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EDN: ONJTWU DOI: 10.21285/achb. 930
Physico-chemical characteristics of melanins culture medium Inocutis dryophila (Berk) Flasson & Niemela
Tatyana G. Gornostai
Siberian Institute of Plant Physiology and Biochemistry SB RAS, Irkutsk, Russian Federation
Abstract. The purpose of the study was to investigate the effect of light treatment on the synthesis of endomelanins from the mycelium of Inocutis dryophila in a liquid culture medium. To identify the effect of light on the synthesis of melanins, the mycelium of Inocutis dryophila was cultivated in a liquid medium in the dark and using blue light treatment. As a result, two types of melanins IDM-1 and IDM-2 were obtained. It has been established that the light factor influences the quantitative release of melanins from the mycelium of Inocutis dryophila into the culture liquid. The use of blue light resulted in lower melanin content in the culture fluid than in the dark. Ultraviolet and infrared spectrometry showed that both types of melanins have typical spectra and graphs for fungal melanins. Infrared spectrometry showed that treatment of mycelium with blue light led to deformation of IDM-1. It was revealed that IDM-2 melanins exhibit greater antiradical activity than IDM-1 melanins. Thus, light treatment of Inocutis dryophila mycelium in a liquid medium affects the quantitative release of melanins into the culture liquid, promotes a change in the structure, as well as manifestation of a biological effect.
Keywords: Inocutis dryophila, culture medium, melanin, blue light, UV-, IR-spectrometry.
Acknowledgment. For the study, the equipment of the Center for Collective Use "Bioanalitika" of the Siberian Institute of Plant Physiology and Biochemistry of the Siberian Branch of the Russian Academy of Sciences (Irkutsk) was used.
Funding. Ministry of Science and Higher Education of the Russian Federation supported this study within the framework of the state task for Siberian Institute of Plant Physiology and Biochemistry SB RAS (Project State Registration no. 122041100049-0).
For citation: Gornostai T.G. Physico-chemical characteristics of melanins culture medium Inocutis dryophila (Berk) Flasson & Niemela. Proceedings of Universities. Applied Chemistry and Biotechnology. 2024;14(3):416-420. (In Russian). DOI: 10.21285/achb.930. EDN: ONJTWU.
КРАТКИЕ СООБЩЕНИЯ
УДК 582.284
Физико-химическая характеристика меланинов культуральной среды Inocutis dryophila (Berk) Flasson & Niemela
Т.Г. Горностай
Сибирский институт физиологии и биохимии растений СО РАН, Иркутск, Российская Федерация
Аннотация. Целью настоящего исследования явилось изучение влияния световой обработки на синтез эндоме-ланинов мицелия Inocutis dryophila в жидкую культуральную среду. Для выявления действия света на синтез меланинов проводили культивирование мицелия Inocutis dryophila на жидкой среде в темноте и с применением обработки синими светодиодами. В результате были получены два вида меланинов IDM-1 и IDM-2. Установлено, что световой фактор оказывает влияние на количественный выход меланинов из мицелия Inocutis dryophila в культуральную жидкость. Применение синего света приводило к меньшему содержанию меланина в культуральной жидкости по сравнению с количеством меланина, полученного в темноте. Методом ультрафиолетовой и инфракрасной спектрометрии выявлено, что оба вида меланинов имеют типичные спектры и графики для
© Gornostai T.G., 2024
грибных меланинов. В ходе инфракрасной спектрометрии было обнаружено, что обработка мицелия синим светом приводила к деформации молекул меланинов ЮМ-1. Установлено, что меланины ЮМ-2 проявляли большую антирадикальную активность, чем меланины ЮМ-1. Таким образом, при глубинном культивировании световая обработка мицелия ¡посияв dryophila оказывает влияние на количественный выход меланинов в культуральную жидкость, вносит вклад в изменение структуры, а также проявление биологического эффекта.
Ключевые слова: /поси^ dryophila, культуральная жидкость, меланины, синий свет, ультрафиолетовая и инфракрасная спектрометрия
Благодарности. В работе использовано оборудование Центра коллективного пользования «Биоаналитика» Сибирского института физиологии и биохимии растений СО РАН (г. Иркутск, Российская Федерация).
Финансирование. Исследование выполнено в рамках государственного задания Минобрнауки России для Федерального государственного бюджетного учреждения науки Сибирского института физиологии и биохимии растений Сибирского отделения Российской академии наук (рег. № НИОКТР - 122041100049-0).
Для цитирования: Горностай Т.Г. Физико-химическая характеристика меланинов культуральной среды ¡поси^э бгуорШа // Известия вузов. Прикладная химия и биотехнология. 2024. Т. 14. N 3. С. 416-420. DOI: 10.21285/ ас1"|Ь.930. EDN: ONJTWU.
INTRODUCTION
Melanins are biopolymers, widespread in nature, having diverse origins, structures and functions, and are currently actively used and introduced into various areas of industry [1, 2]. Considering the complexity of the melanins structure of, currently the number of fungi used to obtain melanins remains small, so the search and characterization of new species of fungi as a potential source of melanins is an urgent task. Basidial fungi have already proven themselves as a source of highly active melanins [3, 4]. The most extensively studied genera include the melanins of Inonotus obliquus [5-7], revealing a wide range of activities [7-9]. The genus Inocutis is a closely related taxon to Inonotus [10], whose representatives have been little studied, but they may probably be no less promising objects for obtaining biologically active substances. We previously discovered that Inocutis dryophila is characterized by a high rate of mycelial growth on solid and liquid media. This study examines the possibility of obtaining fungal melanins from the culture liquid of the basidiomycete fungus I. dryophila using lighting.
EXPERIMENTAL
We used a pure culture of the basidiomycete fungus Inocutis dryophila (Berk) Flasson & Niemela strain 1422. Mycelium cultivation was carried out in 250 ml flasks containing 160 ml of wort-agarized liquid medium of the following composition: water 120 ml, unhopped light wort 40 ml, sucrose 1,6 g, glucose 1,6 g. The duration of cultivation was 30 days at a temperature of 25 °C, on a shaker at 140 rpm, under continuous illumination with blue LEDs with a luminous flux intensity of 12,8 W/m2, and in the dark. Afterwards, filtration was carried out using a vacuum pump to separate the mycelium from the culture medium, followed by the isolation of melanin from the culture medium, as described in the method [11] and obtaining two types of melanins: IDM-1 - melanin from the culture liquid of I. dryophila, obtained on blue light and IDM-2 - melanin from the culture liquid of I. dryophila, obtained in the dark.
Samples for IR spectroscopy were dissolved in the 10% NH3 solution, the solution was applied to thallium-bromine-iodine glass plates (KRS-5) and dried in vacuum.
IR spectra were recorded on a Spectrum 100 IR-Fourier spectrometer (Perkin-Elmer, USA) in film in the range of 4000-450 cm-1.
Antiradical activity was determined with ABTS radical according to the method [12].
The experiment was carried out in three biological replicates. Data were expressed as the mean and their standard deviation. The results were processed using Microsoft Excel. Differences between experimental data were considered statistically significant at p < 0.05.
RESULTS AND DISCUSSION
As a result of isolating mycelium from the culture medium, melanin IDM-1 and IDM-2 were obtained, which is an amorphous substance of dark brown color, insoluble in water, acids and organic solvents, but highly soluble in aqueous solutions of alkalis.
It was shown that the light factor affects the yield of melanin in the culture liquid of I. dryophila, so the use of blue light led to a decrease in melanin content compared to darkness during mycelium cultivation, the yield of IDM-1 and IDM-2 was 1,443 and 1,663 g/l, respectively. Regulation of melanin synthesis was previously shown for I. obliquus; however, the opposite pattern in its accumulation is observed [13].
The UV spectrum of melanins alkaline solutions is typical for representatives of this class of compounds; it lacked absorption bands in the visible wavelength range with a characteristic linear dependence of the optical density logarithm on wavelength (Fig. 1).
Regression analysis showed that in the equations of this dependence (log D = ak + b) the values of the coefficient of determination r2 were in the range of 0,9706-0,9759, and the values of the regression analysis were - 0,0039 to -0,0042, this is consistent with previously published data on fungal melanins [14]. The chromatic coefficients E465/E665 of melanins were 6,24 and 6,59, indicating the presence of aliphatic and O-containing functional groups.
During the analysis of IR spectrometry, it was found that the graphs typical for fungal melanins are in the wavelength ranges from 3600 to 3000 cm-1, from 1650 to 1600 cm-1 and from 1500 to 1400 cm-1 [15, 16] (Fig. 2).
Fig. 1. Dependency graphs for melani ns Inocutis dryophila IDM-1 (blue light) (a) and IDM-2 (dark light) (b); 1 - absorption spectrum of 0,002% solutions of Inocutis dryophila melanins; 2 - dependence of the logarithm of optical density on wavelength; 3 - linear regression graph for the A - log D dependence
Рис. 1. Графики зависимостей для меланинов Inocutis dryophila IDM-1 (синий свет) (а) и IDM-2 (темнота) (Ь): 1 - спектр поглощения 0,002%-х растворов меланинов Inocutis dryophila; 2 - зависимость логарифма оптической плотности от длины волны; 3 - график линейной регрессии для зависимости А - lg D
v.crrr1
Fig. 2. IR spectrum of Inocutis dryophila melanins IDM-1 (blue light) and IDM-2 (dark light)
Рис. 2. ИК-спектр меланинов Inocutis dryophila IDM-1 (синий свет) и IDM-2 (темнота)
Broad bands are in the region of 3000-3300 cm1, due to stretching vibrations of OH bound to NH [17]. The existence of aliphatic fragments (C=H-, CH2-, CH3-) was confirmed by bands in the region of 2913-2020 cm1 and 1439-1448 cm"1 [6]. Strong bands at 1663-1656 cm1 are corresponding to the stretching vibrations of 0=0 and NH groups of secondary amides. Vibrations of the aromatic regions of the carboxyl function C=C and C=0 are usually associated with a strong, characteristic absorption band between 1650 and 1600 cm1 [18]. The bands in the 1600 cm1 region are associated with vibrations on the CH = CH bond plane [19]. The peak of
NH vibration and the peak of stretching vibration of CN- are at 1582-1586 cm1 and 1400 cm1, which indicates the melanin structure typical of indole [20]. The presence of vibrations of the OH- group (tertiary alcohols) in the region of 1402-1310 cm1 was also characteristic. The bands in the spectral region 1070-1047 cm1 belong to hydroxyl, alcohol and phenol groups, respectively [19]. There are vibrations at 750-650 cm1 OH of the bound group, OS at 700-600 cm1, O-NO at 600 cm1 [20]. IR spectrometry showed that irradiation with blue light leads to deformation of IDM-1 melanin molecules. During the analysis of the antiradical activity of the obtained melanins using 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS+ method) it was revealed that the light factor influences the degree of manifestation of this type of activity. Melanins content of I. dryophila obtained in the dark is higher. It was established that the activity of IDM-2 was 1050 = 1,21 |jg/ml, IDM-11050 = 1,51 |jg/ml, both values are higher than those indicated in the literature for /. obliquus melanins, obtained by the same method [6]. All data indicated for I. dryophila are presented for the first time.
CONCLUSION
Thus, the production of melanins from the culture liquid of /. dryophila is a promising source of highly active melanin, and it has been shown that cultivation in the dark has greater yield and activity, which is undoubtedly a big advantage for economically beneficial mass production of melanin.
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11. Ilyicheva T.N., Anan'ko G.G., Kosogova T.A., Olkin S Ye., Omigov V.V., Taranov O.S., et al. Antiviral activity of the melanin from birch fungus (Inonotus obliquus) obtained by cultivating F-1244 strain isolating to pure culture. Chemistry of plant raw material. 2020;2:283-289. (In Russian). DOI: 10.14258/ jcprm.2020025167. EDN: OPSRWL.
12. Ding H.-Y., Chou T.-H., Liang C-H. Antioxidant and antimelanogenic properties of rosmarinic acid methyl ester from Origanum vulgare. Food Chemistry 2010;123(2):254-262. DOI: 10.1016/j.foodchem.2010.04.025.
13. Poyedinok N., Mykhaylova O., Tugay T., Tugay A., Negriyko A., Dudka I. Effect of light wavelengths and coherence on growth, enzymes activity, and melanin accumulation of liquid-cultured Inonotus obliquus (Ach.:Pers.) Pilat. Applied Biochemistry and Biotechnology. 2015;176:333-343. DOI: 10.1007/s12010-015-1577-3.
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15. Babitskaya V.G., Shcherba V.V., Ikonnikova V.L. Melanin complex of the fungus Inonotus obliquus. Applied Biochemistry and Microbiology. 2000;36:377-381. DOI: 10.1007/BF02738046.
16. Ribera J., Panzarasa G., Stobbe, A., Osypova A., Rupper P., Klose D., Schwarze F.W.M.R. Scalable biosynthesis of melanin by the basidiomycete Armillaria cepistipes. Journal of Agricultural and Food Chemistry. 2018;67(1):132-139. DOI: 10.1021/acs.jafc.8b05071.
17. Hou R., Liu X., Xiang K., Chen L., Wu X., Lin W., et al. Characterization of the physicochemical properties and extraction optimization of natural melanin from Inonotus hispidus mushroom. Food Chemistry. 2019;277:533-542. DOI: 10.1016/j.foodchem.2018.11.002.
18. Wang L., Li Y., Li Y. Metal ions driven production, characterization and bioactivity of extracellular melanin from Streptomyces sp. ZL-24. International Journal of Biological Macromolecules. 2019;123:521-530. DOI: 10.1016/j. ijbiomac.2018.11.061.
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СПИСОК И
1. Mavridi-Printezi A., Guernelli M., Menichetti A., Montalti M. Bio-applications of multifunctional melanin nanoparticles: from nanomedicine to nanocosmetics // Nanomaterials. 2020. Vol. 10, no. 11. P. 2276. DOI: 10.3390/nano10112276.
2. Guo L., Li W., Gu Z., Wang L., Guo L., Ma S., et al. Recent advances and progress on melanin: from source to application // International Journal of Molecular Sciences. 2023. Vol. 24, no. 5. P. 4360. DOI: 10.3390/ijms24054360.
3. Liu R., Meng X., Mo C., Wei X., Ma A. Melanin of fungi: from classification to application // World Journal of Microbiology and Biotechnology. 2022. Vol. 38. P. 228. DOI: 10.1007/s11274-022-03415-0.
4. Suthar M., Dufosse L., Singh S.K. The enigmatic world of fungal melanin: a comprehensive review // Journal of Fungi. 2023. Vol. 9, no. 9. P. 891. DOI: 10.3390/jof9090891.
5. Kukulyanskaya T.A., Kurchenko N.V., Kurchenko V.P., Babitskaya V.G. Physicochemical properties of melanins produced by the sterile form of Inonotus obliquus ("Chagi") in natural and cultivated fungus // Applied Bio-
chemistry and Microbiology. 2002. Vol. 38. P. 58-61. DOI: 10.1023/A:1013204706055.
6. Olennikov D.N., Tankhaeva M., Rokhin A.V., Agafonova S.V. Physicochemical properties and antioxidant activity of melanin fractions from Inonotus obliquus sclerotia // Chemistry of Natural Compounds. 2012. Vol. 48. P. 396-403. DOI: 10.1007/s10600-012-0260-y.
7. Wold C.W., Gerwick W.H., Wangensteen H., Inngjerdingen K.T. Bioactive triterpenoids and water-soluble melanin from Inonotus obliquus (Chaga) with immunomodulatory activity // Journal of Functional Foods. 2020. Vol. 71. P. 104025. DOI: 10.1016/j.jff.2020.104025.
8. Burmasova M.A., Utebaeva A.A., Sysoeva E.V., Sysoeva M.A. Melanins of Inonotus obliquus: bifidogenic and antioxidant properties // Biomolecules. 2019. Vol. 9, no. 6. P. 248. DOI: 10.3390/biom9060248.
9. Parfenov A.A., Vyshtakalyuk A.B., Sysoeva M.A., Sysoeva E.V., Latipova A.D., Gumarova L.F., Zobov V.V. Hepatoprotective effect of Inonotus obliquus melanins:
in vitro and in vivo studies // BioNanoScience. 2019. Vol. 9. P. 528-538. DOI: 10.1007/s12668-019-0595-y.
10. Спирин В.А., Змитрович И.В., Малышева В.Ф. К систематике Phellinus s. l. и Inonotus s. l. (Mucronopo-raceae, Hymenochaetales) // Новости систематики низших растений. 2006. Т. 40. С. 153-188. EDN: KHNDHJ.
11. Ильичева Т.Н., Ананько Г.Г., Косогова Т.А., Олькин С.Е., Омигов В.В., Таранов О.С. [и др.]. Противовирусная активность меланина из Чаги (Inonotus obliquus), полученного на основе культивирования штамма F-1244, выделенного в чистую культуру // Химия растительного сырья. 2020. N 2. С. 283-289. DOI: 10.14258/jcprm.2020025167. EDN: OPSRWL.
12. Ding H.-Y., Chou T.-H., Liang C-H. Antioxidant and antimelanogenic properties of rosmarinic acid methyl ester from Origanum vulgare// Food Chemistry. 2010. Vol. 123, no. 2. P. 254-262. DOI: 10.1016/j.foodchem.2010.04.025.
13. Poyedinok N., Mykhaylova O., Tugay T., Tugay A., Negriyko A., Dudka I. Effect of light wavelengths and coherence on growth, enzymes activity, and melanin accumulation of liquid-cultured Inonotus obliquus (Ach.:Pers.) Pilat // Applied Biochemistry and Biotechnology. 2015. Vol. 176. P. 333-343. DOI: 10.1007/s12010-015-1577-3.
14. Suryanarayanan T.S., Ravishankar J.P., Venka-tesan G., Murali T.S. Characterization of the melanin pigment of a cosmopolitan fungal endophyte // Myco-logical Research. 2004. Vol. 108, no. 8. P. 974-978. DOI: 10.1017/s0953756204000619.
INFORMATION ABOUT THE AUTHOR
Tatyana G. Gornostai,
Cand. Sci. (Pharmaceutics), Researcher,
Siberian Institute of Plant Physiology
and Biochemistry SB RAS,
132, Lermontov St., Irkutsk, 664033,
Russian Federation,
https://orcid.org/0000-0002-1120-2148
Contribution of the author
The author performed the research, made a generalization on the basis of the results obtained and prepared the copyright for publication.
Conflict interests
Author declares no conflict of interests regarding the publication of this article.
The final manuscript has been read and approved by the author.
Information about the article
The article was submitted 22.05.2024. Approved after reviewing 04.07.2024. Accepted for publication 30.08.2024.
15. Babitskaya V.G., Shcherba V.V., Ikonnikova V.L. Melanin complex of the fungus Inonotus obliquus // Applied Biochemistry and Microbiology. 2000. Vol. 36. P. 377-381. DOI: 10.1007/BF02738046.
16. Ribera J., Panzarasa G., Stobbe, A., Osypova A., Rupper P., Klose D., Schwarze F.W.M.R. Scalable biosynthesis of melanin by the basidiomycete Armillaria cepistipes// Journal of Agricultural and Food Chemistry. 2018. Vol. 67, no. 1. P. 132-139. DOI: 10.1021/acs.jafc.8b05071.
17. Hou R., Liu X., Xiang K., Chen L., Wu X., Lin W., et al. Characterization of the physicochemical properties and extraction optimization of natural melanin from Inonotus hispidus mushroom // Food Chemistry. 2019. Vol. 277. P. 533-542. DOI: 10.1016/j.foodchem.2018.11.002.
18. Wang L., Li Y., Li Y. Metal ions driven production, characterization and bioactivity of extracellular melanin from Streptomyces sp. ZL-24 // International Journal of Biological Macromolecules. 2019. Vol. 123. P. 521-530. DOI: 10.1016/j.ijbiomac.2018.11.061.
19. Bilinska B. Progress of infrared investigations of melanin structures // Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. 1996. Vol. 52, no. 9. P. 1157-1162. DOI: 10.1016/0584-8539(96)01691-1.
20. Ye M., Guo G., Lu Y., Song S., Wang H., Yang L. Purification, structure and anti-radiation activity of melanin from Lachnum YM404 // International Journal of Biological Macromolecules. 2014. Vol. 63. P. 170-176. DOI: 10.1016/j. ijbiomac.2013.10.046.
ИНФОРМАЦИЯ ОБ АВТОРЕ
Горностай Татьяна Геннадьевна,
к.фарм.н., научный сотрудник, Сибирский институт физиологии и биохимии растений СО РАН, 664033, г. Иркутск, ул. Лермонтова, 132, Российская Федерация, [email protected] https://orcid.org/0000-0002-1120-2148
Вклад автора
Автор выполнил исследовательскую работу, на основании полученных результатов провел обобщение, подготовил рукопись к печати.
Конфликт интересов
Автор заявляет об отсутствии конфликта интересов.
Автор прочел и одобрил окончательный вариант рукописи.
Информация о статье
Поступила в редакцию 22.05.2024. Одобрена после рецензирования 04.07.2024. Принята к публикации 30.08.2024.
