CN103627695A - Method for improving poria cocos mycelium protein content and liquid fermentation biomass - Google Patents
Method for improving poria cocos mycelium protein content and liquid fermentation biomass Download PDFInfo
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- CN103627695A CN103627695A CN201310612653.0A CN201310612653A CN103627695A CN 103627695 A CN103627695 A CN 103627695A CN 201310612653 A CN201310612653 A CN 201310612653A CN 103627695 A CN103627695 A CN 103627695A
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- poria cocos
- pinus massoniana
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Abstract
The invention relates to a bio-pharmaceuticals production technology, and in particular relates to a method for improving poria cocos mycelium protein content and liquid fermentation biomass. The method comprises the following steps: (1) preparing a poria cocos basidiospore suspension liquid; (2) mutating the poria cocos basidiospore through Co60-gamma radiation; (3) pairing basidiospore strains; (4) inoculating a pinus massoniana steep solid culture medium bevel, wherein the culture medium is composed of pinus massoniana extractive, potato extractive and glucose; and (5) inoculating a pinus massoniana extractive liquid fermentation culture medium, wherein the culture medium is composed of pinus massoniana extractive, microcrystal fiber, glucose, glycerinum, ammonium sulfate, magnesium sulfate, potassium hydroxide and phosphoric acid. The poria cocos strain with high mycelium protein content is firstly obtained through mutation, the adopted pinus massoniana steep solid culture medium bevel is in favor of the rapid growth of the poria cocos mycelium in comparison with the potato glucose culture medium, and the adopted pinus massoniana extractive liquid fermentation culture medium is in favor of obtaining high biomass.
Description
Technical field
The invention belongs to production technology field of biological pharmacy, is mainly a kind of method that improves Poria mycelium protein content and liquid fermenting biomass.
Background technology
Poria cocos belongs to the traditional rare traditional Chinese medicine of medicinal fungi ，Shi China and time-honored food.Taste is sweet, light, flat, has the effects such as promoting diuresis to eliminate damp pathogen, invigorating the spleen bowl spares, tranquillizing by calming the heart, and has antibacterial, anti-ageing, antitumor, anti-inflammatory, the effect such as antiviral.Poria mycelium protein content affects its speed of growth, improve Poria mycelium protein content and can promote mycelial growth rate, and liquid submerged fermentation technology is the important channel that obtains the main pharmacological active substance of Poria cocos, but Poria cocos Hyphae Growth speed is slow, the biomass producing after fermentation is few, affect the output of active substance in mycelium, with need to not conforming in a large number of Poria cocos.At present the research of Poria cocos is concentrated on to Pachymose aspect, be not yet improved the research of Poria mycelium protein content height and liquid fermenting biomass aspect.
Summary of the invention
The object of this invention is to provide a kind of method that improves Poria mycelium protein content and liquid fermenting biomass, solve current Poria mycelium protein content low, the speed of growth is slow, the problem that biomass is low.
Object of the present invention realizes approach: collect Poria cocos spore, through the mutagenesis of Co60 – gamma-radiation, carry on a shoulder pole the pairing of spore bacterial strain, obtain mycelium protein content high, the fast bacterial strain of growing, by this inoculation in containing on the PDA substratum of Pinus massoniana Lamb immersion liquid, obtain bacterium piece, bacterium piece is inoculated in Pinus massoniana Lamb immersion liquid fermention medium to 25 ℃～29 ℃, 80～120 r/min shaking tables are cultivated 5～7 days, obtain biomass;
Mainly comprise the following steps:
(1) collect Poria cocos sporidium: with stroke-physiological saline solution modulation spore concentration, be 10
4～10
6the Co60 – gamma-radiation mutagenesis Poria cocos sporidium that individual/ml is 300Gy～900Gy through radiation dose;
(2) pairing: by 10～100 times of mutagenesis Poria cocos sporidium suspension dilutions, in containing Pinus massoniana Lamb immersion liquid potato culture plate, cultivate 5～6 days for 26～28 ℃, different mycelia are connected on to same PDA culture plate between two central, cultivate 4～6 days for 26～28 ℃, mycelia is combined together, gets growth and combines faster, obtains mutagenic strain;
(3) the Poria cocos bacterial strain of mutagenesis is seeded in containing on Pinus massoniana Lamb immersion liquid PDA substratum, this substratum consists of Pinus massoniana Lamb immersion liquid 1.0%～4.0%, potato water cooking liquid 15%～20%, glucose 0.8%～1.2%, agar 1.5%～2.0%, cultivates 5～7 days for 25 ℃～29 ℃;
(4) mycelium liquid fermentation culture: the bacterium piece of inoculation mutagenic strain is in Pinus massoniana Lamb immersion liquid liquid fermentation medium, this substratum consists of Pinus massoniana Lamb immersion liquid 1.0%～4.0%, microcrystalline cellulose 0.8%～3.5%, glucose 1.0%～3.5%, glycerine 0.01%～0.05%, ammonium sulfate 0.1%～0.4%, magnesium sulfate 0.1%～0.3%, potassium hydroxide 0.02%～0.06%, phosphoric acid 0.02%～0.05%, 5.5,25 ℃～29 ℃ of pH, 80～120 r/min shaking tables are cultivated 5～7 days;
(5) separating bio amount: fermented liquid 8 * 10
4pa vacuum filtration 10 min, sterilized water washing is collected thalline 2～3 times, dries to constant weight for 60 ℃;
(6) mycelium protein assay: according to protein content in the first method Kjeldahl nitrogen determination Poria mycelium in the mensuration > > of GB 5009.5-2010 < < Protein in Food;
(7) bacterial strain preservation: mutagenic strain be preserved in paraffin oil or sand pipe in, 4 ℃ of preservations, each year goes down to posterity once;
The present invention adopts Co60 – gamma-radiation mutagenesis Poria cocos spore, carry on a shoulder pole the pairing of spore bacterial strain, obtained the high bacterial strain of mycelium protein content, through passing through after Pinus massoniana Lamb immersion liquid liquid fermenting, solve the problem that in prior art, Poria mycelium protein content is low, the speed of growth is slow, biomass is low, reached object of the present invention:
(1) the present invention passes through that mycelium protein content is high, the Poria cocos bacterial strain of fast growth first mutagenic obtained;
(2) the PDA substratum containing Pinus massoniana Lamb immersion liquid adopting is more conducive to the Fast Growth of Poria mycelium compared with potato dextrose agar;
(3) the Pinus massoniana Lamb immersion liquid liquid fermentation medium adopting is more conducive to obtain higher biomass.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail:
Embodiment 1:
1. bacterial classification: Poria cocos (
wolfiporia cocos) sclerotium, purchased from Poria cocos market, quiet state, Hunan, be numbered: Xiang Jing " 28 ";
2. solid medium consists of: Pinus massoniana Lamb immersion liquid 1.0%～4.0%, potato water cooking liquid 15%～20%, glucose 0.8～1.2%, agar 1.5%～2.0%;
3. liquid fermentation medium forms: Pinus massoniana Lamb immersion liquid 1.0%～4.0%, microcrystalline cellulose 0.8%～3.5%, glucose 1.0%～3.5%, glycerine 0.01%～0.05%, ammonium sulfate 0.1%～0.4%, magnesium sulfate 0.1%～0.3%, potassium hydroxide 0.02%～0.06%, phosphoric acid 0.02%～0.05%, pH 5.5;
4. bacterial strain mutagenesis and liquid fermentation and culture: maturescent poria cocos sclerotium is cleaned, on cracks surface, mark long 3.0 cm, wide 1.5 cm, the opening of dark 1.0 cm, in humidity, be 90%, temperature is at 26 ℃, to cultivate 15 days, and the mycelium kink that wound grows forms after cellular sporophore, temperature is adjusted to 28 ℃, in dry environment, collects spore.With stroke-physiological saline solution modulation spore concentration, being 104～106/ml, is 300 Gray through radiation dose, the Co60 – gamma-radiation mutagenesis Poria cocos sporidium of 600 Gray and 900 Gray.Mutagenesis Poria cocos sporidium suspension is diluted respectively to 10,100,1000 times, in solid culture flat board, cultivate 7 days for 27 ℃, different mycelia are connected on to the dull and stereotyped central authorities of same solid culture between two, cultivate 5 days for 26 ℃, mycelia is combined together, gets growth and combines faster, obtains mutagenic strain.Mutagenic strain is inoculated in culture medium slant, cultivates 7 days for 26 ℃, get inclined-plane bacterium piece and be inoculated in liquid fermentation medium, 27 ℃, 100 r/min shaking tables are cultivated 7 days, fermented liquid 8 * 10
4pa vacuum filtration 10 min, sterilized water washing is collected thalline 2～3 times, dries to constant weight for 60 ℃;
5. mycelium protein detects: Poria mycelium protein content is according to the first method Kjeldahl nitrogen determination in the mensuration > > of GB 5009.5-2010 < < Protein in Food;
Result shows, Poria cocos mutagenic strain is after liquid fermentation medium is cultivated, and obtaining biomass is the dry g/100 ml of 1.2～1.5(), former the bacterial strain that adopts common liq fermention medium to cultivate improved 15%～30%.Poria cocos mutagenic strain is through liquid fermentation and culture, and mycelium protein content is 1.96%, with former bacterial strain mycelium protein content under condition, is 1.48%, and more former bacterial strain mycelium protein content of mutagenic strain has improved 32.43%; Poria cocos mutagenic strain is not through containing the liquid fermentation and culture of Pinus massoniana Lamb immersion liquid, and mycelium protein content is 1.89%, with former bacterial strain mycelium protein content under condition, is 1.15%, and more former bacterial strain mycelium protein content of mutagenic strain has improved 64.34%.
Claims (3)
1. improve a method for Poria fungus silk-protein content and liquid fermenting biomass, it is characterized in that concrete operation step is as follows:
(1) collecting Poria cocos sporidium, is 10 with stroke-physiological saline solution modulation spore concentration
4～10
6individual/ml is the Co60 – gamma-radiation mutagenesis Poria cocos sporidium of 300Gray～900 Gray through radiation dose;
(2) by 10～100 times of mutagenesis Poria cocos sporidium suspension dilutions, in solid medium flat board, cultivate 5～6 days for 26～28 ℃, different mycelia are connected on to the dull and stereotyped central authorities of same solid medium between two, cultivate 4～6 days for 26～28 ℃, mycelia is combined together, gets growth and combines faster, obtains mutagenic strain;
(3) the Poria cocos bacterial strain of mutagenesis is seeded on solid medium, this substratum consists of Pinus massoniana Lamb immersion liquid 1.0%～4.0%, potato water cooking liquid 15%～20%, and glucose 0.8%～1.2%, agar 1.5%～2.0%, cultivates 5～7 days for 25 ℃～29 ℃;
(4) the bacterium piece of inoculation mutagenic strain is in liquid fermentation medium, and this substratum consists of Pinus massoniana Lamb immersion liquid 1.0%～4.0%, microcrystalline cellulose 0.8%～3.5%, glucose 1.0%～3.5%, glycerine 0.01%～0.05%, ammonium sulfate 0.1%～0.4%, magnesium sulfate 0.1%～0.3%, potassium hydroxide 0.02%～0.06%, phosphoric acid 0.02%～0.05%, water 96.25%～87.16%, pH 5.5,25 ℃～29 ℃, 80～120 r/min shaking tables are cultivated 5～7 days;
(5) fermented liquid 8 * 10
4pa vacuum filtration 10 min, sterilized water washing is collected thalline 2～3 times, dries to constant weight for 60 ℃;
(6) mutagenic strain be preserved in paraffin oil or sand pipe in, 4 ℃ of preservations, each year goes down to posterity once.
2. a kind of method that improves Poria mycelium protein content and one fermentation biomass according to claim 1, is characterized in that through radiation dose being the Co60 – gamma-radiation mutagenesis Poria cocos sporidium of 300Gray～900 Gray.
3. a kind of method that improves Poria mycelium protein content and one fermentation biomass according to claim 1, its feature at its nutrient media components is:
(1) the PDA substratum of Pinus massoniana Lamb immersion liquid: Pinus massoniana Lamb immersion liquid 1.0%～4.0%, potato water cooking liquid 15%～20%, glucose 0.8～1.2%, agar 1.5%～2.0%;
(2) Pinus massoniana Lamb immersion liquid liquid fermentation medium: Pinus massoniana Lamb immersion liquid 1.0%～4.0%, microcrystalline cellulose 0.8%～3.5%, glucose 1.0%～3.5%, glycerine 0.01%～0.05%, ammonium sulfate 0.1%～0.4%, magnesium sulfate 0.1%～0.3%, potassium hydroxide 0.02%～0.06%, phosphoric acid 0.02%～0.05%, pH 5.5.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106399420A (en) * | 2016-08-31 | 2017-02-15 | 怀化学院 | Poria health care yogurt and preparation method thereof |
| CN106676032A (en) * | 2016-11-14 | 2017-05-17 | 怀化学院 | Method for increasing yield of pachymic acid of poria cocos liquid fermented mycelium |
| CN106880588A (en) * | 2017-02-19 | 2017-06-23 | 哈尔滨伟平科技开发有限公司 | A kind of preparation method of dried orange peel poria oral liquor |
| CN110184200A (en) * | 2019-06-14 | 2019-08-30 | 福建农林大学 | A kind of high yield Sparassis crispa mycelia fermentation base and preparation method |
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| CN1076825A (en) * | 1993-03-29 | 1993-10-06 | 王汉培 | Method with living pine parasitic cultivation Poria cocos |
| CN103315362A (en) * | 2013-07-16 | 2013-09-25 | 河北大学 | Poria cocos solid state fermentation functional beverage and its preparation method |
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2013
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Patent Citations (2)
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| CN1076825A (en) * | 1993-03-29 | 1993-10-06 | 王汉培 | Method with living pine parasitic cultivation Poria cocos |
| CN103315362A (en) * | 2013-07-16 | 2013-09-25 | 河北大学 | Poria cocos solid state fermentation functional beverage and its preparation method |
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| 吴红娟 等: "松根液对发酵茯苓菌丝产量和多糖成分的影响", 《中国中医药信息杂志》 * |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106399420A (en) * | 2016-08-31 | 2017-02-15 | 怀化学院 | Poria health care yogurt and preparation method thereof |
| CN106676032A (en) * | 2016-11-14 | 2017-05-17 | 怀化学院 | Method for increasing yield of pachymic acid of poria cocos liquid fermented mycelium |
| CN106676032B (en) * | 2016-11-14 | 2020-11-03 | 怀化学院 | Method for increasing pachymic acid yield in poria cocos liquid fermentation mycelium |
| CN106880588A (en) * | 2017-02-19 | 2017-06-23 | 哈尔滨伟平科技开发有限公司 | A kind of preparation method of dried orange peel poria oral liquor |
| CN110184200A (en) * | 2019-06-14 | 2019-08-30 | 福建农林大学 | A kind of high yield Sparassis crispa mycelia fermentation base and preparation method |
| CN110184200B (en) * | 2019-06-14 | 2021-04-27 | 福建农林大学 | High-yield sparassis crispa mycelium fermentation medium and preparation method thereof |
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Application publication date: 20140312 |