CN105754867A - Method for producing selenium-rich grifola frondosa hyphae - Google Patents
Method for producing selenium-rich grifola frondosa hyphae Download PDFInfo
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Abstract
The invention discloses a method for producing selenium-rich grifola frondosa hyphae, and relates to the field of technologies for applying food microorganisms.The method has the advantages that liquid complete rice bran and wheat bran novel cultivation media with added selenium, added cysteine hydrochloride and added sodium copper chlorophyllin are fermented by the aid of novel grifola frondosa strains which are obtained by means of ultraviolet mutagenesis, rice bran and wheat bran are efficiently converted into selenium-rich grifola frondosa hypha raw materials and large quantities of extracellular selenium crude polysaccharides, accordingly, the production cost can be reduced, the yield can be increased, and the selenium-rich grifola frondosa hypha raw materials with high quality can be manufactured; the extracellular selenium crude polysaccharides can be reserved to be further used as deep processing treatment raw materials; the rice bran and the wheat bran which have low value are converted into the selenium-rich grifola frondosa hypha raw materials with diversified healthcare functions, accordingly, beneficial social and economical effects of reducing the production cost, efficient and reasonably utilizing low-end resources and protecting the public health can be realized, and the method is practical.
Description
Technical field
The present invention relates to food microorganisms applied technical field, particularly relating to the method that the mutagenic obtained Grifola frondosa new strains of a kind of use produces selenium-rich Grifola frondosa mycelia raw material, new strains carries out fermentation in the addition of the Testa oryzae new culture medium of wheat bran complete feed of liquid of cysteine hydrochloride, sodium copper chlorophllin and sodium selenite and obtains selenium-rich Grifola frondosa mycelia raw material in the method.
Background technology
Medicine-food two-purpose fungus has long use history in China and uses crowd widely.Modern science discloses, existing frequently-used medicine-food two-purpose the fungus such as mycelium of Ganoderma, Hericium erinaceus (Bull. Ex Fr.) Pers., Cordyceps, Grifola frondosa etc., sporophore or spore can produce the various active compositions such as such as aminoacid, protein, vitamin, polysaccharide, ucleosides, flavonoid and antibiotic or enrichment various trace elements such as selenium, zinc etc., there is the multiple efficacies such as raising body immunity, antitumor, enhancing liver function, antioxidation.Just because of this, the enthusiasm of international and domestic research and development medicine-food two-purpose fungus is in the ascendant, has many researcheres and developer at field of medicaments, field of food, Traditional Chinese health field, agricultural and field of industrial production, it is thus achieved that numerous achievements in research or product.Being used for for food with regard to Rare edible fungus, domestic market has edible mushroom health-care food such as LINGZHI JIAONANG, Grifola frondosa capsule and LINGZHI JIAONANG etc. in fast sale, and their economic worth is significantly high.Cookies adds Hericium erinaceus (Bull. Ex Fr.) Pers. and is then made for Hericium erinaceus (Bull. Ex Fr.) Pers. cookies, have become as bread and cheese supermarket at home and sell.New Zealand, Japan, the U.S., Korea S etc. all produce in the world similar edible mushroom health-care food product.
For Grifola frondosa, research for it shows: Grifola frondosa has effect (lists of references: 1. Chen Shi is good such as regulating immunity, adjunct antineoplastic treatment, treatment hepatitis, blood fat reducing, anti AIDS virus, deferring senility preferably, the research [D] of medicinal fungi Submerged Culture of Grifola frondosa technology and antitumor polysaccharide thereof, Southern Yangtze University, 2000；2. Cui Feng is outstanding, the research [D] of Submerged Culture of Grifola frondosa condition optimizing and mycelium antitumor glycopeptide thereof, Southern Yangtze University, and 2006；3. Lee is little fixed, the structure of grifolan and biological activity [D] thereof, Hua Zhong Agriculture University, and 2002；4. thunder duckweed, Effects of Extracts of Grifola frondosa on Active on the impact of Immune Function of Mice with Spleen Deficiency and study on mechanism [D], Liaoning University of TCM, 2011；5. NanbaH., Maitakemushroomimmunetherapytopreventfromcancergrowthand metastasis [J], Explore1995,6 (1): 74-78；6. KuboK., NanbaH., Anti-hyperliposiseffectofMaitakefruitbody (Grifolafrondosa) [J], Biological&PharmaceuticalBulletin, 1997,20 (7): 781-785.).
Grifola frondosa is a kind of Rare edible fungus, has cultivation on the Zhejiang of China, Hebei, Fujian, Sichuan and other places, and also there is cultivation in Japan.Its sporophore fragrance is dense, nutritious.Grifola frondosa, containing albumen, aminoacid, fat, crude fibre, carbohydrate, trace element etc., has multiple physiological active functions.The acquisition mode of present Grifola frondosa is mainly artificial culture, is used as medicine with sporophore or spore powder, but artificial culture Grifola frondosa cycle length, production efficiency are not high, labor intensity big, is limited by season, environment etc., subjects to pest and disease damage, and quality is unstable with yield.Edible fungus liquid submerged fermentation technology, can overcome the deficiency that traditional sporophore is cultivated, adopt the method for liquid or solid fermentation produce Cordyceps mycelium and in addition deep processing application meet the thinking of development of innovation driving, there is good prospect.The progress that the research and development of maitake mushroom mycelia is obtained all will promote the application of Grifola frondosa further, brings the Social and economic benef@of reality.Just because of this, just it is necessary to study the production method of new maitake mushroom mycelia and deep processed product thereof, such as consider Grifola frondosa mycelia raw material that some low side processing of farm products byproduct sources such as Testa oryzaes, wheat bran Efficient Conversion are high value, it is expected the production cost reducing Grifolas frondosa germ powder raw material thus reducing the price of finished product Grifola frondosa class health food, ordinary people can afford to consume, promoting health of masses, this is also one of patent focus of the present invention.
China is the big producing country of Oryza sativa L. and Semen Tritici aestivi, Testa oryzae and wheat bran aboundresources.Testa oryzae and wheat bran as the by-product of Oryza glutinosa or wheat processing, rich in nutrition content, cheap.Containing the abundant protein of high-quality, active polysaccharide, fat and the significant active substance of the physiological function such as tocotrienol, tocopherol in Testa oryzae, wheat bran is rich in nutritional labelings such as fat, protein, mineral, vitamin and celluloses, wherein cellulosic content is up to more than the 18% of wheat bran total amount, so, Testa oryzae and wheat bran can provide the carbon source of abundance, nitrogenous source, vitamin and mineral for Grifola frondosa growth.Under the effect of Grifola frondosa cellulase and other enzyme system, Testa oryzae and wheat bran can be changed into desired nutritional material and carry out growth metabolism, synthesis maitake mushroom mycelia and adenosine class functional materials by Grifola frondosa.Therefore, use agricultural byproducts Testa oryzae, wheat bran as whole carbon sources and nitrogenous source, carry out Grifola frondosa liquid fermentation growth Grifola frondosa class healthy food material, there is technical feasibility, and good Social and economic benef@can be brought.
Most powder of edible fungus Liquid-state fermentation production method adopt glucose, such as starch, Rhizoma Solani tuber osi or analysis for soybean powder etc. are as culture medium for grain class raw material, even if using agricultural byproducts such as Testa oryzae, wheat bran etc., being also as auxiliary element and adding.The bacterial strains such as original edible fungi Cordyceps, Ganoderma, Grifola frondosa are being not best without upgrowth situation on the Testa oryzae wheat bran complete feed liquid culture medium of glucose or other grain class raw materials, in addition Testa oryzae, bran feedstock is likely to may transfer in mycelium such as lead, arsenic, aflatoxin etc. containing some harmful substances, for stoping this transfer, therefore need the ferment rice bran wheat bran liquid complete feed method of this type of bacterium is carried out innovation research.
Selenium is the trace element of needed by human, and selenium plays a part balance oxidation reduction atmosphere in human body, has important physiological action.The health care of selenium is recognized by people, and Chinese market has occurred such as health-related food and the food material such as Se-enriched egg, selenium-enriched rice such as Beijing University's selenium-rich health, selenium Wei Kang, selemium nutrition element supplement.Investigation shows, the selenium intake of China 2/3 area crowd is lower than minimum recommended value, and edible fungi is the good carrier of Rich in Trace Element selenium, it may be considered that utilize the Enriching Seleniums such as Rare edible fungus such as Ganoderma, Cordyceps, Grifola frondosa, nutrition and effect of improving edible fungi raw materials are worth.Inorganic selenium can be converted into the organic selenium compounds that toxicity is relatively low, with better function by edible fungi Enriching Selenium so that selenium-enriched edible mushroom raw material is more suitably used as the raw material of health food.Owing to being considered as healthy food material, it is necessary to the highest dosis tolerata of the selenium of attention everyone every daily ingestion of adult is 400 μ g, it is recommended that intake is 50 μ g, so, need the addition of selenium is made rational selection when edible fungi Enriching Selenium.How selenium is effectively enriched in Grifola frondosa mycelia raw material, and ensure to use new mutagenic strain when fermenting in the liquid Testa oryzae new culture medium of wheat bran complete feed can high yield, be also one of present invention difficulties of needing to solve.
The research group of the inventor herein Liu Wei people during the last ten years to Grifola frondosa, Ganoderma, Cordyceps liquid fermentation carried out substantial amounts of research, successively formed multiple achievement such as Master's thesis and patent of invention.The Master's thesis that Liu Weimin instructs has: (1) Yang Suohua, frondosa fermentation Testa oryzae prepares polysaccharide (2006)；(2) Gu Huimin, Grifola frondosa liquid in Testa oryzae culture medium cultivates the research (2009) producing polysaccharide and enrichment organic selenium；(3) opening and build, Physical mutation Grifola frondosa liquid fermentation Testa oryzae wheat bran produces the research (2010) of polysaccharide；(4) Guo Chunmei, the induction mutation of bacterium of Grifola frondosa, liquid fermentation Testa oryzae wheat bran produce polysaccharide and selenium-rich research (2011)；(5) Li Yanan, Grifola frondosa induction mutation of bacterium and fermentation and performance study (2013)；(6) Liu Lili, high level converts Ganoderma lucidum submerged fermentation and the induction mutation of bacterium (2012) of Testa oryzae wheat bran；(7) full Testa oryzae wheat bran research (2013) of Guo Tianlong, Ganderma lucidum strain mutation and the high-valued conversion of liquid fermentation；(8) Chen Jing, Cordyceps liquid fermentation higher value application Testa oryzae wheat bran produces the research (2014) of polysaccharide；(9) Zhang Xiaofei, the research (2014) of the full Testa oryzae wheat bran of ganoderma strain capable (GanodermalucidumCFCC6043) liquid fermentation and active polysaccharide, etc..Liu Weimin has as the patent of invention of the first inventor: (1) uses the method that Testa oryzae wheat bran compound material and Grifola frondosa mutagenic strain produce polysaccharide, and 20101010579048.4；(2) Grifola frondosa strain of polysaccharide is produced for Testa oryzae and wheat bran compound material, 201010579078.5；(3) bacterial strain of grifolan is produced for ferment rice bran and wheat bran extracting solution, 201110150888.3；(4) bacterial strain of grifolan is produced for Testa oryzae and wheat bran complete feed liquid fermentation, 201310274913.8；(5) a kind of method that ganoderma lucidum mutant strain liquid fermentation Testa oryzae wheat bran complete feed produces polysaccharide, 201310275061.4；(6) a kind of method that liquid fermentation Testa oryzae wheat bran complete feed produces Cordyceps polysaccharides, 201310274914.2；(7) a kind of method that Grifola frondosa mutagenic fungi liquid fermentation Testa oryzae wheat bran complete feed produces polysaccharide, 201310274911.9；(8) bacterial strain of ganoderan is produced for Testa oryzae and wheat bran complete feed liquid fermentation, 201310274915.7, the production method of (9) a kind of grifolan selenium compound, 201110150889.8, etc..As seen from the above description, the inventor herein Liu Wei people study all around utilizing Testa oryzae and the efficient high-valued production Rare edible fungus mycelia of wheat bran and this special topic of functional component thereof, realize the imagination of innovation and creation, so the ganoderma strain capable of energy proposition mutation is added the Testa oryzae wheat bran complete feed liquid fermentation of selenium, efficient high-valued conversion Testa oryzae wheat bran produces selenium-rich Grifola frondosa mycelia raw material, and can be used for being deeply processed into health food by this mycelia raw material.
Just because of the research deep in this field, and the problem that follow-up recent studies on is disclosed, it has been found that also need to frondosa fermentation liquid Testa oryzae wheat bran complete feed is reformed.Our research has reached before frondosa fermentation Testa oryzae and the maximum fermentation concentration of wheat bran complete feed culture medium are Testa oryzae 8.0g/100mL, wheat bran 8.0g/100mL, both sums is 16.0g/100mL, and the dry weight concentrations of Grifola frondosa mycelia is up to for 6.4g/100mL culture medium.Our research further demonstrates the maximum fermentation concentration of the new Grifola frondosa strain ferment rice bran and wheat bran complete feed culture medium that again pass by mutation selection and is alternatively Testa oryzae 8.0g/100mL, wheat bran 8.0g/100mL, both also can reach 16.0g/100mL at sum, the dry weight concentrations of obtained pure mycelia can reach 7.5g/100mL culture medium, also can obtain the crude extracellular polysaccharide product in the fermentation liquid of higher concentration simultaneously.This new Grifola frondosa strain illustrating to select to obtain higher mycelial yield and more crude extracellular polysaccharide by mutation, is worth further investigation.Studying through mutagenesis testing, obtained desired Grifola frondosa new strains, achievement in research and this patent have declared national inventing patent simultaneously.The Grifola frondosa new strains that institute the obtains fermenting property when adding selenium fermentation, selenium-rich mycelial yield and the conversion capability to Testa oryzae wheat bran complete feed, and obtain a kind of selenium-rich, Grifola frondosa mycelia raw material containing grifolan, for one of the innovative content of this patent.
The patent inventor Liu Wei people of the present invention recognize that in years of researches Testa oryzae and wheat bran complete feed also need to be transformed as fermentation medium gradually, to stop the harmful substances such as lead in former Testa oryzae and wheat bran, arsenic, hydrargyrum, cadmium to be transferred in mycelia as far as possible, this problem was not also studied before making the present invention.Soil, deifferent regions.China lead, arsenic, hydrargyrum pollution situation different, lead contained by water produced rice and Semen Tritici aestivi, arsenic, mercury quantity also differ, for preventing these harmful elements from transferring in mycelia by Testa oryzae and wheat bran, Testa oryzae used and wheat bran should be carried out preliminary inspection and process, and lead, arsenic, hydrargyrum limitation Testa oryzae within certain span of control and wheat bran are just used as fermentation medium.It is likely to during the fermentation due to edible fungi be enriched with some harmful elements, therefore it is considered as introduction of competition when designing culture medium and suppresses the food additive of enrichment harmful element, such as cysteine hydrochloride etc., reduce harmful element by the competitive binding of the elements such as its sulfydryl and lead, arsenic, hydrargyrum and enter the chance of mycelia, thus obtaining qualified mycelia, but the enrichment of selenium will be produced retroaction by this inhibitory action, the method for the transfer therefore controlling harmful element lead, arsenic, hydrargyrum etc. and the enrichment realizing selenium is one of Important Problems of patent of the present invention needs solution.
Due to Oryza sativa L. and Semen Tritici aestivi harvesting link and storage link likely can go mouldy and machined Testa oryzae and wheat bran storage improper, produce aflatoxin, so using Testa oryzae and also should test and process during wheat bran, Aspergillus flavus limitation Testa oryzae in certain span of control and wheat bran are just used as fermentation medium.It is likely to during the fermentation due to edible fungi transfer in mycelia by aflatoxin, is therefore considered as equally when designing culture medium introducing the food additive suppressing aflatoxin transfer, such as sodium copper chlorophllin etc., to obtain qualified mycelia.Chlorophyll copper sodium salt pair plane fragrance carcinogen has complexing, can suppress carcinogenic activity, degradable carcinogen, have scavenging free radicals and antioxidation.Oryza sativa L. and Semen Tritici aestivi can be used pesticide in planting process, residual pesticide in Testa oryzae and wheat bran needs also exist for being controlled by by ingredient inspection and process, edible fungi utilizes the enzyme system of self can also degrade some harmful substances during the fermentation, the transfer preventing pesticide residues is also had beneficial effect by simultaneously affiliated sodium copper chlorophllin etc., and these 3 can prevent pesticide residues from transferring in fermentation mycelium.Therefore, two to the problem that the control of harmful substance aflatoxin and pesticide residues solves for patent of the present invention needs.
After by mutagenic obtained Grifola frondosa new strains, utilize the imagination of Testa oryzae and bran feedstock according to above-mentioned Efficient Conversion and realize selenium-rich and control the method for pollutant, research new strains is needed to the addition of selenium, cysteine hydrochloride, the Testa oryzae new culture medium of wheat bran complete feed of the liquid of sodium copper chlorophllin carries out fermentation and obtains desired mycelial method, and new method obtained convert Testa oryzae wheat bran ability should be higher than that existing achievement in research, Grifola frondosa mycelia is because of containing grifolan, the functional components such as organic selenium, and pollutant load is less than Limited Doses, its quality should be improved.Namely this patent provides desired method.
In sum, the key issue that the invention solves the problems that be provide obtained a kind of have no the Grifola frondosa new strains of report in the newly-designed Testa oryzae wheat bran new culture medium of complete feed liquid that with the addition of selenium effectively growth Efficient Conversion Testa oryzae wheat bran complete feed be the method for maitake mushroom mycelia, this new method must provide composition and the fermentation process of the newly-designed culture medium that with the addition of selenium, cysteine hydrochloride and sodium copper chlorophllin.Therefore patent of the present invention gives sufficient consideration when design to authorizing necessary to patent of invention unique, to obtain prominent substantive distinguishing features and marked improvement innovation and practicality.
The present invention utilizes by a mutagenic obtained strain Grifola frondosa new strains, the newly-designed Testa oryzae wheat bran complete feed liquid culture medium that with the addition of selenium that ferments manufactures quality good selenium-rich Grifola frondosa mycelia raw material, and this mycelia raw material may be used for being deeply processed into as health food.Selenium polysaccharide in the method fermentation liquid can stay the raw material being for further processing with deep processing.
Special instruction: the selenium Testa oryzae wheat bran complete feed liquid fermentation culture medium that adds of the present invention refers in particular to " processing the Testa oryzae reaching requirement and wheat bran as unique in culture medium carbon source, nitrogenous source through inspection; without other carbon sources, nitrogenous source; and Testa oryzae, wheat bran add water into liquid state; do not filter; namely complete feed is used, the desired amount of sodium selenite solution after the addition sterilizing of suitable stage fermented ".New culture medium refers to " with the addition of cysteine hydrochloride, sodium copper chlorophllin to stop what harmful substance possible in raw material proceeded to mycelia to add selenium Testa oryzae wheat bran complete feed liquid culture medium ".
The fermentation that the present invention utilizes adds the new Grifola frondosa strain of the selenium Testa oryzae wheat bran new culture medium of complete feed liquid for being invented acquisition first by ultraviolet mutagenesis.
The present invention provides the effective ways of the new culture medium manufacture selenium-rich Grifola frondosa mycelia raw material of Testa oryzae wheat bran complete feed of above-mentioned Grifola frondosa new strains fermentation liquid, including the measured value of the particular make-up of culture medium, fermentation condition, selenium-rich mycelial yield and the selenium-rich mycelia index of quality.The selenium-rich Grifola frondosa mycelia raw material obtained is through inspection, and lead, arsenic, mercury content are below GB16740-2014 limit index value, and aflatoxin content is lower than the limit index value of GB2761-2011 Oryza glutinosa class and goods thereof.
Summary of the invention
The present invention Testa oryzae new culture medium of wheat bran complete feed adding selenium of a kind of Grifola frondosa new strains fermentation liquid obtained by ultraviolet mutagenesis, efficient high-valued conversion Testa oryzae wheat bran is Grifola frondosa mycelia raw material and the outer selenium crude polysaccharides of more born of the same parents of selenium-rich, reduce production cost, expand yield, produce quality good selenium-rich Grifola frondosa mycelia raw material.The outer selenium crude polysaccharides of born of the same parents then gives over to the raw material processed for further deep processing.
The technical solution used in the present invention is as follows: utilize the Grifola frondosa new strains that a kind of ultraviolet mutagenesis obtains, and ferments in the Testa oryzae new culture medium of wheat bran complete feed adding selenium of liquid, produces the maitake mushroom mycelia raw material of selenium-rich.
A kind of method producing selenium-rich Grifola frondosa mycelia, carries out as steps described below:
(1) Testa oryzae and wheat bran are added 121 DEG C of sterilizing 30min of the desired amount of water；
null(2) using Testa oryzae and wheat bran complete feed as carbon source、Nitrogenous source is directly used in Grifola frondosa strain GrifolafrondosaCCTCCM2015065 fermentation medium，Testa oryzae concentration is 5-8g/100mL，Wheat bran concentration is 5-8g/100mL，The total concentration of Testa oryzae and wheat bran is 10.0-16.0g/100mL，Add potassium dihydrogen phosphate 0.015-0.36g/100mL，Magnesium sulfate heptahydrate 0.015-0.36g/100mL，Cysteine hydrochloride 20-50mg/100mL，Sodium copper chlorophllin 5-10mg/100mL，PH is natural，250mL shaking flask charging 100mL，Fermentation tank filling rate 70-75%，Inoculum concentration 8-10%，Cultivation temperature 24-28 DEG C，Shaking speed 150-180r/min or fermentation stirring slurry rotating speed 50-150r/min，The sodium selenite solution of the sterilizing of 0.45-2.68mg/100mL is added after fermentation 2d，Total fermentation time is 5-12d；
(3) mycelium containing a small amount of Testa oryzae wheat bran of liquid culture gained is isolated mycelia through 20 order metal gauzes in water, and wash twice with water；
(4) gained wet mycelium is carried out lyophilization, obtain mycelium powder finished product；
(5) take the concentration of clear liquid vacuum, spray drying after gained fermentation liquor filter cleaner, obtain the dry in fermentation liquid, can use as the raw material of deep development；
(6) with 90 DEG C of hot water extraction 4h after the grinding of gained mycelium powder, cross leaching clear liquid, after deproteinization, precipitate with ethanol, centrifugation, adopt Phenol sulfuric acid procedure to measure mycelia polysaccharide；
(7) gained fermentation liquor standing, deproteinization, take a certain amount of clear liquid after precipitate with ethanol, adopt Phenol sulfuric acid procedure to measure crude extracellular polysaccharide；
(8), after gained mycelium powder takes a certain amount of sample micro-wave digestion, icp ms is adopted to measure the content of selenium；
(9) with reference to the assay method of GB/T5009.12, GB/T5009.11, GB/T5009.17 lead, arsenic, the assay method of hydrargyrum and GB/T18979 aflatoxin, the content of pollutant lead, arsenic, hydrargyrum and aflatoxin in mensuration Grifola frondosa lyophilizing mycelium powder.
The Grifola frondosa new strains GrifolafrondosaCCTCCM2015065 used in step of the present invention (2), it is that Grifola frondosa strain GrifolafrondosaCCTCCM2013286 is through Uv-induced screening gained, the China typical culture collection center (CCTCC) being deposited in the Wuhan University of Wuhan, China on January 23rd, 2015, bacterial strain deposit number is CCTCCM2015065, and name is called Grifola frondosa CCTCCM2015065(latin name: GrifolafrondosaCCTCCM2015065).
In step of the present invention (2) during shaking flask charge is shaking flask volume 40%, inoculum concentration is the 8-10% of stocking volume, rotating speed 150-180r/min, and incubation time is 5-12d.
In step of the present invention (2), during upper tank fermentation, sample-loading amount is the 70-75% of fermenter volume, and during upper tank, ventilation is tinning liquid volume 1:0.8v/v/mim, cultivation temperature 24-28 DEG C, stir speed (S.S.) 50-150r/min, upper tank fermentation 5-12d.
The cryodesiccated operation pressure of step of the present invention (4) is 5Pa, and the temperature of hot plate is 30 DEG C, and freeze-drying time is 48-72h.
Beneficial effects of the present invention
Utilize described bacterial strain Grifola frondosa CCTCCM2015065 fermenting and producing maitake mushroom mycelia healthy food material in the Testa oryzae wheat bran new culture medium of complete feed liquid add selenium, when in the Testa oryzae wheat bran new culture medium of complete feed liquid, the total concentration of Testa oryzae and wheat bran reaches 16.0g/100mL, KH2PO4For 0.36g/100mL, MgSO4‘7H2O is 0.36g/100mL, cysteine hydrochloride is 50mg/100mL, sodium copper chlorophllin is 10mg/100mL, when sodium selenite is 2.68mg/100mL, dry mycelial weight, mycelia polysaccharide, extracellular polysaccharide, mycelia respectively reach during selenium concentration shake flask fermentation 5.8g/100mL, 266.1mg/100mL, 684.2mg/100mL and the 52.91 dry mycelia of μ g/g, during the fermentation of upper tank, then can reach the level of 5.4g/100mL, 252.8mg/100mL, 623.7mg/100mL and the 56.73 dry mycelia of μ g/g.Through check shake flask fermentation gained frondosa fermentation product mycelium in lead, arsenic, hydrargyrum content be 1.55mg/kg, 0.56mg/kg, 0.04mg/kg, it is below GB16740-2014 limit index value, aflatoxin content is 11.8 μ g/kg, lower than the limit index value of GB2761-2011 Oryza glutinosa class and goods thereof.Upper tank fermentation frondosa fermentation product mycelium in lead, arsenic, hydrargyrum content be 1.66mg/kg, 0.61mg/kg, 0.02mg/kg, it is below GB16740-2014 limit index value, aflatoxin content is 9.2 μ g/kg, lower than the limit index value of GB2761-2011 Oryza glutinosa class and goods thereof.
The present invention realizes described Grifola frondosa strain first and converts the method that the liquid Testa oryzae new culture medium of wheat bran complete feed is selenium-rich maitake mushroom mycelia raw material adding selenium.The bacterial strain GrifolafrondosaCCTCCM2015065 adopted is the strain Grifola frondosa new strains that inventor oneself mutation obtains, this bacterial strain effectively can grow in the liquid Testa oryzae new culture medium of wheat bran complete feed adding selenium, and Efficient Conversion Testa oryzae wheat bran is maitake mushroom mycelia, this bacterial strain has uniqueness, thus the present invention also has uniqueness.
The present invention also achieves the novelty of substantive distinguishing features and marked improvement, reason mainly have two: one be because new strains in the liquid Testa oryzae new culture medium of wheat bran complete feed during efficient growth nuisance transfer to mycelial degree and be reduced, this point did not occur in existing open source literature；nullTwo be GrifolafrondosaCCTCCM2015065 the Testa oryzae wheat bran new culture medium of complete feed liquid in the total concentration of Testa oryzae and wheat bran up to 16.0g/100mL，When adding the fermentation of a certain amount of selenium，The dry weight concentrations of pure mycelia、Mycelia polysaccharide concentration、Crude polysaccharides concentration in fermentation liquid respectively reaches 5.8g/100mL、266.1mg/100mL、684.2mg/100mL，Frondosa fermentation adds the level of this achievement of the Testa oryzae bran feedstock of selenium for obtain first，Open source literature has no report，And the content of selenium is the 52.91 dry mycelia of μ g/g in mycelia，Proper for manufacturing health food，Because the selenium that adult daily intakes is recommended less than 50 μ g，The highest less than 200 μ g，So mycelia is suitable for preparation health food，Under the premise ensureing mycelia polysaccharide amount，The daily intake of selenium can be taken into account again.
The Testa oryzae wheat bran of low value is changed into the selenium-rich maitake mushroom mycelia raw material with plurality of health care functions of high level by the present invention; to reducing production cost, the reasonable higher value application of low side resource high-efficiency, protection health of masses; all there is useful society and economic effect, thus there is practicality.
In sum, the present invention has had been provided with authorizing the necessary uniqueness of patent of invention, achieving the innovation and practicality of substantive distinguishing features and marked improvement, creates technology, economic and social beneficial effect that patent of invention should have.
Accompanying drawing explanation
Fig. 1 is the wet mycelia that the Testa oryzae new culture medium of wheat bran complete feed that is that bacterial strain Grifola frondosa CCTCCM2015065 shaking flask liquid fermentation adds selenium and that add cysteine hydrochloride and sodium copper chlorophllin is separating obtained.
Fig. 2 is born of the same parents' foreign object dried object of the new culture medium gained of Testa oryzae wheat bran complete feed that is that bacterial strain Grifola frondosa CCTCCM2015065 liquid fermentation adds selenium and that add cysteine hydrochloride and sodium copper chlorophllin.
Detailed description of the invention
The invention provides by ultraviolet mutagenesis, the method for the selection-breeding mutagenic strain GrifolafrondosaCCTCCM2015065 that the speed of growth is faster on Testa oryzae wheat bran complete feed liquid culture medium, polysaccharide yield is higher, described method comprises the following steps:
The GrifolafrondosaCCTCCM2013286 taking Laboratories Accession is starting strain；
In PDA culture medium, activation culture is carried out by being taken out an inoculation；Spore suspension is prepared with physiological saline solution eluting after yeast culture；
By the spore suspension required multiple of dilution, irradiating after carrying out mutation under uviol lamp, being connected in Testa oryzae wheat bran screening culture medium lucifuge cultivates, and just filters out the bacterial strain that growth rate is very fast, more stable；The bacterial strain of primary dcreening operation is carried out hereditary stability test, filters out the bacterial strain that growth rate is very fast, more stable again；
Carrying out shake flask fermentation with Testa oryzae wheat bran complete feed liquid culture medium, three filter out the bacterial strain that growth rate is high and polysaccharide yield is high；
Carry out antagonistic effect；
In one embodiment, PDA culture medium used is Rhizoma Solani tuber osi 200g/L, glucose 20g/L, peptone 5g/L, potassium dihydrogen phosphate 1.5g/L, magnesium sulfate 0.75g/L, and agar 20g/L, pH are natural.
In one embodiment, described constant temperature is 28 DEG C.
In one embodiment, described ultraviolet mutagenesis adopts HONGGUANG secretly to operate, and irradiates 40s respectively under the uviol lamp 20cm that distance power is 30W.
In one embodiment, described lucifuge is cultivated and is cultivated 5 days for lucifuge at 28 DEG C.
In one embodiment, it is Testa oryzae, wheat bran solid plate culture medium that described lucifuge cultivates used medium: Testa oryzae 30g/L, wheat bran 30g/L, potassium dihydrogen phosphate 1.5g/L, magnesium sulfate 0.75g/L, and agar 20g/L, pH are natural, and Testa oryzae, wheat bran complete feed use).
In one embodiment, described screening technique is plate diameter algoscopy.
In one embodiment, described primary dcreening operation step is: from the flat board that ultraviolet mutagenesis lucifuge is cultivated, the well-grown single bacterium colony of picking is seeded in new Testa oryzae wheat bran solid plate culture medium respectively, 10 strain fast growths and dense mutagenic fungi is picked out from ultraviolet mutagenesis, it is carried out 3 generation Secondary Culture, therefrom selects bacterial strain fast relative to starting strain growth, that shapeliness, stability are high.
In one embodiment, the described step of sieve again is: more excellent bacterial strain scalping selected carries out 5 generation flat board Secondary Culture respectively, the well-grown single bacterium colony of picking is seeded in new Testa oryzae wheat bran solid plate culture medium respectively, picks out fast growth, stalwartness, pure variant.
In one embodiment, described three sieve steps are: the Seedling height speed variant determined using multiple sieve, as the objects of three sieves, carries out shake flask fermentation screening so that it is determined that the bacterial strain of variant merit stably express together with starting strain.The bacterial strain filtered out again and starting strain GrifolafrondosaCCTCCM2013286 are carried out shake flask fermentation test, continuous fermentation 5 generation, determines purpose mutagenic fungi by index.Fig. 1 is the wet mycelia that the Testa oryzae new culture medium of wheat bran complete feed that is that bacterial strain Grifola frondosa CCTCCM2015065 shaking flask liquid fermentation adds selenium and that add cysteine hydrochloride and sodium copper chlorophllin is separating obtained.Fig. 2 is born of the same parents' foreign object dried object of the new culture medium gained of Testa oryzae wheat bran complete feed that is that bacterial strain Grifola frondosa CCTCCM2015065 liquid fermentation adds selenium and that add cysteine hydrochloride and sodium copper chlorophllin.
In one embodiment, described GrifolafrondosaCCTCCM2015065 growth rate on multiple sieve the 5th generation flat board is 1.1mm/h, is a growth rate strain bacterial strain faster.
In one embodiment, described shaking flask is 250mL conical flask.
In one embodiment, described Testa oryzae, wheat bran liquid fermentation medium are: Testa oryzae 8.0g/100mL, wheat bran 8.0g/100mL, potassium dihydrogen phosphate 0.36g/100mL, magnesium sulfate 0.36g/100mL, cysteine hydrochloride 50mg/100mL, sodium copper chlorophllin 10mg/100mL, pH are natural.
In one embodiment, described index is the pure dry mycelial weight of 100mL fermentation volume, mycelia polysaccharide weight and fermentation liquid crude extracellular polysaccharide weight.
In one embodiment, characterizing hereditary character with antimicrobial experiment, the growth performance of GrifolafrondosaCCTCCM2015065 is better than the performance of starting strain, and hereditary character there occurs change.
In one embodiment, the fermentation results that the new culture medium when Grifola frondosa new strains GrifolafrondosaCCTCCM2015065 fermentation that described screening obtains does not add selenium obtains is: the crude polysaccharides concentration in the dry weight concentrations of pure mycelia, mycelia polysaccharide concentration and fermentation liquid respectively reaches 7.9g/100mL, 524.6mg/100mL and 763.2mg/100mL.
In one embodiment, the dry mycelial weight concentration of new strains increases by 23.4%, and mycelia polysaccharide concentration adds 15.9%, illustrates that mutation gained new strains has the ability of higher ferment rice bran and wheat bran, and the fermenting property of bacterial strain there occurs change positive significantly.
In one embodiment, through inspection, in the mycelia of new strains fermentation gained, constituent content respectively 1.72mg/kg, 0.71mg/kg, the 0.07mg/kg such as lead, arsenic, hydrargyrum, it is below GB16740-2014 limit index value, aflatoxin content is 15.5 μ g/kg, and lower than the limit index value of GB2761-2011 Oryza glutinosa class and goods thereof, gained mycelia meets healthy food material requirement.
In one embodiment, adding the sodium selenite solution of the sterilizing of 2.68mg/100mL after fermentation 2d, total fermentation time is 7d.
In one embodiment, the result adding selenium new fermentation medium is: dry mycelial weight 5.8g/100mL culture medium, mycelia polysaccharide is 266.1mg/100mL culture medium, extracellular polysaccharide is 684.2mg/100mL culture medium, in mycelia, the content of selenium is the 52.91 dry mycelia of μ g/g, plumbous content 1.55mg/kg, the content 0.56mg/kg of arsenic, the content 0.04mg/kg of hydrargyrum, is below GB16740-2014 limit index value.The content 11.8 μ g/kg of aflatoxin, lower than the limit index value of GB2761-2011 Oryza glutinosa class and goods thereof.
The present invention utilizes Grifola frondosa CCTCCM2015065 fermenting and producing maitake mushroom mycelia raw material in the Testa oryzae new culture medium of wheat bran complete feed adding selenium of liquid, the China typical culture collection center (CCTCC) that described bacterial strain Grifola frondosa CCTCCM2015065 has been deposited in the Wuhan University of Wuhan, China on January 23rd, 2015, preservation strain is numbered CCTCCM2015065, and name is called that Grifola frondosa CCTCCM2015065(latin name is GrifolafrondosaCCTCCM2015065).The invention provides a kind of adopt Grifola frondosa CCTCCM2015065 liquid add in the Testa oryzae new culture medium of wheat bran complete feed of selenium produce maitake mushroom mycelia healthy food material production method.
In a specific embodiment, add water 121 DEG C of sterilizing 30min by Testa oryzae and wheat bran, is used for fermenting as culture medium.
Embodiment one
Grifola frondosa strain adopts Grifola frondosa CCTCCM2015065.Testa oryzae makes consumption be 8.0g/100mL culture medium, and the consumption that makes of wheat bran is 8.0g/100mL culture medium, adds KH2PO40.36g/100mL, MgSO4‘7H2O0.36g/100mL, cysteine hydrochloride 50mg/100mL, sodium copper chlorophllin 10mg/100mL, pH are natural, add water to required volume, and 121 DEG C of sterilizing 30min are used for fermenting as culture medium, and shaking flask sample-loading amount is 40%, and inoculum concentration is 10%, cultivation temperature 28oC, rotating speed 180r/min, add the sodium selenite solution of the sterilizing of 2.68mg/100mL after fermentation 2d, total fermentation time is 7d.Hereinafter press step (3)～(9) in technical scheme described by foregoing summary to implement.Result is: dry mycelial weight 5.8g/100mL culture medium, mycelia polysaccharide is 266.1mg/100mL culture medium, extracellular polysaccharide is 684.2mg/100mL culture medium, in mycelia, the content of selenium is the 52.91 dry mycelia of μ g/g, plumbous content 1.55mg/kg, the content 0.56mg/kg of arsenic, the content 0.04mg/kg of hydrargyrum, be below GB16740-2014 limit index value.The content 11.8 μ g/kg of aflatoxin, lower than the limit index value of GB2761-2011 Oryza glutinosa class and goods thereof.
Embodiment two
Grifola frondosa strain adopts Grifola frondosa CCTCCM2015065.Testa oryzae makes consumption be 5.0g/100mL culture medium, and the consumption that makes of wheat bran is 5.0g/100mL culture medium, adds KH2PO40.015g/100mL, MgSO4‘7H2O0.015g/100mL, cysteine hydrochloride 20mg/100mL, sodium copper chlorophllin 5mg/100mL, pH are natural, add water to required volume, and 121 DEG C of sterilizing 30min are used for fermenting as culture medium, and shaking flask sample-loading amount is 40%, and inoculum concentration is 8%, cultivation temperature 24oC, rotating speed 150r/min, add the sodium selenite solution of the sterilizing of 0.45mg/100mL after fermentation 2d, total fermentation time is 5d.Hereinafter press step (3)～(9) in technical scheme described by foregoing summary to implement.Result is: dry mycelial weight 3.5g/100mL culture medium, mycelia polysaccharide is 223.8mg/100mL culture medium, extracellular polysaccharide is 312.6mg/100mL culture medium, in mycelia, the content of selenium is the 22.36 dry mycelia of μ g/g, plumbous content 0.68mg/kg, the content 0.35mg/kg of arsenic, the content 0.02mg/kg of hydrargyrum, be below GB16740-2014 limit index value.The content 6.2 μ g/kg of aflatoxin, lower than the limit index value of GB2761-2011 Oryza glutinosa class and goods thereof.
Embodiment three
Grifola frondosa strain adopts Grifola frondosa CCTCCM2015065.Testa oryzae makes consumption be 6.4g/100mL culture medium, and the consumption that makes of wheat bran is 6.4g/100mL culture medium, adds KH2PO40.15g/100mL, MgSO4‘7H2O0.15g/100mL, cysteine hydrochloride 33mg/100mL, sodium copper chlorophllin 7.8mg/100mL, pH are natural, add water to required volume, and 121 DEG C of sterilizing 30min are used for fermenting as culture medium, and shaking flask sample-loading amount is 40%, and inoculum concentration is 9%, cultivation temperature 26oC, rotating speed 168r/min, add the sodium selenite solution of the sterilizing of 1.2mg/100mL after fermentation 2d, total fermentation time is 7d.Hereinafter press step (3)～(9) in technical scheme described by foregoing summary to implement.Result is: dry mycelial weight 4.1g/100mL culture medium, mycelia polysaccharide is 219.2mg/100mL culture medium, extracellular polysaccharide is 478.5mg/100mL culture medium, in mycelia, the content of selenium is the 39.06 dry mycelia of μ g/g, plumbous content 1.26mg/kg, the content 0.56mg/kg of arsenic, the content 0.02mg/kg of hydrargyrum, be below GB16740-2014 limit index value.The content 9.4 μ g/kg of aflatoxin, lower than the limit index value of GB2761-2011 Oryza glutinosa class and goods thereof.
Embodiment four
Grifola frondosa strain adopts Grifola frondosa CCTCCM2015065.Testa oryzae makes consumption be 5.2g/100mL culture medium, and the consumption that makes of wheat bran is 6.8g/100mL culture medium, adds KH2PO40.16g/100mL, MgSO4‘7H2O0.07g/100mL, cysteine hydrochloride 42mg/100mL, sodium copper chlorophllin 7.8mg/100mL, pH are natural, add water to required volume, and 121 DEG C of sterilizing 30min are used for fermenting as culture medium, and shaking flask sample-loading amount is 40%, and inoculum concentration is 8%, cultivation temperature 28oC, rotating speed 150r/min, add the sodium selenite solution of the sterilizing of 0.86mg/100mL after fermentation 2d, total fermentation time is 8d.Hereinafter press step (3)～(9) in technical scheme described by foregoing summary to implement.Result is: dry mycelial weight 3.2g/100mL culture medium, mycelia polysaccharide is 201.6mg/100mL culture medium, extracellular polysaccharide is 444.6mg/100mL culture medium, in mycelia, the content of selenium is the 26.58 dry mycelia of μ g/g, plumbous content 1.25mg/kg, the content 0.39mg/kg of arsenic, the content 0.02mg/kg of hydrargyrum, be below GB16740-2014 limit index value.The content 10.65 μ g/kg of aflatoxin, lower than the limit index value of GB2761-2011 Oryza glutinosa class and goods thereof.
Embodiment five
Grifola frondosa strain adopts Grifola frondosa CCTCCM2015065.Testa oryzae makes consumption be 7.0g/100mL culture medium, and the consumption that makes of wheat bran is 6.0g/100mL culture medium, adds KH2PO40.020g/100mL, MgSO4‘7H2O0.28g/100mL, cysteine hydrochloride 38mg/100mL, sodium copper chlorophllin 10.0mg/100mL, pH are natural, add water to required volume, and 121 DEG C of sterilizing 30min are used for fermenting as culture medium, and shaking flask sample-loading amount is 40%, and inoculum concentration is 10%, cultivation temperature 27oC, rotating speed 170r/min, add the sodium selenite solution of the sterilizing of 2.2mg/100mL after fermentation 2d, total fermentation time is 8d.Hereinafter press step (3)～(9) in technical scheme described by foregoing summary to implement.Result is: dry mycelial weight 3.7g/100mL culture medium, mycelia polysaccharide is 182.6mg/100mL culture medium, extracellular polysaccharide is 424.9mg/100mL culture medium, in mycelia, the content of selenium is the 39.56 dry mycelia of μ g/g, plumbous content 1.37mg/kg, the content 0.39mg/kg of arsenic, the content 0.03mg/kg of hydrargyrum, be below GB16740-2014 limit index value.The content 9.5 μ g/kg of aflatoxin, lower than the limit index value of GB2761-2011 Oryza glutinosa class and goods thereof.
Embodiment six
Grifola frondosa strain adopts Grifola frondosa CCTCCM2015065.Testa oryzae makes consumption be 5.8g/100mL culture medium, and the consumption that makes of wheat bran is 5.8g/100mL culture medium, adds KH2PO40.12g/100mL, MgSO4‘7H2O0.10g/100mL, cysteine hydrochloride 48mg/100mL, sodium copper chlorophllin 8.8mg/100mL, pH are natural, add water to required volume, and 121 DEG C of sterilizing 30min are used for fermenting as culture medium, and shaking flask sample-loading amount is 40%, and inoculum concentration is 10%, cultivation temperature 28oC, rotating speed 150r/min, add the sodium selenite solution of the sterilizing of 1.1mg/100mL after fermentation 2d, total fermentation time is 8d.Hereinafter press step (3)～(9) in technical scheme described by foregoing summary to implement.Result is: dry mycelial weight 3.7g/100mL culture medium, mycelia polysaccharide is 178.5mg/100mL culture medium, extracellular polysaccharide is 410.6mg/100mL culture medium, in mycelia, the content of selenium is the 45.1 dry mycelia of μ g/g, plumbous content 1.25mg/kg, the content 0.49mg/kg of arsenic, the content 0.02mg/kg of hydrargyrum, be below GB16740-2014 limit index value.The content 10.2 μ g/kg of aflatoxin, lower than the limit index value of GB2761-2011 Oryza glutinosa class and goods thereof.
Embodiment seven
Grifola frondosa strain adopts Grifola frondosa CCTCCM2015065.Fermentation tank sample-loading amount is the 75% of fermenter volume, and fermentation temperature is 28 DEG C, ventilation 1:0.8v/v/mim, mixing speed 90r/min, tank gauge pressure 0.05MPa, inoculum concentration 10%, and Testa oryzae is 8.0g/100mL culture medium, and wheat bran is 8.0g/100mL culture medium, adds KH2PO40.36g/100mL, MgSO4‘7H2O0.36g/100mL, cysteine hydrochloride 50mg/100mL, sodium copper chlorophllin 10mg/100mL, pH is natural, add water to required volume, 121 DEG C of sterilizing 30min, add the sodium selenite solution of the sterilizing of 2.68mg/100mL after fermentation 2d, total fermentation time is 7d, below presses step (3)～(9) in technical scheme described by foregoing summary and implements.Result is: dry mycelial weight 5.4g/100mL culture medium, mycelia polysaccharide is 252.8mg/100mL culture medium, extracellular polysaccharide is 623.7mg/100mL culture medium, in mycelia, the content of selenium is the 56.73 dry mycelia of μ g/g, plumbous content 1.66mg/kg, the content 0.61mg/kg of arsenic, the content 0.02mg/kg of hydrargyrum, be below GB16740-2014 limit index value.The content 9.2 μ g/kg of aflatoxin, lower than the limit index value of GB2761-2011 Oryza glutinosa class and goods thereof.
Embodiment eight
Grifola frondosa strain adopts Grifola frondosa CCTCCM2015065.Fermentation tank sample-loading amount is the 70% of fermenter volume, and fermentation temperature is 24 DEG C, ventilation 1:0.8v/v/mim, mixing speed 50r/min, tank gauge pressure 0.05MPa, inoculum concentration 8%, and Testa oryzae is 5.0g/100mL culture medium, and wheat bran is 5.0g/100mL culture medium, adds KH2PO40.015g/100mL, MgSO4‘7H2O0.015g/100mL, cysteine hydrochloride 20mg/100mL, sodium copper chlorophllin 5mg/100mL, pH is natural, add water to required volume, 121 DEG C of sterilizing 30min, add the sodium selenite solution of the sterilizing of 0.45mg/100mL after fermentation 2d, total fermentation time is 5d, below presses step (3)～(9) in technical scheme described by foregoing summary and implements.Result is: dry mycelial weight 3.4g/100mL culture medium, mycelia polysaccharide is 233.8mg/100mL culture medium, extracellular polysaccharide is 307.5mg/100mL culture medium, in mycelia, the content of selenium is the 29.55 dry mycelia of μ g/g, plumbous content 0.87mg/kg, the content 0.46mg/kg of arsenic, the content 0.01mg/kg of hydrargyrum, be below GB16740-2014 limit index value.The content 2.1 μ g/kg of aflatoxin, lower than the limit index value of GB2761-2011 Oryza glutinosa class and goods thereof.
Embodiment nine
Grifola frondosa strain adopts Grifola frondosa CCTCCM2015065.Fermentation tank sample-loading amount is the 72% of fermenter volume, and fermentation temperature is 26 DEG C, ventilation 1:0.8v/v/mim, mixing speed 80r/min, tank gauge pressure 0.05MPa, inoculum concentration 9%, Testa oryzae makes consumption be 6.4g/100mL culture medium, and the consumption that makes of wheat bran is 6.4g/100mL culture medium, adds KH2PO40.15g/100mL, MgSO4‘7H2O0.15g/100mL, cysteine hydrochloride 33mg/100mL, sodium copper chlorophllin 7.8mg/100mL, pH is natural, add water to required volume, 121 DEG C of sterilizing 30min, add the sodium selenite solution of the sterilizing of 1.20mg/100mL after fermentation 2d, total fermentation time is 10d, below presses step (3)～(9) in technical scheme described by foregoing summary and implements.Result is: dry mycelial weight 3.9g/100mL culture medium, mycelia polysaccharide is 208.5mg/100mL culture medium, extracellular polysaccharide is 495.6mg/100mL culture medium, in mycelia, the content of selenium is the 36.48 dry mycelia of μ g/g, plumbous content 1.36mg/kg, the content 0.65mg/kg of arsenic, the content 0.01mg/kg of hydrargyrum, be below GB16740-2014 limit index value.The content 5.5 μ g/kg of aflatoxin, lower than the limit index value of GB2761-2011 Oryza glutinosa class and goods thereof.
Claims (5)
1. the method producing selenium-rich Grifola frondosa mycelia, it is characterised in that carry out as steps described below:
(1) Testa oryzae and wheat bran are added 121 DEG C of sterilizing 30min of the desired amount of water；
null(2) using Testa oryzae and wheat bran complete feed as carbon source、Nitrogenous source is directly used in Grifola frondosa strain GrifolafrondosaCCTCCM2015065 fermentation medium，Testa oryzae concentration is 5-8g/100mL，Wheat bran concentration is 5-8g/100mL，The total concentration of Testa oryzae and wheat bran is 10.0-16.0g/100mL，Add potassium dihydrogen phosphate 0.015-0.36g/100mL，Magnesium sulfate heptahydrate 0.015-0.36g/100mL，Cysteine hydrochloride 20-50mg/100mL，Sodium copper chlorophllin 5-10mg/100mL，PH is natural，250mL shaking flask charging 100mL，Fermentation tank filling rate 70-75%，Inoculum concentration 8-10%，Cultivation temperature 24-28 DEG C，Shaking speed 150-180r/min or fermentation stirring slurry rotating speed 50-150r/min，The sodium selenite solution of the sterilizing of 0.45-2.68mg/100mL is added after fermentation 2d，Total fermentation time is 5-12d；
(3) mycelium containing a small amount of Testa oryzae wheat bran of liquid culture gained is isolated mycelia through 20 order metal gauzes in water, and wash twice with water；
(4) gained wet mycelium is carried out lyophilization, obtain mycelium powder finished product；
(5) take the concentration of clear liquid vacuum, spray drying after gained fermentation liquor filter cleaner, obtain the dry in fermentation liquid, can use as the raw material of deep development；
(6) with 90 DEG C of hot water extraction 4h after the grinding of gained mycelium powder, cross leaching clear liquid, after deproteinization, precipitate with ethanol, centrifugation, adopt Phenol sulfuric acid procedure to measure mycelia polysaccharide；
(7) gained fermentation liquor standing, deproteinization, take a certain amount of clear liquid after precipitate with ethanol, adopt Phenol sulfuric acid procedure to measure crude extracellular polysaccharide；
(8), after gained mycelium powder takes a certain amount of sample micro-wave digestion, icp ms is adopted to measure the content of selenium；
(9) with reference to the assay method of GB/T5009.12, GB/T5009.11, GB/T5009.17 lead, arsenic, the assay method of hydrargyrum and GB/T18979 aflatoxin, the content of pollutant lead, arsenic, hydrargyrum and aflatoxin in mensuration Grifola frondosa lyophilizing mycelium powder.
2. a kind of method producing selenium-rich Grifola frondosa mycelia according to claim 1, it is characterised in that the Grifola frondosa strain GrifolafrondosaCCTCCM2015065 used in step (2), bacterial strain deposit number is CCTCCM2015065.
3. a kind of method producing selenium-rich Grifola frondosa mycelia according to claim 1, it is characterised in that in step (2) during shaking flask charge is shaking flask volume 40%, inoculum concentration is the 8-10% of stocking volume, rotating speed 150-180r/min, and incubation time is 5-12d.
4. a kind of method producing selenium-rich Grifola frondosa mycelia according to claim 1, it is characterized in that during upper tank fermentation in step (2), sample-loading amount is the 70-75% of fermenter volume, during upper tank, ventilation is tinning liquid volume 1:0.8v/v/mim, cultivation temperature 24-28 DEG C, stir speed (S.S.) 50-150r/min, upper tank fermentation 5-12d.
5. a kind of method producing selenium-rich Grifola frondosa mycelia according to claim 1, it is characterised in that the cryodesiccated operation pressure of step (4) is 5Pa, and the temperature of hot plate is 30 DEG C, and freeze-drying time is 48-72h.
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