海藻是許多抗癌、抗發炎和抗氧化特性生物活性化合物的重要來源。因此，研究海藻內生物活性化合物對食品科學和生物技術行業具重大意義。本次實驗中所研究的生物活性化合物為生物活性海藻蛋白。實驗用的海藻為台灣墾丁採集的匍枝馬尾藻Sargassum polycystum。海藻樣品保存在-20⁰ C保鮮，然後在-80⁰ C冷凍的海藻樣品進行預先處理，之後將等分試樣冷凍樣品在液態氮 (LN2)中進行研磨。剩餘的冷凍樣品將被冷凍乾燥並磨碎。接下來我們將對新鮮、液氮冷凍和凍乾樣品進行水分測定並根據水分含量結果計算樣品的乾重。隨後利用凱氏定氮法測定出匍枝馬尾藻樣品的粗蛋白百分比為 4.51%。通過計算我們可以得出乾燥樣品內可提取的粗蛋白之比重。此研究包括了五種萃取法。首先，冷凍乾燥的樣品提取方法為改良版三氯乙酸 (TCA)/丙酮沉澱法，標記為1a. 經液氮研磨所提取的海藻樣品則標記為1b。由鹽析（硫酸銨，（NH4）2SO4，分餾法）法從凍乾樣品中獲得蛋白質提取物標記為2a；由新鮮的樣品提取蛋白質的方法則標記為 2b。硫酸銨分餾法的樣本根據不同的飽和百分比分別標記為（20%-2a/2b20、40%-2a/2b40、60%-2a/2b60和80%-2a/2b80）。第三種從凍乾樣品中提取蛋白質的方法為經優化超音波震盪輔助蛋白質提取法，標記為 3a。第四種方法為混合萃取法, 採用了部分超音波震盪與鹽析法的萃取方式，標記為 4a。第五種方法同為混合萃取法，整合了鹽析、三氯乙酸/丙酮沉澱法和超音波震盪法的萃取方式，標記為4b。兩組混合萃取法所用的樣品均經液氮研磨處理。混合萃取法的飽和度百分比分別標記為(20%-4a/4b20、40%-4a/4b40、60%-4a/4b60和80%-4a/4b80)。我們用 Thermo Scientific™ NanoDrop 2000 對每種方法的所得蛋白質萃取物進行蛋白質的濃度測定。通過該數據計算蛋白質產率，並比較蛋白質濃度與可提取的總粗蛋白質。接著，我們利用ABTS法 (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid))和DPPH (1, 1-Diphenyl-2- Picrylhydrazyl) 來測定蛋白質的抗氧化活性。然後，經由計算得出樣本的半抑制值IC50 以用於ABTS檢定。最後，使用 SDS-PAGE（十二烷基硫酸鈉-聚丙烯酰胺凝膠電泳）對蛋白質萃取物進行分子量測定。實驗後我們對所有結果進行統計分析與顯著性差異檢定。在這項研究中，第一種組合方法 4a和超音波震盪法3a所萃取物中的蛋白質濃度與其他方法相比有顯著提升。根據實驗結果， 4a20樣品中的蛋白質濃度為 28.9 mg/mL，3a 樣品中的蛋白質濃度則為 16.6 mg/mL。TCA/丙酮沉澱法、第一綜合萃取法和第二綜合萃取法皆呈現高蛋白質產率。1b、4a40和4b40 樣品的產量分別為51.6%、39.1%和36.3%，因此得出以上樣品所採用的方法能有效萃取蛋白質。此外，ABTS 和DPPH 測定結果顯示第二種混合萃取法的蛋白質萃取物與其他方法所獲得的萃取物相比擁有更高的抗氧化活性。 ABTS 測定中樣品1a、4b20、4b60及4b80的抑制率各為 98.2%、 99.1%、98.7%及98.5%。此外，4b80 樣品的IC50 ABTS 測定值0.067 mg/mL為測試組中最低，這表明該蛋白質萃取物樣品能有效清除ABTS 自由基。雖然抑制率不如4b80那麼顯著，但就總體而言樣品4b20、4b40、4b60 和1a均呈現低 IC50值。反之從其他方法獲得的樣品均呈現較高的IC50值。第二種混合萃取法的樣品4b40、4b60及4b80在 DPPH 測定中均呈現高抑制率，分別為87.2%、88.6%及93.0%。實驗中唯一經SDS-PAGE電泳分析的樣品為80% 硫酸銨飽和度下的萃取物，估算出分子量為43 kDa。本研究的宗旨為通過比較在五種萃取法中尋找溫和有效的生物活性蛋白萃取法。此研究成功證明了所萃取的蛋白質具有效抗氧化能力，並有希望在食品生物技術行業中進行實際應用。
關鍵字：ABTS、生物活性蛋白、DPPH、匍枝馬尾藻、SDS-PAGE

Seaweed is a source of many important bioactive compounds that exhibit anti-cancer, anti-inflammatory, and antioxidant properties. Thus, the compounds responsible for these attributes are important to the Food Science and Biotechnology industries. The bioactive compounds of interest in this study are bioactive seaweed proteins. Therefore, samples of the brown seaweed species, Sargassum polycystum, were collected from Kenting, Taiwan. The seaweed sample was primarily kept fresh at -20⁰ C, and the seaweed sample frozen at -80⁰ C was later pretreated. An aliquot of the frozen sample was crushed in liquid nitrogen (LN2). The remaining frozen sample was freeze-dried and ground. Then, the moisture analysis of the fresh, liquid nitrogen frozen, and freeze-dried samples was conducted. The dry weight of the samples was calculated from the moisture content results. Subsequently, the Kjeldahl method determined the crude protein percent of the Sargassum polycystum sample, which was 4.51%. The total crude protein calculations revealed the crude protein available for extraction based on the dry weight of samples. Five distinct methods were employed to extract the available proteins. First, the modified trichloroacetic acid (TCA)/Acetone method was labeled 1a provided that the extraction sample was freeze-dried. If the seaweed sample for extraction was pretreated with LN2, then the method was labeled 1b. Second, the salting-out (ammonium sulfate, (NH₄)₂SO₄, fractionation) method attaining protein extracts from the freeze-dried samples was labeled 2a; if the samples were fresh, the method was labeled 2b. The ammonium sulfate fractionation method was additionally labeled according to the saturation percentages (20%-2a/2b20, 40%-2a/2b40, 60%-2a/2b60, and 80%-2a/2b80). Third, the optimized ultrasound-assisted method, labeled 3a, extracted protein from the freeze-dried samples. Fourth, the first combined method, which incorporated some processes from the ultrasound-assisted and salting-out methods, was labeled 4a. Fifth, the second combined method, labeled 4b, integrated different processes from the salting out, TCA/acetone, and ultrasound-assisted methods. The combined methods both extracted protein from samples pretreated with LN2. The combined methods were further labeled according to the saturation percentages (20%-4a/4b20, 40%-4a/4b40, 60%-4a/4b60, and 80%-4a/4b80). The resulting protein extracts derived from each respective method were quantified using a Thermo Scientific™ NanoDrop 2000 to determine the concentration of proteins within a sample. The protein yield was then calculated and indicated the concentration of protein extracted by each method compared to the total crude protein available for extraction. Subsequently, the antioxidant activity of the protein extracts produced by each method was investigated by ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) radical scavenging and DPPH (1, 1-Diphenyl-2-Picrylhydrazyl) assays. Then, the IC50 values of samples were calculated for the ABTS assay. Finally, the protein extracts were visualized using SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) to determine the molecular weight of extracted proteins. The statistical analysis for all the results was conducted, and the significant differences were determined. In this study, the first combined method, 4a, and the ultrasound-assisted method, 3a, resulted in higher protein concentrations in samples compared to the other protein extraction protocols. The results revealed protein concentrations of 28.9 mg/mL in 4a20 samples and 16.6 mg/mL in 3a samples. The TCA/acetone, first combined, and second combined methods exhibited high protein yield. Since these methods producing 1b, 4a40, and 4b40 samples revealed yields of 51.6%, 39.1%, and 36.3% respectively, then the methods employed were effective in protein extraction. Furthermore, results from the ABTS and DPPH assays conducted on the protein extracts from the second combined method indicated that these samples had high antioxidant activity compared to the samples derived from the other methods. The inhibition percentages of samples in the ABTS assay was 98.2% for 1a, 99.1% for 4b20, 98.7% for 4b60, and 98.5% for 4b80. Furthermore, the 4b80 sample displayed the lowest IC50 value for the ABTS assay, 0.067 mg/mL, which indicated that this protein extract sample can effectively scavenge the ABTS radicals. Similarly, however not at the same capacity, the 4b20, 4b40, 4b60, and 1a samples exhibited low IC50 values. Conversely, samples obtained from all other methods resulted in higher IC50 values. The samples of the second combined method also attained the highest inhibition percentage in the DPPH assay of 87.2% for 4b40, 88.6% for 4b60, and 93.0% for 4b80. The protein extracts derived from the combined method at 80% ammonium sulfate saturation was the only sample visualized in SDS-PAGE analysis, with an estimated molecular weight of 43 kDa. The premise of this study, to deduce the gentle and effective method for extracting bioactive protein among the five compared methods, has been achieved. The study revealed the capacity of the extracted proteins functioning as beneficial antioxidants, which has potential application in the Food Biotechnology industry.
Keywords: ABTS, bioactive proteins, DPPH, Sargassum polycystum, SDS-PAGE

References
Abdollahi, M., Axelsson, J., Carlsson, N.-G., Nylund, G. M., Albers, E., and I. Undeland. 2019. Effect of stabilization Method and Freeze/Thaw-Aided Precipitation on Structural and Functional Properties of Proteins recovered from Brown Seaweed (Saccharina latissima). Food Hydrocolloids 96:140-150.
Admassu, H., Gasmalla, M. A. A., Yang, R., and W. Zhao. 2018. Bioactive Peptides derived from Seaweed Protein and Their Health Benefits: Antihypertensive, Antioxidant, and Antidiabetic Properties. Journal of Food Science 83(1):6-16.
Ahmad, A., Usman, H., Natsir, H. and A. Karim. 2014. Isolation and Characterization of Bioactive Protein from Green Algae Halimeda macrobola Acting as Antioxidant and Anticancer Agent. American Journal of Biomedical and Life Sciences 2(5):134-140.
Ames, B. N., Gold, L. S., and W. C. Willett. 1995. The Causes and Prevention of Cancer. Proceedings of the National Academy of Sciences 92(12):5258-5265.
Aksamitiene, E., Hoek, J. B., Kholodenko, B., and A. Kiyatkin. 2007. Multistrip Western blotting to Increase Quantitative Data Output. Electrophoresis 28(18):3163-3173.
Badmus, U. O., Taggart, M. A., and K. G. Boyd. 2019. The Effect of Different Drying Methods on Certain Nutritionally Important Chemical Constituents in Edible Brown Seaweeds. Journal of Applied Phycology 31(6):3883-3897.
Biancarosa, I., Espe, M., Bruckner, C. G., Heesch, S., Liland, N., Waagbø, R., and E. J. Lock. 2017. Amino Acid Composition, Protein Content, and Nitrogen-to-Protein Conversion Factors of 21 Seaweed Species from Norwegian Waters. Journal of Applied Phycology 29(2):1001-1009.
Birben, E., Sahiner, U. M., Sackesen, C., Erzurum, S., and O. J. Kalayci. 2012. Oxidative Stress and Antioxidant Defense. World Allergy Organization Journal 5(1):9-19.
Bode, A. M., and Z. Dong. 2009. Cancer Prevention Research-Then and Now. Nature Reviews Cancer 9(7):508-516.
Buschmann, A. H., Camus, C., Infante, J., Neori, A., Israel, Á., Hernández-González, M. C., Tadmor-Shalev, N., and A. T. Critchley. 2017. Seaweed Production: Overview of the Global State of Exploitation, Farming and Emerging Research Activity. European Journal of Phycology 52(4):391-406.
Černá, M. 2011. Seaweed Proteins and Amino Acids as Nutraceuticals. Advances in Food and Nutrition Research 64:297-312.
Chakraborty, K., Maneesh, A., and Makkar, F. 2017. Antioxidant Activity of Brown Seaweeds. Journal of Aquatic Food Product Technology 26(4):406-419.
Chia, Y. Y., Kanthimathi, M., Khoo, K. S., Rajarajeswaran, J., Cheng, H. M., and W. S. Yap. 2015. Antioxidant and Cytotoxic Activities of Three Species of Tropical Seaweeds. BMC Complementary and Alternative Medicine 15(1):1-14.
Cho, S. H., Kang, S. E., Cho, J. Y., Kim, A. R., Park, S. M., Hong, Y. K., and D. H. Ahn. 2007. The Antioxidant Properties of Brown Seaweed (Sargassum siliquastrum) Extracts. Journal of Medicinal Food 10(3):479-485.
Contreras, L., Dennett, G., Palma, E., Moenne, A., and J. A. Correa. 2007. Molecular and Morphologically Distinct Scytosiphon Species (Scytosiphonales, Phaeophyceae) Display Similar Antioxidant Capacities. Journal of Phycology 43(6):1320-1328.

Contreras, L., Ritter, A., Dennett, G., Boehmwald, F., Guitton, N., Pineau, C., Moenne, A., Potin, P. & Correa, J. 2008. Two Dimensional Gel Electrophoresis Analysis of Brown Algal Protein Extracts. Journal of Phycology 44:1315-1321.
Contreras-Porcia, L., and C. López-Cristoffanini. 2012. Proteomics in Seaweeds: Ecological Interpretations. Gel Electrophoresis-Advanced Techniques: InTech: 21-52.
Diplock A., Charleux J., Crozier-Willi G., Kok, J. F., Rice-Evans, C., Roberfroid, M., Stahl, W., and J. Viña-Ribes. 1998. Functional Food Science and Defense Against Reactive Oxygen Species. British Journal of Nutrition 80(1):77-112.
Doll, R., and R. Peto. 1981. The Causes of Cancer: Quantitative Estimates of Avoidable Risks of Cancer in the United States Today. JNCI: Journal of the National Cancer Institute 66(6):1192-1308.
Elias, R. J., Kellerby, S. S., and E. A. Decker. 2008. Antioxidant Activity of Proteins and Peptides. Critical Reviews in Food Science and Nutrition 48(5):430-441.
Ferdouse, F., Holdt, S. L., Smith, R., Murúa, P., and Z. Yang. 2018. The Global Status of Seaweed Production, Trade and Utilization. FAO Globefish Research Programme 124:120.
Firmansyah, S. B., Firmansyah, R. A., and N. J. Hayati. 2017. Antioxidant Activity and Antibacterial Seaweed Methanol Extract (Sargassum Duplicatum J. Agardh) and it’s Potential as a Natural Preservative Alternative to Salted Eggs. Journal of Natural Sciences And Mathematics Research 2(1):133-142.
Fleurence, J. 1999. Seaweed Proteins: Biochemical, Nutritional Aspects and Potential Uses. Trends in Food Science & Technology 10(1):25-28.
Gallagher, S. 2006. One‐dimensional SDS Gel Electrophoresis of Proteins. Current Protocols in Molecular Biology 75(1):10.12. 11-10.12 A. 37.
Gany, S. A., Tan, S. C., and S. Y. Gan. 2015. Antioxidative, Anticholinesterase and Anti-Neuroinflammatory Properties of Malaysian Brown and green Seaweeds. International Journal of Industrial and Manufacturing Engineering 8(11):1269-1275.
Garfin, D. E. 1990. One-dimensional Gel Electrophoresis. Methods in Enzymology Elsevier 182:425-441.
Harrysson, H., Hayes, M., Eimer, F., Carlsson, N. G., Toth, G. B., and I. Undeland. 2018. Production of Protein Extracts from Swedish Red, Green, and Brown Seaweeds, Porphyra umbilicalis Kützing, Ulva lactuca Linnaeus, and Saccharina latissima (Linnaeus) JV Lamouroux using Three Different Methods. Journal of Applied Phycology 30(6):3565-3580.
Hussain, S. P., Hofseth, L. J., and C. C. Harris. 2003. Radical Causes of Cancer. Nature Reviews Cancer 3(4):276–285.
Johnson, M., Kanimozhi, S. A., Malar, T. R. J. J., Shibila, T., Freitas, P. R., Tintino, S. R., and H. D. M. Coutinho. 2019. The antioxidative effects of Bioactive Products from Sargassum polycystum C. Agardh and Sargassum duplicatum J. Agardh against Inflammation and Other Pathological Issues. Complementary Therapies in Medicine 46:19-23.
Kadam, S., O'Donnell, C., Rai, D., Hossain, M., Burgess, C., Walsh, D., and B. Tiwari. 2015. Laminarin from Irish Brown Seaweeds Ascophyllum nodosum and Laminaria hyperborea: Ultrasound Assisted Extraction, Characterization and Bioactivity. Marine Drugs 13 (7):4270-4280.
Karim, H., Ahmad, A., Natzir, R., Massi, M. N., Arfah, R., Asmi, N., and A. Karim. 2019. Isolation and Identification of Bioactive Proteins from the Brown Algae Sargassum, Sp. and Their Potential as Anticancer Agents. Journal of Physics: Conference Series 1341:032009.
Kristiansen, K. A., Potthast, A., and B. E. Christensen. 2010. Periodate Oxidation of Polysaccharides for Modification of Chemical and Physical Properties. Carbohydrate Research 345(10):1264–1271.
Hurd, C.L., Harrison, P.J., Bischof, K. and C. S. Lobban. 2014. Seaweed Ecology and Physiology. 2nd ed. Cambridge University Press, Cambridge.
Lobo, V., Patil, A., Phatak, A., and N. J. Chandra. 2010. Free Radicals, Antioxidants and Functional Foods: Impact on Human Health. Pharmacognosy Reviews, 4(8):118-126.
Lourenço, S. O., Barbarino, E., De‐Paula, J. C., Pereira, L. O. D. S., and U. M. L. Marquez. 2002. Amino Acid Composition, Protein Content and Calculation of Nitrogen‐To‐Protein Conversion Factors for 19 Tropical Seaweeds. Phycological Research, 50(3):233-241.
Mabeau, S., and J. Fleurence. 1993. Seaweed in Food Products: Biochemical and Nutritional Aspects. Trends in Food Science & Technology 4(4):103-107.
Mellouk, Z., Benammar, I., Krouf, D., Goudjil, M., Okbi, M., and W. Malaisse. 2017. Antioxidant Properties of the Red Alga Asparagopsis taxiformis Collected on The North West Algerian Coast. Experimental and Therapeutic Medicine 13(6):3281-3290.
Mhadhebi, L., Mhadhebi, A., Robert, J., and A. Bouraoui. 2014. Antioxidant, Anti-Inflammatory and Antiproliferative Effects of Aqueous Extracts of Three Mediterranean Brown Seaweeds of the Genus Cystoseira. Iranian Journal of Pharmaceutical Research: IJPR, 13(1):207-220.
Mohd Rosni, S., Fisal, A., Azwan, A., Chye, F. Y., and P. Matanjun. 2015. Crude Proteins, Total Soluble Proteins, Total Phenolic Contents and SDS-PAGE Profile of Fifteen Varieties of Seaweed from Semporna, Sabah, Malaysia. International Food Research Journal 22(4):1483-1493.
Nisizawa, K., Noda, H., Kikuchi, R., and T. J. Watanabe. 1987. The Main Seaweed Foods in Japan. Hydrobiologia 151(1):5-29.
Nayar, S., & K. Bott. 2014. Current Status of Global Cultivated Seaweed Production and Markets. World Aquaculture 45(2):32-37.
Niu, L., Zhang, H., Wu, Z., Wang, Y., Liu, H., Wu, X., W. Wang. 2018. Modified TCA/acetone Precipitation of Plant Proteins for Proteomic Analysis. PLoS ONE 13(12):e0202238.
Nurjanah, M. N., Anwar, E., Luthfiyana, N., and T. Hidayat. 2018. Identification of Bioactive Compounds of Seaweed Sargassum sp. and Eucheuma cottonii Doty as a Raw Sunscreen Cream. Proceedings of the Pakistan Academy of Sciences: B. Life and Environmental Sciences 54(4):311-318.
Perumal, B., Chitra, R., Maruthupandian, A., and M. Viji. 2019. Nutritional Assessment and Bioactive Potential of Sargassum polycystum C. Agardh (Brown Seaweed). International Journal on Multicultural Societies (IJMS) Vol.48(04):492-498.
Rattaya, S., Benjakul, S., and T. Prodpran. 2015. Extraction, Antioxidative, and Antimicrobial Activities of Brown Seaweed Extracts, Turbinaria ornata and Sargassum polycystum, Grown in Thailand. International Aquatic Research 7(1):1-16.
Rawel, H. M., Meidtner, K., and J. J. Kroll. 2005. Binding of Selected Phenolic Compounds to Proteins. Journal of Agricultural and Food Chemistry 53(10):4228-4235.
Roosdiana, A., & N. L. Rahmah. 2012. The Potency of Sargassum duplicatum Bory Extract on Inflammatory Bowel Disease Therapy in Rattus norvegicus. Journal of Life Sciences 6(2):144-154.
Salosso, Y. 2019. Nutrient and Alginate Content of Macroalgae Sargassum sp. from Kupang Bay Waters, East Nusa Tenggara, Indonesia. Aquaculture, Aquarium, Conservation & Legislation 12(6):2130-2136.
Shahidi, F. 2000. Antioxidants in food and food antioxidants. Food/Nahrung 44(3):158-163.
Shaikh, W., Shameel, M., Usmanghani, K., and V. J. Ahmad. 1991. Phycochemical Examination of Padina tetrastromatica (Dictyotales, Phaeophyta). Pakistan Journal of Pharmaceutical Sciences 4(1):55-61.
Sun, Y., Rukeya, J., Tao, W., Sun, P., and X. Ye .2017. Bioactive compounds and antioxidant activity of wolfberry infusion. Scientific Reports, 7(1):1-8.
Sung, H., Ferlay, J., Siegel, R. L., Laversanne, M., Soerjomataram, I., Jemal, A., and F. Bray. 2021. Global cancer statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA: A Cancer Journal for Clinicians 71(3):209-249.
Supendy, R., Taridala, S. A. A., Hafid, H., Sifatu, W. O., Sailan, Z., and L. Niampe. 2018. Income of Seaweed Farming Households: A Case Study from Lemo of Indonesia. IOP Conference Series: Earth and Environmental Science 175(1):012221.
Trigueros, E., Sanz, M. T., Alonso-Riaño, P., Beltrán, S., Ramos, C., and R. Melgosa. 2021. Recovery of the Protein Fraction with High Antioxidant Activity from Red Seaweed Industrial Solid Residue after Agar Extraction by Subcritical Water Treatment. Journal of Applied Phycology 33(2):1181-1194.
Vadas, R. L. 1979. Seaweeds: An Overview; Ecological and economic Importance. Experientia 35(4):429-433.
Valko, M., Leibfritz, D., Moncol, J., Cronin, M. T., Mazur, M., and J. Telser. 2007. Free radicals and Antioxidants in Normal Physiological Functions and Human Disease. International Journal of Biochemistry & Cell Biology 39(1):44-84.
Vilg, J. V., and I. Undeland. 2017. pH-driven Solubilization and Isoelectric Precipitation of Proteins from The Brown Seaweed Saccharina latissima—Effects of Osmotic Shock, Water Volume and Temperature. Journal of Applied Phycology 29(1):585-593.
Waks, A. G., and E. P. Winer. 2019. Breast Cancer Treatment:A Review. JAMA 321(3):288-300.
Wang, W., Vignani, R., Scali, M., and M. Cresti. 2006 A Universal and Rapid Protocol for Protein Extraction from Recalcitrant Plant Tissues for Proteomic Analysis. Electrophoresis 27(13):2782-2786.
Walther, B., & R. Sieber. 2011. Bioactive Proteins and Peptides in Foods. International Journal for Vitamin and Nutrition Research 81(2):181-192.
Weinberg, R. A. 1996. How Cancer Arises. Scientific American 275(3):62-70.
Wong, K., and P. Chikeung Cheung. 2001 Influence of Drying Treatment On Three Sargassum Species 2. Protein Extractability, In Vitro Protein Digestibility and Amino Acid Profile of Protein Concentrates. Journal of Applied Phycology 13 (1):51-58.
Yadav, A., Kumari, R., Yadav, A., Mishra, J., Srivatva, S., and S. Prabha. 2016. Antioxidants and its Functions in Human Body-A Review. Research in Environment and Life Sciences 9(11):1328-1331.
YuCETEPE, A., SArOglu, O., Bildik, F., Ozcelik, B., and C. Daskaya-Dikmen. 2018. Optimization of Ultrasound-Assisted Extraction of Protein from Spirulina platensis using RSM. Czech Journal of Food Sciences 36(1):98-108.
Zhuang, C., Itoh, H., Mizuno, T., and H. Ito. 1995. Antitumor Active Fucoidan from the Brown Seaweed, umitoranoo (Sargassum thunbergii). Bioscience, Biotechnology, and Biochemistry 59(4):563-567.