CN110423788A - A method of grifolan is produced using the Grifola frondosa strain that mutagenesis generates - Google Patents
A method of grifolan is produced using the Grifola frondosa strain that mutagenesis generates Download PDFInfo
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- CN110423788A CN110423788A CN201910601981.8A CN201910601981A CN110423788A CN 110423788 A CN110423788 A CN 110423788A CN 201910601981 A CN201910601981 A CN 201910601981A CN 110423788 A CN110423788 A CN 110423788A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
Abstract
The invention discloses a kind of methods of Grifola frondosa strain production grifolan generated using mutagenesis, are related to food microorganisms applied technical field.One plant of grifola frondosus new strains is obtained by ultraviolet mutagenesis, deposit number is CCTCC NO:M 2018907, the rice bran wheat bran complete feed culture medium for meeting contaminants in foods is being examined to carry out liquid state fermentation, Efficient Conversion rice bran wheat bran is maitake mushroom mycelia raw material and more crude extracellular polysaccharides.Tunning removes the miscellaneous grifolan product for obtaining qualification through multiple frequency ultrasonic extraction, freeze concentration, citric acid extraction displacement heavy metal ion, alcohol precipitation alcohol.The method that the present invention realizes grifola frondosus new strains production grifolan for the first time; the rice bran wheat bran of low value is converted to the grifolan with plurality of health care functions of high level by the present invention simultaneously; to production cost, the reasonable higher value application of low side resource high-efficiency, protection health of masses is reduced, all there is beneficial society and economic effect.
Description
Technical field
The present invention relates to food microorganisms applied technical field more particularly to a kind of mutagenic obtained new strains is used to produce
The method of polysaccharide, new strains are in the liquid culture for meeting the rice bran wheat bran complete feed of contaminants in foods through examining in the method
It ferments in base, it is dense by multiple frequency ultrasonic assisted extraction, freezing after the fermentation material for producing mycelium raw material and fermentation liquid
Contracting, citric acid extraction displacement heavy metal ion, alcohol precipitation alcohol wash, be lyophilized and obtains the polysaccharide product of qualification.
Background technique
Many dual-purpose of drug and food fungies or medicinal fungi have long usage history in China and widely use crowd.It is existing
Generation science discloses, the mycelium of existing frequently-used rare fungi such as grifola frondosus, Hericium erinaceus, ganoderma lucidum, Inonotus obliquus, cordyceps sinensis etc.,
Amino acid, protein, vitamin, polysaccharide, ucleosides, terpenoid, flavonoids etc. can be generated in fructification or spore
A variety of active ingredients or enrichment various trace elements such as selenium, zinc etc. have and improve the immunity of the human body, is antitumor, enhancing liver function
The multiple efficacies such as energy, anti-oxidant.Just because of this, international and domestic all kinds of personnel research and develop the with keen interest of this kind of fungi,
In field of medicaments, field of food, Traditional Chinese health field, agricultural and field of industrial production, all there are many researcher and developers, obtain
Obtain numerous research achievement or product, such as ganoderma lucidum capsule, grifola frondosus capsule and Paecilomyces hepiali chen capsule, their economic valence
It is worth higher.Hericium erinaceus being added in biscuit, Hericium erinaceus biscuit being then made, having become ordinary food, supermarket is sold at home.
New Zealand, Japan, the U.S., South Korea etc., which produce, in the world similar rare fungi health food or pharmaceutical products.
For grifola frondosus, for its research shows that: grifola frondosus it is antitumor, antiviral, hypoglycemic, delay senescence,
Strengthen immunity etc. all has certain bioactivity, medical value with higher (bibliography: 1. He Y, Li X,
Hao C,et al.Grifola frondosa polysaccharide:a review of antitumor and other
biological activity studies in China[J].Discovery Medicine,2018,25(138):159-
176.②Mao G H,Ren Y,Feng W W,et al.Antitumor and immunomodulatory activity of
a water-soluble polysaccharide from Grifola frondosa[J].Carbohydr Polym,2015,
134:406-412.③Chan Y Y,Chan E,Chan S W,et al.Enhancement of in vitro and in
vivo anticancer activities of polysaccharide peptide from Grifola frondosa by
chemical modifications[J].Pharmaceutical Biology,2011,49(11):1114-20.④He X,
Wang X,Fang J,et al.Polysaccharides in Grifola frondosa mushroom and their
health promoting properties:A review[J].International journal of biological
5. Zhang Zongqi, Liu Liping grifolan design feature and its bioactivity are ground by macromolecules, 2017,101:910.
Study carefully the brewing of progress [J] China, 2018 (5)).
Grifola frondosus (Grifola frondosa) is a kind of dual-purpose of drug and food fungi of preciousness, is under the jurisdiction of Basidiomycota, layer bacterium
Guiding principle, Polyporaceae, sets flower Pseudomonas also known as polyporus frondosus, thousand Buddhist bacterium, lotus flower bacterium, chestnut mushroom etc. at Aphyllophorales, according to its life
The difference in long place, grifola frondosus have different alias, are known as lotus flower bacterium in southwest, are called chestnut mushroom, In in the Northeast
Southwest mountainous areas of Zhejiang province is called " cloud gill fungus ", and Japan is referred to as " grifola frondosa " (Maitake), and the U.S. is referred to as " jungle fowl " (hen of the
woods).Grifola frondosus is grown in high mountain broad-leaf forest Oak Tree, chestnut or evergreen needle, in wealthy mixed forest more.The root of trees is parasitized,
It is typical forest whiterot fungi.Yunnan, Sichuan, Hebei, Heilungkiang, Jilin, Guangxi, Tibet, Fujian are distributed mainly in China
Etc. ground.Wild Grifola frondosa mainly originates under the broad-leaf forest of 800~1400m of height above sea level, likes warm weather and wetland,
Form can be divided into mycelium and fructification two large divisions, wherein product of the mycelium as Submerged Culture of Grifola frondosa, and color is cream
White, form are in spherical shape of uniform size；And the fructification as edible part is a kind of rich in protein, vitamin E and more
The nutraceutical of kind mineral element.The artificial domesticating cultivation of grifola frondosus has entered industrialization, and regional culture is basic in south China
It is northern then buried based on soil cultivation with generation material for substituting stuff cultivation.For Grifola Frondosa sporophore grey to taupe to canescence, edge has one
The irregular point of circle is convex, and bacterial context white, meat is tender and crisp, has handle and handle is short in coralliform branch, be overlapped into clump.Its appearance is laminated seemingly
Chrysanthemum, remote to see like cloud sheet layer by layer, the name of " cloud gill fungus " therefore obtains.The acquisition modes of present grifola frondosus are mainly artificial cultivation, with son
Entity or conidia powder are used as medicine, but the artificial cultivation grifola frondosus period is long, production efficiency is not high, large labor intensity, by season, environment etc.
Limitation, is subject to pest and disease damage, quality is unstable with yield.Rare fungi fermentation technology can overcome traditional fructification to cultivate not
Foot produces frondosa fermentation object using liquid or the method for solid state fermentation and is subject to the development that deep processing application meets innovation driving
Thinking has good prospect.The progress obtained to the research and development of frondosa fermentation object all will further push grifola frondosus
Application, bring actual social and economic benefit.Just because of this, just it is necessary to study new frondosa fermentation object and its depths
The production method of converted products, such as consider some low side processing of farm products byproduct sources such as wheat bran, rice bran Efficient Conversion
For the frondosa fermentation raw material of high value, it is expected to efficiently utilize low-grade agricultural resource, reduce frondosa fermentation object
To reduce the price of finished product such as grifola frondosus class health food or drug, interest concessions promote public strong production cost in ordinary people
Health, this is also one of the invention patent focus.
China is the big producer of rice and wheat, and rice bran and wheat bran are resourceful, and annual output rice bran is about 10,000,000 tons,
Wheat bran (this specification wheat bran refers both to wheat bran) is about 30,000,000 tons.Rice bran and wheat bran are as paddy or the pair of wheat processing
Product, rich in nutrition content are cheap.Contain in rice bran and enriches good protein, active polysaccharide, fat and fertility triolefin
The significant active material of the physiological functions such as phenol, tocopherol, wheat bran is rich in fat, protein, minerals, vitamin and cellulose etc.
Nutritional ingredient, wherein the content of cellulose is up to 18% or more of wheat bran total amount, so, rice bran and wheat bran can be ash tree flower growth
Sufficient carbon source, nitrogen source, vitamin and mineral are provided.It, can be by rice under the action of grifola frondosus cellulase and other enzyme systems
Chaff and wheat bran are converted to needed nutrient matter and carry out growth metabolism, synthesize maitake mushroom mycelia and types of functionality substance.Therefore,
With agricultural and sideline product rice bran, wheat bran as whole carbon source and nitrogen source, carries out grifola frondosus liquid state fermentation and produce frondosa fermentation object
And deep processing has technical feasibility at health food or drug, and can bring preferable social and economic benefit.
Most edible mushroom Liquid-state fermentation production methods use glucose, grain class raw material such as starch, potato or soybean powder
Deng be used as culture medium, even if agricultural and sideline product such as rice bran, wheat bran etc. can be used, and as auxiliary element be added.Original
Grifola frondosus upgrowth situation on the rice bran wheat bran complete feed liquid culture medium for not adding glucose or other grain class raw materials is not to be
Preferably, in addition rice bran, contain some harmful substances such as lead, arsenic, aflatoxin, vomitoxin, pesticide residue in bran feedstock
Object etc. influences final products quality, therefore the fermentation wheat bran rice bran liquid complete feed to grifola frondosus is needed to produce frondosa fermentation object
Method carries out innovation research.
The research group of this patent first invention people Liu Weimin is during the last ten years to the liquid of ganoderma lucidum, grifola frondosus, cordyceps sinensis etc.
State fermentation has carried out a large amount of research, successively forms multiple achievements such as Master's thesis and patent of invention.The master of Liu Weimin guidance
Paper has: (1) Yang Suohua, and frondosa fermentation rice bran prepares polysaccharide (2006 years)；(2) Gu Huimin, grifola frondosus is in rice bran culture medium
Middle liquid culture produces polysaccharide and the research (2009) for being enriched with organic selenium；(3) it opens and builds, physical method mutagenesis grifola frondosus liquid fermentation rice
The research (2010) of bran skin production polysaccharide；(4) Guo Chunmei, induction mutation of bacterium, the liquid fermentation rice bran wheat bran of grifola frondosus produce polysaccharide
And selenium-rich research (2011)；(5) Li Yanan, grifola frondosus induction mutation of bacterium and fermentation and performance study (2013)；(6) Liu Li
It is beautiful, the Ganoderma lucidum submerged fermentation and induction mutation of bacterium (2012) of high level conversion rice bran wheat bran；(7) Guo Tianlong, lucidum strain mutagenesis and
The full rice bran wheat bran research (2013) of the high-valued conversion of liquid state fermentation；(8) Chen Jing, cordyceps sinensis liquid fermentation higher value application rice
The research (2014) of bran skin production polysaccharide；(9) Zhang Xiaofei, ganoderma strain (Ganoderma lucidum CFCC6043) liquid
It ferments the research (2014) of full rice bran wheat bran and active polysaccharide；(10) Cao Xun, grifola frondosus mutagenesis and the fermentation of new and old strain liquid
Rice bran wheat bran studies (2015)；(11) Jin Lei, ganoderma lucidum mutagenesis and new and old strain liquid ferment rice bran and wheat bran research (2015
Year)；(12) Wang Xuemei, with the comparative studies (2016) of Cordyceps strain liquid fermentation rice bran wheat bran complete feed；(13) Li Ting
It is graceful, Cordyceps strain mutagenesis and the research (2017) of the new culture medium of new strains liquid state fermentation rice bran wheat bran complete feed.Liu Weimin
Patent of invention as inventor has: (1) method for producing polysaccharide using rice bran wheat bran compound material and grifola frondosus mutagenic strain,
20101010579048.4；(2) Grifola frondosa strain for rice bran and wheat bran compound material production polysaccharide,
201010579078.5；(3) bacterial strain for ferment rice bran and wheat bran extracting solution production grifolan,
201110150888.3；(4) bacterial strain for rice bran and wheat bran complete feed liquid state fermentation production grifolan,
201310274913.8；(5) a kind of method of ganoderma lucidum mutant strain liquid state fermentation rice bran wheat bran complete feed production polysaccharide,
201310275061.4；(6) a kind of method of liquid state fermentation rice bran wheat bran complete feed production Chinese caterpillar fungus polysaccharide,
201310274914.2；(7) a kind of method of grifola frondosus mutagenic fungi liquid state fermentation rice bran wheat bran complete feed production polysaccharide,
201310274911.9；(8) bacterial strain for rice bran and wheat bran complete feed liquid state fermentation production ganoderma lucidum polysaccharide,
201310274915.7；(9) production method of Se-rich lucid ganoderma mycelia raw material, 201510996120.6；(10) one plants of production ash trees
Spend mycelial grifola frondosus mutagenic strain, 201510990344.6；(11) the cordyceps sinensis mutagenesis of aweto mycelium is produced
Bacterial strain, 201510996118.9 etc..
As seen from the above description, inventor herein Liu Weimin is efficiently high-valued all around trans-utilization rice bran and wheat bran
Production this special topic of Rare edible fungus fermentation material is studied, and realizes the imagination of innovation and creation, and is continuously improved various rare true
The production method of bacterium fermentation material.
The inventor Liu Wei people have participated in 13 emphasis research and development plan special project of country, and " modern food processing and grain store up fortune
In technology and equipment " in project " large rice made products appropriateness processing key technical equipment research and development and demonstration (2017YFD0401105) "
The research of project " foodization of Rice producing by-product utilizes new technology and new model ", assumes responsibility for Task " rice therein
Chaff semi-solid ferment key technology and new-product development ".Liu Weimin combination the studies above task, has combed the master instructed and has ground
Study carefully raw paper and in the past patent of invention relevant to the invention patent, considering can not when transferring the possession of with enterprise negotiation related ends
The problem of answer, such as pervious strain provenance, illustrate the requirement for being unsatisfactory for enterprise, enterprise thus the hope for carrying out industrialization development
It is not strong etc., have been found that Industrialization to be moved towards, there are still many problems to need for relevant patented technology and research paper
It researchs and solves and is innovated.These problems include have the rare fungus in high efficiency new strains of different performance acquisition and confirmation and
Exploitation, the formulation of rice bran bran feedstock standard, the innovation of rare fungal fermentate fermentation process, rare fungal fermentate isolate and purify
New method uses and these need to innovate the combination etc. of object.Patent of invention is in design to Grant Patent Right for Invention institute
Necessary unique, acquirement substantive distinguishing features outstanding and the innovation and practicality of marked improvement will give abundant consideration.
So the combined innovation of above-mentioned these problems should be ensured that relevant patent of invention reaches necessary to Grant Patent Right for Invention solely
Characteristic, innovation and practicality.
Inventor based on the above considerations and the research to frondosa fermentation object production method, proposes the invention patent
Application, be utilized the Grifola Frondosa sporophore picked from production scene isolated Grifola frondosa strain source different from the past therefore
The different grifola frondosus original strain of fermenting property carries out mutagenic obtained new strains, and the ash tree that the production of ferment rice bran wheat bran is qualified
Flower polysaccharide product.
In conclusion field of the present invention is that be used to ferment by new strains wheat bran and rice bran complete feed produces fermentation material simultaneously
Become qualified products after deep processing is handled.Be different from the achievement of previous research and acquirement: the invention solves key
One of problem is the method needed through mutagenesis, promotes the fermentability of original strain, obtains a kind of new strains having not been reported,
And gene sequencing can be carried out to this new strains, it is ensured that strain used is correct；The invention solves critical issue two to make
Wheat bran raw rice bran standard, because wheat bran and rice bran source are complicated, wherein pollutant kind and its level are different, before inventor
Research and patent do not give enough concerns in this regard, to subsequent product exploitation bring indefinite consequence；The invention solves
Critical issue three production methods for becoming qualified products after deep processing is handled for fermentation material mycelia and fermentation liquid.With
Upper three promise the invention patent and all kinds of achievements of other inventors and the present inventor Liu Weimin research team are different, tool
There is unique, innovation and practicality.
The reason of proposing for the critical issue in terms of above three is further explained.It is proposed the invention solves
One of critical issue proposes the method needed through mutagenesis, promote the fermentability of original strain, obtains one kind and does not appear in the newspapers
The new strains in road, and gene sequencing can be carried out to this new strains, it is ensured that strain used is correct.The background of this problem is to invent
People team it is expected to obtain one plant from unused and with good performance bacterial strain, and it is expected that it can be in wheat bran rice bran bioconversion
Aspect has good behaviour, therefore considers the method for research fermentation wheat bran rice bran production fermentation material.It is passed through according to the previous research of inventor
It tests, this kind of rare fungi fermentation wheat bran rice bran is firstly the need of the adaptability problem for solving strain.It would therefore be desirable to this all one's life
The fermentability of strain in production method is investigated, and improves its fermenting property by the method for induction mutation of bacterium.In addition it is grinding
During studying carefully, to guarantee that mistake does not occur in strain after a large amount of strain processing step, it should carry out gene sequencing.This is previous
Research in do not pay attention to this thing.In this way, this patent dig-ins the measurement of gene series from the beginning, guarantee that bacterial strain is correct.
It is proposed the invention solves critical issue two be make wheat bran raw rice bran standard background be it is former we
Research and patent do not give enough concerns in this regard, to subsequent product exploitation bring unexpected consequence sometimes.With
Preceding we always wish that studied achievement is capable of handling various wheat brans and rice bran, therefore relax use intentionally to wheat bran raw rice bran
Limitation does not carry out raw material standard formulation, but this have the consequence that occurs not in end product quality characterization in research model
Enclose interior some exceptional values, such as certain determining heavy metals result excessive problem that previous studies do not measure, trace it to its cause be because
Complicated for wheat bran and raw rice bran source, quality is difficult to keep similar level, and unknown pollutant eventually enters into end product.In this way
Originally the invention production method designed for certain specific pollutants and its possible content, it is possible to occur it in the product
Its pollutant copes with the new pollutant being likely to occur so to redesign the production method of fermentation material.In this way, wheat bran and
The control of raw rice bran quality and standard formulation problem just become the problem of new invention Patent design must be taken into consideration.The present invention proposes
The use standard of raw material wheat bran and rice bran.
It is proposed the invention solves critical issue three for fermentation material carry out deep processing processing become qualification
The production method of product, background are that the conjunction of entire fermentation material production method is considered on the basis of the first two critical issue solves
Manage reasonability.Since wheat bran raw rice bran is possible to bring unknown pollutant into, it is dense that freezing is devised to tunning after fermentation
The process for separation and purification such as contracting, citric acid extract displacement heavy metal ion, alcohol precipitation alcohol is washed, it is final to obtain qualified grifola frondosus Thick many candies
Product.Based on this understanding, the invention patent designs more reasonable fermentation material production and processing method.
The present invention needs to obtain new strains by the method for mutagenesis.The selection of microorganism mainly has physical mutagenesis, changes
Learn mutagenesis, genetic recombination etc., the present invention is to avoid using poisonous and hazardous chemical mutagen and also not public in field of food
The gene engineering method of approval will utilize relatively easy easy ultraviolet mutagenesis technology, starting strain made to be in the extreme of mutagenesis
A possibility that environment expands the range in mutational site to greatest extent, and raising obtains direct mutation bacterial strain.The present invention uses ultraviolet mutagenesis
This common method of mutagenesis, although the method does not have novelty, novelty is had occurred in the bacterial strain performance that the method obtains
Variation, the unique, innovation and practicality when fermenting wheat bran rice bran complete feed culture medium.
Inventor mainly solves the critical new problem of above three, constructs the contents of the present invention.
The new strains of gained fermentation wheat bran rice bran complete feed liquid culture medium of the invention are that invention obtains for the first time.
Wheat bran of the invention is to execute the former Ministry of Agriculture to eat feed wheat bran standard NY/T 3218-2018 using standard, outside
Add the level of pollution not more than 3mg/kg of Heavy Metals lead, arsenic, mercury, cadmium, chromium, applying pesticides accord with during wheat planting
Agrotechnical specification is closed, is done harm in wheat growth without serious head blight, harvesting season causes wheat rotten without continuous rainwater, small Meccah
Wheat bran storage and transportation not will lead to mildew after work.Rice bran is to execute the former agricultural additional main weight of ministerial standard NY/T122-1989 using standard
Metallic lead, arsenic, mercury, cadmium, chromium level of pollution limit the quantity not more than 3mg/kg, applying pesticides meet agriculture during Rice Cropping
Skill specification, paddy growth is in the process without serious plant disease, and harvesting season causes paddy rotten without continuous rainwater, rice bran after paddy processing
Storage and transportation not will lead to mildew.
The present invention provides above-mentioned new strains through examining the liquid for meeting the rice bran wheat bran complete feed of contaminants in foods to train
It supports and ferments in base, the method for preparing polysaccharide, concrete composition, fermentation condition, extraction and impurity-removing method, bacterium including culture medium
The measured value of silk and polysaccharide yield and the polysaccharide index of quality.The mycotoxin aflatoxin B1s of total starches obtained, B2 and
Limitation, heavy metal lead, arsenic, mercury, cadmium, the limitation of chromium and pesticide carbendazim, avermectin, the residual of the butachlor limit of vomitoxin
Amount not higher than Limited Doses as defined in GB2761-2017, GB2762-2017, GB2763-2016 or is not detected by measurement.
Summary of the invention
A kind of method for producing grifolan by mutagenic obtained grifola frondosus new strains of the present invention, in the method
Grifola frondosus new strains ferment in the liquid culture medium through examining the rice bran wheat bran complete feed for meeting contaminants in foods, raw
After the fermentation material of output maitake mushroom mycelia raw material and fermentation liquid, by multiple frequency ultrasonic assisted extraction, freeze concentration, lemon acidleach
Displacement heavy metal ion, alcohol precipitation alcohol is mentioned to wash, be lyophilized and obtain the grifolan product of qualification.
The technical solution used in the present invention is as follows: using a kind of grifola frondosus new strains obtained through ultraviolet mutagenesis, passing through
It examines and ferments in the liquid culture medium for the rice bran wheat bran complete feed for meeting contaminants in foods, produce maitake mushroom mycelia
After the fermentation material of raw material and fermentation liquid, by multiple frequency ultrasonic assisted extraction, freeze concentration, citric acid extraction displacement heavy metal from
Son, alcohol precipitation alcohol wash, be lyophilized obtain qualification grifolan product.
In one aspect of the invention, the method for the grifola frondosus new strains production grifolan generated using mutagenesis,
It carries out as steps described below:
(1) examine wheat bran and rice bran, Heavy Metals lead, arsenic, mercury, cadmium, chromium level of pollution not more than 3mg/kg,
Remaining corresponds with the former Ministry of Agriculture and eats feed wheat bran standard NY/T 3218-2018 and rice bran standard NY/T122-1989, small
Applying pesticides meet agrotechnical specification during wheat seeds are planted, and do harm in wheat growth without serious head blight, gather in season without continuous
Rainwater causes wheat rotten, and wheat bran storage and transportation not will lead to mildew after wheat processing, and applying pesticides meet agriculture during Rice Cropping
Skill specification, paddy growth is in the process without serious plant disease, and harvesting season causes paddy rotten without continuous rainwater, rice bran after paddy processing
Storage and transportation not will lead to mildew；
(2) rice bran and wheat bran are added into 121 DEG C of sterilizing 30min of the desired amount of water；
(3) rice bran and wheat bran complete feed are directly used in grifola frondosus new strains as carbon source, nitrogen source and save number CCTCC NO:
The fermentation medium of the bacterial strain of M 2018907, rice bran concentration are 0.1-10g/100mL, and wheat bran concentration is 0.1-10g/100mL,
Rice bran and wheat bran concentration sum of the two are up to 10.1g/100mL, add potassium dihydrogen phosphate 0.015-0.25g/100mL, seven water
Magnesium sulfate 0.015-0.25g/100mL, pH 100mL, fermentor filling rate 75%, inoculum concentration naturally, 250mL shaking flask charges
10%, 20-25 DEG C of cultivation temperature, shaking speed 150r/min or fermentation rotating speed of agitator 50-150r/min, fermentation time 7-
10d；
(4) liquid culture is centrifugally separating to obtain mycelium and fermentation liquid, and measures the production of exocellular polysaccharide in fermentation liquid
Amount；
(5) the ultrasonic wave added liquid circulation of 35kHz and 1.5kW is added to extract 1.5h 70 DEG C of hot water of gained mycelium, from
Heart separation takes clear liquid, and measures mycelia polysaccharide yield；
(6) gained clear liquid will be centrifugated twice to merge, freeze concentration, citric acid extraction displacement heavy metal ion, alcohol precipitation
Alcohol washes, is lyophilized and obtains qualified grifolan product；
(7) gained grifola frondosus mycelia polysaccharide and exocellular polysaccharide are all made of Phenol sulfuric acid procedure measurement, and gained grifolan is
The sum of mycelia polysaccharide and exocellular polysaccharide are total starches.
(8) referring to national standard, pollutant lead, the arsenic, mercury, cadmium, chromium, aflatoxin B1, deoxidation in grifolan are measured
Nivalenol, carbendazim, avermectin, the content of butachlor.
In one aspect of the invention, grifola frondosus new strains used in step (3) of the present invention, Latin are entitled
Grifolafrondosa, it is the ash that inventor picks from grifola frondosus production scene that saving number, which is CCTCC NO:M 2018907,
The Grifola frondosa strain that tree beggar's entity separation obtains is through obtained by Uv-induced screening, Jing Shenggong bioengineering (Shanghai) share is limited
Company's DNA sequencing is determined as one plant of grifola frondosus new strains, and the grifola frondosus new strains are in place in preservation on December 19 in 2018
In Wuhan, China Wuhan University China typical culture collection center (CCTCC), deposit number is CCTCC NO:M 2018907,
It is recommended that classification naming be Grifola frondosa strain JSULIUWANG18Grifola frondosa JSULIUWANG18, institute of the present invention
It states grifola frondosus new strains and refers both to this bacterial strain.
In one aspect of the invention, the charge in step (3) of the present invention when shaking flask is the 40% of shaking flask volume, is connect
Kind amount is the 10% of stocking volume, revolving speed 150r/min, incubation time 7-10d.
In one aspect of the invention, sample-loading amount is fermenter volume when upper tank fermentation in step (3) of the present invention
75%, ventilation quantity is that the ratio between ventilation volume and tinning liquid volume per minute are 1:0.8, cultivation temperature under aeration condition when upper tank
20-25 DEG C, stirring rate 50-150r/min, upper tank fermentation 7-10d.
Beneficial effects of the present invention
Using the grifola frondosus new strains, through examining the liquid for meeting the rice bran wheat bran complete feed of contaminants in foods to train
Fermenting and producing grifolan in base is supported, rice bran is 7.0g/100mL, wheat bran 3.1g/ in rice bran wheat bran complete feed culture medium
100mL, potassium dihydrogen phosphate 0.25g/100mL, magnesium sulfate 0.15g/100mL, the dry weight concentrations of mycelia, mycelia Thick many candies are dense
Degree, crude extracellular polysaccharide concentration and total Thick many candies concentration respectively reach 3.151g/100mL, 156.861mg/100mL,
331.876mg/100mL and 488.737mg/100mL.And it then can reach 3.821g/100mL, 183.021mg/ when the fermentation of upper tank
The level of 100mL, 247.665mg/100mL and 430.686mg/100mL.Through detecting, what shake flask fermentation and upper tank fermented
The mycotoxin aflatoxin B1 of total starches, the limitation of B2 and deoxynivalenol, heavy metal lead, arsenic, mercury, cadmium,
The limitation and pesticide carbendazim of chromium, avermectin, butachlor residue limits be not higher than GB2761-2017, GB2762-
2017, it Limited Doses as defined in GB2763-2016 or is not detected.
The method that the present invention realizes the grifola frondosus new strains production grifolan for the first time, grifola frondosus is new in the method
Bacterial strain ferments in the liquid culture medium through examining the rice bran wheat bran complete feed for meeting contaminants in foods, tunning warp
It is more that multiple frequency ultrasonic extraction, freeze concentration, citric acid extract and replace heavy metal ion, alcohol precipitation alcohol removes the miscellaneous grifola frondosus for obtaining qualification
Sugared product.Used grifola frondosus new strains are one plant of grifola frondosus new strains that inventor oneself mutagenesis obtains, which can be
The new culture medium of liquid rice bran wheat bran complete feed is effectively grown, and Efficient Conversion rice bran wheat bran is grifolan, which has only
Characteristic, thus the present invention is also unique.
The present invention also achieves the novelty of substantive distinguishing features and marked improvement.The dry weight of grifola frondosus new strains mycelia is dense
Degree, mycelia Thick many candies concentration, crude extracellular polysaccharide concentration and total Thick many candies concentration respectively reach 3.151g/100mL,
156.861mg/100mL, 331.876mg/100mL and 488.737mg/100mL.Total starches are that final grifolan produces
Object reaches the production level of 488.737mg/100mL, and in grifolan pollutant content it is low, meet raw-food material
It is required that illustrating that grifola frondosus new strains fermenting property in Optimal Medium is good and high yield grifolan, the invention patent obtain
Novelty with substantive distinguishing features and marked improvement.
The rice bran wheat bran of low value is converted to the grifolan with plurality of health care functions of high level by the present invention, to reduction
Production cost, the reasonable higher value application of low side resource high-efficiency, protection health of masses, all have beneficial society and economic effect,
Thus there is practicability.
In conclusion the present invention has had been provided with uniqueness necessary to authorizing patent of invention, has achieved substantive distinguishing features
With the innovation and practicality of marked improvement, technology, the beneficial effect of economy and society that patent of invention should have are produced.
Detailed description of the invention
When Fig. 1 is 2018907 shaking flask liquid state fermentation rice bran wheat bran complete feed of bacterial strain grifola frondosus CCTCC NO:M new culture medium
Outside drawing.
Fig. 2 is that bacterial strain grifola frondosus CCTCC NO:M 2018907 is resulting in the new culture medium of liquid state fermentation rice bran wheat bran complete feed
Mycelia product.
Fig. 3 be grifola frondosus new strains and chadogram.
Specific embodiment
The present invention provides by ultraviolet mutagenesis, the speed of growth is more on rice bran and wheat bran complete feed liquid culture medium for breeding
Fastly, the method for the higher grifola frondosus new strains of polysaccharide yield, the method includes the following steps:
The original strain Grifola frondosa of laboratory preservation is taken, is starting strain；
Taken starting strain is inoculated in PDA culture medium and carries out activation culture；
It is eluted after thallus culture with sterile saline and spore suspension is made；
Multiple needed for spore suspension is diluted after irradiation carries out mutagenesis in the UV lamp, is connected to rice bran and wheat bran screening
Culture is protected from light on culture medium, initial screening goes out the bacterial strain that growth rate is very fast, more stable；The bacterial strain of primary dcreening operation is subjected to inheritance stability
Property test, secondary screening selects the bacterial strain that growth rate is very fast, more stable；
Shake flask fermentation is carried out with rice bran and wheat bran complete feed liquid culture medium, growth rate height is filtered out and polysaccharide yield is high
Bacterial strain；
The new strains filtered out carry out antagonistic effect and gene sequencing.
In one embodiment, PDA culture medium used is potato 200g/L, glucose 20g/L, peptone 5g/
L, potassium dihydrogen phosphate 1.5g/L, epsom salt 0.75g/L, agar 20g/L, pH are natural.
In one embodiment, the constant temperature is 23 DEG C.
In one embodiment, the ultraviolet mutagenesis is secretly operated using feux rouges, in the ultraviolet lamp for being 20W apart from power
40s is irradiated under 20cm respectively.
In one embodiment, described to be protected from light culture to be protected from light culture 7 days at 23 DEG C.
In one embodiment, the culture used medium that is protected from light is rice bran, wheat bran solid plate culture medium: rice bran
20g/L, wheat bran 30g/L, potassium dihydrogen phosphate 1.5g/L, epsom salt 0.75g/L, agar 20g/L, pH are naturally, rice bran, wheat bran
Complete feed uses, and rice bran and wheat bran have to comply with standard.
In one embodiment, the screening technique is plate diameter measuring method.
In one embodiment, the primary dcreening operation step are as follows: it is good to be protected from light picking growth on the plate of culture from ultraviolet mutagenesis
Good single colonie is seeded to respectively in new rice bran and wheat bran solid plate culture medium, is therefrom selected and is grown relative to starting strain
Fastly, the high bacterial strain of shapeliness, stability.
In one embodiment, the secondary screening step are as follows: the more excellent bacterial strain that scalping is selected is subjected to 5 generation plates respectively and is passed
It is commissioned to train feeding, the well-grown single colonie of picking is seeded to respectively in new rice bran and wheat bran solid plate culture medium, selects birth
Long speed is fast, healthy and strong, pure variant.
In one embodiment, the three sieves step are as follows: using the Seedling height rate variant that secondary screening determines as three sieves
Object, together with starting strain carry out shake flask fermentation screening so that it is determined that variant merit stablizes the bacterial strain of expression.It will
The grifola frondosus new strains and original starting strain Grifola frondosa that secondary screening is selected carry out shake flask fermentation test, continuously ferment
In 5 generations, determined target mutagenic fungi by index.
In one embodiment, the grifola frondosus mutagenesis new strains are No. 6 bacterial strain, on the 5th generation plate of secondary screening
Growth rate is 1.738cm/d, hence it is evident that is faster than the growth rate of No. 0 bacterial strain of starting strain.
In one embodiment, the shaking flask is 250mL conical flask.
In one embodiment, the rice bran, wheat bran liquid fermentation medium are as follows: rice bran 7.0g/100mL, wheat bran
1.6g/100mL, potassium dihydrogen phosphate 0.25g/100mL, magnesium sulfate 0.25g/100mL, pH are natural.
In one embodiment, required volume, 121 DEG C of sterilizing 30min, as culture medium for fermenting are added water to.
In one embodiment, shaking flask sample-loading amount is 40%, inoculum concentration 10%.
In one embodiment, 23 DEG C of cultivation temperature, revolving speed 150r/min, incubation time 8d.
In one embodiment, the index be the pure dry mycelial weight of 100mL fermentation volume, mycelia Thick many candies weight and
Fermentation liquid crude extracellular polysaccharide weight.
In one embodiment, mycelia Thick many candies and crude extracellular polysaccharide are the product before separation of polysaccharides, using phenol sulphur
Acid system measurement.
In one embodiment, inhereditary feature is characterized with antimicrobial experiment, result is as shown in Fig. 2, and antagonism line is bright
Aobvious, the grifola frondosus mutagenesis new strains growth on the right is dense, thick and solid, it is seen that its growth performance is better than the performance of starting strain, heredity
Beneficial variation has occurred in character.
In one embodiment, grifola frondosus new strains, saving number is CCTCC NO:M 2018907, through raw work biology
Engineering (Shanghai) limited liability company DNA sequencing is determined as one plant of grifola frondosus new strains.The grifola frondosus new strains are in 2018
On December 19, in is deposited in positioned at Wuhan, China Wuhan University China typical culture collection center (CCTCC), and deposit number is
CCTCC NO:M 2018907, it is proposed that classification naming be Grifola frondosa strain JSULIUWANG18 Grifola frondosa
JSULIUWANG18, grifola frondosus new strains of the present invention refer both to this bacterial strain (see Fig. 3).
The present invention provides a kind of methods using mutagenic obtained grifola frondosus new strains production grifolan, in the party
Grifola frondosus new strains are sent out in the liquid culture medium through examining the rice bran wheat bran complete feed for meeting contaminants in foods in method
Ferment, after the fermentation material for producing maitake mushroom mycelia raw material and fermentation liquid, by multiple frequency ultrasonic assisted extraction, freeze concentration, lemon
Lemon acidleach mentions displacement heavy metal ion, alcohol precipitation alcohol and washes, be lyophilized and obtain the grifolan product of qualification, and the method includes following
Step:
In one embodiment, examine wheat bran and rice bran, Heavy Metals lead, arsenic, mercury, cadmium, chromium level of pollution most
Height is no more than 3mg/kg, remaining corresponds with the former Ministry of Agriculture and eats feed wheat bran standard NY/T 3218-2018 and rice bran standard
NY/T122-1989, applying pesticides meet agrotechnical specification during wheat planting, without serious head blight evil in wheat growth,
Harvesting season causes wheat rotten without continuous rainwater, and wheat bran storage and transportation is applied during not will lead to mildew Rice Cropping after wheat processing
Meet agrotechnical specification with pesticide, paddy growth is in the process without serious plant disease, and harvesting season causes paddy rotten without continuous rainwater, rice
Rice bran storage and transportation not will lead to mildew after paddy processing；
In one embodiment, grifola frondosus new strains CCTCC NO:M 2018907 is through raw work bioengineering (Shanghai) stock
Part Co., Ltd DNA sequencing is determined as one plant of grifola frondosus new strains, is used as fermentation strain；
In one embodiment, the rice bran, wheat bran liquid fermentation medium are as follows: rice bran concentration is 0.1-10g/
100mL, wheat bran concentration are 0.1-10g/100mL, and rice bran and wheat bran concentration sum of the two are up to 10.1g/100mL addition phosphoric acid
Potassium dihydrogen 0.015-0.25g/100mL, epsom salt 0.015-0.25g/100mL, pH are naturally, 250mL shaking flask charges
100mL, fermentor filling rate 75%, inoculum concentration 10%, 20-25 DEG C of cultivation temperature, shaking speed 150r/min or fermentation are stirred
Starch revolving speed 50-150r/min, fermentation time 7-10d；
In one embodiment, the resulting maitake mushroom mycelia of Liquid Culture is centrifuged, respectively obtains bacterium
Filament and separating liquid measure crude extracellular polysaccharide to separation clear liquid Phenol sulfuric acid procedure；
In one embodiment, by maitake mushroom mycelia extract polysaccharide operation, with 60 DEG C of hot water add 35kHz and
The ultrasonic wave added liquid circulation of 1.5kW extracts 1.5h, and centrifuge separation takes clear liquid, and measures mycelia polysaccharide yield with phenol sulfuric acid；
In one embodiment, gained fermentation liquid and mycelia extracting solution merge, and set by freeze concentration, citric acid extraction
Change heavy metal ion, alcohol precipitation alcohol is washed, the method that is freeze-dried obtains qualified exocellular polysaccharide；
In one embodiment, the fermentation knot for screening obtained grifola frondosus new strains and fermentation medium being utilized to obtain
Fruit are as follows: dry weight concentrations, mycelia Thick many candies concentration, crude extracellular polysaccharide concentration and the total Thick many candies concentration of mycelia are respectively reached and reached respectively
To 3.151g/100mL, 156.861mg/100mL, 331.876mg/100mL and 488.737mg/100mL.
In one embodiment, grifolan yield reaches 488.737mg/100mL, illustrates that grifola frondosus new strains exist
Fermenting property is good in Optimal Medium and high yield grifolan, the invention patent achieve with substantive distinguishing features and significantly into
The novelty of step.
In one embodiment, through detecting, mycotoxin aflatoxin B1, B2 and the deoxynivalenol of total starches
Limitation, heavy metal lead, arsenic, mercury, cadmium, the limitation of chromium and the pesticide carbendazim of bacterium enol, avermectin, butachlor residue limits
It is not higher than Limited Doses as defined in GB2761-2017, GB2762-2017, GB2763-2016 or is not detected.
Embodiment 1
The grifola frondosus new strains that Grifola frondosa strain is obtained using the present invention.It is former using the rice bran and wheat bran of standard using reaching
Material.Rice bran usage amount is 7.0g/100mL culture medium, and the usage amount of wheat bran is 3.1g/100mL culture medium, adds potassium dihydrogen phosphate
0.25g/100mL, epsom salt 0.15g/100mL, pH are naturally, add water to required volume, 121 DEG C of sterilizing 30min, as
Culture medium is for fermenting, and shaking flask sample-loading amount is 40%, inoculum concentration 10%, and 23 DEG C of cultivation temperature, revolving speed 150r/min, when culture
Between 7d.Implement the step of pressing preceding solution below (4)~(8).It as a result is that the dry weight concentrations of mycelia, mycelia Thick many candies are dense
Degree, crude extracellular polysaccharide concentration and total Thick many candies concentration respectively reach 3.151g/100mL, 156.861mg/100mL,
Rotten sickle is avenged in 331.876mg/100mL and 488.737mg/100mL, mycotoxin aflatoxin B1, B2 and the deoxidation of total starches
Limitation, heavy metal lead, arsenic, mercury, cadmium, the limitation of chromium and pesticide carbendazim, avermectin, the residual of the butachlor limit of knife bacterium enol
Amount not higher than Limited Doses as defined in GB2761-2017, GB2762-2017, GB2763-2016 or is not detected by measurement.
Embodiment 2
The grifola frondosus new strains that Grifola frondosa strain is obtained using the present invention.It is former using the rice bran and wheat bran of standard using reaching
Material.Rice bran usage amount is 5.0g/100mL culture medium, and the usage amount of wheat bran is 5.1g/100mL culture medium, adds KH2PO4
0.25g/100mL, MgSO4·7H2Naturally, adding water to required volume, 121 DEG C of sterilizing 30min make by O 0.15g/100mL, pH
It is culture medium for fermenting, shaking flask sample-loading amount is 40%, inoculum concentration 10%, 23 DEG C of cultivation temperature, revolving speed 150r/min, culture
Time 8d.Implement below by step (4)~(8) in technical solution described by foregoing summary.It as a result is the dry weight of mycelia
Concentration, mycelia Thick many candies concentration, crude extracellular polysaccharide concentration and total Thick many candies concentration respectively reach 2.789g/100mL,
160.045mg/100mL, 364.586mg/100mL and 524.631mg/100mL, the mycotoxin aflatoxin of total starches
The limitation of B1, B2 and deoxynivalenol, heavy metal lead, arsenic, mercury, cadmium, the limitation of chromium and pesticide carbendazim, Avermectin
Element, butachlor residue limits be not higher than Limited Doses as defined in GB2761-2017, GB2762-2017, GB2763-2016 or
It is not detected.
Embodiment 3
The grifola frondosus new strains that Grifola frondosa strain is obtained using the present invention.It is former using the rice bran and wheat bran of standard using reaching
Material.Rice bran usage amount is 10.0g/100mL culture medium, and the usage amount of wheat bran is 0.1g/100mL culture medium, adds KH2PO4
0.015g/100mL, MgSO4·7H2O 0.015g/100mL, pH are naturally, add water to required volume, 121 DEG C of sterilizing 30min,
As culture medium for fermenting, shaking flask sample-loading amount is 40%, inoculum concentration 10%, 25 DEG C of cultivation temperature, revolving speed 150r/min, training
Support time 10d.Implement below by step (4)~(8) in technical solution described by foregoing summary.As a result it is, mycelia
Dry weight concentrations, mycelia Thick many candies concentration, crude extracellular polysaccharide concentration and total Thick many candies concentration respectively reach 3.206g/
100mL, 124.712mg/100mL, 163.722mg/100mL and 288.434mg/100mL, the mycotoxin aspergillus flavus of total starches
The limitation of toxin B1, B2 and deoxynivalenol, heavy metal lead, arsenic, mercury, cadmium, the limitation of chromium and pesticide carbendazim, Ah
Tie up rhzomorph, the residue limits of butachlor are not higher than limitation as defined in GB2761-2017, GB2762-2017, GB2763-2016
It is worth or is not detected.
Embodiment 4
The grifola frondosus new strains that Grifola frondosa strain is obtained using the present invention.It is former using the rice bran and wheat bran of standard using reaching
Material.Rice bran usage amount is 3.1g/100mL culture medium, and the usage amount of wheat bran is 7.0g/100mL culture medium, adds KH2PO4
0.15g/100mL, MgSO4·7H2Naturally, adding water to required volume, 121 DEG C of sterilizing 30min make by O 0.075g/100mL, pH
It is culture medium for fermenting, shaking flask sample-loading amount is 40%, inoculum concentration 10%, 20 DEG C of cultivation temperature, revolving speed 150r/min, culture
Time 7d.Implement below by step (4)~(8) in technical solution described by foregoing summary.It as a result is the dry weight of mycelia
Concentration, mycelia Thick many candies concentration, crude extracellular polysaccharide concentration and total Thick many candies concentration respectively reach 2.117g/100mL,
145.143mg/100mL, 397.297mg/100mL and 542.44mg/100mL, the mycotoxin aflatoxin B1 of total starches,
The limitation of B2 and deoxynivalenol, heavy metal lead, arsenic, mercury, cadmium, the limitation of chromium and pesticide carbendazim, avermectin,
The residue limits of butachlor are not higher than Limited Doses as defined in GB2761-2017, GB2762-2017, GB2763-2016 or do not examine
Out.
Embodiment 5
The grifola frondosus new strains that Grifola frondosa strain is obtained using the present invention.It is former using the rice bran and wheat bran of standard using reaching
Material.Rice bran usage amount is 0.1g/100mL culture medium, and the usage amount of wheat bran is 10.0g/100mL culture medium, adds KH2PO4
0.25g/100mL, MgSO4·7H2Naturally, adding water to required volume, 121 DEG C of sterilizing 30min make by O 0.25g/100mL, pH
It is culture medium for fermenting, shaking flask sample-loading amount is 40%, inoculum concentration 10%, 23 DEG C of cultivation temperature, revolving speed 150r/min, culture
Time 9d.Implement below by step (4)~(8) in technical solution described by foregoing summary.It as a result is the dry weight of mycelia
Concentration, mycelia Thick many candies concentration, crude extracellular polysaccharide concentration and total Thick many candies concentration respectively reach 1.481g/100mL,
106.819mg/100mL, 344.594mg/100mL and 451.413mg/100mL, the mycotoxin aflatoxin of total starches
The limitation of B1, B2 and deoxynivalenol, heavy metal lead, arsenic, mercury, cadmium, the limitation of chromium and pesticide carbendazim, Avermectin
Element, butachlor residue limits be not higher than Limited Doses as defined in GB2761-2017, GB2762-2017, GB2763-2016 or
It is not detected.
Embodiment 6
The grifola frondosus new strains that Grifola frondosa strain is obtained using the present invention.It is former using the rice bran and wheat bran of standard using reaching
Material.Rice bran usage amount is 10.0g/100mL culture medium, and the usage amount of wheat bran is 0.1g/100mL culture medium, adds KH2PO4
0.075g/100mL, MgSO4·7H2O 0.075g/100mL, pH are naturally, add water to required volume, 121 DEG C of sterilizing 30min,
As culture medium for fermenting, shaking flask sample-loading amount is 40%, inoculum concentration 10%, 23 DEG C of cultivation temperature, revolving speed 150r/min, training
Support time 8d.Implement below by step (4)~(8) in technical solution described by foregoing summary.It as a result is that mycelia does
Weight concentration, mycelia Thick many candies concentration, crude extracellular polysaccharide concentration and total Thick many candies concentration respectively reach 3.386g/
100mL, 181.027mg/100mL, 282.810mg/100mL and 463.837mg/100mL, the mycotoxin aspergillus flavus of total starches
The limitation of toxin B1, B2 and deoxynivalenol, heavy metal lead, arsenic, mercury, cadmium, the limitation of chromium and pesticide carbendazim, Ah
Tie up rhzomorph, the residue limits of butachlor are not higher than limitation as defined in GB2761-2017, GB2762-2017, GB2763-2016
It is worth or is not detected.
Embodiment 7
The grifola frondosus new strains that Grifola frondosa strain is obtained using the present invention.It is former using the rice bran and wheat bran of standard using reaching
Material.Rice bran usage amount is 0.1g/100mL culture medium, and the usage amount of wheat bran is 10.0g/100mL culture medium, adds KH2PO4
0.15g/100mL, MgSO4·7H2Naturally, adding water to required volume, 121 DEG C of sterilizing 30min make by O 0.075g/100mL, pH
It is culture medium for fermenting, shaking flask sample-loading amount is 40%, inoculum concentration 10%, 23 DEG C of cultivation temperature, revolving speed 150r/min, culture
Time 9d.Implement below by step (4)~(8) in technical solution described by foregoing summary.It as a result is the dry weight of mycelia
Concentration, mycelia Thick many candies concentration, crude extracellular polysaccharide concentration and total Thick many candies concentration respectively reach 0.912g/100mL,
84.413mg/100mL, 358.737mg/100mL and 443.151mg/100mL, the mycotoxin aflatoxin B1 of total starches,
The limitation of B2 and deoxynivalenol, heavy metal lead, arsenic, mercury, cadmium, the limitation of chromium and pesticide carbendazim, avermectin,
The residue limits of butachlor are not higher than Limited Doses as defined in GB2761-2017, GB2762-2017, GB2763-2016 or do not examine
Out.
Embodiment 8
Using grifola frondosus new strains.Wheat bran and rice bran is examined to meet use standard of the invention.Fermentor sample-loading amount is
The 75% of fermenter volume, fermentation temperature are 20 DEG C, and ventilation quantity is ventilation volume and tinning liquid bulk per minute under aeration condition
The ratio between product is 1:0.8, mixing speed 50r/min, tank gauge pressure 0.05MPa, inoculum concentration 10%, incubation time 7d, fermentation medium
For rice bran 10.0g/100mL, wheat bran 0.1g/100mL, KH2PO40.015g/100mL, MgSO4·7H2O 0.015g/100mL,
PH is natural.Implement below by step (4)~(8) in technical solution described by foregoing summary.As a result are as follows: dry mycelial weight
3.424g/100mL, mycelia polysaccharide 176.315mg/100mL, exocellular polysaccharide are 330.521mg/100mL culture medium, grifola frondosus
Polysaccharide is 506.836mg/100mL, mycotoxin aflatoxin B1, the limit of B2 and deoxynivalenol of total starches
Amount, heavy metal lead, arsenic, mercury, cadmium, the limitation of chromium and pesticide carbendazim, avermectin, butachlor residue limits be not higher than
Limited Doses as defined in GB2761-2017, GB2762-2017, GB2763-2016 are not detected.
Embodiment 9
Using grifola frondosus new strains.Wheat bran and rice bran is examined to meet use standard of the invention.Fermentor sample-loading amount is
The 75% of fermenter volume, fermentation temperature are 25 DEG C, and ventilation quantity is ventilation volume and tinning liquid bulk per minute under aeration condition
The ratio between product is 1:0.8, mixing speed 150r/min, tank gauge pressure 0.05MPa, inoculum concentration 10%, incubation time 10d, fermented and cultured
Base is rice bran 0.1g/100mL, wheat bran 10.0g/100mL, KH2PO40.25g/100mL, MgSO4·7H2O 0.25g/100mL,
PH is natural.Implement below by step (4)~(8) in technical solution described by foregoing summary.As a result are as follows: dry mycelial weight
1.819g/100mL, mycelia polysaccharide 112.396mg/100mL, exocellular polysaccharide 258.248mg/100mL, grifolan are
370.644mg/100mL, the mycotoxin aflatoxin B1s of total starches, the limitation of B2 and deoxynivalenol, again
Metallic lead, arsenic, mercury, cadmium, the limitation of chromium and pesticide carbendazim, avermectin, butachlor residue limits be not higher than GB2761-
2017, it Limited Doses as defined in GB2762-2017, GB2763-2016 or is not detected.
Claims (1)
1. using mutagenesis generate grifola frondosus new strains production grifolan method, it is characterised in that as steps described below into
Row:
(1) wheat bran and rice bran are examined,
(2) rice bran and wheat bran are added into 121 DEG C of sterilizing 30min of the desired amount of water；
(3) rice bran and wheat bran complete feed are directly used in grifola frondosus new strains as carbon source, nitrogen source and save number CCTCC NO:M
The fermentation medium of 2018907 bacterial strain, rice bran concentration are 0.1-10g/100mL, and wheat bran concentration is 0.1-10g/100mL, rice
Chaff and wheat bran concentration sum of the two are up to 10.1g/100mL, add potassium dihydrogen phosphate 0.015-0.25g/100mL, seven water sulphur
Sour magnesium 0.015-0.25g/100mL, the pH 100mL naturally, 250mL shaking flask charges, fermentor filling rate 75%, inoculum concentration 10%,
20-25 DEG C of cultivation temperature, shaking speed 150r/min or fermentation rotating speed of agitator 50-150r/min, fermentation time 7-10d；
(4) liquid culture is centrifugally separating to obtain mycelium and fermentation liquid, and measures the yield of exocellular polysaccharide in fermentation liquid；
(5) the ultrasonic wave added liquid circulation of 35kHz and 1.5kW is added to extract 1.5h, centrifugation point 60 DEG C of hot water of gained mycelium
From taking clear liquid, and measure mycelia polysaccharide yield；
(6) gained clear liquid will be centrifugated twice to merge, freeze concentration, citric acid extract displacement heavy metal ion, alcohol precipitation alcohol is washed,
Freeze-drying obtains qualified grifolan product.
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| CN114921348B (en) * | 2022-06-14 | 2023-09-01 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | New high-yield polysaccharide grifola frondosa strain W151021 and molecular marker thereof |
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