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Method to obtain C-phycocyanin of high purity
Abstract
A new approach is made for the purification of C-phycocyanin (C-PC), which is simple and more efficient than existing methods. The proposed method involves two steps: aqueous two phase extraction and ion-exchange chromatography. Crude extract of C-phycocyanin, of purity 1.18, obtained from Spirulina platensis is subjected to aqueous two phase extraction. C-phycocyanin obtained from this process showed a purity of 5.22, which is higher than the reported value till date. In order to explore the possibility of further purification, C-phycocyanin is subjected to ion-exchange chromatography and found that the purity increased from 5.22 to 6.69. The fluorescence, intactness of structure and purity of C-phycocyanin are confirmed using spectrofluorometry, circular dichroism spectra and sodium dodecyl sulfate-polyacrylamide gel, respectively.
... Purity values ≥ 0.7, 3.0-3.9, and ≥4.0 are the recognized standards for food-grade, reaction grade, and analytical grade C-PC, respectively, and determine the price and application of extract (Patil et al., 2006). Analytical grade C-PC can cost up to US $15.00 per milligram, nearly 100 times that of food-grade (Güroy et al., 2017). ...
... As evident from the above results regarding extract from batch culture, the purity of obtained crude C-PC extracts is usually lower than or close to food-grade quality, therefore, subsequent purification steps are required to improve C-PC purity. However, these processes are time-consuming and expensive, and the costs account for 50-90% of the purified products (Patil et al., 2006). In the present study, the purity of C-PC extract from two-stage culture reached the reaction grade (A 620 /A 280 = 3.16) after ammonium sulfate precipitation, suggesting that the purity of C-PC products could be effectively enhanced by increasing the C-PC content in Arthrospira and thus, eliminating expensive purification procedures, such as ultrafiltration, chromatographic method and aqueous two-phase extraction (Fabre et al., 2022;García-López et al., 2020;Khazi et al., 2018;Lee et al., 2016). ...
... Amarante et al. (2020) [200] obtained a phycocyanin purity between 3.5 and 4.2 and recoveries between 32% and 49% using a single IEC step with pH gradient elution. Patil et al. (2006) [207] described a purification method for phycocyanin using ion-exchange chromatography, which resulted in a purity of 5.22. ...
... Amarante et al. (2020) [200] obtained a phycocyanin purity between 3.5 and 4.2 and recoveries between 32% and 49% using a single IEC step with pH gradient elution. Patil et al. (2006) [207] described a purification method for phycocyanin using ion-exchange chromatography, which resulted in a purity of 5.22. ...
Large-scale production of microalgae and their bioactive compounds has steadily increased in response to global demand for natural compounds. Spirulina, in particular, has been used due to its high nutritional value, especially its high protein content. Promising biological functions have been associated with Spirulina extracts, mainly related to its high value added blue pigment, phycocyanin. Phycocyanin is used in several industries such as food, cosmetics, and pharmaceuticals, which increases its market value. Due to the worldwide interest and the need to replace synthetic compounds with natural ones, efforts have been made to optimize large-scale production processes and maintain phycocyanin stability, which is a highly unstable protein. The aim of this review is to update the scientific knowledge on phycocyanin applications and to describe the reported production, extraction, and purification methods, including the main physical and chemical parameters that may affect the purity, recovery, and stability of phycocyanin. By implementing different techniques such as complete cell disruption, extraction at temperatures below 45 °C and a pH of 5.5–6.0, purification through ammonium sulfate, and filtration and chromatography, both the purity and stability of phycocyanin have been significantly improved. Moreover, the use of saccharides, crosslinkers, or natural polymers as preservatives has contributed to the increased market value of phycocyanin.
... The solution was centrifuged again in the same conditions and the precipitate was dissolved in DW, dialyzed to remove ammonium sulfate residues, and freeze-dried. The purity ratio of the PBP was quantified spectrally using synergy H1 microplate reader (Biotek, Winooski, VT, USA) based on a previously published method by Patil et al. (2006) [36]. The protein content was analyzed with CHNS elemental analyzer (Flash 2000, Thermo-scientific, Milan, Italy) based on previ- ...
... The solution was centrifuged again in the same conditions and the precipitate was dissolved in DW, dialyzed to remove ammonium sulfate residues, and freeze-dried. The purity ratio of the PBP was quantified spectrally using synergy H1 microplate reader (Biotek, Winooski, VT, USA) based on a previously published method by Patil et al. (2006) [36]. The protein content was analyzed with CHNS elemental analyzer (Flash 2000, Thermo-scientific, Milan, Italy) based on previ- ...
The utilization of natural blue pigments in foods is difficult as they are usually unstable during processing and the commonly applied pH. The current study focuses on natural blue pigment, possessing antioxidant properties, found in Arthrospira platensis (spirulina), and phycobiliproteins (PBP). These pigments are a complex of conjugated protein and non-protein components, known as phycocyanobilin. PBP has low stability during pasteurization (high-pressure or heat treatments), resulting in protein denaturation and color deterioration that limits the application. The phycocyanobilin pigment might also be liable to oxidation during pasteurization and storage, resulting in color deterioration. Yet, the instability of the pigment phycocyanobilin during the pasteurization process and storage conditions was never studied before, limiting the comprehensive understanding of the reasons for PBP instability. In this study, the stability of phycocyanobilin under high-pressure and high-temperature conditions was compared to the stability of phycobiliproteins. We revealed that phycobiliproteins have a higher color deterioration rate at 70–80 °C than at high-pressure (300–600 MPa) whereas phycocyanobilin remained stable during high-pressure and heat processing. During storage at pH 7, phycocyanobilin was oxidized, and the oxidation rate increased with increasing pH, while at lower pH phycocyanobilin had low solubility and resulted in aggregation.
... It is therefore commercially interesting and important to develop a rapid and efficient R-PE purification technique that produces biologically active R-PE in high yield under mild conditions. An affinity precipitation technique (APT) based on chitosan (CS) and AC is a potential alternative to existing methods that can address the above-mentioned objectives and limitations (Patil et al. 2006;Gupta and Sainis 2010;Liao et al. 2011;Lee et al. 2016). This method offers some advantages over conventional methods, such as time savings, simplicity, safety (non-toxic biosorbents), and the ability to physically and efficiently adsorb impurities (Kaixian et al. 1993;Kanatt et al. 2013;Dastkhoon et al. 2015). ...
... The low FI of purified R-PE, particularly R-PE purified by the ASP method, could be due to unfavorable changes in PE's spatial structure during purification processes, but it was higher in R-PE purified by PECE + AC + IE. The study findings on phycocyanin were consistent with the results of Patil et al. (2006) and Fekrat et al. (2019) who found that the FI reduced when the native form of phycocyanin was manipulated. Finally, it can be concluded that the AA and FI of PC have a linear regression relationship. ...
Phycoerythrin (R-PE) is a naturally occurring colorant with excellent fluorescent and antioxidant properties with a wide range of applications. This study aimed to compare the use of different purification methods, which are activated charcoal (AC), ammonium sulfate precipitation (ASP), or anion-exchange chromatography (IE) alone and a combination of AC, ASP, and IE, for R-PE extraction from the Caspian Sea red macroalga (Osmundea caspica). Response surface methodology (RSM) was employed for preliminary purification, with the independent variables being activated charcoal content (0.1-1% w/v) and stirring time (2-10 min). With an AC content of 0.4% and a stirring time of 2 min, R-PE purity and concentration were optimized. The predicted values from the equations agreed well with the experimental values, demonstrating the model's robustness. A three-step increase in the ionic strength of IE was also evaluated at three different treatments (PECE, PECE + AC + IE, and ASP + IE). The highest R-PE purity and lowest Fluorescence intensity and antioxidant activity were obtained in the fraction of AE-200 (second fraction) with an index of 2.8 and a recovery yield of 48% in ASP + IE, whereas an index purity of 2 and the highest recovery yield of 67% were obtained using PECE + AC + IE.
... The concentration of phycocyanin (C-PC), allophycocyanin (APC), and phycoerythrin (PE) were calculated using Equations (1)-(3), which were described by Bennett and Bogorad [67]. The purity of C-PC, APC, and PE was determined using Equations (4)-(6) proposed by Patil [68] and Antello et al. [69], where each of the phycobiliprotein is divided by the total content of proteins measured at 280 nm ...
... The purity of C-PC, APC, and PE was determined using Equations (4)-(6) proposed by Patil [68] and Antello et al. [69], where each of the phycobiliprotein is divided by the total content of proteins measured at 280 nm ...
This study evaluates the role of different LED lights (white, blue/red), intensity (µmol m−2 s−1), and photoperiod in the production of biomass and phycocyanin-C, allophycocyanin and phycoerythrin (C-PC, APC, and PE respectively) from a novel thermotolerant strain of Oscillatoria sp. Results show that a mixture of white with blue/red LEDs can effectively double the biomass concentration up to 1.3 g/L, while the concentration of the selected phycobiliproteins increased proportionally to biomass. Results also indicate that high light intensities (>120 µmol m−2 s−1) can diminish the final concentration of C-PC, APC, and PE, significantly reducing the overall biomass produced. Finally, the photoperiod analysis showed that longer light exposure times (18:6 h) improved both biomass and phycobiliproteins concentration. These results demonstrate that the application of LEDs to produce a novel strain of Oscillatoria sp can double the biomass concentration, and the photoperiod regulation can eventually enhance the final concentration of specific phycobiliproteins such as APC and PE.
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... [ DOI:10.52547/fsct.19.123.145 ] [ DOR: 20.1001.1.20088787.1401.19.123.24.9 ][ Downloaded from fsct.modares.ac.ir on 2022-[11][12][13][14][15][16][17][18][19][20][21][22][23][24] ...
... Following a successful extraction, various purification steps might be needed to obtain high purity PC (de Amarante et al. 2020). The most commonly applied purification methods include ammonium sulphate precipitation (Boussiba and Richmond 1979), membrane dialysis (Kaur et al. 2019), gel filtration (Minkova et al. 2003), ion-exchange chromatography (Jian-Feng et al. 2007), activated charcoal-chitosan treatment (Fekrat et al. 2019) and aqueous two phase system (ATPS) (Patil et al. 2006;Mogany et al. 2019). These methods can also be applied in combination to increase the final purity of the PC for certain applications such as nutrient supplements and natural food colorants (Patil et al. 2006). ...
... The most commonly applied purification methods include ammonium sulphate precipitation (Boussiba and Richmond 1979), membrane dialysis (Kaur et al. 2019), gel filtration (Minkova et al. 2003), ion-exchange chromatography (Jian-Feng et al. 2007), activated charcoal-chitosan treatment (Fekrat et al. 2019) and aqueous two phase system (ATPS) (Patil et al. 2006;Mogany et al. 2019). These methods can also be applied in combination to increase the final purity of the PC for certain applications such as nutrient supplements and natural food colorants (Patil et al. 2006). ...
Phycocyanin is a water-soluble, blue-colored phycobiliprotein mainly found in cyanobacteria and Rhodophyta. Major commercial interest exists on phycocyanin as an important ingredient for food, pharmaceutical and cosmetics applications. Despite the common interest from industry, there is no dedicated method for optimized extraction of phycocyanin from different species. Yet, biomass type and choice of pretreatment have major influences on the extraction yield and final purity of phycocyanin. Various impurities such as cell debris and other photosynthetic pigments also decrease the quality of extracted phycocyanin. In this study, wet and lyophilized biomass samples from several cyanobacteria (including a local isolate) and a red microalga species were harvested and pretreated with bead-beating, mortar and pestle homogenization, freeze–thaw cycling and sonication techniques. High concentration yielding phycocyanin extracted from Phormidium sp., Synechocystis sp., Desertifilum tharense, Nostoc sp., and Galdieria sulphuraria were purified by ammonium sulfate fractionation combined with acetate buffer elution method. Bead-beating was found to be the most efficient pretreatment technique for the extraction of phycocyanin from the majority of tested biomass samples. Phycocyanin from Synechocystis sp. and Phormidium sp. was further purified with anion exchange chromatography. Overall, food grade phycocyanin (purity ratio A620/A280 > 0.7) extraction was achieved for all tested biomass samples except Scytonema sp. Biomass samples from Synechocystis sp. yielded analytical grade phycocyanin (purity ratio > 4), one of the highest values observed in literature.
... January 2022 | Volume 10 | Article 774760 PC concentration in the crude extract was determined by the methods of Bennet and Bogorad (1973). PC purity in extracts was measured as the ratio of the optical absorbances at 620 and 280 nm (Patil et al., 2006). PC yields were calculated as mg PC g AFDW mg PC ml p extraction volume (ml) AFDW of biomass g Phycocyanobilin (PCB) was extracted from the unwashed and lyophilized outdoor pond RABR biomass as described previously (Ito, et al., 2013) using 0.05 M sodium phosphate buffer (pH 7) as phycocyanin extraction buffer. ...
... The phycocyanin yield from the two materials differed greatly with the cotton belt material displaying 47.0 mg PC/g AFDW when compared to the cotton rope material at 3.4 mg PC/g AFDW (Table 2). Similarly, the crude phycocyanin extract purity was 5.6 times higher with the cotton belt material, however not to food grade purity level of 0.7 without further purification (Patil et al., 2006). Phycocyanobilin (PCB) content was evaluated solely in the biomass from the cotton belt material due to the low phycocyanin levels and purity in the biomass from the cotton rope material. ...
Production and enhancement of high value phycocyanin pigment from microalgae biofilms cultured on oilfield and natural gas produced wastewater were investigated. Cyanobacteria isolated from Logan City, Utah, wastewater treatment Lagoons (LLC2) was cultured in produced water using rotating algal biofilm reactors (RABRs). The RABRs were operated under “low” and “high” light conditions and biomass and phycocyanin content were compared. Phycocyanin content was enhanced by growth under low light conditions to a maximum yield of 31.7 mg/g ash-free dry weight (AFDW) biomass for an 87.6% increase in phycocyanin yield. Phycocyanin productivity was equivalent for both the low and high light treatments (327 ± 81 and 305 ± 39 mg/m ² /day, respectively), due to the significantly lower AFDW biomass productivity of the low light treatment (2.7 ± 0.4 g/m ² -day). An indoor laboratory evaluation of 14 substrata for biofilm growth showed that cotton rope and cotton belt material provided the highest biomass yields. Further evaluation in a pilot-scale outdoor produced wastewater pond showed that the biomass characteristics from the two substrata differed. The corrugated surface area of the cotton rope cultured a biofilm with a large community of non-photosynthetic organisms with an autotrophic index of 507 and a low phycocyanin yield of 3.4 mg/g AFDW. However, the cotton belt substratum cultured a healthy photosynthetic biofilm with an autotrophic index of 127 and a phycocyanin yield of 47.0 mg/g AFDW. These results demonstrate the cultivation of microalgae biomass and valorization of oilfield and natural gas produced wastewater through the design and management of algal-based biofilm photobioreactors.
... Sodium azide and dithiothreitol are commonly used as preservatives of PE for analytical purpose, but they are toxic (Mishra et al. 2010); so for developing process of food grade PE only edible preservatives with unique properties can be used (Mishra et al. 2010). It is therefore, desired to develop a simple, but more well-organized method for the separation, purification and stabilization of the food grade PE from cyanobacteria (Patil et al. 2006). Food additives have been used by mankind for centuries, e.g. ...
Cyanobacteria have many bioactive compounds. In the present study, we investigated the degree of purification and free radical scavenging ability of phycocyanin (PC) and phycoerythrin (PE), and compare their stability against selected preservatives at different temperatures with the aim of achieving the best and most stable preservative in increasing shelf life of PC and PE. After collecting and culturing Nostoc sp. strains FSN and ASN in BG-110 medium, the pigments phycocyanin and phycoerythrin were extracted and purified with 56% ammonium sulfate followed by dialysis. The antioxidant activity of pigments was evaluated by DPPH and ABTS assays. Their stability was compared with food-grade preservatives citric acid, sodium chloride, sucrose, and calcium chloride at two temperatures of 5 °C and 35 °C over time period from 3 to 30 days of cultivation. The results showed that the concentration and purity of the pigments increased after the dialysis, the pigments had antioxidant properties and were more stable at 5 °C. In addition, among different preservatives, citric acid caused more stability over time.
... The grade of phycocyanin is determined using the purity ratio values, ≥ 0.7 is food grade phycocyanin, ≥ 3.9 is reagent grade phycocyanin, and ≥ 4.0 is analytical grade phycocyanin (Patil et al., 2006). ...
Phycocyanin is a blue accessory protein pigment that is abundant in Spirulina sp. and has gained popularity due to its diverse applications in various industries. Phycocyanin is rich in natural properties that can be used as a nutraceutical, combining food and pharmaceutical sectors. It has been explored in the pharmaceutical industry by combining with disease-specific drugs and has been experimented against various conditions such as cancer, anemia, inflammation, diabetes, obesity, and neurodegenerative disorders. Additionally, phycocyanin has been used as a natural alternative to artificial food colorants, which imparts color to food and boosts the nutrient value of the food. The market value of phycocyanin is projected to reach $279.6 million by 2030, with a CAGR of 28.1%. Despite its various benefits, phycocyanin has some drawbacks, including instability towards light, pH, and temperature, lower yield, and higher production costs, which limit the expansion of the industry. This comprehensive review provides an overview of different aspects of phycocyanin production and utilization, including Spirulina cultivation, various extraction and purification strategies, and its applications predominantly in food industries and other allied sectors.
... For the supernatant, the concentration of the organic acids acetate, formate, propionate, butyrate, succinate, and lactate were measured using an UltiMate 3000 HPLC system (ThermoFisher Scientific, USA) equipped with an Aminex HPX-87H column and a UV detector, as previously described [86]. The phycocyanin and total protein concentration in the supernatants were measured as absorption at 620 nm and 280 nm respectively [87] using an Evolution 260 Bio UV-Visible Spectrophotometer (ThermoFisher Scientific, USA), with a standard curve prepared from laboratory-grade phycocyanin (Sigma-Aldrich, USA). Bright-field microscope images were taken using a Zeiss Axio Imager A2 Microscope (Carl Zeiss AG, Germany). ...
Cyanobacteria form dense multicellular communities that experience transient conditions in terms of access to light and oxygen. These systems are productive but also undergo substantial biomass turnover through cell death, supplementing heightened heterotrophic respiration. Here we use metagenomics and metaproteomics to survey the molecular response of a mat-forming cyanobacterium undergoing mass cell lysis after exposure to dark and anoxic conditions. A lack of evidence for viral, bacterial, or eukaryotic antagonism contradicts commonly held beliefs on the causative agent for cyanobacterial death during dense growth. Instead, proteogenomics data indicated that lysis likely resulted from a genetically programmed response triggered by a failure to maintain osmotic pressure in the wake of severe energy limitation. Cyanobacterial DNA was rapidly degraded, yet cyanobacterial proteins remained abundant. A subset of proteins, including enzymes involved in amino acid metabolism, peptidases, toxin-antitoxin systems, and a potentially self-targeting CRISPR-Cas system, were upregulated upon lysis, indicating possible involvement in the programmed cell death response. We propose this natural form of cell death could provide new pathways for controlling harmful algal blooms and for sustainable bioproduct production.
... Although UAE gives many advantages, there is no report available on biphasic extraction from Spirulina using this technique, to date. In fact, almost all the methods reported in the literature for the purification of phycocyanin require numerous of expensive and time-consuming steps, such as precipitation, centrifugation, dialysis, ion-exchange chromatography, gel filtration chromatography [24][25][26]. Moreover, their purification cost ranges between 50 and 90 % of the entire production [27]. ...
To meet the criteria of modern extraction, it is essential to use solvents that simultaneously reach the standards of green chemistry and allow the extraction of a wide spectrum of bio-compounds. Natural Deep Eutectic Solvents (NaDES), as a green solution, meet this difficult task and represent a satisfactory alternative to organic solvents. Microalgae are recognized as a sustainable resource to produce polar and non-polar metabolites, such as ca-rotenoids, free fatty acids (FFA) and phycobiliproteins. Among them, Spirulina (Arthrospira platensis) is one of the most studied, due to its strong potential for food, health and cosmetic. In this work, two biorefinery scenarios were investigated using polar and non-polar NaDES. The first one is a combination of a polar/non-polar sequential solid/liquid extraction together with a liquid-liquid extraction of the polar extract. The second scenario implements an innovative triphasic solid/liquid/liquid approach, based on the simultaneous use of polar and non-polar NaDES in the solid/liquid extraction step. Several NaDES were screened in this study to identify the best polar/non-polar NaDES pair. According to the results, the use of the triphasic approach allowed an increase of the productivity by a factor 4 for chlorophylls and FFA, and by a factor 2 for carotenoids. It was also interesting to note that the phycocyanin-enriched polar fraction achieves much higher levels of purity than the corresponding single-phase extraction for both applied biorefinery scenarios. These results pave the way for the first time for a microalgae biorefinery entirely based on the use of NaDES and industrially credible.
... Although UAE gives many advantages, there is no report available on biphasic extraction from Spirulina using this technique, to date. In fact, almost all the methods reported in the literature for the purification of phycocyanin require numerous of expensive and time-consuming steps, such as precipitation, centrifugation, dialysis, ion-exchange chromatography, gel filtration chromatography [24][25][26]. Moreover, their purification cost ranges between 50 and 90 % of the entire production [27]. ...
... C-PC should be purified depending on the field of use [33]. The purity of C-PC is typically estimated according to the absorbance ratio of A620/A280, wherein a purity of 0.7 is considered a food grade, 3.9 as a reactive grade, and values greater than 4.0 as an analytical grade [34]. ...
Cyano-phycocyanin (C-PC) is a light-absorbing biliprotein found in cyanobacteria, commonly known as blue-green algae. Due to its antioxidative, anti-inflammatory, and anticancer properties, this protein is a promising substance in medicine and pharmaceuticals. However, cyanobacteria tend to bind heavy metals from the environment, making it necessary to ensure the safety of C-PC for the development of pharmaceutical products, with C-PC isolated from naturally collected cyanobacterial biomass. This study aimed to determine the content of the most toxic heavy metals, arsenic (As), cadmium (Cd), mercury (Hg), and lead (Pb) in C-PC isolated from different cyanobacterial biomasses collected in the Kaunas Lagoon during 2019–2022, and compare them with the content of heavy metals in C-PC isolated from cultivated Spirulina platensis (S. platensis). Cyanobacteria of Aphanizomenon flos-aquae (A. flos-aquae) dominated the biomass collected in 2019, while the genus Microcystis dominated the biomasses collected in the years 2020 and 2022. Heavy metals were determined using inductively coupled plasma mass spectrometry (ICP-MS). ICP-MS analysis revealed higher levels of the most investigated heavy metals (Pb, Cd, and As) in C-PC isolated from the biomass with the dominant Microcystis spp. compared to C-PC isolated from the biomass with the predominant A. flos-aquae. Meanwhile, C-PC isolated from cultivated S. platensis exhibited lower concentrations of As and Pb than C-PC isolated from naturally collected cyanobacterial biomass.
... The protein part of C-phycocyanin is a trimeric (αβ) 6 or a hexameric (αβ) 3 consisting of an α subunit with a molecular weight of 18 kDa and a β subunit of 20 kDa (Patil et al. 2006;Galland-Irmouli et al. 2000). The only phycobilin to which it is attached is phycocyanobilin. ...
Pigment is an important food additive that plays a major role in the sensory impact of food. And natural sources, healthy and non-toxic edible pigments are receiving a lot of attention. Algae is an important source of natural pigments, and contain chlorophyll, phycoerythrin, carotene, and other natural pigments. Besides staining, the pigment also has powerful physiological activities such as antioxidants, anti-inflammatory, anti-obesity, and lipid-lowering. In this paper, three pigments in seaweed were reviewed, and their main structural properties and functions are presented, filling the gap in the review of pigments with seaweed as the main object of introduction. This review provides research basis for the development of new health foods, a new direction for the use of seaweed chlorophyll in the food and pharmaceutical industries. Graphical Abstract
... Absorbance was measured at 760 nm using a glass cuvette. The quantitative results were calculated using a standard curve of gallic acid and are expressed as mg of gallic acid equivalents per kg of OMW sample [44]. ...
The aim of this study was to formulate nanodispersions to encapsulate antioxidants extracted from olive mill wastewater (OMW) and phycocyanin extracted from Spirulina maxima to act as enhancers for the skin’s protection against UV radiation. For this purpose, two water-in-oil nanoemulsions were prepared using a low-energy homogenization method. Both systems were based on isopropyl myristate as the continuous phase, while water or a mixture of glycerol and water was used as the dispersed phase. Then, antioxidants extracted from OMW and phycocyanin from Spirulina maxima were encapsulated in the water core of the nanoemulsions. The empty and antioxidant-loaded systems were then structurally studied using dynamic light scattering for the detection of their droplet size and stability over time. Electron paramagnetic resonance (EPR) spectroscopy using adequate probes was applied for the characterization of the surfactants’ monolayer in the presence and absence of antioxidants. It was found that the mean droplet diameter of the emulsions was 200 nm. The nanoemulsions remained stable for over 2 months. The encapsulated antioxidants were assessed for their scavenging activity of a model stable radical by applying EPR spectroscopy. It was found that the loaded systems exhibited an increased antioxidant capacity compared with the empty ones. Finally, the most stable system was added to commercial sunscreen lotions and the overall sun protection factor (SPF) was assessed. The sunscreen lotions that contained the nanoemulsions loaded with OMW extracts or phycocyanin showed an increase in their SPF value.
... Therefore, there has been no indication of Cphycocyanin's stability loss. A similar pattern has been reported in other works (Patil et al., 2006). It was observed that the secondary structure of the standard sample was completely consistent with that of the purified C-phycocyanin using the ATPS method. ...
C-phycocyanin is a high market value compound derived from cyanobacterium Spirulina platensis and has a wide range of applications in pharmaceutical and food industries as a useful biochemically active compound. In this study, essentially an aqueous two-phase system (ATPS) is presented for purification of C-phycocyanin. The phase behavior of ATPSs that include Pluronic copolymers and salts was evaluated systematically. After that, the effects of different parameters such as salt type, copolymer structure, pH of solution, tie line length (TLL), system’s temperature, and volume ratio for purification of C-phycocyanin were investigated. The results revealed that Pluronic 10R5/potassium phosphate buffer system is the most appropriate system for promoting the C-phycocyanin separation from other contaminants. Besides, the purity index of C-phycocyanin was enhanced by up to 3.92 at TLL of 46.31%, pH = 6, volume ratio of 0.34, and temperature of 35 °C without any loss of stability. At these conditions, the obtained C-phycocyanin recovery was 90%. After that, the purity factor reached to 5.9 by applying the ultrafiltration method. In addition, the results of circular dichroism (CD) spectroscopy proved that the C-phycocyanin structure remained intact during the purification step. Finally, 78% of 10R5 copolymer was recovered by increasing the temperature above 57 °C (micelle formation (which can be used in new ATPS.
... The purity of phycocyanin is assessed by measuring the absorbance ratio A620/A280. The absorbance at A620 corresponds to pigment whereas the absorbance at A280 represents total protein content 37 . An absorbance ratio value lesser than 0.7 is considered food grade, a ratio in the range 0.7 -3.9 is considered as reagent grade; and a ratio greater than 4.0 is considered as analytical grade 2, 21, 37 . ...
Cyanobacteria are simple, photosynthetic, prokaryotic blue-green algae. They can thrive in fresh water, terrestrial, glacial, aerial, brackish and marine regions. Cyanobacteria have been exploited in recent decades due to the presence of compounds with potential activity like pigments, vitamins, proteins, carbohydrates, minerals etc. These potential compounds have valuable applications as pharmaceuticals, nutraceuticals and cosmeceuticals and also as health supplements. This review focuses on the phycocyanin pigment of cyanobacteria and its pharmaceutical applications. Phycocyanin is a blue-colored pigment protein that is abundant in cyanobacteria. Phycocyanin is exploited due to its antioxidant properties, antidiabetic, anti-inflammatory and anticancer activity. This pigment can be used as a nutrient supplement in order to treat age-related diseases and other oxidative stress-related diseases.
... According to this purity rate, the usage area of phycocyanin is determined. If the purity rate is 0.7 and above, it is considered suitable for food, 3.9 is reactive, and if it is 4 and above, it is considered to be analytical purity (Patil et al., 2006). Furthermore, phycocyanin has the ability to dissolve in water can facilitate the puri cation procedure, allowing these molecules to be separated from other oil-soluble pigments. ...
Blue-green cyanobacteria, which have attracted great interest in recent years due to their potential applications in biotechnology, have an essential ability to synthesize natural pigments of different colors. The chosen study is aimed at investigating the strain with the highest phycocyanin yield among the five cyanobacterial strains isolated from the wastewaters of Porsuk river, thermal springs, and boron-bearing waters of Eskişehir county of Turkey, and optimized studies, purification, and characterization for increasing the phycocyanin yield. At the initial stage, the phycocyanin yield was analyzed by applying four different extraction methods - freeze-thawing, sonication, homogenization, and treatment with lysozyme - to the selected strains. The most appropriate strain sonication method was selected and thermophilic Geitlerema sp was obtained with 116 ± 0.18 mg g − 1 phycocyanin. The crude extract obtained following the optimized studies was first purified by solid ammonium sulfate precipitation/dialysis at 50% and 75% saturation, and then by ion-exchange chromatography using two anionic resins (DEAE -cellulose, and Q -sepharose). The purity rate determined in the spectrum was recorded as 4.12 with 48.9% recovery (A 620 /A 280 ). The molecular weights of the α and β subunits were found to be 17 kDa to 19 kDa, respectively, by characterization using the SDS-PAGE method. In addition, the effect of purified thermal phycocyanin at different temperatures was examined, the CR value of its pigment was determined to be stable by resisting at high temperatures up to 60Cº. Generally, in the study, a thermostable C-phycocyanin was purified from Geitlerema sp, which may be differentiated commercially from other mesophilic species.
... When the molecule is exposed to light, heat, or pH changes it loses its color, and thus its nutraceutical properties [7,8]. Considering this, new alternatives have been proposed to extract it from the cell matrix such as enzymatic digestion, high-pressure processing, ion-exchange chromatography, mortar grinding, pulsed electric fields, ultrasonication, and ultrafiltration [5,6,9,10]. It has been suggested, that ultrasonication extracts more intact phycocyanin-C in comparison to water extraction [11]. ...
Several phycocyanin extraction methods have been proposed, however, most of them present economical or productive barriers. One of the most promising methods that has been suggested is ultrasonication. We have analyzed here the effect of operational conditions and additives on the extraction and purity of phycocyanin from Arthrospira maxima. We followed three experimental designs to determine the best combination of buffered pH solutions, additives, fresh and lyophilized biomass. We have found that additives such as citric acid and/or disaccharides could be beneficial to the extraction process. We concluded that the biomass–solvent ratio is a determining factor to obtain high extraction and purity ratios with short ultrasonication times.
... In another study using a new rivanol treatment method 45.7% PC was recovered from S. fusiformis with a PF of 4.3 [152]. Chen et al. [50] [176] demonstrated that aqueous two-phase extraction (ATPE) combined with ion-exchange chromatography a highly pure C-PC with a yield of 73.0%. In addition, this method produced a highly pure product with fewer contaminating proteins without the need for multiple steps. ...
Increasing awareness of the harmful effects of synthetic colorants has led consumers to favor the use of natural alternatives such as plant or microbial pigments in food and cosmetics. Cyanobacteria are a rich source of many natural biopigments that are of high commercial value. In the market, bio-based pigments are usually sold as extracts to reduce purification costs. Various cell disruption methods are used for pigment extraction, such as sonication, homogenization, high pressure, supercritical CO2 extraction, enzymatic extraction, as well as other promising novel extraction methods that make the production of cyanobacterial pigments economically viable. In addition, a continuous cultivation system is considered the most suitable cultivation mode for large-scale biomass production. However, a major limitation in the large-scale production of cyanobacterial pigments is the installation and operation costs. Thus, basic and applied research is still needed to overcome such limitations and enable cyanobacteria to enter the global market. This review focuses on various cyanobacterial pigments, their applications, and current biotechnological approaches to increase the production of biopigments for their potential use in the pharmaceutical, food, and cosmetic industries. The current state of production technologies based on either open pond systems or closed photobioreactors was compared. The potential of scientific and technological advances to increase yield and reduce production costs of cyanobacteria biomass-based pigments was also discussed.
... The low FI of puri ed PE, particularly PE puri ed by the ASP method, could be due to unfavorable changes in PE's spatial structure during puri cation processes, but it was higher in PE puri ed by AC + IE. The study ndings on phycocyanin were consistent with the results ofPatil et al. (2006) andFekrat et al. (2018) who found that the FI reduced when the native form of phycocyanin was manipulated. Finally, it can be concluded that the AA and FI of PC have a linear regression relationship. ...
Phycoerythrin (red pigment) (PE) is a naturally occurring colorant with excellent fluorescent and antioxidant properties that has a wide range of applications. More studies are needed, however, to improve the purification yield and chemical properties of PE. This study aimed to compare the use of different purification methods, which are activated charcoal (AC), ammonium sulfate precipitation (ASP), or anion-exchange chromatography (IE) alone and a combination of AC, ASP, and IE, for PE extraction from the Caspian Sea red macroalgae ( Osmundea caspica ). Response surface methodology (RSM) was employed for preliminary purification, with the independent variables being activated charcoal content (0.1–1% w/v) and stirring time (2–10 min). With an AC content of 0.4% and a stirring time of 2 min, PE purity and concentration were optimized. The predicted values from the equations agreed well with the experimental values, demonstrating the model's robustness. A three-step increase in the ionic strength of IE was also evaluated at three different treatments (PECE, PECE + AC + IE, and ASP + IE). The highest PE purity and lowest Fluorescence intensity (FI) and Antioxidant activity (AA) were obtained in the fraction of AE-200 (second fraction) with an index of 2.8 and a recovery yield of 48% in ASP + IE, whereas an index purity of 2 and the highest recovery yield of 67% were obtained using PECE + AC + IE.
... During extraction, their stability can be improved by adding high concentrations of sugars and salts, such as glucose and sodium chloride, in order to maintain their bright blue color and antioxidant properties. [43] . It has also been shown that light emitting diode lamps and the use of lamp color changes can significantly improve the production of blue pigments [44] . ...
Natural edible blue pigment is a scarce pigment among natural pigments, mainly derived from animal and plant tissues, microbial fermentation metabolites. Among the plant natural blue pigments, the ones that account for the widest market of edible natural blue pigments are algal blue pigment and gardenia blue pigment. The sources of microbial natural blue pigment include some fungi and bacteria in addition to microalgae, mainly focusing on the study of Streptomyces, Pseudomonas, Pseudoalteromonas, purple non-sulfur bacteria, Dourollerella, and sprouting short-stalked mold. The natural blue pigment is unstable, so it is of far-reaching significance to improve the extraction process, especially the research on the isolation and purification of blue pigment produced by microbial fermentation. High-speed frozen centrifugation, solid-phase extraction, high performance liquid chromatography, nuclear magnetic resonance spectroscopy, mass spectrometry and other methods are commonly used for the separation and purification of natural blue pigments and analytical identification. Some natural blue pigments have antioxidant, antitumor, hypolipidemic, hypoglycemic, anti-inflammatory, anti-cancer, immunomodulatory activities, etc. There will be a broad development prospect for the future research of natural blue pigments.
... The absorbance of phycoerythrin at 280 nm and 545 nm were measured using TU-1810 UV-Visible spectrophotometer. The optical density at the wavelength of 280 nm and 545 nm were used to represent the concentration of crude total protein and B-phycoerythrin, respectively [30][31][32][33]. The purity of B-phycoerythrin was expressed by the following equation: ...
Phycoerythrin, a special photosynthetic pigment, is widely used as fluorescent dye and has lots of underlying beneficial effects on health. A marine red microalga Porphyridium is considered as the potential feedstock for phycoerythrin production. However, the phycoerythrin-related properties of Porphyridium have not been systematically evaluated, especially between the species of P. cruentum and P. purpureum. The present study aimed to evaluate the production and fluorescence characteristics of phycoerythrin of three strains of Porphyridium. The results showed that P. purpureum SCS-02 presented the highest biomass, phycoerythrin content and yield were 6.43 g L−1, 9.18% DW and 0.288 g L−1, respectively. There was no significant difference between P. purpureum and P. cruentum in α and β subunits amino acid sequences of phycoerythrin and in fluorescence characteristics. The high gene expression level of the key enzymes in phycoerythrobilin synthesis (porphobilinogen synthase and oxygen-dependent coproporphyrinogen-III oxidase) could be related to the high phycoerythrin content of Porphyridium. Based on systematic evaluation, P. purpureum SCS-02 was selected due to its high biomass and phycoerythrin yield. P. purpureum and P. cruentum were highly similar in the phylogenetic tree, as well as in fluorescence characteristics; therefore, it was speculated that they might be the same Porphyridium species.
... An aqueous two-phase system extraction was performed following a previous report [18] by using Q-Sepharose Fast Flow column (GE Healthcare, Chicago, USA). Further technical details are provided in Supplementary Methods. ...
Background Ischemic stroke produces a large health impact worldwide, with scarce therapeutic options. Objective This study aimed to reveal the role of NADPH oxidase and neuroinflammatory genes on the cerebral anti-ischemic effects of C-Phycocyanin (C-PC), the chief biliprotein of Spirulina platensis. Methods : Rats with either focal cerebral ischemia/reperfusion (I/R) or acute brain hypoperfusion, received C-PC at different doses, or a vehicle, for up to 6 h post-stroke. Neurological, behavioral and histochemical parameters were assessed in I/R rats at 24 h. Cerebral gene expression and hippocampal neuron viability were evaluated in hypoperfused rats at acute (24 h) or chronic phases (30 days), respectively. A molecular docking analysis between NOX2 and C-PC-derived Phycocyanobilin (PCB) was also performed. Results C-PC, obtained with a purity of 4.342, significantly reduced the infarct volume and neurologic deficit in a dose-dependent manner, and improved the exploratory activity of the I/R rats. This biliprotein inhibited NOX2 expression, a crucial NAPDH oxidase isoform in the brain, and the superoxide increase produced by the ischemic event. Moreover, C-PC-derived PCB showed a high binding affinity in silico with NOX2. C-PC downregulated the expression of pro-inflammatory genes (IFN-γ, IL-6, IL-17A, CD74, CCL12) and upregulated immune suppressive genes (Foxp3, IL-4, TGF-β) in hypoperfused brain areas. This compound also decreased chronic neuronal death in the hippocampus of hypoperfused rats. Conclusion These results suggest that the inhibition of cerebral NADPH oxidase and the improvement of neuroinflammation are key mechanisms mediating the neuroprotective actions of C-PC against brain ischemia.
... The concentration of C-PC, APC, and PE was calculated using the Equations (1)-(3) described by Bennett and Bogorad [29]. The purity of C-PC, APC, and PE was determined using the Equations (4)-(6) proposed by Patil [30] and Antello et al. [31]. The average of the results obtained for each experiment was used to perform the ANOVA analysis according to the Design-Expert ® software. ...
The present study evaluated the effect of multiple variables (drying time, drying temperature, biomass/solvent ratio, glass beads/biomass ratio, extraction time, and extraction speed) in the solubilization of three different phycobiliproteins (C-PC, APC, and PE) from a thermotolerant Oscillatoria sp. The strain was grown in BG11 media (28 °C, light: dark cycle of 12:12 h at 100 µmol·m−2·s−1, 20 days) and the experiments were conducted according to a two-level randomized factorial design with six center points (38 runs). Results show that biomass/solvent ratio, glass beads/biomass ratio, and extraction time, are the most significant variables in the extraction of all three proteins, whereas the glass beads/biomass ratio and extraction time significantly affect their purity. The optimized conditions allow a statistical increase in the concentration of C-PC, APC, and PE extracted from the biomass; however, the purity was lower in comparison with the expected value. The latter occurs due to a larger biomass/solvent ratio and longer extraction times, which enhanced the solubility of other hydrophilic metabolites (proteins and carbohydrates, etc.).
... and as analytical grade with the ratio value of A620/A280 is ≥4.0. 19,[23][24][25] The ATPS-D fraction was chosen for the next research. The effect of PEG 6000 on the levels and purity of CPC was further examined. ...
Several substrates have been used for the determination of protease activity of pancreatin. However, there are still weaknesses of substrates such as insolubility, preparation, and a number of reagents used. Therefore, the alternative finding for the substrate for protease activity assay of pancreatin is still openly investigated. This study was aimed to isolate and characterize C-phycocyanin (CPC) from Spirulina platensis and its application as a substrate in protease activity assay. The research includes isolation, purification and characterization of CPC, the interaction of CPC with pancreatin, determining the relationship between initial rate of reaction and protease activity and study of pancreatin protease kinetics. From the experimental results, the ATPS-D fraction with a CPC content of 138.5 ± 0.06 μg/mL and a CPC purity of 0.74 ± 0.00 was used as a substrate solution. The results of the interaction of CPC with pancreatin showed a decrease in CPC content in the incubation time used. The relationship between the initial rate of reaction and protease activity (5 – 30 IU/mL) showed a linear curve, especially at an incubation time of 5 -30 minutes with R2 > 0.9. Enzyme kinetics study showed that the Vmax and Km values for reaction incubation time of 10, 20, 30 minutes were 2.59 g/mL/minute and 91.66 g/mL; 1.47 g/mL/minute and 70.86 g/mL; 1.31 g/mL/minute and 79.22 g/mL/minute, respectively. In conclusion, CPC can be used as a new substrate candidate for the determination of pancreatin protease activity.
... In addition, it is a natural food pigment with high nutritional value. [31][32][33] Several studies have shown that C-PC possesses various biological activities, including antioxidant, 34 anti-inflammatory, 35,36 anti-cancer, 37,38 immunomodulatory hepatoprotective and antiplatelet activities. [39][40][41] Several mechanisms for the anti-inflammatory activity of C-PC have been reported, including selective inhibition of COX-2, 42 inhibition of NF-κB activation, and consequent inhibition of the release of inflammatory mediators, 43 eliminating reactive oxygen species (ROS), and reducing oxidative stress. ...
Background Seawater drowning-induced acute lung injury (ALI) is a severe clinical condition characterized by increased alveolar-capillary permeability, excessive inflammatory response, and refractory hypoxemia. C-phycocyanin (C-PC), a biliprotein found in blue-green algae such as spirulina platensis, is widely used in the food and dietary nutritional supplement fields due to its beneficial pharmacological effects. Previous studies have revealed that C-PC has anti-inflammatory, antioxidant, and anti-apoptotic activities. Purpose Therefore, this study investigated the protective effect and underlying mechanisms of C-PC on lipopolysaccharide (LPS) and seawater (SW) induced ALI (SW and LPS-induced ALI). Methods An SW and LPS mouse model of ALI mice was established through intratracheal administration of 5mg/kg LPS and 25% SW. Different doses of C-PC (100, 200 and 400 mg/kg) were administered by intraperitoneal injection for seven days. In addition, gap junction communication in RAW264.7 and MLE-12 cells was determined following stimulation with 25% SW and 10 μg/ml LPS after treatment with C-PC (120 μg/ml). Moreover, the arterial partial pressure of oxygen, lung wet/dry weight ratios, total protein content and MPO levels in the bronchoalveolar lavage fluid (BALF), and the histopathologic and ultrastructure staining of the lung tissues were determined. The oxidative stress index, levels of the pro-inflammatory mediators, epithelial cell viability and apoptosis, and the regulatory effect of C-PC on the NF-κB/NLRP3 axis were investigated. Results The results showed that C-PC significantly alleviated pathological damages, suppressed oxidative stress, inflammation and apoptosis, and enhanced the viability of epithelial cells in the lung tissues. Furthermore, C-PC was shown to inhibit activation of the NF-κB/NLRP3 pathway and the formation of the NLRP3 inflammasome complex. Conclusions In conclusion, C-PC shows promising therapeutic value in SW and LPS-induced ALI/ARDS, providing new insight into ALI/ARDS treatment.
... The functionality of C-PC in terms of fluorescence property is owing to the presence of covalently linked linear tetrapyrrole chromophore phycocyanobilin (PCB) (Rastogi et al. 2015). Phycocyanin is a red fluorescent protein which fluoresces over a wavelength range from 637 to 647 nm when excited with specific wavelength (MacColl and Guard-Frair 1987;Patil et al. 2006). The distinct peak detected at 645 nm in both the strains illustrated the structural integrity of the apoprotein-chromophore interaction (ESM_3Cite ESM..pdf and ESM_4Cite ESM..pdf). ...
C-phycocyanin (C-PC), an algal biopigment, has a fairly big market demand in food and other industries including pharmaceuticals for its valuable properties. However, its application in diverse fields is affected by the cost associated with its production and downstream processing. Most of the present studies are focussed on improvement in C-PC biosynthetic capacity of existing commercial strain, Arthrospira platensis; nonetheless, many cyanobacterial strains might present in nature that can naturally produce C-PC in high amount. The current study aimed at selecting high C-PC producer from diverse cyanobacteria isolated from different regions of India. Three cyanobacteria that stood out to be high producers are Phormidium sp. CCC317, Plectonema sp. CCC316 and Phormidium sp. CCC112 with C-PC contents of 191.1 ± 4.42, 158.1 ± 4.13 and 140.6 ± 4.04 mg g⁻¹ cell dry weight, respectively. The results were significant, and Phormidium sp. CCC317 has shown C-PC production comparable to A. platensis under normal growth condition. Further, a simple improved purification protocol employing weak anion exchanger and two NaCl concentrations (0.12 and 0.18 M) yielded analytical grade purity (A620/A280 ratio) of 5.56 and 5.63 with 56.41% and 32.41% C-PC recovery for Phormidium sp. CCC317 and Plectonema sp. CCC316, respectively. Interestingly, analytical grade purity of 4.25 with 95.32% recovery was obtained in Phormidium sp. CCC317 with only (NH4)2SO4 precipitation which can reduce the pigment production cost remarkably. Higher antioxidant activity of purified C-PC of both isolates is further attributed to their source of isolation. Hence, this work highlights the importance of better strain and its commercial prospect for C-PC production securing good recovery and higher purity.
... Estimation of the amount of C-PC and purity were determined as described by (42,43). ...
C-phycocyanin (C-PC), the integral blue-green algae (BGA) constituent has been substantially delineated for its biological attributes. Numerous reports have illustrated differential extraction and purification techniques for C-PC, however, there exists paucity in a broadly accepted process of its isolation. In the present study, we reported a highly selective C-PC purification and characterization method from nontoxic, filamentous and non-heterocystous cyanobacterium Plectonema sp. C-PC was extracted by freeze-thawing, desalted and purified using ion-exchange chromatography. The purity of C-PC along with its concentration was found to be 4.12 and 245 µg/ml respectively. Comparative characterization of standard and purified C-PC was performed using diverse spectroscopic techniques namely Ultra Violet-visible, fluorescence spectroscopy and Fourier transform infrared (FT-IR). Sharp peaks at 620 nm and 350 nm with UV-visible and FT-IR spectroscopy respectively, confirmed amide I bands at around 1638 cm-1 (C=O stretching) whereas circular dichroism (CD) spectra exhibited α-helix content of secondary structure of standard 80.59% and 84.59% of column purified C-PC. SDS-PAGE exhibited two bands of α and β subunits 17 and 19 kDa respectively. HPLC evaluation of purified C-PC also indicated a close resemblance of retention peak time (1.465 min, 1.234 min, 1.097 min and 0.905 min) with standard C-PC having retention peak timing of 1.448 min, 1.233 min and 0.925 min. As a cautious approach, the purified C-PC was further lyophilized to extend its shelf life as compared to its liquid isoform. To evaluate the bioactive potential of the purified C-PC in silico approach was attempted. The molecular docking technique was carried out of C-PC as a ligand-protein with free radicals and α-amylase, α-glucosidase, glycogen synthase kinase-3 and glycogen phosphorylase enzymes as receptors to predict the free radical scavenging (antioxidant) and to target antidiabetic property of C-PC. In both receptors free radicals and enzymes, ligand C-PC plays an important role in establishing interactions within the cavity of active sites. These results established the antioxidant potential of C-PC and also give a clue towards its antidiabetic potential warranting further research.
... This highlights the purity of the protein compared to that of the contaminating proteins (non-specific absorbance at 280 nm). In general, a purity ratio between 0.7 and 3.9 indicates that phycocyanin is provided as food grade and a higher purity ratio indicates reagent/analytical grade [16]. High purity can be obtained when the ratio is >4 [26]. ...
The cyanobacterium Arthrospira platensis, spirulina, is a source of pigments such as phycobiliprotein, phycocyanin. Phycocyanin is used in the food, cosmetic, and pharmaceutical industries because of its antioxidant, anti-inflammatory, and anticancer properties. The different steps involved in extraction and purification of this protein can alter the final properties. In this review, the stability of phycocyanin (pH, temperature, and light) is discussed, considering the physicochemical parameters of kinetic modeling. The optimal working pH range for phycocyanin is between 5.5 and 6.0 and it remains stable up to 45 °C; however, exposure to relatively high temperatures or acidic pH decreases its half-life and increases the degradation kinetic constant. Phycobiliproteins are sensitive to light and preservatives such as mono- and di-saccharides, citric acid, or sodium chloride appear to be effective stabilizing agents. Encapsulation within nano- or micro-structured materials such as nanofibers, microparticles, or nanoparticles, can also preserve or enhance its stability.
... Solvents such as phosphate buffer [70,134], potassium phosphate buffer [96], or phosphate buffer with ammonium sulfate [135] were used to extract PC from red seaweeds. Purification methods existent to obtain pure PC from algae extracts include aqueous two phase extraction [136], ammonium sulfate precipitation, polyacrylamide gel electrophoresis [99], gel filtration chromatography, high-performance liquid chromatography [99], and ion-exchange chromatography [20], which can be applied combined (e.g., aqueous two-phase extraction and ion-exchange chromatography [137] or gel filtration followed by ion exchange chromatography [134]). These methods have been mostly applied to recover C-PC from microalgae, and as long as cyanobacteria continue to satisfy the industry as the main provider of PC, red seaweeds will fall behind in comparison, and dedicated studies focusing on PC obtained from these macroalgae may not be easily acknowledged, followed, or promoted in the near future. ...
Algae taxa are notably diverse regarding pigment diversity and composition, red seaweeds (Rhodophyta) being a valuable source of phycobiliproteins (phycoerythrins, phycocyanin, and allophycocyanin), carotenes (carotenoids and xanthophylls), and chlorophyll a. These pigments have a considerable biotechnological potential, which has been translated into several registered patents and commercial applications. However, challenges remain regarding the optimization and subsequent scale-up of extraction and purification methodologies, especially when considering the quality and quantity needs, from an industrial and commercial point of view. This review aims to provide the state-of-the-art information on each of the aforementioned groups of pigments that can be found within Rhodophyta. An outline of the chemical biodiversity within pigment groups, current extraction and purification methodologies and challenges, and an overview of commercially available products and registered patents, will be provided. Thus, the current biotechnological applications of red seaweeds pigments will be highlighted, from a sustainable and economical perspective, as well as their integration in the Blue Economy
This research sought to purify C-phycocyanin (C-PC) from Spirulina platensis and investigate its potential in enhancing the quality parameters and in vivo fertility of ram semen subjected to cooled storage at 5 ◦C, when using a skim milk (SM) based semen extender. The purification process of C-PC involved cold maceration, prepurification using chitosan and activated charcoal, followed by purification through aqueous two-phase extraction (ATPE) and ion-exchange chromatography. Afterward, fifty ejaculates were collected from 4 fertile Boujaad rams and extended using the SM extender at 37 ◦C, enriched with 0 μg/mL (control), 1.2 μg/mL, 2.4 μg/mL, 3.6 μg/mL, or 4.8 μg/mL of C-PC. The diluted semen was subsequently cooled to 5 ◦C using a controlled cooling process, with a gradual cooling rate of approximately 0.5 ◦C per minute, and its quality parameters were evaluated after 0, 4, 8, and 24 h of cooling storage. Then, its fertilization ability after 4 h of cooling storage was evaluated using artificial insemination. The adopted purification process yielded a grade analytical purity of 4.06. Additionally, semen extended in SM with a 2.4 μg/mL C-PC supplement displayed significant (P < 0.0001) enhancement in total motility, progressive motility, curvilinear velocity, straight-line velocity, average path velocity, viability and lipid peroxidation of ram semen at 0, 4, 8, and 24 h of cooling storage. These improvements were observed in direct comparison to both the control group and the other C-PC concentrations. Regarding fertility rates, semen extended in SM with a 2.4 μg/mL C-PC recorded a 76 % rate, a notable increment from the 63 % observed in ewes inseminated by semen extended in SM alone, although the difference was not statistically significant (p > 0.05). These findings underscore the promising potential of C-PC as a natural supplement for enhancing semen quality, warranting further investigations.
Phycobiliproteins (PBPs), one of the functional proteins from algae, are natural pigment–protein complex containing various amino acids and phycobilins. It has various activities, such as anti-inflammatory and antioxidant properties. And are potential for applications in food, cosmetics, and biomedicine. Improving their metabolic yield is of great interest. Microalgaes are one of the important sources of PBPs, with high growth rate and have the potential for large-scale production. The key to large-scale PBPs production depends on accumulation and recovery of massive productive alga in the upstream stage and the efficiency of microalgae cells breakup and extract PBPs in the downstream stage. Therefore, we reviewed the status quo in the research and development of PBPs production, summarized the advances in each stage and the feasibility of scaled-up production, and demonstrated challenges and future directions in this field.
Phycobiliproteins are photosynthetic light-harvesting pigments isolated from microalgae with fluorescent, colorimetric and biological properties, making them a potential commodity in the pharmaceutical, cosmetic and food industries. Hence, improving their metabolic yield is of great interest. In this regard, the present review aimed, first, to provide a detailed and thorough overview of the optimization of culture media elements, as well as various physical parameters, to improve the large-scale manufacturing of such bioactive molecules. The second section of the review offers systematic, deep and detailed data about the current main features of phycobiliproteins. In the ultimate section, the health and nutritional claims related to these bioactive pigments, explaining their noticeable potential for biotechnological uses in various fields, are examined.
This study evaluated a tertiary wastewater treatment technology using cyanobacteria to recover value-added phycobiliproteins. The presence of contaminants of emerging concern (CECs) in wastewater, cyanobacteria biomass and pigments recovered were also analyzed. For this, a wastewater-borne cyanobacterium (Synechocystis sp. R2020) was used to treat secondary effluent from a municipal wastewater treatment plant, with and without nutrients supplementation. Then, the stability of phycobiliprotein production was assessed by operating the photobioreactor in semi-continuous mode. Results showed similar biomass productivity with and without nutrients supplementation (153.5 and 146.7 mg L-1 d-1, respectively). Upon semi-continuous operation, the phycobiliprotein content was stable and reached up to 74.7 mg gDW-1. The phycocyanin purity ratio ranged from 0.5 to 0.8, corresponding to food grade (> 0.7). Out of 22 CECs detected in secondary effluent, only 3 were present in the phycobiliprotein extract. In order to identify applications, prospective research should focus on CECs removal during pigment purification.
Phycocyanin (PCY), as a natural bioactive and pigment, is susceptible to heat and light in aqueous solutions. The aim of this study was to produce PCY-loaded nanoliposomes (NLPs) coated with chitosan (NLPC) to extend PCY stability as well as examining its physicochemical properties and encapsulation efficiency (EE). After finding the optimal concentration of PCY (3 mg/mL), NLPs were coated with various levels of chitosan (0, 0.1, 0.2, 0.4 and 0.6 mg/mL), and the prepared NLPs were investigated in terms of physicochemical, antioxidant, brightness, and thermal properties, crystallinity, chemical structure and morphology. SDS-PAGE results indicated the presence of α-chain and β-chain (phycocyanobilin) bands in the range of 63–35 kDa. The coating of NLPs with chitosan (especially at the optimal concentration of 0.4 mg/mL) led to an increase in the particle size (from 92.1 to 212.7 nm) and a change in their zeta potential (from −12.3 to 16.2 mV) (P < 0.05). Also, the antioxidant activity of NLPs (32.7 %) increased after loading with PCY (54.5 %) and coating with chitosan (48.2 %). On the other hand, chitosan coating led to an increase in physical stability and preservation of EE after 28 days of storage at ambient and refrigerator temperatures. The morphological characteristics (SEM) confirmed the results of DLS and the existence of relatively spherical particles with uneven surfaces. The results of differential scanning calorimetry (DSC), X-ray diffraction (XRD), and Chemical (FTIR) analysis demonstrated the formation of PCY-NLP complexes and the successful encapsulation of PCY in the NLPs.
Drying the biomass produced is one of the critical steps to avoid cell degradation; however, its high energy cost is a significant technological barrier to improving this type of bioprocess’s technical and economic feasibility. This work explores the impact of the biomass drying method of a strain of Potamosiphon sp. on the extraction efficiency of a phycoerythrin-rich protein extract. To achieve the above, the effect of time (12–24 h), temperature (40–70 °C), and drying method (convection oven and dehydrator) were determined using an I-best design with a response surface. According to the statistical results, the factors that most influence the extraction and purity of phycoerythrin are temperature and moisture removal by dehydration. The latter demonstrates that gentle drying of the biomass allows removing the most significant amount of moisture from the biomass without affecting the concentration or quality of temperature-sensitive proteins.
Phycobiliproteins, carotenoids and fucoxanthin are photosynthetic pigments extracted from microalgae and cyanobacteria with great potential biotechnological applications, as healthy food colorants and cosmetics. Phycocyanin possesses a brilliant blue color, with fluorescent properties making it useful as a reagent for immunological essays. The most important source of phycocyanin is the cyanobacterium Arthrospira platensis, however, recently, the Rhodophyta Galdieria sulphuraria has also been identified as such. The main obstacle to the commercialization of phycocyanin is represented by its chemical instability, strongly reducing its shelf-life. Moreover, the high level of purity needed for pharmaceutical applications requires several steps which increase both the production time and cost. Microalgae ( Chlorella , Dunaliella , Nannochloropsis , Scenedesmus ) produce several light harvesting carotenoids, and are able to manage with oxidative stress, due to their free radical scavenging properties, which makes them suitable for use as source of natural antioxidants. Many studies focused on the selection of the most promising strains producing valuable carotenoids and on their extraction and purification. Among carotenoids produced by marine microalgae, fucoxanthin is the most abundant, representing more than 10% of total carotenoids. Despite the abundance and diversity of fucoxanthin producing microalgae only a few species have been studied for commercial production, the most relevant being Phaeodactylum tricornutum . Due to its antioxidant activity, fucoxanthin can bring various potential benefits to the prevention and treatment of lifestyle-related diseases. In this review, we update the main results achieved in the production, extraction, purification, and commercialization of these important pigments, motivating the cultivation of microalgae as a source of natural pigments. Graphical abstract
Spirulina, a photosynthetic cyanobacteria, has bioactive molecules such as phycobiliproteins which are useful in bioindustries. Aqueous two-phase extraction, an industrially effective process, have been applied to extract invaluable molecules from Spirulina, but mainly focused on optimal extraction of C-Phycocyanin. This research is to selectively remove chlorophyll a-pertaining photosystem polypeptides by stably forming interfacial layer between two phases of polyethylene glycol/potassium phosphate. Under an optimal condition of aqueous two-phase extraction, a stable interfacial layer was formed, while preserving purification efficiency of C-Phycocyanin. Through UV, fluorescence, and gel electrophoresis, chlorophyll a-pertaining photosystem polypeptides were ascertained to be extracted in the interfacial layer. C-Phycocyanin was mainly present in the top phase and allophycocyanin was in the bottom phase. In addition, the safety of the purified C-Phycocyanin was confirmed by examining cytotoxicity of C-Phycocyanin. In conclusion, a stable formation of interfacial layer was proved to be helpful in understanding the selective extraction of chlorophyll a-pertaining photosystem polypeptides, and the purified C-Phycocyanin can be used without cytotoxicity.
Phycocyanin is a natural blue pigment produced by cyanobacteria and is a valuable compound for food and cosmetic industries. At present, phycocyanin is manufactured with expensive and resource-heavy biotechnology, impeding its widespread use as a blue dye substitute. Here we show that cells of an alkaliphilic cyanobacterium lyse spontaneously in dark incubations mimicking natural soda lake environments, releasing concentrated phycocyanin. Proteogenomics showed that lysis likely resulted from a programmed response triggered by a failure to maintain osmotic pressure in the wake of severe energy limitation. This response explains the high turnover rates of cyanobacterial cells observed in soda lakes. Cells of Arthrospira platensis (Spirulina), currently used worldwide for phycocyanin production, lyse and release their pigments in the same manner. We propose this natural form of programmed cell death could reduce the costs and resources needed to produce phycocyanin, enabling displacement of current artificial blue colourants associated with adverse health effects. One-Sentence Summary Failure to maintain osmotic balance in the dark forces blue-green algae to share their bounty with the world.
Cultivation of Cyanobacteria is preferable for CO2 fixation process due to its efficiency and production of beneficial byproducts like phycocyanin. In this study, Thermosynechococcus sp. CL-1 (TCL-1) was cultivated in a 30 L flat panel photobioreactor using a 3-fold-modified Fitzgerald medium with 113.2 mM dissolved inorganic carbon. The highest CO2 fixation rate of 21.98 ± 1.52 mg/L/h was followed by higher lipid content (49.91 % dry weight content or %dwc) than the generated carbohydrate (24.22 %dwc). TCL-1 also potentially produced phycocyanin that was dominated by C-phycocyanin (98.10 ± 6.67 mg/g) along with a lower amount of allophycocyanin and phycoerythrin under extraction using various types of solvent. Stability of phycocyanin extract was further examined during storage under various temperatures and light illuminations. Extraction with 36 % glucose solvent presented a protective effect to phycocyanin from heat and photo-damage which was proven by the kinetics study of phycocyanin degradation in this study.
Phycocyanin is a water-soluble blue pigment biliprotein that has been utilized as a nutritional supplement for its potent antioxidant and anti-inflammation activity. In this study, three cyanobacterial species from the North-Eastern India, Calothrix sp. (BTA-476), Calothrix sp. (BTA-857), and Westiellopsis sp. (BTA-801) are taxonomically and molecularly identified using 16s rRNA gene sequencing. The cyanobacteria with the highest phycocyanin content were chosen, and phycocyanin was extracted. The purity was determined to be 2.3, which was almost 5 times purer than the crude extract, with a recovery rate of 47.47 %. Purified phycocyanin has strong cytotoxic potential with high anti-proliferative activity in breast cancer cell line MDA-231, resulting in chromatin condensation and the formation of apoptotic bodies. Apoptotic cell death was validated by caspase 3 activation where phycocyanin IC50 120 μg mL⁻¹ is equivalent to 31.37 nM AMC caspase 3 cleavage of DEVD-AMC substrate.
The need for cell disruption to obtain high value–added products has limited the large-scale production of commercial biotechnological products of intracellular origin. Cell disruption is a crucial step in the passage of intracellular biomolecules, aiming at high-yield extraction. Therefore it is necessary to explore more about the techniques used to carry out this process in natural matrices, such as plants and microalgae, for example. Mechanical and nonmechanical processes are used for cell disruption. Mechanical methods include ultrasound, high-pressure homogenization, ball milling, microwaves, and pulsed electric fields. On the other hand, nonmechanical methods use chemical or biological materials that interact directly with cell walls; these methods include enzymatic lysis, freezing-thawing, osmotic shock, treatment with organic solvents, surfactants, acids, alkalis, ionic liquids, and deep eutectic solvents. After obtaining the extract of the bioproduct, the isolation step is carried out, which can be carried out by different techniques. In this context, the chapter addresses technologies widely used for cell disruption and isolation of high value–added bioproducts such as biofuels, biopolymers, enzymes, fatty acids, phytohormones, and pigments, presenting the advantages and disadvantages of each technique, as well as the perspectives for application of these technologies to reducing costs and environmental impacts.
Cyano-phycocyanin is one of the active pigments of the blue-green algae and is usually isolated from the filamentous cyanobacteria Arthrospira platensis Gomont (Spirulina). Due to its multiple physiological functions and non-toxicity, cyano-phycocyanin may be a potential substance for the topical treatment of various skin diseases. Considering that the conventional medicine faces drug resistance, insufficient efficacy and side effects, the plant origin compounds can act as an alternative option. Thus, the aim of this paper was to review the wound healing, antimicrobial, antioxidative, anti-inflammatory, antimelanogenic and anticancer properties and mechanisms of cyano-phycocyanin topical activities on human skin. Moreover, possible applications and biotechnological requirements for pharmaceutical forms of cyano-phycocyanin for the treatment of various skin diseases are discussed in this review.
Arthrospira maxima is a natural source of fine chemicals for multiple biotechnological applications. We determined the optimal environmental conditions for A. maxima by measuring its relative growth rate (RGR), pigment yield, and photosynthetic performance under different pH and temperature conditions. RGR was highest at pH 7–9 and 30 °C. Chlorophyll a, phycocyanin, maximal quantum yield (Fv/Fm), relative maximal electron transport rate (rETRmax), and effective quantum yield (ΦPSII) were highest at pH 7–8 and 25 °C. Interestingly, phycoerythrin and allophycocyanin content was highest at 15 °C, which may be the lowest optimum temperature reported for phycobiliprotein production in the Arthrospira species. A threestep purification of phycocyanin (PC) by ultrafiltration, ion-exchange chromatography, and gel filtration resulted in a 97.6% purity of PC.
C-phycocyanin is a highly valuable phycobiliprotein from Arthrospira platensis. However, its extraction, purification and conservation currently limit its commercial use. We review here the most common techniques and less conventional methods. Simple incubation in phosphate buffer at neutral pH can give high yields (> 100 mg/g) and even high protein purity (> 0.7) if the parameters (buffer concentration, temperature, incubation time, Arthrospira platensis. state etc.) are chosen correctly. This method is preferable to incubation in distilled water or acid solutions. Some mechanical, physical or thermal treatments can improve the extraction yield and accelerate the release of C-phycocyanin. Ultrasound-assisted extraction has been widely studied and probes generally give higher extraction yields than baths (> 100 mg/g) in less than 30 min. This technique can be coupled with others, such as freeze–thaw methods, to improve protein release. Indeed, freeze–thaw cycles are an efficient destructuring technique that can be used alone or as a pretreatment. Bead mills or pulsed electric fields can also be used to extract C-phycocyanin, but there is room for improvement in the choice of operating parameters. For purification of the extracted pigments, salting out or aqueous two-phase extraction can be used to increase phycocyanin grade. More advanced purification methods, mostly based on chromatography, can provide addition improvement. C-phycocyanin stability is dependent principally on pH and temperature, and is higher between pH 5 and pH 7 and at temperatures below 40 °C, but the use of various preservatives or conditioning can increases its lifetime.
Phycobiliproteins are brilliantly colored, highly fluorescent components of the photosynthetic light-harvesting antenna complexes of cyanobacteria (blue-green algae), red algae and cryptomonads. These proteins carry covalently attached linear tetrapyrrole pigments related structurally to biliverdin. Phycobiliproteins, purified from certain organisms, are isolated as either trimers, ()3, of approximatelyM r 110–120103 (e.g., allophycocyanins), or hexamers, ()6, of aboutM r 250103 (certain phycoerythrins). Three phycobiliproteins R-phycoerythrin, B-phycoerythrin, and allophycocyanin serve as valuable fluorescent tags with numerous applications in flow cytometry, fluorescence activated cell sorting, histochemistry and, to a limited degree, in immunoassay and detection of reactive oxygen species. These applications exploit the unique physical and spectroscopic properties of phycobiliproteins.
This chapter discusses aqueous two-phase extraction (ATPE) for downstream processing of enzymes or proteins. Downstream processing in many fermentation processes accounts for a large share of the total cost. The ATPE appears to be a promising technique for efficient downstream processing and some successful applications of ATPE on large/industrial scale have been demonstrated. In order to develop effective downstream processing methods employing ATPSs, an interdisciplinary effort involving a combination of microbiological, biochemical, and engineering aspects is very essential. In addition, enough attention has been paid to microbiological and biochemical aspects. Mathematical modeling, which is highlighted in this chapter, is of immense use in predicting the protein enzyme partitioning without the measurement of an inordinately large number of parameters. Although, in principle, ATPE offers the advantage of easy adaptation of the extraction equipment used in the chemical or pharmaceutical industry to achieve efficient extraction, the drop dynamics and mass transfer aspects of this contacting equipment should be studied in detail employing various ATPSs and real systems involving the actual fermentation broths. Even if some of these aspects are addressed in greater depth by future researchers, the objective of this chapter can be considered fulfilled in view of the recognized scientific and industrial potential of ATPE.
A number of drying methods studied for the processing of Spirulina (crossflow dried, spray dried and oven dried) resulted in approximately 50% loss of phycocyanin. Therefore fresh biomass was suitable for phycocyanin extraction. Of the extraction methods tested, freezing and thawing of cells, homogenisation using a mortar and pestle in the presence of abrasive material and homogenisation using a blender at 10 000 rpm yielded 19.4±0.4 mg phycocyanin per 100 mg dry weight of Spirulina while water extraction was a slow process. Acid treatment also resulted in phycocyanin leaching. Phycocyanin was stable over a pH range of 5–7.5 at 9±1°C, whereas temperature beyond 40°C lead to instability. The pigment phycocyanobilin was separated from the phycocyanin.
A novel process for the recovery of c‐phycocyanin from Spirulina maxima exploiting aqueous two‐phase systems (ATPS), ultrafiltration and precipitation was developed in order to reduce the number of unit operations and benefit from an increased yield of the protein product. The evaluation of system parameters such as PEG molecular mass, concentration of PEG as well as salt, system pH and volume ratio was carried out to determine under which conditions the c‐phycocyanin and contaminants concentrate to opposite phases. PEG1450–phosphate ATPS proved to be suitable for the recovery of c‐phycocyanin because the target protein concentrated in the top phase whilst the cell debris concentrated in the bottom phase. A two‐stage ATPS process with a phase volume ratio ( V r ) equal to 0.3, PEG1450 7% (w/w), phosphate 20% (w/w) and system pH of 6.5 allowed c‐phycocyanin recovery with a purity of 2.4 (estimated as the relationship of the 620 nm to 280 nm absorbances). The use of ultrafiltration (with a 30 kDa membrane cut‐off) and precipitation (with ammonium sulfate) resulted in a recovery process that produced a protein purity of 3.8 ± 0.1 and an overall product yield of 29.5% (w/w). The results reported here demonstrated the practical implementation of ATPS for the design of a prototype recovery process as a first step for the commercial purification of c‐phycocyanin produced by Spirulina maxima . © 2001 Society of Chemical Industry
Scale-up of aqueous two-phase extraction, which is useful in the isolation and purification of certain bioproducts, is limited by the slow demixing rates of the two aqueous phases. Electrokinetic demixing has been shown to increase by more than 5-fold the demixing rates of systems up to 100 mL in volume in a manner that depends on field strength, field polarity, pH, and phase composition. The present study is an attempt to relate demixing rates to droplet electrokinetic mobilities which were measured microscopically and inferred from demixing data. A clear dependence of demixing rate was observed on drop electrophoretic mobility and pH. The electrophoretic mobility of individual phase droplets suspended in the other phase was measured for poly(ethylene glycol) /Dextran systems using a microelectrophoresis unit and compared with mobilities predicted by electrokinetic theory. We confirmed earlier reports that the droplet electrophoretic mobility increased with increasing drop diameter and explained this increase on the basis of an internal electroosmotic flow model. Effective electrophoretic mobilities were estimated from electrokinetic demixing data in a 100-mL column and compared with predicted as well as experimentally measured values of electrophoretic mobility. The mobilties increased with increased phosphate ionization due to change in pH irrespective of the sign (or polarity) of the applied electric field. The electroosmotic flow model could explain satisfactorily the following two paradoxes: (1) the direction of migration of drops is the opposite of that predicted by colloid electrokinetics and (2) the phase demixing rate increased irrespective of the sign of the applied electric field.
c-Phycocyanin and allophycocyanin were separated and purified from Spirulina platensis by precipitation with ammonium sulphate, ion exchange chromatography and gel filtration chromatography. Pure c-phycocyanin and allophycocyanin were finally obtained with an A620/A280value of 5.06 and an A655/A280 value of 5.34, respectively.
Two main biliproteins c-phycocyanin and allophycocyanin were identified and characterized in the blue-green alga Spirulina platensis. The specific absorbance, fluorescence maxima, sub-unit make-up and amino acid composition of the biliproteins in Spirulina platensis resemble those reported for other blue-green algae. However, the minimum molecular weights (44,000 for c-phycocyanin and 38,000 for the allophycocyanin) and the specific extinction coefficients (73, and 58 for c-phycocyanin and allophycocyanin respectively) of these biliproteins were different from these values in other blue-green algae.
Spirulina biomass was separated into two fractions which may have various uses. A phycocyanin fraction may provide a food colourant and biomarkers, and a protein-rich leftover may be useful as aquaculture feed. Activated charcoal adsorption, ultrafiltration and spray drying were used effectively to produce a high quality colourant grade phycocyanin, while activated charcoal adsorption, ammonium sulphate precipitation, dialysis and chromatography were effective in preparing reagent grade phycocyanin.
The article reviews the current status of the application of aqueous two-phase systems for the extractive purification of enzymes, especially with regard to large-scale processing. The method can be used for the separation of proteins from cell debris as well as for further purification. The latter can be performed by a series of single step partitions, and apparently also by continuous multistage processes. The specificity and selectivity of extraction can be enhanced by introducing specific or general ligands. Scale-up of extractive enzyme purification is relatively simple utilizing commercially available equipment and machinery common in the chemical industry. Besides the technical performance, economic considerations also indicate the feasibility of the method at production scale.
Publisher Summary Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) is an excellent method to identify and monitor proteins during purification and to assess the homogeneity of purified fractions. The movements of the particles are retarded by interactions with the surrounding gel matrix, which acts as a molecular sieve. The opposing interactions of the electrical force and molecular sieving result in differential migration rates for the constituent proteins of a sample. SDS–PAGE overcomes the limitations of native polyacrylamide gel electrophoresis (PAGE) by imposing uniform hydrodynamic and charge characteristics on all the proteins in a sample mixture. During sample preparation, proteins are treated with hot sodium dodecyl sulfate (SDS). The anionic detergent binds tightly to most proteins at about 1.4 mg of SDS/mg of protein, imparting a negative charge to the resultant complexes. Most electrophoresis is done in vertical chambers in gel slabs formed between two glass plates. The slab format provides uniformity, so that different samples can be directly compared in the same gel.
A new technique to speed up the phase separation of aqueous two-phase systems is described. The technique is based on the addition of magnetically susceptible additives (ferrofluids or iron oxide particles). In a magnetic field such additives will induce a faster phase separation. In one approach, dextran-stabilized ferrofluid was added to an aqueous two-phase system containing polyethylene glycol and dextran. The ferrofluid was totally partitioned to the dextran phase. After mixing of the two-phase system, it was possible to reduce the separation time by a factor of 35 by applying a magnetic field to the system. Another approach involved the use of 1-micron iron oxide particles instead of ferrofluid. In this case also, the phase-separation time was reduced, by a factor of about 70, when the system was placed in a magnetic field. The addition of ferrofluid and/or iron oxide particles was shown to have no influence on enzyme partitioning or on enzyme activity. The partitioning of chloroplasts, on the other hand, was influenced unless the ferrofluid used had been treated with epoxysilane. A column system comprising 15 magnetic separation stages was constructed and was used for semicontinuous separation of enzyme mixtures.
Publisher Summary This chapter discusses some recent developments in the estimation of various conformations in a protein molecule from its circular dichroism (CD) spectrum in the ultraviolet region. Optical rotatory dispersion (ORD) and CD are two chiroptical phenomena, which differentiate two enantiomers. They are caused by different interactions of left- and right-circularly polarized light with chiral molecules. Chirality is a geometric property of molecules; the corresponding substances are therefore optically active. At present CD has replaced ORD for the conformational analysis of proteins because CD bands that are characteristic of various secondary structures can be directly observed. The very simplicity of the CD method and the short time required for the measurements are extremely attractive. It is often advantageous to compare the CD analysis with empirical predictions of secondary structure of proteins from sequences, if such are available. All current methods for CD analysis are developed to determine not only the helix and β-form but also the β-turn and unordered forms.
An enzyme immunoassay (EIA) for phycocyanin in foods was developed. Anti-phycocyanin monoclonal antibodies were obtained from A/J mice immunized with phycocyanin. The phycocyanin in a food was extracted by dissolving the sample in a borate buffer solution, pH 8.0 (BBS) and adjusting the pH value of this solution to 8.0 with NaOH. The extract was then diluted more than 10 fold with 1% gelatin in BBS. Phycocyanin was determined by avidin-biotin sandwich EIA, using the P26-8 monoclonal antibody as the solid-phase antibody and the P277-4 monoclonal antibody as the enzyme-labeled antibody. The working range for a quantitative analysis was 100-1000 ng/ml, and the detection limit was 10 micrograms/g of the original sample. Recoveries of phycocyanin from foods by this assay were > 71% for candy, and > 66% for ice cream and sherbet. Phycocyanin was assayed in 22 blue-, green-, purple-, and brown-colored commercial foods, and detected in one green colored-jelly at 49 micrograms/g.
Aqueous two-phase extraction has been recognized as a versatile downstream processing technique for the recovery of biomolecules. A major deterrent to its industrial exploitation is the slow demixing of the two aqueous phases after extraction, due to their similar physical properties. A method to decrease the demixing times of these systems, employing a travelling acoustic wave field, is reported. The effects of phase composition and microbial cells on demixing in a polyethylene glycol/potassium phosphate two-phase system are studied in detail. As phase composition increased, demixing time decreased gradually. Phase volume ratio was found to have a significant effect on demixing time at low phase compositions. However, at intermediate and high phase compositions, only a small effect on demixing time was observed. The effect of phase composition and volume ratio on demixing behavior was explained based on the droplet size of the dispersed phase, which is the resultant effect of the physical properties of the phases. At all the phase compositions studied, the acoustically assisted process decreased the demixing time by 17-60% when compared to demixing under gravity alone. Increasing the cell concentration increased the demixing time markedly in case of yeast cells. However, it remained practically constant in the case of Lactobacillus casei cells. Application of an acoustic field reduced the demixing times up to 60% and 40% in the case of yeast and L. casei cells, respectively. Visual observations indicated that ultrasonication caused mild circulation currents in the phase dispersion enhancing droplet-droplet interaction, which in turn enhanced the rate of coalescence, eventually resulting in an enhanced demixing rate.
Peroxynitrite (ONOO(-)) is known to inactivate important cellular targets and also mediate oxidative damage in DNA. The present study has demonstrated that phycocyanin, a biliprotein from spirulina platensis and its chromophore, phycocyanobilin (PCB), efficiently scavenge ONOO(-), a potent physiological inorganic toxin. Scavenging of ONOO(-) by phycocyanin and PCB was established by studying their interaction with ONOO(-) and quantified by using competition kinetics of pyrogallol red bleaching assay. The relative antioxidant ratio and IC(50) value clearly indicate that phycocyanin is a more efficient ONOO(-) scavenger than PCB. The present study has also shown that PCB significantly inhibits the ONOO(-)-mediated single-strand breaks in supercoiled plasmid DNA in a dose-dependent manner with an IC(50) value of 2.9 +/- 0.6 microM. These results suggest that phycocyanin, has the ability to inhibit the ONOO(-)-mediated deleterious biological effects and hence has the potential to be used as a therapeutic agent.
Tobacco has long been considered as a host to produce large quantity of high-valued recombinant proteins. However, dealing with large quantities of biomass is a challenge for downstream processing. Aqueous two-phase extraction (ATPE) has been widely used in purifying proteins from various sources. It is a protein-friendly process and can be scaled up easily. In this paper, ATPE was studied for its applicability to recombinant protein purification from tobacco with egg white lysozyme as the model protein. Separate experiments with poly(ethylene glycol) (PEG)-salt-tobacco extract and PEG-salt-lysozyme were carried out to determine the partition behavior of tobacco protein and lysozyme, respectively. Two-level fractional factorial designs were used to study the effects of factors such as, PEG molecular mass, PEG concentration, the concentration of phase forming salt, sodium chloride concentration and pH, on protein partitioning. The results showed that, among the studied systems, PEG-sodium sulfate system was most suitable for lysozyme purification. Detailed experiments were conducted by spiking lysozyme into the tobacco extract. The conditions with highest selectivity of lysozyme over native tobacco protein were determined using a response surface design. The purification factor was further improved by decreasing the phase ratio along the tie line corresponding to the phase compositions with the highest selectivity. Under selected conditions the lysozyme yield was predicted to be 87% with a purification factor of 4 and concentration factor of 14. From this study, ATPE was shown to be suitable for initial protein recovery and partial purification from transgenic tobacco.
The present paper describes an efficient single step chromatographic method for purification of C-Phycocyanin from three cyanobacterial species, i.e., Spirulina sp. (freshwater), Phormidium sp. (marine water) and Lyngbya sp. (marine water). C-Phycocyanin from these cyanobacterial species was purified to homogeneity and some of their properties were investigated. The purification involves a multistep treatment of the crude extract by fractional precipitation with ammonium sulfate, followed by ion-exchange chromatography on DEAE-Sepharose CL-6B column. Pure C-Phycocyanin was finally obtained from Spirulina, Phormidium, and Lyngbya spp. with purity ratio (A620/A280) 4.42, 4.43, and 4.59, respectively, further the purity and homogeneity were confirmed by native and SDS-PAGE. The estimated molecular weights of purified C-PC from Spirulina, Phormidium, and Lyngbya spp. were 112, 131, and 81 kDa, respectively. SDS-PAGE of pure C-Phycocyanin yielded two bands corresponding to alpha and beta subunits. The results of SDS-PAGE demonstrate the same molecular weight of beta subunits (24.4 kDa) for all the three cyanobacterial species, whereas the molecular weight of the alpha subunit is different for all (17 kDa Spirulina sp., 19.1 kDa Phormidium sp., 15.2 kDa Lyngbya sp.). Thus, the C-Phycocyanin was characterized as (alphabeta)3 for Spirulina and Phormidium spp., while as (alphabeta)2 for Lyngbya sp.
