JP2002315570A - Method for culturing phellinus linteus producing sod- like substance and method for producing sod-like substance - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for culturing Phellinus linteus producing a superoxide dismutase(SOD)-like substance in large amounts and a method for producing the SOD-like substance. SOLUTION: (1) This method for culturing a mycelium of Phellinus linteus producing the SOD-like substance is characterized by inoculating the mycelium of Phellinus linteus into a liquid medium and culturing the mycelium under conditions of the following (a) and (b): (a) feeding oxygen to the liquid medium during culture; (b) irradiating the liquid medium with light. (2) This SOD-like substance is obtained from the mycelium and the culture solution.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a Phelli which produces a large amount of superoxide dismutase (SOD) -like substance.
The present invention relates to a method for culturing nus linteus and a method for producing an SOD-like substance.

[0002]

2. Description of the Related Art SOD (superoxide dismutase) is an enzyme that eliminates (reduces) superoxide (O2...), One of active oxygen and free radicals, and converts it into hydrogen peroxide (H2 O2). 2. Description of the Related Art Conventionally, a wide variety of diseases related to the fate of active oxygen, such as singlet oxygen (1O2), superoxide, hydrogen peroxide, and hydroxyl radical (HO.), Have been known. The development of pharmaceuticals and foods that eliminate active oxygen has been awaited.

[0003]

[Problems to be Solved] The present inventors, Phellinus lint
When studying a method to obtain a large amount of eus (meshimakobu) mycelium in a liquid medium, culturing while irradiating the medium with light produces a large amount of SOD-like substance in the mycelium and the culture solution And completed the present invention.

[0004]

The present invention provides the following (1).
To (6). Claim 1: Phellinus linteus inoculating a mycelium of Phellinus linteus in a liquid medium and culturing it under the following conditions (a) and (b), wherein Phellinus linteus produces a SOD (superoxide dismutase) -like substance. Culture method of mycelium. (A) Supplying oxygen to the liquid medium being cultured. (B) irradiating the liquid medium with light. Claim 2: Phellinus lint producing a SOD-like substance, wherein the mycelium of Phellinus linteus is inoculated into a liquid medium and cultured under the following conditions (a) to (c).
Eus mycelium culture method. (A) Supplying oxygen to the liquid medium being cultured. (B) irradiating the liquid medium with light. (C) Convection of the liquid medium. Claim 3: An SOD-like substance obtained from the mycelium of Phellinus linteus obtained by the culture method of claim 1 or claim 2. Claim 4: A SOD-like substance obtained from the culture solution of Phellinus linteus obtained by the culture method of claim 1 or 2. Claim 5: A method for producing an SOD-like substance, wherein the mycelium of Phellinus linteus obtained by the culture method of claim 1 or 2 is purified by the following steps. Step A: Step of extracting mycelium with ethanol and removing ethanol Step B: Step of extracting the substance obtained in Step A with ethyl acetate Claim 6: Obtained by the culture method of Claim 1 or Claim 2 A method for producing a SOD-like substance, comprising extracting and purifying a culture solution of Phellinus linteus with ethyl acetate.

[0005]

DESCRIPTION OF THE PREFERRED EMBODIMENTS (a) Phellinu used in the present invention
s linteus was collected in October 1998 at Suki-mura, Nishimoro-prefecture, Miyazaki Prefecture, Japan.
PL-
The strain stored as 08 strain was used. This strain has a yellow-brown bristle body, which is peculiar to the fruit body of Mesimakob, based on the identification of the fruiting body by Dr. Yasuhisa Abe of the Rot and Disease Laboratory, Forestry and Forestry Department, Forestry and Forestry Agency, Ministry of Agriculture, Forestry and Fisheries. And the form identified from the above as Mesimakobu was used. The preculture of the test strain was carried out by storing the mycelium stored at a low temperature of 5 ° C. in a Petri dish having an inner diameter of 90 mm.
Inoculate Potato Dextrose Agar medium (Difco),
The surface was cultured in the dark at 25 ° C. for 15 days. This cultured mycelium was cut off with a cork borer having an inner diameter of 5 mm (corresponding to a dry mycelium weight of 0.35 mg) and subjected to a test.

(1) Phellinus linteus
The effect of light on mycelium growth of E. coli was investigated. Culture conditions are as follows. 300ml of the following medium
Into a 500 ml Erlenmeyer flask sterilized by dispensing
Inoculated with linus linteus (Meshimakobu), 0.22 μm
Sterile air passed through the filter was ventilated at a rate of 0.5 l / min, and the amount of grown Sesamekob was measured. (A) Medium composition Glucose: 4%, Yeast extract: 0.3%, Peptone: 0.3%, KH2 PO4: 0.05%, Na2 HPO4: 0.05% (pH 5.5) (b) Culture conditions (A) Culture temperature: 25 ° C (b) Culture scale: 500 ml Erlenmeyer flask, medium 300 ml (c) Aeration rate: 0.5 l / min (d) Culture days: 40 days (e) Irradiation conditions: Light source: EYELA LED light source, red LED (660 nm ) Illuminance: 8 categories of 0 to 4000 LUX below 0, 100, 500, 1000, 1500, 2000, 3000, 4000 (LUX) Irradiation time: 8 hours / DAY,

The results are shown in FIG. 1 (Effect of light irradiation on growth of mycelium of Phellinus linteus).
According to FIG. 1, it is found that when the irradiation intensity exceeds 1000 (LUX), the bacterial cell yield decreases. The light source used in the present invention is not limited to the above light source, and it is sufficient to irradiate ordinary visible light.
(470 nm), the growth of mycelium is slowed down and tends to brown (age).
(735 nm) tended to have slow mycelium growth.

[0008] (2) Phellinus linteus is cultured under the conditions of the medium composition of the above (a) and the following (b).
A dried mycelium of (Meshimakobu) was obtained (10.2 g /
L). The obtained mycelium is hereinafter referred to as “PL-L”. (A) Medium composition: Same as (A) above. (B) Culture conditions (a) Culture temperature: 25 ° C (b) Culture scale: 1000 L (see FIG. 2) (c) Aeration rate: 35 L / min (d) Culture days: 20 days (e) Irradiation conditions: Light source: EYELA LED light source, red LED (660 nm) Illumination: 1000 LUX Irradiation time: 8 hours / DAY Illumination: 1000 LUX Meanwhile, the same medium composition and culture conditions (a) to (d) as described above
Was used to culture Phellinus linteus (meshimakobu) without irradiating light to obtain a dried mycelium (8.3 g / ml).
L). The obtained mycelium is hereinafter referred to as “PL-nonL”
Is displayed. Also, for example, "PL-nonL-20da
"y" represents a mycelium cultured for 20 days without light irradiation.

(C) The mycelium cultured under the above conditions was separated by a centrifuge, ethanol was added thereto, the mycelium was crushed by a mixer, and the mixture was allowed to stand for 24 hours. Thereafter, an ethanol fraction was collected by a centrifugal separator, ethanol was removed, and the residue was extracted with ethyl acetate. Then, ethyl acetate was removed to obtain a sample for measuring an antioxidant active substance. In addition, the sample for antioxidant active substance measurement was prepared also about what processed the culture medium (medium) before inoculation of meshimakobu with ethanol and ethyl acetate like mycelium. Two samples of the mycelium ("PL-nonL-20day"),
The ethanol solutions of “PL-L-20day”) and “medium” were subjected to electron spin resonance (ESR) to determine the superoxide (O 2.
Behavior). The measurement of superoxide (O 2 .-) by the electron spin resonance (ESR) method is performed using a spin trapping agent, DMPO (5,5-dimethyl-pyrroline-N-oxid).
Method e) (edited by Kazuo Ouchi, edited by Laboratory for Biopharmaceutical Chemistry, Inflammation and Allergy, p. 218-p. 223 (Hirokawa Shoten), BullL. Chem. Soc. Jpn., 63, 187-191 (1990), BIOC
HEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL Vol.4
2, No. 1, P. 35-44, (1997)).

FIG. 3 shows the results. According to FIG. 3, Phellinus linteus (meshimakobu) cultured by irradiating light
It can be seen that the mycelium (PL-L-20day) has a higher superoxide (O2.-) elimination rate than the mycelium (PL-nonL-20day) cultured without irradiating light. .

(3) Phellinus linteus in paragraph 0008
(Meshimakobu) The concentration test of the SOD-like substance was performed on the mycelium and the culture filtrate obtained by the culture (irradiated with light). (A) For mycelium, "PL-
F1 "," PL-F2 "," PL-F3 "," PL-F
4 "was obtained. (B) Further, regarding the culture filtrate from which the mycelium was removed, the “PL-F5”, “PL-F6”, “PL-F6”
-F7 "was obtained.

These "PL-F1" to "PL-F7"
In the same manner as in paragraph 0009, the seven fraction components of
2) The erasure rate (SOD-like action) was determined. The result is shown in FIG. According to FIG. 6, the superoxide (O 2.
) The value of the erasure rate is “PL-F2” and “PL-F6”.
It can be seen that the value of the two fraction components is larger than the other components.

FIGS. 7 to 18 show actual measurement charts of superoxide (O 2 · −) by the electron spin resonance (ESR) method. 7 to 10 show the measurement results that are the basis for calculating the superoxide erasure rate (%) shown in FIG. 3, and FIGS. 11 to 18 show the calculation basis for the superoxide erasure rate (%) shown in FIG. FIG.

[0014]

As described above, according to the method for culturing Phellinus linteus (meshimakobu) and the method for producing the SOD-like substance of the present invention, which produce the SOD-like substance, as described above.
nteus (meshimakobu) mycelium culture and its culture solution can produce a large amount of SOD-like substances,
This has the effect that the produced SOD-like substance can be efficiently concentrated.

[Brief description of the drawings]

FIG. 1 is a view showing the effect of light irradiation on the growth of mycelium of Phellinus linteus (Phellinus linteus).

FIG. 2 is a schematic view of an apparatus for culturing Phellinus linteus (Phellinus linteus).

FIG. 3 is a graph showing values of superoxide (O 2 · −) erasure rate.

FIG. 4 is a schematic diagram showing a procedure for concentrating SOD-like substances from mycelium.

FIG. 5 is a schematic diagram showing a procedure for concentrating a SOD-like substance from a culture solution.

FIG. 6 is a diagram showing values of a superoxide (O 2 · −) erasing rate.

FIG. 7 is a diagram (control) in which a sample-free superoxide is measured by an electron spin resonance (ESR) method.

FIG. 8 is a diagram showing the superoxide erasing ability of “medium” determined by an electron spin resonance (ESR) method.

FIG. 9 is a view showing a superoxide erasing ability of “PL-nonL-20day” measured by an electron spin resonance (ESR) method.

FIG. 10 is a view showing a superoxide erasing ability of “PL-L-20day” measured by an electron spin resonance (ESR) method.

FIG. 11 is a diagram (control) in which a sample-free superoxide is measured by an electron spin resonance (ESR) method.

FIG. 12 is a view showing a superoxide erasing ability of “PL-F1” measured by an electron spin resonance (ESR) method.

FIG. 13 is a view showing a superoxide erasing ability of “PL-F2” measured by an electron spin resonance (ESR) method.

FIG. 14 is a view showing the superoxide erasing ability of “PL-F3” measured by an electron spin resonance (ESR) method.

FIG. 15 is a view showing the superoxide erasing ability of “PL-F4” measured by an electron spin resonance (ESR) method.

FIG. 16 is a view showing a superoxide erasing ability of “PL-F5” measured by an electron spin resonance (ESR) method.

FIG. 17 is a view showing a superoxide erasing ability of “PL-F6” measured by an electron spin resonance (ESR) method.

FIG. 18 is a view showing a superoxide erasing ability of “PL-F7” measured by an electron spin resonance (ESR) method.

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 4B050 CC01 DD03 EE04 4B065 AA71X AC14 BA22 BC06 BC08 BC26 BC48 CA28

Claims (6)

[Claims] 1. A Phellinus linteus producing a SOD (superoxide dismutase) -like substance, wherein a mycelium of Phellinus linteus is inoculated into a liquid medium and cultured under the following conditions (a) and (b). Culture method of mycelium. (A) Supplying oxygen to the liquid medium being cultured. (B) irradiating the liquid medium with light. 2. A Phellinus producing a SOD-like substance, wherein a mycelium of Phellinus linteus is inoculated into a liquid medium and cultured under the following conditions (a) to (c).
Culture method of linteus mycelium. (A) Supplying oxygen to the liquid medium being cultured. (B) irradiating the liquid medium with light. (C) Convection of the liquid medium. 3. An SO obtained from the mycelium of Phellinuslinteus obtained by the culture method according to claim 1 or 2.
D-like substance. 4. An SO obtained from a culture of Phellinuslinteus obtained by the culture method according to claim 1 or 2.
D-like substance. 5. A method for producing an SOD-like substance, wherein the mycelium of Phellinuslinteus obtained by the culture method according to claim 1 or 2 is purified by the following steps. Step A: Step of extracting mycelium with ethanol and removing ethanol Step B: Step of extracting the product obtained in Step A with ethyl acetate 6. A method for producing a SOD-like substance, comprising extracting a culture of Phellinuslinteus obtained by the culture method according to claim 1 or 2 with ethyl acetate and purifying the extract.