spectively. Prior to analysis, they incubated the phosphate/ concentration of dilute phycocyanin solutions, since it EDTA/lyzozyme suspended sample overnight at 37°C, operates at ambient temperature and at atmospheric followed by centrifugation and precipitation (Campanella pressure without causing any heat or shear damage to the et al. 2000). A method employing HPLC coupled with product. It also reduces the amount of water load on electrospary ionization mass spectrometry to determine subsequent processing steps, e.g. freeze-drying, to obtain APC and PC were explained. Pigments were harvested the final product in powder form. A one-step procedure for from Synechocystis PCC 6803 cells by centrifugation and extraction and purification of this protein from Corallina disruption in a low-ionic-strength buffer in a Braun elongata, a Mediterranean red alga, using freeze dried cells homogenizer. The separation took 31 min to complete and and involving chromatography on hydroxylapatite column, resulted in two peaks for each protein. Most of these was performed. Approximately 15 mg of pure phycoery- methods are not well suited to routine analysis of natural thrin was obtained from 25 g of dried cells. A potential for specimens due to their time-consuming and sometimes labor- scaling up this extraction and purification procedure was intensive procedures (Zolla and Bianchetti 2001). A rapid also indicated (Babu et al. 2006). In a one-step chromato- 118 J Appl Phycol (2008) 20:113–136 graphic technique for purification of R-phycoerythrin from rate of production was maintained for more than 45 days. the marine red alga Polysiphonia urceolata, a DEAE- Phycobiliprotein content in the culture kept at a density of Sepharose Fast Flow chromatography column was used and 1.5 g L−1 reached 14% of the total biomass. Nostoc sp. as a a pH gradient was applied to elute the pigment. Until now, possible organism for pigment production were demon- very different methodologies have been proposed for strated by cultivating it in 1-L glass air-lift reactor as well purifying phycobiliproteins from microalgae but only some as 17-L polyethylene bags and purification by ultra of them are useful for scale-up (Lu et al. 2005b). filtration followed by gel filtration and ion-exchange chromatography (Reis et al. 1998). A commercial process Organisms and yield of open-tank mass cultivation of a marine cyanobacterium Phormidium valderianum BDU 30501 was also developed The major producers of phycobiliproteins (i.e., phycoery- for producing phycocyanin (Sekar and Subramanian 1998). thrin and phycocyanin) are the cyanobacterium Arthrospira The average pigment yield was 20% on (dwt) basis. and the rhodophyte Porphyridium (Roman et al. 2002). The Optimization of mass cultivation media for the production nature of culture conditions employed, particularly nitrogen of biomass and natural colourants from marine cyanobac- and carbon sources, determines the content of phycobili- teria by a mathematical design of experiments were also proteins. Supply of 3% CO2 and 97% N2 in gaseous form performed (Sekar et al. 2000). Using Phormidium tenue supported 20.1% of C-phycocyanin yield in a laboratory BDU 46241, increase in the production of biomass and culture of Nostoc sp. (Silva et al. 1989). In Anabaena sp., phycoerythrin was obtained. Similarly, in P. valderianum the yield of C-phycocyanin was 8.3% of cell (dwt) BDU 30501, the medium was optimized for the production (Rodriguez et al. 1989). A screening process indicated that of biomass and phycocyanin. the content of C-phycocyanin was 17% of (dwt) in some The effects of major media constituents of Porphyridium strains of Anabaena and Nostoc and 10% of (dwt) in some spp. was studied using response surface methodology Nostoc sp. (Moreno et al. 1995). Similarly, glucose and (RSM) on biomass yield, total phycobiliprotein and the acetate enhanced cell growth and phycocyanin production production of phycoerythrin. The independent variables of S. platensis. The highest specific growth rate, cell such as concentration of NaCl, MgS04,NaN03 and concentration and phycocyanin production were respective- K2HPO4 influenced the total phycobiliproteins and phyco- ly 0.62 day−1, 2.66 g Lj1 and 322 mg Lj1 on glucose and erythrin production. The optimum condition showed that 0.52 day−1, 1.81 g Lj1 and 246 mg Lj1 on acetate (Chen et total phycobiliproteins was 4.8% at the concentration of j1 _ _ al. 1996). The feasibility of phycoerythrin production by (in g L ) NaCl - 26.1, MgSO4 5.23, NaNO3 1.56, and batch cultures of an autoflocculent microalga Rhodosorus K2HPO4-0.034. In the case of optimum PE production marinus was studied. In a vertical tubular photoreactor (3.3%), the corresponding values are 29.62, 6.11, 1.59 and equipped with a gaz-lift system, it achieved a growth rate of 0.076 g Lj1, respectively. PE production depends greatly 0.029 h−1 with a maximum biomass yield of 2 g L−1 (dwt) on the concentration of chloride, nitrate, and sulphate as (Dupre et al. 1995). well as phosphate of which the former possess the Gloeotrichia natans, a nitrogen fixing cyanobacterium, maximum effect (Kathiresan et al. 2006). as a possible source of phycobiliproteins was accessed (Boussiba 1991). Under optimal growth conditions (38°C, pH 8.0, no carbon enrichment), the specific growth rate of Patent analysis the rice-field isolate of Gloeotrichia natans was 0.076 h-1. The pH of the medium (between 6.5 and 9.0) did not One of the most insightful means of analyzing the research influence the growth rate, but it did affect phycobiliprotein trends is to examine the concurrent patenting trends. In content, as reflected by a change of the color of the order to analyze the potentials of phycobiliproteins, patent cultures. At pH 7.0, the culture was green-brown with databases of various countries available through internet phycobiliproteins constituting up to 10% of the total were mined, including United States of America, Europe, protein, while at pH 9.0 the culture was brownish-black Japan, South Korea, New Zealand, Australia, Singapore, and the content of these pigments was as high as 28% of China, Thailand, Taiwan, Germany, United Kingdom and the total protein. In outdoor cultures, the specific growth Canada. Strategic mining of patents was performed by rate was directly related to cell density in the range of 0.7 framing search keywords as well as mining data from the to 1.5 g (dwt) L−1 at a rate of stirring of 30 rpm, and database in choosing fields of search. In terms of search inversely related to cell density at half this rate. At a stirring keywords, seven major keywords were used: phycobili- rate of 30 rpm, daily production of outdoor cultures har- some, phycobiliprotein, phycobilin, phycocyanin, phycoer- vested to maintain cell densities of 0.7, 1.15 and 1.5 g L−1 ythrin, phycoerythrocyanin and allophycocyanin. In were 14.7, 17.1 and 18.1 g m−2 day−1, respectively. This addition, other keywords such as ‘chromatic adaptation’, J Appl Phycol (2008) 20:113–136 119

‘natural color’ and ‘food color’ were also used. In term of Table 1 Patents on fluorescence based applications of Phycobiliproteins fields of search, all field, abstracts and claims of the patents Group wise details of patents were used for United States Patent and Trademark Office (USPTO). Title and abstract of patents as available were As fluorescent labels, tags and markers (Total patents 122) used for rest of countries mentioned above. The patent hits US 7,067,258 (Esser et al. June 27, 2006) obtained from each keyword were corroborated with one US 7,052,859 (Batra et al. May 30, 2006) US 7,049,151 (Ngugen and Song May 23, 2006) another and overlaps if present were removed. Overlaps of US 6,977,148 (Dean et al. Dec 20, 2005) the patents were identified by sorting in MS-Excel software. US 6,960,478 (Watkins and Edwards Nov 1, 2005) The entire patent hits obtained after removal of overlaps US 6,911,313 (Houwen et al. June 28, 2005) were critically analyzed and those found relevant were US 6,900,023 (Houwen et al. May 31, 2005) selected. This search and analysis of patents was performed US 6,893,822 (Schweitzer et al. May 17, 2005) as described previously (Sekar and Paulraj 2007). US 6,828,109 (Kaplan Dec 7, 2004) United States Patent and Trademark Office (USPTO), US 6,825,176 (White and Yoakim Nov 30, 2004) US 6,824,986 (Finkelman and Morris Nov 30, 2004) European Patent Office (EPO) and Japan Patent Office US 6,821,740 (Darzynkiewicz et al. Nov 2, 2004) (JPO) contain the vast majority of patents. Using those of US 6,811,970 (Sluis-Cremer et al. Nov 2, 2004) seven major keywords, a total patent of 180 from USPTO, US 6,770,448 (Glabe and Rodriguez Aug 3, 2004) 48 from JPO and 69 from EPO database were obtained. The US 6,753,156 (Mathis et al. June 22, 2004) patent hits obtained from Australia, South Korea, Germany, US 6,740,756 (Chan and Hertzberg May 25, 2004) China, Canada and keywords like ‘chromatic adaptation’, US 6,696,243 (Siiman Feb 24, 2004) ‘natural colour’ and ‘food colour’ were found to exist US 6,632,924 (Bogan and Lodish Oct 14, 2003) already in the patent hits and hence they were excluded. US 6,613,531 (Burgess et al. Sep 2, 2003) US 6,600,017 (Glabe and Rodriguez Jul 29, 2003) Whereas database of countries such as Singapore, Thailand US 6,596,501 (Roth Jul 22, 2003) and Taiwan contains no data to the keywords used. The US 6,586,190 (Bernasconi et al. July 1, 2003) entire text of 297 patents obtained by this strategic mining US 6,573,043 (Cohen et al. June 3, 2003) was critically analyzed and the information categorized into US 6,548,254 (Beckman and Mancebo Apr 15, 2003) three groups: US 6,540,977 (van de Winkel April 1, 2003) US 6,503,702 (Stewart Jan 7, 2003) 1) Patents on fluorescence based applications of phycobi- US 6,423,549 (Knight and Del July 23, 2002a) liproteins (Table 1) US 6,351,712 (Stoughton and Dai Feb 26, 2002) 2) Patents on the general applications of phycobiliproteins US 6,342,389 (Cubicciotti Jan 20, 2002) (Table 2) US 6,329,158 (Hoffman and Frey Dec 11, 2001) 3) Patents on the production of phycobiliproteins US 6,319,668 (Nova et al. Nov 20, 2001) (Table 3) US 6,294,381 (Olweus and Lund-Johansen Sep 25, 2001) US 6,287,791 (Terstappen and Chen Sep 11, 2001) US 6,284,541 (Auer et al. Sep 4, 2001) US 6,280,618 (Watkins and Edwards Aug 28, 2001) Patents on fluorescence based applications US 6,270,971 (Ferguson-Smith et al. Aug 7, 2001) US 6,265,551 (Duke-Cohan et al. July 24, 2001) of phycobiliproteins US 6,210,875 (Patterson et al. April 3, 2001) US 6,153,384 (Lynch et al. Nov 28, 2000) Patents on fluorescent based application of phycobilipro- US 6,096,540 (Olweus et al. Aug 1, 2000) teins (Table 1) were on the use of phycobiliproteins as a US 6,060,240 (Kamb and Feldhaus May 9, 2000) moiety in fluorescent energy transfer, fluorescent labels, US 6,046,014 (Lagarias and Murphy April 4, 2000) tags, tracers and markers which are useful in flowcytom- US 6,008,052 (Davis and Ward Dec 28, 1999) etry, fluorescent immunoassays, immunophenotyping and US 6,007,996 (McNamara et al. Dec 28, 1999) such other fluorescent studies. For example, dual nucleic US 5,976,802 (Ansorge et al. Nov 2, 1999) US 5,945,291 (Bolton and Koester Aug 31, 1999) acid probes with resonance energy transfer moieties are US 5,891,738 (Soini et al. April 6, 1999) provided in which the acceptor moiety was phycobilipro- US 5,863,401 (Chen Jan 26, 1999) tein selected from B-phycoerythrin or R-phycoerythrin and US 5,861,253 (Asgari et al. Jan 19, 1999b) the donor moiety was lanthanide chelate. They hybridize US 5,858,649 (Asgari et al. Jan 12, 1999a) sufficiently near each other on a subject nucleic acid to US 5,853,984 (Davis et al. Dec 29, 1998) generate an observable interaction, thereby providing US 5,840,587 (Stewart et al. Nov 24, 1998) detectable signals for rapid, specific and sensitive hybrid- US 5,837,547 (Schwartz Nov 17, 1998) US 5,834,196 (Reutelingsperger Nov 10, 1998) ization determination in vivo. They are also used selectively US 5,830,679 (Bianchi and Weinschenk Nov 3, 1998) to impregnate biological structures and enhance images of 120 J Appl Phycol (2008) 20:113–136

Table 1 (continued) Table 1 (continued)

Group wise details of patents Group wise details of patents

US 5,814,468 (Siiman et al. Sep 29, 1998) US 4,520,110 (Stryer et al. May 28, 1985) US 5,807,879 (Rosebrough Sep 15, 1998) CN 1657912 (Jue August 24, 2005) US 5,807,536 (Morcos Sep 15, 1998) CN 1417228 (Gong May 14, 2003) US 5,786,160 (Anderson and Vivier July 28, 1998) CN 1382973 (Liu and Zhu Dec 4, 2002) US 5,780,227 (Sheridan et al. July 14, 1998) CN 1117973 (Yuanzhen and Zhaoqi Mar 6, 1996) US 5,776,785 (Lin and Wilson July 7, 1998) WO 02073202 (Knight and Del Sep 19, 2002b) US 5,766,843 (Asgari et al. June 16, 1998) WO 0196383 (Morseman and Moss Dec 2, 2001) US 5,763,201 (Tomer 1998) CA 2343700 (Pawliszyn and Wu Oct 13, 2001) US 5,716,829 (Rosok et al. Feb 10, 1998) CA 1222705 (Dattagupta and Crothers Sep 6, 1987) US 5,714,386 (Roederer Feb 3, 1998) EP 0735360 (Davis and Bishop Oct 2, 1996) US 5,712,095 (Britschgi and Cangelosi Jan 27, 1998) JP 135476 (Kamoda and Harasawa June 7, 1988) US 5,695,928 (Stewart Dec 9, 1997) As a moiety in fluorescent energy transfer (Total patents 25) US 5,695,990 (Cubicciotti Dec 9, 1997) US 7,081,336 (Bao et al. July 25, 2006) US 5,665,540 (Lebo Sep 9 1997) US 7,041,642 (Desjardins et al. May 9, 2006) US 5,627,037 (Ward et al. May 6, 1997) US 6,939,721 (Mauze and Yang Sep 6, 2005) US 5,620,842 (Davis and Bishop April 15, 1997) US 6,903,196 (Roben and Stevens June 7, 2005) US 5,597,688 (Connelly et al. Jan 28, 1997) US 6,852,206 (Pawliszyn et al. Feb 3, 2005) US 5,583,033 (Terstappen and Picker Dec 10, 1996) US 6,821,952 (Desjardins et al. Nov 23, 2004d) US 5,571,680 (Chen Nov 5, 1996) US 6,815,423 (Desjardins et al. Nov 9, 2004c) US 5,538,855 (Orfao de Matos Correira and Vale, July 23, 1996) US 6,680,367 (Desjardins et al. Jan 20, 2004b) US 5,536,382 (Sunzeri July 16, 1996) US 6,677,430 (Desjardins et al. Jan 13, 2004a) US 5,523,210 (Paulus June 4, 1996) US 6,054,557 (Faure et al. April 25, 2000) US 5,466,610 (Bright et al. 1995) US 5,824,772 (Vincent et al. Oct 20, 1998) US 5,464,748 (Sommadossi Nov 14, 1995) US 5,824,478 (Muller Oct 20, 1998) US 5,439,830 (Sakashita et al. Aug 8, 1995) US 5,716,784 (Di Cesare Feb 10, 1998) US 5,380,663 (Schwartz and Hetzel Jan 10, 1995) US 5,693,679 (Vincent et al. Dec 2, 1997) US 5,356,779 (Mozes and Pecht Oct 18, 1994) US 5,652,093 (Cubbage et al. July 29, 1997) US 5,326,696 (Chang July 5, 1994) US 5,627,074 (Mathis et al. May 6, 1997) US 5,314,824 (Schwartz May 24, 1994) US 5,501,952 (Cubbage et al. Mar 26, 1996) US 5,256,542 (Chang Oct 26, 1993b) US 5,457,184 (Lehn et al. Oct 10, 1995) US 5,234,816 (Terstappen Aug 10, 1993) US 5,439,797 (Tsien et al. Aug 8, 1995) US 5,213,960 (Chang May 25, 1993a) US 5,434,088 (Ikeda et al. July 18, 1995) US 5,156,951 (Bach et al. Oct 20, 1992) US 5,206,178 (Monji and Cole Oct 25, 1993) US 5,153,117 (Simons Oct 6, 1992) US 4,822,733 (Morrison April 18, 1989) US 5,108,904 (Landay April 28, 1992) US 4,780,409 (Monji et al. Oct 25, 1988) US 5,137,809 (Loken et al. Aug 11, 1992) US 4,666,862 (Chan 1987) US 5,098,849 (Hilerio and Kirchanski March 24, 1992) IT 11230953 (Voyta and Brooks Nov 8, 1991) US 5,093,234 (Schwartz March 3, 1992) As signal generators and image contrast agents (Total patents 13) US 5,085,985 (Maino and Janszen Feb 4, 1992) US 7,072,036 (Jones et al. July 4, 2006) US 5,084,394 (Vogt and Schwartz Jan 28, 1992) US 7,014,839 (Klaveness et al. Mar 21, 2006) US 5,073,497 (Schwartz Dec 17, 1991) US 6,540,981 (Klaveness et al. April 1, 2003) US 5,068,178 (Nowinski Nov 26, 1991) US 6,593,101 (Richards-Kortum et al. July 15, 2003) US 5,057,413 (Terstappen et al. Oct 15, 1991) US 6,159,445 (Klaveness et al. Dec 12, 2000) US 5,047,321 (Loken and Terstappen Sep 10, 1991) US 6,123,923 (Unger and Wu Sep 26, 2000) US 4,959,309 (Dattagupta and Crothors Sep 25, 1990) US 6,110,682 (Dellinger et al. Aug 29, 2000b) US 4,895,796 (Lanier et al. Jan 23, 1990) US 6,103,474 (Dellinger et al. Aug 15, 2000a) US 4,857,451 (Schwartz Aug 15, 1989) US 5,661,040 (Huff et al. Aug 26, 1997) US 4,812,394 (Dolbeare and Gray Mar 14, 1989) US 5,512,493 (Mathis et al. Apr 30, 1996) US 4,774,189 (Schwartz Sep 21, 1988) US 5,260,004 (Samuelson et al. Nov 9, 1993) US 4,764,462 (Bredehorst et al. Aug 16, 1988) CN 1588231 (Gong and Gu Mar 2, 2005) US 4,745,285 (Recktenwald and Chen May 17, 1988b) WO 0017650 (Haugland and Haugland Mar 30, 2000) US 4,737,454 (Dattagupta and Crothers Apr 12, 1988) As diagnostic tools (Total patents 12) US 4,727,020 (Recktenwald Feb 23, 1988a) JP 235568 (Nakajima et al. Aug 26, 2003) US 4,708,931 (Christian Nov 24, 1987) JP 257823 (Han Sep 11, 2002) US 4,607,007 (Lanier and Warner Aug 19, 1986) JP 338030 (Inuitakashi et al. Dec 8, 2000) US 4,599,304 (Lanier and Phillips July 8, 1986) JP 009733 (Ishikara et al. Jan 14, 2000) J Appl Phycol (2008) 20:113–136 121

Table 1 (continued) Table 1 (continued)

Group wise details of patents Group wise details of patents

JP 326323 (Berend et al. 26 Nov, 1999) Intact phycobilisome preparation for fluorescent applications JP 232232 (Okutomi Sep 2, 1998) (Total patents 3) JP 035100 (Higgs Feb 5, 1990) CN 1654627 (Zhang and Zhang Aug 17, 2005a) JP 112147 (Watanabe Apr 28, 1989) CN 1654628 (Zhang and Zhang Aug 17, 2005b) JP 298761 (Yasuda et al. Dec 25, 1987) CN 1654629 (Zhang and Zhang Aug 17, 2005c) CN 1715924 (Wu Jan 4, 2006) WO 03093795 (Trainer Nov 13, 2003) AU - Austria, CA -Canada,CN -China,EP -Europe,GR - Germany, EP 0509718 (Rao Oct 21, 1992) IT -Italy,JP -Japan,US - United States of America, WO - World Intellectual Property Organization. In bioluminescent novelty items (Total patents 4) US 6,232,107 (Bryan and Szent-Gyorgyi May 15, 2001) US 6,152,358 (Bryan Nov 28, 2000b) all structures such as arterial wall thickness, atherosclerotic US 6,113,886 (Bryan Sep 5, 2000a) plaque, luminal boundaries and to better delineate tumors US 5,876,995 (Bryan Mar 2, 1999) mass outlines. The use of phycobilins and phycobiliproteins For conjugation with other molecules (Total patents 24) US 6,387,622 (Siiman et al. May 14, 2002) as light imaging contrast agents has also been proposed. US 6,372,445 (Davis et al. April 16, 2002) And many patents pertain phycobiliproteins as a diagnostic US 6,146,837 (van de Winkel Nov 14, 2000) tool. Methods for detecting tobacco mosaic virus using US 6,133,429 (Davis et al. Oct 17, 2000) phycoerythrin as a fluorescent probe have been patented in US 6,020,212 (Mathis Feb 1, 2000a) which the secondary antibody is labeled with phycoerythrin US 5,994,089 (Siiman et al. Nov 30, 1999b) and thereby aids in the detection of the virus. And other US 5,891,741 (Siiman et al. April 6, 1999a) such methods for detection of microbes in the clinical US 5,783,673 (Gupta July 21, 1998) samples were described in few patents. US 5,736,624 (Bieniarz et al. April 7, 1998) US 5,272,257 (Gupta Dec 21, 1993c) Phycobiliproteins are used in the novelty items designed US 5,171,846 (Gupta Dec 15, 1992) for entertainment, recreation and amusements, including US 5,055,556 (Stryer et al. Oct 8, 1991) toys, paints, slimy play material, textiles particularly US 4,867,908 (Recktenwald et al. Sep 1989) clothing, bubbles in bubble making, personal items such US 4,861,728 (Wagner Aug 29, 1989) as cosmetics, bath powders, body lotions, toothpastes and US 4,859,582 (Stryer et al. Aug 22, 1989) other dentrifrices, soaps, foods such as gelatins, icings and US 4,857,474 (Waterbury et al. Aug 15, 1989) frostings, beverages such as beer, wine, champagne, soft US 4,542,104 (Stryer and Glazer Sep 17, 1985) WO 9852040 (Siiman and Burshteyn Nov 19, 1998) drinks, and ice fountains, including liquid fireworks and WO 9405701 (Gupta Mar 17, 1994) other such jets or sprays. In addition, there is a range of WO 9303062 (Gupta Feb 18, 1993b) patents dealing with combinations of two fluorescent dyes AU 685062B (Gupta Jan 15, 1998) and methods for preferential labeling of a phycobiliproteins AU 665930B (Gupta Jan 25, 1996) with amine reactive dyes were also disclosed. For example, GR 3032423T (Mathis May 31, 2000b) phycoerythrin or allophycocyanin is conjugated with an CA 2114837 (Gupta Feb 18, 1993a) amine-reactive dye such as Texas Red or corboxyfluo- Recombinant constructs for diverse end-uses (Total patents 13) rescein succinimidyl ester, in the presence of a selective salt CN 1721446 (Zhang Jan 18, 2006) CN 1618803 (Qin and Lin May 25, 2005) which causes a hydrophobic intramolecular rearrangement CN 1443849 (Zhao and Ran Sep 24, 2003) of the phycobiliprotein thereby exposing more hydrophobic CN 1434057(Zhao Aug 6, 2003) sites for binding to the amine-reactive dye (Gupta 1998a,b). CN 1157324 (Qin and Li Aug 20, 1997) The conjugates prepared according to the invention are WO 2005093040 (Block and Mittmann Oct 6, 2005) useful in multiple color fluorescence assays without WO 03012448 (Glazer and Toole Feb 13, 2003) requiring the use of multiple exciting sources. Methods WO 0146395 (Glazer and Yuping June 28, 2001) for molecular manipulation of cyanobacteria and red algae WO 0196871 (Allnutt and Toole Dec 20, 2001) to express of phycobiliproteins and phycobilisomes linker US 6,740,507 (Glazer et al. May 25, 2004) US 6,649,376 (Glazer and Cai Nov 18, 2003) fusion protein and their utilization as phycobiliproteins and US 5,518,897 (Stevens, Jr. and Murphy May 21, 1996) phycobilisomes and subassembly based reagents were also US 4,956,280 (Buzby et al. Sep 11, 1990) patented. Recombinant cells which express a fluorescent holo-phycobiliproteins fusion protein and method of use were described. In addition, processes for preparing and changing intact phycobilisome for application in super- sensitive biomedicine detection were also patented. 122 J Appl Phycol (2008) 20:113–136

Table 2 Patents on the general Application Patent number applications of Phycobilipro- teins Therapeutic applications (Total patents 17) Anti inflammatory US 7,025,965 (Pieloch April 11, 2006) JP 256478 (Hirabashi et al. Sep 16, 2004) Liver Protecting CN 1633889 (Ke and Suo Jul 6, 2005) Anti viral CN 1524574 (Que Sep 1, 2004) US 6,346,408 (Chueh Feb 12, 2002) Anti tumour CN 1478552 (Jue and Jue Mar 3, 2004) CN 1325729 (Wang and Li Dec 12, 2001) CN 1091976 (Shu and Xinhan Sep 14, 1994) US 5,163,898 (Morcos and Henry Nov 17, 1992) JP 065216 (Iijima et al. Apr 18, 1983) Treatment of atherosclerosis US 4,886,831 (Morcos and Henry Dec 12, 1989) Lipase activity inhibitor JP 359638 (Koda and Okuda Dec 24, 2004) Serum lipid reducing agent JP 137805 (Nagaoka et al. May 14, 2003) Skin function activation factor JP 036744 (Fujikawa and Matsushima Feb 9, 2006) Anti oxidant JP 330733 (Oho Nov 19, 2002) As an agent that obstructs absorption of JP 157559 (Yoneda June 12, 2001) environmental WO 2005112667 (Omori Jan 12, 2005) pollutant deposition in the body. Cosmetic uses (Total patents 2) US 6,136,329 (Boratyn Ovt 24, 2000) JP 063911 (Tanabe and Shimamatsu May 30, 1981) In drink/beverage compositions CN 1127611(Zhongliang and Changlan Jul 31, 1996) (Total patents 3) CN 1096178 (Xu Dec 14, 1994) WO 03099039 (Keillar Dec 4, 2003) Miscellaneous applications (Total patents 8) Light protection in packaging US 6,686,004 (Daziano and van Agthoven Feb 3, 2004) As a fabric dye JP 166480 (Kimura Jun 27, 1995) Quantification of U-V US 5,075,557 (Harasawa et al. Dec 24, 1991) For coating on Laminaria JP 134977 (Tanabe et al. Oct 22, 1981) As a ingredient in tissue culture medium EP 0247881 (Shinhara and Kotanino Dec 2, 1987b) CN - China, EP - Europe, JP - Japan, US - United States of JP 278979 (Shinhara and Kotanino Dec 3 1987a) America, WO - World Intel- As a plant growth regulator JP 000408 (Harasawa et al. Jan 6, 1987) lectual Property Organization. As a molecular weight marker JP 222029 (Sakakibara et al. Dec 23, 1983)

Patents on the general applications of phycobiliproteins rosis or cancer were described. Once injected, phycocyanin was selectively taken up into atherosclerotic plaques or A number of other applications of phycobiliproteins are cancer cells, and upon subsequent irradiation destruction of also patented (Table 2). In these cases, extracts obtained the atherosclerotic plaques or cancer cells occur. And new- from blue green algae (phycobiliproteins) exhibiting vari- type laser photosensitizer for curing carcinosis were ous activities such as anti-inflammatory, antioxidant, anti- patented in which phycocyanin was used as photosensitizer. viral and anti tumor activities were patented. For example., Phycocyanin use as lipase activity inhibitor, serum lipid as an anti inflammatory agent, phycocyanin has been found reducing agent and skin function activator were patented. to function as Cox-2 inhibitor that is effective in treating As an active lipase activity inhibitor, they act on substrate- arthritis and other inflammatory conditions in animals. This binding sites of a lipase such as pancreatic lipase. extract is administered in a form that is pharmaceutically Therefore, lipolysis of lipase is checked, disassembly of acceptable and palatable for animals (Pieloch 2006). A fat is controlled which ultimately reduces obesity (Koda method of allophycocyanin inhibition of enterovirus and and Okuda 2004). As a serum lipid reducing agent, they are influenza virus reproduction has been patented in which effective in prevention and therapy of hyperlipidemia, allophycocyanin as an active ingredient has the capability ischemic heart diseases or cerebral apoplexy by promoting of inhibiting enterovirus and influenza virus reproduction, blowdown and improving serum lipid while controlling thereby preventing a cytopathic effect (Chueh 2002). absorption of the cholesterol from the intestinal tract Further patents on medical uses were also found. For (Nagaoka et al 2003). Phycocyanin containing healthy food example, the uses of phycocyanin for treating atheroscle- composition capable of preventing environmental pollu- J Appl Phycol (2008) 20:113–136 123

Table 3 Patents on the production of phycobiliproteins tants from being deposited in human body has been Group wise details of patents disclosed (Yoneda 2001). As a result, harmful heavy metals or harmful minerals present with the food of everyday Extraction and purification processes (Total patents 23) intake are effectively excreted from the body. CN 1563083 (Liu and Shen Jan 12, 2005) A cosmetic containing hard-soluble phycocyanin has CN 1587275 (Chen and Wang Mar 2, 2005) been patented. This was obtained by making a water-soluble CN 1687440 (Zhu and Li Oct 26, 2005) CN 1680435 (Zhang and Liu Oct 12, 2005) phycocyanin extracted from an alga Spirulina with an CN 1417227 (Gong and Zeng May 14, 2003) aqueous solvent insoluble and by treating with an organic CN 1344723 (Wang and Sun Apr 17, 2002) solvent (ethanol). This shows high durability to water such CN 1295080 (Zheng May 16, 2001) as sweat and high safety to human bodies and is therefore CN 1281727 (Liu and Ding Jan 31, 2001) used for eye shadow, eyeliner or lipstick. Phycobiliproteins CN 1268516 (Jin and Liu Oct 4, 2000) role as plant growth regulators in greenhouse horticulture CN 1291616 (Qian and Li Apr 18, 2001) have been patented, in which they are used as a photo- JP 075851 (Fukuda et al. March 24, 2005) active substance absorbing green light in incident light and JP 027041 (Fukuda et al. Jan 29, 2004) JP 292815 (Sumi Oct 15, 2003b) emitting the light having the wavelength of the yellow to JP 342489 (Fukuda et al. Dec 3, 2003) red region. Phycocyanin is used as dyeing material for JP 271783 (Ishii et al. Sep 27, 1994) cloths, such as silk, cotton and rayon. Patents on the use of JP 109787 (Koyano and Kawate May 14, 1988) phycobiliproteins as media ingredients were documented, JP 086958 (Kato and Shimamatsu Jul 15, 1981) where they are used as an alternate for serum. Human and IN 186297 (Pillai and Raghavarao July 28, 2001) various other animal cells such as myeloma cell strain, etc., IN 184767 (Sarada and Sathuluri Sep 30, 2000) are proliferated in a medium containing phycocyanin as a FR 2690452 (Corine and Dominique Oct 29, 1993) BG 96440 (Chernov and Minkova May 31, 1995) cell proliferation promoting substance. Patents on the use of RU 2055842 (Gordeev Oct 3, 1996) phycocyanin and phycoerythrin in drink and beverage US 5,358,858 (Chiang and Chou, October 25, 1994) preparation were mentioned and they impart unique color Culture methods (Total patents 2) to the drink and good eating enjoyment. JP 092935 (Maeda Nov 2, 2003) JP 315569 (Hara Oct 29, 2002) Patents on the production of phycobiliproteins Overall production technologies (Total patents 26) JP 295829 (Hara Oct 27, 2005) JP 166704 (Chiang et al. 2004) Although varied applications of phycobiliproteins are JP 231821 (Sumi Aug 19, 2003a) patented, it is essential to have the technology for the JP 190244 (Eto July 17, 2001) cultivation of cyanobacterium. There are a range of patents JP 299450 (Kato and Nagatsuka Nov 2, 1999) dealing with varied cultivation, harvesting systems, extrac- JP 294596 (Hirano et al. Nov 18, 1997) tion, purification and production processes for phycobili- JP 046993 (Wake et al. Feb 21, 1995) proteins (Table 3). The purification of phycoerythrin JP 118555 (Kato and Fukuda May 9, 1995) included distilled water leaching, staged precipitation with JP 277075 (Miyazaki et al. Oct 4, 1994) ammonium sulfate and ionic exchange column chromatog- JP 146961 (Miyagawa et al. June 8, 1989a) JP 123865 (Miyagawa et al. May 16, 1989b) raphy, thereby phycoerythrin of purity up to 4.85 and total JP 263095 (Tunaka Oct 31, 1988) yield of 52% were obtained. Treatment of the raw extract JP 094990 (Matsunga Apr 26, 1988) with 0.001–1.0% aqueous solution of rivanol (6, 9- JP 006691 (Kumano et al. Jan 13, 1987) diamino-2-ethoxyacridinium lactate), centrifuging of the JP 077890 (Shimamatsu and Kato Jun 12, 1980) mixture at 2,000–8,000 rpm, desalinization of the superna- JP 018449 (Shimamatsu and Hamada Feb 8, 1980) tant by 50% ammonium sulphate, subsequent centrifuga- CN 1520718 (Jiang and Zhou Aug 18, 2004a) tion, dissolution with acetic acid, centrifugation and CN 1521185 (Jiang and Zhou Aug 18, 2004c) CN 1521186 (Jiang Aug 18, 2004) desalination of the solution were also patented for pure B- CN 1521187 (Jiang and Zhou Aug 18, 2004b) phycoerythrin production (Chernov and Minkova 1995). CN 1438240 (Xiang and He Aug 27, 2003) Algal pigment materials with excellent color-tone and TW 222463B (Chiang and Chou Oct 21, 2004a) thermal stability for use in foods were patented, in which TW 222999B (Chiang and Chou Nov 1, 2004b) algal pigment material is obtained by evaporating to TW 223000B (Chiang and Chou Nov 21, 2004d) dryness an aqueous solution essentially containing trehalose TW 224135B (Chiang and Chou Nov 21, 2004c) (10 wt.%) and algal pigment(s) such as phycocyanin or EP 1591518 (Lu and Chiang Nov 21, 2005a) phycoerythrin. This algal pigment material has broad BU - Bulgaria, CN - China, EP - Europe, FR - France, IN - India, JP - applicability, being used preferably as a colorant for food Japan, RU - Russia, US - United States of America, TW - Taiwan such as ice cream (Kato and Nagatsuka 1999). 124 J Appl Phycol (2008) 20:113–136

Table 4 Products marketed using phycobiliproteins

Company Products

Cyanotech Corporation (http://www.phycobiliprotein.com) Allophycocyanin(APC), B-Phycoerythrin(B-PE), C-Phycocyanin(C-PC), R-Phycoerythrin(R-PE) and Cross-linked Allophycocyanin PROzyme Inc. (http://www.prozyme.com/phycolink/pj-kits.html) Phycolink® - Allophycocyanin (APC) conjugation kit, Phycolink® - R-Phycoerythrin (R-PE) conjugation kit, Phycolink® - B-Phycoerythrin (B-PE) conjugation kit, C-Phycocyanin (C-PC), GT5 Allophycocyanin, Crosslinked Allophycocyanin, R-Phycocyanin (R-PC), Y-Phycoerythrin (Y-PE) Pierce Biotechnology Inc. (http://www.piercenet.com/products/ R-Phycoerythrin(R-PE) browse.afm?) Vectors laboratories (http://www.vectorlabs.com/products.asp? Phycoerythrin Avidin D, Phycoerythrin Streptavidin catID-34)

Dojindo Molecular Technologies (http://www.dojindo.com/ B-Phycoerythrin (B-PE) - Labeling kit - NH2 labeling/labelingkits.html) B-Phycoerythrin (B-PE) - Labeling kit - SH

R - Phycoerythrin (R-PE) - Labeling kit-NH2 Phycoerythrin (R-PE) - Labeling kit-SH

Allophycocyanin (APC) - Labeling kit-NH2 Allophycocyanin (APC) - Labeling kit-SH Flogen® (http://www.febico.com.tw/flogen/english/product- Allophycocyanin(APC), flogen.htm) B-Phycoerythrin(B-PE), R-Phycoerythrin(R-PE) Cross-linked Allophycocyanin (CL-APC) ANAspec Inc. (http://www.anaspec.com/products/product. Allophycocyanin (APC), category.asp?id=350) AnaTag™ APC.protein labeling kit, AnaTag™ B-Phycoerythrin (B-PE) protein labeling kit, AnaTag™ R-Phycoerythrin (R-PE) protein labeling kit, B-Phycoerythrin (B-PE), R-Phycoerythrin (R-PE), Crosslinked Allophycocyanin (CL-APC) Martek Bioscience Corporation (http://www.fluorescent.martek. R-Phycoerythrin (R-PE), com/technicaloverview) B-Phycoerythrin (B-PE), SureLight® Allophycocyanin (APC), Sensilight™ dyes (Stabilized phycobilisomes) PBXL-1 PBXL-3 P3L Phycoerythrin conjugates SensiLight™ P3L conjugates PBXL-1 conjugates PBXL-3 conjugates APC conjugates Invitrogen-Molecular probes (http://www.probes.invitrogen.com/ Phycoerythrin (R-PE), servlets/pricelist?id=32093) B-Phycoerythrin (B-PE), Allophycocyanin (APC), Crosslinked Allophycocyanin (APXL) R-Phycoerythrin (R-PE)- streptavidin conjugates, B-Phycoerythrin (B-PE) - streptavidin conjugates, Alllophycocyanin (APC) streptavidin conjugates. Europa Bioproducts Ltd (http://www.europa-bioproduct.com/ PhycoPro™ C-Phycocyanin, PhycoPro™ Crossliked Allophycocyanin (CL-APC), catalogue /44) PhycoPro™ B-Phycoerythrin (B-PE), PhycoPro™ R-Phycoerythrin (R-PE), Phycolink®-FLAG® R-Phycoerythrin (R-PE) conjugate, Streptavidin - R-Phycoerythrin (R-PE), Chromaprobe Inc. (http://www.chromaprobe.com/products.html) R-Phycoerythrin (R-PE), Allophycocyanin (APC) J Appl Phycol (2008) 20:113–136 125

To industrially cultivate large amounts of algae at all times Methods for increasing the yield of phycocyanin by of the year without being affected by culturing site with good introducing a plasmid, capable of participating in phycocy- efficiency using a low cost method have been patented, in anin synthesis were demonstrated (Matsunga 1988). In which the proliferation of algae is promoted by irradiating an addition, methods for increasing phycocyanin yield by the algae-culturing liquid with a monochromatic light having a use of urea type or amino type water soluble nitrogen wavelength exhibiting ≥600 nm specific absorbance by compounds along with other nutrients necessary for culture chlorophyll. This monochromatic light is the one having were also patented (Shimamatsu and Hamada 1980) 630–690 nm wavelength (red light) and/or the one having 400–460 nm wavelength (blue light or violet light). The photosynthesis of carotenoid was promoted in the algae- Commercialization culturing liquid by the irradiation with 400–500 nm mono- chromatic light (violet, blue and green) emitted by the light Several multinational companies exploit these phycobili- emitting diode, and the photosynthesis of phycocyanin proteins as a commercial commodity (Table 4). Cyanotech promoted by the irradiation with 500–630 nm monochro- is developing micro-algae based technologies and produc- matic light (green, yellow and orange) emitted by the light ing micro alga derived products. They also produce emitting diode (Hara 2002). A Method for changing the phycobiliproteins, which are sold to the medical and color tone of laver products from the red to black color is biotechnology research industry. Their special properties provided, by installing a filter selectively transmitting a red make them useful as tags or markers in many kinds of color light having ≥600 nm wavelength at the upper part of a biological assay such as flow cytometry, fluorescence net to which the thalluses of Porphyra are attached and immunoassay and fluorescent microcopy. Cyanotech pro- reducing phycoerythrin of phycobilin by having the wave duces a line of four phycobiliprotein products with various length of the light to be absorbed in the thalluses to increase spectral properties. R- Phycoerythrin (R-PE) is a red phycocyanin. Thereby, the color tone of laver nerve were pigment used primarily in flow cytometry, allophycocyanin made into black (blue:red:green:yellow=7:9:3:1) from red (APC) is a blue pigment also used in flow cytometry, but (base:base:red colouing matter:blue green yellow coloring typically in combination with R-PE to form a fluorescent matter=9:7:3:1) (Maeda 2003). Cultivation of cyanobac- tandem phycobiliprotein conjugate which improves sensi- teria for the production of phycobiliproteins under a tivity. Cross-linked (XL-APC) is a stabilized form of APC magnetic field was patented in which a photosynthetic alga, which can be used in much diluted solution without the for, e.g., blue-green alga (Spirulina), red alga (Colarina)or problem of degradation. C-phycocyanin is also a blue cryptoalga, is charged in a test tube and the test tube is pigment, although not used externally in cytometry, has placed between the N- and S-poles of a magnet so that the potential application in food and cosmetic colouring. The both poles oppose each other on both sides of the tube. The company’s phycobiliproteins currently range in price from test tube is irradiated with a fluorescent lamp with an US$5,000-$33,000/g illuminance of 800–8,000 lux and the culture is continued Prozyme produces R-phycocyanin, C-phycocyanin, GT5 at 24°C for 480 h to produce phycobiliproteins (for, e.g., allophycocyanin, crosslinked allophycocyanin and has phycocyanin) (Hirano et al 1997). recently added a new cyanobacterial phycobiliprotein, to

Table 5 Type of products covered in patents, applied research and commercial sector

Properties reported in patents Applied research pertaining to this property Commercial distributors

Phycobiliproteins as fluorescent labels,tags and markers Waggoner (2006) Cyanotech Corporation Sohn and Sautter (1991) PROzyme Inc. Pierce Biotechnology Inc. Dojindo Molecular technologies Flogen® ANAspec Inc. Martek Bioscience Corporation Invitrogen-Molecular probes Phycobiliproteins conjugates Kronick and Grossman (1983) Vector Laboratories Oi et al. (1982) Martek Bioscience Corporation Invitrogen-Molecular probes Europa Bioproducts Stabilized phycobilisome Telford et al. (2001a,b) Martek Bioscience Corporation 126 J Appl Phycol (2008) 20:113–136

Table 6 Properties reported in both patents and applied research that Martek Biosciences Corporation produces a variety of are not commercialized fluorescent pigments from algae, includes R-Phycocyanin Properties reported in patents Applied research pertaining (R-PC) and B-Phycoerythrin from Porphyridium cruentum, to this property a red alga, and R-Phycoerythrin (R-PE) from Porphyra yezoensis, C-Phycocyanin (C-PC) and allophycocyanin Anti-inflammatory Benedetti et al. (2004) (APC) from Spirulina platensis. They have also developed Antioxidant Romay et al. (2003) Romay et al. (1998) stabilized phycobilisomes, a line of proprietary pigments Liver protecting Gonzalez et al. (2003) from the photosystem II antennae complex of red algae and Remirez et al. (2002) cyanobacteria which includes PBXL-1, PBXL-3 and P3L Antiviral Shih et al. (2003) named SensiLight™ dyes. The PBXL™ pigments are water Anti tumor Liu et al. (2000) soluble and have been successfully applied to different Lipase activity inhibitor and serum Nagaoka et al. (2005) immunodiagnostic formats (e.g., sandwich, competitive lipid reducing agent displacement assays, microtiter, flow cytometry, microsco- py and paramagnetic beads). In addition to their proprietary which they have assigned the designation Y-phycoerythrin PBXL dye conjugates, they developed phycoerythrin (Y-PE) in recognition of the shift of its fluorescence conjugates. B-Phycoerythrin and R-Phycocyanin streptavi- emission toward the yellow, relative to R-and B-phycoer- din conjugates have been favorably evaluated for use on ythrins. Its absorbance and excitation maxima are located at microarray imaging and immunohistochemistry. ∼495 nm, making it particularly suitable for excitation with a 488-nm laser. Its shorter emission wavelength (∼563 nm), relative to other phycoerythrins (575 nm) makes it a good Trends and prospects candidate for multicolour fluorescence applications, where separation from higher wavelength emission is desired. In This analysis amply demonstrates that phycobiliproteins addition, preliminary results suggest that Y-PE may be a have potential and diverse applications. The use of more efficient donor in fluorescence resonance energy phycobiliproteins for coloring purposes in foods is well transfer (FRET) applications. The shift in its spectral exploited in confectioneries, dairy products and ice creams, characteristics, relative to other phycoerythrins, reflects a soft drinks, beverages and cake icing. In food application, high content of the phycourobilin chromophore. Indeed, there is an increasing interest in the production of light- their properties (high molar absorbance coefficients, high fluorescent products. The scope for the use of phycobili- fluorescence quantum yield, large Stokes shift, high proteins in textile and printing dyes is in the offing. Further, oligomer stability and high photo stability) make them it is likely to have applications as colorant in pharmaceu- very powerful and highly sensitive fluorescent reagents. tical products like pills and syrups. However, use of Dojindo offers two types of R-PE, B-PE and allophyco- phycobiliproteins for food purposes has to be further cyanin labeling kits. For example, in R-PE, one is NH2- supported with toxicity testing. Only in the case of C- reactive labeling kit (NH2 type) and other is SH-reactive phycocyanin, categorical reports on the safety for use in labeling kit (SH type). NH2 type reacts with a primary or food is available (Dufosse et al. 2005). In relation to other secondary amine group of target molecules, and SH type reacts with thiol groups. They are useful in immunoblotting and Immunostaining. Flogen® phycobiliproteins have appli- Table 7 Properties reported only in patents that are not commercialized cations in high sensitivity direct fluorescence detection in Novel Properties flow cytometry, fluorescence in situ hybridization, fluores- cence activated cell sorting (FACS), receptor binding in Treatment of atherosclerosis fluorescence resonance energy transfer (FRET), fluores- Skin function activation factor cence immunoassays, fluorescence microscopy, multi color As a agent that obstructs absorption of environmental pollutant deposition in the body immunofluorescence and other imaging techniques. The Cosmetic use properties such as excellent stability, high water solubility, In drink/beverage compositions extremely high quantum efficiencies, easy to link to anti- Light protection in packaging bodies and other proteins, large Stokes shifts-excitation As a fabric dye and emission bands at visible wavelengths, intense long Quantification of U-V wavelength excitation and emission (relatively free of For coating on Laminaria interference from other biological materials), stability of As a ingredient in tissue culture medium As a plant growth regulator the chromophore after binding to their ligands and target, As a molecular weight marker make phycobiliproteins a valuable commercial commodity. J Appl Phycol (2008) 20:113–136 127

Table 8 Yield of organisms employed for phycobiliproteins production as reported in applied research

Organisms Nature of pigment Yield (Dry weight %) Reference

Cyanobacteria Anabaena sp. Phycocyanin 8.3 (Rodriguez et al. 1989) Nostoc sp. Phycocyanin 20.0 (Silva et al. 1989) Phormidium valderianum Phycocyanin 20.0 (Sekar and Subramanian 1998) Spirulina fusiformis C-phycocyanin 46.0 (Minkova et al. 2003) Spirulina platensis C-phycocyanin 9.6 (Zhang and Chen 1999) Allophycocyanin 9.5 Red algae Rhodosorus marinus Phycoerythrin 8.0 (Dupre et al. 1995) Phorphyridium cruentum B-phycoerythrin 32.7 (Roman et al. 2002) R-phycocyanin 11.9

pigments, toxicological evaluation has to be performed. used and their yield obtained was up to 32.7% (Table 8). There is a need to improve thermo stability, aqueous stability, The success in the production of phycobiliproteins depends pH stability, alcohol resistant, light stability and shelf life of on the nature of organisms, its growth characteristics, the pigments. In addition, smaller size seems to be advanta- availability of mass cultivation technology, the extent of geous and hence lot of scope exists with cryptomonads which accumulation of pigments, etc. It also depends on the also harbor novel phycobiliproteins and phycobilins. More- efficacy of downstream processing. Further research is over, suitability of separated phycobilins for application needed to develop fast growing organisms adapted to suit purposes was not looked into. Further, exploration of conditions in mass cultivation like resistant to photobleach- phycoerythrocyanin, finding new and novel phycobilins and ing, contaminants, variation in temperature are required. phycobiliproteins as well as making color combination using Further, technologies for the high accumulation of phyco- phycobiliproteins should also be investigated. biliproteins using organismal and genetic modification are Phycocyanin is extensively used as a colorant rather than required. Efforts should also be made to isolate high phycoerythrin. However, phycoerythrin is very much used pigment producers too. in fluorescent applications. Patent analysis revealed that A perusal of patent and research literature shows the most of the US patents are related to fluorescent application trends in extraction and purification methods. The down- while most of the Japanese patents are related to the stream processing comprises of various steps, viz. cell production, purification and application for therapeutic and disruption, primary isolation and purification and drying. A diagnostic purposes. It also covers the novel use of variety of physical and chemical methods are encountered phycobiliprotein as lipase activity inhibitor, serum lipid for cell disruption according to the nature of organisms. reducing agent, skin function activation factor and reagent Physical methods like sonication, cavitation, osmotic to obstruct the environmental pollutant deposition in the shock, and repeated freeze thawing are commonly encoun- body, fabric dye, ingredient in tissue culture media, plant tered. In chemical methods, usage of acids, alkali, deter- growth regulator and molecular weight maker. gents, enzymes and their combination thereof are reported. A comparison of patents, applied research and commer- In general, combinations of a variety of physical and cial activities indicate the common use of phycobiliprotein chemical methods are exploited for cell breakage. After cell for fluorescent purposes (Table 5). Further, biomedical breakage, clarification by centrifugation was performed and properties such as anti-inflammatory, anti-oxidant, liver the product is primarily isolated from the supernatant. It protection, anti-tumor, lipase activity inhibitor, serum lipid comprises of fractionation using ammonium sulphate, reducing agent is documented both in patents and applied dialysis and polyethylene glycol precipitation. Further research that are not commercially ventured (Table 6). purification is usually achieved by column chromatographic Similarly, a number of other novel properties are reported methods using either adsorbents, molecular sieves, anion only in patents (Table 7). Some of these properties exchangers or their combinations (Table 9). According to documented in patents and applied research can be looked the nature of organisms, a combination of physical and for commercialization. chemical methods with primary isolation and purification of In relation to the nature of organisms and yield potential, the product is adapted. For drying the pigment, only freeze wide variations are encountered. Phycocyanin is commonly drying was found to be satisfactory and no other methods produced using cyanobacteria and their yield is reported as seem to be suitable. It is up to the technologist to choose high as 46%. For phycoerythrin production, red algae are any of these methods and their combination to achieve 128 J Appl Phycol (2008) 20:113–136

Table 9 Extraction and purification methods exploited in applied research and patents

Cell disruption Primary isolation Column chromatographic Physical methods Chemical methods purification

Sonication 0.05% or 0.15% digitonin Fractionation using Adsorbents (Myers and Kratz 1955) (Viskari and Colyer 2003) Ammonium sulphate (Bennett and Bogorad 1971) Sonication with sand 3% Chaps & 0.3% asolectin Brushite (Siegelman (Wiltshire et al 2000) (Viskari and Colyer 2003) and Kycia 1978) French press cell 1% cellulase, 0.1% pecto lyase Hydroxy apatite (Bryant et al 1976) (with or without Triton X-100) (Boussiba and (Viskari and Colyer 2003) Richmond 1979 Nitrogen cavitation 5 mM phosphate buffer (pH 7) Molecular cut-off by dialysis Molecular sieves (Viskari and Colyer 2003) with 0.1 or 1.0% Triton X-100 (Boussiba and Richmond (Viskari and Colyer 2003) 1979) Repeated freeze thawing 250 mM Trizma,10 mM EDTA Sephadex G -25 in phosphate or acetate buffer & 2 mg/ml lysozyme (Bhaskar et al (Ranjitha and Kaushik 2005) (with or without Triton X-100) 2005) (Viskari and Colyer 2003) Repeated freeze thawing 10 mM NaOH followed by Sephadex G -100 in liquid nitrogen 10 mM HCL (Kilpatrick 1985) (Bremjo 2006) (Stewart and Farmer 1984) Freezing in liquid nitrogen 25 mM phosphate buffer,10 mg/ml Selective precipitation Anion exchanger & grinding using mortar lysozyme(pH 7) (Kilpatrick 1985) of phycobilisomes using and pestle (Stewart polyethylene glycol and Farmer 1984) (Rigbi et al 1980) Freeze thawing 100 mM phosphate buffer, DEAE - Cellulose in distilled water 200 mM NaCl,10 mM EDTA, (Bennett and (Padgett and 0.02% Triton X-100 & 1 mg/ml Bogarad 1971) Krogmann 1987) lysozyme (Vernet et al 1990) Using mechanical tissue 1 M Tris (pH 8.0), 0.5 M EDTA, DEAE-sepharose homogenizers 20% sucrose CL-6B (Zhang (Kao et al 1975) (w/v) with 5 mg/ml lysozyme and Chen 1999) (Bhaskar et al 2005) Extraction of dried 1% rivanol (Minkova et al 2003) Streamline - DEAE bio mass at 4°C (Bremjo 2006) (Doker 2005) Q-Sepharose (Sajilata and Singhal 2006) maximum recovery of the product. This is promoted by the and will certainly continue for a long time to hold captivating amenability of the organisms for the extraction of phyco- interest for mankind. The number of cyanobacterial and red biliproteins in addition to the designing of equipment for algal strains that are currently amenable to phycobiliproteins down streaming process. productions is miniscule; none of the characterized strains are significant producers of the products. Basic research is needed to expand the range of organisms represented in Conclusion culture collections beyond the fast-growing strains that currently make up the bulk of available cultures. Screening For more than 150 years, phycobiliproteins and phycobili- of additional cyanobacteria for the presence of biliproteins somes were extensively studied and revealed a lot of their with desirable characteristics will result in additional mystery hidden behind their glowing colors. Their remark- discoveries of this nature. It is very clear that existence of able physio-chemical properties, and their wonderful struc- patents in this field is huge and the applied research is also ture, so precisely fitted to harvest and transfer light energy to gaining upper hand, the problem lies in the commercializa- the photosynthetic reaction centres, still fascinate researchers tion. The future depends on the expansion of the range of J Appl Phycol (2008) 20:113–136 129 products to be manufactured as well as improvements in Bernasconi P, Carpenter RD, Powell GK, Petrovich RM, White KA, bioprocess engineering. Hunt JA (2003) Parallel high throughput method and kit. US Patent 6,586,190, 1 July 2003 Bhaskar US, Gopalswamy G, Raghu R (2005) A simple method for Acknowledgement The authors acknowledge the financial support efficient extraction and purification of C-Phycocyanin from extended by the Department of Science and Technology, New Delhi, Spirulina platensis Geitler. 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