International Journal of Food Engineering

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1 International Journal of Food Engineering Volume 1, Issue Article 2 An Improved and Efficient Method for the Extraction of Phycocyanin from Spirulina sp Jayant Mahadev Doke Jr., University of Pune Recommended Citation: Doke, Jayant Mahadev Jr. (2005) "An Improved and Efficient Method for the Extraction of Phycocyanin from Spirulina sp," International Journal of Food Engineering: Vol. 1: Iss. 5, Article 2. DOI: / by the authors. All rights reserved.

2 An Improved and Efficient Method for the Extraction of Phycocyanin from Spirulina sp Jayant Mahadev Doke Jr. Abstract This paper describes an improved drying method and efficient procedure for the optimum extraction of phycocyanin from the Spirulina sp. We observed that, when Spirulina biomass is dried at 250C under shadow by air circulation and extraction of phycocyanin at 40C for 24 h in phosphate buffer at ph 7.0 (0.1 M) yield maximum phycocyanin (80 mg/g). This also shows relatively highest purity ratio of 1.8. When extraction of phycocyanin with other available methods were carried out, showed purity ratio of 0.45 to The extraction of phycocyanin in hydrochloric acid (2.0 N to 10.0 N) showed the contamination of chlorophyll in the phycocyanin extract. The proposed method of air-drying and extraction suggested in this paper showed only 5 to 7% loss of phycocyanin during drying process. KEYWORDS: Phycocyanin, drying method, spirulina, phosphate buffer, extraction Author Notes: The authors wish to thank CSIR, (Council of Scientific and Industrial Research), New Delhi, India for financial support. We also wish to thank Dr V. R. Gunale, and Dr T. D. Nikam, Department of Botany, University of Pune, Pune, , India. for their help in the present work.

3 1. INTRODUCTION Doke: An improved and efficient method for the extraction of phycocyani The Cyanobacteria Spirulina has been commercialized in several countries for its use as health food and for therapeutic purposes due to its valuable constituents particularly proteins and vitamins (Venkataraman, 1985; Benneman, 1988). It is also a rich and inexpensive source of the pigment like phycocyanin (Richmond, 1986; 1988). The growing awareness of importance of natural colours especially in food and cosmetics colorants has placed great demand on biological sources of natural colours. Cyanobactaria and algae possess a wide range of colored components including carotenoids, chlorophyll and phycocyanin (Gantt, 1981). The phycocyanin is arranged into particles named phycobilisomes, which is attached in regular array to the external surface of the thylakoid membrane and act as major light harvesting pigments in cyanobactaria and red algae (Romay et al., 1998). Phycocyanin has a significant antioxidant, anti-inflammatory, hepatoprotective and radical scavenging properties; even it is used in food colouring, and in cosmetics, as they are nontoxic and noncarcinogenic (Henrikson, 1989; Romay et al., 2000). Phycocyanin is used in chewing gums, dairy products, ice creams, jellies (Cohen, 1986; Yoshida et al., 1996), also in biomedical research (Glazer, 1994). It is used as a potential therapeutic agent in oxidative stress induced disease (Romay, 1998; Bhat, 2001). Phycobiliproteins are easily isolated as pigment protein complex, which is soluble in water and are very fluorescent (Glazer, 1981). It has gained importance in the development of phycofluor probes for immunodiagnostics (Kronik and Grossman, 1983). Pharmaceutical industries demands highly pure phycocyanin with A 620 /A 280 ratio of 4 and food industry a ratio of 2 (Bhaskar et al., 2005). For the extraction of phycocyanin from Spirulina biomass, a number of drying methods were ported such as spray dried and oven dried which resulted in approximately 50% loss of phycocyanin (Sarada et al., 1999). The improved drying method is important to store maximum amount of phycocyanin in the biomass during drying process. In view of great demand for phycocyanin at commercial level, it is therefore important to develop a simple, an improved drying method and more efficient procedure for the maximum extraction with relatively high purity ratio of phycocyanin from Spirulina sp. 2. MATERIAL AND METHODS 2.1 Algal strain and cultivation conditions The cyanobacteria Spirulina sp. was isolated from local area. Algae were grown in batch culture under the following growth conditions. A modified CFTRI (Central Food Technology Research Institute) medium containing the following constituents (all in g per liter, NaHCO g; K 2 HPO 4 0.5g; NaNO 3 1.5g; NaCl 1.0g; K 2 SO 4 1.0g; MgSO 4 7H 2 O 0.2g; FeSO 4 7H 2 O 0.01g; CaCl 2 2H 2 O 0.04g). ph was used to grow the Spirulina culture (Venkataraman et al., 1982). 1

4 The culture International was maintained Journal at of Food 30+2Engineering, o C with an Vol. illumination 1 [2005], Iss. of 5, 3-5 Art. 2K. lux light intensity provided by cool white fluorescent tubes with a dark and light cycle of 12:12 hours. The culture was grown for a period of 8 days. The growth was monitored spectrophotometrically at 560 nm. All experiments of drying and extraction were carried out in triplicates. 2.2 Analytical Procedure Phycocyanin concentration: The phycocyanin concentration in the supernatants was calculated by spectrophotometrically by measuring the absorbance at 620 nm using following equation (Baussiba and Richmond, 1979). Derivation of pure C-Phycocyanin (CPC) % Pure CPC = A 620 *10 ml * * mg sample * (% dry weight) Where 7.3 is the Extinction coefficient of CPC at 620 nm Purification factor: The purity of phycocyanin extract was monitored spectrophotometrically by the A 620 / A 280 ratio (Bennett and Bogorad, 1973). The algal biomass was determined by measurement of absorption at 560 nm (Digital Photo colorimeter), which was plotted against dry weight (g/l) on a standard curve. The ph was monitored by using digital ph meter (Elico model Li- 120). 2.3 Extraction Procedures: Phycocyanin was extracted from the wet biomass using following methods. 1. Water extraction: Harvested biomass was suspended in distilled water and kept it at 4 0 C for 24h. The extract was centrifuged at 6000 rpm (Remi India, model R8C Laboratory Centrifuge) for 10 min. Then, the supernatant was subjected to phycocyanin estimation by spectrophotometric method. 2. Homogenization of cells in homogenizer: Harvested biomass was homogenized in homogenizer (Remi India, model RQ-127A) for 5 min. in presence of phosphate buffer at ph 7.0. The extract was centrifuged at 6000 rpm for 10 min. Then, the supernatant was subjected to phycocyanin estimation by spectrophotometric method. 3. Freezing and thawing: The wet biomass was frozen at 21 0 C and thawed at 4 0 C repeatedly for 4 h in 10 ml of phosphate buffer at ph 7.0, The extract was centrifuged at 6000 rpm (Remi India, model R8C Laboratory Centrifuge) for 10 min. Then, the supernatant was subjected to phycocyanin estimation by spectrophotometric method. DOI: /

5 2.4 Drying Doke: methods An improved and efficient method for the extraction of phycocyani The harvested biomass of Spirulina was subjected to different drying methods and subsequently followed by incubation at 4 0 C for 24 h in phosphate buffer at ph 7.0, (0.1 M) for phycocyanin extraction. 1. Drying in water bath: 1g of wet biomass of Spirulina was transferred 50 ml beaker and kept in water bath at 50 0 C for one hour. The dried biomass was then grinded in mortar, pestle and sieved through 120-mesh size sieve. 2. Drying in direct sun light: 1g of wet biomass of Spirulina were dried in direct sunlight for one hour, when ambient temperature was 35 0 C. (Recorded humidity is 38%) The dried powder was then grinded in mortar, pestle and sieved through 120-mesh size sieve. 3. Air-drying: 1g of wet biomass of Spirulina were dried at 25 0 C under air circulation for 1h in shadow (without exposure of direct sunlight). The dried powder was then grinded in mortar, pestle and sieved through 120-mesh size sieve. 2.5 Solubility study of Phycocyanin The solubility of phycocyanin for air-dried biomass was studied using different buffers at ambient temperature ( C) and at low temperature (4 to 9 0 C). The experiment were carried with ph ranging from 2.0 to 12.0 in Distilled water, Phosphate buffer at ph range 6.0 to 8.0, citrate buffer at ph range 3.0 to 6.0 and hydrochloric acid from 2.0 N to 10.0 N were studied. 2.6 Stability study of Phycocyanin The stability of extracted phycocyanin was studied at ambient temperature ( C) and at low temperature (4 to 9 0 C). The extracted phycocyanin was kept for eight days at ambient temperature and at lower temperature (4 to 9 0 C) conditions. After every 24 h time interval, the concentration of phycocyanin was determined by measuring absorbance at 620 nm. The stability study of phycocyanin at lower temperature was continued for 4 months. 4. RESULTS AND DISCUSSION In the present study, an improved drying method to store maximum amount of phycocyanin in the biomass and its efficient extraction procedures is reported. Techniques used for maximum extraction of phycocyanin that work well for one Spirulina sp may not be efficient for the extraction of corresponding phycobiliproteins form another Spirulina sp. Some cyanobacteria species are able to synthesize phycocyanin (λmax 620 nm), phycoerythrins (λmax 540 to 570 nm), allophycocyanin (λmax 650 to 655 nm) or phycoerythrocyanin (λmax 575 nm) in response to ecological and growth conditions (Sidler, 1994). For this reason, the 3

6 best procedure International to obtain Journal a maximum of Food Engineering, extraction with Vol. 1 relatively [2005], Iss. high 5, Art. purity 2 ratio of phycocyanin from wet and dry Spirulina biomass was attempted (Plate 1). For the comparative study of different extraction methods was carried out for the phycocyanin extraction from Spirulina sp. three extraction methods were used for wet Spirulina biomass. Their efficiency was determined by phycocyanin concentration and its purity ratio (Expressed as A 620 / A 280 ratio). In wet biomass, the freezing at 21 0 C and thawing at 4 0 C repeatedly for 4 h obtained highest phycocyanin concentration 86.3 mg/g with purity ratio 1.34 (Table 1). The method such as water extraction and homogenization of cells shows less recovery of phycocyanin 13.4 mg/g and 82.1 mg/g respectively and purity ratio 0.45 and 0.62 respectively (Table 1). Other methods reported for extraction of phycocyanin by freezing in liquid nitrogen or by ultrasonication indicated poor recovery and purity ratio 0.18 and 0.1 respectively (Abalde et al., 1998). The wet biomass have some disadvantages for example, it cannot be stored and transportable for a longer time and not convenient for handling. The wet biomass is immediately be utilized by bacteria and starts degradation because of its chemical composition. To avoid these problems, the use of dried biomass is suitable and convenient for the extraction of phycocyanin. As per the Sarada et al. (1999), considerable loss of phycocyanin concentration was observed when wet biomass was dried at elevated temperature. Absorbance Chlorophyll Phycocyanin Wavelength (nm) Fig. 1 Absorption spectra of phycocyanin extracted from S. platensis biomass in phosphate buffer at ph 7.0 (0.1 M) and in 10.0 N HCl. DOI: /

7 Table 1 Comparisons Doke: An improved of and different efficient methods for the for extraction extraction of phycocyani of phycocyanin from wet Spirulina biomass Extraction method Phycocyanin (mg/g) Purity ratio (A 620 / A 280 ) Water extraction Homogenization (by using Homogeniser) Frozen at 21 0 C and thawed at 4 0 C repeatedly for 4 h Table 2 Comparison of different drying methods for extraction of phycocyanin at 4 0 C for 24 h in phosphate buffer at ph 7.0 (0.1 M) Drying Methods Drying temperature Phycocyanin conc. (mg/g) Purity ratio (A 620 / A 280 ) Water bath 50 0 C for 1h Sun dried Ambient temperature was 35 0 C+ 2 0 C for 1h Air dried By air current at C for 1h

8 International Journal of Food Engineering, Vol. 1 [2005], Iss. 5, Art. 2 Plate 1 Blue colored phycocyanin with red fluorescence extracted from S. platensis. The Spirulina biomass was subjected to various drying methods as depicted in Table 2 The phycocyanin extracted from these dried samples showed variations in the phycocyanin content and purity ratio. The Spirulina biomass dried at C under shadow by air-circulation shows maximum quantity of phycocyanin recovery (80 mg/g) with relatively highest purity ratio 1.8. The biomass dried in water bath at 50 0 C (16.5 mg/g), and sun-dried method at ambient temperature 35 0 C (64.8 mg/g) shows purity ratio 0.95 and 0.85 respectively (Table 2). These results confirm that the temperature plays an important role on phycocyanin concentration during the drying process of Spirulina biomass and the recovery of phycocyanin. The significant loss of phycocyanin in dried sample was observed when, biomass are subjected to higher temperature that could be due to its peripheral position in phycobilisomes on the thylakoid membrane and attributable to its sensitivity to temperature (Gantt, 1981). Therefore drying process is very important step to store maximum phycocyanin content in biomass and efficient extraction method for maximum recovery with relatively high purity ratio. DOI: /

9 As Doke: per the An improved Yi-Ming and Zhang efficient and method Feng for Chen the extraction (1999) of and phycocyani Minkova et al. (2003) the absorbance ratio of the phycocyanin fraction (A 620 /A 280 ) was 4.5 and 4.3 respectively were achieved by using various chromatography techniques. As shown in Table 1 and 2, the air-dried powder shows purity ratio of 1.8, which is higher than those are obtained using wet biomass. As described in the present paper phycocyanin extracted from air-dried biomass shows relatively higher purity ratio than those reported in the literature for cell extract 1.14 to 0.95 (Yi-Ming Zhang and Feng Chen, 1999; Minkova et al., 2003). The powder obtained by different drying methods (Table 2) were also tried for different extraction solutions/buffers to obtain maximum recovery and relatively higher purity ratio. The results showed that the highest concentration was obtained with the phosphate buffer at ph 7.0 (80 mg/g) as compared to ph 6.0 (57.9 mg/g) and ph 8.0 (42.3 mg/g) (Fig. 2). The extraction medium like distilled water (70.2 mg/g) and citrate buffer ph range from 3.0 to 6.0 yielded poor results than those obtained with phosphate buffer at ph 7.0 (Fig. 3). At lower temperature (4 to 9 0 C) extraction and purity ratio of phycocyanin was found to be much higher than the ambient temperature. Phycocyanin (mg/g) C Ambient temperature ph 6.0 ph 7.0 ph 8.0 Phosphate buffer Fig. 2 Extraction of phycocyanin from air-dried Spirulina biomass in phosphate buffer. Phycocyanin extracted in phosphate buffer at ph 7.0 shows only single peak at 620 nm, the absorption spectra were confirm the presence of phycocyanin in extract in relatively pure form (Fig. 1). The same observation was reported by Yamanaka (1980) and Abalde et al. (1998). 7

10 International Journal of Food Engineering, Vol. 1 [2005], Iss. 5, Art Phycocyanin (mg/g) C Ambient temperature ph 3.0 ph 4.0 ph 5.0 ph 6.0 Citrate buffer Fig. 3 Extraction of phycocyanin from air-dried Spirulina biomass in citrate buffer. In acid extraction, the increasing normality of HCl directly affects the cell wall morphology. The results obtained with increasing concentration of HCl on extraction of phycocyanin at lower (4 to 9 0 C) and at ambient temperature are shown in Fig. 4. The maximum recovery of phycocyanin was achieved between 8.0 to 10.0 N HCl (45.1 mg/g to 68.2 mg/g respectively) with purity ratio 1. The spectra of the phycocyanin extracted in 10 N HCl shows two peaks at 620 nm and 663 nm. Absorption at 663 nm confirms chlorophyll contamination in phycocyanin extract (Fig. 1). The microscopic observation shows that the acidic conditions damage the cell morphology; Sarada et al. (1999) also reported similar findings. Phycocyanin (mg/g) C Ambient temperature 2.0 N 4.0 N 6.0 N 8.0 N 10.0 N Normality of HCl Fig. 4 Extraction of phycocyanin from air-dried Spirulina biomass in HCl. The phycocyanin is protenious in nature; microorganisms at room temperature can easily utilize it. The results obtained during stability study indicated that, the extracted phycocyanin is more stable for more than 4 months when stored at low temperature from 4 to 9 0 C (Fig. 5). As per the results, the frozen at 21 0 C and thawed at 4 0 C repeatedly for 4 h. procedure for extraction of phycocyanin gives maximum recovery but relatively less purity ratio. The procedure is also not suitable and convenient for commercial purpose. Therefore, DOI: /

11 our reported Doke: improved An improved drying and method efficient method and extraction for the extraction procedure of phycocyani of phycocyanin is more suitable, efficient and convenient for commercial production point of view. 2.5 Optical density (620 nm) t (days) 4-9 C Ambient temperature Fig. 5 Stability of phycocyanin at different temperature conditions in phosphate buffer at ph CONCLUSIONS For the maximum recovery of phycocyanin from Spirulina biomass, different steps viz; different drying methods, efficient extraction procedure and various buffer do greatly affect the pigment recovery and purity ratio of phycocyanin. The highest concentration of phycocyanin was obtained with wet Spirulina biomass by freezing at 21 o C and thawing at 4 o C with relatively purity ratio Our finding shows that, when Spirulina biomass is dried at 25 o C under shadow by air circulation yield maximum phycocyanin (80 mg/g). This also shows highest purity ratio of 1.8. In our proposed method, there is only 5 to 7% loss of phycocyanin as compare to other methods. Powder obtained by air-dried method is more convenient to store, easily transportable and suitable for maximum recovery of phycocyanin. It is also possible to preserve phycocyanin more than four months at lower temperature (4-9 o C). Therefore, it can be concluded that the powder obtained by proposed drying method is most suitable and efficient to obtain maximum yield and purity ratio. This method being simple, efficient and convenient could be exploited at commercial level. 6. ACKNOWLEDGEMENT The authors wish to thank CSIR, (Council of Scientific and Industrial Research), New Delhi, India for financial support. We also wish to thank Dr V. R. Gunale, and Dr T. D. Nikam, Department of Botany, University of Pune, Pune, , India. for their help in the present work. 9

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