بهینه سازی شرایط استخراج آنزیمی پروتئین و شناسایی پروتئین های ریزجلبک سندسموس آبلیکوس
محورهای موضوعی : میکروبیولوژی مواد غذاییمطهره سادات امیری 1 , سید ابراهیم حسینی 2 , بابک خیام باشی 3 , غلامحسن اسدی 4
1 - دانشجو دکتری علوم و صنایع غذایی، واحد علوم و تحقیقات، دانشگاه آزاد اسلامی، تهران، ایران
2 - دانشیار گروه علوم و صنایع غذایی، واحد علوم و تحقیقات، دانشگاه آزاد اسلامی، تهران، ایران
3 - استادیار گروه تحقیقات خاک و آب ، مرکز تحقیقات و آموزش کشاورزی و منابع طبیعی اصفهان، اصفهان، ایران
4 - استادیار گروه علوم و صنایع غذایی، واحد علوم و تحقیقات، دانشگاه آزاد اسلامی، تهران، ایران
کلید واژه: استخراج, الکتروفورز, سلولاز, شناسایی پروتئین, مولتی آنزیم,
چکیده مقاله :
مقدمه: هدف از این مقاله بهینه سازی شرایط استخراج پروتئین ریزجلبک سندسموس به روش آنزیمی با استفاده از تکنیک سطح پاسخ و شناسایی پروتئین‌های استخراج شده از این ریزجلبک می‌باشد. مواد و روش‌ها: بهینه سازی شرایط استخراج آنزیمی پروتئین به کمک نرم‌افزار Minitab به روش سطح پاسخ طرح مرکب مرکزی، برای فاکتورهای نسبت آنزیم به سوبسترا و زمان اثر آنزیم (آنزیم‌های سلولاز و مولتی آنزیم تحت شرایط بهینه pH) طراحی شد. سپس بهترین تیمار از نظر بیشترین راندمان استخراج در کمترین زمان بهینه سازی شد و در پایان پروتئین‌های استخراج شده به روش الکتروفورز شناسایی شدند. یافته‌ها: نتایج نشان داد هرچه غلظت آنزیم افزایش یابد درصد استخراج و راندمان استخراج پروتئین بیشترمی‌شود به طوری که با افزایش غلظت از 2 به 6 میکرولیتر آنزیم بر میلی لیتر سوبسترا راندمان استخراج به کمک آنزیم مولتی آنزیم از 28/15 به 87/27 درصد و برای سلولاز از 88/16 به 42/21 درصد به ترتیب افزایش یافته است. بهینه شرایط محاسبه شده در بالاترین غلظت و کمترین زمان پاسخگوئی برابر 84/6 میکروگرم بر میلی لیتر به مدت 34/2 ساعت به ازای 5/0 گرم وزن خشک بدست آمد. نتایج بررسی‌های الکتروفورز به روش SDS- page نشان داد پروتئین‌های استخراج شده از این ریزجلبک حاوی 8 باند پروتئینی با وزن ملکولی بین 110-20 کیلودالتون (kDa) می‌باشد. همچنین شاخص شیمیایی پروتئین که معیاری است برای ارزیابی کیفیت پروتئین برای این ریز جلبک به میزان 4/0-2/0 محاسبه شد. نتیجه‌گیری: استخراج پروتئین به کمک تیمار آنزیمی در مقایسه با تیمار شیمیایی راندمان استخراج بالاتر داشته همچنین به دلیل مقرون به صرفه بودن از نظر اقتصادی، جهت استخراج پروتئین از سوبسترا پیشنهاد می‌گردد. باتوجه به نزدیک بودن شاخص شیمیایی پروتئین‌های این ترکیب با پروتئین غلات، این ترکیب به عنوان یک منبع پروتئینی پیشنهاد می‌گردد.
Introduction: The aim of this research work is to optimize the extraction conditions of Scenedesmus Obliquus algae protein by enzymatic and alkaline methods using response surface technique and to investigate the extracted protein’s functional properties. Materials and Methods: Optimization of enzymatic protein extraction using Minitab software with central composite response surface methodolog was designed for enzyme to substrate ratio factors and enzyme’s effective time (cellulose enzymes and multi-enzymes under optimum pH conditions) followed by optimization of the best treatment for the maximum extraction efficiency in the shortest time and finally the extracted proteins were identified by electrophoresis. Results: The results showed that, the higher the enzyme concentration, the higher the extraction rate and the protein extraction efficiency, therefore with an increase in the concentration from 4 to 6 μl/ml, the efficiency of the extraction is increased from 15.28 to 27.87 percent using multienzyme, and it increased from 16.88 to 21.42 percent using cellulase. The optimum conditions calculated at the highest concentration and lowest response time were 4 μg / ml for 2.34 hours. The results obtained from the chemical extraction of samples indicated that, the extraction efficiency was calculated as 19.13 percent. The results of electrophoresis analysis showed that the proteins extracted from this microalga contained 8 protein bands with molecular weight ranging from 20 to 110 kDa. Conclusion: Enzymatic extraction of proteins indicated a better yield as compared to chemical extraction and also considering the economical aspects this method of extraction is suggested.
Aiking, H. (2011). Future protein supply. Trends in Food Science and Technology 22, 112-120.
Al-Shamsi, K., Mudgil, P., Hassan, H. M. & Maqsood, S. (2018). Camel milk protein hydrolysates with improved technofunctional properties and enhanced antioxidant potential in in vitro and in food model systems. American Dairy Science, 101,1-14.
Al-Zuhair, S. & Ashraf, S. (2016). Enzymatic Pre-treatment of Microalgae Cells for Enhanced Extraction of Proteins. Engineering in Life Sciences, 1-22.
Doucha, J. & Livansky, K. (2014). Influence of processing parameters on disintegration of Chlorella cells in various types of homogenizers. Applied Microbiology and Biotechnology, 81, 431–440.
Estrada, J. E. P., Bescos, P.B. & Fresno, A. M. V. (2001). Antioxidant activity of different fractions of Spirulina platensis protean extract. Il Farmaco, 56, 497-500.
FAO/WHO. (2002). Protein quality. Report of Joint FAO/WHO expert consultation.
Fleurence, J. (1999). The enzymatic degradation of algal cell walls: a useful approach for improving protein accessibility? Applied Phycology, 11, 313–314.
Furuki, T., Maeda, S., Imajo, S., Hiroi, T., Amaya, T. & Hirokawa, T. (2003). Rapid and selective extraction of phycocyanin from Spirulina platensis with ultrasonic cell disruption. Applied Phycology, 15, 319–324.
Guil-Guerrero, G. L. & Navarro-Juarez, R. (2004). Functional properties of the biomass of three microalgal species. Food Engineering, 65, 511-517.
Guil-Guillermo, J. L., Navarro-Juarez, R., Lopez-Martinez, J. C., Campra-Madrid, P. & Rebolloso-Fuentes, M. M. (2004). Functional properties of the biomass of three microalgal species. Food Engineering, 65, 511-517.
Laemmli, U. K. (1970). "Cleavage of structural proteins during the assembly of the head of bacteriophage T4." Nature. 227, 680–685.
Lee, J. Y., Yoo, C., Jun, S. Y. & Ahn, H. M. (2010). Comparison of several methods for effective lipid extraction from microalgae. Bioresource Technology, 101, S75–S77.
Lourenço, O., Barbarino, E., Joel, C., De-Paula Pereira, L. & Ursula, M. (2002). Amino acid composition, protein content and calculation of nitrogen-to-protein conversion factors for 19 tropical seaweeds. Phycological Research, 233–241.
Maehre, H. K., Jensen, I. J. & Eilertsen, K. E. (2014). characteristic of protein lipid and mineral content in common Norwegian seaweed and evaluation of thaire potential as food and feed. Science Food Agriculture, 94, 3281-3290.
Maehre, H. K., Jensen, I. J. & Eilertsen, K. E. (2016). Enzymatic pre treatment increases protein bio accessibility and extractability in dulse (palmaria palmate). Drugs, 14, 196.
Mokni ghribi, A., Maklouf gafci, I. & Blecker, C. (2015). Effect of drying methods on physico-chemical and functional properties of chickpea protein concentrates. Food Engineering, 1-42.
Moure, A., Domínguez, H. & Parajó, J. C. (2006). Antioxidant properties of ultrafiltrationrecovered soy protein fractions from industrial effluents and their hydrolysates. Process Biochemistry, 41, 447–456.
Ngo, D. H., Vo, T. S., Ngo, D. N., Wijesekara, I. & Kim, S. K. (2012). Biological activities and potential health benefits bioactive peptides derived from marine organisms. International Journal of Biological Macromolecules, 51, 378–383.
Ogbonda, K. H., Aminigo, R. E. & Abu, G. O. (2007). Influence of temperature and pH on biomass production and protein biosynthesis in a putative Spirulina sp. Bioresour Technology, 98, 2207–2211.
Padraigin, A., Harnedy, R. & FitzGerald, J. (2013). Extraction of protein from the macroalga Palmaria palmata. Food Science and Technology, 51, 375-382.
Rajapakse, N., Mendis, E., Jung, W. K., Je, J. Y. & Kim, S. K. (2005). Purification of a radical scavenging peptide from fermented mussel sauce and its antioxidant properties.
Food International Research, 38, 175–182.
Rangel-Yagui, C. O., Danesi, E. D. G., Carvalho J. C. M. & Sato, S. (2004). Chlorophyll production from Spirulina platensis: cultivation with urea addition by fed-batch process. Bioresour Technology, 92, 133–141.
Rebolloso-Fuentes, M. M., Acien-Fernandez, F. G. & Sanchez-Perez, J. (2000). Biomass Nutrient Profiles of the microalga Porphyridium cruentum. Food Chemistry, 70, 345–353.
Rebolloso-Fuentes, M. M., Garca-Camacho, F., Navarro Garcıa, A. & Guil-Guerrero, J. L. (2001). Biomass nutrient profiles of the microalga Nannochloropsis spp. Agricultural and Food Chemistry, 49, 2966–2972.
Safi, C. & Violeta, A. (2014). Aqueous extraction of proteins from microalgae: Effect of different cell disruption methods. Algal Research, 3, 61–65.
Sari, Y. W., Bruins, M. E. & Sanders, J. P. M. (2013). Enzyme assisted protein extraction from rapeseed, soybean, and microalgae meals. Industrial Crops and Products, 43, 78–83.
Sarmadi, B. H. & Ismail, A. (2010). Antioxidative peptides from food proteins: A review. Peptides, 31, 1949-1956.
Schwenzfeier, A. (2013). Physico-chemical and techno-functional properties of proteins isolated from the green microalgae Tetraselmis sp. thesis, Netherland: Wageningen University.
Seneviratene, M. & Jea-Young, J. (2010). Enzymatic extract from edible red algae, porphyra tenera and their antioxidunt, anti acetylcholinesterase, and anti inflammatory activities. Food Science, 19, 1551-1557.
Waghmare, A. G., Salve, M. K., LeBlanc, J. G. & Arya, S. S. (2016). Concentration and characterization of microalgae proteins from Chlorella pyrenoidosa. Bioresour and Bioprocessing, 3, 1-11.
Yang, Z. K., Ma, Y., Zheng, J., Yang, W., Liu, J. & Li, H. (2013). Proteomics to reveal metabolic network shifts towards lipid accumulation following nitrogen deprivation in the diatom Phaeodactylum tricornutum. open access at Springer, 1-10.
Zheng, H., Yin, J., Gao, V., Huang, H. & Dou, C. (2011). Disruption of Chlorella vulgaris cells for the release of biodiesel-producing lipids: a comparison of grinding, ultrasonication, bead milling, enzymatic lysis, and microwaves. Applied Biochemistry and Biotechnology, 164, 1215–1224.
_||_Aiking, H. (2011). Future protein supply. Trends in Food Science and Technology 22, 112-120.
Al-Shamsi, K., Mudgil, P., Hassan, H. M. & Maqsood, S. (2018). Camel milk protein hydrolysates with improved technofunctional properties and enhanced antioxidant potential in in vitro and in food model systems. American Dairy Science, 101,1-14.
Al-Zuhair, S. & Ashraf, S. (2016). Enzymatic Pre-treatment of Microalgae Cells for Enhanced Extraction of Proteins. Engineering in Life Sciences, 1-22.
Doucha, J. & Livansky, K. (2014). Influence of processing parameters on disintegration of Chlorella cells in various types of homogenizers. Applied Microbiology and Biotechnology, 81, 431–440.
Estrada, J. E. P., Bescos, P.B. & Fresno, A. M. V. (2001). Antioxidant activity of different fractions of Spirulina platensis protean extract. Il Farmaco, 56, 497-500.
FAO/WHO. (2002). Protein quality. Report of Joint FAO/WHO expert consultation.
Fleurence, J. (1999). The enzymatic degradation of algal cell walls: a useful approach for improving protein accessibility? Applied Phycology, 11, 313–314.
Furuki, T., Maeda, S., Imajo, S., Hiroi, T., Amaya, T. & Hirokawa, T. (2003). Rapid and selective extraction of phycocyanin from Spirulina platensis with ultrasonic cell disruption. Applied Phycology, 15, 319–324.
Guil-Guerrero, G. L. & Navarro-Juarez, R. (2004). Functional properties of the biomass of three microalgal species. Food Engineering, 65, 511-517.
Guil-Guillermo, J. L., Navarro-Juarez, R., Lopez-Martinez, J. C., Campra-Madrid, P. & Rebolloso-Fuentes, M. M. (2004). Functional properties of the biomass of three microalgal species. Food Engineering, 65, 511-517.
Laemmli, U. K. (1970). "Cleavage of structural proteins during the assembly of the head of bacteriophage T4." Nature. 227, 680–685.
Lee, J. Y., Yoo, C., Jun, S. Y. & Ahn, H. M. (2010). Comparison of several methods for effective lipid extraction from microalgae. Bioresource Technology, 101, S75–S77.
Lourenço, O., Barbarino, E., Joel, C., De-Paula Pereira, L. & Ursula, M. (2002). Amino acid composition, protein content and calculation of nitrogen-to-protein conversion factors for 19 tropical seaweeds. Phycological Research, 233–241.
Maehre, H. K., Jensen, I. J. & Eilertsen, K. E. (2014). characteristic of protein lipid and mineral content in common Norwegian seaweed and evaluation of thaire potential as food and feed. Science Food Agriculture, 94, 3281-3290.
Maehre, H. K., Jensen, I. J. & Eilertsen, K. E. (2016). Enzymatic pre treatment increases protein bio accessibility and extractability in dulse (palmaria palmate). Drugs, 14, 196.
Mokni ghribi, A., Maklouf gafci, I. & Blecker, C. (2015). Effect of drying methods on physico-chemical and functional properties of chickpea protein concentrates. Food Engineering, 1-42.
Moure, A., Domínguez, H. & Parajó, J. C. (2006). Antioxidant properties of ultrafiltrationrecovered soy protein fractions from industrial effluents and their hydrolysates. Process Biochemistry, 41, 447–456.
Ngo, D. H., Vo, T. S., Ngo, D. N., Wijesekara, I. & Kim, S. K. (2012). Biological activities and potential health benefits bioactive peptides derived from marine organisms. International Journal of Biological Macromolecules, 51, 378–383.
Ogbonda, K. H., Aminigo, R. E. & Abu, G. O. (2007). Influence of temperature and pH on biomass production and protein biosynthesis in a putative Spirulina sp. Bioresour Technology, 98, 2207–2211.
Padraigin, A., Harnedy, R. & FitzGerald, J. (2013). Extraction of protein from the macroalga Palmaria palmata. Food Science and Technology, 51, 375-382.
Rajapakse, N., Mendis, E., Jung, W. K., Je, J. Y. & Kim, S. K. (2005). Purification of a radical scavenging peptide from fermented mussel sauce and its antioxidant properties.
Food International Research, 38, 175–182.
Rangel-Yagui, C. O., Danesi, E. D. G., Carvalho J. C. M. & Sato, S. (2004). Chlorophyll production from Spirulina platensis: cultivation with urea addition by fed-batch process. Bioresour Technology, 92, 133–141.
Rebolloso-Fuentes, M. M., Acien-Fernandez, F. G. & Sanchez-Perez, J. (2000). Biomass Nutrient Profiles of the microalga Porphyridium cruentum. Food Chemistry, 70, 345–353.
Rebolloso-Fuentes, M. M., Garca-Camacho, F., Navarro Garcıa, A. & Guil-Guerrero, J. L. (2001). Biomass nutrient profiles of the microalga Nannochloropsis spp. Agricultural and Food Chemistry, 49, 2966–2972.
Safi, C. & Violeta, A. (2014). Aqueous extraction of proteins from microalgae: Effect of different cell disruption methods. Algal Research, 3, 61–65.
Sari, Y. W., Bruins, M. E. & Sanders, J. P. M. (2013). Enzyme assisted protein extraction from rapeseed, soybean, and microalgae meals. Industrial Crops and Products, 43, 78–83.
Sarmadi, B. H. & Ismail, A. (2010). Antioxidative peptides from food proteins: A review. Peptides, 31, 1949-1956.
Schwenzfeier, A. (2013). Physico-chemical and techno-functional properties of proteins isolated from the green microalgae Tetraselmis sp. thesis, Netherland: Wageningen University.
Seneviratene, M. & Jea-Young, J. (2010). Enzymatic extract from edible red algae, porphyra tenera and their antioxidunt, anti acetylcholinesterase, and anti inflammatory activities. Food Science, 19, 1551-1557.
Waghmare, A. G., Salve, M. K., LeBlanc, J. G. & Arya, S. S. (2016). Concentration and characterization of microalgae proteins from Chlorella pyrenoidosa. Bioresour and Bioprocessing, 3, 1-11.
Yang, Z. K., Ma, Y., Zheng, J., Yang, W., Liu, J. & Li, H. (2013). Proteomics to reveal metabolic network shifts towards lipid accumulation following nitrogen deprivation in the diatom Phaeodactylum tricornutum. open access at Springer, 1-10.
Zheng, H., Yin, J., Gao, V., Huang, H. & Dou, C. (2011). Disruption of Chlorella vulgaris cells for the release of biodiesel-producing lipids: a comparison of grinding, ultrasonication, bead milling, enzymatic lysis, and microwaves. Applied Biochemistry and Biotechnology, 164, 1215–1224.
