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UDC 579.22: 582.28 https://doi.org/10.15407/biotech12.04.034
INFLUENCE OF NITROGEN CONCENTRATION ON PHOTOINDUCED GROWTH, ENZYMATIC ACTIVITY AND MELANINE SYNTHESIS BY Inonotus obliquus (Ach.:Pers.) Pilat
N. L. Poyedinok1 institute of Food Biotechnology and Genomics of
T. I. Tugay2 the National Academy of Sciences of Ukraine, Kyiv
A. V. Tugay2 2Zabolotny Institute of Microbiology and Virology
O. B. Mykchaylova3 of the National Academy of Sciences of Ukraine, Kyiv
N. N. Sergiichuk 3Kholodny Institute of Botany of the National Academy
A. M. Negriyko5 of Sciences of Ukraine, Kyiv
4Open International University of Human Development "Ukraine", Kyiv 5Institute of Physics of the National Academy of Sciences of Ukraine, Kyiv
E-mail: [email protected]
Received 09.06.2019 Revised 17.07.2019 Accepted 30.08.2019
The aim of the work was to study the effect of nitrogen concentrations on photo-induction of growth, enzymatic activity and synthesis of melanin by the medicinal mushroom Inonotus obliquus (Ach.: Pers.) Pilat. Irradiated by light of low intensity light, different coherence and in different wavelength ranges, mycelium was cultivated in a dynamic mode on a glucose-peptone medium with different concentrations of total nitrogen. The concentration of the nitrogen source was not shown to significantly affect the photo-induced stimulation of the I. obliquus growth. The increase in biomass accumulation of mycelium photoactivated in different modes was almost the same in all variants of the experiment, compared with the biomass of not-irradiated mycelium. A reliable dependence of the photo-stimulation of melanin synthesis on the concentration of nitrogen in the medium was established. Reduced nitrogen concentration more than twice increased the stimulating effect of low-intensity laser radiation with a wavelength of 488 nm. Using substrate with a reduced content of the nitrogen source is advisable to increase the photo-induced stimulating effect in the production of extracellular catalase, tyrosinase and polyphenol oxidase, intracellular peroxidase.
Thus, the cultivation parameters of I. obliquus and the light treatment regimes of the inoculum should be adjusted according to the target bioactive components.
Key words: Inonotus obliquus, photoinduction, nitrogen, melanin, catalase, tyrosinase, polyphenol oxidase, peroxidase, growth activity.
For several hundreds of years, Inonotus obliquus has been used, rather successfully, in traditional medicine to treat cancer and other illnesses. Its chemical composition is known by now. Thus, the mushroom is recognized as a promising producer of not only phenolic compounds, melanins and triterpenoids, but also of other components with anti-oxidant, antitumor, antiviral, hepatoprotective and immunomodulation properties [1-4].
Previously we've established that the low-intensity light of different coherence in the visible part of the spectrum can be effectively utilized to regulate the growth and biosynthesis activity of this mushroom [5, 6]. However, the way the mushroom reacts to light exposure depends on various factors. For many of them, the mechanisms of action are hardly studied at all. The published data mostly touches on the processes of photoreception and photo-regulation of the microfungi metabolism
[7-13]. For Hypocrea atroviridis it is shown that the composition of medium and culturing regime determine the photostimulation levels of growth and conidia production. In Trichoderma reesei, photo-induced activity of enzymes, involved in plant cell destruction, also depends on the nutrient source [13]. The photoreception and expression of Aspergillus enzimes is sometimes linked with carbohydrate metabolism [14]. Other researchers think that there is a relationship between light and the metabolism of nitrogen [15, 16]. We have previously revealed the dependence of photo-activation levels of mycelial growth in five species of fungi on the carbon concentration in substrate [17]. There is no statistically significant evidence of the effect of nitrogen source concentration on the photo-induction of growth stimulation and biomass accumulation in those fungi. However, the nitrogen concentration can affect the photo-induction of secondary metabolites in those species, which does not necessarily correlate with the growth activity [17-20].
To test that hypothesis, we've studied the biosynthetic activity of I. оbliquus on media with different concentrations of nitrogen (2 and 4 g/l of peptone).
Materials and Methods
Study object was pure culture of Inonotus obliqus (Ach.: Pers.) Pilát 1877 from the mushroom culture collection (IBK) of M. G. Kholodny Institute of Botany of the National Academy of Sciences of Ukraine.
The medium used to study the effect of nitrogen concentration on photo-induced biosynthetic activity was the glucose-peptone medium (Bacteriological Peptone (laboratorios CONDA), with 30 g/l glucose and different peptone concentrations (2 and 4 g/l, equal to 282 and 564 mg/l of total nitrogen). The mycelium, surface-grown on wort-agar at 25-26 °C, was irradiated. Immediately after that, disks of mycelium were cut out, 5 mm in diameter. Mycelial disks were put in groups of five in Erlenmeyer flasks with 150 ml of nutrient medium, and cultured in dynamic mode (180 rpm) at same temperature for 12 days. The biomass was dried at 60 °C to constant weight. The change in growth indices after irradiation with low-intensity light was calculated in% compared to control.
The sources of coherent visible low-intensity laser radiation (LILR) were gas lasers: heliumneon laser LGN-215 with a wavelength of 632.8 nm (red light) ("Poliaron", Lviv,
Ukraine), and argon ion laser with a wavelength of 488.0 nm (blue light) (modified LGN-106M1, "Plasma", Russia). The laser ray was defocused with a lens to the size of a Petri dish.
The light-emitting diodes (LED) matrix was the source of low-coherent light. It was produced with 21 high power LED units based on AlGaInN (China Young Sun Led Technology Ltd). Each diode block included two microchips emitting light with wavelengths of 463 nm (blue) and 625 nm (red). The electrical power of each microchip was 1 W. Radiation intensity was regulated from zero to maximum independently for each spectral range, that is separately for the blue, green and red lights by adjusting the current running through the diodes [6].
Radiation power density was measured using a PM-100D digital optical power and energy meter, Thorlabs Inc. with standard photodiode power sensor S120C, operating range of 400-1100 nm. The radiation dose was determined as the product of the power density and the exposure time, and was 230 mJ/cm2 in all variants of the experiment. We chose the modes of irradiation based on our previous results [5, 6, 18, 20]. Light processing of inoculum was carried out in the complete absence of other light sources.
Methods of melanin extraction from biomass and its quantitative determination in mycelium and culture medium were described in [6], together with methods used for the determination of enzyme activity.
Statistical processing of the results was performed using Excel 2007 software program. All experiments were performed in 4-5 replicates. Statistical processing of the results was calculated in Statistica 6.0 software program. The results of study on the biosynthetic activity of I. obliquus on media with different concentrations of nitrogen were statistically significant at P < 0.05 according to the t-student criterion.
Results and Discussion
Our results did not reveal any significant effect of the nitrogen concentration on the photoinduction of I. оbliquus growth (Fig. 1). The increase in biomass accumulation by mycelium, photoactivated in different modes, was almost the same in all variants of the experiment compared with control (non-irradiated mycelium). We've obtained similar data for other species of fungi [5]. This allows us to assume that there are common mechanisms of photo-regulation of growth activity in the studied species of fungi.
Fig. 1. Growth of photo-activated mycelium of I. obliquus on media with different concentrations of nitrogen
(control = 100%, 0 on the figure)
The results presented on Fig. 2 demonstrate that there is a statistically significant dependence of photo-stimulation of melanin synthesis on the concentration of nitrogen in medium. Reducing the concentration of nitrogen increases the stimulating effect of LILR with a wavelength of 488 nm by more than 2 times.
In this regard, the study of the effect of nitrogen on photo-induced changes under the activity of I. obliquus enzymes which were established earlier [6] was of particular interest, specifically catalyzing the synthesis of melanin, extracellular and intracellular tyrosinase, polyphenol oxidase, as well as catalase and peroxidase, which provide antioxidant cell protection and maintaining the concentration of reactive oxygen species at the physiological level.
Limiting the nitrogen concentration in the nutrient medium lead to increased stimulating effect of both laser and LED irradiation only for the extracellular tyrosinase (Fig. 3). The tyrosinase activity increased by 42.7% and 65.0% on a medium with 284 mg/l of nitrogen after irradiation of the seed mycelium with light with the wavelength of 632.8 nm and 488.0 nm, respectively, compared to cultivation on medium containing 568 mg/l nitrogen. The tyrosinase is known to catalyze the synthesis of melanin, thus a parallel twofold increase in the synthesis of this pigment in the same variant of the experiment is quite explicable. The results suggest that nitrogen metabolism does not affect the biosynthesis of intracellular tyrosinase.
Similar study was conducted on the changes
Fig. 2. Effect of nitrogen concentration (282 and 564 mg/l) on photoinduction of melanin in I. obliquus:
* statistically significant differences of melanin synthesis on media with different nitrogen concentrations (P < 0.05), results presented as М±n, n = 4 to 5
А
B
Fig. 3. Effect of nitrogen concentrations (282 with compared to 564 mg/l) on activity of tyrosinase
in I. obliquus
Here and after (A) extracellular, (B) intracellular enzyme, results presented as М±n, n = 4 to 5. * Statistically significant differences of tyrosinase activity on media with different nitrogen concentrations
(P <0.05).
in the activity of polyphenol oxidase, another enzyme responsible for the synthesis of melanin in I. obliquus (Fig. 4). It was observed that decreasing the concentration of nitrogen in the culture medium lead, on the contrary, to 42.4% decrease in the level of intracellular enzyme activity if mycelium irradiated with blue laser light were used as seed material. A significant decrease in the activity of polyphenol oxidase in mycelium was also noted when using other modes of irradiation, but not as substantial.
In a medium with reduced nitrogen content, the activity of extracellular polyphenol oxidase increased more than twice when the seed mycelium was activated by red low-coherent light (Fig. 4).
Higher extracellular peroxidase activity in I. obliquus was observed on a medium containing 284 mg/l of nitrogen for non-irradiated inoculum (control). Irradiation
reduced enzymatic activity on media with both concentrations of nitrogen (Fig. 5).
Reduction of nitrogen concentration did not affect the activity of intracellular peroxidase in control and after irradiation of mycelium with non-coherent light. Irradiation with both blue and red coherent light caused an increase in peroxidase activity in the mycelium of I. obliquus. However, changes in the concentration of nitrogen in the medium affected the photo-induction of this enzyme differently. For example, cultivating mycelium irradiated with light with a wavelength of 632.8 nm on a medium with 284 mg/l of total nitrogen resulted in lower enzymatic activity than when cultivating it on a medium with 568 mg/l of nitrogen. When those media were inoculated with mycelium activated by blue coherent light, the opposite reaction was observed. We can only assume that light of
A
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Fig. 4. Effect of nitrogen concentrations (282 with compared to 564 mg/l) on activity of polyphenol oxidase in I. obliquus: * Statistically significant differences of polyphenol oxidase activity on media with different
nitrogen concentrations (P < 0.05)
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Fig. 6. Effect of nitrogen concentrations (282 with compared to 564 mg/l) on activity of catalase in I. obliquus:
* Statistically significant differences of catalase activity on media with different nitrogen concentrations
(P < 0.05)
different wavelengths and coherence induces various changes in metabolism, changing the peroxidase activity of mycelium. Studying the mechanisms of such changes is undoubtedly of practical and scientific interest and deserves due attention.
Regarding the effect of nitrogen concentrations on the realization of photoinduced changes in catalase activity, our studies unequivocally showed the advantages of using media with a reduced content of nitrogen source to culture the photoactivated inoculum, for an additional stimulating effect (Fig. 6).
The activity of extracellular catalase after irradiation with red and blue coherent light on the medium with 284 mg/l of nitrogen was 2.6 and 10.0 times higher, respectively, than when cultivated on a medium with higher nitrogen content. The activity of intracellular catalase during cultivation on a medium
with low nitrogen content was 11.1 to 29.4% higher than with higher nitrogen content in all variants of the experiment, including control. It can be assumed that photo-induction of intracellular catalase does not affect the nitrogen metabolism in I. obliquus.
Thus, usinge nutrient media with reduced nitrogen content is advisable to enhance the photo-induced stimulating effect in the production of melanin, intracellular peroxidase, and extracellular catalase, tyrosinase, and polyphenol oxidase.
When cultivating I. obliquus, the parameters and modes of light treatment of the seed mycelium should be adjusted in accordance with the target bioactive components. Regulating the mechanisms of metabolic pathways of bioactive components with light of different wavelength ranges and coherence should be subject to systematic control depending on specific biotechnological problems.
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ВПЛИВ КОШЦЕШТРАЦП АЗОТУ НА ФОТО1ШДУКОВАШУ РОСТОВУ, ЕНЗИМАТИЧНУ АКТИВШСТЬ I СИНТЕЗ МЕЛАШШУ Inonotus obliquus (Ach.:Pers.) Pilât
Н. Л. Поединок1, Т. I. Тугай2, А. В. Тугай2, О. Б. Михайлова2, Н. М. Сергшчук4, А. М. Негрийко5
1ДУ «1нститут xap40B0Ï бмтехнологп
та геномши НАН Украши», Киïв 21нститут мшробмлоги i вiрусологiï iM. Д. К. Заболотного НАН Украши, Киïв Институт ботанiки iM. М. Г. Холодного
НАН Украши, Киïв 4Вiдкритий мiжнародний унiверситет
розвитку людини «Украша», Кшв 51нститут фiзики НАН Украïни, Кшв
E-mail: [email protected]
Метою роботи було вивчити вплив концентраций азоту на фотошдущю ростовоï, ензимноï активносм та синтез меланiну лiкувального гриба Inonotus obliquus (Ach.: Pers.) Pilat. з Ко-лекцiï культур шапинкових грибiв 1нституту ботанiки iM. М. Г. Холодного НАН Украши. Опромшений свгглом низькоï iнтенсивностi рiзноï когерентносм та в рiзних дiапазонах довжин хвиль мщелш культивували в дина-мiчному режимi на глюкозо-пептонному серед-овищi з рiзними концентрацiями загального азоту.
Отримаш результати дослiдження не вияви-ли достовiрного впливу концентрацiï азоту на фотошдуковану стимуляцiю росту I. dbliquus. Збшьшення накопичення бiомаси фотоактиво-ваним в рiзних режимах мiцелieм, порiвняно з неопромiненим, було практично однаковим у вих варiантах дослщу. Встановлено достовiрну залежнiсть фотостимуляцiï синтезу меланшу вiд концентрацiï азоту в середовищь Зниження концентрацiï азоту збшьшуе стимулювальний ефект низькоiнтенсивного лазерного випромь нювання з довжиною хвилi 488 нм бшьш нiж у 2 рази. Використання живильних середовищ зi зниженим вмятом джерела азоту е доцiльним для пiдвишення фотоiндукованого стимулю-вального ефекту за отримання позаклiтинноï каталази, тирозинази i полiфенолоксiдази, вну-трiшньоклiтинноï пероксидази.
Таким чином, параметри культивування I. оbliquus i режими свгглового оброблення по-сiвного мщелт слiд коригувати вiдповiдно до щльових бiологiчно активних компонентiв.
Ключовi слова: Inonotus obliquus, фотошдук-цiя, азот, меланш, каталаза, тирозиназа, полiфенолоксидаза, пероксидаза, ростова актившсть.
ВЛИЯНИЕ КОНЦЕНТРАЦИИ АЗОТА НА ФОТОИНДУЦИРОВАННУЮ РОСТОВУЮ, ЭНЗИМАТИЧЕСКУЮ АКТИВНОСТЬ И СИНТЕЗ МЕЛАНИНА Inonotus obliquus (Ach.:Pers.) Pilat
Н. Л. Поединок1, Т. И. Тугай2,
А. В. Тугай2, О. Б. Михайлова2, Н. Н. Сергийчук4, А. М. Негрийко5
1ГУ «Институт пищевой биотехнологии
и геномики НАН Украины», Киев 2Институт микробиологии и вирусологии им. Д. К. Заболотного НАН Украины, Киев 3Институт ботаники им. Н. Г. Холодного
НАН Украины, Киев 4Открытый международный университет развития человека «Украина», Киев
5Институт физики НАН Украины, Киев
E-mail: [email protected]
Целью работы было изучение влияния концентрации азота на фотоиндуцию ростовой, эн-зиматической активности и синтез меланина лекарственного гриба Inonotus obliquus (Ach.: Pers.) Pilat. из Коллекции культур шляпочных грибов Института ботаники им. Н. Г. Холодного НАН Украины. Облученный светом низкой интенсивности разной когерентности и в разных диапазонах длин волн мицелий культивировали в динамическом режиме на глюкозо-пептонной среде с разными концентрациями общего азота.
Полученные результаты исследования не выявили достоверного влияния концентрации азота на фотоиндуцированную стимуляцию роста I. dbliquus. Увеличение накопления биомассы фотоактивированным в разных режимах мицелием, по сравнению с необлученным, было практически одинаковым во всех вариантах опыта. Установлена достоверная зависимость фотостимуляции синтеза меланина от концентрации азота в среде. Снижение концентрации азота увеличивает стимулирующий эффект низкоинтенсивного лазерного излучения с длиной волны 488 нм более чем в 2 раза. Использование питательных сред с пониженным содержанием азота целесообразно для повышения фотоиндуцированного стимулирующего эффекта при получении внеклеточной каталазы, тирозиназы и полифенолоксидазы, внутриклеточной пероксидазы.
Таким образом, параметры культивирования I. оbliquus и режимы световой обработки посевного мицелия следует корректировать в соответствии целевыми биологически активными компонентами.
Ключевые слова: Inonotus obliquus, фотоиндукция, азот, меланин, каталаза, тирозиназа, полифенолоксидаза, пероксидаза, ростовая активность.
