CN103130550B - Culture medium and culture method of male agaric mycelium - Google Patents

Abstract

The invention provides a culture medium and a preparation method of male agaric mycelium, and a method for culturing the male agaric mycelium by using the culture medium. The culture medium comprises rice and a culture solution. As rice is used as the raw material of the culture medium, the raw material is available and is economic, the operation is simple, and the effect is good.

Description

A kind of substratum of phellinus igniarius mycelium and cultural method

Technical field

The present invention relates to edible mushrooms and cultivate field, relate to a kind of substratum and cultural method of phellinus igniarius mycelium specifically.

Background technology

Bao nurse Phellinus (Phellinus baumii), is commonly called as Phellinus mushroom, mulberry ear, is the perennial medicinal fungi of one developed in recent years, records the earliest in LI Shi-Zhen Compendium of Material Medica.Traditional Chinese Medicine thinks that Phellinus is sweet, flat, bitter, pungent, return liver, urinary bladder channel, be used for the treatment of metrorrhagia, blood drenches, prolapse of the anus rushes down blood, under band, amenorrhoea, diarrhea due to hypofunction of the spleen etc.Numerous experimental studies shows, the polysaccharide in Phellinus and flavonoid compound have important biological activity, plays an important role in Therapeutic cancer, liver protecting, enhancing immunity, CO2 laser weld.

Free radical is the product of human metabolism, and generally, human body has the ability to remove the free radical self produced, but along with the aging of human body, the reduced capability of scavenging free radicals.If the free radical produced can not be removed in time, will body aging be accelerated, even make human body generation pathology etc.Now there are some researches show: the polysaccharide in Medicinal Fungus Phellinus igniarius and Flavonoid substances have good antioxygenation; But present research focuses mostly in the research of Phellinus fruiting body extract and liquid fermenting, and less to the research of solid fermentation phellinus igniarius mycelium and antioxygenation thereof.Wild sporophore is mainly distributed in the forest zone such as Heilungkiang, Shaanxi, and resource standing stock are rare, cause expensive owing to yielding poorly.Current Phellinus sporophore mainly obtains by artificial culture, but the artificial culture cycle is long, needs larger manpower financial capacity to drop into.By contrast, fermentation technique has cycle short, the advantage such as controllability good, experimental situation is simple, is the good approach obtaining Phellinus secondary metabolite.And liquid fermenting easily pollutes in the process of cultivating, the biomass of expection can not be reached in certain incubation time, badly in culturing process to control artificially.

Summary of the invention

An object of the present invention is the substratum providing a kind of phellinus igniarius mycelium, and this substratum comprises following component:

Rice and nutrient solution, wherein the weight (g) of rice and the volume (ml) of nutrient solution are than being x:(x+(0-30), wherein x >=10;

Wherein nutrient solution contains: 2.5% glucose, 0.3%KH ₂pO ₃and 0.15%MgSO ₄7H ₂o.

Above-mentioned substratum is prepared by the following method:

Configuration nutrient solution (2.5% glucose, 0.3%KH ₂pO ₃, 0.15%MgSO ₄7H ₂o), rice is placed in nutrient solution, autoclaving.

Present invention also offers a kind of method utilizing above-mentioned culture medium culturing phellinus igniarius mycelium, the method comprises the steps:

Phellinus mother is planted and is inoculated on above-mentioned substratum, be placed in 26 DEG C of biochemical cultivation cases and cultivate, growth cycle 25-30 days;

Cultured mycelium is dried as in 30 DEG C of convection oven together with substratum.

Phellinus igniarius mycelium after above-mentioned rice medium fermentation has oxidation resistant biological action, can be used as foodstuff additive application, improves Nutritive value of food.

Innovation of the present invention:

1, use substratum of the present invention to cultivate, relative liquid cultivates the advantage having convenient and easy, save energy, not easily pollute.

2, substratum of the present invention, adopt rice as raw material not only material easily get, and very economical, respond well.Very large in the energy consumption of liquid culture medium power, and the present invention cultivates bacterial classification and only needs quiescent culture at a certain temperature, save the energy, and growth conditions is also better than liquid culture.Found by anti-oxidant experiment, the mycelium of cultivation has stronger antioxidant effect simultaneously.Nowadays foodstuff additive are subject to extensive concern, and rice is as a kind of food of nutrient safe, and the mycelium of cultivation can be used as additive to improve Nutritive value of food.

Embodiment

Phellinus bacterial classification (Phellinus baummii Pilat from edible mushrooms branch center, Shanghai, Chinese microorganism strain preservation center, is numbered: Phellinus baumii Pilat3249).

Embodiment 1-4 prepares substratum

Configure 2.5% glucose, 0.3%KH ₂pO ₃, 0.15%MgSO ₄7H ₂the nutrient solution of O, gets 60ml respectively in 250ml culturing bottle, then take 30 respectively, 40,50, the rice of 60g is placed in nutrient solution, soaks 6 hours, 121 DEG C of autoclaving 30min.Rice (the fragrant round-grained rice in the profit village) is provided by Shang Hairun village agricultural science and technology company limited.

Table 1 rice loading amount is on the impact of biomass

Note: the rate of recovery of biomass refers to the ratio of the mycelium after finally drying and rice loading amount.

Embodiment 5 substratum of embodiment 3 is cultivated

Be longer than by Phellinus bacterial classification on PDA inclined-plane, growth cycle is one week.

Get above-mentioned Phellinus strain inoculation on the substratum of embodiment 1, be placed in 26 DEG C of biochemical cultivation cases and cultivate, growth cycle 25-30 days.

Take out phellinus igniarius mycelium, dry as in the convection oven of 30 DEG C.

The mensuration of flavones content in embodiment 6 mycelium alcohol extract

The mycelium of oven dry is added 80% ethanol of 10 times of volumes, soak 24h, filter; Collect filtrate, residue spends the night by 80% alcohol immersion of 10 times of volumes again, repeats aforesaid operations, merges twice filtrate, concentrated, obtains phellinus igniarius mycelium alcohol extract.

Use NaNO ₂-Al (NO ₃) ₃colorimetric method for determining flavones content, in the mycelium alcohol extract after oven dry, the weight percent content of flavones is 40%.

The mensuration of embodiment 7 phellinus igniarius mycelium anti-oxidant activity

The source of phellinus igniarius mycelium alcohol extract is with in embodiment 6; Measure the anti-oxidant activity of phellinus igniarius mycelium alcohol extract, mainly carry out removing hydrogen peroxide, superoxide anion and DPPH experiment.

Concrete grammar is as follows

One, H is removed ₂o ₂free radical is tested

Alcohol extract 70% ethanol is mixed with four concentration, is 1 μ g/mL respectively, 10 μ g/mL, 20 μ g/mL, 50 μ g/mL.To H ₂o ₂removing experiment use luminol,3-aminophthalic acid cyclic hydrazide-H ₂o ₂luminous reaction system, with 80% ethanol as blank.Get 5 μ L respectively and add 96 orifice plates, pump into 150 μ L luminol,3-aminophthalic acid cyclic hydrazides-CBS solution (pH9.5) by pump 1, pump 2 pumps into 10 μ L6%H ₂o ₂, start luminous reaction at 20 DEG C, every 0.6s records a luminous value, continuous recording 30s.Light-emitting procedure is as table 3-1:

Table 3-1H ₂o ₂the light-emitting procedure of free radical

H ₂o ₂free radical scavenging activity calculation formula is as follows

Result shows, the phellinus igniarius mycelium alcohol extract of high density is to H ₂o ₂the clearance rate of free radical reaches 80%.

Two, superoxide anion experiment is removed

Alcohol extract 80% dissolve with ethanol is mixed with different concentration, i.e. 500 μ g/mL, 800 μ g/mL, 1000 μ g/mL, 2000 μ g/mL, do blank with 80% ethanol.Adopt the chemoluminescence method of luminol,3-aminophthalic acid cyclic hydrazide-pyrogallol-CBS system.Each concentration samples is got 10 μ L respectively and is added 96 orifice plates.6.25 × 10 are pumped into by pump 1 ^-4mol/L pyrogallol solution, pump 2 pumps into 150 μ L luminol,3-aminophthalic acid cyclic hydrazides-CBS solution (pH10.2).Start luminous reaction at 20 DEG C, every 0.6S collects an optical signal, collects 30S continuously.

Light-emitting procedure is as table 3-2:

Table 3-2 removes the light-emitting procedure of superoxide anion

The clearance rate formula of superoxide anion is with removing H ₂o ₂free radical scavenging activity formula

Result shows, when lower concentration 0.5mg/mL, just show the ability of higher removing superoxide anion, concentration is when 2mg/mL, and clearance rate is close to 100%.

Three, the experiment of DPPH free radical is removed

Get 6 × 10 ^-4the storing solution 100 μ L of mol/L DPPH adds 900 μ L80% ethanol, is diluted to 6 × 10 ^-5mol/L DPPH working fluid.With 80% ethanol, mycelium alcohol extract sample preparation is become different concns (1 μ g/mL, 20 μ g/mL, 100 μ g/mL, 200 μ g/mL).The sample getting 500 μ L different concns respectively adds in 1mL DPPH working fluid, mixing, measures light absorption value, using 80% ethanol as blank after leaving standstill 20min in 517nm place.DPPH free radical scavenging activity is calculated as follows:

Result shows, the clearance rate of alcohol extract to DPPH free radical of high density reaches 40%.

Claims (3)

1. a substratum for phellinus igniarius mycelium, is characterized in that this substratum comprises following component:

Rice and nutrient solution, wherein the weight (g) of rice and the volume (ml) of nutrient solution are than being x:(x+ (0 ~ 30)), wherein x=30 ~ 60;

Wherein nutrient solution contains: 2.5% glucose, 0.3%KH ₂pO ₃and 0.15%MgSO ₄7H ₂o.

2. prepare a method for substratum described in claim 1, it is characterized in that the method is:

Configuration nutrient solution, is placed in nutrient solution by rice, autoclaving.

3. utilize a method for culture medium culturing phellinus igniarius mycelium described in claim 1, the method is:

Phellinus mother being planted is inoculated on substratum, is placed in 26 DEG C of biochemical cultivation cases and cultivates, growth cycle 25-30 days;

Cultured mycelium is dried as in 30 DEG C of convection oven together with substratum.