KR100773126B1 - Medium for mycelial growth using fomitopsis pinicola - Google Patents

Abstract

A culture medium composition and a method for producing hypha and extracellular polysaccharides are provided to mass-produce the hypha and extracellular polysaccharides of Fomitopsis pinicola under optimum culturing conditions. A culture medium composition for producing hypha of Fomitopsis pinicola comprises 3-4 weight/volume% of glucose, 0.4-0.6 weight/volume% of yeast extract, 0.05-0.15 weight/volume% of malt extract, 0.05-0.15 weight/volume% of K2HPO4 and 0.03-0.05 weight/volume% of MgSO4.7H2O. A method for producing hypha and extracellular polysaccharide comprises a step of culturing the Fomitopsis pinicola in the culture medium at a temperature of 25±0.5 deg.C under the pH of 5.8-6.2 using a shaking incubator with the speed of 140-150 rpm.

Description

Medium composition for pine myrtle mycelium production and method for producing mycelium and extracellular polysaccharides using the same {Medium for Mycelial growth using Fomitopsis pinicola}

1 shows a procedure of the DNS method.

Figure 2a shows the mycelial growth of Fomitopsis pinicola with temperature in PDA medium.

Figure 2b shows the mycelial growth and extracellular polysaccharide production of Fomitopsis pinicola according to the shaking speed at 25 ℃, initial pH 6.0.

Figure 3a shows the mycelial growth and extracellular polysaccharide production of Fomitopsis pinicola according to glucose concentration.

Figure 3b shows the mycelial growth and extracellular polysaccharide production of Fomitopsis pinicola according to yeast extract concentration.

Figure 3c shows the mycelial growth and extracellular polysaccharide production of Fomitopsis pinicola according to malt extract concentration.

Figure 3d shows the mycelial growth and extracellular polysaccharide production of Fomitopsis pinicola according to K ₂ HPO ₄ concentration.

Figure 3e shows the mycelial growth and extracellular polysaccharide production of Fomitopsis pinicola according to the concentration of MgSO ₄ · 7H ₂ O.

Figure 4a shows the mycelial growth and extracellular polysaccharide production of Fomitopsis pinicola according to agitation speed (jar fermentation: 25 ℃, pH 6.0, 0.5 vvm)

Figure 4b shows the mycelial growth and extracellular polysaccharide production of Fomitopsis pinicola according to the aeration rate (jar fermentation at 25 ℃, pH 6.0 and 200 rpm).

Figure 5 shows the difference between mycelial growth and extracellular polysaccharide production of Fomitopsis pinicola (jar fermentation: 25 ℃, pH 6.0, 1.5 vvm, 200 rpm).

6a shows the change of specific growth rate according to the change of cell mass.

6B shows the change in specific production rate according to the change in specific growth rate.

Figure 7a shows the difference in mycelial growth and extracellular polysaccharide production of Fomitopsis pinicola in air-Bubble bioreactor, 25 ℃, pH 6.0, 0.5 vvm conditions.

Figure 7b shows the difference in mycelial growth and extracellular polysaccharide production of Fomitopsis pinicola in air-Bubble bioreactor, 25 ℃, pH 6.0, 1.0 vvm conditions.

Figure 7c shows the difference in mycelial growth and extracellular polysaccharide production of Fomitopsis pinicola in air-Bubble bioreactor, 25 ℃, pH 6.0, 1.5 vvm conditions.

Figure 7d shows the difference in mycelial growth and extracellular polysaccharide production of Fomitopsis pinicola in air-Bubble bioreactor, 25 ℃, pH 6.0, 2.0 vvm conditions.

FIG. 8 shows the results of mycelial growth in FIGS. 7A to 7D.

The present invention relates to a medium composition for the production of mycelia of pine needles mushrooms and a method for producing the mycelium and extracellular polysaccharides using the same, more specifically, the optimum culture of liquid culture for mycelial growth and the production of extracellular polysaccharides of pine needles mushrooms It relates to a medium composition and a method for producing mycelium and extracellular polysaccharides using the same.

Pinus edible mushrooms occur on the trunks of trees and trees of various conifers in the family of pines. Without sacks, the lampshade is attached to the tree trunk in a shelf shape, forming a semicircular shape. Gad is a large mushroom up to 50 cm in diameter and 15 cm thick. The upper surface is covered with a thick epidermis, hard, the surface is flat, black or reddish brown, and has concentric ridges. Often a reddish brown strip is circumscribed around the lampshade. The underside is yellowish white with fine pores. The flesh is pale yellow white and hard wood. Its distribution is north and south of Korea, Japan and northern hemisphere. Korea grows in the dead pine tissues in alpine areas more than 300m above sea level. Pine needles are known to have effects on stomach cancer, heart disease, anti-tumor, liver cancer, cholesterol, etc., and are known to have excellent effects on pancreas and diabetes. Folk medicine is used to treat pneumonia, lung cancer, and antipyretics of colds.

Mushrooms are low in calories, sodium, fat, and cholesterol, while proteins, carbohydrates, fibers, vitamins, and Minerals and other abundant nutritional properties, it is a very good health food. Since ancient times, mushrooms have been known to have pharmacological and immune enhancing effects such as nourishing tonic, urea, blood lipid lowering, expectoration, coronary artery blood flow and blood pressure lowering. Research on mushrooms has been actively conducted in China, Japan, and Korea. In Japan, shiitake mycelium extract has already been generalized as a functional food. In addition, since the polymer polysaccharide extracted from Agaricus blzei fruiting body in 1992 is known to be effective in inhibiting cancer cell proliferation, it is effective in all diseases caused by weakened immune function such as rheumatoid arthritis, chronic bronchitis, and gastritis. It is recently commercialized as a functional food. Among the components of the mushroom, it is known that it is a polysaccharide that exhibits immuno-enhancing action. In general, although it is a single substance having a branched structure of β-1,6 glucan in the skeleton of β-1,3 glucan, it is known to have various effects on ecological function. . Recent studies on exo-polysaccharides of cells from mushrooms have been reported for biological activities. Several anti-cancer substances produced from the extract of mushrooms such as Lentinus edodes of Lentinan, Schizophyllum commune of Schizophyllan, and Coriolus Versicolor of Krestin are being currently used commercially.

Artificial production of mushrooms has increased rapidly over the last few decades, and their production and consumption is mainly in Asia, especially in China, Japan and Korea. In spite of this situation, the improvement of mushroom productivity is somewhat limited because the physiological and environmental culture conditions for the solid culture of mushrooms have not been extensively studied.

In the meantime, most of basidomycetes use fruit body and are grown and produced by solid culture, but it consumes much labor and time. Therefore, efficient production through mycelial culture in fermenter by liquid culture is desirable. . In addition, liquid culture is less risk of contamination by mold and has the advantage of mass production of mycelia in a compact space and in a short time. Although many researchers have studied to obtain optimal liquid culture conditions for producing mycelia and producing bioactive substances (extracellular polysaccharides) from various mushrooms, nutritional equirement and There is no wide range of culture conditions.

Pachyman, a polysaccharide in Fukryeong, was effective in restoring leukopenia, and Hll, a protein-bound polysaccharide, had anticancer effects. Profuramine, an acid peptide obtained from an enoki mushroom, has been considered for practical use as an anticancer agent. In addition, Calvacin is known as a peptide anticancer substance from Calvatia gigantea , erinacin from Hericiem erinacius, and sparasin from Sparassis crispa .

To date, a variety of useful substances have been reported in mushrooms, and the anticancer activity of more than 600 mushrooms has proved to be effective in fruiting bodies and mycelial culture. Therefore, more in-depth study of the function of these materials using various mushrooms is needed and the development of new biologics based on them.

The present invention has been proposed to solve the problems of the prior art as described above, an object of the present invention is to provide a medium composition for the production of mycelium of the pinnacle mushroom.

Another object of the present invention is to provide a method for producing pine myrtle mushroom mycelium and extracellular polysaccharides using the optimum medium composition of liquid culture for mycelial growth and extracellular polysaccharide production.

The present invention for achieving the above object is glucose 3 ~ 4% (w / v), yeast extract (yeast extract) 0.4 ~ 0.6% (w / v), malt extract (malt extract) 0.05 ~ 0.15% (w / v), K ₂ HPO ₄ 0.05 ~ 0.15% (w / v) and MgSO ₄ · 7H ₂ O 0.03 ~ 0.05% (w / v) provides a medium composition for the production of mycelium of pine zanbi mushroom.

The present invention also provides a method for producing mycelium and extracellular polysaccharides by cultivating pine beetle mushrooms in the medium.

Hereinafter, the present invention will be described in detail.

The present invention investigated the optimum culture conditions and medium composition of liquid culture for mycelial growth and extracellular polysaccharide production of Fomitopsis pinicola .

Medium composition for mycelial production of pine needles mushrooms of the present invention, glucose 3 ~ 4% (w / v), yeast extract 0.4 ~ 0.6% (w / v), malt extract 0.05 ~ 0.15% (w / v), K ₂ HPO ₄ 0.05-0.15% (w / v) and MgSO ₄ .7H ₂ O 0.03-0.05% (w / v). More preferably, glucose 4% (w / v), yeast extract 0.5% (w / v), malt extract 0.1% (w / v), K ₂ HPO ₄ 0.1% (w / v) and MgSO ₄ A medium composition of 7H ₂ O 0.05% (w / v) is suitable for mycelial production of pine needles.

In another aspect, the present invention provides a method for producing mycelium and extracellular polysaccharides by culturing the pine needles mushrooms in the medium of claim 1.

In the above, the pH of the medium is preferably 6.0 ± 0.2, the optimum pH of the medium is preferably 6.0, the culture temperature is preferably 25 ± 0.5 ℃, more preferably 25 ℃.

In the above, the culture of pine needles butterfly may be agitated culture, fermenter (Jar fermenter) or a bio-reactor (Air-bioreactor).

When cultivating pine needles mushrooms in the medium, when using a stirring incubator, the stirring speed is preferably 140 ~ 150rpm, more preferably 150rpm. In addition, when using a fermenter (Jar fermenter) or a bio-reactor (Air-bioreactor), the stirring speed is preferably 190 ~ 200rpm, more preferably 200rpm, the aeration amount is preferably cultured at 1.4 ~ 1.5 vvm, It is more preferable to incubate at 1.5vvm.

Hereinafter, the contents of the present invention will be described in more detail with reference to Examples. However, these examples are only presented to understand the contents of the present invention, but the scope of the present invention is not limited to these embodiments.

Example 1 Strains and Inoculum

The strains used in this study were obtained from Fomitopsis pinicola mushrooms in the Gaya Baeksong spawn culture center in Chungnam, Korea, and cultured in Patoto dextrose agar (Difco ^TM ) medium at 25 ° C for 7 days and then stored at 4 ° C. It was used in the experiment while subcultured weekly.

In preparation of the inoculum, in the case of solid culture, mycelium disk obtained by cold storage strain was cut with 5 mm cork borer at the center of PDA medium (Difco ^TM : potatoes 4 g / L, dextrose 20.0 g / L, agar 15 g / L). Was incubated in an incubator at 25 ° C. and used for experiments. For liquid culture, a 50 ml base medium (YMG: yeast extract 8 g / L, malt extract 20 g / L, glucose 8 g / L) in a 300 ml flask was used. After autoclaving at 121 ℃ for 15 minutes, inoculate 4-5 pieces of mycelium disk with 5mm cork borer, incubate at 25 ℃ for 7 days, and then incubate the culture solution aseptically for 30 seconds in a clean bench. Used as.

Example 2 Investigation of the Effect of Culture Conditions

Analysis method:

The measurement of mycelial growth in solid media was done by drawing a vertical line and a horizontal line perpendicular to the center of the inoculated mycelial section with a planetary pen on the underside of a petri-dish plate. The two diameters were averaged to determine the growth diameter of the hyphae. Dry cell weight in liquid culture was centrifuged at 5,000 rpm for 15 minutes, the precipitated mycelium was washed with distilled water two or three times, dried at 60 ° C for 24 hours, and left to dry in a deciccator until dry. The weight was measured, and the supernatant was added to 4 times of 95% ethanol and left at 4 ° C. for one day. The precipitated extracellular polysaccharides were separated by centrifugation at 5,000 rpm for 15 minutes and dried at 60 ° C. for one day. It was left until it became a fixed quantity and dry weight was measured. Glucose in the substrate was quantified according to the DNS (di-nitrosalicyclic acid) method (Fig. 1).

DNS sample was prepared by dissolving 16 g of NaOH, 1 g of DNS, 300 g of C ₄ H ₄ KNaO ₆ .4H ₂ O (Rochelle salt) in 1000 ml of distilled water. 1 ml of the sample was added to the test tube, and 3 ml of the sample prepared as described above was added and mixed well. The mixture was heated in a water bath at 90 ° C. for 10 minutes, and the mixture became reddish brown. After heating, the mixture was cooled to room temperature and absorbance was measured at 570 nm using a spectrophotometer (UV mini 1240, SHIMAZU).

(1) influence of temperature

In order to investigate the best temperature for mycelial growth of pine needles butterfly, PDA medium was prepared and autoclaved at 121 ° C for 15 minutes, and then, 20 ml of each petri-dish was hardened, inoculated and inoculated at 20 ° C and 25 ° C. , 12 days incubation in a thermostat adjusted to a temperature range of 30 ℃ was investigated the growth of mycelia at daily intervals. As a result, as shown in FIG. 2a, it can be seen that the mycelial growth of pine grasshopper mushrooms is rapidly lowered at 20 ° C. or lower and 30 ° C., and the optimum culture temperature is 25 ° C. due to the best mycelial growth at 25 ° C. .

(2) Effect of initial pH

In order to investigate the optimum initial pH of mycelial growth, the basic medium was dispensed in 300 ml triangle flasks, each with 50 ml of 1 N-HCl and 1N-NaOH. 5% (v / v) of the aseptic homogenized inoculum was inoculated by autoclaving and incubated at 25 ° C. and 100 rpm for 7 days. As a result, as shown in Table 1, mycelial growth and extracellular polysaccharide production were good in the pH range of 5.5-6.0, and mycelial growth and extracellular polysaccharide production tended to be somewhat suppressed in the pH range of 4.0-5.0. At initial pH 6.0, mycelial growth and extracellular polysaccharide production were 5.45 g / L and 1.24 g / L, respectively. However, when examined in terms of mycelial growth and the production of extracellular polysaccharides, it can be seen that the optimum initial pH of the mycelia of pine pine fern mushroom is 6.0. In addition, as shown in Table 1, the change in pH after the experimental results at the initial pH of pine beetle mushroom showed that the mycelial growth and extracellular polysaccharide production tended to increase proportionally.

Table 1. Fomitopsis by Initial pH pinicola Mycelial growth and Exo-polysaccharide production

Initial pH Mycelial growth (g / L) Exopolysaccharide concentration (g / L) △ pH 4.0 3.88 0.58 1.8 4.5 3.90 0.65 2.2 5.0 4.27 0.80 2.56 5.5 5.28 1.17 2.75 6.0 5.45 1.24 3.4 6.5 4.82 1.12 3.15 7.0 4.3 1.07 2.5 7.5 3.95 1.02 2.25 8.0 3.65 1.00 2.7

(3) Effect of air on volume

In order to investigate the effect of air by volume, adjust the pH to 6.0 by varying the volume of the 50 ~ 150mL volume of the basic medium in a 300mL Erlenmeyer flask, and autoclave at 121 ℃ for 15 minutes, and then inoculate 25 ± 1 C was incubated at 100 rpm for 7 days. As a result, as shown in Table 2, the smaller the culture volume, the smoother the oxygen supply, so that the mycelial growth and extracellular polysaccharide production are much improved. The maximum mycelial growth (5.35 g / L) and extracellular polysaccharide production are shown. The culture volume showing (1.51 g / L) was 50 ml. These results show that it is more effective to maintain proper culture volume for smoothing the rate of oxygen dissolution and the movement speed in liquid for the mycelial growth and extracellular polysaccharide production of pine needles.

Table 2. Effect of Air on Volume

Volume (mL) Mycelial growth (g / L) Exo-polysaccharide (g / L) 50 5.35 1.51 100 5.05 1.34 150 4.78 1.17

(4) Effect of Rotation Speed of Stirrer

In order to investigate the optimum number of rotations of mycelial growth in agitated incubator, the 50ml basic medium was adjusted to 6.0 in a 300ml Erlenmeyer flask, followed by autoclaving at 121 ° C for 15 minutes, and then the inoculum was maintained at 25 ° C for 50 days for 7 days. Incubation was carried out at different speeds at 200 rpm.

Viscosity in the culture medium increases due to accumulation in the culture medium such as extracellular polysaccharide products as well as mycelial growth during the culture period. Therefore, efficient mixing to facilitate mass transfer is a major environmental factor that can increase mycelial growth and increase the yield of extracellular polysaccharides. Therefore, the effects on the mycelial growth and extracellular polysaccharide production were investigated. The results of mycelial growth and extracellular polysaccharide production according to the number of rotations were obtained at the shaking speed of 150 rpm as shown in FIG. 2B. The maximum mycelial growth and extracellular polysaccharide production were obtained. Polysaccharide production was shown. These results indicate that the more the shaking speed increases, the more effective the mycelial growth and extracellular polysaccharide production is. However, mycelium growth and extracellular polysaccharide production tended to be somewhat lower than those at 100 rpm, which is thought to be a factor that inhibits mycelial growth due to increased shear stress on the mycelium in culture. . Therefore, it can be seen that the optimal rotation speed in the flask culture is 150 rpm.

Example 3 Nutritional Needs Investigation

(1) Selection and optimal concentration of carbon source

To investigate mycelial growth and extracellular polysaccharide production according to carbon source, 2% (w / v) of glucose and 8 kinds of sugars were added, and the pH of the medium was adjusted to 6.0. After autoclaving at 121 ℃ for 15 minutes, the inoculum was inoculated at 5% (w / v) and shaken at 25 ℃ and 150 rpm for 7 days, and the effect of the selected optimal carbon source concentration was investigated. To do this, the range of 0-9% (w / v) by 0.5% (w / v) was investigated in the same manner as the carbon source selection experiment.

  The carbon source is an essential nutrient source for the growth of strains as a source of synthesis and energy for carbohydrates, proteins, lipids and nucleic acids in fungi. As a result of incubating 9 kinds of carbon sources on the growth of pine myrtle mushroom mycelium at 2% (w / v) each, it was the best in the monosaccharide glucose (1.93g / L) addition group for mycelial growth as shown in Table 3. The disaccharide was sucrose (1.64g / L), sugar alcohol manitol (0.92g / L) in order. In extracellular polysaccharide production, glucose (0.55g / L) added group was the best, followed by sucrose (0.34 g / L).

As a result of investigating the effect on the mycelial growth and extracellular polysaccharide production according to the concentration of glucose selected as the optimal carbon source, the maximum mycelial growth and extracellular polysaccharide production of pine myrtle mushroom mycelium in Fig. 3a was 4% (w / v) Mycelial growth and extracellular polysaccharide production were poor at the addition or below 4% (w / v) concentration. Therefore, as shown in Table 3, it can be seen that the glucose concentration for mycelial growth and the production of extracellular polysaccharides is 4% (w / v).

Table 3. Mycelial Growth and Extracellular Polysaccharide Production by Carbon Sources

Carbon source Mycelial dry weight (g / L) Exo-polysaccharide (g / L) control 0.29 0.03 glucose 1.93 0.55 fructose 0.93 0.24 galatose 0.90 0.22 arabinose 0.73 0.19 maltose 0.89 - sucrose 1.64 0.44 dextrin 1.52 - starch 0.79 - manitol 0.92 -

(2) Selection of nitrogen source and optimal concentration

Nitrogen source is an essential nutrient for the synthesis of the major components of the cytoplasm. Therefore, to investigate the effect of nitrogen source on mycelial growth and extracellular polysaccharide production of pine beetle, 4% (w / v) glucose was added. 12 kinds of organic nitrogen sources and inorganic nitrogen sources were added to the medium (pH 6.0). As a result, as shown in Table 4, yeast extract and malt extract added as organic nitrogen sources showed the best mycelial growth and extracellular polysaccharide production. In the case of inorganic nitrogen source, the production yield was relatively lower than that of organic nitrogen source.

In order to investigate the optimal yeast extract concentration for mycelial growth and extracellular polysaccharide production, glucose 4% (w / v) was increased by varying the yeast extract concentration from 0 to 1.25% (w / v) by 0.25% (w / v). Investigation was added to the medium contained. As a result, as shown in Figure 3b yeast extract 0.5% (w / v) addition showed the maximum mycelial growth and extracellular polysaccharide production. In the mycelial growth and extracellular polysaccharide production, yeast extract added concentration increased up to 0.5%, but the production yield of mycelial growth and extracellular polysaccharides was not significantly different at high concentrations above 0.75% (w / v), resulting in an optimal concentration of 0.5%. (w / v) was selected.

To investigate the effects of malt extract on mycelial growth and extracellular polysaccharide production, glucose concentrations of 4.0% (w / v) and yeast extract 0.5 were varied by varying the concentration of malt extract by 0.025% from 0 to 0.15% (w / v). Irradiation was added to the medium containing% (w / v). As a result, as shown in FIG. 3C, mycelial growth and extracellular polysaccharide production were more mycelial growth when 0.8% (w / v) yeast extract was added than when only 1.0% (w / v) polypeptone was added. And extracellular polysaccharide production yielded much better yields. Therefore, it can be seen that the optimum concentrations of yeast extract and malt extract, which are the optimal nitrogen sources, are 0.5% (w / v) and 0.1% (w / v), respectively.

Table 4. Mycelial Growth and Extracellular Polysaccharide Production by Nitrogen Sources

Nitrogen source Mycelial dry weight (g / L) Exo-polysaccharide (g / L) Control (none) 2.42 0.42 Ca (NO ₃ ) ₂ 2.51 0.44 NaNO ₃ 2.39 - (NH ₄ ) ₂ SO ₄ 2.38 - NH ₄ NO ₃ 2.35 - NH ₄ H ₂ PO ₄ 2.64 0.54 (NH ₄ ) ₂ HPO ₄ 2.51 0.51 KNO ₃ 2.55 0.58 Malt extract 3.61 0.82 Eptone 3.02 0.62 Tryptone 3.23 0.72 Yeast extract 3.74 0.97 Polypeptone 3.12 0.77

(4) Selection of inorganic salts and optimal concentration

To investigate the effect of inorganic salts on mycelial growth and extracellular polysaccharide production, medium containing 4% glucose (w / v), 0.5% (w / v) yeast extract, and 0.1% (w / v) malt extract Eight kinds of inorganic salts were investigated by adding 0.15% (w / v), respectively. As a result, as shown in Table 5, inorganic salts were effective for mycelial growth of pine grass butterfly. Among them, K ₂ HPO ₄ and The mycelial growth and extracellular polysaccharide production were the best in MgSO ₄ · 7H ₂ O addition. In addition, the production of mycelial growth and extracellular polysaccharides was enhanced by the effects of PO ₄ ^3- , which acts as a buffering reagent, K ⁺ , which forms a cell structure, and Mg ²⁺ , which affects the biosynthesis and ion permeability of cell walls of fungi. It is considered to be. In order to investigate the effect of K ₂ HPO ₄ added as the optimal inorganic salt, glucose 4% (w / v), yeast extract 0.5% (w / v), malt extract 0.1% (w / v) As a result of adding 0 to 0.2% (w / v) to the medium, the maximum mycelial growth (5.12 g / L) and extracellular polysaccharides were observed at 0.1% (w / v) K ₂ HPO ₄ addition as shown in FIG. Production (1.47 g / L).

In addition, 4% (w / v) glucose, 0.5% (w / v) yeast extract, 0.5% (w / v) yeast extract, and 0.1% (w / v) malt extract were investigated to investigate mycelial growth and extracellular polysaccharide production by addition of MgSO ₄ · 7H ₂ O. _{v), K 2 HPO 4 0.1} % (w / v) mycelial growth, as shown in Figure 3e After a review by the addition by 0~0.1% (w / v) in the culture medium is contained _{MgSO 4 · 7H 2 O 0.05%} ( The yield of polysaccharide production of cells in the test group to which w / v) concentration was added showed better yield than the extracellular polysaccharide production shown in the experimental results according to the concentration of K ₂ HPO ₄ . Therefore, the optimum concentrations of K ₂ HPO ₄ and MgSO ₄ · 7H ₂ O, which are the optimal inorganic salts for mycelial growth and the production of extracellular polysaccharides, are 0.1% (w / v) and 0.05% (w / v).

Table 5. Effect of Inorganic Salts on Mycelial Growth and Extracellular Polysaccharide Production

Mineral source Mycelial dry weight (g / L) Exo-polysaccharide (g / L) Control (none) 4.150 1.130 MgSO ₄ 5.304 1.446 CaCl _{2 ㅇ} 2H ₂ O 4.380 1.038 ZnSO4, 7H ₂ O 0.696 - MgCl ₂ 5.142 0.888 MgSO ₄ 7H ₂ O 5.634 1.872 Na ₂ HPO ₄ 4.392 0.762 FeSO ₄ 7H ₂ O 4.584 0.612 K ₂ HPO ₄ 5.952 2.010 KH ₂ PO ₄ 4.986 1.464

Example 4 Fermenter Culture

In the fermenter culture, the fermenter is a 3L (working volume 2L) Jar fer-memter (Kobiotech, Korea) equipped with a thermostat, agitation speed controller, dissolved oxygen concentration (DO) sensor and pH sensor. Oxygen was supplied to the fermentor via a filtration filter using an air compressor, and Antifoam 204 (SIGMA ALDRICH) was used to remove bubbles generated during incubation.

(1) Effect of Agitation Speed

The effects of agitation speed on mycelial growth and the production of extracellular polysaccharides of pine needles were investigated at 100 rpm, 200 rpm and 300 rpm in 5 L jar fermintion. The medium used for the culture of the fermenter was an optimal medium obtained through nutrient composition experiments in the flask culture, and the culture temperature, pH, and flow rate were maintained at 25 ° C., pH 6.5, and 0.5 vvm, respectively, during the incubation period. 11 days of incubation and mycelial growth and extracellular polysaccharide production were examined. Figure 4a relates to mycelial growth and extracellular polysaccharide production during incubation period under different agitation speed, as can be seen that the agitation speed affects mycelial growth and extracellular polysaccharide production. Increasing the agitation speed to more than 200 rpm decreases mycelial growth and extracellular polysaccharide production. These results suggest that as the agitation speed increases, the strong shear stress caused by the impeller causes damage to the mycelia in the fermenter, which negatively affects the mycelial growth and the production of polysaccharides in the cells. As shown in FIG. 4A, the maximum mycelial growth and the production of extracellular polysaccharides cultured for 8 days were obtained under an agitation speed of 200 rpm, and the yields were 7.05 g / L and 2.59 g / L, respectively. Therefore, it can be seen that the optimum agitation speed is 200 rpm.

(2) Influence of Flow

In order to investigate the effect of flow rate on mycelial growth and extracellular polysaccharide production in jar fermenter, the flow rate was investigated at 0.5 vvm and 1.5 vvm, respectively. Incubation temperature, pH, and agitation speed were adjusted to 25 ℃, pH6.5, and 200rpm to maintain constant during the incubation period.

As shown in FIG. 4B, the maximum mycelial growth and extracellular polysaccharide production obtained for 8 days were obtained under flow conditions of 1.5 vvm, and the maximum mycelial growth (7.63 g / L) and extracellular polysaccharide production (2.71 g / L) were obtained. Indicated. These results suggest that as the flow rate increases, the oxygen transfer rate in the culture increases, creating a favorable environment for mycelial growth of Baekyoung mushrooms, which in turn has a positive effect on the yield of mycelial growth and the production of extracellular polysaccharides.

Example 5 time profile under optimal culture conditions

Optimal medium obtained from nutritional requirements in Flask culture (glucose 4% (w / v), yeast extract 0.5% (w / v), malt extract 0.1% (w / v), K ₂ HPO ₄ 0.1% (w) / v) and MgSO ₄ · 7H ₂ O 0.05% (w / v)) 2.0L in 5L jar fermintation and incubated for 12 days at 25 ℃, pH 6.5, agitation speed 200rpm, and areation 1.5vvm conditions The extracellular polysaccharide production and mycelial glucose consumption of mycelial growth were investigated over time.

Figure 5 shows the mycelial growth and extracellular polysaccharide production and residual glucose concentration of pine needles butterfly according to the incubation time. As shown in the results, the mycelial growth was sharply increased after 3 days of incubation time, showing the maximum mycelial growth (12.84 g / L) at 11 days of incubation time. The extracellular polysaccharide began to show production of 2.78 g / L at 4 days of culture time and the maximum extracellular polysaccharide production (4.85 g / L) at 11 days of culture, after which the extracellular polysaccharides were inhibited by mycelial growth. It can be seen that polysaccharide production is also declining. In addition, when the mycelial growth of pine beetle mycelium began to increase rapidly, the use of glucose, a substrate in the medium, was rapidly increasing. During the incubation period, the glucose concentration in the medium decreased from 50 g / L to 21.3 g / L.

Example 6 Development of Mycelial and Extracellular Polysaccharide Growth Production Model

In this study, logistic model was used as a model to express mycelial growth in Jar fermenter. This model was used to represent growth models for Ganoderma lucidum and other basidiomycetes. The growth characteristics of pine leaf butterfly are similar to that of ganoderma lucidum mushroom, so the logostic model is used.

In formulas (1) to (3), X is the cell mass, P is the extracellular polysaccharide concentration, μ is the specific growth rate, α is the substrate consumption coefficient of proliferative linkage, and β is the polysaccharide production coefficient of non-proliferative linkage. Each model variable μ _max , χ _m , α and β values were calculated from the FIG. 6A batch data. The change in specific growth rate according to the change in cell mass is shown in FIG. 6A. As a result of the experiment, it can be seen that the specific growth rate decreases with the increase of cell mass, and their correlation can be expressed as eq. (2). μ _max and χ _m are 0.46 / hr and 11.5g / L, respectively.

α and β were calculated from the relationship between specific growth rate and specific production rate. Figure 6b shows the change in specific production rate with the change in specific growth rate. Here, it can be seen that as the specific growth rate increases, the specific production rate increases proportionally. The slope α is 1.17, and extracellular polysaccharides can be considered to depend only on mycelial growth, so β was concluded to be zero.

Example 7 Air-Bubble bioreactor

Optimal medium obtained from nutritional requirements in Flask culture (glucose 4% (w / v), yeast extract 0.5% (w / v), malt extract 0.1% (w / v), K ₂ HPO ₄ 0.1% (w / v) and MgSO ₄ · 7H ₂ O 0.05% (w / v)) 2.0 L into 3L air-bubble bioreactor and incubated for 11 days under areation 0.0 ~ 2.0 vvm at 25 ℃ and pH 6.0 Extracellular polysaccharide production of mycelial growth was investigated.

Figure 7a ~ 7d shows the mycelial growth and extracellular polysaccharide production of pine mushrooms according to the incubation time. FIG. 8 shows the results of mycelial growth in FIGS. 7A to 7D. As shown in the results, the mycelial growth was rapid from 4 days after the incubation time, showing the maximum mycelial growth (8.5 g / L) at about 10 days of incubation time, after which the mycelial growth was somewhat suppressed. In addition, the production of 2.95 g / L at about 11 days of the extracellular polysaccharide incubation time, the production of extracellular polysaccharides was hardly produced as the mycelial growth was suppressed thereafter. In addition, as shown in FIG. 8, mycelial growth was active at relatively low aeration from incubation day 4 to 4 days after inoculation, but mycelial growth was good at 7 days as the growth rate of mycelium was relatively high. From the time of growth, relatively high aeration volume is required.

As described above, the present invention provides a medium composition for cultivating pine myrtle mushrooms and culture optimum conditions, thereby enabling the production of pine myrtle mushroom mycelium and extracellular polysaccharides in large quantities.

Claims (5)

Glucose 3 ~ 4% (w / v), yeast extract 0.4 ~ 0.6% (w / v), malt extract 0.05 ~ 0.15% (w / v), K ₂ HPO ₄ Medium composition medium composition for the production of mycelium of pine needles butterfly, characterized in that 0.05 ~ 0.15% (w / v) and MgSO ₄ · 7H ₂ O 0.03 ~ 0.05% (w / v). A method for producing mycelium and extracellular polysaccharides by cultivating pine needles mushrooms in the medium of claim 1. The method of claim 2, wherein the pH of the medium is 6.0 ± 0.2, the culture temperature is 25 ± 0.5 ℃ method for producing mycelium and extracellular polysaccharides. The method for producing mycelium and extracellular polysaccharides according to claim 2, wherein the culturing is at a stirring speed of 140 to 150 rpm in a stirring incubator. The method of claim 2, wherein the culture is a fermentation or bioreactor production method of mycelium and extracellular polysaccharides, characterized in that the culture at agitation speed 190 ~ 200rpm, aeration amount 1.4 ~ 1.5 vvm.

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