CN110396476A - One plant of Grifola frondosa strain obtained by ultraviolet mutagenesis - Google Patents

Abstract

The Grifola frondosa strain obtained the invention discloses one plant by ultraviolet mutagenesis, is related to food microorganisms technical applications.The mutagenic strain is deposited in Wuhan China typical culture collection center (CCTCC) on December 19th, 2018, number is CCTCC M 2018907, it is recommended that classification naming be Grifola frondosa strain JSULIUWANG18, latin name be Grifola frondosa JSULIUWANG18.The grifola frondosus new strains are for liquid state fermentation through examining the wheat bran and rice bran complete feed culture medium as main component that reach pollutant limitation and use standard, it after the fermentation material for producing maitake mushroom mycelia raw material and fermentation liquid, washed by multiple frequency ultrasonic assisted extraction, freeze concentration, citric acid extraction displacement heavy metal ion, alcohol precipitation alcohol, be lyophilized and obtain the grifolan product of qualification.

Description

One plant of Grifola frondosa strain obtained by ultraviolet mutagenesis

Technical field

The present invention relates to food microorganisms technical applications more particularly to one plant of grifola frondosus obtained by ultraviolet mutagenesis New strains, for liquid state fermentation through examining the wheat bran and the training as main component of rice bran complete feed that reach pollutant limitation using standard Support base, after the fermentation material for producing maitake mushroom mycelia raw material and fermentation liquid, by multiple frequency ultrasonic assisted extraction, freeze concentration, Citric acid extraction displacement heavy metal ion, alcohol precipitation alcohol washes, be lyophilized and obtains the grifolan 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.China be distributed mainly on Yunnan, Sichuan, Hebei, Heilungkiang, Jilin, Guangxi, Tibet, Fujian, Zhejiang and other places.Wild Grifola frondosa mainly originates under the broad-leaf forest of 800~1400m of height above sea level, likes warm weather and moist soil Earth, form can be divided into mycelium and fructification two large divisions, wherein product of the mycelium as Submerged Culture of Grifola frondosa, color For milky, form is in spherical shape of uniform size；And the fructification as edible part is a kind of rich in protein, vitamin E With the nutraceutical of a variety of mineral elements.The artificial domesticating cultivation of grifola frondosus has entered industrialization, in south China regional culture Essentially substituting stuff cultivation, it is northern then with generation material bury soil cultivation based on.Grifola Frondosa sporophore grey is to taupe to canescence, edge There is the irregular point of a circle convex, bacterial context white, meat is tender and crisp, has handle and handle is short in coralliform branch, be overlapped into clump.The stacking of its appearance Like chrysanthemum, remote to see like cloud sheet layer by layer, the name of " cloud gill fungus " is therefore obtained.The acquisition modes of present grifola frondosus are mainly artificial cultivation, with Fructification 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 Deng limitation, it is subject to pest and disease damage, quality is unstable with yield.Rare fungi fermentation technology, can overcome traditional fructification to cultivate Deficiency produces frondosa fermentation object using liquid or the method for solid state fermentation and is subject to the hair that deep processing application meets innovation driving Thinking is opened up, there is good prospect.The progress obtained to the research and development of frondosa fermentation object all will further push ash tree Colored application brings actual social and economic benefit.Just because of this, just it is necessary to study new frondosa fermentation object and its The production method of deep processed product, such as consider efficiently to turn some low side processing of farm products byproduct sources such as wheat brans, rice bran Turn to the frondosa fermentation raw material of high value, it is expected to efficiently utilize low-grade agricultural resource, reduce frondosa fermentation object Production cost to reduce the price of finished product such as grifola frondosus class health food or drug, interest concessions promote public 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 is about 30,000,000 tons.Wheat bran refers both to wheat bran hereinafter.Rice bran and wheat bran as paddy or the by-product of wheat processing, Rich in nutrition content, it is cheap.Contain in rice bran and enriches good protein, active polysaccharide, fat and tocotrienols, life The significant active material of the physiological functions such as phenol is educated, wheat bran is rich in nutrition such as fat, protein, minerals, vitamin and celluloses Ingredient, wherein the content of cellulose is up to 18% or more of wheat bran total amount, so, rice bran and wheat bran can provide for ash tree flower growth Carbon source, the nitrogen source, vitamin and mineral of abundance.Under the action of grifola frondosus cellulase and other enzyme systems, can by rice bran and Wheat bran is converted to needed nutrient matter and carries out growth metabolism, synthesizes maitake mushroom mycelia and types of functionality substance.Therefore, it uses As whole carbon source and nitrogen source, progress grifola frondosus liquid state fermentation production frondosa fermentation object is simultaneously deep for agricultural and sideline product rice bran, wheat bran It is processed into health food or drug, there is technical feasibility, and preferable social and economic benefit can be brought.

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 all around trans-utilization rice bran and Wheat bran efficiently studied by this special topic of high-valued production Rare edible fungus fermentation material, realizes the imagination of innovation and creation, and constantly Improve the production method of various rare fungal fermentates.

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 hope of the requirement that the problem of answer, such as pervious strain provenance are unsatisfactory for enterprise, enterprise thus progress industrialization development is not strong It is strong etc., have been found that Industrialization to be moved towards, there are still many problems to need to study for relevant patented technology and research paper It solves and is innovated.These problems include having the acquisition of the rare fungus in high efficiency new strains of different performance and confirmation and opening Hair, the formulation of rice bran bran feedstock standard, the innovation of rare fungal fermentate fermentation process, rare fungal fermentate isolate and purify newly Method uses and these need to innovate the combination etc. of object.Patent of invention must to Grant Patent Right for Invention in design Uniqueness, acquirement substantive distinguishing features outstanding and the innovation and practicality of marked improvement of palpus will give abundant consideration.Institute With the combined innovation of above-mentioned these problems should be ensured that relevant patent of invention reaches uniqueness necessary to Grant Patent Right for Invention Property, 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 the wheat bran and rice bran for being used to ferment by selection by grifola frondosus new strains Complete feed produces frondosa fermentation object and becomes qualified products after deep processing is handled.Be different from previous research and acquirement at Fruit, the invention solves one of critical issue to need the method by mutagenesis, promote the fermentability of original strain, obtain A kind of grifola frondosus 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 present invention The two of the critical issue to be solved are to make wheat bran raw rice bran standard, because wheat bran and rice bran source are complicated, wherein pollutant Type and its horizontal difference, inventor's previous studies and patent do not give enough concerns in this regard, open to subsequent product Hair band carrys out indefinite consequence；The invention solves critical issue three handled through deep processing for fermentation material mycelia and fermentation liquid Become the production method of qualified products afterwards.Above three promise the invention patent and other inventors and the present inventor Liu Weimin All kinds of achievements of research team are different, 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 grifola frondosus 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 It is that inventor team it is expected to obtain one plant from unused and with good performance Grifola frondosa strain, and it is expected that it can be in bran There is good behaviour in terms of micromicron chaff bioconversion, therefore considers the side of research frondosa fermentation wheat bran rice bran production frondosa fermentation object Method.According to the previous research experience of inventor, this kind of rare fungi fermentation wheat bran rice bran is firstly the need of the adaptability for solving strain Problem.It would therefore be desirable to be investigated to the fermentability of the grifola frondosus strain in this production method, and lured by strain The method of change improves its fermenting property.In addition in the course of the research, to guarantee that strain does not go out after a large amount of strain processing step Existing mistake, it should carry out gene sequencing.This does not attach great importance to this thing in previous research.In this way, this patent is from the beginning The measurement of grifola frondosus gene series is just dig-inned, guarantees 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 grifola frondosus new strains by the method for mutagenesis.The selection of microorganism mainly has physics to lure Change, chemical mutagenesis, genetic recombination etc., the present invention be avoid using poisonous and hazardous chemical mutagen and field of food also not by The gene engineering method that masses approve will utilize relatively easy easy ultraviolet mutagenesis technology, starting strain made to be in mutagenesis A possibility that extreme environment expands the range in mutational site to greatest extent, and raising obtains direct mutation bacterial strain.The present invention is using ultraviolet This common method of mutagenesis of mutagenesis, although the method does not have novelty, the bacterial strain performance that the method obtains is created The variation of new property, the unique, innovation and practicality when fermenting wheat bran rice bran complete feed culture medium.Gained fermentation of the invention The grifola frondosus new strains of wheat bran rice bran complete feed liquid culture medium are that invention obtains for the first time.

Summary of the invention

The present invention is in order to which using the Grifola frondosa strain of preferable performance, efficiently high-valued conversion liquid rice bran wheat bran complete feed is ash The fermentation materials such as flower mycelium are set, mutagenesis screening is needed to go out to be suitble to the grifola frondosus grown on the new culture medium of rice bran wheat bran complete feed liquid New strains.To avoid using poisonous and hazardous chemical mutagen and in the genetic engineering side that field of food is not approved by masses also Method considers that ultraviolet physical mutagenesis is technically simple easy, therefore the method for using ultraviolet mutagenesis carries out mutagenesis, and by a series of steady The screening techniques such as qualitative, heredity guarantee the merit of grifola frondosus new strains, are the high-valued conversion low cost of industrial high efficiency Wheat bran rice bran be that establish strain basic for the frondosa fermentation object of quality qualification.The present invention is used wheat bran raw rice bran system Raw material standard is determined, has obtained good fermentation material for fermentation and establish raw material basis, heretofore described wheat bran rice bran is unless there are spy Different explanation refers both to meet wheat bran and rice bran that this raw material uses standard.Fermentation material grifola frondosus mycelia of the invention and extracellular useful hair Ferment object needs to purify by further extracting and developing, finally obtains the fermentation material finished product of quality qualification, for later derivative production Product production, present invention grifola frondosus new strains obtained are determined as one plant of grifola frondosus new strains through specialized company's DNA sequencing.

The technical solution used in the present invention is as follows:

Bacterial strain grifola frondosus new strains JSULIUWANG18 provided by the invention, can be with for obtained by ultraviolet mutagenesis one plant The grifola frondosus new strains of fast-growth and high yield fermentation material, Jing Shenggong bioengineering in wheat bran rice bran complete feed liquid culture medium (Shanghai) limited liability company DNA sequencing is determined as one plant of grifola frondosus new strains；The grifola frondosus new strains are in 2018 12 The moon is deposited in positioned at Wuhan, China Wuhan University China typical culture collection center (CCTCC), deposit number CCTCC on the 19th 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.

In one aspect of the invention, above-mentioned Grifola frondosa is provided, deposit number is CCTCC NO:M 2018907 purposes produces frondosa fermentation object for ferment rice bran wheat bran complete feed liquid culture medium；

In one aspect of the invention, grifola frondosus new strains obtained reach use through examining for liquid state fermentation The wheat bran and raw rice bran complete feed culture medium as main component of standard, produce the hair of maitake mushroom mycelia raw material and fermentation liquid Ferment object；

In one aspect of the invention, the fermentation material for producing maitake mushroom mycelia raw material and fermentation liquid is auxiliary by ultrasound Extraction, freeze concentration, citric acid extraction displacement heavy metal ion, alcohol precipitation alcohol is helped to remove the miscellaneous grifolan for obtaining qualification and produce Object；

In one aspect of the invention, wheat bran is to execute the former Ministry of Agriculture to eat feed wheat bran standard NY/T using standard 3218-2018, additional Heavy Metals lead, arsenic, mercury, cadmium, chromium level of pollution not more than 3mg/kg, wheat planting process Middle applying pesticides meet agrotechnical specification, do harm in wheat growth without serious head blight, harvesting season causes small without continuous rainwater Wheat is rotten, and wheat bran storage and transportation not will lead to mildew after wheat processing；

In one aspect of the invention, rice bran is to execute the former agricultural additional master of ministerial standard NY/T122-1989 using standard The level of pollution limitation not more than 3mg/kg of heavy metal lead, arsenic, mercury, cadmium, chromium is wanted, applying pesticides accord with during Rice Cropping Agrotechnical specification is closed, paddy growth is in the process without serious plant disease, and harvesting season causes paddy rotten without continuous rainwater, after paddy processing Rice bran storage and transportation not will lead to mildew.

Beneficial effects of the present invention

To adapt to the requirement that subsequent fermentation object is used to produce health food, the present invention uses one plant of physical mutagenesis technology breeding Grifola frondosus new strains, original Grifola frondosa strain JSULIUWANG0 is from Zhejiang Province, China Qingyuan County grifola frondosus production base Grifolas frondosa germ Strain fructification is separately cultured to obtain, and is determined as Grifolas frondosa germ through Sangon Biotech's DNA sequencing Strain, the entitled Grifola frondosa of the Latin of the bacterial strain confirm provenance, ensure that strain is correct.Thus original strain is made For starting strain, ultraviolet mutagenesis is carried out, is screened using growth rate, pure dry mycelial weight and mycelia polysaccharide as index, is finally obtained Grifola frondosus new strains JSULIUWANG18 is obtained, is deposited in positioned at Wuhan, China Wuhan University China typical culture collection center (the mechanism english abbreviation is CCTCC), deposit number is CCTCC NO:M 2018907.The new strains be not added other carbon sources and Nitrogen source meets raw material using grifola frondosus mycelia raw material is produced in the rice bran and wheat bran complete feed liquid culture medium of standard, with grifola frondosus Original strain is higher compared to production capacity, and pure mycelial yield is improved, and mycelia polysaccharide and the polysaccharide in fermentation liquid are also mutually deserved To raising.

When shake flask fermentation, a kind of rice bran used in grifola frondosus new strains and wheat bran complete feed liquid culture medium composition are rice bran and bran Skin concentration is respectively 7.0g/100mL (referring to culture volume, rear all herewith to understand) and 1.6g/100mL, the dry weight of pure mycelia Concentration, mycelia Thick many candies concentration, crude extracellular polysaccharide concentration and total Thick many candies concentration respectively reach 3.367g/100mL, 175.076mg/100mL, 269.385mg/100mL and 444.461mg/100mL.And original strain Grifola frondosa exists The dry weight concentrations of pure mycelia, mycelia Thick many candies concentration, crude extracellular polysaccharide concentration and total Thick many candies concentration are respectively under the same terms 2.236g/100mL, 138.437mg/100mL, 230.562mg/100mL and 368.999mg/100mL.The mycelia of new strains is dry Weight concentration increases by 50.6%, and mycelia polysaccharide concentration increases 26.5%, and exocellular polysaccharide concentration increases 16.8%, total starches concentration 20.5% is increased, illustrates the ability that new strains obtained by mutagenesis have stronger ferment rice bran and wheat bran, the fermenting property of bacterial strain Significant positive variation has occurred.Final products total starches are the polysaccharide separated after impurity elimination, are clean products, different from the past Product is only calculated with mycelia polysaccharide, also achieves the novelty of conspicuousness progress.

The present invention is finally obtained one plant of grifola frondosus new strains, deposit number by Uv-induced screening research repeatedly For CCTCC M NO:2018907, the new strains are that meeting for other carbon sources and nitrogen source is not added is complete using the rice bran and wheat bran of standard Expect in liquid culture medium the speed of growth faster, the ability of ferment rice bran and wheat bran it is stronger so that the mycelia in specified fermentation volume Amount and mycelia polysaccharide yield are higher, and the fermentation character of grifola frondosus new strains changes compared with original strain, embodies distinctness Uniqueness.Based on this plant of grifola frondosus new strains, the side of new ferment rice bran and wheat bran complete feed liquid culture medium is formd Wheat bran and rice bran complete feed are subject to liquid state fermentation application by method, this method, and have comprehensively been more than original on capacity and output Bacterial strain, it is seen that the present invention grifola frondosus new strains achieve " novelty for obtaining substantive distinguishing features outstanding and marked improvement ". Cheap rice bran and wheat bran is efficiently utilized in the invention patent, can reduce production cost, reduces resource consumption, hence it is evident that increases and produces Amount, and in grifolan pollutant content it is low, meet the requirement of raw-food material, have practicability.

In conclusion the present invention using had been provided with the uniqueness of patent of invention, obtain substantive distinguishing features outstanding and The innovation and practicality of marked improvement, produces beneficial effect.

Detailed description of the invention

Fig. 1 is the flow chart of the ultraviolet mutagenesis selection of present invention new strains obtained.

Fig. 2 is the plate culture figure that grifola frondosus new strains deposit number is CCTCC NO:M 2018907.

Fig. 3 be grifola frondosus new strains and chadogram.

Specific embodiment

The present invention attached process shown in FIG. 1 to specifications, provides through ultraviolet mutagenesis, breeding is complete in rice bran and wheat bran Expect liquid culture medium on the speed of growth faster, the method for the higher grifola frondosus new strains of polysaccharide yield, the method includes following Step:

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 JSULIUWANG18Grifola frondosa JSULIUWANG18, grifola frondosus new strains of the present invention refer both to this bacterial strain.(see Fig. 3)

In one embodiment, the obtained grifola frondosus mutagenesis new strains that screen utilize rice bran and wheat bran fermented and cultured The fermentation results obtained after base fermentation completely are as follows: the dry weight concentrations of pure mycelia, mycelia Thick many candies concentration, crude extracellular polysaccharide concentration and Total Thick many candies concentration respectively reach 3.367g/100mL, 175.076mg/100mL, 269.385mg/100mL and 444.461mg/100mL。

In one embodiment, the dry weight concentrations of pure mycelia, mycelia are slightly more under the same conditions for original Grifola frondosa strain Sugared concentration, crude extracellular polysaccharide concentration and total Thick many candies concentration be respectively 2.236g/100mL, 138.437mg/100mL, 230.562mg/100mL and 368.999mg/100mL.

In one embodiment, compared with original strain, the dry mycelial weight concentration of grifola frondosus new strains increases 50.6%, mycelia polysaccharide concentration increases 26.5%, and exocellular polysaccharide concentration increases 16.8%, and total starches concentration increases 20.5%, illustrate the ability that new strains obtained by mutagenesis have stronger fermentation wheat bran and rice bran, the fermenting property of bacterial strain has occurred Significant positive variation.

In one embodiment, the grifola frondosus mycelia that is obtained after fermentation by centrifuge separation, multiple frequency ultrasonic assisted extraction, The method that freeze concentration, citric acid extract displacement heavy metal ion, alcohol precipitation alcohol is washed, are freeze-dried obtains qualified mycelia polysaccharide.

In one embodiment, the fermentation liquid obtained after fermentation is by freeze concentration, citric acid extraction displacement heavy metal The method that ion, alcohol precipitation alcohol washes, is freeze-dried obtains qualified exocellular polysaccharide.

In one embodiment, qualified mycelia polysaccharide and qualified exocellular polysaccharide sum of the two are total starches.

In one embodiment, mycotoxin aflatoxin B1, B2 and the deoxynivalenol of total starches Limitation, heavy metal lead, arsenic, mercury, cadmium, the limitation of chromium and pesticide carbendazim, avermectin, butachlor residue limits through survey Fixed to be not higher than Limited Doses as defined in GB2761-2017, GB2762-2017, GB2763-2016 or be not detected, final products are total Polysaccharide is the polysaccharide separated after impurity elimination, is clean products, and product different from the past is only calculated with mycelia polysaccharide, is also achieved significant The novelty of property progress.

Embodiment 1

The grifola frondosus new strains that Grifola frondosa strain is obtained using the present invention, deposit number CCTCC NO:M 2018907.Rice Chaff usage amount is 5.0g/100mL culture medium, and the usage amount of wheat bran is 5.0g/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 8d.Implement below by step (1)~(5): (1) by Liquid Culture 60 mesh of the resulting mycelium containing a small amount of rice bran wheat bran After metallic sieve separation, and it is washed twice with water；(2) gained wet mycelium is carried out multiple frequency ultrasonic auxiliary warm water to extract, water temperature No more than 60 DEG C, clear liquid is filtered to take, chilled concentration, citric acid extraction displacement heavy metal ion, alcohol precipitation, centrifugal filtration, alcohol wash, Freeze-drying obtains qualified mycelia polysaccharide product；(3) gained fermentation liquid is through filtering, freeze concentration, a citric acid extraction displacement huge sum of money Belong to ion, alcohol precipitation, centrifugal filtration, alcohol are washed, are freeze-dried and are obtained qualified exocellular polysaccharide product；(4) gained polyose is using title Weight method measures polyoses content, as a result are as follows: the dry weight concentrations of pure mycelia, mycelia Thick many candies concentration, crude extracellular polysaccharide concentration and total thick Polysaccharide concentration respectively reach 2.789g/100mL, 160.045mg/100mL, 364.586mg/100mL and 524.631mg/100mL；(5) through detecting, mycotoxin aflatoxin B1, B2 and the deoxynivalenol of total starches Limitation, heavy metal lead, arsenic, mercury, cadmium, the limitation of chromium and pesticide carbendazim, avermectin, butachlor residue limits through survey It is fixed to be not higher than Limited Doses as defined in GB2761-2017, GB2762-2017, GB2763-2016 or be not detected.

Claims (1)

1. one plant by ultraviolet mutagenesis obtain Grifola frondosa strain, deposit number be CCTCC NO:M 2018907, it is proposed that point Class is named as Grifola frondosa strain JSULIUWANG18, and latin name is Grifola frondosa JSULIUWANG18.