Laetiporus Sulphureus (Bull.) Murrill - Carotenoid Producer”

VYTAUTAS MAGNUS UNIVERSITY FACULTY OF NATURAL SCIENCES DEPARTMENT OF BIOLOGY

ANASTASIIA SHELEST

“METHODS OF INCREASING BIOSYNTHETIC ACTIVITY OF THE STRAIN LS-0917 LAETIPORUS SULPHUREUS (BULL.) MURRILL - CAROTENOID PRODUCER”

Master Thesis

Molecular biology and biotechnology study program, state code 6211DX012 Molecular biology studies the direction

Supervisor: Prof. Dr. Algimantas Paulauskas ______(signature) (date) Defence: Prof. Dr. Saulius Mickevičius______(signature) (date)

KAUNAS, 2020 TABLE OF CONTENTS

SANTRAUKA...... 4

ABSTRACT...... 5

LIST OF ABBREVIATION...... 6

LIST OF TABLES ……………………………………………………………………….………. 7

LIST OF FIGURES…………………………………………………………..……….………….. 8

INTRODUCTION...... 9

1.LITERATURE REVIEW...... 11

1.1 General characteristics of carotenoids……………….…………………………….. 11 1.2 General characteristics of Basidium fungi ……………...………………………….. 16 1.3 Previous studies of carotenoids content in basidiocarps of some species of Basidiomycetes…………………………………………………………………………….. 16 1.4 Effect of monochromatic irradiation on living organisms…………………………. 19

2.MATERIALS AND METHODS...... 21

3.EXPERIMENTAL PART AND DISCUSSION OF RESULTS………….………………….. 28

3.1 Screening of carotenoid producing strains………………………………………….. 28 3.1.1 Daily increase of mycelium of L. sulphureus strains on PGA……………………. 28 3.1.2 The maximum accumulation of ADB by mycelium with L. sulphureus strains on GPM……………………………………………………………………………. 29 3.1.3 Dynamics of carotenoid pigment increase in mycelia of L. sulphureus strains on GPM……………………………………………………………………………. 29 3.2 Investigation of the effect of laser irradiation on the growth and biosynthetic parameters of L. sulphureus strains……………………………………………………… 31 3.2.1 Daily increase in mycelium of L. sulphureus strains on PGA due to different irradiation spectra……………………………………………………...………. 31 3.2.2 Dynamics of accumulation of carotenoid pigments in mycelia of strains of L. sulphureus on GPM due to different irradiation spectra………………………………. 32 3.2.3 Accumulation dry mycelial biomass by the mycelium of L. sulphureus strains on GPM due to different irradiation spectra…………………………………...……….. 32

3.3 Investigation of the effect of laser irradiation on the growth and biosynthetic parameters of L. sulphureus strains at reduced glucose concentrations…………... 33 3.3.1. Daily increase in mycelium of L. sulphureus strains on PGA due to different irradiation spectra at reduced glucose concentrations………………………….…... 33 3.3.2 Dynamics of carotenoid pigment accumulation in mycelium of strains of L. sulphureus cultivated on GPM at different irradiation spectra at low glucose concentrations……………………………………………………………………...…. 37

3.4 Discussion of results………………………………………………………………. 40

CONCLUSIONS...... 42

REFERENCES...... 44

SUPPLEMENT ……………………………………………………………………….……… 51

SANTRAUKA

Bakalauro darbo autorius: Anastasiia Shelest Bakalauro darbo pavadinimas: Karotinoidų gamintojo - Laetiporus sulphureus (bul.) Murrill linijos Ls-0917 biosintetinio aktyvumo didinimo metodai

Vadovas: Prof. Dr Algimantas Paulauskas

Darbas pristatytas: Vytauto Didžiojo Universitetas, Gamtos mokslų fakultetas,

Puslapių skaičius: 52

Lentelių skaičius: 3

Paveikslų skaičius: 21

Magistro darbe ištirti karotinogenezės augimo tempai, švitinimo poveikis karotenoidų kaupimuisi grybiena, filtrato kultūroje ir absoliučiai sausos biomasės kaupimuisi bei problemos, susijusios su metodų, kaip padidinti kamieno biosentitinį aktyvumą, kaupimasis. Laetiporus sulphureus (Bull.) Murrill - karotinoidų gamintojas. Tikslas buvo pasiektas naudojant skirtingą spektro švitinimą monochromatine LED lazerių šviesa L. sulphureus paderme. Taip pat maistinė terpė buvo optimizuota mažinant gliukozės koncentraciją - taip sumažinant auginimo sąnaudas. Ypatingas dėmesys skiriamas tiriamo objekto (Ls-0917) biosintetiniam aktyvumui.

ABSTRACT

Author of Master Thesis: Anastasiia Shelest Full title of Master Thesis: Methods of increasing biosynthetic activity of the culture Ls-17 Laetiporus sulphureus (Bull.) Murrill - carotenoid producer Supervisor: Prof. Dr Algimantas Paulauskas Presented at: Vytautas Magnus University, Faculty of Natural Science, Kaunas, 2020

Number of pages: 52

Number of tables: 3

Number of pictures: 21

This research investigated the growth rates of carotenogenesis, the effect of irradiation on the accumulation of carotenoids in the mycelia, culture filtrates; the accumulation of absolutely dry biomass, and methods of increasing the biosynthetic activity of the strains Laetiporus sulphureus (Bull.) Murrill - carotenoid producer. The goal was achieved due using different spectra of monochromatic light irradiation by LED lasers on strains L. sulphureus. Also, we optimized nutrient medium due to reducing glucose concentrations. It was a way to decrease the cost of cultivation. Particular attention focused on the biosynthetic activity of the object (Ls-0917) during study.

LIST OF ABBREVIATION

ADB – absolutely dry biomass

CF – culture filtrate

GPM – Glucose-Peptone Medium

PGA – Potato Glucose Agar

WA – Wort Agar

LIST OF TABLES Table 1. The components PGA for surface cultivation of Laetiporus sulphureus Table 2. The composition GPM for surface cultivation of Laetiporus sulphureus Table 3. The concentration of carotenoids in different fungi

LIST OF FIGURES Figure 1. Chemical structures and molecular formulas of α- and β-carotenoids Figure 2. Total content of carotenoids in a wildly growing basidiocarps of Polyporales Figure 3. Total content of carotenoids in basidiocarps of Agaricales Figure 4. The fruiting body of the fungus Laetiporus sulphureus Figure 5. Agar slants with obtained cultures of Ls-0917, Ls-0918 and Ls-0919 Figure 6. The mycelium of Ls-0917 cultivated on PGA in Petri dishes Figure 7. Observed strains cultivated in Erlenmeyer flasks on a GPM Figure 8. Daily growth of L. sulphureus strain in PGA Figure 9. Accumulation dry mycelial biomass from of L. sulphureus in GPM Figure 10. Dynamics of carotenoid pigment accumulation in mycelium of L. sulphureus strains in GPM Figure 11. Daily increase in mycelium of L. sulphureus strains on PGA due different irradiation spectra Figure 12. Dynamics of carotenoid pigment accumulation in mycelium of L. sulphureus strains on GPM due different irradiation spectra Figure 13. The accumulation dry mycelial biomass by of L. sulphureus strains on GPM due different irradiation spectra Figure 14. Daily increase in mycelium of L. sulphureus strains on PGA at different glucose concentrations Figure 15. The average daily increase in mycelium of L. sulphureus strains on PGA due red laser irradiation and at different glucose concentration Figure 16. The average daily increase in mycelium of L. sulphureus strains on PGA due green laser irradiation and at different glucose concentrations Figure 17. The average daily increase in mycelium of L. sulphureus strains on PGA due to blue laser irradiation and at different glucose concentrations Figure 18. Dynamics of accumulation of carotenoid pigments in mycelium of strains of L. sulphureus on GPM at different glucose concentrations Figure 19. Dynamics of carotenoid pigment accumulation in mycelium of L. sulphureus strains on the GPM by irradiation with green spectrum of monochromatic light on media at different glucose concentrations Figure 20. Dynamics of carotenoid pigment accumulation in mycelium of L. sulphureus strains on the GPM by irradiation with red spectrum of monochromatic light on media at different glucose concentrations Figure 21. Dynamics of carotenoid pigment accumulation in mycelium of L. sulphureus strains on the GPM by irradiation with blue spectrum of monochromatic light on media at different glucose concentrations 8

INTRODUCTION

There are many biologically active substances and one of the most popular are carotenoids. They are 40-carbon isoprenoid molecules that produce the orange, red, and yellow colouration found in nature. (Takashi Maoka, 2020). Furthermore, they are the most widely spread pigments in nature and are present in photosynthetic bacteria, some species of archaea and fungi, algae and microalgae, plants, and animals. (Yiguang at al., 2018). Except carotenoid’s role as natural pigments, this biological substances have been important in diet because some of them function as provitamin A, and data has assembled over the last 30 years that they may contribute to decreasing the risk of developing various non-communicable diseases including several types of cancers, can prevent manifestations of atherosclerosis, cardiovascular disease, arthrosis, infections and skin or eye disorders, among others (Britton G., et al., 2011, Krinsky et al., 2004, 2005, Rodriguez-Concepcion et al., 2018). In addition, they have immunomodulatory, anti‐mutagenic, anti-inflammatory and antioxidant biological properties (Gammone at al., 2015, Bee at al., 2019, Bhuvaneswari at al., 2005) Carotenoids create a number of positive effects on the body. But with a chronic lack of these pigments in the daily diet it can occurs many pathological processes in the body (Moore et al., 2011). That is why the creation of biologically active additives, perfumes and cosmetics containing carotenoids, has become so popular and demanded direction for studies (Antonio Meléndez-Martínez at al., 2019) . In the modern world there is an active search for new sources of these biologically active substances (Torregrosa-Crespo at al., 2018). Fungi are increasing importance in modern nutrition and medicine, due to the presence of metabolites with pharmacological potential in their mycelia (Ribeiro at al., 2011). Among new sources an important place occupied basidial fungi. Most of the basidiomycetes are unpretentious in the composition of the nutrient media, they are edible and non- toxic, which justifies their involvement in microbiological production (Wasser, 2010, Bukhalo, 1988). Carotenoids content was investigated in the fruiting bodies of higher basidial fungi of the Genera Hygrophorus, Fistulina, Cantharellus, Boletus, Suillus etc (Ribeiro at al., 2011). However, the available data give an insufficiently formed picture of the qualitative and quantitative content of carotenoids in basidiomycetes, their mycelia, and culture filtrates during their cultivation. That's why further screening works are necessitates in this direction. (Veligodska, 2012). Among medicinal mushrooms, one of the most perspective fungus is the basidiomycete L. sulphureus, a producer of carotenoids (Velygodska at al., 2016). This fungus can be widely used to obtain preparations with antioxidant protection (Becker, 1988).

MAIN GOAL OF THE WORK is to study the total content of carotenoids in mycelium and culture filtrate of the fungus L. sulphureus and to find ways to improve carotenoid biosynthesis. To achieve this goal, we are going to perform following TASKS: 1. To investigate daily growth rates and average radial growth rate. 2. To study indicators of accumulation of absolutely dry biomass under the conditions of surface cultivation on Glucose-Peptone Medium. 3. To investigate the dynamics carotenoid’s biosynthesis increase in mycelia and culture filtrates and determine the most productive strain. 4. To investigate the effects of monochromatic light radiation on daily radial growth of mycelium, absolutely dry biomass accumulation and the carotenoid’s increase in mycelia and culture filtrates of the most productive strain. 5. To increase the biosynthetic activity of the most productive strain by laser irradiation and optimize the nutrient medium by reducing the glucose concentration simultaneously. Objects of the study: processes of carotinogenesis of L. sulphureus in conditions of surface cultivation. Subject of study: peculiar properties of regulation of carotenogenesis of L. sulphureus with the possibility of further application as a perspective object of biotechnology. Research methods: biotechnological, mycological, physico-biochemical and statistical. Cultivation and biosynthetic characteristics of strains; spectrophotometric determination of carotenoids. Scientific novelty of the obtained results: carotenoids content in cultures of basidium fungus strains (L. sulphureus: Ls-0917, Ls-0918, Ls-0919) was established for the first time. Based on the obtained data, strains - perspective producers of carotenoids. The effect of laser irradiation on the processes of carotenogenesis of L. sulphureus strains was first studied and optimized the nutrient medium by reducing glucose concentration and laser irradiation were performed. The practical significance of the obtained results: - to identify the most productive strain of of L. sulphureus and to use it as a promising biotechnology object for the secretion of carotenoids; - to investigate the influence of laser irradiation that can increase the amount of target product; - to optimize the nutrient medium by reducing glucose concentration that can reduce economic costs. Master's work was carried out in the scientific laboratories of the Department of Plant Physiology and Biochemistry of Vasyl' Stus Donetsk National University in 2019.

1.LITERATURE REVIEW

1.1 General characteristics of carotenoids

Carotenoids form one of the most important classes of plant pigments and take a place in defining colour parameters of fruit and vegetables (Van den Berg, at al., 2016). These natural pigments are responsible for dying plant leaves, fruits, and flowers. Also, carotenoids present in bird’s plumage, fish squama, crustacean’s and insect’s exoskeletons in the orange, red, and yellow tones. There are few well-known examples of carotenoid coloration. At first, it is the orange colour of carrots and some citrus fruits and another illustration could be the red colour of peppers and tomatoes (Pfander, 1992). Mostly, they are found in plants, algae, microalgae and photosynthetic bacteria, where carotenoids, as well as chlorophyll, play important role in the photosynthetic process. Also, they are presented in some non-photosynthetic bacterias, fungi, yeasts, and molds, where these pigments may carry out a protective function against damage by light and oxygen (Ong, at al., 1992, Britton, 1995). More than 600 carotenoids are known, but only about 50 of them are related to provitamin A production (Paliwal at al., 2016). It should be noted that animals cannot synthesise carotenoids, so they need some source of these substances. Usually, they get sufficient consumption with food. But, if there are not enough carotenoids in someone's diet, it could cause different pathologies in their organisms (Paul, at al., 2004). Carotenoids can be broken down into multiple categories, including carotenes and xanthophylls. Carotenes are typically orange, red and yellow pigments. These pigments are derivatives of cyclic or acyclic isoprenoids that play diverse roles in nature. Carotenoids are readily soluble in many organic solvents: hydrogen sulphide, dichloroethane, chloroform, isopropanol, benzene and boiling petroleum ether, in hexane, ethanol (Kapitanov at al., 2016). Particularly, most of the carotenoids are tetraterpenoids, including 40 carbon atoms. And these atoms are originated from eight isoprene molecules. (Harrison at al., 2016). The carotenoid molecule has a polyene chain consisted of four isoprene units at the base with cyclohexene or other aliphatic residues at the end (Figure 1).

Figure 1. Chemical structures and molecular formulas of α- and β-carotenoids (Fiedor at al., 2014))

Carotenoids take a great important place in nature. They contain the typical cyclohexene cycle in two isomeric forms - α- and β - ionic structures (Figure 1). The substituents X and X 'in the carotenoid molecules may be different, for example, α-, γ- and δ-carotene contain two different terminal fragments. The highest biological activity of such combinations is β-carotene, which contains two β-ion fragments. All such compounds are hydrocarbons and are attributed to the carotene group. Synthetic β-apo-8'-carotinal, β-apo-12'-carotinal and ethyl ester of β-apo-8'-carotene are widely used as pigments in the food industry. Enzymatic transformation of β-carotene is important for animals and humans. It mainly occurs in the mucous membrane of the small intestine. Among all types of carotenoids, many biologists and physicians focus their attention on β-carotene because it is possible to divide one molecule of β-carotene into two equal parts that are vitamins A. The central double bond of the β- carotene wform an unstable four-carbohydrate heterocycle that, which decomposes to form two molecules of retinal. This process relates to a dioxygenase reaction, and the corresponding enzyme was isolated from the intestinal mucosa of some mammals. It has been proved that carotenoidase belongs to thiol enzymes (participation in the reaction of SH-groups) and depends on iron ions. Similarly, for this reaction, it is necessary to have a minimal presence of bile (Brody at al., 1989). However, the real mechanism for the conversion of β-carotene to retinal and retinol is complicated, and involves oxidative degradation starting from one end of the molecule through the formation of apo-carotinals (Rao at al., 2007). Carotenoid biosynthesis in the early stages involves chain elongation by the addition of isoprenoid retinol - vitamin A fragments to the C20 framework, whose dimerization leads to phytoin formation (containing 4 double bonds less than lycopene). The next transformations can be combined into a simplified scheme, the following processes occur: 1) increasing of unsaturation - formation of unsaturated C = C-bond; 2) cyclization of the final fragment; 12

3) hydroxylation at position 3 (or 3 ') of the cyclohexane moiety; 4) epoxidation (usually double bonded at position 5); 5) rearrangement of epoxy compounds in furanoxide structure: Hydroxy derivatives are synthesized in the hydroxylation of the terminal fragments, which together with the keto and various epoxy or furanoxy derivatives form a huge group of carotenoids, called xanthophylls, more common than the carotene of the flora. Accordingly, the introduction of oxygen into the cyclohexene moiety usually deprives the carotenoid of provitamin activity (Baker at al., 1999). Carotenoids that plays a role as forerunners of vitamin A are characterized by their structure ring of β-ionone (3,4-dehydroionone), connected with an aliphatic chain that contents conjugated double bonds. Such carotenoids as β-carotene, α-carotene, γ-carotene, and cryptoxanthin are most valuable. The researchers established that they can use some carotenoid functions and properties such as: non-harmless food coloring (Е160), which gives tones to the food; therapeutic and prophylactic properties; which protects the body from ionizing radiation, chemical carcinogens and other harmful factors; antioxidant function, which neutralizes the active radicals that are formed in the body, and also helps to prolong the shelf life of food. The main raw materials for carotene are carrots, pumpkins, sea buckthorns, alfalfa, but the current advances in biotechnology make it possible to solve the large problems of carotenoids production from other sources. Such sources could be presented by unicellular algae, mycelium fungi, bacteria (Veligodska, 2012). Qualitative and quantitative analysis of carotenoids can be determined by the intensity of the maximum light absorption in the blue-violet region of the spectrum by photocolorimetric or spectrophotometric methods, as well as by chromatography. Carotene is used as a food additive for coloring butter, margarine, cheese, mayonnaise, yoghurts, condensed milk, pastry and flour products, macaroni, bakery products. Also, it is used in small quantities as a vitamin supplement. Expanding the scope of natural carotenoids and expanding the range of existing β-carotene products requires increased volumes and improved industrial production of carotene from vegetable raw materials grown in world. Photosynthesis is the most important function of many pigments in plants. Typically, in the first stage of photosynthesis, chlorophyll molecules act as antennas that absorb light energy, and then, in subsequent steps, play the role of energy transmitters. Another important function of plant pigments is photosensitivity. Plants absorb energy of light and "capture" almost all visible light, as well as some of ultraviolet and infrared spectra using pigments. Much of this energy is spent on chemical processes in plants, as well as spent on heating. This is very important because the rate of chemical reactions is temperature dependent. A very important function performed by carotenoids is 13 the neutralization of free radicals that disrupts the flow of biochemical processes in plants (Manuel at al., 2017). The first and most active way to use carotenoids was to use them for dyeing fabrics, furniture, and creating paints. Subsequently, these dyes were replaced by more resistant synthetic paints. Plant pigments are non-toxic and natural, so they are mostly used to color the most popular foods. There is no harm, especially if it is related to food (Milne, 2005). Another direction of pigment using is valuable in pharmacology. According to the literature, carotenoids slow down aging, inhibit the growth of cancers, regulate the permeability of blood vessels, accelerate their recovery, have anti-inflammatory action (in such medications as Kaleflon, Flacarbin, Flamin and many others). Chlorophylls possess strong antiseptic, antibacterial properties, they limit the growth of several kinds of bacteria not by directly destroying them, but by providing an environment which stops their growth. That makes it possible to use chlorophylls in the manufacture of the antibacterial drug chlorophyllipt. Some of the plant pigments are precursors of vitamins. One of the most important and common precursor of vitamin A is carotene. It plays an important role in the mechanism of vision as a natural antioxidant. Also, it stimulates the function of the reproductive glands, increases the immune system, protects against photodermatosis. During consuming a large amount of carotenoids, hypervitaminosis was not observed (Britton et al., 2011, Krinsky et al., 2004, 2005, Rodriguez-Concepcion et al., 2018). Carotenoids take a special place for maintaining the health of the visual organ. Nature has awarded a humanity with an extraordinary gif: color vision. Under the influence of absorbed light, carotenoid molecules change their shape, that affects biochemical processes that play a role in color perception (Nancy E Moran et al., 2018). Beta-carotene (provitamin A) is important for maintaining good vision. Vitamin A is best known as a micronutrient, essential for maintaining vision function. Retinol is a part of the rhodopsins, a light-sensing protein sensor. Various forms of rhodopsin are found both in retinal cones and rods. Cones are retinal photoreceptors that provide color vision and rods are responsible for highly sensitive black and white vision, including twilight vision. In addition, vitamin A also promotes moisturizing of the eyes. Vitamin A deficiency causes a disorder of visual adaptation in the dark or twilight vision ("chicken blindness"). Night blindness or inability to see clearly in dim light is a major symptom of vitamin A deficiency. Symptoms of vitamin A deficiency could be presented with an itch or burning in the eyes, slight redness. One of the very early markers of decreasing of vitamin A is the accumulation of mucus in the inner corners of the eyes. Vitamin A deficiency can cause xerophthalmia, softening and ulcer formation, even up to the rupture of the cornea. Vitamin A and B-carotene are used in the treatment of numerous diseases of the organ of vision: keratitis, xerosis, keratomalacia, eye burns, 14 blepharitis, recurrent barley, retinal degeneration, optic atrophy, corneal diseases. The pathogenetic mechanism that causes the damage of eyes is based on the oxidation increase depended on influence of adverse effects of environmental factors and destructive effects of blue light, which leads to the depletion of antioxidant protection in the end (Tanumihardjo et al., 2016). It should be mentioned that carotenoids have a positive influence on the cardiovascular system. It was noticed after Mediterranean diet surveys. One of the main features of the Mediterranean diet is increased daily consumption of vegetables, fruits, whole grains, and healthy fats. For example, tomatoes are very popular in the Mediterranean zone. Greeks eat averagely 2 tomatoes per day that is 4 times more than Americans and most of Europeans do. The study of the chemical composition of the tomato showed that it is rich with carotenoids. In recent years, intensive studies of such carotenoid as phytoin have been carried out in several countries. Results have shown high therapeutic properties of this carotenoid (Tanumihardjo et al., 2019). Carotenoids can be absorbed in the intestine without biotransformation. They include in the composition of lipoproteins and transport to adipose tissue, liver, adrenal glands, ovaries and other organs (Nancy E Moran et al., 2018). Free radical modification of molecules and components of biological membranes is a common component of the vast majority of biochemical processes that involve carotenoids. In living systems, Red-Ox reactions are carried out by one-electron transfer and form free radicals. (Halliwel B., et al., 1995). The reasons for the free radical increase in the pro- and eukaryotic cells may be ionizing radiation, products of activation of macrophages (in animals), products of metabolism of some drugs, disturbances in the work of the antioxidant system of the organism. Any organic substrates, including amino acids, proteins, lipids, carbohydrates, DNA and RNA, are exposed to this oxidative action (Halliwel et al., 2015). Lipid molecules, which have radicals of easily oxidised unsaturated fatty acids, are often targeted with oxidative action. It should be noticed that lipid peroxidation is one of the most common free radical processes in the body. Carotenoids neutralize peroxide radicals and prevent the peroxidation of lipid components of cell membranes. Synergism of the antioxidant action of carotenoids, together with other fat-soluble antioxidants such as α- tocopherol and coenzyme Q10, was revealed. (Sy et al., 2015). The antioxidant properties of many carotenoids provide them radioprotective, antimutagenic, immunomodulatory, anti-infective, anticarcinogenic properties. Recent studies have shown that astaxanthin, 3,3'-dihydroxy-β, β'-carotene-4,4-dione, synthesized by the marine microalga Haematococcus pluvialis Flotow are more effective antioxidants than such known free radical scavengers such as α-tocopherol (vitamin E), β-carotene, lycopene, lutein and others. Another important finding was the identification of a specific role of lutein and zeaxanthin (dihydroxy derivatives of α- and β-carotene, respectively) in preventing age-related vision loss. Lycopene is a 15 bright red carotenoid found in tomatoes, products of their processing and other red fruits and vegetables. A series of studies on the biological activity of lycopene had shown an inverse correlation between the consumption of the carotenoid and cancer manifestations. (Md. Mahfuzur R. Shah et al., 2016). Considering all the beneficial properties of carotenoids, further exploration and the search for new objects to excretion these natural pigments are extremely important and promising direction in the fields connected with biotechnology, pharmacy and medicine.

1.2 General characteristics of Basidium fungi

Basidiomycota, large and various kind of fungi (kingdom Fungi). It includes several numbers of species such as jelly and shelf fungi; certain yeasts; mushrooms, puffballs, and stinkhorns; and the rusts and smuts. Basidiomycota are typically filamentous fungi formed of hyphae. Most representatives of this group reproduce with a club-shaped spore-bearing organ (basidium) that usually produces four spores (basidiospores). Earlier the Basidiomycota were called Basidiomycetes. The main feature of this group is the structure that is characterized by the fruiting bodies presence in their composition. These fungi are usually called "mushrooms" in daily life. Most Basidiomycota have single-celled basidia (holobasidia), but sometimes, basidia could be multicellular (a phragmobasidia). (Britton, 1983). Compared to the number of other groups of living organisms such as bacteria, plants, and animals, fungi including species Basidiomycetes and the asexual Deuteromycetes produce high levels of carotenoids. Some other biological groups of organisms that can produce such high concentrations of carotenoid in their biomass are microalgae such as Dunaliella and Haematococcus species and related green microalgae. However, an opposition to microalgae, fungi can be intensively cultured heterotrophically in fermentors to achieve high biomass and product yields. Taking into account the fact of close phylogenetic relation of certain fungi to humans, fungi could serve as excellent model systems to study the roles of carotenoids in health and aging (Echavarri- Erasun at. al,. 2002).

1.3 Previous studies of carotenoids content in basidiocarps of some species of Basidiomycetes

Previous studies evaluated the total carotenoids content in 225 carpophores of 27 polypolar basidiomycetes species and in 220 carpophores of 23 agaric basidiomycetes species. Generalized results of this study are presented in Supplement No. 1, which also provides data on the systematic position of fungi and the number of tested basidiocarps samples (Velygodska at al., 2016).

Phellinus pomaceus 1,9 Phellinus igniarius 3,72 Inonotus obliquus 2,05 Ganoderma lucidum 8,9 Ganoderma applanatum 55,04 Laetiporus sulphureus 50,14 Polyporus squamosus 2,3 Piptoporus betulinus 1,5 Daedalea quercina 0,9 Fomitopsis pinicola 0,9 Heterobasidion annosum 1,33 Fomes fomentarius 5,83 Trametes zonatus 0,64 Trametes versicolor 0,61

Species Species Trametes campestris 2,01 Trametes squalens 1,5 Hydnum ochraceum 1 Amyloporia lenis 1,5 Irpex lacteus 2,67 Tyromyces undosus 1,03 Tyromyces revolutus 1,2 Tyromyces lacteus 1,6 Fibuloporia mollusca 1,05 Sparassis crispa 0,25 Chaetoporus ambiquus 0,1 Laeticorticium roseum 0,7 Auricularia auricula-judae 0,85

0 10 20 30 40 50 60 70 Carotenoid content, mg / g

Figure 2. Total content of carotenoids in a wildly growing basidiocarps of Polyporales

Tricholoma sejunctum * 3,14 Tricholoma flavovirens * 9,35 Lyophyllum connatum * 2,04 Lyophyllum loricatum * 2,13 Stropharia rugosoannulata ** 5,95 Stropharia aeruginosa * 3,25 Sсhizophyllum commune * 0,1 Pholiota squarrosa * 1,2 Pholiota aurivella * 1,8 Kuehneromyces mutabilis * 4,88 Pleurotus ostreatus var.Florida ** 5,3 Pleurotus ostreatus ** 5,56 Pleurotus ostreatus * 0,94

Pleurotus eryngii ** 1,5 Species Species Pleurotus citrinopileatus ** 3,75 Marasmius oreades * 3,7 Lentinus edodes ** 0,81 Flammulina velutipes ** 6,5 Flammulina velutipes * 25,28 Fistulina hepatica * 40,74 Coprinus micaceus * 2,5 Coprinus comatus * 2,5 Agrocybe cylindracea ** 16,1 Agaricus campestris * 2,34 Agaricus bisporus ** 4,15 Agaricus arvensis * 2,45

0 5 10 15 20 25 30 35 40 45 Carotenoid content, mg / g

Figure 3. Total content of carotenoids in basidiocarps of Agaricales "*" is a wildly growing basidiocarps, "**" is a commercial basidiocarps

As can be seen from the obtained data (Figure 2, 3), there are significant variations in the total carotenoids content in samples of fungal fruiting bodies in some species (eg, F. velutipes and P. ostreatus). In addition, there are significant variations in the total carotenoids content in samples of fruiting bodies in different species. These outcomes show the necessity of further findings the biosynthetically productive strains. The highest content of carotenoids have displayed by fruiting bodies of G. applanatum and L. sulphureus with 50.14 mg/g and 55.04 mg/g of absolutely dry biomass. The highest total content of carotenoids from 16.10 to 40.74 mg/g was detected in the fruiting bodies of 3 species such as A. cylindracea, F. velutipes and F. hepatica. However, these indicators are in 1.4 times lower than the content of carotenoids in the fruiting bodies of L. sulphureus. It should be noted, the average carotenoid content of Blakeslea trispora mycelium is 18–36 mg/g in absolutely dry biomass (Gessler at al., 2003). The obtained data shows that wild-breed L. sulphureus could become a perspective source of carotenoids.

1.4 Effect of monochromatic irradiation on living organisms

One of the important factors required for the fungal proper growth and development is light. It is an environmentally friendly growth factor and has a significant effect on the life of fungi. It is known that different types of mushrooms respond individually to the effect of light of various spectral compositions. Although, fungi are not phototrophic organisms, and light plays an important role in regulating their vital functions. The nature of the light influence depends on its spectral characteristics and duration of illumination (Kamada et al., 2010). As of now, it has been proven that fungi can perceive not only blue, but also near-UV, green, and red light. It is possible because of using up to 11 different photoreceptors (Herrera–Estrella et al., 2007; Zhenzhong et al., 2018). Basidial macromycetes Coprinopsis cinerea, Lentinula edodes and Pleurotus ostreatus contain genes encoding receptors responsible for the perception of blue light. The study of the genome of these fungi also revealed photoreceptor genes that encode proteins sensitive to red light (Galagan et al., 2003; Kamada et al., 2010). Sensitivity to a red light is realized through phytochrome - a protein molecule that causes morphogenetic responses of various organisms to light. Sensitivity to the rays of the blue part of the spectrum is provided by a photoreceptor based on flavin. A green light is perceived by retinal-based opsin systems, which biological functions is still unclear (Zhenzhong et al., 2018).

The use of artificial light for the stimulation of biological processes for fungal growth is a currently limited. This limitation is due to methods that require long-term illumination of cultures at different stages of morphogenesis. It leads to additional energy expenditure. However, studies conducted by T.Y. Karu showed that short-term (within a few seconds) irradiation of various objects with low-intensity laser light of a certain wavelength in relatively small doses (102–103 J / m2) cause the effects that stay for a long time. (Karu, 1986; Karu, 2008). At present, the influence of low-intensity light on the linear growth and accumulation of biomass is known for several species of micromycetes: Agaricus bisporus, Ganoderma lucidium, Hericium erinaceus, Inonotus obliquus, Lentinula edodes and others. (Poyedinok et al., 2000, 2003; Poyedinok, 2001; Poedinok, Bisko, 2005). So far, the positive effect of UV and γ-irradiation on the yield of the fungus P. ostreatus have studied . So, we know that irradiation with laser light with a wavelength of 632.8 nm at doses of 45–230 mJ/cm2 stimulates spore germination and mycelial growth in Hericium erinaceus (Poedinok, 2013). According to N.L Poyedinok, basidial macromycetes at different stages of ontogenesis are sensitive to low-intensity light in the visible wavelength range with different spectral and energy characteristics (Poedinok et al., 2015). In addition, it was studied that changes in the growth activity of spores and vegetative mycelium of fungi, which are caused by short-term influence by low- intensity light, are transmitted to next phases of ontogenesis and do not demand further activation by light (Poyedinok et al., 2000). According to the literature of fungal photoreception, we can conclude that the feasibility of using of light for regulation the morphogenesis and biological activity of fungi can be the basis for creating more effective technologies for their cultivation. It should be noted that the use of helium-neon and argon lasers, which have large dimensions and considerable energy intensity, complicates the technology of stimulating the growth and development of fungi. We used LED lasers that in comparing to helium-neon and argon lasers have higher efficiency (up to 50%), speed (up to 10-11 seconds), convenience of excitation and small dimensions. In addition, they intensify the metabolic processes of macromycetes is much more efficiently (Vasiura, 1998).Other their advantages are low cost and small amount of required energy. According to the literature, the understanding of LED lasers effects on fungal growth parameters is insufficient, so this issue needs further investigation. In view of this, the aim of our work was to investigate the effect of irradiation of LED lasers on growth parameters, fruiting time and yield of L. sulphureus fungi during solid phase cultivation on PGA with different glucose concentration. Therefore, research that studies effects of the light spectral composition on mycological objects has environmental importance. 20

2.MATERIALS AND METHODS

Materials of the study were based on strains of fungus Laetiporus sulphureus. The mycelia of the three strains of L. sulphureus (Bull.) Murrill Ls-0917, Ls-0918 and Ls-0919 were isolated from wild fruiting bodies (Figure 4). Carpophores were collected within the Vinnytsia city limits (Ukraine). Strains were obtained by the conventional method of isolating pure cultures of basidiomycetes from wild fruit bodies (Bisko at al., 1983). Ls-0917, Ls-0918 and Ls-0919 were found growing on deciduous tree Fraxinus lanceolata. Fruit bodies grown at a height of 1 m above the soil surface. These fungi were located on the wood in groups. Each group were composed with several fungi that superimposed one on top of another. Form of fruit bodies looked like fan-shaped outgrowths of pseudo caps. Each of them had a size of 200-500 mm. Hymenophore had tubular shape with small pores with yellow colour. The pulp was soft and fleshy with a yellowish tone. The systematic position of basidiomycetes research has been established according to scientific literature (Kirk at al., 2001).

Figure 4. The fruiting body of the fungus Laetiporus sulphureus

Scientific classification Kingdom: Fungi Division: Basidiomycota Class: Agaricomycetes Subclass: Agaricomycetidae Order: Polyporales Family: Polyporaceae Genus: Laetiporus Species: L. sulphureus For the purpose to obtain pure culture, carpophores were washed, dried and milled to a particle size of 0.5 ± 0.1 mm. For studying the culture and morphological characteristics of strains, they were cultured on Wort Agar (WA) in tube slants and Potato Glucose Agar (PGA) in Petri dishes, and on liquid Glucose-Peptone Medium (GPM) in Erlenmeyer flasks. The prepared nutrient mediums were sterilized in an autoclave at 126 ± 2 ° C for 45 minutes. The inoculum was presented be a 10-day mycelial cultures of strains grown on WA. Cultivation period lasted 10, 15 and 20 days at a temperature of 27.5 ° C. The optimal cultivation regime was determined experimentally. The isolation of pure cultures of basidiomycetes was carried out by seeding a sterile slice (5 × 5 mm) of vegetative mycelium on nutrient medium. Cultivation of fungus included several stages: 1. Finding of wild fungus. 2. Preparation nutrient medium. 3. Isolation and obtaining pure cultures on agar nutrient media. The cultivation of fungi for the purpose of biomass accumulation or to determine the products of metabolism we need contained following steps: 1. Presentation of a pure culture. 2. Preparation of standard seed material. 3. Determination of conditions that are necessary for the growth of fungi and the manifestation of their synthetic activity. Method of isolation pure cultures. Fungi were isolated in a pure culture and observed in vitro. The isolation of pure cultures makes it possible: ● to determine the nature of growth and spore formation in fungi, morphogenesis features; ● to identify fruiting bodies and spore-bearing species; 22

● to establish the ratio of fungi to environmental factors (temperature, humidity, light sources, acidic environment, radiation, substrate components); ● to determine the biosynthetic activity of metabolic products (enzymes, growth regulators, vitamins) fungi; ● to identify the ratio of fungi to fungicides, drugs; ● to conduct a comparative analysis of isolates of species of fungi; ● to conduct population studies; ● to establish the relationship of fungi between each other; ● to identify the degree of parasitism, etc. Specifically for our research, we performed the method of isolation pure cultures for evaluating the fungal growth dependence on environmental factors (temperature, light sources) and describe morphogenesis features. Pure cultures of the fungi were obtained by transferring them to agar slants. Fungal pure culture is obtained by inoculating a single piece of mycelium on the agar medium in tube slants. They were placed in an incubator for a couple of days at a temperature of 27 ± 1 °С. After this term, the agar plates and slants shown the fungal culture growth (Figure 5).

Figure 5. Agar slants with obtained cultures of Ls-0917, Ls-0918 and Ls-0919

For determination the linear growth, strains were cultivated on PGA in Petri dishes. Composition of PGA is following: ● potato broth – 200,0 ml/liter; ● agar-agar – 20 gm/liter; ● glucose – 20 gm/liter.

The mycelia were spread on agar medium equally. According to the classification of J. Stalpers, presented mycelia were defined as a powder type (Dudka at al., 1982). The colonies of the fungus had a round shape, with higher density in the centre (Figure 6.A.). For assessment of the linear growth, we measured the diameter of each colony (from the center to the edge of the mycelial growth zone) in Petri dishes (Figure 6.B.). They were measured every day since third to ninth day of growth. For this aim the studied fungi were sown in the centre of a dense nutrient medium surface (Becker, 1988). The diameter of the colony was measured in two mutually perpendicular directions. The number of measurements depended on the growth rate of the fungus. Measurements were not carried out if fungus overgrown a Petri dish. To assess the growth of fungal cultures, we used a formula based on the measuring the radius increase of the colonies during the cultivation.

The radial growth rate (Vr) is calculated by the formula (Buhalo, 1988): 푎 − 푏 푉푟 = , 푡1 − 푡0 where: a - the radius of the colony at the end of growth, mm; b - the radius of the colony at the beginning of the phase of linear growth, mm; t1 – t0 - duration of the linear growth, days.

Figure 6. The mycelium of Ls-0917 cultivated on PGA in Petri dishes. A. Surface of the strain. B. Measurement of growth rate of the fungus

Composition of GPM is following: ● Glucose – 10,0 gm/liter; ● peptone – 3,0 gm/liter;

● КН2РО4 – 0,6 gm/liter;

● К2НРО4 – 0,4 gm/liter;

● MgSO4x7H2O – 0,5 gm/liter;

● CaCl2 – 0,05 gm/liter;

● ZnSO4 x7H2O – 0,001 gm/liter (Voloshko et al., 2011, Dudka at al., 1982). The test strains were cultivated on the surface in Erlenmeyer flasks with a capacity of 100 ml on a GPM with pH of 6.5 ± 0.2 units (Figure 7). The hydrogen index was set by potentiometric method, for example, with pH meter "pH-150MI". Erlenmeyer flasks contain 50 ml of medium.

Figure 7. Observed strains cultivated in Erlenmeyer flasks on a GPM

At the end of the cultivation period, the mycelium was separated from the culture fluid by filtration to obtain culture filtrate (CF). Obtained mycelium was additionally dried on filter paper. The prepared mycelium was homogenized by grinding in a mortar with the gradual addition of the extractant. It performed for obtaining a mycelial homogenate. Accumulation of ADB of mycelium was determined by weight method. The mycelium was dried in calibrated boxes at 105 ° C to permanent weight and weighed for this purpose (Dudka at al., 1982). Determination of carotenoid content. For determination the total carotenoid content, the mycelium was homogenized by grinding in a sterile mortar and extracted with ethanol 96% in a 1:10 ratio (Saleba et al., 2016). The mixture was centrifuged at 2000 g for 10 minutes. The amount of carotenoids was determined per unit mass, g in mycelium and per unit volume, cm3 in the CF by spectrophotometric method and was calculated according to Wettstein method (1957). For this aim, we measured absorbance at 3 wavelengths of 440, 644 and 662 nm using spectrophotometer. We determined concentrations of the pigment fractions (Chl a, Chl b and carotenoids) as mg ml-1 using the following equations. Chlorophyll content is calculated by the formula: Ca (mg/L) = 9.784 x E662 – 0.990 x E644 (1), Cb (mg/L) = 21.426 x E644 – 4.650 x E662 (2), C(a+b)(mg/L) = 5.134 x E662 + 20.436 x E644 (3),

Carotenoids content is calculated by the formula: Carotenoids (mg/L) = 4.695 x E440 – 0.268 x C(a+b) (4), where: E662, E644 and E440 are the results of measuring chlorophyll color at wavelengths of 662 nm, 644 nm and 440 nm; Ca, Cb, Ca+b are accordingly chlorophyll content a, b and total. Then, the pigment content in the test material is calculated (Agarwal and Rao, 1998): 퐶⋅푉 퐴 = 푥 = (5), 푛⋅1000 where: A - is the pigment content in the prototype; C - is the pigment concentration found by the Wetstein formula, mg / dm3; V - is the volume of the extract, cm3; n - is the sample of the prototype, g; 1000 - coefficient for conversion of pigment concentration to 1 cm3. To study the effect of laser irradiation on the growth and morphological and cultural characteristics of the fungus L. sulphureus, the mycelium of strain Ls-17 was cultured for 7 days on potato-glucose agar medium in standard Petri dishes (9 cm in diameter). Subsequently, a 5 mm diameter micellar disc was excised from the mother culture using a sterile steel tube. Before sowing on the substrate, they were irradiated with the help of LED lasers. Four variants of irradiation were used in the studies: control without irradiation and single laser irradiation for 15 sec. (red, blue and green spectrum). The BRP – 3010–5 LED lasers with a red spectrum with a wavelength of 635 nm, BBP – 3010–5 with a blue spectrum with a wavelength of 405 nm and BGP – 3010–5 with the emission of a green spectrum with a wavelength of 532 nm were used for irradiation (manufacturer BOB LASER Co., China). The power of each laser was 100 mW. The energy density of the laser irradiation was calculated by Vakarchuk (2012). The energy dose of irradiation (light energy per unit area) was defined as the product of energy density and irradiation time. The irradiation energy in all variants of the experiment was 77,3 mJ / cm2. There were used non-irradiated mycelium in the control group. The cultivation was carried out at a temperature of 27 ± 1 ° C until complete overgrowth of the Petri dish with mycelium in the thermostat. All experiments were repeated three time. To determine the likelihood of laser irradiation, analysis of variance was used. The comparisons of the average meanings were performed by the Dunnett method (through a comparison between control measurements and the other measurements (Prisedsky, 1999). The processing was performed using a package of statistical programs created at the Department of Plant Physiology, Donetsk National Vasyl Stus University (Prisedsky, 1999).

3.EXPERIMENTAL PART AND DISCUSSION OF RESULTS

3.1 Screening of carotenoid producing strains

3.1.1 Daily increase of mycelium of L. sulphureus strains on PGA

The first stage of our research was undertaken to the cultivation of strains Ls-0917, Ls-0918, Ls-0919 on PGA for 9 days. According to evaluation of daily growth of mycelium, the best results were shown by strain Ls-0919 with its maximum value on the 8th day of cultivation (Figure 8). This period also corresponded to the largest daily increase of strain Ls-0917.

Figure 8. Daily growth of L. sulphureus strain in PGA

The maximum growth rate on Potato Glucose Agar (PGA) was also observed for the cultivation of strain Ls-0919, however, strain Ls-0917 also showed quite positive results, which were only 8-10% lower than the culture of Ls-0919 (Figure 8). Strain Ls-0918 showed the lowest rates of both daily growth and average radial growth rate, which is probably due to the low suitability of the used agar medium for cultivation of this strain.

3.1.2 The maximum accumulation of ADB by mycelium with L. sulphureus strains on GPM

A perspective direction for protein production is the use of fungi biomass. It is known that the biological value of proteins of microbial and fungal biomass exceeds the value of proteins of cereals and legumes. The production of mycelium by the technology of microbiological industries can reduce the duration of the process by 10-15 times, and the output of the product from raw materials to increase by 2-3 times (Velygodska at al., 2012). The next step was the periodic cultivation of strains Ls-0919 and Ls-0917 on standard GPM for 20 days. Our aim was to identify the day of maximum accumulation of biomass. As can be seen, the maximum accumulation of biomass was observed on day 15th for both strains, by the 20th day of accumulation it stopped. This is likely due to the depletion of a certain amount of nutritional resources (Figure 9). This research shows that cultivated strains demonstrated the same result as the previously studied strain-producer Ls-08 (Velygodska at al., 2012).

Figure 9. Accumulation dry mycelial biomass from of L. sulphureus in GPM

3.1.3 Dynamics of carotenoid pigment increase in mycelia of L. sulphureus strains on GPM

In industrial poultry farming involves a large physiological load on the body of various stress factors (high accumulation, hypodynamia, vaccinations, use of specific diets with high content of 29 proteins and fats, lack of vitamins and amino acids), influence of pathogens. One of the ways to solve these problems is to create fundamentally new physiologically functional preparations of immunostimulating antioxidant action. In this regard, it is promising to use carotenoids as substances that perform in the body a number of specific and vital functions. Carotenoids perform more than 20 biological functions - from photoreception to protecting the body from lipid peroxidation. In poultry, beta-carotene, along with vitamins A, E and C, are part of an effective antioxidant complex. Producers of lipophilic bioantioxidants are of particular interest. Among the medicinal mushrooms, one of the most promising is the basidial fungus Laetiporus sulphureus, a carotenoid producer. This mushroom can be widely used for the preparation of drugs having antioxidant protection (Becker, 1988). The study of carotenoid content shows that cultures can accumulate pigments throughout the cultivation period. The maximum carotenoid content was observed for strain Ls-0917 on day 15th (Figure 10).

Figure 10. Dynamics of carotenoid pigment accumulation in mycelium of L. sulphureus strains in GPM

Thus, in terms of daily growth of mycelium and average radial growth rate, the leader is the strain L. sulphureus Ls-0919 with its maximum value on the 8th day of cultivation. The maximum accumulation of mycelium biomass on GPM was observed for 15 days for both strains. The study demonstrates that strains are more productive comparing to the previously studied strain-producer Ls-08 (Velygodska at al., 2012).

On this basis, they are promising cultures for further studies to obtain carotenoids of fungal origin.

3.2 Investigation of the effect of laser irradiation on the growth and biosynthetic parameters of L. sulphureus strains

3.2.1 Daily increase in mycelium of L. sulphureus strains on PGA due to different irradiation spectra

Further research was conducted with strain Ls-0917. We studied the influence of laser irradiation on daily mycelium growth. At first we discovered that laser irradiation has a positive effect on mycelium growth. Depending on the irradiation spectrum, the linear radial growth rate of the strain also differed. The best results of daily radial growth of mycelium were demostrated after using the red monochromatic light with its maximum value on the 4th day of cultivation (Figure 11).

6,71 6,58 6,6 6 6,25 6,1 5,8 5 5,36

3 Daily Daily increase, mm/day 2

0 Control Red Green Blue Red+Green Blue+Green Red+Blue Irradiation spectra

Figure 11. Daily increase in mycelium of L. sulphureus strains on PGA due to different irradiation spectra

Mycelium irradiated by a combination of wavelengths in the blue and green spectra also showed a good result. The worst result was observed with the strain irradiated by the combination of wavelengths in the red and green spectra.

3.2.2 Dynamics of accumulation of carotenoid pigments in mycelia of strains of L. sulphureus on GPM due to different irradiation spectra

Another important indicator for us was the carotenoids accumulation in the mycelium. Here we can see, that the strain Ls-0917 irradiated with the green spectrum of monochromatic light showed the best result. However, the culture that was exposed to red light demonstrated poor results (Figure 12).

6 5,8163 5 5,35

4 4,32255 4,18801

2 Carotenoid content, content, mg/g Carotenoid

0 Control Green Blue Red

Iradiation spectra

Figure 12. Dynamics of carotenoid pigment accumulation in mycelium of L. sulphureus strains on GPM due to different irradiation spectra

In the study of carotenoid accumulation in the culture filtrate, the best result was obtained by irradiation with red monochromatic light.

3.2.3 Accumulation dry mycelial biomass by the mycelium of L. sulphureus strains on GPM due to different irradiation spectra

The maximum biomass accumulation was observed on 15th days for the strains irradiated with both green and blue monochromatic light (Figure 13). But the results were slightly below than control’s, what may be explained by the fact that mycelium resources due to this irradiation are aimed at increasing the synthesis of carotenoids.

0,14

0,12

0,11 0,1

0,09 0,09 0,08 0,085

0,06

0,04 Accumulation dry biomass, dry g/l micelial Accumulation

0,02

0 Control Green Blue Red Iradiation spectra

Figure 13. The accumulation dry mycelial biomass by of L. sulphureus strains on GPM due to different irradiation spectra

3.3 Investigation of the effect of laser irradiation on the growth and biosynthetic parameters of L. sulphureus strains at reduced glucose concentrations

3.2.1. Daily increase in mycelium of L. sulphureus strains on PGA due to different irradiation spectra at reduced glucose concentrations

Further studies were performed with strain Ls-0917 on PGA with different glucose concentrations (Table 1).

Table 1. The components PGA for surface cultivation of Laetiporus sulphureus

Components of nutrient medium Variant of research Potato broth, ml/liter Agar-agar, gm/liter Glucose, gm/liter

1 (control) 20,0

2 10,0 200,0 20,0

3 8,0

4 6,0

According to the results of the studies, the best growth of L. sulphureus mycelium was observed during cultivation on medium containing 20 grams of glucose per liter, the rate of growth of mycelium on the substrate with 10 grams of glucose per liter was 35% lower. The smallest growth of mycelium was recorded on the substrate with 6 grams of glucose content (Figure 14).

6 5,9375 5

4 3,8593 3 3,1011 2,9997

2 micelium, mm/day micelium,

1 The average daily increase in increase daily average The

0 20 10 8 6 Glucose concentration, g/l

Figure 14. Daily increase in mycelium of L. sulphureus strains on PGA at different glucose concentrations

In the following stages, we investigated the daily increase in mycelium due to different spectra of irradiation with monochromatic light at PGA with different variants of glucose concentration: 10, 8 and 6 g/l. Irradiation with red light significantly improved the rate of mycelium growth on the test substrates. The best reaction was observed in response to the influence of irradiation on cultivation on the substrate with 10 grams of glucose content - 60% better than control (Figure 15).

9,5

8 7,98 7,1309 6 5,9375

2 The average daily mm/day micelium, in increase daily average The

0 Control 10 8 6 Glucose concentration, g/l

Figure 15. The average daily increase in mycelium of L. sulphureus strains on PGA due to irradiation with a red laser and at different glucose concentration

During cultivation for other glucose content, the growth rate during this irradiation increased from 20.1% to 34.4%, respectively (Figs. 16, 17).

6,1

5,9 5,9375

5,8 5,8

5,7 mm/day mm/day 5,68 5,7 5,6

5,5

The average daily micelium, in increase daily average The 5,4 Control 10 8 6 Glucose concentration, g/l

Figure 16. The average daily increase in mycelium of L. sulphureus strains on PGA due to green laser irradiation at different glucose concentrations

6,1

5,9 5,9375 5,9016 5,8769 5,8

5,7

mm/day mm/day 5,6 5,6545

5,5

5,4

The average daily increase daily average micelium, in The 5,3 Control 10 8 6 Glucose concentration, g/l

Figure 17. The average daily increase in mycelium of L. sulphureus strains on PGA due to blue laser irradiation at different glucose concentrations

At the same time, irradiation with green and blue light caused slight changes in the rate of mycelium growth (Figs. 16, 17).

Irradiation of mycelium with red light for 15 seconds helped to reduce the time of fouling at all glucose concentrations from 45 to 90%.

3.3.2 Dynamics of carotenoid pigment accumulation in mycelium of strains of L. sulphureus cultivated on GPM at different irradiation spectra at low glucose concentrations

Further studies were conducted with strain Ls-0917 on GPM at different values of glucose concentration (Table 2).

Table 2. The composition GPM for surface cultivation of Laetiporus sulphureus

Components of nutrient medium, gm/liter

Variant of

research 2

x7H

РО

x7H

2 2 4

НРО

CaCl

Peptone

Glucose

КН

ZnSO MgSO

1 (control) 10,0

2 3,0 0,6 0,4 0,5 0,05 0,001 8,0

3 6,0

The best result of carotenoid accumulation was observed on a medium with a glucose content of 10 grams per litter. With a very small difference from the control of the amount of carotenoids in the mycelium was observed on a medium at a glucose content of 8 grams per litter. The lowest result was observed on the medium with glucose content of 6 grams per litter (Figure 18).

7 6 5 5,55 5,52 4,9 4 3 2

1 Carotenoid content, mg/g mg/g content, Carotenoid 0 Control 8 6 Glucose concentration, g/l

Figure 18. Dynamics of accumulation of carotenoid pigments in mycelia of strains of L. sulphureus on GPM at different glucose content

In the study of carotenoid accumulation in the culture filtrate, the best result was observed in the control. In the following stages, we investigated the carotenoid content of the mycelium at different spectra of irradiation with monochromatic light on the GPM at two glucose concentrations (8 and 6 grams). The best result of carotenoid accumulation was observed by irradiation with the green spectrum of monochromatic light on the GPM at glucose content of 8 grams per liter, which is 20% higher than the control (Figure 19). 8 7 6,66 6 5,55 5,57 5 4 3 2

1 Carotenoid content, mg/g mg/g Carotenoidcontent, 0 Control 8 6 Glucose concentration, g/l

Figure 19. Dynamics of carotenoid pigment accumulation in mycelia of L. sulphureus strains on the GPM by irradiation with green spectrum of monochromatic light on media at different glucose content 38

The lowest accumulation was observed by irradiation with red monochromatic light on a medium with a glucose content of 6 grams per litter (Figure 20).

5,565 5,56 5,56 5,555 5,55 5,55 5,545 5,54 5,54 5,535

5,53 Carotenoid content, content, mg/g Carotenoid 5,525 5,52 Control 8 6 Glucose concentration, g/l

Figure 20. Dynamics of carotenoid pigment accumulation in mycelium of L. sulphureus strains on the GPM by irradiation with red spectrum of monochromatic light on media at different glucose content

Irradiation with the blue spectrum of monochromatic light differs from the control by 4.5% (Figure 21).

6 5,9 5,8 5,79 5,7 5,6 5,5 5,55 5,57 5,4

Carotenoid content, mg/g mg/g content, Carotenoid 5,3 5,2 Control 8 6 Glucose concentration, g/l

Figure 21. Dynamics of carotenoid pigment accumulation in mycelium of L. sulphureus strains on the GPM by irradiation with blue spectrum of monochromatic light on media at different glucose content 39

3.4 Discussion of results

Table 3. The concentration of carotenoids in different fungi

Authors Fungi Yield Ginka I. et al., 2003 Rhodotorula rubra 2.1 mg/g Tinoi J. et al., 2005 Rhodotorula glutinis 3.5 mg/g Velygodska at al., 2012 Laetiporus sulphureus-08 5,13 mg/g Velygodska at al., 2012 Fomes fomentarius Ff-1201 3,02 mg/g Vustin at al., 2010 Rhodosporidium diobovatum VKPM Y-3158 2,5-3,5 mg/g Avchieva at al., 1998 Rhodotorula glutinis VKPM U-2210 3-4 mg/g Laetiporus sulphureus-0917 6,6 mg/g (due to green irradiation spectra)

According to the table 3, the strain Ls-0917 due to the influence of the green irradiation spectrum, cultivated on media with low glucose concentrations (8g/l) (Figure 19), shows better results in comparison with the previously studied fungi. The known yeast strain Rhodosporidium diobovatum VKPM Y-3158 is also a producer of carotenoids. This strain is cultivated on a nutrient medium containing sources of Carbon, Nitrogen and mineral salts. Its biomass contained carotenoids in the amount of 3-4 mg/g of dry biomass, including carotene 2,5-3,5 mg/g, ie up to 90% (Vustin at al., 2010). There are no provided data about the content of carotenoids in cultures of basidiomycetes. In addition, yeast cultures can cause occupational diseases of service personnel when used as producers in the microbiological industry (Mansdorf, 2019). Known yeast strain Rhodotorula glutinis VKPM U-2210 is producer of carotenoids. The content of carotenoids is 3-4 mg/g (Avchieva at al., 1998). No data are provided on the content of carotenoids in basidiomycete cultures. Yeast cultures can cause occupational diseases of service personnel when used as producers in the microbiological industry (Mansdorf, 2019). There are data on strains of the yeast Blakeslea trispora KP 74+ and KR 86-producers of beta- carotene. The carotenoid content is 190 mg/100 ml of medium (Kunshchikova at al., 2001). There are no provided data about the content of carotenoids in cultures of basidiomycetes. Through, using as a carotenoid producer has some disadvantages. Minus of this strains are that cultures of lower fungi can cause occupational diseases of service personnel when it used as producers in the microbiological industry (Mansdorf, 2019)..

The closest analogue to Ls-0917 is the strain Laetiporus sulphureus (Bull.) Murrill Ls-08 with a content of carotenoids in the mycelium - up to 5.13 mg/g on the 12th day of cultivation on GPM (Velygodska at al., 2012). However, the strain Ls-0917 has a different area of origin and has a more intense biosynthesis of carotenoids in the mycelium (Figure 9, 10). The practical significance of the obtained results: the most productive strain is identified and could become a source of carotenoids. These strains are more productive compared to the previously studied strain-producer Ls-08 (Ginka I. et al., 2003, Tinoi J. et al., 2005, Velygodska at al., 2012). Despite the widespread of carotenoids in nature and particularly in fruits, vegetables, and algae, using the fungus as a producent of carotenoids L. sulphureus has several advantages: - the medicinal fungus L. sulphureus is widely distributed worldwide (Qiang Li at al., 2018); - these fungi usually have low nutritional requirements during their growth and cultivation, which reduces the production costs; - the maximum carotenoid content is produced already on 15th day; - production of carotenoids from fungus has no problems as plants with seasonal and geographic variability that cannot be regulated (Chang ST, et al., 2004).

CONCLUSIONS

According to the results of the study we can draw the following conclusions:

1. According to evaluation of daily growth of mycelium, strain Ls-0919 displayed the best results with its maximum value on the 8th day of cultivation. This period also corresponded to the largest daily increase of strain Ls-0917. The maximum growth rate on Potato Glucose Agar (PGA) was also observed for the cultivation of strain Ls-0919. However, strain Ls-0917 also demonstrated satisfactory results, which were only from 8 to 10% lower than the culture of Ls-0919. Strain Ls-0918 illustrated the lowest rates of both daily growth and average radial growth rate, which is probably due to the low suitability of the used agar medium for cultivation of this strain. 2. The maximum accumulation of absolutely dry biomass was observed on 15th day of cultivation for Ls-0919 and Ls-0917 strains. Measurements were not performed by the 20th day of growth. This is likely due to the depletion of a certain amount of nutritional resources of medium. 3. The study of carotenoid content shows that cultures can accumulate pigments throughout the cultivation period. The maximum carotenoid content was observed for strain Ls-0917 on 15th day. We can conclude that strain Ls-0917 is the most productive carotenoid producer. 4. Laser irradiation had a positive effect on mycelium growth. Depending on the irradiation spectrum, the linear radial growth rate of the strain was different. According to evaluation of daily radial growth of mycelium due to different light influences, the best results were shown by using the red monochromatic light with its maximum value on the 4th day of cultivation. The maximum biomass accumulation was observed on 15th days for the strains irradiated with both green and blue monochromatic light. However, the results were slightly below the control ones, what may be explained by the fact that mycelium resources due to this irradiation increased the synthesis of carotenoids but not a biomass accumulation. The strain Ls-0917 was irradiated with the green spectrum of monochromatic light. It showed the best result of the carotenoids accumulation in the mycelium. The least accumulation was observed by the strain irradiated with the red monochromatic light. 5. According to the results of the studies of increasing the biosynthetic activity of the strain Ls- 0917 by laser irradiation and optimisation the nutrient medium by reducing the glucose concentration the daily increase in mycelium at PGA by irradiation with red light significantly improved the rate of mycelium growth on the test substrates. The best reaction was observed in response to the influence of irradiation on cultivation on

42 the substrate with 10 grams of glucose content - 60% better than control. During cultivation for other glucose content, the growth rate during this irradiation increased from 20.1% to 34.4%. At the same time, irradiation with green and blue light caused slight changes in the rate of mycelium growth. The best result of carotenoid accumulation was observed by irradiation with the green spectrum of monochromatic light on the GPM with glucose content of 8 grams per liter. It is 20% higher than the control. Irradiation with the blue spectrum of monochromatic light differs from the control by 4.5%. The lowest accumulation was observed by irradiation with red monochromatic light on a medium with a glucose content of 6 grams per litter. Summarizing, the studies proved the possibility of a positive irradiation effect of certain spectra of monochromatic light on the carotenoids synthesis in the mycelium of investigated strains. Thus, the studies made it possible to determine the most productive strain and the most effective mode of stimulation the growth and biosynthetic processes of the fungus L. sulphureus by laser irradiation. The obtained results indicate the feasibility of using laser irradiation during the cultivation of L. sulphureus mycelium. L. sulphureus can be used in pharmacy, cosmetics, dietary supplements production that need carotenoids content. Moreover, L. Sulphureus strains are reliable microorganisms to produce carotenoids.

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Supplement 1. Total content of carotenoids in basidiocarps of some species of Basidiomycetes

Species The number of tested basidiocarps samples Carotenoid content, mg/g Polyporales Auricularia auricula-judae * 12 0,85 ± 0,05 Laeticorticium roseum * 3 0,70 ± 0,01 Chaetoporus ambiquus * 6 0,10 ± 0,01 Sparassis crispa * 9 0,25 ± 0,02 Fibuloporia mollusca * 6 1,05 ± 0,03 Tyromyces lacteus * 9 1,60 ± 0,02 Tyromyces revolutus * 3 1,20 ± 0,01 Tyromyces undosus * 6 1,03 ± 0,01 Irpex lacteus * 9 2,67 ± 0,03 Amyloporia lenis * 3 1,50 ± 0,02 Hydnum ochraceum * 3 1,00 ± 0,02 Trametes squalens * 6 1,50 ± 0,02 Trametes campestris * 6 2,01 ± 0,02 Trametes versicolor * 15 0,61 ± 0,11 Trametes zonatus * 9 0,64 ± 0,04 Fomes fomentarius * 12 5,83 ± 0,49 Heterobasidion annosum * 12 1,33 ± 0,06 Fomitopsis pinicola * 6 0,90 ± 0,41 Daedalea quercina * 6 0,90 ± 0,01 Piptoporus betulinus * 12 1,50 ± 0,10 Polyporus squamosus * 9 2,30 ± 0,07 Laetiporus sulphureus * 9 50,14 ± 10,74 Ganoderma applanatum * 9 55,04 ± 7,35 Ganoderma lucidum * 15 8,90 ± 0,15 Inonotus obliquus * 12 2,05 ± 0,03 Phellinus igniarius * 9 3,72 ± 0,76 Phellinus pomaceus * 9 1,90 ± 0,05

Species The number of tested basidiocarps samples Carotenoid content, mg/g Agaricales Agaricus arvensis * 5 2,45 ± 0,40 Agaricus bisporus ** 9 4,15 ± 0,16 Agaricus campestris * 5 2,34 ± 0,01 Agrocybe cylindracea ** 9 16,10 ± 0,30 Coprinus comatus * 15 2,50 ± 0,05 Coprinus micaceus * 15 2,50 ± 0,05 Fistulina hepatica * 9 40,74 ± 1,20 Flammulina velutipes * 27 25,28 ± 5,31 Flammulina velutipes ** 3 6,50 ± 0,09 Lentinus edodes ** 9 0,81 ± 0,08 Marasmius oreades * 3 3,70 ± 0,05 Pleurotus citrinopileatus ** 3 3,75 ± 0,01 Pleurotus eryngii ** 6 1,50 ± 0,02 Pleurotus ostreatus * 34 0,94 ± 0,45 Pleurotus ostreatus ** 3 5,56 ± 0,03 Pleurotus ostreatus 3 5,30 ± 0,01 var.Florida ** Kuehneromyces mutabilis * 9 4,88 ± 0,13 Pholiota aurivella * 3 1,80 ± 0,05 Pholiota squarrosa * 3 1,20 ± 0,05 Sсhizophyllum commune * 21 0,10 ± 0,04 Stropharia aeruginosa * 3 3,25 ± 0,05 Stropharia rugosoannulata 6 5,95 ± 0,05 ** Lyophyllum loricatum * 5 2,13 ± 0,03 Lyophyllum connatum * 5 2,04 ± 0,12 Tricholoma flavovirens * 5 9,35 ± 3,08 Tricholoma sejunctum * 5 3,14 ± 0,02

Note: "*" is a wildly growing basidiocarps, "**" is a commercial basidiocarps.