CN103695316B - A kind of liquid fermentation process improving phellinus igniarius mycelium yield of flavone - Google Patents
A kind of liquid fermentation process improving phellinus igniarius mycelium yield of flavone Download PDFInfo
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- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 claims abstract description 8
- 229910001425 magnesium ion Inorganic materials 0.000 claims abstract description 8
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Abstract
The invention discloses a kind of liquid fermentation process improving phellinus igniarius mycelium yield of flavone, containing following steps: the cultivation of (1) Phellinus liquid fermenting bacterial classification: the Phellinus solid spawn of slant preservation is inoculated on PDA plate culture medium, after activation culture, get Phellinus bacterium block to be inoculated in the triangular flask that PD liquid nutrient medium is housed and to carry out fermentation culture, regulate shaking speed, culture temperature and incubation time, adopt low-intensity ultrasonic irradiation every day; (2) liquid fermentation and culture of Phellinus: by cultured Phellinus liquid fermenting bacterial classification homogeneous; then get bacterium liquid be inoculated in add magnesium ion PD liquid fermentation medium in carry out fermentation culture; regulate shaking speed; culture temperature and incubation time; adopt low-intensity ultrasonic irradiation every day, obtain the phellinus igniarius mycelium of high flavones content.The method is with short production cycle, is affected by the external environment little, and technique is simple, can increase substantially phellinus igniarius mycelium yield of flavone, and can suitability for industrialized production.
Description
Technical field
The invention belongs to technical field of bioengineering, be specifically related to a kind of liquid fermentation process improving phellinus igniarius mycelium yield of flavone.
Background technology
Phellinus (
phellinusigniarius(L.exFr.) Quel.) also known as Sang Chen, mulberry ear, Phellinus mushroom etc., be a kind of medicinal fungi of preciousness, main parasitic, on the trunk of the xylophytas such as mulberry, poplar, birch, pine, all has distribution on China northeast, North China, northwest and other places.Phellinus sporophore is used as medicine, and mildly bitter flavor is cold in nature, returns liver, kidney channel, and its earliest medicinal is recorded in Compendium of Material Medica: " sharp the five internal organs, a surname's flatus, toxin expelling gas ".China's Traditional Chinese Medicine often uses Phellinus under treating uterine bleeding band, the diseases such as diarrhea due to hypofunction of the spleen.Modern pharmacology research confirms, Phellinus has the effects such as antitumor, anti-oxidant, reducing blood-fat, strengthening immunity, is one of internationally recognized best anticancer epiphyte.Chemical composition analysis shows, the materials such as polysaccharide, flavones and terpene are the key active ingredients of Phellinus.Wherein, flavonoid compound has anti-oxidant, reducing blood-fat and antitumor isoreactivity, generally extracts from natural phant and obtains, and Phellinus is one of rare medicinal fungi being rich in flavonoid compound.But, along with Phellinus pharmaceutical use day by day accept by people, the output of wild Phellinus becomes the important bottleneck of restriction Phellinus industrialized development.
Because wild Phellinus is subject to the restriction of its special physiological state and complicated growing environment, at the wild Phellinus sporophore rare numbers that occurring in nature is formed.And artificial culture is extremely difficult, culture condition is harsh, and growth cycle is longer; form pharmaceutically acceptable sporophore and generally need 3 ~ 4 years; the research of Phellinus is restricted, also constrains the further exploitation of Phellinus product, be difficult to meet the market requirement day by day expanded.Therefore, a kind of method finding out effective flavonoids from phellinus of production fast, to solve the contradiction between prior art and Production requirement, becomes one of current Phellinus research and development field problem in the urgent need to address.Early-stage Study shows, also containing flavonoid compound in Phellinus liquid fermenting mycelium, has the effects such as anti-oxidant, antitumor equally.Utilize liquid submerged fermentation to cultivate and obtain phellinus igniarius mycelium and active substance thereof in a large number, because it is with short production cycle, it is little to be affected by the external environment, save the advantages such as labor force, become a kind of effective way of artificial production Phellinus.
Summary of the invention
The object of the present invention is to provide a kind of liquid fermentation process improving phellinus igniarius mycelium yield of flavone, the method is with short production cycle, is affected by the external environment little, and technique is simple, can increase substantially phellinus igniarius mycelium yield of flavone, and can suitability for industrialized production.
Above-mentioned purpose of the present invention can realize by following technical solution: a kind of liquid fermentation process improving phellinus igniarius mycelium yield of flavone, comprises the following steps:
(1) cultivation of Phellinus liquid fermenting bacterial classification: the Phellinus solid spawn of slant preservation is inoculated on PDA plate culture medium, activation culture, culture temperature 25 ~ 30 DEG C, incubation time 5 ~ 7 days, then get Phellinus bacterium block to be inoculated in the triangular flask that PD liquid nutrient medium is housed and to carry out fermentation culture, shaking speed 150 ~ 200r/min, culture temperature 25 ~ 30 DEG C, incubation time 5 ~ 7 days, adopts low-intensity ultrasonic irradiation every day 1 ~ 3 time;
(2) liquid fermentation and culture of Phellinus: by cultured Phellinus liquid fermenting bacterial classification homogeneous; then get liquid-spawn inoculation and carry out fermentation culture in the PD liquid fermentation medium that with the addition of magnesium ion; shaking speed 100 ~ 150r/min; culture temperature 25 ~ 30 DEG C; incubation time 5 ~ 10 days; adopt low-intensity ultrasonic irradiation every day 3 ~ 5 times, obtain the phellinus igniarius mycelium of high flavones content.
In the liquid fermentation process of above-mentioned raising phellinus igniarius mycelium yield of flavone:
The ultrasonic frequency adopted in step of the present invention (1) is 10 ~ 30KHz, and ultrasonic power is 2 ~ 10W, and every day, ultrasonic number of times was 1 ~ 3 time, and each ultrasonic time is 5 ~ 10s, and each interval time is 10 ~ 30s.
The ultrasonic frequency adopted in step of the present invention (2) is 10 ~ 50KHz, and ultrasonic power is 5 ~ 20W, and every day, ultrasonic number of times was 3 ~ 5 times, and each ultrasonic time is 10 ~ 30s, and each interval time is 30 ~ 60s.
A kind of fermentation process improving yield of flavone provided by the invention; just being of most critical carries out irradiation by low intensive ultrasonic wave to fermented bacterium; ultrasonic intensity is crossed conference and is killed bacterial classification; intensity is too small, acts on not obvious; the present inventor finds through a large amount of tests; adopt ultrasonic frequency and the power of above-mentioned restriction, higher yield of flavone can be obtained from phellinus igniarius mycelium.
Magnesium ion described in step of the present invention (2) derives from magnesium sulfate (MgSO
4) or magnesium chloride (MgCl
2).
In step of the present invention (2), in PD liquid fermentation medium, the concentration of magnesium ion is 0.01 ~ 0.03mol/L.
Compared with prior art, the present invention has following effect:
(1) low intensity ultrasound field is applied to the liquid fermenting production of phellinus igniarius mycelium flavones by the present invention, and increased substantially phellinus igniarius mycelium yield of flavone, output can reach more than 600mg/L, is more than 5 times of control group;
(2) the ultrasonic equipment wide material sources adopted in the present invention, existing a large amount of commerical prod is sold, and most commercial ultrasonic generator all can be applied to present method, can obtain good effect, reproducible;
(3) Phellinus liquid fermentation process of the present invention is simple, and raw materials is cheap and easy to get, and Be very effective, instrument are easy and simple to handle, and the whole fermenting process of what is more important is controlled, affects very little by external environmental condition, is applicable to suitability for industrialized production;
(4) flavonoids from phellinus extracted from the phellinus igniarius mycelium that the present invention prepares, its output is high, and the flavonoids from phellinus of acquisition can be used for preparing anti-oxidant, antitumor, antidotal medicine and additive etc.
Embodiment
The present invention is further illustrated below in conjunction with embodiment, but not as limitation of the present invention.
embodiment 1
Bacterial classification: Phellinus be common cultivation kind (
phellinusigniarius), purchased from China General Microbiological culture presevation administrative center, CGMCCNo.5.00095.
The liquid fermentation process of the raising phellinus igniarius mycelium yield of flavone that the present embodiment provides, comprises the following steps:
(1) cultivation of Phellinus liquid fermenting bacterial classification
The Phellinus solid spawn of slant preservation is inoculated on PDA plate culture medium (potato dextrose agar), inoculum size can be the Phellinus bacterium block 3 ~ 5 pieces of cut-off footpath 4 ~ 6mm, be inoculated in the triangular flask that PD liquid nutrient medium is housed, the bottled PD liquid nutrient medium of every 500mL triangle 200 ~ 250mL, activation culture, culture temperature 25 DEG C, incubation time 7 days, be the bacterium block 3 pieces of 4mm in colony edge cut-off footpath with aseptic punch tool, be inoculated in the triangular flask that PD liquid nutrient medium (removing agar component in the component for PDA substratum to form) is housed and carry out fermentation culture, every 500mL triangle bottled PD liquid nutrient medium 200mL, shaking speed 150r/min, culture temperature 25 DEG C, incubation time 5 days, frequency is adopted every day to be 10KHz, power is the low-intensity ultrasonic irradiation 1 time of 10W, each ultrasonic time 5s, each interval time 10s, when being inoculated into liquid from solid spawn, the bacterium block of about 5mm diameter can be inoculated, be not construed as limiting herein, only give to illustrate, those skilled in the art can adjust as required, in from liquid-spawn inoculation to nutrient solution, the amount of inoculation is approximately the 5%(mass percentage of liquid culture base unit weight), be all common inoculum size, do not need to limit, be only described herein,
(2) liquid fermentation and culture of Phellinus
By cultured Phellinus liquid fermenting bacterial classification homogeneous, then get liquid spawn (inoculating 3mL bacterial classification in every 100mL liquid nutrient medium) and be inoculated in interpolation magnesium sulfate (MgSO
4) PD liquid fermentation medium (adding magnesium sulfate 0.01mol in every 1000mL liquid nutrient medium) in carry out fermentation culture; shaking speed 100r/min; culture temperature 25 DEG C; incubation time 10 days; adopt every day frequency to be 10KHz, power is the low-intensity ultrasonic irradiation 3 times of 20W, each ultrasonic time 10s; each interval time, 30s, obtained the phellinus igniarius mycelium of high flavones content.
Liquid Phellinus bacterial classification after homogeneous; be inoculated in and with the addition of in the PD liquid fermentation medium of magnesium ion; 3 ~ 5mL Phellinus bacterial classification can be inoculated in every 100mL liquid fermentation medium; liquid nutrient medium is loaded in triangular flask; the bottled liquid nutrient medium 200 ~ 250mL of every 500mL triangle; only carrying out general explanation to the inoculation situation of liquid Phellinus bacterial classification herein, is not limitation of the invention.
(3) mensuration of flavonoids from phellinus
By gained phellinus igniarius mycelium in 60 DEG C of oven dry; pulverize; get phellinus igniarius mycelium powder 1.0g, add 30mL60% ethanol, in 60 DEG C of refluxing extraction 24h; filter; filtrate reduced in volume, residue repeats extraction 3 times, united extraction liquid; aluminum nitrate-Sodium Nitrite colorimetric method for determining Phellinus general flavone content is adopted to be 65.2mg/g mycelium (dry weight), yield of flavone 601.6mg/L fermented liquid.
embodiment 2
Bacterial classification: Phellinus be common cultivation kind (
phellinusigniarius), purchased from China General Microbiological culture presevation administrative center, CGMCCNo.5.00095.
Improve the liquid fermentation process of phellinus igniarius mycelium yield of flavone, comprise the following steps:
(1) cultivation of Phellinus liquid fermenting bacterial classification
The Phellinus solid spawn of slant preservation is inoculated on PDA plate culture medium, activation culture, culture temperature 28 DEG C, incubation time 6 days, be the bacterium block 4 pieces of 5mm in colony edge cut-off footpath with aseptic punch tool, be inoculated in the triangular flask that PD liquid nutrient medium is housed and carry out fermentation culture, every 500mL triangle bottled PD liquid nutrient medium 225mL, shaking speed 180r/min, culture temperature 28 DEG C, incubation time 6 days, frequency is adopted every day to be 20KHz, power is the low-intensity ultrasonic irradiation 2 times of 6W, each ultrasonic time 7s, each interval time 20s;
(2) liquid fermentation and culture of Phellinus
By cultured Phellinus liquid fermenting bacterial classification homogeneous, then get liquid spawn (inoculating 4mL bacterial classification in every 100mL liquid nutrient medium) and be inoculated in and be added with magnesium sulfate (MgSO
4) PD liquid fermentation medium in (in every 1000mL liquid nutrient medium, adding magnesium sulfate 0.02mol) carry out fermentation culture; shaking speed 120r/min; culture temperature 28 DEG C; incubation time 7 days; adopt every day frequency to be 30KHz, power is the low-intensity ultrasonic irradiation 4 times of 12W, each ultrasonic time 20s; each interval time, 45s, obtained phellinus igniarius mycelium.
(3) mensuration of flavonoids from phellinus
By gained phellinus igniarius mycelium in 60 DEG C of oven dry; pulverize; get phellinus igniarius mycelium powder 1.0g, add 30mL60% ethanol, in 60 DEG C of refluxing extraction 24h; filter; filtrate reduced in volume, residue repeats extraction 3 times, united extraction liquid; aluminum nitrate-Sodium Nitrite colorimetric method for determining Phellinus general flavone content is adopted to be 78.8mg/g mycelium (dry weight), yield of flavone 730.4mg/L fermented liquid.
embodiment 3
Bacterial classification: Phellinus be common cultivation kind (
phellinusigniarius), purchased from China General Microbiological culture presevation administrative center, CGMCCNo.5.00095.
Improve the liquid fermentation process of phellinus igniarius mycelium yield of flavone, comprise the following steps:
(1) cultivation of Phellinus liquid fermenting bacterial classification
The Phellinus solid spawn of slant preservation is inoculated on PDA plate culture medium, activation culture, culture temperature 30 DEG C, incubation time 5 days, be the bacterium block 5 pieces of 6mm in colony edge cut-off footpath with aseptic punch tool, be inoculated in the triangular flask that PD liquid nutrient medium is housed and carry out fermentation culture, every 500mL triangle bottled PD liquid nutrient medium 250mL, shaking speed 200r/min, culture temperature 30 DEG C, incubation time 7 days, frequency is adopted every day to be 30KHz, power is the low-intensity ultrasonic irradiation 3 times of 2W, each ultrasonic time 10s, each interval time 30s;
(2) liquid fermentation and culture of Phellinus
By cultured Phellinus liquid fermenting bacterial classification homogeneous, then get liquid spawn (inoculating 5mL bacterial classification in every 100mL liquid nutrient medium) and be inoculated in and be added with magnesium chloride (MgCl
2) PD liquid fermentation medium in (in every 1000mL liquid nutrient medium, adding magnesium chloride 0.03mol) carry out fermentation culture; shaking speed 150r/min; culture temperature 30 DEG C; incubation time 5 days; adopt every day frequency to be 50KHz, power is the low-intensity ultrasonic irradiation 5 times of 5W, each ultrasonic time 30s; each interval time, 60s, obtained phellinus igniarius mycelium.
(3) mensuration of flavonoids from phellinus
By gained phellinus igniarius mycelium in 60 DEG C of oven dry; pulverize; get phellinus igniarius mycelium powder 1.0g, add 30mL60% ethanol, in 60 DEG C of refluxing extraction 24h; filter; filtrate reduced in volume, residue repeats extraction 3 times, united extraction liquid; aluminum nitrate-Sodium Nitrite colorimetric method for determining Phellinus general flavone content is adopted to be 82.2mg/g mycelium (dry weight), yield of flavone 665.8mg/L fermented liquid.
control group
Bacterial classification: Phellinus be common cultivation kind (
phellinusigniarius), purchased from China General Microbiological culture presevation administrative center, CGMCCNo.5.00095.
Normal control group Phellinus liquid fermentation process, comprises the following steps:
(1) cultivation of Phellinus liquid fermenting bacterial classification
The Phellinus solid spawn of slant preservation is inoculated on PDA plate culture medium, activation culture, culture temperature 28 DEG C, incubation time 6 days is the bacterium block 4 pieces of 5mm in colony edge cut-off footpath with aseptic punch tool, be inoculated in the triangular flask that PD liquid nutrient medium is housed and carry out fermentation culture, every 500mL triangle bottled PD liquid nutrient medium 225mL, shaking speed 180r/min, culture temperature 28 DEG C, incubation time 6 days, obtains Phellinus liquid fermenting bacterial classification;
(2) liquid fermentation and culture of Phellinus
By cultured Phellinus liquid fermenting bacterial classification homogeneous; then get liquid spawn (inoculating 4mL bacterial classification in every 100mL liquid nutrient medium) to be inoculated in PD liquid fermentation medium and to carry out fermentation culture, shaking speed 120r/min, culture temperature 28 DEG C; incubation time 7 days, obtains phellinus igniarius mycelium.
(3) mensuration of flavonoids from phellinus
By gained phellinus igniarius mycelium in 60 DEG C of oven dry; pulverize; get phellinus igniarius mycelium powder 1.0g, add 30mL60% ethanol, in 60 DEG C of refluxing extraction 24h; filter; filtrate reduced in volume, residue repeats extraction 3 times, united extraction liquid; aluminum nitrate-Sodium Nitrite colorimetric method for determining Phellinus general flavone content is adopted to be 18.8mg/g mycelium (dry weight), yield of flavone 118.3mg/L.
Phellinus bacterial classification of the present invention also can adopt other common cultivation kinds.
Above embodiment is only for setting forth the present invention, and protection scope of the present invention is not only confined to above embodiment.The those of ordinary skill of described technical field, according to above content disclosed by the invention and scope that each parameter is got, all can realize object of the present invention.
Claims (3)
1. improve a liquid fermentation process for phellinus igniarius mycelium yield of flavone, it is characterized in that comprising the following steps:
(1) cultivation of Phellinus liquid fermenting bacterial classification
The Phellinus solid spawn of slant preservation is inoculated on PDA plate culture medium, activation culture, culture temperature 25 ~ 30 DEG C, incubation time 5 ~ 7 days, then get Phellinus bacterium block to be inoculated in the triangular flask that PD liquid nutrient medium is housed and to carry out fermentation culture, shaking speed 150 ~ 200r/min, culture temperature 25 ~ 30 DEG C, incubation time 5 ~ 7 days, adopts low-intensity ultrasonic irradiation every day;
(2) liquid fermentation and culture of Phellinus
By cultured Phellinus liquid fermenting bacterial classification homogeneous, then get bacterium liquid be inoculated in add magnesium ion PD liquid fermentation medium in carry out fermentation culture, shaking speed 100 ~ 150r/min, culture temperature 25 ~ 30 DEG C, incubation time 5 ~ 10 days, adopt low-intensity ultrasonic irradiation every day, obtain the phellinus igniarius mycelium of high flavones content;
The ultrasonic frequency adopted in step (1) is 10 ~ 30KHz, and ultrasonic power is 2 ~ 10W, and every day, ultrasonic number of times was 1 ~ 3 time, and each ultrasonic time is 5 ~ 10s, and each interval time is 10 ~ 30s;
The ultrasonic frequency adopted in step (2) is 10 ~ 50KHz, and ultrasonic power is 5 ~ 20W, and every day, ultrasonic number of times was 3 ~ 5 times, and each ultrasonic time is 10 ~ 30s, and each interval time is 30 ~ 60s.
2. the liquid fermentation process of raising phellinus igniarius mycelium yield of flavone according to claim 1, is characterized in that, the magnesium ion described in step (2) is from magnesium sulfate or magnesium chloride.
3. the liquid fermentation process of raising phellinus igniarius mycelium yield of flavone according to claim 1, is characterized in that, in step (2), in PD liquid fermentation medium, the concentration of magnesium ion is 0.01 ~ 0.03mol/L.
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| CN112063663A (en) * | 2020-08-24 | 2020-12-11 | 浙江省农业科学院 | Fermentation method for high-yield phellinus igniarius extracellular flavonoids |
| CN112656820A (en) * | 2021-01-14 | 2021-04-16 | 鲁东大学 | A high-efficiency preparation method for Phellinus linteus polyphenol from submerged fermentation mycelium of Phellinus linteus |
| CN112899109A (en) * | 2021-04-23 | 2021-06-04 | 长江师范学院 | Preparation method of white peach mulberry yellow wine |
| CN114958620B (en) * | 2022-05-30 | 2024-02-20 | 杭州市农业科学研究院 | Phellinus linteus mycelium liquid fermentation medium and fermentation culture method |
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