SCREENING OF CONTENT AND DYNAMIC OF ACCUMULATION OF POLYPHENOLS IN SOME BASIDIOMYCETES SPECIES

Целью работы было изучение общего содержания полифенольных веществ в плодовых телах 50 видов базидиомицетов из которых 27 принадлежат к порядку Polyporales и 23 - порядка Agaricales . Интродуцировано 23 штаммы 8 видов базидиальных грибов. Методы. Собранные дикорастущие карпофоры высушивали и измельчали ​​до размера частиц 0,1±0,01 мм, а исследуемые штаммы культивировали поверхностно в колбах Ерленмейера на стандартной глюкозо-пептонной питательной среде. Определение общего содержания полифенольных веществ проводили в спиртовых вытяжках микологического материала по модифицированной методике Фолина-Чокальтеу. Абсолютно сухую биомассу плодовых тел и мицелия определяли весовым методом. Результаты. Обнаруженные виды трутовых грибов - Ganoderma applanatum , Ganoderma lucidum , Laetiporus sulphureus и Fomes fomentarius и виды агариковых грибов - Stropharia rugosoannulata , Agrocybe cylindracea , Tricholoma flavovirens , Flammulina velutipes , Pleurotus ostreatus и Fistulina hepatica с высоким содержанием полифенольных веществ – более 60 мг / г абсолютно сухой биомассы. Для интродуцированных штаммов установлена динамика роста и накопления полифенольных веществ в мицелии и культуральном фильтрате при ферментации на глюкозо-пептонной среде. Все культуры достигают максимума накопления биомассы на 12-е сутки роста. Здесь самые продуктивные – штаммы Sсhizophyllum commune Sc-1101 и Sc-10 и штаммы F. velutipes F-202. Самые низкие значения накопления абсолютно сухой биомассы зафиксированы для штамма P. ostreatus P-192 и штамма F. fomentarius Ff-09. Установлено, что исследованные культуры имеют индивидуальные значения роста – накопление биомассы в примененных условиях культивирования, что, вероятно, отражает пригодность этих условий для их роста и генотипические особенности. Штаммы в подавляющем большинстве способны к накоплению полифенольных веществ как в мицелии, так и в культуральной жидкости в течение всего срока культивирования. Максимум содержания полифенолов в мицелии для 96%, и в культуральной жидкости - для 91% от общего количества штаммов совпадал с окончанием дванадцятидобового срока их культивирования. Вычисления коэффициента корреляции между содержанием полифенолов в мицелии и культуральной жидкости одновозрастных культур показало, что наблюдается очень высокая положительная корреляция в 73,2%, высокая положительная – в 17,4% и средняя – в 4,5% опытов. Вывод. Отобраны штаммы видов Sсhizophyllum commune , Pleurotus ostreatus , Fistulina hepatica и Laetiporus sulphureus – перспективные для дальнейших исследований с целью получения полифенолов мицелиальными и внеклеточного происхождения. Ключевые слова: полифенолы, базидиомицеты, карпофоры, мицелий, культуральный фильтрат

Introduction. In the last decades an actual problem is searching for new biologically active substances (BAS's) and their producers for the purpose of development and manufacturing application of modern drug and therapeutic products (Запрометов, 1993; Никитина, 2007).
In particular, polyphenol compounds, which are natural antioxidants preventing development of different pathogenic effects in a cell and as a result of different diseases, are the desired substances in different branches of industry and medicine (Запрометов, 1993;Федотов at al., 2012Федотов at al., Asatiani at al., 2010. These include phenolic acids and aldehyde derivatives, substances of polyphenoloxycarbon complex, carotenoids, flavonoids, melanins, tannins, etc. (Asatiani at al., 2010;Wasser, 2010).
It is established that these substances are synthesised by almost all plant and fungi organisms (Никитина, 2007  . However, these studies give a vague idea about the qualitative and quantitative content of polyphenols in higher basidial fungi and mycological material when cultivating them that makes it necessary to carry out further screening operations in this field. The interest for basidiomycetes, including wooddestroying ones, is firstly associated with their ability to synthesise numerous BAS's. Especially while destructing lignin-cellulose complex, they produce antioxidant substances -oxidoreductases, vitamins, polyphenols, free radical blockers et al., which provide adaptive mechanisms for antioxidant protection of xylotrophs (Peyrat-Maillard at al., 2000; Fedotov, 500 Біологічні системи. Т. 5. Вип. 4. 2013 2007; Wasser, 2010). Secondly, mycelium cultures of these organisms are not demanding for a nutrient media composition, most of them are edible and non-poisonous, and can be used in the microbiological production of BAS's.
The aim of this study was to determine total polyphenols in the carpophores and mycelium and in the culture filtrate of some basidiomycetes species.
Materials and methods. The carpophores, mycelium and culture filtrate of 50 macromycetes species, 27 of which belong to order Polyporales and 23 to order Agaricales, division Basidiomycetes were used as the materials for this investigation. To determine total poliphenols, the collected FBs were dried and ground to the particle size of 0.10.01 mm, and the strains under investigation were cultivated superficially in Erlenmeyer flasks of 250 ml in a glucose-peptone growth medium (GPM, рН0 6.5±0.2) of 50 ml with the following composition (g/l): glucose -10.0; peptone -3.0; КН2РО4 -0.6; К2НРО4 -0.4; MgSO4 · 7H2O -0.5; CaCl2 -0.05; ZnSO4 · 7H2O -0.001. 10-day mycelial strain cultures on wort agar were used as inoculums. The incubation temperature was 27.5°С. The cultivation period was 6, 9 and 12 days. Upon completion of the cultivation period, a mycelium was separated from the culture broth by filtration at 51°С. The mycelium obtained was additionally dried a little on filtration paper and cooled down to 10.5°С. The prepared mycelium was homogenized by grinding in a chilled mortar. The ground carpophores (GCs), homogenized mycelium (HM) and culture filtrate (CF) were used for further tests.
Absolutely dry biomass (ADB) of the GCs and mycelium was determined by gravimetric method (Государственная фармакопея, 1987).
The determination of total (W) polyphenols (PPs) was carried out in the alcoholic extracts of the mycological material using modified Folin-Ciocalteu method (Мусиенко at al., 2001) and calculated by the standart formula.
The tests were performed in three replications. Statistical manipulation was done using the programs for statistical manipulation of biological test results. A correlation analysis was performed to determine correlation level between PP concentration in the mycelium and CF of one-year cultures. A difference was considered as significant at confidence level Р>0.95 (Приседський, 1999).
Results and their discussion. Total polyphenols in 225 carpophores of 27 species of Polyporales fungi and in 220 ones of 23 species of Agaricales fungi were evaluated at the first step of investigation ( Table 1).
The analysis of polyphenols in the carpophores of Polyporales fungi showed the following. Most of their FBs (85%) contain little polyphenols being within the range of 9 mg/g (D. quercina) to 39 mg/g (F. pinicola). The other group comprises 3 species of Polyporales fungi (G. lucidum, L. sulphureus and G. applanatum) containing polyphenols in their FBs in the amount of 89 mg/g to 161 mg/g (ADB). The fruit bodies of tinder fungus F. fomentarius have the highest concentration of poliphenols -more than 248 mg/g (ADB). To compare the results obtained, it should be noted that the method of reactant extraction from the carpophores of shelf fungus Inonotus obliquus (Ach. ex Pers.) Pil. with the maximum PP concentration of 140 mg/g (ADB) by water extraction has been patented (Сысоева at al., 2005; Патент 2448721 ).
The analysis of polyphenols in the carpophores of Agaricales fungi showed that most of them (74%) contain little polyphenols in their FBs but it is less compared to Polyporales fungi. Here they are within the range of 12 (P. squarrosa) to 37 mg/g (P. citrinopileatus). 5 species of Agaricales fungi (P. ostreatus, S. rugosoannulata, T. flavovirens, F. velutipes and A. cylindracea) can be put into a group with the medium polyphenol concentration of 53 to 101 mg/g in the fruit bodies. The highest polyphenol concentration of about 172 mg/g was registered in the wild fruit bodies of F. hepatica. However, this parameter is more than 1.5 times lower than the concentration of phenolic substances in the fruit bodies of tinder fungus F. fomentarius. In contrast, it should be noted that the mean concentration of polyphenols amounts to 450 mg/g in the plant raw material of Camellia sinensis, and 70 mg/g in the my- S. rugosoannulata, A. cylindracea, T. flavovirens, F. velutipes, P. ostreatus and F. hepatica having the high concen-tration of these substances of more than 60 mg/g (ADB). The next step of the investigation was to isolate pure cultures from the carpophores as well as to study the growth and synthesis rate of polyphenols of some of them when cultivating in the GPM.  The results of ADB accumulation by the strains while their growing (on the 6 th , 9 th and 12 th day of cultivation) are shown on Figure 1. As can be seen, all cultures reach the maximum of this parameter on the 12 th day of their growth. The most productive strains here are those of S. commune Sc-1101 and Sc-10 and that of F. velutipes F-202. The lowest values of ADB accumulation were registered for P. ostreatus strain P-192 and F. fomentarius strain Ff-09. Thus, the investigated cultures have their individual growth valuesbiomass accumulation in the cultivation conditions used that probably reflects suitability of these conditions for their growth.
It was established that most of strains were able to accumulate polyphenols both in the mycelium and CF during the whole cultivation period. The maximum of PP concentration in the mycelium for 96% and in the CF for 91% of the total strains coincided with the termination of their cultivation period. The investigation results of total polyphenols in the mycelium and culture filtrate while growing some basidiomycetes strains are shown on Figure 2    The trend of polyphenols in the mycelium of the strains under investigation has the following characteristics. The highest concentration of these substances within the range of 107.9 to 129.4 mg/g was registered for P. ostreatus strains Р-039 and Р-208, and S. commune strain Sc-1102 on the 12 th day of their growth. The lowest values of PP concentration of 29.9 to 50.1 mg/g were registered for F. velutipes strains F-1, F-202 and F-03, and P. ostreatus strain P-192 at the end of their cultivation period.
The trend analysis of polyphenols in the culture filtrate of the strains under investigation showed the following. The highest PP concentration was registered on the 12 th day of their growth within the range of 5.4 to 6.8 mg/ml for the strains of F. hepatica Fh-18, L. sulphureus Ls-08 and P. ostreatus Р-01, and the lowest (0.9 to 1.7 mg/ml) -for the strains of F. hepatica Fh-08, P. ostreatus P-192 and F. velutipes F-1.
In all cases the concentration of polyphenols in the mycelium was much higher than the concentration of these substances in the culture filtrate and varied on the 12 th day of cultivation from 11.4 times for L. sulphureus strain Ls-08 to 52.9 times for F hepatica strain Fh-08. A considerable difference between ability of strains to synthesise and accumulate PPs in the mycelium and CF may be most likely explained by their genotype realization under test conditions. The calculation of a correlation coefficient between PP concentration in the mycelium and CF of one-year cultures showed the following. A very high positive correlation is observed in 73.2%, high positive onein 17.4% and medium onein 4.5% of tests.
Thus, the test results of total polyphenols in some basidiomycetes species enable us to make the follow-ing conclusions. The species of tinder fungi -Ganoderma applanatum, Ganoderma lucidum, Laetiporus sulphureus and Fomes fomentarius, and the species of Agaricales fungi -Stropharia rugosoannulata, Agrocybe cylindracea, Tricholoma flavovirens, Flammulina velutipes, Pleurotus ostreatus and Fistulina hepatica are characterized by the highest concentration of polyphenols in their carpophores. Most of introduced strains can accumulate polyphenols both in the mycelium and CF during the whole period of their cultivation. The strains of P. ostreatus Р-01, F. hepatica Fh-18 and L. sulphureus Ls-08 are perspective for further investigations aimed to obtain polyphenols of extracellular origin and the strains of S. commune Sc-1102, and P. ostreatus Р-039 and Р-208those of mycelial one. References: