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Mustafin Kairat,
Almaty University of Power Engineering & Telecommunications,
Lead Researcher, Ph.D., Almaty, Kazakhstan Akhmetsadykov Nurlan, LLP Antigen, professor, Almaty, Kazakhstan Narmuratova Zhanar, MSc., Researcher, Department of Biochemistry, LLP Antigen, Almaty, Kazakhstan Zhakipbekova Aigerim, MSc., Junior Researcher, Department of Biochemistry, LLP Antigen Tapenbayeva Inkar, MSc., Junior Researcher Department of Biochemistry, LLP Antigen E-mail: gnj40@inbox.ru
SELECTION OF OPTIMAL CARBON AND NITROGEN SOURCES FOR ENHANCED POLYSACCHARIDES PRODUCTION BY LENTINUS EDODES
Abstract. In this paper the effect of carbon and nitrogen sources on the endopolysaccharides production by Lentinus edodes 2541 strain was investigated. Medium composition is the most important aspect to take into consideration when growing any microorganism. It was found that cellobiose was the best carbon source for fungal growth and endopolysaccharides production by L. edodes 2541 strain. Maximal endopolysaccharides production (5.5%) was detected in media with cellobiose as a carbon source. Among the various nitrogen sources used, peptone was a suitable nitrogen source for maximal endopolysaccharides production by L. edodes 2541. The medium had the following composition g/l: cellobiose -30; KH2PO4-1; K2HPO4-1; MgSO4-0.25; peptone - 3.5; yeast extract - 20 ml. Selection of carbon and nitrogen sources allowed increase polysaccharides production by Lentinus edodes.
Keywords: Lentinus edodes, Polysaccharides, Selection.
Introduction. Mushrooms such as Ganoderma lucidum (Reishi), Lentinus edodes (Shiitake), Inonotus obliquus (Chaga) and many others are extensively known for their immunomodulatory, hepatoprotective, antinociceptive, antidiabetic, antiviral, and antimicrobial properties [1; 2]. Shiitake is the common Japanese name for Lentinus edodes, and is also the common name now used in the West. Indigenous to Asia, shiitake is now cultivated and is the second most commonly produced edible mushroom in the world. Besides being a culinary delicacy, there is a long tradition of use of shiitake as medicine in Asia, dating back > 2000 years. Shiitake contains protein (26% of dry weight), lipids (primarily linoleic acid); carbohydrate; fiber; minerals; vitamins B-1, B-2, and C; and ergosterols [3]. Besides its nutritive content several important compounds have been isolated from shiitake that have immunomodulatory, lipid-lowering, and antimicrobial properties. These include lentinan, Lentinus edodes mycelium (LEM), KS-2, and eritadenine [4].
Another compound isolated from shiitake is eritadenine, which has been shown to lower serum cholesterol and lipid concentrations in various studies in rodents [5]. Besides the well-studied compounds other potentially beneficial compounds have been found in shiitake.
In spite of many researchers' efforts for the production of bioactive metabolites by mushrooms, the physiological and engineering aspects of submerged cultures (production kinetics, structural features, biological activity and biosynthesis control methods of some bioactive compounds) are still far from being thoroughly studied. Existing studies of the artificial cultivation of higher mushrooms are concerned to increase in the yield of the target products (biomass, protein, amino acids), the development of new cheap and simple nutrient media for the biosynthesis of biologically active compounds. In this regard, the primary role is given to the composition of the nutrient medium (carbon and nitrogen sources), which
Section 2. Medical science
influence both the constructive exchange of cultures and the synthesis of polysaccharides.
Materials and methods. The object of present research was the L. edodes 2541 strain from the collection of higher mushrooms of the Institute of Botany, Kiev, Ukraine. Extracellular endopolysaccharides were produced in synthetic glucose-peptone-yeast medium. Mycelia biomass was be assessed after 7 days of submerged cultivation in 250 ml flasks containing 50 ml of medium. The mycelia biomass was separated by centrifugation (4 °C, 3000 rpm, 30 min), washed by dH2O, dried at 50 °C until constant weight was obtained, and measured as g L-1 of the medium. Biomass production was estimated at 3, 5, 7 days of cultivation in liquid medium. Biomass and endopolysaccharides production were evaluated in artificial culture media with different carbon and nitrogen sources.
The effect ofvarious carbon and nitrogen sources on the endopolysaccharides production was determined. Mono-, di-, and
polysaccharides such as glucose, arabinose, xylose, galactose, mannose, fructose, lactose, maltose, sucrose, mannitol, sorbitol, and cellobiose in concentration equal to 30 g/l glucose were used as carbon sources. Inorganic and organic sources such as NaNO3, NH4NO3, NH4Cl, (NH4)2SO4, peptone, asparagine and urea in concentration equal to the amount of nitrogen in 2g of NaNO3, were used as nitrogen sources.
Total content of polysaccharides was determined by the phenol-sulfuric acid method [6]. The content of polysaccharides was calculated in% of absolutely dry biomass (a.s.m.). All the analyses were performed in triplicate, and the results were expressed as mean SD values of the three sets of observations. The mean values and standard deviation will be calculated using STATISTICA 6 [7].
Results and discussion. At the first stage, the effect of the carbon sources on the endopolysaccharides production in L. edodes 2541 strain was studied (Table 1).
Table 1. - The effect of various carbon sources on endopolysaccharides production by L. edodes 2541 in submerged cultivation
Carbon source Biomass, g/l Endopolysaccharides,% a.s.m.
Glucose 13.5 ± 0.2 4.5 ± 0.2
Arabinose 3.0 ± 0.3 2.8 ± 0.2
Galactose 3.5 ± 0.4 2.5 ± 0.1
Xylose 3.2 ± 0.2 2.9 ± 0.3
Mannose 4.6 ± 0.4 2.0 ± 0.2
Fructose 3.3 ± 0.2 2.3 ± 0.1
Lactose 6.2 ± 0.3 3.2 ± 0.3
Maltose 6.4 ± 0.2 2.8 ± 0.2
Sucrose 5.8 ± 0.2 2.8 ± 0.2
Cellobiose 14.8 ± 0.4 5.5 ± 0.3
Mannitol 3.2 ± 0.3 2.5 ± 0.1
Sorbitol 4.5 ± 0.2 2.0 ± 0.2
The results indicate that the best carbon source for the endopolysaccharides production in L. edodes 2541 was cellobiose. The yield of endopolysaccharides in the medium with cellobiose was 5.5%.
Further experiments were aimed at studying the effect of various nitrogen sources for endopolysaccharides production in L. edodes 2541 strain. The data obtained are presented in (Table 2).
Nitrogen source Biomass, g/l Endopolysaccharides,% a.s.m.
NaNO3 4.4 ± 0.3 2.9 ± 0.1
NH NO, 5.2 ± 0.2 2.5 ± 0.1
NH^Cl 4.9 ± 0.1 2.7 ± 0.2
(NHj,SO, 6.0 ± 0.3 3.5 ± 0.1
Peptone 13.5 ± 0.2 4.5 ± 0.2
Asparagine 6.2 ± 0.4 2.5 ± 0.2
Urea 2.7 ± 0.1 2.4 ± 0.1
Table 2. - Effect of nitrogen sources on endopolysaccharides production by L. edodes 2541 strain in submerged cultivation
As can be seen from the data presented in (Table 2), peptone found to be optimal for the endopolysaccharides production by L. edodes 2541.
In conclusion, the optimal carbon and nitrogen sources in the nutrient medium were selected. The medium had the following composition (g/l): cellobiose - 30; KH2PO4-1; K2HPO4-1; MgSO4-0.25; peptone - 3.5; yeast extract - 20 ml. Quantitative assessment of the influence of nutrient op-
timization and cultivation conditions on the efficiency of the growth process and polysaccharide biosynthesis by L. edodes 2541 strain indicates a significant increase in the content of endopolysaccharides in biomass by 60% as a result of optimization of biotechnological cultivation processes.
Funding: This research work was funded by Ministry of Education and Sciences of the Republic of Kazakhstan. Grant № AP05130493.
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