TECHNOLOGY OF DEEP CULTIVATION OF MEDICINAL FUNGUS SCHIZOPHYLLUM COMMUNE

ABSTRACT

This article investigates the formation of biomass in the deep cultivation of medicinal mushrooms S.commune. Deep cultivation of the culture contains information about the results obtained, in particular, the study of the influence of pH, temperature and aeration of the medium on the formation of biomass.

АННОТАЦИЯ

В данной статье исследовано формирование биомассы при глубинном культивировании лекарственных грибов S.commune. Глубинное культивирование культуры содержит информацию о полученных результатах, в частности, изучение влияния рН, температуры и аэрации среды на формирование биомассы.

Keywords: Schizophyllan, polysaccharide, isolate, Erlenmeyer flask, sterilization, inoculation, macromycete, pH value, aeration.

Ключевые слова: Шизофиллан, полисахарид, изолят, колба Эрленмейера, стерилизация, инокуляция, макромицет, значение pH, аэрация.

Introduction

Medicinal fungus synthesize a large amount of biologically active substances. These biologically active substances are important in the pharmaceutical industry today. Growing fungi in solid nutrient condition has several disadvantages. It is difficult to control the fruit body during growth. Most of the fungi grow in selective nutrient condition, and it takes a long time to choose a substrate with an optimal nutrient condition for them. Difficulties arise in separating biologically active substances formed outside the cells of fungi grown in a solid nutrient condition [1].

Growth of fungi in a liquid nutrient condition allows easy isolation of pure biologically active substances free from the above defects. In addition, many factors affecting the growth of fungi, including the morphology of fungi, the time of mycelium formation, the average composition of the feed, temperature, pH, light, etc. allows to manage factors [2].

For the first time, the selection of nutrient media for the cultivation of the medicinal fungus Schizophyllum commune in liquid nutrient condition and the work of planting mycelium in liquid nutrient condition were carried out by scientists Hu et al. in 2003 and Survase in 2006 [3, 4].

Yifeng Zhang found that the medicinal fungus S.commune synthesizes schizophyllan polysaccharide when grown in a liquid nutrient condition [5].

Alam et al., [6] isolated vitamins B, D, K, A and C from isolates of S. commune grown in liquid nutrient medium. Adejoye et al., [7] fully separated the proteins, vitamins, lipids and U P, Mg, K mineral elements contained in the fungus.

J.P. Sherkulova and E.Y. Eshonkulov [8, 9] have conducted scientific research on the cultivation of S.commune macrofungus in a solid medium in the conditions of Uzbekistan.

The main purpose of the study is to investigate the formation of biomass by the S.commune culture at different pH, temperature and aeration of the medium on the formation of biomass in submerged cultivation.

Materials and method

A microbiological shaker (multi-functional orbital shaker PSU-20i) was used to obtain the mycelial biomass of S. commune. In this case, thick mycelial films are formed on the surface and in the depth of the layer of liquid medium. Erlenmeyer flasks with a capacity of 250 ml are used for surface sowing. For this purpose, the mycelium mixture was placed in a liquid nutrient medium and grown in oscillators at an average vibration speed of 150 and 250 for 30 days. Our next experiments were conducted in a laboratory fermenter with a capacity of 6 l. Experiments were carried out 3 times in 500 cm3 Erlenmeyer flasks. 200 ml of the prepared nutrient media were poured into each flask and sterilized. For inoculation, seven-day-old mycelia of rock-grown macromycetes were sown. (A. Dudka, S. P. Wasser, I. A. Ellanskaya and dr. 1982) [10].

To study the effect of pH on growth, strains were grown in all media with a pH value of 3.5; 4.5; 5.5; Experiments were performed between 6.5 and 7.5. Changing the acidity of the medium was achieved by adding citric acid to the medium. The control variant had a pH value of 7.5, that is, slightly alkaline, and the transition to high acidity occurred after the addition of citric acid. Cultivation was carried out in a TS-80 thermostat at a temperature of 28±1 °C during the experiment. (Bukhalo A.S. 1988) [11].

The following nutrient media were selected for growing S commune fungus in a liquid nutrient conition:

1) liquid nutrient condition with Potatoes-glucose: (potatoes 400, glucose 100, agar 2 g);

2) Chapeka liquid nutrient medium: magnesium sulfate 0.2 g, anhydrous potassium phosphate 0.1 g, potassium chloride 0.5 g, iron sulfate 0.01 g, sodium nitrate 0.1 g, glucose 20 g, agar 2 g, water 1 l.;

3) Wort (beer yeast): water up to 70 ⁰C according to Balling hydrometer is added to beer wort. 2 g of agar-agar is added to the resulting wort and heated.

4) Liquid nutrient medium with yeast: (g / l): 7-10 g of dried yeast per 1 liter of distilled water is boiled for 20-30 minutes; It is left in the refrigerator for 12 hours, filtered, filled with one liter of water; It is boiled for 30 min and 10% sucrose, 2 g agar and 0.5% NaCl are added to the medium;

5) Cabbage liquid nutrient medium, (g/l): 200 g of cabbage is boiled for 10 minutes in 1 liter of water. The squeezed water is brought to 1 liter, then diluted 2 times and 2 g of agar and 2% glucose are added to the filtrate.

Results and discussion

During the cultivation of the medicinal fungus S.commune in a liquid nutrient medium, the experiments were carried out in 5 replicates and continuously monitored in the selected optimal nutrient mediums (Fig. 1, 2).

S.commune fungus was changed by pH effect in liquid nutrient condition. It grows well in weakly acidic and neutral environments. Initially, biomass production was 31.6±0.1 g/L in acidic medium with a maximum pH of 6.5 in potato-glucose and Suslo (brewer's yeast) liquid nutrient media. As a result of the change in pH from this point of growth of the isolates, mycelium formation decreased in the liquid nutrient media with yeast, Chapeka and Cabbage. For S.commune, mycelium formation increased at slightly more acidic than neutral condition pH (Fig.3).

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Figure 3.  Effect of pH on biomass formation of S.commune fungus in liquid nutrient medium

Temperature is the most important factor in the formation of mycelial biomass of S.commune fungus. The most favorable temperature for the formation of biomass in all liquid nutrient environments of the fungus was 30ºC, and biomass was formed at 31.9±0.1 g/L. When this temperature is exceeded or decreased (26-35ºC), the formation of mycelium in yeast and cabbage liquid nutrient media is reduced. (Fig.4)

Figure 4.  The effect of temperature on the formation of biomass in the liquid nutrient medium of S.commune fungus

In our research, the effect of aeration, the main factor of the fermentation process, in the formation of mycelial biomass of  S.commune fungus was significant. During the cultivation of the fungus in liquid nutrient medium, the amount of dissolved oxygen and filamentous growth affected the morphology of the mycelium. Branched hyphae of S.commune fungus were observed in potato-glucose and chapeka liquid nutrient media at 150 (rpm) vibration rotation, while in yeast, cabbage and Suslo (beer yeast) liquid nutrient media, the mycelium was moved at a rotational speed of 250 (rpm) and when shot, spherical, compact mycelium appeared (Fig.5)

Figure 5. Growing mycelia of S.commune fungus: a), b) Chapeka; c) appearance of branched hyphae in potato-glucose liquid nutrient media at 150 rpm; d) yeast; e) Cabbage; g) Appearance of spherical mycelia at 250 (rpm) mycelial rotation speed in wort (brewer's yeast) liquid nutrient condition

The mycelium of S.commune fungus caused damage to the hyphal granules during 350 vibration cycles, in which hyphae disintegration from the outer surface of the mycelium caused it to break into pieces. The highest biomass achieved as a result of increasing the vibration power of aeration was 32.8±0.2 at 150 (rpm) vibration rotation in potato-glucose and Suslo (brewer's yeast) liquid nutrient media. In yeast, Cabbage and Chapeka liquid nutrient media, when the mycelium was stirred at a rotation speed of 180 (rpm), the mycelium produced an average of 29.6±0.1 (Fig. 6).

Figure 6. On biomass formation of S.commune fungus in liquid nutrient medium effect of aeration

In conclusion, as a result of our scientific research, prepared potato-glucose, yeast, chapeka, Suslo (beer yeast) and Cabbage liquid nutrient media were selected to study the formation of biomass of the medicinal fungus S.commune in the liquid nutrient medium. Biomass production was 31.6±0.1 g/L in potato-glucose and Suslo (brewer's yeast) liquid nutrient mediums with a maximum pH of 6.5. The most favorable temperature for the formation of biomass in all liquid nutrient environments of  S.commune fungus was 30ºC, and biomass was formed at 31.9±0.1 g/L. Branched hyphae of the fungus S.commune were observed in potato-glucose and chapeka liquid nutrient media at 150 (rpm) vibration rotation, while in yeast, cabbage, and Suslo (beer yeast) liquid nutrient media, the mycelium was spherical in vibration rotation at 250 (rpm) rotation speed, formation of compact mycelia was observed. The highest biomass achieved as a result of increasing the vibration power of aeration, 32.8 ± 0.2 biomass was obtained in potato-glucose and Suslo (brewer's yeast) liquid nutrients at 150 (rpm) vibration rotation. Mycelium produced an average of 29.6±0.1 biomass when shaken at 180 (rpm) rotation speed in yeast, cabbage and chapeka liquid nutrient media. The most optimal for growing S.commune fungus in the liquid nutrient condition can be shown the potato-glucose and wort (brewer's yeast) liquid nutrient condition.

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