بررسی محتوای پروتئینی و ارتباط فعالیت آنزیمی با اثر ضد میکروبی نمونه‌های‌ عسل ایرانی

نویسندگان
محدثه نقادیان مقدم
الهه محمودی
گروه زیست شناسی سلولی و مولکولی، دانشکده شیمی، دانشگاه کاشان، کاشان، ایران.
10.52547/fsct.18.114.319
چکیده
عسل محصولی طبیعی است که از دیرباز علاوه بر تغذیه، برای درمان نیز کاربرد داشته است. در این ارتباط، اثر ضد میکروبی چند عسل ایرانی علیه سویه­های باکتریایی مقاوم به آنتی­بیوتیک و ارتباط این اثر با فعالیت آنزیمی نمونه­های عسل مورد ارزیابی قرار گرفت. در این مطالعه، محتوای پروتئینی 20 نمونه عسل قبل و بعد از استخراج تعیین و با تکنیک SDS-PAGE بررسی گردید. سپس میزان فعالیت آنزیم­های گلوکزاکسیداز، آمیلاز، اینورتاز و کاتالاز هر عسل اندازه­گیری شد. مهار سویه­های باکتریایی مورد آزمون در حضور عسل با روش انتشار از چاهک­ بررسی شد و ارتباط فعالیت آنزیمی با اثر ضد باکتریایی و تازگی عسل ارزیابی گردید. محتوای پروتئینی نمونه­ها قبل از استخراج در محدوده 06/0±4/0 تا 19/64±0/1 میلی­گرم بر گرم عسل قرار داشت و پس از استخراج این میزان حدود 50% یا کمتر کاهش یافت. در نمای پروتئینی همه عسل­ها پس از الکتروفورز، 3 باند مربوط به آنزیم­های آمیلاز، اینورتاز و گلوکزاکسیداز قابل مشاهده بود. میزان فعالیت آنزیم­ها به ترتیب به گلوکزاکسیداز> اینورتاز> آمیلاز> کاتالاز اختصاص داشت. نمونه­های عسل منتخب فاقد اثر بر سویه­ی Staphylococcus epidermidis و دارای کمترین اثر بر سویه­ی Pseudomonas aeruginosa بودند. الگوی مهاری سویه­های Escherichia coli و Enterococcus faecalis مشابه بود، اما الگوی مهاری متفاوتی از سویه­ی Staphylococcus aureus پس از تلقیح عسل­ها مشاهده شد. این بررسی نشان داد که آنزیم گلوکزاکسیداز نقش مهمی در مهار باکتری­ها دارد، اما این نقش در سویه­های کاتالاز مثبت دچار نوسان است. همچنین آنزیم­های آمیلاز و اینورتاز می­توانند معیاری از تازگی عسل­ها باشند.
کلیدواژه‌ها
عسل‌های ایرانی
فعالیت ضد باکتریایی
گلوکزاکسیداز
اینورتاز
آمیلاز

موضوعات

عنوان مقاله English

Investigation of protein content and the relationship between enzymatic activity and antimicrobial effect of Iranian honey samples

نویسندگان English

Mohadese Naghadian Moghadam
elahe mahmoodi
Department of Cell and Molecular Biology, Faculty of Chemistry, University of Kashan, Kashan, Iran
چکیده English

Honey is a natural product that has long been used for treatment in addition to nutrition. In this regard, the antimicrobial effect of some Iranian kinds of honey against antibiotic-resistant bacterial strains and the relationship between this effect and the enzymatic activity of honey samples were evaluated. In this study, the protein content of 20 honey samples before and after extraction was determined and analyzed by SDS-PAGE technique. Then the activity of glucose oxidase, amylase, invertase, and catalase enzymes of each honey was measured. Inhibition of the tested bacterial strains in the presence of honey was investigated by ager well diffusion method and the relationship between enzymatic activity with antibacterial effect and freshness of honey was evaluated. The protein content of the samples before extraction ranged from 0.4 06 0.06 to 1.0 64 64.19 mg/g honey and after extraction this amount decreased by about 50% or less. In the protein profile of all kinds of honey after electrophoresis, 3 bands related to amylase, invertase, and glucosidase enzymes were visible. The activity of enzymes was assigned to glucose oxidase> invertase> amylase> catalase, respectively. The selected honey samples did not affect Staphylococcus epidermidis and had the least effect on Pseudomonas aeruginosa. After honey inoculation, the inhibitory pattern of Escherichia coli and Enterococcus faecalis were similar, but a different inhibitory pattern was observed from Staphylococcus aureus strain. This study showed that glucose oxidase plays an important role in bacterial inhibition, but this role fluctuates in catalase-positive strains. Also, Amylase and invertase enzymes can be a measure of the freshness of honey.

کلیدواژه‌ها English

Iranian honeys
Antibacterial activity
Glucose oxidase
Invertase
Amylase
[1] Sherlock, O., Dolan, A., Athman, R., Power, A., Gethin, G., Cowman, S., & Humphreys, H. (2010). Comparison of the antimicrobial activity of Ulmo honey from Chile and Manuka honey against methicillin-resistant Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. BMC complementary and alternative medicine,10(1), 1-5.
[2] Bogdanov, S., Jurendic, T., Sieber, R., & Gallmann, P. (2008). Honey for nutrition and health: a review. Journal of the American College of Nutrition, 27(6), 677-89.
[3] Adams, C. J., Boult, C. H., Deadman, B. J., Farr, J. M., Grainger, M. N., Manley-Harris, M., & Snow, M. J. (2008). Isolation by HPLC and characterisation of the bioactive fraction of New Zealand manuka (Leptospermum scoparium) honey. Carbohydrate Research, 343(4), 651-9.
[4] Cantarelli, M. A., Pellerano, R. G., Marchevsky, E.J., & Camiña, J.M. (2008). Quality of honey from Argentina: study of chemical composition and trace elements. Journal of the Argentine Chemical Society, 96(1-2), 33-41.
[5] Sahin, H., Kolayli, S., & Beykaya, M. (2020). Investigation of Variations of Invertase and Glucose Oxidase Degrees against Heating and Timing Options in Raw Honeys. Journal of Chemistry, 2020, 1-7.
[6] Leyva-Jimenez, F. J., Lozano-Sanchez, J., Borras-Linares, I., de la Luz Cadiz-Gurrea, M., & Mahmoodi-Khaledi, E. (2019). Potential antimicrobial activity of honey phenolic compounds against Gram positive and Gram negative bacteria. LWT, 101, 236-45.
[7] Huidobro, J. F., Sánchez, M. P., Muniategui, S., & Sancho, M.T. (2005). Precise method for the measurement of catalase activity in honey. Journal of AOAC International, 88(3), 800-4.
[8] Ohashi, K., Natori, S., & Kubo, T. (1999). Expression of amylase and glucose oxidase in the hypopharyngeal gland with an age‐dependent role change of the worker honeybee (Apis mellifera L.). European Journal of Biochemistry, 265(1), 127-33.
[9] Mahmoodi-Khaledi, E., Kashef, N., Habibi-Rezaei, M., & Moosavi-Movahedi, A.A. (2015). In vitro characterization of antibacterial potential of Iranian honey samples against wound bacteria. European Food Research and Technology, 241(3), 329-39.
[10] Levy, S.B., & Marshall, B. (2004). Antibacterial resistance worldwide: causes, challenges and responses. Nature medicine,10(12), S122-S9.
[11] Wasihun, A.G., & Kasa, B. G. (2016). Evaluation of antibacterial activity of honey against multidrug resistant bacteria in Ayder Referral and Teaching Hospital, Northern Ethiopia. SpringerPlus, 5(1), 842.
[12] Zwayen, G. F., & Mahmoodi-Khaledi, E. (2020). Antimicrobial effect of different types of honey derived from Iraqi flora on clinical strains of Proteus mirabilis, Proteus vulgaris and Klebsiella pneumonia. Annals of Tropical Medicine and Public Health, 23, 23-1122.
[13] Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical biochemistry, 72(1-2), 248-54.
[14] Won, S-R., Lee, D-C., Ko, S. H., Kim, J-W., & Rhee, H-I. (2008). Honey major protein characterization and its application to adulteration detection. Food Research International, 41(10), 952-6.
[15] Ahmed, H. (2004). Principles and reactions of protein extraction, purification, and characterization, CRC press.
[16] Hou, X., Liu, B., Deng, X., Zhang, B., Chen, H., & Luo, R. (2007). Covalent immobilization of glucose oxidase onto poly (styrene-co-glycidyl methacrylate) monodisperse fluorescent microspheres synthesized by dispersion polymerization. Analytical biochemistry, 368(1), 100-10.
[17] Ghosh, S., Ahire, M., Patil, S., Jabgunde, A., Bhat Dusane, M., Joshi, B. N., Pardesi, K., Jachak, S., Dhavale, D. D., Chopade, B. A. (2012). Antidiabetic activity of Gnidia glauca and Dioscorea bulbifera: potent amylase and glucosidase inhibitors. Evidence-Based Complementary and Alternative Medicine, 2012, 1-10.
[18] Hong, J-M., Lee, K-A., Kim, J., & Park, I. (1990). Production and properties of invertase from Aspergillus niger. Journal of the Korean Society of Food Science and Nutrition, 19, 577-87.
[19] Aebi, H. (1974). Catalase, Methods of enzymatic analysis, Elsevier, 673-84.
[20] Basualdo, C., Sgroy, V., Finola, M. S., & Marioli, J. M. (2007). Comparison of the antibacterial activity of honey from different provenance against bacteria usually isolated from skin wounds. Veterinary microbiology, 124(3-4), 375-81.
[21] French, V. M., Cooper, R. A., & Molan, P. C. (2005). The antibacterial activity of honey against coagulase-negative staphylococci. Journal of Antimicrobial Chemotherapy, 56(1), 228-31.
[22] Bocian, A., Buczkowicz, J., Jaromin, M., Hus, K. K., & Legáth, J. (2019). An Effective Method of Isolating Honey Proteins. Molecules, 24(13), 2399.
[23] da C Azeredo, L., Azeredo, M., De Souza, S., & Dutra, V. (2003). Protein contents and physicochemical properties in honey samples of Apis mellifera of different floral origins. Food chemistry, 80(2), 249-54.
[24] Belay, A., Haki, G. D., Birringer, M., Borck, H., Lee, Y-C., Kim, K-T., Baye, K., Melaku, S. (2017). Enzyme activity, amino acid profiles and hydroxymethylfurfural content in Ethiopian monofloral honey. Journal of food science and technology, 54(9), 2769-78.
[25] Bucekova, M., Buriova, M., Pekarik, L., Majtan, V., & Majtan, J. (2018). Phytochemicals-mediated production of hydrogen peroxide is crucial for high antibacterial activity of honeydew honey. Scientific reports, 8(1), 1-9.
[26] Serrano, S., Espejo, R., Villarejo, M., & Jodral, M. L. (2007). Diastase and invertase activities in Andalusian honeys. International journal of food science & technology, 42(1), 76-9.
[27] Brudzynski, K. (2020). A current perspective on hydrogen peroxide production in honey. A review. Food Chemistry, 127229.
[28] Dastouri, M. R., Fakhimzadeh, K., Shayeg, J., Dolgari-Sharaf, J., Valilou, M. R., & Maheri-Sis, N. (2008). Evaluating antibacterial activity of the Iranian honey through MIC method on some dermal and intestinal pathogenic bacteria. Journal of animal and veterinary advances, 7(4), 409-12.
[29] Taormina, P. J., Niemira, B. A., & Beuchat, L. R. (2001). Inhibitory activity of honey against foodborne pathogens as influenced by the presence of hydrogen peroxide and level of antioxidant power. International journal of food microbiology, 69(3), 217-25.
[30] Alonso-Torre, S. R., Cavia, M. M., Fernández-Muiño, M. A., Moreno, G., Huidobro, J. F., Sancho, M. T. (2006). Evolution of acid phosphatase activity of honeys from different climates. Food chemistry, 97(4), 750-5.
[31] Moniruzzaman, M., Khalil, M., Sulaiman, S., & Gan, S. (2012). Advances in the analytical methods for determining the antioxidant properties of honey: a review. African Journal of Traditional, Complementary and Alternative Medicines, 9(1), 36-42.
[32] Mahmoodi-Khaledi, E., Lozano-Sánchez, J., Bakhouche, A., Habibi-Rezaei, M., Sadeghian, I., Segura-Carretero, A. (2017). Physicochemical properties and biological activities of honeys from different geographical and botanical origins in Iran. European Food Research and Technology, 243(6), 1019-30.
[33] Shah, T., Ali, N., Shah, Z., Hayat, A. (2019). Antibacterial Activity of Pakistani Honey. Pakistan Journal of Scientific and Industrial Research Series B, 2019;62(2):97-100.
[34] White, J. r. J. W., Subers, M. H., & Schepartz, A. I. (1963). The identification of inhibine, the antibacterial factor in honey, as hydrogen peroxide and its origin in a honey glucose-oxidase system. Biochimica et Biophysica Acta, 73(1), 57-70.