Investigation of a simple method for extraction c phycocyanin from the blue green microalgae arthrospira platensis

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY -  - GRADUATION THESIS TITLE: “INVESTIGATION OF A SIMPLE METHOD FOR EXTRACTION C-PHYCOCYANIN FROM THE BLUEGREEN MICROALGAE ARTHROSPIRA PLATENSIS” Hanoi, January 2023 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY -  - GRADUATION THESIS TITLE: “INVESTIGATION OF A SIMPLE METHOD FOR EXTRACTION C-PHYCOCYANIN FROM THE BLUEGREEN MICROALGAE ARTHROSPIRA PLATENSIS” Student name : Duong Le Long Student code : 637042 Class : K63CNSHE Faculty : Biotechnology Supervisor : Assoc.Prof.Dr Nguyen Duc Bach Hanoi, January 2023 COMMITMENT This thesis has not been submitted for academic credit at any other higher education institution I declare that the work presented in this thesis is original and solely my own To my knowledge, the contents of this thesis not include any previously published material unless appropriately cited Hanoi, January 10th, 2023 Student Duong Le Long i ACKNOWLEDGEMENTS First of all, I extend my sincere gratitude to the director of the Vietnam National University of Agriculture, the board of deans, and all the instructors in the Faculty of Biotechnology for providing me with the opportunity to complete this thesis During my graduation thesis completion, I would like to extend my sincere appreciation to my thesis advisor, Assoc Prof Dr Nguyen Duc Bach - Director of the Institute for Microalgae and Pharmacosmetics, Viet Nam National University of Agriculture, for providing me with invaluable support and guidance throughout my academic pursuits Their dedication, thoughtfulness, and expertise played a significant role in ensuring the success of my thesis At the same time, I would like to thank the staff who are studying and doing research at the Institute for Microalgae and Pharmacosmetics for their enthusiastic guidance and support during the process of making my thesis graduation project Finally, I would like to extend my sincere gratitude to my parents, siblings, relatives, and friends for their unwavering support, blessings, and constant source of inspiration throughout my academic journey Once again, thank you for everything! Sincerely, Hanoi, January 10th, 2023 Student Duong Le Long ii CONTENTS CHAPTER INTRODUCTION 1.1 Preface 1.2 Objectives 1.3 Requirements CHAPTER LITERATURE REVIEW 2.1 An overview of Arthrospira platensis 2.1.1 Introduction about Arthrospira platensis 2.1.2 Taxonomy of Arthrospira platensis 2.1.3 Chemical composition of the algae Arthrospira platensis 2.1.4 The applications of Arthrospira platensis 2.1.4.1 For human health 2.1.4.2 In the cosmetics industry 10 2.1.4.3 Some studies show positive results 10 2.2 An overview of C-phycocyanin 11 2.2.1 Introduction about C-phycocyanin 11 2.2.2 Characteristics of C-phycocyanin 12 2.2.3 Applications of C-phycocyanin 13 2.2.4 Some studies on the use of C-phycocyanin 14 2.3 Studies on C-phycocyanin extract in the world and Vietnam 18 2.3.1 Studies on C-phycocyanin extract in the World 18 2.3.2 Studies on C-phycocyanin extract in Vietnam 18 CHAPTER 3: MATERIALS AND EXPERIMENTAL METHODS 20 iii 3.1 Materials 20 3.1.1 The origin 20 3.1.2 Place and time of experiment 20 3.2 Chemicals and equipments 20 3.3 Research methods 21 3.3.1 Extraction method of C-PC 21 3.3.2 Factors affecting the extraction of C-PC 23 3.3.3 Investigate the method of processing C-PC solution into dry powder 24 3.3.3.1 Treatment of C-PC solution into dry powder by freeze-drying method 24 3.3.3.2 Treatment of C-PC solution into dry powder by natural evaporation method 25 3.3.4 Analytical method 26 3.3.5 Data processing method 26 CHAPTER 4: RESULTS AND DISCUSSION 27 4.1 The results of selecting the optimal conditions for the extraction process 27 4.1.1 Choose the optimal solvent 27 4.1.2 Choose the optimal salt concentration 33 4.1.3 Choose the optimal extraction time 36 4.1.4 Choose the ratio of algae biomass-solvent volume 38 4.2 Survey results on the method of processing C-PC solution into dry powder 42 iv 4.2.1 Experiment results images 42 4.2.2 The quality comparison between freeze-drying method and natural drying 43 4.2.3 Recovery efficiency of the refining process 44 CHAPTER 5: CONCLUSION AND SUGGESTION 46 5.1 Conclusion 46 5.2 Suggestion 46 REFERENCES 48 v LIST OF TABLES Table 2.1 Scientific classification of Arthrospira platensis Table 2.2 Amino acid composition in Arthrospira platensis Table 2.3 Vitamin composition in Arthrospira platensis Table 2.4 Pigmentation composition in Arthrospira platensis Table 3.1 List of equipments used in the experiment 20 Table 4.1 Survey results of C-PC extraction with different solvents 30 Table 4.2 Survey results of C-PC extraction at different salt concentrations 33 Table 4.3 Survey results of C-PC extraction over time course 36 Table 4.4 Survey results of C-PC extraction with different solvent volume ratios 39 Table 4.5 Investigate the quality of C-PC when using freeze-drying and natural drying methods 44 Table 4.6 Recovery efficiency of the whole process of refining C-PC to powder form 45 vi LIST OF FIGURES Figure 4.1 Image of C-PC solution after being extracted in different solvents 28 Figure 4.2 Images of algae structure before and after soaking for hours, observed under a microscope with 40x magnification 29 Figure 4.3 The change of C-PC content according to the solvent 31 Figure 4.4 The change of purity of C-PC with solvent 32 Figure 4.5 The change of C-PC content according to salt concentration 35 Figure 4.6 The change of purity of C-PC with salt concentration 35 Figure 4.7 The change in the content of C-PC over the time course 37 Figure 4.8 The change in the purity of C-PC over the time course 38 Figure 4.9 Influence of solvent ratio on the content of extracted C-PC 40 Figure 4.10 Influence of solvent ratio on the purity of extracted C-PC 41 vii LIST OF ABBREVIATIONS A platensis : Arthrospira platensis C-PC : C-phycocyanin UV-VIS : Ultravioliet – visible W/v : Weight/volume WHO : World Health Organization viii Survey results C-PC extraction with different solvent volume ratios directly affects the efficiency of the extraction process If the extraction ratio is not suitable, the amount of C-PC extracted is not high and the raw material is wasted Experimental results with different solvent ratios are shown in table (4.4) Table 4.4 Survey results of C-PC extraction with different solvent volume ratios Solvent ratio (w/v; gmL) Content of PC (mg/mL) Purity of PC (A620nm/A280nm) 0.0375 0.0500 0.0625 0.0750 0.0875 2.94 3.08 3.15 3.19 3.20 0.68 0.65 0.63 0.54 0.52 The results show that a significant impact on the C-PC extraction process can be achieved by increasing the ratio of biomass weight When increasing the biomass/solvent ratio, due to the concentration of the suspension in the solution, the amount of C-PC was easily extracted and peaked at 3.20 (mg/mL) In contrast, low suspension density will help the amount of C-PC to be extracted almost completely, so the yield is very high, reaching 0.68 Therefore, the achieved purity is almost inversely related to the concentration of C-PC, as high suspension density results in the presence of numerous substances or proteins that decrease the purity index The purity of C-PC at concentrations of 0.6 and 0.8 is considered to be pure, leading to the selection of an optimal ratio of 0.0625 Figure (4.9) shows the correlation of the influence of different solvents on the content of C-PC 39 Figure 4.9 Influence of solvent ratio on the content of extracted C-PC From the graph in Figure 4.9, it can be seen that the amount of C-PC extracted at the rate of 0.0375 reached 2.94 mg/mL the lowest, the highest when using the solvent ratio 0.075 reached 3.19 mg/mL Figure (4.10) shows the correlation effect of different solvent concentration ratio on the purity of C-PC 40 From the graph of figure (4.10), it can be seen that the purity of C-PC extracted at the rate of 0.0875 reaching 0.52 is the lowest, the highest when using the solvent ratio of 0.0375 reaching 0.68 Figure 4.10 Influence of solvent ratio on the purity of extracted C-PC From the above experiments, the optimal extraction conditions can be derived, resulting in the following as:  Solvent: NaCl  Solvent concentration: 0.9%  Extraction time: (12 hours)  Biomass/volume ratio: 0.0625 (equivalent to 2.5 grams of dry algae for 40 mL of solvent) 41 4.2 Survey results on the method of processing C-PC solution into dry powder 4.2.1 Experiment results images After extraction, C-PC solution was treated by freeze-drying (1) (2) (3) (4) Figure 4.11 Image of the experimental process of maintaining C-PC by freeze-drying method Note: Images are for illustrative purposes only and cannot be the main result C-PC solution is spread evenly on petri dishes Sublimation Dryer HT-FD6 C-PC solution after being sublimated to remove water C-PC obtained as solid 42 After extraction, solvent C-PC was spread in a thin layer on a metal dish, then stored in cold conditions and protected from light (1) (2) (3) (4) Figure 4.12 Image of the experimental process of maintaining C-PC by natural evaporation method Note: The C-PC solution is spread evenly and thinly on the metal tray The tray is sealed to avoid direct light and is kept in a cold room condition C-PC solution after evaporating all natural water C-PC obtained as solid 4.2.2 The quality comparison between freeze-drying method and natural drying The solution containing C-PC after being obtained of the two methods was dissolved with distilled water and measured OD at different wavelengths: A620 nm; A650nm and A280nm for concentration and purity testing 43 The results after calculation are compared with a 40 (mL) solvent containing C-PC as a standard with a C-PC content of 3.15 (mg/mL) and purity of 0.63 Table 4.5 Investigate the quality of C-PC when using freeze-drying and natural drying methods Content of PC (mg/mL) (A620nm/A280nm) C-PC content in dried algae (mg/g) Freeze-drying method 2.86 0.61 45.76 Natural drying method 1.92 0.51 30.72 Method Purity of PC Experimental results show that, when using the freeze-drying method to maintain C-PC, the results are superior to that of natural evaporation After sublimation drying, the content and purity of C-PC did not change too much from the initial level of 2.86 (mg/mL) with a purity of 0.61 Meanwhile, the natural evaporation method only reached 1.92 (mg/mL) and the purity was very low 0.51 compared to the original solution measured 3.15 (mg/mL) and 0.63 The reason for this difference is because when drying with sublimation drying technology, almost all the water in the C-PC solution is removed without destroying the structure Therefore, the content as well as the purity quality of C-PC remains almost the same In contrast, when using the natural evaporation method to preserve C-PC, due to the high exposure to air, C-PC is easily oxidized, leading to reduced content and quality 4.2.3 Recovery efficiency of the refining process In this study, the efficiency of the C-phycocyanin purification process was evaluated using the standard addition method The procedure involved 44 accurately weighing 2.5 (g) of pure C-PC, mixing it with 40 mL of 0.9% NaCl solution, extracting it for 12 hours at 4°C, and then centrifuging it at 1900 (rpm) for 20 minutes The solution was then purified into a powder through freezedrying and weighed Table 4.6 Recovery efficiency of the whole process of refining C-PC to powder form Theoretical mass (g) Actual weight (g) Recovery efficiency(%) 2.50 2.03 81.2% There are several other studies around the world that have tested different methods to extract C-phycocyanin from algae Here are some research results cited and compared with the results of the above study: Study by H Dhakal et al., (2018) used ultrasonic mixed extraction method with ethanol and water to extract C-phycocyanin from Spirulina The results show that the maximum extraction efficiency is 17.3% The study by O.P Ruiz et al., (2019) used a high-pressure treatment to extract C-phycocyanin from the algae Arthrospira platensis The results show that the maximum extraction efficiency is 85.4% The study by S.K Basha, et al., (2019) used saline extraction (NaCl) to extract C-phycocyanin from Spirulina The results show that the maximum extraction efficiency is 20.8% Compared with the above research results, the above study results show that the mechanical extraction method is the simplest and lowest cost method and the C-phycocyanin extraction efficiency is 81.2% 45 CHAPTER 5: CONCLUSION AND SUGGESTION 5.1 Conclusion Through the study of simple C-phycocyanin extraction method from the greenmicroalgae Arthrospira platensis, the following conclusions were drawn: The mechanical extraction method, which involves breaking algae cells to release C-PC, is a straightforward and low-cost approach that is easy to implement Despite having a low concentration and purity, the resulting product still satisfies the safety requirements for use In this study, C-phycocyanin extraction procedure was extracted with the following conditions: 0.9% NaCl buffer solution; the algal mass-volume solvent ratio is 0.0625; extraction time is 14 hours Centrifuge the solution at 1900 (rpm) for 20 minutes at 4°C The product after extraction by the optimized process reached a concentration of 3.15 (mg/mL) with purity A620nm/A620nm = 0.63, the efficiency of the entire process including the extraction process and make powder products of Cphycocyanin is 81.2% The superiority of freeze drying method in making dry solid products compared to natural evaporation method has been shown In particular, this method does not significantly reduce the quality and purity of C-PC 5.2 Suggestion Based on the results obtained from the experimental process and some limitations of the study, recommendations were made as follows: Conducted surveys with various other methods to improve C-PC content as well as purity Some promising methods such as high-pressure extraction, ultrasonic extraction or lyzozyme treatment have great applications 46 Conduct a survey on methods to increase the purity of C-PC extract such as using ammonium sulphate salt, using activated carbon, chitosan In addition, some other C-PC purification methods such as sepandex gel filtration chromatography, high performance liquid chromatography (HPLC) or simpler can use multiple extraction methods 47 REFERENCES A Herrera, A Napoleone, A Hohlberg (1989), “Recovery of Cphycocyanin from the cyanobacterium Spirulina maxima”, Journal of Applied Phycology, 1, pp.325-331 A Vonshak, (1997), “Spirulina platensis (Athrospira): physiology, Cell Biology and Biotechnology”, Taylor and Francis,1, pp.230-233 C D‟souza, H.N Pradeep, C.A Nayak (2018), “Extraction of phycocyanin from Spirulina plantesis using sonication”, pp.27974-27978 C Romay, R González1, N Ledón1, D Remirez1, V Rimbau (2003), “Phycocyanin: A Biliprotein with Antioxidant, Anti Inflammatory and Neuroprotective Effects Current Protein and Peptide Science”, 1, pp.207-216 C Sili, G Torzillo, A Vonshak, (2012), “Ecology of Cyanobacteria II: Their Diversity in Space and Time”, Springer Dordrecht Heidelberg New York London, (25), pp.677-705 D Kumar, D.W Dhar, S Pabbi, N Kumar, S Walia, (2014), “Extraction and purification of C-phycocyanin from Spirulina platensis”, Indian J Plant Physiol, 19, pp.184-188 D.P Jaeschke, I.R Teixeira, L.D.F Marczak, G.D Mercali (2021), “Phycocyanin from Spirulina: A review of extraction methods and stability”, Affiliations expand, PMID: 33992333 DOI: 10.1016/j.foodres.2021.110314 H Dhakal, B.R Pokharel, B Shrestha, P Poudel, A Wagle, N Wagle, (2018), “Extraction of C-phycocyanin from Spirulina platensis using ultrasound-assisted extraction”, Journal of Applied Research on Medicinal and Aromatic Plants, 9, pp.55-61 48 H.T.H Tran, T.T.N Nguyen, T.M Hoang, T.T Nguyen, T.N Do, (2020), “Extraction of C-phycocyanin from Spirulina platensis using highspeed centrifugation”, Journal of cosmetic dermatology, 19(2), pp.245-252 J Abalde, L Betancourt, E Torres, A Cid, C Barwell (1998),“ Purification and characterization of phycocyanin from the marine cyanobacterium Synechococcus sp” Plant Science, 136, 1, pp.109-120 J Léonard, P Compère, (1967), “Spirulina platensis blue alga of great nutritional value due to its high protein content”, (37), pp.1–23 J.C Dillon, A.P Phuc, J.P Dubacq, (1995), “Nutritional value of the algae Spirulina”, World Rev nutr diet, 77, pp.32-46 J.C Lee, M.F Hou, H.W Huang, F.R Chang, C.C Yeh, Tang, J.Y Chang, H.W Marine, (2013), “Algal natural products with anti-oxidative, antiinflammatory, and anti-cancer properties”, Cancer Cell Int, 13, pp.55-61 L Chen, M Qiu, Y Guo, J Chen (2017), “Extraction of C-phycocyanin from Spirulina platensis using high-speed centrifugation and ultrasonic extraction”, Marine drugs, pp.331-334 L.V Lăng, (1999), “Spirulina - nuôi trồng sử dụng y dược dinh dưỡng”, NXB Y học M Kuddus, P Singh, G Thomas, A.A Hazimi (2013), “Recent Developments in Production and Biotechnological Applications of Cphycocyanin”, pp.1-9 M Qiu, L Chen, Y Guo, J Chen, (2016), “Extraction of Cphycocyanin from Spirulina platensis using microwave and ultrasonic treatments”, Marine drugs, 14(2), pp.40-41 49 M.A.B Habib, M Parvin, T.C Huntington, M.R Hasan (2008), “A review on culture, production and use of Spirulina as food for humans and feeds for domestic animals”, F.A.O Fisheries and Aquaculture Circular, 1, pp.33-34 M.R Palomares, L Nunez, D Amador, (2001), “Practical application of aqueous two‐ phase systems for the development of a prototype process for C‐phycocyanin recovery from Spirulina maxima”, Journal of Chemical Technology & Biotechnology, 76(12), pp.1273-1280 N Mallick, L.C Rai, (2007), “Inhibition of Fe (III)-EDTAphotodegradation of C-phycocyanin, the pigment-protein complex from Spirulina platensis, by UV-B radiation”, Process Biochemistry, 42(2), pp.253257 N Seyidoglu, S Inan, C Aydin, (2017), “A prominent superfood: Spirulina platensis, superfood and functional food - The development of superfoods and their roles as medicine”, IntechOpen, DOI: 10.5772/66118 N.T Eriksen, (2008), “Production of phycocyanin-a pigment with applications in biology, biotechnology, foods and medicine”, Appl Microbiol Biotechnol, 80, pp.1-14 Nguy n c B ch, Nguy n Phan Khu , Ph Th C m Mi n, Kim Anh Tu n, Nguy n Th Hi n, (2020), “Nghi n c u nh hư ng c a n LED n sinh trư ng, h m lượng s c t v kh th ch ng c a m t s ch ng t o xo n Arthrospira platensis m a ng mi n b c i t Nam”, T p ch Khoa học N ng nghi p Vi t Nam, 18(8), pp.637-648 O.P Ruiz, V.M.M Juarez, M.R.R Burgueno, (2019), “Extraction of Cphycocyanin from Arthrospira platensis by high-pressure homogenization”, Journal of Applied Phycology, 31(6), pp.3481-3491 50 P.D Karkos, S.C Leong, C.D Karkos, N Sivaji, D.A Assimakopoulos (2011), „„Spirulina in Clinical Practice: Evidence-Based Human Applications”, Evidence-Based Complementary and Alternative Medicine, pp.1-4 R González, S Rodríguez, C Romay, O Ancheta, A González, J Armesto, D Remirez, N Merino, (1999), “Anti-inflammatory activity of phycocyanin extract in acetic acid-induced colitis in rats”, Pharmacol Res, 39(1), pp.45-59 R Sathasivam, R Radhakrishnan, A Hashem, P Ganesan, (2018), “Green synthesis of C-phycocyanin from Spirulina platensis and its anticancer potential”, Journal of Photochemistry and Photobiology, 178, pp.251-258 R.A Lewin, (1953), “The cell structure and growth of Spirulina platensis in culture”, Annals of the New York Academy of Sciences”, 56(5), pp.867-880 R.G Fisher, N.E Woods, H.E Fuchs, R.M Sweet, (1980), “Three Dimensional Structures of C-phycocyanin and B-Phycoerythrin at 5-A Resolution”, The Journal of Biological Chemistry, 255(11), pp.5082-5089 S Aishwarya, S Rajeshwari, P Ramasamy, (2017), “Comparative study on the extraction of C-phycocyanin from Arthrospira platensis using different methods”, Journal of Microbiology and Biotechnology Research, 7(4), pp.8489 S Ayehunie, A Belay, T.W Baba, R.M Ruprecht, (1998), “ Inhibition of HIV-1 replication by an aqueous extract of Spirulina platensis (Arthrospira platensis)”, 18(1), pp.7-12, DOI: 10.1097/00042560-199805010-00002 S Boussiba (1979) “Isolation and characterization of phycocyanins from the Blue-Green alga Spirulina platensis” Arch microbiol, 120, pp.155–159 51 S.M Hosseini, S Shahbazizadeh, K.D Khosravi, M.R Mozafari, (2013), “Spirulina paltensis: Food and Function”, Curr Nutr Food Sci, 9, pp.189–193 S.P Kamble, R.B Gaikar, R.B Padalia, K.D Shinde, (2013), “Extraction and purification of C-phycocyanin from dry Spirulina powder and evaluating its antioxidant, anticoagulation and prevention of DNA damage activity”, Journal of Applied Pharmaceutical Science, 3(08), pp.149-153 T.M Hoang, T.T.N Nguyen, T.T.H Nguyen, H.T.H Tran, (2018), “Protective effect of C-phycocyanin extracted from Spirulina platensis against UV-B radiation in vitro”, Journal of cosmetic dermatology, 17(4), pp.514-520 W Song, C Zhao, S Wang, (2013) “A large-scale preparation method of high purity C-phycocyanin”, International Journal of Bioscience, Biochemistry Bioinformatics, 3(4), pp.293-297 W Song, C Zhao, S Wang, (2013), “A Large-Scale Preparation Method of High Purity C-phycocyanin”, International Journal of Bioscience, Biochemistry and Bioinformatics, 3(4), pp.294-297 W.S Park, H.J Kim, M Li, D.H Lim, J Kim, J.J Kwak, C.M Kang, M.G Ferrruzi, M.J Ahn (2018), “Two classes of pigments, carotenoids and Cphycocyanin, in Spirulina powder and their antioxidant activities”, Molecules, 23(8), DOI: 10.3390/molecules23082065 Y.K Kim, H.K Kim, Y.C Kim, (2015), “Extraction of C-phycocyanin from Spirulina using ultrasonic and heat treatments”, Journal of food science, 80(2), pp.339-344 Z Khan, P Bhadouria, P.S Bisen, (2005), “Nutritional and therapeutic potential of Spirulina”, Curr Pharm Biotechnol, 6, pp.373-379 52 S.K Basha, D Prabaharan, R.P Singh, N Selvaraju, R Raja, (2019), “Extraction of C-phycocyanin from Spirulina platensis using saline extraction method”, Journal of Applied Phycology, 31(6), pp.3585-3594 L Zhang, W Liu, X Wang, X Zhang, L Zhao, (2018), “Extraction of C-phycocyanin from Spirulina platensis using aqueous two-phase flotation combined with ultrasound and macroporous resin purification Bioresource technology”, 254, pp.168-174 D Singh, I Rawat, B Adhikari, R.K Rai, (2018), “Efficient extraction of C-phycocyanin from Spirulina platensis using ammonium sulphate precipitation followed by sonication”, Journal of Applied Phycology, 30(1), pp.201-210 R Ktari, (2020), “Extraction of C-phycocyanin from Spirulina using salt solution: Optimization by response surface methodology and product characterization”, Journal of Food Science and Technology, 57(1), pp.61-69 H.T Phuc, (2020), “Optimization of C-phycocyanin extraction from Spirulina platensis using response surface methodology”, Journal of Applied Phycology, 32(4), pp.2347-2361 Y Jiang, (2019), “Efficient extraction of C-phycocyanin from Spirulina platensis and its stability and antioxidant activity”, Food Science & Nutrition, 7(7), pp.2352-2361 X Han, (2021), “Green extraction and characterization of phycocyanin from Spirulina platensis using water as the solvent”, Journal of Food Process Engineering, 44(5), pp13525-13528 53