بهینه‌سازی شرایط تولید آنزیم فسفولیپاز در محیط‌کشت غوطه‌وری توسط سویه Trichoderma atroviride sp. ZB-ZH292

نویسندگان
1 گروه علوم و مهندسی صنایع غذایی، واحد تهران شمال، دانشگاه آزاد اسلامی، تهران، ایران
2 گروه علوم و صنایع غذایی، دانشکده کشاورزی،، دانشگاه تربیت مدرس، تهران، ایران
3 گروه تحقیقات صنایع غذایی، انستیتو تحقیقات تغذیه ای و صنایع غذایی کشور، دانشگاه علوم پزشکی شهید بهشتی،
چکیده


فسفولیپازها گروه مهمی از آنزیم­ها با کاربردهای گسترده در صنایع مختلف هستند. در این پژوهش بهینه­سازی دو مرحله­ای شامل استفاده از طرح غربالگری پلاکت- برمن و سپس بهینه­ سازی سطح پاسخ با هدف افزایش تولید فسفولیپاز سویه منتخب جهش ­یافته Trichoderma atroviride sp.ZB-ZH292 انجام شد. در مرحله اول جهت غربالگری و انتخاب مهمترین فاکتورها بر میزان توده زیستی و فعالیت فسفولیپاز، از طرح پلاکت - برمن با هفت متغیر دما، زمان، میزان فسفولیپید سویا (به عنوان منبع کربنی)، میزان پپتون (به عنوان منبع نیتروژنی)، نسبت مساوی از منو و دی پتاسیم ­هیدروژن فسفات (به عنوان منبع فسفر)، درصد تلقیح و سن تلقیح، در دو سطح استفاده گردید. بر اساس نتایج حاصل از غربالگری متغیرهای زمان گرمخانه­گذاری (0/01>P) ، دما گرمخانه­ گذاری (0/01>P) و فسفولیپید سویا (0/05>P) به عنوان منبع کربنی اثر معنی­ داری بر میزان فعالیت آنزیمی داشتند و در نتیجه جهت اجرای بهینه­ سازی نهایی به عنوان متغیرهای مستقل در طرح مرکب­مرکزی به روش آماری سطح پاسخ وارد شدند. بر این اساس طی 20 آزمایش طـراحی شده، اثر سه متغـیر دمای گرمخانه­ گذاری (°C 30-20)، زمان گرمخانه­ گـذاری (5-3 روز) و فسـفولیپید سویا (% 9-3) بر میزان فعالیت آنزیم مورد بررسی قرار گرفت. بر اساس نتایج آنالیز واریانس (ANOVA) میزان %4/32 فسفولیپید، دما °C 29/73و زمان 101/76 ساعت گرمخانه­گذاری به عنوان شرایط بهینه در تولید فسفولیپاز پیش­بینی شدند. در این شرایط، میزان فعالیت فسفولیپاز (U/ml) 3/57 تعیین گـردید که با نتیجه پیش­بینی­شده توسط مدل U/ml) 3/56) مطابقت داشت.
کلیدواژه‌ها

موضوعات


عنوان مقاله English

Optimization of phospholipase production condition in submerged medium for Trichoderma atroviride sp. ZB-ZH292

نویسندگان English

Zahra BeigMohammadi 1
Zohreh Hamidi Esfahani 2
Kiyanoosh Khosravi Darani 3
1 Department of Food Science and Technology, North Tehran Branch, Islamic Azad University, Tehran, Iran
2 Food Science and Technology Department, Agriculture Faculty, Tarbiat Modares University, Tehran, Iran
3 Department of Food Technology Research, National Nutrition and Food Technology Research Institute, Shahid Beheshti University of Medical Sciences, Tehran, Iran
چکیده English

Phospholipases are important groups of enzyme with wide applications in various industries. In this study, two-step optimization including Plackett-Burman screening design and response surface methodology (RSM) optimization were done with the goal of higher production level of phospholipase by selected mutant strains of Trichoderma atroviride sp.ZB-ZH292. First step was done by screening and evaluation of seven factors affecting the enzyme activity and biomass production by selected mutant, using Plackett-Burman design at two levels each namely temperature, time, amount of soybean phospholipids (as a carbon source), peptone level (as nitrogen source), equal ratio of mono and di-potassium hydrogen phosphate (as phosphorus source), seed size, and seed age. According to the result of screening design, incubation time (P<0.01), incubation temperature (P<0.01), and soybean phospholipids as a carbon source (P<0.05), had significant effect on the enzyme activity, so they were selected and used as independent variables in central composite design (CCD) under response surface methodology (RSM). In the analysis of 20 experimental runs, the effects of three independent variables including incubation temperature (20-30 °C), incubation time (3-5 day), and phospholipid concentration (3-9%) were evaluated on phospholipase activity. Analysis of variance (ANOVA) showed that the optimum values of soybean phospholipid, incubation temperature and incubation time were 4.32%, 29.73°C, and 101.76 h, respectively. At this optimum point, the phospholipase activity was found to be 3.57 (U/ml) which is in good agreement with the predicted value (3.56 (U/ml)) by the model.

کلیدواژه‌ها English

Phospholipase
Tricoderma atroviride
Plackett-Burman Design
Optimization
response surface methodology
[1] Guo, Z., Vikbjerg, A.F., Xu, X. (2005). Enzymatic modification of phospholipids for functional applications and human nutrition. Biotechnology Advances, 23(3), 203–259.
[2] Richmond, G.S., Smith, T.K. (2011). Phospholipases A1. International Journal of Molecular Science, 12(1), 588–612.
[3] Song, J.K., Han, J.J., Rhee, J.S. (2005). Phospholipases: occurrence and production in microorganisms, assay for high-throughput screening, and gene discovery from natural and man-made diversity. Journal of the American Oil Chemists’ Society, 82(10), 691-705.
[4] Song, J.K., Rhee, J.S. (2000). Simultaneous enhancement of thermo-stability and catalytic activity of phospholipase A1 by evolutionary molecular engineering. Applied and Environmental Microbiology., 66(3), 890–894.
[5] Burkert, J.F, Maugeri, F., Rodrigues, M.I. (2004). Optimization of extracellular lipase production by Geotrichum sp. using factorial design. Bioresource Technology, 91(1), 77-84.
[6] Acikel, U., Ersana, M., Acikel, y.s. (2010). Optimization of critical medium components using response surface methodology for lipase production by Rhizopus delemar. Food and Bioproducts Processing. 88(1), 31-39.
[7] Plackett, R.L., Burman, J.P. (1946). The design of optimum multifactorial experiments, Biometrika., 33(4), 305.
[8] Farbeh, F., Rezazad Bari, M., Alizadeh Khaledabad, M. (2010). Optimization of pectinlyase production from date pulp by Aspergillus niger using response surface methodology, Food Research 3(2), 13-22.
[9] Annapurna Kumari, A., Mahapatra, P., Banerjee, R. (2009). Statistical optimization of culture conditions by response surface methodology for synthesis of lipase with Enterobacter aerogenes. Brazilian Archives of Biology and Technology, 52(6), 1349-1356.
[10] Yasmeen, Q., Asgher, M., Sheikh, M., Nawaz, H. (2013). Optimization of ligninolytic enzymes production through response surface methodology. BioResources, 8(1), 944-968.
[11] Montgomery, D.C. (1991). Design and analysis of experiment. 3td ed., John Wiley, New York. pp: 427-510.
[12] Beig Mohammadi, Z., Hamidi Esfahani, Z., Sahari, M.A., Khosravi Darani, K. (2015). Isolation and identification of a strain producing the phospholipase enzyme from waste oil industry and its mutant. Journal of Food Science and new technologies, Inpress.
[13] Alp, S., Arikan, S. (2008). Investigation of extracellular elastase, acid proteinase and phospholipase activities as putative virulence factors in clinical isolates of Aspergillus species. Journal of Basic Microbiology, 48(5), 331-337.
[14] Jiang, F., Wang, J., Kaleem, I., Dai, D., Zhou, X., Li. C. (2011). Degumming of vegetable oils by a novel phospholipase B from Pseudomonas fluorescens BIT-18. Bioresource Technology 102(17), 8052-8056.
[15] Jiang, X., Chang, M., Wang, X., Jin, Q., Wang, X. (2014). The effect of ultrasound on enzymatic degumming process of rapeseed oil by the use of phospholipase A1. Ultrasonic Sonochemistry., 21(1), 142–148.
[16] Hosseni, S. M., Khosravi-Darani, K., Mohammadifar, M. A., Nikoopour, H. (2009). Production of mycoprotein by Fusarium venenatum growth on modified vogel medium. Asian Journal of Chemistry, 21(5), 4017-4022.
[17] Fatemi, S.S., Shojaosadati, S.A. Experimental design optimization of citric acid production by Aspergillus niger in solid state fermentation (2001). Amirkabir Journal of Science and Technology,11: 314–319.
[18] Khosravi Darani, K., Zoghi, A., Alavi, S.A., Fatemi, S.S. Application of Plackett Burman Design for Citric Acid Production from Pretreated and Untreated Wheat Straw (2008). International Journal of Chemistry and Chemical Engineering, 22:91-104
[19] Singh, A., Shahid, M., Srivastava, M., Pandey, S., Sharma, A. (2014). Optimal physical parameters for growth of Trichoderma Species at varying pH, temperature and agitation. Virology Mycology, 3:127. doi: 10.4172/2161-0517.1000127.
[20] Singh, A., Shahid, M., Pandey, N.K., Kumar, S., Srivastava, M., Biswas, S.K. (2011). Influence of temperature, pH and media for growth and sporulation of Trichoderma atroviride and its shelf life study in different carrier based formulation. Journal of Plant Disease Sciences, 6(1), 32-34.
[21] Honardoost, S., Fooladi, J., Azin, M.,Ghadam, P. (2012). Effect of different lipidic carbon and nitrogen sources on production of lipase by native strain Geobacillus stearothermophilus. Second International Congress of Microbiology. 554-568.
[22] Pera, L.M., Romero, C.M., Baigori, M.D., Castro, G.R. (2006). Catalytic Properties of Lipase Extracts from Aspergillus niger. Journal of Food Technology and Biotechnology, 44(2), 247–252.
[23] Falony, G., Armas, J.C., Dustet Mendoza, J.C., Martínez Hernández, J.L. (2006). Production of Extracellular Lipase from Aspergillus niger by solid-state fermentation. Journal of Food Technology and Biotechnology, 44(2), 235–240.