بهینه‌سازی اثر امواج فراصوت بر تولید گانودریک اسید در Ganoderma adspersum با روش سطح پاسخ

نوع مقاله : مقاله کامل علمی پژوهشی

نویسندگان

1 دانشجوی دکتری ، جنگل‌شناسی و اکولوژی جنگل، دانشکده علوم جنگل، دانشگاه علوم کشاورزی و منابع طبیعی گرگان، گرگان، ایران.

2 دانشیار دانشکده علوم جنگل، دانشگاه علوم کشاورزی و منابع طبیعی گرگان، گرگان، ایران.

3 استاد دانشکده داروسازی، دانشگاه علوم پزشکی مشهد، مشهد، ایران.

چکیده

سابقه و هدف: گانودریک اسید (GA) نوعی از تری‌ترپنوئیدها است که توسط گونه‌های مختلف گانودرما تولید می‌شود. این متابولیت ثانویه به دلیل عملکردهای دارویی فوق‌العاده در سال‌های اخیر توجه زیادی را به خود معطوف نموده و باوجوداینکه گانودرما لوسیدیوم معروفیت زیادی به خاطر داشتن این ترکیب دارد اما گونه‌های دیگر نیز به‌عنوان جایگزین موردتوجه هستند. یکی از مهم‌ترین گونه‌های گانودرما قارچ Ganoderma adspersum است که به‌صورت پارازیت یا ساپروفیت روی درختان زنده یا برخی اوقات روی کنده‌های درختان یافت می‌شود و بازیدیوکارپ آن بسیار شباهت به گونه applanatum G. دارد و اغلب اشتباه گرفته می‌شود. خواص دارویی مختلفی چون کاهش فشار و قند خون، تقویت سیستم ایمنی، خاصیت ضدویروسی و ضد باکتریایی و ضدالتهابی را برای این‌گونه ذکر کرده‌اند و از منابع اصلی تولید گنودریک اسید است. در پژوهش حاضر ضمن شناسایی مولکولی قارچ و حصول اطمینان از گونه، از امواج فراصوت به‌عنوان محرک و با هدف افزایش گانودریک اسید کل در شرایط این‌ویترو استفاده و در دو حالت درون‌سلولی و برون سلولی نتایج بررسی شد.
مواد و روش‌ها: نمونه‌برداری از قارچ در سه سایز کوچک، متوسط و بزرگ انجام و پس از خالص‌سازی، مورد شناسایی مورفولوژیکی و مولکولی قرار گرفت. نمونه‌ها در محیط PDA کشت شده و پس از 14 روز در محیط کشت سوسپانسیون (PDB)، در معرض محرک امواج فراصوت با متغیرهای تعداد (1 تا 3 بار صوت دهی)، زمان (60، 180 و 300 ثانیه) و دمای صوت دهی (30، 40 و 50 درجه سانتی‌گراد) قرار گرفتند. سپس از میسلیوم و محیط کشت قارچ موجود در کشت سوسپانسیون عصاره‌گیری و گانودریک اسید درون‌سلولی و برون سلولی اندازه‌گیری شد. جهت انجام کار از روش سطح پاسخ RSM و طرح باکس بنکن استفاده و 17 آزمایش توسط نرم‌افزار Design expert 7. طراحی گردید و مقدار بهینه متغیرها برای حداکثر تولید گانودریک اسید، به کمک نرم‌افزار تعیین شد.
یافته‌ها: طبق شناسایی مورفولوژیکی و مولکولی گونه انتخابی گانودرما، ادسپرسوم نام دارد. وجود گانودریک اسید در این قارچ به اثبات رسید مشخص شد در بین سه سایز انتخاب‌شده قارچ، سایز بزرگ‌تر (21 × 16 سانتی‌متر) گانودریک اسید بیشتری نسبت به دو سایز دیگر داشت و از همین سایز برای نمونه‌گیری و القا محرک استفاده شد. بیشترین میزان گانودریک اسید کل مربوط به دومرتبه صوت دهی به مدت 282 ثانیه و دمای 30 درجه سانتی‌گراد بود (21/79 میلی‌گرم بر گرم) که باعث افزایش 6/1 برابری گانودریک اسید کل نسبت به شاهد گردید و این در حالی است که این میزان در قارچ طبیعی 48/25 میلی‌گرم بر گرم بوده است.
نتیجه‌گیری: کشت‌ در محیط این‌ویترو به‌جای برداشت از جنگل یک روش پایدار برای استخراج متابولیت‌های ثانویه گیاهی می‌تواند مورداستفاده قرار گیرد. گانودریک اسید یکی از مهم‌ترین متابولیت‌های ضدالتهاب و ضدویروس است که در گونه‌های قارچ گانودرما وجود دارد. با توجه به نیاز به برداشت وسیع از جنگل و عدم امکان دسترسی مداوم به جنگل، جایگزین نمودن روش‌های نوین کشت قارچ در شرایط آزمایشگاهی به‌جای جمع‌آوری از جنگل امری ضروری است. پژوهش حاضر در همین راستا و باهدف افزایش مقدار گانودریک اسید برای اولین بار بر روی گانودرما ادسپرسوم صورت گرفت. همچنین این قارچ برای اولین بار بر روی ممرز گزارش می‌شود. با توجه به افزایش بیش از 5/1 برابری گانودریک اسید کل بعد از اعمال تیمار صوت دهی نسبت به نمونه قارچ طبیعی، انجام تحقیقات تکمیلی برای شناسایی جزئی‌تر و نوع گانودریک‌ها پیشنهاد می‌شود.

کلیدواژه‌ها

موضوعات

عنوان مقاله [English]

Optimization of ultrasound waves effects on production of ganoderic acid of Ganoderma adspersum by response surface method

نویسندگان [English]

  • Mansooreh Aghasizadeh Shaarbaf 1
  • vahide payamnoor 2
  • Mohammadreza Kavosi 2
  • Javad Asili 3
1 Doctoral student of forestry and forest ecology, Faculty of Forest Sciences, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran.
2 Associate Professor, Faculty of Forest Sciences, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran.
3 Professor, Faculty of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran.
چکیده [English]

Background and objectives: Ganoderic acid (GA) is a type of triterpenoids produced by various species of Ganoderma. This secondary metabolite has attracted much attention in recent years due to its extraordinary medicinal functions and although Ganoderma Lucidium is famous for having this compound, other species are also considered as alternatives. One of the most important species of Ganoderma is Ganoderma adspersum, which is found as a parasite or saprophyte on living trees or sometimes on tree stumps, and its basidiocarp is very similar to G. applanatum and is often confused. Various medicinal properties such as reducing blood pressure and blood sugar, strengthening the immune system, antiviral, antibacterial and anti-inflammatory properties have been mentioned for this species, and it is one of the main sources of gonodriic acid production. In the present research, while molecular identification of the fungus and ensuring the species, ultrasound waves were used as an elicitor with the aim of increasing total ganoderic acid in in vitro conditions, and the results were examined in both intracellular and extracellular conditions.
Materials and methods: Mushrooms were sampled in three sizes, small, medium and large, and after purification, they were morphologically and molecularly identified. The samples were cultured in PDA medium and after 14 days in suspension culture medium (PDB), exposed to ultrasound stimulation with variable number (1 to 3 times of sound), time (60, 180 and 300 seconds) and sound temperature ( 30, 40 and 50 °C). Then, extracts were extracted from mycelium and mushroom culture medium in the suspension culture, and intracellular and extracellular gonadoric acid was measured. In order to do the work, RSM response surface method and Benken's box design were used and 17 experiments were designed by Design expert 7 software, and the optimal value of the variables for the maximum production of ganoderic acid was determined with the help of the software.
Results: According to morphological and molecular identification, the selected species of Ganoderma is called adspersum. The presence of ganoderic acid in this mushroom was proved. It was found that among the three sizes of mushrooms selected, the larger size (21 x 16 cm) had more ganoderic acid than the other two sizes, and this size was used for sampling and elicitor induction. The highest amount of total ganoderic acid was related to sonication twice for 282 seconds and temperature of 30 degrees Celsius (79.21 mg/g), which increased the amount of total ganoderic acid by 1.6 times compared to the control, while this amount It was 25.48 mg/g in natural mushroom.
Conclusion: Cultivation in an invitro environment instead of harvesting from the forest can be used as a sustainable method for extracting plant secondary metabolites. Ganoderic acid is one of the most important anti-inflammatory and antiviral metabolites found in Ganoderma mushroom species. Considering the need for extensive harvesting from the forest and the impossibility of continuous access to the forest, it is necessary to replace the new methods of growing mushrooms in laboratory conditions instead of collecting them from the forest. In the same direction and with the aim of increasing the amount of ganodriic acid, the present research was conducted for the first time on Ganoderma adspersum. This fungus is reported for the first time on Carpinus betulus. Considering the increase of more than 1.5 times of total ganoderic acid after ultrasound treatment compared to the natural mushroom sample, it is suggested to carry out additional research for more detailed identification and type of ganoderic acid.

کلیدواژه‌ها [English]

  • Ganoderic acid
  • DNA investigation
  • Ganoderma adspersum
  • Secondary metabolite
  • Ultrasonic waves

مراجع

 1.Fei, Y., Li, N., Zhang, D. H., & Xu, J. W. (2019). Increased production of ganoderic acids by overexpression of homologous farnesyl diphosphate synthase and kinetic modeling of ganoderic acid production in Ganoderma lucidum. Microbial Cell Factories. 18, 115. 1-9.
2.Xiao, H., & Zhong, J. J. (2016). Production of useful terpenoids by higher-fungus cell factory and synthetic biology approaches. Trends Biotechnol. 34, 242-55.
3.You, B. J., Tien, N., Lee, M. H., Bao, B. Y., Wu, Y. SH., Hu, T. C., & Lee, H. Z. (2017). Induction of apoptosis and ganoderic acid biosynthesis by cAMP signaling in Ganoderma lucidum. Scientific Reports. 7 (318), 1-13.
4.Tel-Çayan, G., Öztürk, M., Duru, M. E., Rehman, M., Adhikari, A., Türkoðlu, A., & Choudhary, M. I. (2015). Phytochemical investigation, antioxidant and anticholinesterase activities of Ganoderma adspersum. Industrial Crops and Products. 76, 749-754.
5.Omidi, M., & Farzin, N. (2012). Biotechnology solutions in increasing the efficiency of medicinal plants. Modern Genetic J. 7 (3), 220-209.
6.Abdul Malik, N. A., Kumar, I. S., & Nadarajah, K. (2020). Elicitor and receptor molecules: Orchestrators of plant defense and immunity. International J. of Molecular Sciences. 21 (963), 1-34.
7.Naik, P. M., & Al-Khayri, J. M. (2016). Impact of abiotic elicitors on in vitro production of plant secondary metabolites: A Review. J. of Advanced Research in Biotechnology. 1 (2), 7p.
8.Rahmatinia, M., Moradi, M., Ghasemi Omran, V., & Hadadinejad, M. (2018). The effect of different magnetic field duration on direct organogenesis of African violets (Saintpaulia Ionantha) In tissue culture medium with and without pgrs. J. of Crop Breeding. 9 (24), 103-111.
9.Meng, L., Bai, X., Zhang, S., Zhang, M., Zhou, S., Mukhtar, I., Wang, L., Li, Z., & Wang, W. (2019). Enhanced ganoderic acids accumulation and transcriptional responses of biosynthetic genes in Ganoderma lucidum Fruiting bodies by elicitation supplementation. International J. of Molecular Sciences. 20 (2830), 1-11.
10.Esmaeilzadeh, M., kianirad, M., Sheykhinejad, A., Khosravi, A., & Sharifzadeh, A. (2019). Ganoderic acid and exopolysaccharide production by Ganoderma lucidum from semi-solid-state and submerged fermentation. Biological J. of Microorganism. 7 (28), 63-75.
11.Yanru, H., Shakeel, A., Jiawei, L., Biaobiao, L., Zengyan, G., Qiyun, Z., Xiaohua, L., & Xuebo, H. (2017). Improved Ganoderic acids production in Ganoderma lucidum by wood- decaying components. Scientific reports. 7 (46623), 1-10.
12.Wei, Z. H., Liu, L. L., Guo, X. F., Li, Y. J., Hou, B. C., Fan, Q. L., Wang, K. X., Luo, Y. D., & Zhong, J. J. (2016). Sucrose fed-batch strategy enhanced biomass, polysaccharide, and ganoderic acid production in the fermentation of Ganoderma lucidum. Bioprocess and Biosystems Engineering. 39, 37–44.
13.Hu, G., Zhai, M., Niu, R., Xu, X., Liu, Q., & Jia, J. (2018). Optimization of culture condition for ganoderic acid production in Ganoderma lucidum liquid static culture and design of a suitable bioreactor. Molecules. 23 (2563), 1-12.
14.Vasilevski, G. (2003). Perspectives of the application of biophysical methods in sustainable agriculture. Bulgarian
J. Plant Physiology (Special Issue). 179-186.
15.Cui, Y., Lu, J., Liu, C., Chen, S., Ma, C., Liu, Z., Wang, J., & Kang, W. (2020). Ionic Liquid-Based Ultrasonic-Assisted Extraction Coupled with HPLC and Artificial Neural Network Analysis for Ganoderma lucidum. Molecules.
25 (6), 1-16.
16.Maleki, M. (2014). Effect of methyl jasmonate and ultrasonic on secondary metabolite production in cell suspension culture of Aloe barbadensis Mill [Dissertation, Thesis on Agronomy and plant breeding]. Tehran: Tehran University.
17.Sun, L., Liu, L. P., Wang, Y. Z., Yang, L., Zhang, C., Yue, M. X., Dabbour, M., Mintah, B. K., & Wang, L. (2022). Effect of ultrasonication on the metabolome and transcriptome profile changes in the fermentation of Ganoderma lucidum. Microbiological Research. 254 (126916), 1-9.
18.Moradali, M. F., Hedjaroude, G. A., Mostafavi, H., Abbasi, M., Ghods, S., & Sharifi-Tehrani, A. (2007). The genus Ganoderma (Basidiomycota) in Iran. Mycotaxon, 99, 251-269.
19.Sabeti, H. A. (2008). Iran's forests, trees, and shrubs. Yazd University Press, fifth edition, 806p.
20.Aghajani, H., Marvie Mohadjer, M. R., Asef, M., & Shirvany, A. (2014). The relationship between wood-decay fungi abundance and some morphological features of hornbeam (Case study: Kheyroud forest, Noshahr). Forest and range protection research. 12 (1), 55-65.
21.Aghajani, H. (2012). Study on the oak (Quercus castaneifolia) and Hornbeam a (Carpinus betulus) decaying macrofungi in mixed Oak-Hornbeam forest community in Kheyroud forest, North of Iran, M.Sc. thesis, Department of Forestry and Forest Economics, Faculty of Natural resources, Tehran University, 95p.
22.Ryvarden, L. (1991). Genera of polypores, nomenclature, and taxonomy, Synopsis Fungorum 5, Fungoflora, Norway, Oslo.
23.Ryvarden, L., & Gilbertson, R. L. (1993). European polypores, Oslo: Fungiflora, 387p.
24.White, T.J., Bruns, T.D., Lee, S., & Taylor, J. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. a guide to methods and applications. Pp: 315-322.
25.Aghajani, H., Bari, E., Bahmhani, M., Miha, H., Tajick Ghanbary, M. A., Nicholas, D. D., & Zahedian, E. (2018). Influence of relative humidity and temperature on the cultivation of Pleurotus species. Maderas-Ciencia Tecnologia. 20 (4), 571-578.
26.Tamura, K., Stecher, G., Peterson, D., Filipski, A., & Kumar, S. (2013). MEGA6: Molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution. 30, 2725-2729.
27.Nojoki, F., Hatamian-Zarmi, A., Ebrahimi Hosseinzadeh, B., Mir-derikvand, M., Beygom mokhtari-Hosseini, Z., & Kalantari-Dehaghi, S. (2017). Investigation and optimization effects of ultrasound waves to produce Ganoderic acid, anti-cancer mushrooms metabolite. Iranian J. of Medical Microbiology. 11 (1), 58-66.
28.Alzorqi, I., Singh, A., Manickam, S., & F. Al-Qrimli, H. (2017). Optimization of ultrasound-assisted extraction (UAE) of β-d-glucan polysaccharides from Ganoderma lucidum for prospective scale-up. Resource-Efficient Technologies. Resource - Efficient Technologies. 3 (1), 46-54.
29.Payamnoor, V., Lazemi, G., Nazari, J., & Alishah, O. (2020). Evaluation of the effect of elicitors on antioxidant properties and mycelial secondary metabolites of Stereum hirsutum, Hyphodontia paradoxa, and Arthrinium arundinis from Golestan province. Eco-phytochemical J. of Medicinal Plants. 2 (30), 76-88.
30.Fang, Q., & Zhong, J. (2002). Two-stage culture process for improved production of ganoderic acid by liquid fermentation of higher fungus Ganoderma lucidum. Biotechnology Progress. 18 (1), 51-4.
31.Qiang, G., Hua-Xi, X., Xiao, H., Wang, X., Zhao, Y., Zhang, Y., & Ren, G. (2011). Stimulated production of triterpenoids of Ganoderma lucidum by an ether extract from the medicinal insect, catharsis molossus, and identification of the key stimulating active components. Applied Biochemistry Biotechnology. 165 (1), 87-97.
32.Keypour, S., Riahi, H., Asef, M. R., Abdollahzadeh, J., Burhani, A., & Safaie, N. (2020). The true nature of Ganoderma in Iran: Taxonomy based on ITS and mtSSU rDNA. Forest Pathology. 00:e12605. 1-13.
33.Badalyan, S. M., Gharibyan, N. G., Iotti, M., & Zambonelli, A. (2012). Morphological and genetic characteristics of different collections of Ganoderma P. Karst. species. In: Mushroom Science XVIII, Proceedings of the 18th ISMS Congress.
34.Mau, J. L., Tsai, S. Y., Tseng, Y. H., & Huang, S. J. (2005). Antioxidant properties of methanolic extracts from Ganoderma tsugae. Food Chemistry. 93, 641-649.
35.Cör, D., Botić, T., Knez, Z., Gregori, A., & Pohleven, F. (2017). The effects of different solvents on bioactive metabolites and “in vitro” antioxidant and anti-acetylcholinesterase activity of Ganoderma lucidum fruiting body and primordia extracts. Macedonian J. of Chemistry and Chemical Engineering. 36 (1), 1-13.
36.Zhu, W., Zhong, J., & Tang, Y. (2008). Significance of fungal elicitors on the production of ganoderic acid and Ganoderma polysaccharides by the submerged culture of medicinal mushroom Ganoderma lucidum. Process Biochemistry. 43 (1), 1359-1570.
37.Liang, C., Li. Y., Xu, J., Wang, J., Miao, X., & Tang, Y. (2010). Enhanced biosynthetic gene expressions and production of ganoderic acids in static liquid culture of Ganoderma lucidum under phenobarbital induction. Applied Microbiology Biotechnology. 86 (5), 1367-74.
38.Nojoki, F., Hatamian, A. S., Mirderikvand, M., Ebrahimi, B., Mokhtari, Z. B., & Kalantari, S. (2016). Impact of rifampin induction on the fermentation production of ganoderic acids by Medicinal Mushroom Ganoderma lucidum. Applied Food Biotechnology. 3 (2), 91-8.
39.Zhang, J., Zhong, J., & Geng, A. (2014). Improvement of ganoderic acid production by fermentation of Ganoderma lucidum with cellulase as an elicitor. Process Biochemistry. 49 (10), 1580-1586.
40.Heydarian, M. S., Hatamian, A. S., Amoabediny, G., & Yazdian, F. (2015). Doryab, A. Synergistic effect of elicitors in the enhancement of ganoderic acid production: optimization and gene expression studies. Applied Food Biotechnology. 2 (3), 57-62.
41.Xu, J. W., Zhao, W., & Zhong, J. J. (2010). Biotechnological production and application of ganoderic acids. Applied Microbiology and Biotechnology. 87, 457-466.
42.Zhang, H., Ma, H., Liu, W., Pei, J., Wang, Z., Zhou, H., & Yan, J. (2014). Ultrasound enhanced the production and antioxidant activity of polysaccharides from the mycelial fermentation of Phellinus igniarius. Carbohydrate Polymers. 113, 380-387.
43.Yeo, S. K., & Liong, M. T. (2013). Effect of ultrasound on bioconversion of isoflavones and probiotic properties of parent organisms and subsequent passages of Lactobacillus. LWT - Food Science and Technology. 51, 289-295.
44.Avhad, D. N., & Rathod, V. K. (2014). Ultrasound stimulated the production of a fibrinolytic enzyme, Ultrasonics Sonochemistry. 21, 182-188.
45.Wang, F., Ma, A. Z., Guo, C., Zhuang, G. Q., & Liu, C. Z. (2013). Ultrasound-intensified laccase production from Trametes versicolor. Ultrasonics Sonochemistry. 20, 118-124.
46.Taherkhani, T., Asghari Zakaria, R., Omidi, M., & Zare, N. (2017). Effect of ultrasonic waves on crocin and safranal content and expression of their controlling genes in suspension culture of saffron (Crocus sativus L.), Natural Product Research. 10 November 2017, 05:15.
47.Atrashi, M., Tavokoli Dinanie, E., Darzi, M. T., Hashemi, J., Rozbeh, S., & Masumi, A. (2011). Effect of ultrasound on the production of Carvone as a secondary metabolite in callus derived from Bunium persicum Boiss. J. of Herbal Medicine. 2 (2), 129-135.
48.Wu, J., & Lin, L. (2003). Enhancement of taxol production and release in Taxus chinensis cell cultures by ultrasound, methyl jasmonate, and in situ solvent extraction. Appl. Microbiol. Biotechnol. 62 (2-3), 151-155.
49.Rezaei, A., Ghanati, F., & Behmanesh, M. (2012). Stimulation of taxol production by magnetic field in cell culture of hazel (Corylus avellana L.). Iranian J. of Biomedical Engineering. 6, 113-122.
50.Sainz Herrán, N., Casas López, J. L., Sánchez Pérez, J. A., & Chisti, Y. (2008). Effects of ultrasound on the culture of Aspergillus terreus. J. of Chemical Technology and Biotechnology.
83, 593-600.
51.Lu, P., Lou, H., Wei, T., Liu, Z., Jiao, Y., & Chen, Q. (2020). Ultrasound enhanced the production of mycelia and exopolysaccharide by Agaricus bitorquis (Quél.) Sacc. Chaidam. Ultrasonics Sonochemistry. 64, 105040.1-12.
52.Zare, N., Farjaminezhad, R., Asghari-Zakaria, R., & Farjaminezhad, M. (2014). Enhanced thebaine production in Papaver bracteatum cell suspension culture by the combination of elicitation and precursor feeding. Natural Product Research. 28, 711-717
53.Rezaei, A., Ghanati, F., Behmanesh, M., & Mokhtari-Dizaji, M. (2011). Ultrasound-potentiated salicylic acid-induced physiological effects and production of taxol in hazelnut (Corylus avellana L.) cell culture. Ultrasound in Medicine and Biology. 37 (11), 1938-1947.
54.Li, W., Ma, H., He, R., Ren, X., & Zho, C. (2021). Prospects and application of ultrasound and magnetic fields in the fermentation of rare edible fungi. Ultrasonics Sonochemistry. 76 (105613), 1-12.
55.Ojha, K. S., Mason, T. J., O’Donnell, C. P., Kerry, J. P., & Tiwari, B. K. (2017). Ultrasound technology for food fermentation applications. Ultrasonics Sonochemistry. 34, 410-417.
56.Teixeira da Silva, J. A., & Dobranszki, J. (2014). Sonication (ultrasound) affects In Vitro growth of hybrid Cymbidium. Botanica Lithuanica. 20, 121-130.
پژوهش‌های علوم و فناوری چوب و جنگل
دوره 30، شماره 2
تیر 1402
صفحه 15-37

فایل ها

هم رسانی

ارجاع به این مقاله

آمار