CN101702984A - Phellinus igniarius mycelium liquid fermentation method by utilizing fungal elicitor for improving yield of flavone of phellinus igniarius - Google Patents
Phellinus igniarius mycelium liquid fermentation method by utilizing fungal elicitor for improving yield of flavone of phellinus igniarius Download PDFInfo
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Abstract
本发明公开了一种利用真菌诱导子提高桑黄黄酮产量的桑黄菌丝体液体发酵方法，其将真菌诱导子菌种接种活化培养后，取菌块接种培养过滤得到真菌诱导子菌丝体；然后烘干研磨破碎加入乙醇浸泡，过滤并收集滤渣，滤渣依次加入氯仿和正丁醇的混合液、丙酮、水冲洗，风干后干物质加入三氟乙酸，沸水浴0.5-2小时，然后过滤，取滤液中和、静置、0.45μm微孔滤膜过滤除菌，制成真菌诱导子，-10℃～-20℃冷冻保存；将桑黄菌种接种活化培养，后取菌块接种发酵培养；在桑黄液体发酵培养第1～5天，加入真菌诱导子继续培养得到桑黄菌丝体，本发明工艺简单、成本低、效果显著，可大幅度提高桑黄细胞黄酮含量，是一种极具工业化前景的生产方法。The invention discloses a method for liquid fermentation of Phellinus mycelium by using fungal elicitors to increase the output of Phellinus flavonoids. After inoculating and activating the fungal elicitor strains, the fungal elicitor mycelium is obtained by inoculating, culturing and filtering fungal elicitors Then dry, grind and break, add ethanol to soak, filter and collect the filter residue, add the mixed solution of chloroform and n-butanol, acetone, and water to wash the filter residue in turn, add trifluoroacetic acid to the dry matter after air drying, bathe in boiling water for 0.5-2 hours, and then filter, Take the filtrate for neutralization, let it stand, filter and sterilize with a 0.45 μm microporous membrane to make a fungal inducer, and store it in a freezer at -10°C to -20°C; inoculate and activate the Phellinus fungus, and then take the bacteria block to inoculate the fermentation culture ; In the 1st to 5th days of Phellinus liquid fermentation culture, adding fungal elicitors to continue culturing to obtain Phellinus mycelium, the invention has simple process, low cost and remarkable effect, can greatly improve the flavonoid content of Phellinus cells, and is a kind of A production method with great industrialization prospects.
Description
Technical field:
The invention belongs to technical field of bioengineering, be specifically related to a kind of liquid fermentation method that utilizes fungal elicitor to improve flavonoids from phellinus output.
Background technology:
Phellinus (Phellinus igniarius) is Basidiomycotina (Basidiomycotina); Hymenomycetes (Hymenomycetes); Aphyllophorales (Polyporales); rust leather pore fungi section (Hymenochaetacae); Phellinus (Phellinus) fungi is a kind of large-scale medicinal fungi of preciousness.The tradition traditional Chinese medical science is thought sweet, the bitter of Phellinus, is used for the treatment of under metrorrhagia, prolapse of the anus, the band, amenorrhoea, splenasthenic diarrhea etc.Modern pharmacology studies show that, effect such as that Phellinus has is antitumor, enhancing immunity, anti-hepatic fibrosis.The Phellinus main active is polysaccharide, triterpene substance and Flavonoid substances.Wherein flavonoids has anti-oxidant, hemangiectasis, reducing blood lipid and effect such as anticancer, generally is to extract to obtain from natural plants, and Phellinus is the rare medicinal fungi that contains flavonoids.When the Phellinus medical value was extensively disclosed, the output of Phellinus became the limiting factor that suppresses the Phellinus application.Owing to be subjected to particularity and the complexity and the outside factors of physiological status, cause Phellinus to form the fruit body rareness at occurring in nature, particularly forming available fruit body needs for many years.And artificial cultivation is extremely difficult, the condition of culture harshness, and growth cycle reaches 3～4 years, is difficult to satisfy the market demand that expands day by day.Therefore, need seek out a kind of method of effective production flavonoids from phellinus, to overcome prior art and to produce the contradiction that needs.Solution fermentation production Phellinus active ingredient since with short production cycle, labour economize and be subjected to advantages such as external environment influence is little to be considered to a kind of effective method.Utilize fungal elicitor to be used for the Phellinus fermented and cultured, have not yet to see report with the research that improves secondary metabolites such as flavonoids from phellinus output.Therefore, set up the Phellinus liquid fermentation by the biotechnology approach and induce system, further improve the content of cytoflav letones, have important significance for theories and using value.
Summary of the invention:
The objective of the invention is to overcome the deficiency of above-mentioned prior art and provide that a kind of technology is simple, cost is low, effect is remarkable; the fungal elicitor that utilizes that increases substantially flavonoids from phellinus output improves the phellinus igniarius mycelium liquid fermentation method of flavonoids from phellinus output, and this method can be used for the suitability for industrialized production of flavonoids from phellinus.
Purpose of the present invention can reach by following measure: a kind of phellinus igniarius mycelium liquid fermentation method that utilizes fungal elicitor to improve flavonoids from phellinus output is characterized in that comprising the steps:
(1) preparation of fungal elicitor:
1. the cultivation of fungal elicitor bacterial strain: with the fungal elicitor bacterial classification inoculation on the PDA slant medium activation after, be transferred on the PDA plating medium, cultivated 3～5 days for 25～30 ℃, getting the bacterium piece is inoculated in the triangular flask that the PD liquid nutrient medium is housed, after in 25～30 ℃, 100～180r/min shaking table, cultivating 5～7 days, filter that to obtain the fungal elicitor mycelium standby;
2. prepare fungal elicitor: the fungal elicitor mycelium is dried down in 60～70 ℃, grind broken, (g: it is 65～85% ethanol that ml) 1: 2～1: 5 ratio adds concentration to volume ratio by weight, at room temperature soaked 10～15 hours, filter and collect filter residue, filter residue is volume ratio (g: ml) 1: 2～1: 5 the ratio adding chloroform and the mixed liquor flushing of n-butanol by weight, the volume ratio of chloroform and n-butanol is 3: 1～5: 1, wash filter residue so repeatedly 3～5 times, then with filter residue by weight volume ratio (g: ml) 1: 3～1: 5 ratio with acetone rinsing after, filter residue is volume ratio (g: ml) 1: 3～1: 5 ratio water flushing more by weight, natural air drying, dry matter by weight volume ratio (g: it is 5～15% trifluoroacetic acids that ml) 1: 5～1: 7 ratio adds concentration, boiling water bath 0.5～2 hour, filter then, it is 4～7 that filtrate is neutralized to pH with NaOH, under 3 ℃～8 ℃, left standstill 10～15 hours, with 0.45 μ m filtering with microporous membrane degerming, make fungal elicitor ,-10 ℃～-20 ℃ freezing preservations;
(2) liquid fermentation and culture of Phellinus:
With the Phellinus bacterial classification inoculation of slant preservation to the PDA plating medium, carry out activation culture, 25～30 ℃ of cultivation temperature, incubation time 5～10 days, getting the bacterium piece then is inoculated in and carries out fermented and cultured in the triangular flask that the PD liquid fermentation medium is housed, shaking speed 100～180r/min, 25～30 ℃ of temperature, 5～8 days time;
(3) fungal elicitor is to the inducing culture of Phellinus cell:
Phellinus liquid fermentation and culture the 1st～5 day, add fungal elicitor, make the concentration of fungal elicitor reach 40～200 μ g/mL, continue then to cultivate 2～7 days, obtain phellinus igniarius mycelium.
In order further to realize purpose of the present invention, 3～5 of the bacterium pieces of cut-off footpath 0.3～0.6cm are inoculated in the PD liquid nutrient medium the bottled PD liquid nutrient medium of every 500mL triangle 100～200mL in the incubation step of described fungal elicitor bacterial strain.
In order further to realize purpose of the present invention, 3～5 of the bacterium pieces of cut-off footpath 0.3～0.6cm are inoculated in the PD liquid fermentation medium the bottled PD liquid nutrient medium of every 500mL triangle 100～200mL in the liquid fermentation and culture step of described Phellinus.
In order further to realize purpose of the present invention, described fungal elicitor continues to cultivate to the adding in the 1st day of fungal elicitor in the Phellinus inducing culture step in the Phellinus fermented and cultured then.
In order further to realize purpose of the present invention, described fungal elicitor continues to cultivate to the adding in the 3rd day of fungal elicitor in the Phellinus inducing culture step in the Phellinus fermented and cultured then.
In order further to realize purpose of the present invention, described fungal elicitor continues to cultivate to the adding in the 5th day of fungal elicitor in the Phellinus inducing culture step in the Phellinus fermented and cultured then.
The present invention can produce following good effect compared with the prior art:
1, the present invention is used for the liquid fermentation production of flavonoids from phellinus with fungal elicitor, increases substantially flavonoids from phellinus output, and output is reached more than the 340mg/L, is 4.5 times of contrast.This method technology is simple, cost is low, effect is remarkable, and flavonoids from phellinus content height is a kind of production method that has industrial prospect.The flavonoids from phellinus of being produced can be used for preparing anti-oxidant, anti-senile preparation, medicine, the additive of prevention or treatment tumour.
2, fungal elicitor bacterium source of the present invention is extensive, and the elicitor of edible mushroom disease fungus preparation all has and well induces effect mostly, and the elicitor preparation method is easy, good reproducibility.
3, Phellinus liquid fermentation process of the present invention is simple, and the cost of raw material is cheap, and the what is more important whole fermentation process is controlled, not limited by external environment condition, is fit to very much suitability for industrialized production.
Embodiment: following the specific embodiment of the present invention is elaborated:
Embodiment 1:
Bacterial classification: the fungal elicitor bacterial classification is Acremonium fungi top spore (Acremoniumstrictum), is bought by Chinese common micro-organisms culture presevation administrative center, and bacterium numbering is CGMCC No.3.2058.
The Phellinus bacterial classification is common cultivation kind (Phellinus igniarius), available from Chinese common micro-organisms culture presevation administrative center, is numbered CGMCC No.51328.
A kind of phellinus igniarius mycelium liquid fermentation method that utilizes fungal elicitor to improve flavonoids from phellinus output is characterized in that comprising the steps:
(1) preparation of fungal elicitor:
1. the cultivation of fungal elicitor bacterial strain: with the fungal elicitor bacterial classification inoculation on the PDA slant medium activation after, be transferred on the PDA plating medium, cultivated 5 days for 25 ℃, beat 3 of the bacterium pieces of cut-off footpath 0.3cm at colony edge with card punch, be inoculated in the PD liquid nutrient medium, the bottled PD liquid nutrient medium of every 500mL triangle (forming) 100mL for removing agar component in the component of PDA medium, in 25 ℃, 100r/min shaking table, cultivate 7 days after, filter that to obtain the fungal elicitor mycelium standby;
2. fungal elicitor preparation: the fungal elicitor mycelium is dried down in 60 ℃, grind broken, (g: (fungal elicitor: it was 65% ethanol that ratio ethanol) adds concentration to volume ratio in ml) 1: 2 by weight, at room temperature soaked 10 hours, filter and collect filter residue, filter residue is volume ratio (g: ml) 1: 2 (filter residue: the ratio adding chloroform mixed liquor of chloroform and n-butanol) and the mixed liquor flushing of n-butanol by weight, the volume ratio of chloroform and n-butanol is 3: 1, wash filter residue so repeatedly 3 times, then filter residue by weight volume ratio (g: ml) 1: 3 (filter residue: ratio acetone) with acetone rinsing after, filter residue is volume ratio (g: ml) 1: 3 (filter residue: the water flushing again of ratio water) by weight, natural air drying, dry matter by weight volume ratio (g: (dry matter: it was 5% trifluoroacetic acid that trifluoroacetic acid) ratio adds concentration in ml) 1: 5, boiling water bath 0.5 hour, filter then, it is 5.8 that filtrate is neutralized to pH with NaOH, under 3 ℃, left standstill 10 hours, with 0.45 μ m filtering with microporous membrane degerming, make fungal elicitor ,-10 ℃ of freezing preservations;
(2) liquid fermentation and culture of Phellinus:
With the Phellinus bacterial classification inoculation of slant preservation to the PDA plating medium, carry out activation culture, 25 ℃ of cultivation temperature, incubation time 10 days is beaten 3 of the bacterium pieces of cut-off footpath 0.3cm then at colony edge with card punch, be inoculated in the PD liquid nutrient medium (removing agar component for the PDA medium forms) and carry out fermented and cultured, the bottled medium 100mL of every 500mL triangle,, shaking speed 100r/min, 25 ℃ of temperature, 8 days time;
(3) fungal elicitor is to the inducing culture of Phellinus cell:
Phellinus liquid fermentation and culture the 1st day, add fungal elicitor, make the fungal elicitor final concentration reach 40 μ g/mL, continue then to cultivate 7 days, obtain phellinus igniarius mycelium.
(4) extraction of flavonoids from phellinus and detection:
The gained phellinus igniarius mycelium is dried; get phellinus igniarius mycelium powder 100mg, add 2mL 60% ethanol, in room temperature lower sealing ultrasonic Extraction 24h; filter; filtrate sealing put 4 ℃ of refrigerations down, and residue repeats aforesaid operations 3 times, merges three times leaching liquor; be settled to 50ml with 60% ethanol; as liquid to be measured, colorimetric method for determining flavonoids from phellinus content is 10.5mg/100mg mycelia (dry weight), output 270.2mg/L.
Embodiment 2:
Bacterial classification: the fungal elicitor bacterial classification is fungus Trichoderma lignin wood mould (Trichodermaviride), is bought by Chinese common micro-organisms culture presevation administrative center, and bacterium numbering is CGMCC No.3.2196.
The Phellinus bacterial classification is common cultivation kind (Phellinus igniarius), available from Chinese common micro-organisms DSMZ, is numbered CGMCC No.51328.
A kind of phellinus igniarius mycelium liquid fermentation method that utilizes fungal elicitor to improve flavonoids from phellinus output is characterized in that comprising the steps:
(1) preparation of fungal elicitor:
1. the cultivation of fungal elicitor bacterial strain: with the fungal elicitor bacterial classification inoculation on the PDA slant medium activation after, be transferred on the PDA plating medium, cultivated 3 days for 30 ℃, beat 5 of the bacterium pieces of cut-off footpath 0.6cm at colony edge with card punch, be inoculated in the PD liquid nutrient medium, the bottled PD liquid nutrient medium of every 500mL triangle (removing agar component for the PDA medium forms) 200mL, in 30 ℃, 180r/min shaking table, cultivate 5 days after, filter that to obtain the fungal elicitor mycelium standby;
2. fungal elicitor preparation: the fungal elicitor mycelium is dried down in 70 ℃, grind broken, (g: (fungal elicitor: it was 85% ethanol that ratio ethanol) adds concentration to volume ratio in ml) 1: 5 by weight, at room temperature soaked 15 hours, filter and collect filter residue, filter residue is volume ratio (g: ml) 1: 5 (filter residue: the ratio adding chloroform mixed liquor of chloroform and n-butanol) and the mixed liquor flushing of n-butanol by weight, the volume ratio of chloroform and n-butanol is 5: 1, wash filter residue so repeatedly 5 times, then filter residue by weight volume ratio (g: ml) 1: 5 (filter residue: ratio acetone) with acetone rinsing after, filter residue is volume ratio (g: ml) 1: 5 (filter residue: the water flushing again of ratio water) by weight, natural air drying, dry matter by weight volume ratio (g: (dry matter: it was 15% trifluoroacetic acid that trifluoroacetic acid) ratio adds concentration in ml) 1: 7, boiling water bath 2 hours, filter then, it is 4 that filtrate is neutralized to pH with NaOH, under 8 ℃, left standstill 15 hours, with 0.45 μ m filtering with microporous membrane degerming, make fungal elicitor ,-20 ℃ of freezing preservations;
(2) liquid fermentation and culture of Phellinus:
With the Phellinus bacterial classification inoculation of slant preservation to the PDA plating medium, carry out activation culture, 30 ℃ of cultivation temperature, incubation time 5 days is beaten 5 of the bacterium pieces of cut-off footpath 0.6cm with card punch at colony edge then, is inoculated in the PD liquid nutrient medium (for PDA medium removal agar component forms) and carries out fermented and cultured, the bottled medium 200mL of every 500mL triangle, shaking speed 180r/min, 30 ℃ of temperature, 5 days time;
(3) fungal elicitor is to the inducing culture of Phellinus cell:
Phellinus liquid fermentation and culture the 5th day, add fungal elicitor, make the fungal elicitor final concentration reach 200 μ g/mL, continue then to cultivate 2 days, obtain phellinus igniarius mycelium.
(4) extraction of flavonoids from phellinus and detection:
The gained phellinus igniarius mycelium is dried; phellinus igniarius mycelium powder 100mg adds 2mL 60% ethanol, in room temperature lower sealing ultrasonic Extraction 24h; filter; filtrate sealing put 4 ℃ of refrigerations down, and residue repeats aforesaid operations 3 times, merges three times leaching liquor; be settled to 50ml with 60% ethanol; as liquid to be measured, colorimetric method for determining flavonoids from phellinus content is 8.0mg/100mg mycelia (dry weight), output 170mg/L.
Embodiment 3:
Bacterial classification: the fungal elicitor bacterial classification is that Verticillium dahliae belongs to fungi mushroom wheel branch spore (Verticilliumpsalliotae), is bought by Chinese common micro-organisms culture presevation administrative center, and bacterium numbering is CGMCC No.3.4423.
The Phellinus bacterial classification is common cultivation kind (Phellinus igniarius), available from Chinese common micro-organisms culture presevation administrative center, is numbered CGMCC No.51328.
A kind of phellinus igniarius mycelium liquid fermentation method that utilizes fungal elicitor to improve flavonoids from phellinus output is characterized in that comprising the steps:
(1) preparation of fungal elicitor:
1. the cultivation of fungal elicitor bacterial strain: with the fungal elicitor bacterial classification inoculation on the PDA slant medium activation after, be transferred on the PDA plating medium, cultivated 4 days for 28 ℃, beat 4 of the bacterium pieces of cut-off footpath 0.4cm at colony edge with card punch, be inoculated in the PD liquid nutrient medium (removing agar component for the PDA medium forms), the bottled PD liquid nutrient medium of every 500mL triangle 150mL cultivated in 28 ℃, 150r/min shaking table after 6 days, filtered that to obtain the fungal elicitor mycelium standby;
2. fungal elicitor preparation: the fungal elicitor mycelium is dried down in 65 ℃, grind broken, (g: (fungal elicitor: it was 70% ethanol that ratio ethanol) adds concentration to volume ratio in ml) 1: 4 by weight, at room temperature soaked 12 hours, filter and collect filter residue, filter residue is volume ratio (g: ml) 1: 4 (filter residue: the ratio adding chloroform mixed liquor of chloroform and n-butanol) and the mixed liquor flushing of n-butanol by weight, the volume ratio of chloroform and n-butanol is 4: 1, wash filter residue so repeatedly 4 times, then filter residue by weight volume ratio (g: ml) 1: 4 (filter residue: ratio acetone) with acetone rinsing after, filter residue is volume ratio (g: ml) 1: 4 (filter residue: the water flushing again of ratio water) by weight, natural air drying, dry matter by weight volume ratio (g: (dry matter: it was 10% trifluoroacetic acid that trifluoroacetic acid) ratio adds concentration in ml) 1: 6, boiling water bath 1 hour, filter then, it is 7 that filtrate is neutralized to pH with NaOH, under 5 ℃, left standstill 12 hours, with 0.45 μ m filtering with microporous membrane degerming, make fungal elicitor ,-15 ℃ of freezing preservations;
(2) liquid fermentation and culture of Phellinus:
With the Phellinus bacterial classification inoculation of slant preservation to the PDA plating medium, carry out activation culture, 28 ℃ of cultivation temperature, incubation time 8 days is beaten 4 of the bacterium pieces of cut-off footpath 0.4cm with card punch at colony edge then, is inoculated in the PD liquid nutrient medium (for PDA medium removal agar component forms) and carries out fermented and cultured, the bottled medium 150mL of every 500mL triangle, shaking speed 150r/min, 28 ℃ of temperature, 7 days time;
(3) fungal elicitor is to the inducing culture of Phellinus cell:
Phellinus liquid fermentation and culture the 3rd day, add fungal elicitor, make the fungal elicitor final concentration reach 120 μ g/mL, continue then to cultivate 5 days, obtain phellinus igniarius mycelium.
(4) extraction of flavonoids from phellinus and detection:
Phellinus igniarius mycelium is dried; phellinus igniarius mycelium powder 100mg adds 2mL 60% ethanol, in room temperature lower sealing ultrasonic Extraction 24h; filter; filtrate sealing put 4 ℃ of refrigerations down, and residue repeats aforesaid operations 3 times, merges three times leaching liquor; be settled to 50ml with 60% ethanol; as liquid to be measured, colorimetric method for determining flavonoids from phellinus content is 15.5mg/100mg mycelia (dry weight), output 340mg/L.
Fungal elicitor bacterial classification of the present invention also can adopt other similar bacterial classifications, and the Phellinus bacterial classification also can adopt other common cultivation kinds.
Claims (6)
1. a phellinus igniarius mycelium liquid fermentation method that utilizes fungal elicitor to improve flavonoids from phellinus output is characterized in that comprising the steps:
(1) preparation of fungal elicitor:
1. the cultivation of fungal elicitor bacterial strain: with the fungal elicitor bacterial classification inoculation on the PDA slant medium activation after, be transferred on the PDA plating medium, cultivated 3～5 days for 25～30 ℃, getting the bacterium piece is inoculated in the triangular flask that the PD liquid nutrient medium is housed, after in 25～30 ℃, 100～180r/min shaking table, cultivating 5～7 days, filter that to obtain the fungal elicitor mycelium standby;
2. prepare fungal elicitor: the fungal elicitor mycelium is dried down in 60～70 ℃, grind broken, (g: it is 65～85% ethanol that ml) 1: 2～1: 5 ratio adds concentration to volume ratio by weight, at room temperature soaked 10～15 hours, filter and collect filter residue, filter residue is volume ratio (g: ml) 1: 2～1: 5 the ratio adding chloroform and the mixed liquor flushing of n-butanol by weight, the volume ratio of chloroform and n-butanol is 3: 1～5: 1, wash filter residue so repeatedly 3～5 times, then with filter residue by weight volume ratio (g: ml) 1: 3～1: 5 ratio with acetone rinsing after, filter residue is volume ratio (g: ml) 1: 3～1: 5 ratio water flushing more by weight, natural air drying, dry matter by weight volume ratio (g: it is 5～15% trifluoroacetic acids that ml) 1: 5～1: 7 ratio adds concentration, boiling water bath 0.5～2 hour, filter then, it is 4～7 that filtrate is neutralized to pH with NaOH, under 3 ℃～8 ℃, left standstill 10～15 hours, 0.45 μ m filtering with microporous membrane degerming, make fungal elicitor ,-10 ℃～-20 ℃ freezing preservations;
(2) liquid fermentation and culture of Phellinus:
With the Phellinus bacterial classification inoculation of slant preservation to the PDA plating medium, carry out activation culture, 25～30 ℃ of cultivation temperature, incubation time 5～10 days, getting the bacterium piece then is inoculated in and carries out fermented and cultured in the triangular flask that the PD liquid fermentation medium is housed, shaking speed 100～180r/min, 25～30 ℃ of temperature, 5～8 days time;
(3) fungal elicitor is to the inducing culture of Phellinus cell:
Phellinus liquid fermentation and culture the 1st～5 day, add fungal elicitor, make fungal elicitor concentration reach 40～200 μ g/mL, continue then to cultivate 2～7 days, obtain phellinus igniarius mycelium.
2. a kind of phellinus igniarius mycelium liquid fermentation method that utilizes fungal elicitor to improve flavonoids from phellinus output according to claim 1; 3～5 of bacterium pieces that it is characterized in that cut-off footpath 0.3～0.6cm in the incubation step of described fungal elicitor bacterial strain; be inoculated in the PD liquid nutrient medium the bottled PD liquid nutrient medium of every 500mL triangle 100～200mL.
3. a kind of phellinus igniarius mycelium liquid fermentation method that utilizes fungal elicitor to improve flavonoids from phellinus output according to claim 1; 3～5 of bacterium pieces that it is characterized in that cut-off footpath 0.3～0.6cm in the liquid fermentation and culture step of described Phellinus; be inoculated in the PD liquid nutrient medium base the bottled PD liquid nutrient medium of every 500mL triangle 100～200mL.
4. a kind of phellinus igniarius mycelium liquid fermentation method that utilizes fungal elicitor to improve flavonoids from phellinus output according to claim 1; it is characterized in that described fungal elicitor to the 1st day adding of fungal elicitor in the Phellinus cell induction incubation step in the Phellinus fermented and cultured, continues to cultivate then.
5. a kind of phellinus igniarius mycelium liquid fermentation method that utilizes fungal elicitor to improve flavonoids from phellinus output according to claim 1; it is characterized in that described fungal elicitor to the 3rd day adding of fungal elicitor in the Phellinus cell induction incubation step in the Phellinus fermented and cultured, continues to cultivate then.
6. a kind of phellinus igniarius mycelium liquid fermentation method that utilizes fungal elicitor to improve flavonoids from phellinus output according to claim 1; it is characterized in that described fungal elicitor to the 5th day adding of fungal elicitor in the Phellinus cell induction incubation step in the Phellinus fermented and cultured, continues to cultivate then.
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