اثر پیش تیمارهای حرارت و انجماد-یخ زدایی بر هیدرولیز آنزیمی پروتئین عدس به وسیله آنزیم آلکالاز و تولید پپتیدهای آنتی‌اکسیدان
محورهای موضوعی : میکروبیولوژی مواد غذاییپرستو قاسمی 1 , مهتا میرزایی 2 , سعید میردامادی 3
1 - دانش آموخته کارشناسی ارشد، گروه علوم و صنایع غذایی، واحد شهر قدس، دانشگاه آزاد اسلامی، تهران، ایران
2 - استادیار گروه علوم و صنایع غذایی، واحد شهر قدس، دانشگاه آزاد اسلامی، تهران، ایران
3 - استاد پژوهشکده زیست فناوری، سازمان پژوهش های علمی و صنعتی ایران، تهران، ایران
کلید واژه: پیش تیمار حرارت, پیش تیمار انجماد-یخ زدایی, پروتئین عدس, پپتیدهای آنتی‌اکسیدان, هیدرولیز آنزیمی,
چکیده مقاله :
مقدمه: فعالیت زیستی محصولات هیدرولیز پروتئینی تحت تاثیر نوع آنزیم، فعالیت آنزیم و شرایط هیدرولیز آنزیمی نظیر دما، زمان و نسبت آنزیم/ سوبسترا و فرآیند پیش تیمار پروتئین می باشد. فرآیند پیش تیمار می تواند از طریق تاثیر بر ساختار فضایی پروتئین و افزایش دسترسی آنزیم به باندهای پپتیدی، بر پیشرفت هیدرولیز آنزیمی و تولید پپتیدهای آنتی اکسیدان موثر باشد. مواد و روش ها: پروتئین استخراج شده از عدس ابتدا تحت پیش تیمارهای حرارت (65 ،75، 85 درجه سانتی گراد به مدت 15 دقیقه) و انجماد-یخ زدایی (3 سیکل انجماد در دمای 20- درجه سانتی گراد و یخ زدایی در دمای محیط) و هیدرولیز آنزیمی به وسیله آنزیم آلکالاز (با نسبت آنزیم به سوبسترا 90 آنسون / کیلوگرم پروتئین، دمای 55 درجه سانتی گراد، 3 ساعت) قرار گرفت. در طی زمان، پیشرفت هیدرولیز آنزیمی با روش ارتوفتال آلدئید (OPA) و فعالیت آنتی اکسیدانی با روش های مهارکنندگی رادیکال های DPPH و ABTS مورد بررسی قرار گرفتند و با نمونه کنترل (بدون پیش تیمار) مقایسه شدند. یافته ها: پیش تیمار نمونه در دمای 75 درجه سانتی گراد به مدت 180 دقیقه، بیشترین اثر را در افزایش شدت هیدرولیز آنزیمی نسبت به نمونه های کنترل بدون پیش تیمار داشت. حداکثر میزان فعالیت مهارکنندگی رادیکال DPPH (57/63 درصد) مربوط به نمونه پیش تیمار شده در دمای 65 درجه سانتی گراد و حداکثر میزان فعالیت مهار کنندگی رادیکال ABTS (24/36 درصد) مربوط به نمونه پیش تیمار شده بوسیله فرآیند انجماد-یخ زدایی در زمان 180 دقیقه بود. نتیجه گیری: نتایج نشان دادند که پیش تیمار حرارتی و انجماد-یخ زدایی قبل از هیدرولیز آنزیمی اثر مثبتی بر پیشرفت هیدرولیز آنزیمی و تولید پپتیدهای آنتی اکسیدان دارد. بر اساس نتایج حاصل از این تحقیق، فرآیند پیش تیمار حرارتی پروتئین عدس در دمای 65 درجه سانتی گراد یا انجماد-یخ زدایی و هیدرولیز آنزیمی بوسیله آنزیم آلکالاز روشی موثر در تولید محصول هیدرولیز پروتئین عدس برای کاربرد در فرمولاسیون محصولات غذایی فراسودمند شناخته شد.
Introduction: The biological activity of protein hydrolysis products is affected by the enzyme type, protein type and enzymatic hydrolysis conditions including temperature, time and enzyme/substrate ratio and pre-treatment process.The protein pretreatment process can cause improving the enzymatic hydrolysis and production of antioxidant peptides by affecting the spatial structure of the protein and increasing the enzyme access to the peptide bounds. Materials and Methods: In this study, The protein extracted from lentils was first subjected to heat pretreatment (65,75, 85°C, for 15 min) and freeze-thaw (3 freezing cycles at -20 °C and thawing at room temperature). It was then exposed to hydrolysis for 3 hr by alcalase (with an E/S of 90 AU / kg protein, 55°C). Over time, the progress of enzymatic hydrolysis and antioxidant activity were investigated by O-phthaldialdehyde (OPA) assay and DPPH and ABTS radical scavenging methods and compared with the control sample (without pretreatment). Results: Pre-treatment at 75°C causes the highest value of free amino groups .The maximum DPPH (63.57%) and ABTS (36.24%) radical scavenging activity were observed respectively, for samples pre-treated at 65°C and by freeze-thaw process. Conclusion: Heat pretreatment and freezing-thawing before enzymatic hydrolysis have a positive effect on the development of enzymatic hydrolysis and production of antioxidant peptides. Based on the results of this study, the process of heat treatment of lentil protein at 65°C or freezing-thawing and enzymatic hydrolysis by enzyme alcalase was identified as an effective method in the production of lentil protein hydrolysis for use in the formulation of functional foods.
Arcan, I. & Yemenicioğlu, A. (2010). Effects of controlled pepsin hydrolysis on antioxidant potential and fractional changes of chickpea proteins. Food Research International, 43(1), 140-147.
Adjonu, R., Doran, G., Torley, P. & Agboola, S. (2013). Screening of whey protein isolate hydrolysates for their dual functionality: influence of heat pre-treatment and enzyme specificity. Food chemistry, 136(3-4), 1435-1443.
Arrutia, F., Puente, Á., Riera, F. A., Menéndez, C. & González, U. A. (2016). Influence of heat pre-treatment on BSA tryptic hydrolysis and peptide release. Food Chemistry, 202(1), 40-48.
Alizadeh, O. & Aliakbarlu, J. (2020). Effects of ultrasound and ohmic heating pretreatments on hydrolysis, antioxidant and antibacterial activities of whey protein concentrate and its fractions. LWT, 131, 109913.
Bondet, V., Brand-Williams, W. & Berset, C. (1997). Kinetics and mechanisms of
antioxidant activity using the DPPH. free radical method. LWT-Food Science and Technology, 30(6), 609-615.
Church, F. C., Swaisgood, H. E., Porter, D. H. & Catignani, G. L. (1983). Spectrophotometric assay using o-phthaldialdehyde for determination of proteolysis in milk and isolated milk proteins. Journal of Dairy Science, 66(6), 1219-1227.
Chen, L., Chen, J., Ren, J. & Zhao, M. (2011). Modifications of soy protein isolates using combined extrusion pre-treatment and controlled enzymatic hydrolysis for improved emulsifying properties. Food Hydrocolloids, 25(5), 887-897.
Chandrapala, J., Zisu, B., Palmer, M., Kentish, S. & Ashokkumar, M. (2011). Effects of ultrasound on the thermal and structural characteristics of proteins in reconstituted whey protein concentrate. Ultrasonics Sonochemistry, 18(5), 951-957.
Cotabarren, J., Rosso, A. M., Tellechea, M., García-Pardo, J., Rivera, J. L., Obregón, W. D. & Parisi, M. G. (2019). Adding value to the chia (Salvia hispanica L.) expeller: Production of bioactive peptides with antioxidant properties by enzymatic hydrolysis with Papain. Food Chemistry, 274, 848-856.
Dryáková, A., Pihlanto, A., Marnila, P., Čurda, L. & Korhonen, H.J. (2010). Antioxidant properties of whey protein hydrolysates as measured by three methods. European Food Research and Technology, 23(6), 865-874.
DE Castro, R. J. S., Cason, V. G. & Sato, H. H. (2017). Binary mixture of proteases increases the antioxidant properties of white bean (Phaseolus vulgaris L.) protein-derived peptides obtained by enzymatic hydrolysis. Biocatalysis and Agricultural Biotechnology, 10, 291-297.
Elmalimadi, M.B., Jovanović, J.R., Stefanović, A.B., Tanasković, S.J., Djurović, S.B., Bugarski, B.M. & Knežević-Jugović, Z.D. (2017). Controlled enzymatic hydrolysis for improved exploitation of the antioxidant potential of wheat gluten. Industrial Crops and Products, 109, 548-557.
Farrokhi, F., Badii, F., Ehsani, M. R. &Hashemi, M. (2019). Functional and thermal properties of nanofibrillated whey protein isolate as functions of denaturation temperature and solution pH. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 583, 124002.
Hou, R. Z., Yang, Y., Li, G., Huang, Y. B., Wang, H., Liu, Y. J., Xu, L. & Zhang, X. Z. (2006). Synthesis of a precursor dipeptide of RGDS (Arg‐Gly‐Asp‐Ser) catalysed by the industrial protease alcalase. Biotechnology and Applied Biochemistry, 44(2), 73-80.
Joshi, M., Timilsena, Y. & Adhikari, B. (2017). Global production, processing and utilization of lentil: A review. Journal of Integrative Agriculture, 16(12), 2898-2913.
Kittiphattanabawon, P., Benjakul, S., Visessanguan, W. & Shahidi, F. (2012). Gelatin hydrolysate from blacktip shark skin prepared using papaya latex enzyme: Antioxidant activity and its potential in model systems. Food Chemistry, 135(3), 1118-1126.
Lowry, O., Rosebrough, N., Farr, A. & Randall, R. (1951). Total protein estimation by Lowry's method. The Journal of Biological Chemistry, 193(1), 265-275.
Lassoued, I., Mora, L., Nasri, R., Aydi, M., Toldrá, F., Aristoy, M.-C., Barkia, A. & Nasri, M. (2015). Characterization, antioxidative and ACE inhibitory properties of hydrolysates obtained from thornback ray (Raja clavata) muscle. Journal of Proteomics, 128, 458-468.
Morr, C., German, B., Kinsella, J., Regenstein, J., Buren, J.V., Kilara, A., Lewis, B. & Mangino, M. (1985). A collaborative study to develop a standardized food protein solubility procedure. Journal of Food Science, 50(6), 1715-1718.
Muzaifa, M., Safriani, N. & Zakaria, F. (2012). Production of protein hydrolysates from fish by-product prepared by enzymatic hydrolysis. Aquaculture, Aquarium, Conservation & Legislation, 5(1), 36-39.
Moslehishad, M., Mirdamadi, S., Ehsani, M. R., Ezzatpanah, H. & Moosavi‐Movahedi, A. A. (2013). The proteolytic activity of selected lactic acid bacteria in fermenting cow's and camel's milk and the resultant sensory characteristics of the products. International Journal of Dairy Technology, 66(2), 279-285.
Mirdamadi, S., Soliman Zadeh, N., Mirzaei, M. & Motahhari, P. (2017). Bioactive peptides: Production process, health effects and application as natural additives in the production of processed foods. Journal of Food Hygiene, 7(25), 1-20 [In Persian].
Maqsoudlou, A., Mahoonak, A.S., Mora, L., Mohebodini, H., Toldrá, F. & Ghorbani, M. (2019). Peptide identification in alcalase hydrolysated pollen and comparison of its bioactivity with royal jelly. Food Research International, 116, 905-915.
Mercado-Mercado, G., Laura, A. & Alvarez-Parrilla, E. (2020). Effect of pectin on the interactions among phenolic compounds determined by antioxidant capacity. Journal of Molecular Structure, 1199, 126967.
Nourmohammadi, A., Sadeghi Mahonak, A., Shahrampour, D. & Khamiri, M. (2015). Optimization of Hydrolysis of Pumpkin Seed Meal Protein Protein to Achieve Maximum Antioxidant Properties. Electronic Journal of Food Processing and Preservation, 9(1), 1-12 [In Persian].
Pezeshk, S., Ojagh, M., Rezaei, M. & Shabanpour, B. (2017). Optimization of Hydrolyzed Protein with Antioxidant Activity from the Tuna and viscera of Apricot (Thunnus albacares) with Protamox Enzyme. Iranian Journal of Nutrition Sciences & Food Technology, 12(3), 99-108 [In Persian].
Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M. & Rice-Evans, C. (1999). Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biology and Medicine, 26(9), 1231-1237.
Rooni, V., Raud, M. & Kikas, T. (2017). The freezing pre-treatment of lignocellulosic material: A cheap alternative for Nordic countries. Energy, 139, 1-7.
Shekib, L. A., Zoueil, M., Youssef, M. & Mohamed, M. S. (1986). Amino acid composition and in vitro digestibility of lentil and rice proteins and their mixture (Koshary). Food Chemistry, 20(1), 61-67.
Shahidi, F. & Zong, Y. (2008). Bioactive peptides. Journal of AOAC International, 91(4), 914-931.
Sarmadi, B.H. & Ismail, A. (2010). Antioxidative peptides from food proteins: a review. Peptides 31(10), 1949-1956.
Singh, A. & Ramaswamy, H.S. (2014). Effect of high‐pressure treatment on trypsin hydrolysis and antioxidant activity of egg white proteins. International Journal of Food Science & Technology, 49(1), 269-279.
Wang, X., Yu, H., Xing, R., Chen, X., Liu, S. & Li, P. (2017). Optimization of the extraction and stability of antioxidative peptides from mackerel (Pneumatophorus japonicus) protein. BioMed Research International.
Zhang, Y., Olsen, K., Grossi, A. & Otte, J. (2013). Effect of pretreatment on enzymatic hydrolysis of bovine collagen and formation of ACE-inhibitory peptides. Food Chemistry, 141(3), 2343-2354.
_||_Arcan, I. & Yemenicioğlu, A. (2010). Effects of controlled pepsin hydrolysis on antioxidant potential and fractional changes of chickpea proteins. Food Research International, 43(1), 140-147.
Adjonu, R., Doran, G., Torley, P. & Agboola, S. (2013). Screening of whey protein isolate hydrolysates for their dual functionality: influence of heat pre-treatment and enzyme specificity. Food chemistry, 136(3-4), 1435-1443.
Arrutia, F., Puente, Á., Riera, F. A., Menéndez, C. & González, U. A. (2016). Influence of heat pre-treatment on BSA tryptic hydrolysis and peptide release. Food Chemistry, 202(1), 40-48.
Alizadeh, O. & Aliakbarlu, J. (2020). Effects of ultrasound and ohmic heating pretreatments on hydrolysis, antioxidant and antibacterial activities of whey protein concentrate and its fractions. LWT, 131, 109913.
Bondet, V., Brand-Williams, W. & Berset, C. (1997). Kinetics and mechanisms of
antioxidant activity using the DPPH. free radical method. LWT-Food Science and Technology, 30(6), 609-615.
Church, F. C., Swaisgood, H. E., Porter, D. H. & Catignani, G. L. (1983). Spectrophotometric assay using o-phthaldialdehyde for determination of proteolysis in milk and isolated milk proteins. Journal of Dairy Science, 66(6), 1219-1227.
Chen, L., Chen, J., Ren, J. & Zhao, M. (2011). Modifications of soy protein isolates using combined extrusion pre-treatment and controlled enzymatic hydrolysis for improved emulsifying properties. Food Hydrocolloids, 25(5), 887-897.
Chandrapala, J., Zisu, B., Palmer, M., Kentish, S. & Ashokkumar, M. (2011). Effects of ultrasound on the thermal and structural characteristics of proteins in reconstituted whey protein concentrate. Ultrasonics Sonochemistry, 18(5), 951-957.
Cotabarren, J., Rosso, A. M., Tellechea, M., García-Pardo, J., Rivera, J. L., Obregón, W. D. & Parisi, M. G. (2019). Adding value to the chia (Salvia hispanica L.) expeller: Production of bioactive peptides with antioxidant properties by enzymatic hydrolysis with Papain. Food Chemistry, 274, 848-856.
Dryáková, A., Pihlanto, A., Marnila, P., Čurda, L. & Korhonen, H.J. (2010). Antioxidant properties of whey protein hydrolysates as measured by three methods. European Food Research and Technology, 23(6), 865-874.
DE Castro, R. J. S., Cason, V. G. & Sato, H. H. (2017). Binary mixture of proteases increases the antioxidant properties of white bean (Phaseolus vulgaris L.) protein-derived peptides obtained by enzymatic hydrolysis. Biocatalysis and Agricultural Biotechnology, 10, 291-297.
Elmalimadi, M.B., Jovanović, J.R., Stefanović, A.B., Tanasković, S.J., Djurović, S.B., Bugarski, B.M. & Knežević-Jugović, Z.D. (2017). Controlled enzymatic hydrolysis for improved exploitation of the antioxidant potential of wheat gluten. Industrial Crops and Products, 109, 548-557.
Farrokhi, F., Badii, F., Ehsani, M. R. &Hashemi, M. (2019). Functional and thermal properties of nanofibrillated whey protein isolate as functions of denaturation temperature and solution pH. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 583, 124002.
Hou, R. Z., Yang, Y., Li, G., Huang, Y. B., Wang, H., Liu, Y. J., Xu, L. & Zhang, X. Z. (2006). Synthesis of a precursor dipeptide of RGDS (Arg‐Gly‐Asp‐Ser) catalysed by the industrial protease alcalase. Biotechnology and Applied Biochemistry, 44(2), 73-80.
Joshi, M., Timilsena, Y. & Adhikari, B. (2017). Global production, processing and utilization of lentil: A review. Journal of Integrative Agriculture, 16(12), 2898-2913.
Kittiphattanabawon, P., Benjakul, S., Visessanguan, W. & Shahidi, F. (2012). Gelatin hydrolysate from blacktip shark skin prepared using papaya latex enzyme: Antioxidant activity and its potential in model systems. Food Chemistry, 135(3), 1118-1126.
Lowry, O., Rosebrough, N., Farr, A. & Randall, R. (1951). Total protein estimation by Lowry's method. The Journal of Biological Chemistry, 193(1), 265-275.
Lassoued, I., Mora, L., Nasri, R., Aydi, M., Toldrá, F., Aristoy, M.-C., Barkia, A. & Nasri, M. (2015). Characterization, antioxidative and ACE inhibitory properties of hydrolysates obtained from thornback ray (Raja clavata) muscle. Journal of Proteomics, 128, 458-468.
Morr, C., German, B., Kinsella, J., Regenstein, J., Buren, J.V., Kilara, A., Lewis, B. & Mangino, M. (1985). A collaborative study to develop a standardized food protein solubility procedure. Journal of Food Science, 50(6), 1715-1718.
Muzaifa, M., Safriani, N. & Zakaria, F. (2012). Production of protein hydrolysates from fish by-product prepared by enzymatic hydrolysis. Aquaculture, Aquarium, Conservation & Legislation, 5(1), 36-39.
Moslehishad, M., Mirdamadi, S., Ehsani, M. R., Ezzatpanah, H. & Moosavi‐Movahedi, A. A. (2013). The proteolytic activity of selected lactic acid bacteria in fermenting cow's and camel's milk and the resultant sensory characteristics of the products. International Journal of Dairy Technology, 66(2), 279-285.
Mirdamadi, S., Soliman Zadeh, N., Mirzaei, M. & Motahhari, P. (2017). Bioactive peptides: Production process, health effects and application as natural additives in the production of processed foods. Journal of Food Hygiene, 7(25), 1-20 [In Persian].
Maqsoudlou, A., Mahoonak, A.S., Mora, L., Mohebodini, H., Toldrá, F. & Ghorbani, M. (2019). Peptide identification in alcalase hydrolysated pollen and comparison of its bioactivity with royal jelly. Food Research International, 116, 905-915.
Mercado-Mercado, G., Laura, A. & Alvarez-Parrilla, E. (2020). Effect of pectin on the interactions among phenolic compounds determined by antioxidant capacity. Journal of Molecular Structure, 1199, 126967.
Nourmohammadi, A., Sadeghi Mahonak, A., Shahrampour, D. & Khamiri, M. (2015). Optimization of Hydrolysis of Pumpkin Seed Meal Protein Protein to Achieve Maximum Antioxidant Properties. Electronic Journal of Food Processing and Preservation, 9(1), 1-12 [In Persian].
Pezeshk, S., Ojagh, M., Rezaei, M. & Shabanpour, B. (2017). Optimization of Hydrolyzed Protein with Antioxidant Activity from the Tuna and viscera of Apricot (Thunnus albacares) with Protamox Enzyme. Iranian Journal of Nutrition Sciences & Food Technology, 12(3), 99-108 [In Persian].
Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M. & Rice-Evans, C. (1999). Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biology and Medicine, 26(9), 1231-1237.
Rooni, V., Raud, M. & Kikas, T. (2017). The freezing pre-treatment of lignocellulosic material: A cheap alternative for Nordic countries. Energy, 139, 1-7.
Shekib, L. A., Zoueil, M., Youssef, M. & Mohamed, M. S. (1986). Amino acid composition and in vitro digestibility of lentil and rice proteins and their mixture (Koshary). Food Chemistry, 20(1), 61-67.
Shahidi, F. & Zong, Y. (2008). Bioactive peptides. Journal of AOAC International, 91(4), 914-931.
Sarmadi, B.H. & Ismail, A. (2010). Antioxidative peptides from food proteins: a review. Peptides 31(10), 1949-1956.
Singh, A. & Ramaswamy, H.S. (2014). Effect of high‐pressure treatment on trypsin hydrolysis and antioxidant activity of egg white proteins. International Journal of Food Science & Technology, 49(1), 269-279.
Wang, X., Yu, H., Xing, R., Chen, X., Liu, S. & Li, P. (2017). Optimization of the extraction and stability of antioxidative peptides from mackerel (Pneumatophorus japonicus) protein. BioMed Research International.
Zhang, Y., Olsen, K., Grossi, A. & Otte, J. (2013). Effect of pretreatment on enzymatic hydrolysis of bovine collagen and formation of ACE-inhibitory peptides. Food Chemistry, 141(3), 2343-2354.