СОСТОЯНИЕ ИССЛЕДОВАНИЙ В БИОТЕХНОЛОГИИ ЛИШАЙНИКОВ

СОСТОЯНИЕ ИССЛЕДОВАНИЙ В БИОТЕХНОЛОГИИ ЛИШАЙНИКОВ

Научная статья

Теплицкая Л.М.¹, Кириакиди Э.П.^2, *, Семенова Е.Ф.³, Гончаров М.А.⁴

³ ORCID: 0000-0002-7987-6886;

^{1, 2, 3, 4} Медицинская академия им. С.И. Георгиевского Крымского федерального университета им. В.И. Вернадского, Симферополь, Россия

* Корреспондирующий автор (kiriakidi98[at]list.ru)

Аннотация

Проведен контент-анализ научных источников о современном состоянии исследований в биотехнологии лишайников. Природное и хозяйственное значение этих биообъектов многообразно: кормовое, пищевое, ароматическое, медицинское. Лишайники с древних времен применялись в качестве лекарственных средств и включены в фармакопеи различных стран. Однако, лишайники – очень медленно растущие организмы (1-8 мм в год) и для восстановления в естественных условиях произрастания требуется от 10 до 30 лет. Также интерес к лишайникам возрастает в связи с широким использованием индикационного подхода в мониторинговых исследованиях. Поэтому актуальна разработка лихенотехнологий в контролируемых условиях. В настоящее время разработаны методические подходы к культивированию лишайников (роды Usnea, Rhinocarpon, Umbilicara) и их компонентов: фикобионта (роды Nostoc, Chlorella), микобионта (Petrusaria pertiza, Leydea parasema).

Ключевые слова: биотехнология, состояние исследований, использование лишайников, биологически активные вещества, культивирование фикобионта и микобионта.

STATE OF RESEARCH IN LICHEN BIOTECHNOLOGY

Research article

Teplitskaya L.M.¹, Kiriakidi E.P.^2, *, Semenova E.F.³, Goncharov M.A.⁴

³ ORCID: 0000-0002-7987-6886;

^{1, 2, 3, 4} Medical Academy named after S. I. Georgievsky V. I. Vernadsky Crimean Federal University, Simferopol, Russia

* Corresponding author (kiriakidi98[at]list.ru)

Abstract

The content analysis of scientific sources on the current state of research in lichen biotechnology is carried out. The natural and economic significance of these biological objects is diverse: forage, food, aromatic, and medical. Lichens have been used as medicines since ancient times and are included in the pharmacopoeias of various countries. However, lichens are very slow-growing organisms (1-8 mm per year) and it takes from 10 to 30 years to recover in natural conditions of growth. Also, interest in lichens is increasing due to the widespread use of the indicative approach in monitoring studies. Therefore, the development of lichen technologies in controlled conditions is relevant. Currently, methodological approaches to the cultivation of lichens (genus Usnea, Rhinocarpon, Umbilicara) and their components: phycobiont (genus Nostoc, Chlorella), mycobiont (Petrusaria pertiza, Leydea parasema) have been developed.

Keywords: biotechnology, state of research, use of lichens, biologically active substances, cultivation of phycobiont and mycobiont.

Introduction

Lichens are a very interesting and peculiar group of lower plants, whose organism consists of a phycobiont (a single-celled green alga or cyanobacteria) and a mycobiont (usually an ascomycete, less often a basidiomycete), forming a single organism on the basis of symbiosis and, possibly, partial parasitism on the part of the fungus [1]. The natural and economic significance of these biological objects is diverse: forage (Cladonia sp., Cetraria sp.), food (Gyrophora edible or G. esculenta, Aspicilia edible or A. esculenta, Cetraria islandica), aromatic (Evernia prunastri), medical (C. islandica, E. prunastri, Usnea sp. et al.) [2].

Lichens are producers of organic metabolites, many of which have biological activity, in particular, antimicrobial against staphylococci, streptococci, tuberculosis bacillus. In many cases, lichens, due to the peculiarities of their chemical nature, had a positive effect on the patient both as stimulants that raise the tone of the body, and as antibiotics. Since the mid-twentieth century, studies have been conducted on various properties of lichen acids, and formulations of the corresponding drugs have been developed: ewosyna, usniakin, usnin, usnimycena, usno, eihushin, sodul [3].

According to the modern classification, lichens are medicinal plants containing simple C₆-Ce-series phenols that have an antiseptic, anti-burn, regenerating effect. As a medicinal raw material for the production of the sodium salt of usninic acid, which has a phloroglucinic nature, the following lichen species collected during the year on the soil or trunks of various trees and dried layers are used: Cetraria islandica (L.) Ach., C. cucullata (Bell.) Ach., C. nivalis (L.) Ach., Parmelia vagans Nyl. - sem. Parmeliaceae; Cladonia stelldris (Opiz) Pouzar et Vezda = C. alpestris (L.) Rabenh., C. arbus-culata (Wallr.) Flot. = C. sylvatica (L.) Hoffm., C. shapeless C. deformis (L.) Hoffm. - sem. Cladoniaceae; Usnea longissima Ach., U. bearded U. barbata (L.) Weber ex F. H. Wigg. = U. barbata (L.) Wigg. s. l., U. florida (L.) Weber ex F. H. Wigg. = U. florida (L.) G. H. Web. s. L., U. hirta (L.) Weber ex F. H. Wigg. = U. hirta (L.) Hoffm., Alectoria ochroleuca (Hoffm.) A. Massal. = A. ochroleuca (Ehrh.) Nyl., Evernia mesomorpha Nyl. = E. thamnoides (Flot.) Arn., E. esorediosa (Muell. Arg.) Du Rietz., E. prunastri Ach. - sem. Usneaceae [4]. The quality of raw materials of official lichen species in the Russian Federation is regulated by FS 42-766-73, GOST 13727-68, GOST 21565-76.

The raw material contains polysaccharides (about 50 %), consisting mainly of lichenin with a molecular weight of 10000-37000, during acid hydrolysis of which D-glucose is formed. The accompanying substances include bitterness (cetrarin), ascorbic and folic acids, mineral compounds, in particular, calcium oxalate. Other vitamins are produced by lichens (namely phycobionts) in small amounts: biotin (vitamin H), cyanocobalamin (vitamin B₁₂), nicotinic acid (vitamin B₅, or PP), pantothenic acid (vitamin B₃), riboflavin (vitamin B₂), thiamine (vitamin B₁) [5]. Therefore, raw materials and extracts from it are used as an appetite-stimulating, enveloping, anti-inflammatory and homeopathic remedy. The layers are part of the breast and stomach collections (teas), biologically active food additives.

However, lichens are very slow-growing organisms (1-8 mm per year) and it takes from 10 to 30 years to restore the raw material base in natural conditions of growth. Also, interest in lichens is increasing due to their widespread use as bioindicators in monitoring studies. Therefore, it is relevant to develop lichen technologies under controlled conditions that allow to intensify the processes of accumulation of biomass and biologically active compounds produced by phyco-and mycobionts.

Methods

The object of study was the botanical species of lichens of modern ecological and geographical groups and occupying different areas. The main research method is content analysis of patent and scientific literature sources. Also in this work, the original results obtained using morphological, cytological, biochemical, microbiological, biotechnological, and mathematical research methods are systematized and presented.

Results

Lichen Technology as a branch of biotechnology, the biological object of which is lichens and their components, can also be considered a branch of modern botany that studies the morphological, anatomical and physiological features of lichens, the founder of which is the Swedish scientist E. Acharius [6].

In the last 30 years, the study of lichen cultivation under in vitro conditions has been carried out not only in Russia, but also abroad (Japan, Germany, France, Estonia, etc.). The influence of cultivation conditions on the growth of lichen tissue culture has been studied [7]. Lichen thallomas were grown at 20°C in the dark on an agarized medium. Usnea longissima tissues increased by 13 times the initial weight after 12 weeks of cultivation on a Lily-Barnett medium containing 2% mannitol, a yeast extract in the presence of D-asparagine. Interspecific differences of lichens in the composition of sugars and amino acids were found. Exogenous administration of vitamins, phytohormones, and nucleic acid derivatives into the medium did not accelerate tissue growth.

Some Antarctic lichens grown at low temperatures were also studied. At the same time, only three of them - Rhinocarpon flavum, Umbilicara aprina, and U. decunnata - were able to obtain tissue cultures [8].

Fragmentary information is available in the scientific literature on the influence of temperature on the growth rate of lichens in culture in vitro. For comparison, U. nachlinbergii and U. pennaylvanica from Japan were used. All lichens, with the exception of U. aprina, grew better at 15 °C and worse at 5 °C. At the same time, U. aprina grew better at 5 °C. The growth rate of U. decassara is similar to the growth rate of species of this genus from Japan, although they are found in warmer areas than Antarctica [9].

Of great importance at present are studies concerning the cultivation of phyco-and mycobionts, both for the purpose of resynthesis of the original lichen, and for the production of economically valuable metabolites.

In France, the development of pure cultures of mycobionts, in particular, Petrusaria pertiza from asco - and conidiespor, was studied in vitro. On agar, tallom formed multi-lobed vesicles bearing conidia. Some of the mycelium filaments grew away from the bubbles and initiated certain structures. Only mycelial covers were formed on malt agar, and hollow structures were formed on the Lily-Barnett medium. The blades of young Leydea parasema thallomas developed a mycelium that transformed into a thalloma with concentric outgrowths. Cultures of Lobaria laetevirens, L. pulmonaria gave mycelial covers forming a thallom [10].

In Germany, laboratory experiments were conducted on the cultivation of leafy and bushy lichens and their components. The possibility of cultivating the components of leafy lichens on inorganic media was investigated in order to obtain prerequisites for subsequent experiments on resynthesis and to trace the early stages of lichenization, which could not be identified in natural conditions. Both symbionts of Dermacaron niniatum were isolated by cultivation on mineral media [11].

The most common for fixing odors is the resinoid "oak moss", which is obtained from the lichen Evernia prunastri, which grows in the Crimea [8]. The populations of this species are limited and the collection of this plant significantly reduces natural resources. In this regard, the search for new sources of raw materials for obtaining odor fixators is relevant. Biotechnology methods can solve the problem of obtaining resinoid, provide raw materials for the technological process of essential oil production, the pharmaceutical industry, as well as the preservation of natural reserves all year round [12].

Our comparative analysis of potential phycobionts: 73 cultures of blue-green and green algae belonging to the genera Cаlothrix, Cylindrosperum, Anabaena, Nostoc, Spirulina, Chlorella, etc. showed that the amount of synthesized volatile aromatic substances is at the level of 3 mg / L of cultural liquid. It was shown that in a continuous culture of Chlorella vulgaris, Nostoc punktiformei, Spirulina platensis, a large amount of lipids (14.5-81.0 mg / g dry matter) accumulates, a significant part of which is C₁₄-C₃₀ paraffins. The use of the biomass of Chlorella vulgaris, Spirulina platensis, and other microalgae is promising for the production of alcoholic extracts such as the oakmoss resinoid, which is a part of almost all known perfumes as a scent fixer, as well as an independent aromatic and pigment principle. In addition, thick extracts, biologically active food supplements based on unicellular algae can be used for therapeutic and prophylactic purposes [13]. The assessment of the toxicity of pharmaceutical products was carried out: the average lethal concentration LC 50 = 3.86 mg / ml; the average effective concentration of EC 50 = 1.93 mg / ml; LC 100 = 30.8 mg / ml at 48 hour exposure, median survival time LT 50 = 60 min. This gives reason to consider them relatively safe and not showing acute toxicity in the experiment [14].

Conclusion

The conducted content analysis of scientific sources about lichens showed that the economic value of these biological objects is diverse: fodder, food, aromatic, and medical. Since ancient times, lichens have been used as medicines and are included in the pharmacopoeias of various countries (GF RF, BTF, etc.). However, lichens are very slow growing organisms and it takes up to 30 years to restore the raw material base in natural conditions. Therefore, the development of lichen technologies under controlled conditions is relevant. At present, methodological approaches have been developed for the cultivation of lichens (genus Usnea, Rhinocarpon, Umbilicara) and their components: phycobiont (genus Nostoc, Chlorella), mycobiont (Petrusaria pertiza, Leydea parasema). The main biologically active compounds (usnic acid, lichenin, cetrarin, water-soluble vitamins, calcium oxalate) and the pharmacological effects they cause (antimicrobial, appetite-stimulating, enveloping, anti-inflammatory) have also been determined.

Biotechnological approaches can be implemented for species cloning, reproduction, creation of a bank of cell cultures of lichens and their components in order to create collections of producers and develop technologies for obtaining valuable biologically active substances. These in vitro techniques have become in many countries an important component of the strategy for the conservation of the gene pool and the rational use of plant resources.

Конфликт интересов Не указан.

Conflict of Interest None declared.

Список литературы / References

1. Lobakova E.S. Experimental Lichenology / E.S. Lobakova, I.A. Smirnov, 2012. [Electronic resource]. – URL: https://www.intechopen.com/books/advances-in-applied-biotechnology/experimental-lichenology (accessed 12.05.2021)
2. Aftab A. Biotechnological Applications of Lichen / Aftab A., Rizwana K., Shamsul H. et al. // Lichen‐Derived Products: Extraction and Applications / Editor Mohd Y. / Chapter 9. Scrivener Publishing LLC, 2020.
3. Семенова Е.Ф. Фармацевтический словарь-cправочник по биотехнологии / Е.Ф. Семенова, Е.Н. Гаврилова, Е.В. Преснякова. - Пенза: Изд-во ПГУ, 2014. - 147 с.
4. Upreti D. K. Modern Methods and Approaches in Lichen Systematics and Culture Techniques / Upreti D. K., Divakar P. K., Shukla V. et al. // Recent Advances in Lichenology. Springer, 2015. – Vol. 2. - 123 p.
5. Патент № 2603099 РФ Штамм микроводоросли Chlorella Beyerinck. – продуцент смеси душистых соединений, близкой к резиноиду дубового мха / Семенова Е.Ф., Преснякова Е.В., Шпичка А.И., Митишев А.В. (РФ) Заявл. 29.06.2015 (заявка № 2015125893 с датой приоритета изобретения 29.06.2015). Зарегистрировано в Государственном реестре изобретений РФ 28.10.2016. Опубл. 20.11.2016, Бюл. № 32.
6. Теплицкая Л.М. Биотехнология получения резиноида из клеточных культур Evernia prunastri / Л.М. Теплицкая, Е.Ф. Семенова // Материалы III Международной научно-практической конференции «Современные проблемы отечественной медико-биологической и фармацевтической промышленности», Пенза, 21 ноября 2014 г. Пенза: ИИЦ ПГУ, 2014. - С. 160-173.
7. Митишев А.В. Сравнительный анализ штаммов продуцента и инновационного продукта как основных элементов биотехнологии резиноида хлореллы / А.В. Митишев, Е.В. Преснякова, Е.Ф. Семенова и др. // Известия высших учебных заведений. Поволжский регион. Серия «Естественные науки», 2014. - №4 (8). - С. 19-29.
8. Митишев А.В. К вопросу разработки технологии культивирования и переработки Chlоrellа vulgаris для получения резиноида / А.В. Митишев, Е.В. Преснякова, Е.Ф. Семенова // Вестник Воронежского государственного университета. Серия «Химия. Биология. Фармация», 2017. - №4. - С. 60-63.
9. Mitishev A. Nephelometric method for determining parameters of growth of chlorella culture / A. Mitishev, E. Semenova, V. Presnyakova et al. // Materials of the conference " Breakthrough directions of scientific research in MEPhI: prospects for development within the framework of Strategic academic divisions. - Dubai, UAE; Knowledge E, 2018. - P. 206-212.
10. Suzuki M.T. Lichens as natural sources of biotechnologically relevant bacteria / Suzuki M.T., Parrot D., Gabriele Berg et al. // Appl. Microbiol. Biotechnol., 2016. – Vol. 100 (2). - P. 583-595. DOI: 10.1007/s00253-015-7114-z
11. Grimm M. The Lichens’ Microbiota, Still a Mystery? / Grimm M., Grube M., Schiefelbein U. et al. // Front. Microbiol., 2021. DOI: 10.3389/fmicb.2021.623839
12. Semenova E. F. On biotechnology of essential oils based on microbial synthesis / E. F. Semenova, A. I. Shpichka, I. Yu. Moiseeva // European Journal of Natural History, 2012. - No. 4 - P. 29-31
13. Mitishev A.V. Determination of biological activity of chlorella resinoids in relation to Paramecium caudatum / A.V. Mitishev, E. F. Semenova, V. P. Velichko et al. // International Scientific Journal, 2016. - No. 4 (46), Part 5. - Р. 23-27
14. Mitishev A.V. Determination of toxicity of thick chlorella extract / A.V. Mitishev, E. F. Semenova, I. Ya. Moiseeva // Pharmaceutical Journal, 2019. - No. 1. - Р. 98-102

Список литературы на английском языке / References in English

1. Lobakova E.S. Experimental Lichenology / E.S. Lobakova, I.A. Smirnov, 2012. [Electronic resource]. – URL: https://www.intechopen.com/books/advances-in-applied-biotechnology/experimental-lichenology (accessed 12.05.2021)
2. Aftab A. Biotechnological Applications of Lichen / Aftab A., Rizwana K., Shamsul H. et al. // Lichen‐Derived Products: Extraction and Applications / Editor Mohd Y. / Chapter 9. Scrivener Publishing LLC, 2020.
3. Semenova E.F. Pharmatsevticheskiy slovar-spravochnik po biotekhnologii. [Pharmaceutical dictionary-handbook on biotechnology] / E.F. Semenova, E.N. Gavrilova, E.V. Presnyakova. - Penza: Publishing House of PSU, 2014. – 147 p. [in Russian].
4. Upreti D. K. Modern Methods and Approaches in Lichen Systematics and Culture Techniques / Upreti D. K., Divakar P. K., Shukla V. et al. // Recent Advances in Lichenology. Springer, 2015. – Vol. 2. - 123 p.
5. 2603099 Russian Federation. Shtamm mikrovodorosli Chlorella Beyerinck. – producent smesi dushistykh soedinenij, bliskoj k rezinoidu dubovogo mha [Strain of microalgae Chlorella Beyerinck. - producer of a mixture of fragrant compounds close to oak moss resinoid] / Semenova E. F., Presnyakova E. V., Shpichka A. I., Mitishev A.V. (RF) Application 29.06.2015 (No. with the date of priority of the invention 2015125893 application 29.06.2015). Registered in the State Register of Inventions of the Russian Federation 28.10.2016. Publ. 20.11.2016, Bul. No. 32. [in Russian].
6. Teplitskaya L. M. Biotechnologiya polucheniya rezinoida iz kletochnykh kultury Evernia prunastri [Biotechnology of obtaining resinoid from cell culture Evernia prunastri] / L. M. Teplitskaya, E. F. Semenova // Materials for the III International scientific-practical conference "Modern problems of the domestic medical-biological and pharmaceutical industry", Penza, November 21, 2014. Penza: IIC PSU, 2014. - P. 160-173 [in Russian].
7. Mitishev A.V. Sravnitelnyj analis shtammov producenta i innovazionnogo produkta kak osnovnykh elementov biotehnologii rezinoida chlorelly [Comparative analysis of producer and innovative product strains as the main elements of chlorella resinoid biotechnology] / A.V. Mitishev, E. V. Presnyakova, E. F. Semenova et al. // News of higher educational institutions. Volga region. Natural Sciences Series", 2014. - No. 4 (8). – 19-29 [in Russian].
8. Mitishev A.V. K voprosu razrabotki tehnologii kultivirovanija i pererabotki Chlоrellа vulgаris dlya polucheniya rezinoida [On the development of technology for the cultivation and processing of chlorella for the production of resinoid] / A.V. Mitishev, E. V. Presnyakova, E. F. Semenova // Bulletin of the Voronezh State University. Series "Chemistry. Biology. Pharmacy", 2017. - No. 4. - Р. 60-63. [in Russian].
9. Mitishev A. Nephelometric method for determining parameters of growth of chlorella culture / A. Mitishev, E. Semenova, V. Presnyakova et al. // Materials of the conference " Breakthrough directions of scientific research in MEPhI: prospects for development within the framework of Strategic academic divisions. - Dubai, UAE; Knowledge E, 2018. - 206-212.
10. Suzuki M.T. Lichens as natural sources of biotechnologically relevant bacteria / Suzuki M.T., Parrot D., Gabriele Berg et al. // Appl. Microbiol. Biotechnol., 2016. – Vol. 100 (2). - P. 583-595. DOI: 10.1007/s00253-015-7114-z
11. Grimm M. The Lichens’ Microbiota, Still a Mystery? / Grimm M., Grube M., Schiefelbein U. et al. // Front. Microbiol., 2021. DOI: 10.3389/fmicb.2021.623839
12. Semenova E. F. On biotechnology of essential oils based on microbial synthesis / E. F. Semenova, A. I. Shpichka, I. Yu. Moiseeva // European Journal of Natural History, 2012. - No. 4 - P. 29-31
13. Mitishev A.V. Determination of biological activity of chlorella resinoids in relation to Paramecium caudatum / A.V. Mitishev, E. F. Semenova, V. P. Velichko et al. // International Scientific Journal, 2016. - No. 4 (46), Part 5. - Р. 23-27
14. Mitishev A.V. Determination of toxicity of thick chlorella extract / A.V. Mitishev, E. F. Semenova, Ya. Moiseeva // Pharmaceutical Journal, 2019. - No. 1. - Р. 98-102