CN110438010A - One plant of ganoderma strain obtained by ultraviolet mutagenesis - Google Patents

Abstract

The ganoderma strain obtained the invention discloses one plant by ultraviolet mutagenesis, it is related to food microorganisms technical applications, the ganoderma strain is deposited in the China typical culture collection center (CCTCC) positioned at Wuhan, China Wuhan University on December 19th, 2018, deposit number is CCTCC NO:M 2018908, it is recommended that classification naming be ganoderma strain JSU LIUYU18 or latin name is Ganoderma lucidum JSU LIUYU18.This new strains is used 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 ganoderma lucidum mycelium 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 ganoderma lucidum polysaccharide product of qualification.

Description

One plant of ganoderma strain obtained by ultraviolet mutagenesis

Technical field

The present invention relates to food microorganisms technical applications more particularly to one plant of ganoderma lucidum by ultraviolet mutagenesis acquisition are new Bacterial strain, for liquid state fermentation through examining the wheat bran and the culture as main component of rice bran complete feed that reach pollutant limitation using standard Base, after the fermentation material for producing ganoderma lucidum mycelium raw material and fermentation liquid, by multiple frequency ultrasonic assisted extraction, freeze concentration, lemon Acidleach mentions displacement heavy metal ion, alcohol precipitation alcohol and washes, be lyophilized and obtain the ganoderma lucidum 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 Hericium erinaceus, ganoderma lucidum, grifola frondosus, 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, all kinds of personnel research and develop the with keen interest of this kind of fungi, In both at home and abroad All there are many researcher and developers for field of medicaments, field of food, Traditional Chinese health field, agricultural and field of industrial production, obtain Numerous research achievements or product, such as ganoderma lucidum capsule, grifola frondosus capsule and Paecilomyces hepiali chen capsule, their economic value It is higher.Hericium erinaceus being added in biscuit, Hericium erinaceus biscuit being then made, having become ordinary food, supermarket is sold at home.State Ji Shang New Zealand, Japan, the U.S., South Korea etc., which produce, similar rare fungi health food or pharmaceutical products.

For ganoderma lucidum, for its research shows that: ganoderma lucidum in antitumor, anticancer, anti-oxidant, hypoglycemic, enhancing is immune Power, treatment cardiovascular disease etc. all have certain bioactivity, medical value (bibliography: 1. Lee with higher Ying Bo efficiently separates the Shanghai research [D] for preparing antitumor ganodenic acid monomer: East China University of Science from ganoderma lucidum mycelium, 2013.②Yuen J W M,Mak D S Y,Chan E S,et al.Tumor inhibitory effects of intravesical Ganoderma lucidum instillation in the syngeneic orthotopic MB49/ C57bladder cancer mice model[J].Journal of Ethnopharmacology,2018: S0378874118311991.③Yongjun Kan,Tiqiang Chen,Yanbin Wu,Jianguo wu,Jinzhong Wu,et al.Antioxidant activity of polysaccharide extracted from Ganodermalucidum using response surface methodology[J].International Journal The screening of of Biological Macromolecules, the 2015,72:151-157. 4. precious effective hypoglycemic component of pine ganoderma lucidum of Teng And the Shanghai hypoglycemic mechanism study [D]: Fudan University, 2011. 5. Tang W, Liu J W, Zhao W M, et al.Ganoderic acid T from Ganoderma lucidum mycelia induces mitochondria mediated apoptosis in lung cancer cells[J].Life Sciences,2006,80(3):0-211.)。

Ganoderma lucidum (Ganoderma lucidum) belongs to Basidiomycota, Basidiomycetes, agaric subclass, Aphyllophorales, Ganoderma Lucidum Section, Ganoderma are a kind of dual-purpose of drug and food macro fungis, be born in woods by the timber of broad leaf tree more or wood, standing tree, fall wood on, Sometimes it is also born in coniferous tree, cap suberin, kidney shape or semicircle, reddish brown, purple or mulberry, tool paint sample gloss has ring Shape rib and radial wrinkle, size and metamorphosis are very big, and the cap of Large scale individual is 20 × 10cm, thickness about 2cm, general a Body is 4 × 3cm, and 0.5~1cm of thickness has numerous small holes below, and nozzle is white or filbert, has 4~5 in every millimeter, nozzle Circle, inner wall are hymenium, and spore results from load top.Stem side is raw, seldom wilfully, is longer than bacteria cover diameter, puce is extremely Black has paint sample gloss, hard.Spore oval, 8~11 × 7cm, two layers of wall, there is small wart on inner wall brown, surface, and outer wall is saturating It is bright colourless.Ganoderma lucidum Europe, America, Africa, east Asia etc. area be distributed, China ganoderma lucidum be mainly distributed Longquan, Zhejiang Province, There is part on the ground such as Heilungkiang, Jilin, Hebei, Shandong, Huoshan, Jiangsu, Jiangxi, Hunan, Guizhou, Fujian, Guangdong, Guangxi Yield.The ganoderma lucidum speed of growth under wild state is very slow, and price is high.As it can be seen that since Wild ganoderma resource is very rare, because This, it is necessary to solve the problems, such as the scarcity of resources of ganoderma lucidum.The acquisition modes of present ganoderma lucidum are mainly artificial cultivation, but artificial cultivation is all Phase is long, production efficiency is not high, large labor intensity, is limited by season, environment etc., is subject to pest and disease damage, quality is unstable with yield.It is precious Dilute fungi fermentation technology can overcome the shortcomings of traditional fructification cultivation, produce ganoderma lucidum using liquid or the method for solid state fermentation Fermentation material is simultaneously subject to the thinking of development that deep processing application meets innovation driving, has good prospect.Ganoderma fermented product is ground Study carefully and develop the progress obtained all by the further application for pushing ganoderma lucidum, brings actual social and economic benefit.Just because of such as This, just it is necessary to study the production methods of new Ganoderma fermented product and its deep processed product, such as consider some low side agricultural productions Product processed side product raw material such as wheat bran, rice bran Efficient Conversion are the glossy ganoderma fermentation raw material of high value, it is expected to reduce glossy ganoderma fermentation To reduce the price of finished product such as ganoderma lucidum class health food or drug, interest concessions promote public the production cost of object 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 mention for ganoderma lucidum growth For sufficient carbon source, nitrogen source, vitamin and mineral.Under the action of ganoderma fiber element enzyme and other enzyme systems, can by rice bran and Wheat bran is converted to needed nutrient matter and carries out growth metabolism, synthesizes ganoderma lucidum mycelium and types of functionality substance.Therefore, with agriculture Byproduct rice bran, wheat bran as whole carbon source and nitrogen source, carry out ganoderma lucidum liquid fermenting and producing Ganoderma fermented product and deep processing at Health food or drug have technical feasibility, 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 Ganoderma lucidum 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 most It is good, in addition rice bran, contain some harmful substances such as lead, arsenic, aflatoxin, vomitoxin, pesticide residue in bran feedstock Etc. influencing final products quality, thus need the method for fermentation wheat bran rice bran liquid complete feed production Ganoderma fermented product to ganoderma lucidum into Row 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 paper and invention relevant to the invention patent in the past that the inventor Liu Wei people comb instructed Master degree candidate Patent consults institute's unanswerable problem when related ends are transferred the possession of in conjunction with enterprise, can not such as provide bacterial strain qualitative gene map Critical support material is composed, enterprise thus the hope for carrying out industrialization development be not strong etc., have been found that Industrialization to be moved towards, There are still many problems needs to research and solve and be innovated for relevant patented technology and research paper.These problems include having It is the acquisition and confirmation and exploitation of the rare fungus in high efficiency new strains of different performance, the formulation of rice bran bran feedstock standard, rare true The innovation of bacterium fermentation material fermentation process, rare fungal fermentate isolate and purify new method use and these need to innovate the group of object Conjunction mode etc..Patent of invention is in design to uniqueness necessary to Grant Patent Right for Invention, acquirement substantive distinguishing features outstanding Abundant consideration is given with the innovation and practicality of marked improvement.So the combined innovation of above-mentioned these problems should be protected It demonstrate,proves relevant patent of invention and reaches uniqueness, innovation and practicality necessary to Grant Patent Right for Invention.

Inventor based on the above considerations and the research to Ganoderma fermented product production method, proposes the invention patent Application is utilized the different ganoderma lucidum original strain of event fermenting property in ganoderma strain source different from the past, carries out mutagenic obtained new Bacterial strain, and the ganoderma lucidum polysaccharide product that the production of ferment rice bran wheat bran is qualified.

In conclusion field of the present invention be ganoderma lucidum new strains are used to ferment wheat bran by selection and rice bran it is complete Material production Ganoderma fermented product simultaneously becomes qualified products after deep processing is handled.It is different from the achievement of previous research and acquirement, The invention solves one of critical issue to need the method by mutagenesis, promote the fermentability of original strain, obtain one The ganoderma lucidum new strains that kind has 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 will solve The two of critical issue certainly are to make wheat bran raw rice bran standard, because wheat bran and rice bran source are complicated, wherein pollutant kind And its it is horizontal different, inventor's previous studies and patent do not give enough concerns in this regard, develop band to subsequent product Carry out indefinite consequence；The invention solves critical issue three for fermentation material mycelia and fermentation liquid after deep processing is handled at For the production method of qualified products.Above three promise the invention patent and other inventors and the present inventor Liu Wei people study All kinds of achievements of 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 ganoderma lucidum 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, Inventor team it is expected to obtain one plant from unused and with good performance ganoderma strain, and it is expected that it can be in wheat bran rice bran There is good behaviour in terms of bioconversion, therefore considers the method for research glossy ganoderma fermentation wheat bran rice bran production Ganoderma fermented product.According to hair The previous research experience of bright people, this kind of rare fungi fermentation wheat bran rice bran is firstly the need of the adaptability problem for solving strain.Therefore, We need the fermentability to the lucidum strain in this production method to investigate, and are improved by the method for induction mutation of bacterium Its fermenting property.In addition in the course of the research, to guarantee that mistake does not occur in strain after a large amount of strain processing step, it should into Row gene sequencing.This does not pay attention to this thing in previous research.In this way, this patent dig-ins ganoderma lucidum gene series from the beginning Measurement, guarantee bacterial strain it 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 obtain qualified ganoderma lucidum crude polysaccharide as spirit Sesame polysaccharide final product.Based on this understanding, the invention patent designs more reasonable fermentation material production and processing method.

The present invention needs to obtain ganoderma lucidum 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 ganoderma lucidum 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 ganoderma strain of preferable performance, efficiently high-valued conversion liquid rice bran wheat bran complete feed is ganoderma lucidum The fermentation materials such as mycelium need mutagenesis screening to go out to be suitble to the new bacterium of ganoderma lucidum grown on the new culture medium of rice bran wheat bran complete feed liquid Strain.To avoid examining using poisonous and hazardous chemical mutagen and the gene engineering method that do not approved by masses also in field of food The method considered that ultraviolet physical mutagenesis is technically simple easy, therefore use ultraviolet mutagenesis carries out mutagenesis, and by a series of stability, The screening techniques such as heredity guarantee the merit of ganoderma lucidum new strains, are the wheat bran of the high-valued conversion low cost of industrial high efficiency Rice bran is that the Ganoderma fermented product of quality qualification establishes strain basis.The present invention is that used wheat bran raw rice bran has formulated raw material Standard obtains good fermentation material for fermentation and establishes raw material basis, and heretofore described wheat bran rice bran is equal unless there are specified otherwise Refer to and meets wheat bran and rice bran that this raw material uses standard.Fermentation material Ganoderma lucidum mycelium of the invention and extracellular useful fermentation material need through Further extracting and developing purifying is crossed, the fermentation material finished product of quality qualification is finally obtained, is produced for later derived product, this It invents ganoderma lucidum new strains obtained and is determined as one plant of ganoderma lucidum new strains through specialized company's DNA sequencing.

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

Bacterial strain ganoderma lucidum new strains JSU LIUYU18 provided by the invention, can be for obtained by ultraviolet mutagenesis one plant The ganoderma lucidum new strains of fast-growth and high yield fermentation material, Jing Shenggong bioengineering (Shanghai) in wheat bran rice bran complete feed liquid culture medium Limited liability company's DNA sequencing is determined as one plant of ganoderma lucidum new strains；The ganoderma lucidum new strains are in preservation on December 19 in 2018 In the China typical culture collection center (CCTCC) for being located at Wuhan, China Wuhan University, deposit number is CCTCC NO: M2018908, it is proposed that classification naming be ganoderma strain JSU LIUYU18Ganoderma lucidum JSU LIUYU18, this It invents the ganoderma lucidum new strains and refers both to this bacterial strain.

In one aspect of the invention, the purposes of above-mentioned ganoderma lucidum new strains is provided, ferment rice bran wheat bran complete feed liquid is used for State culture medium produces Ganoderma fermented product；

In one aspect of the invention, ganoderma lucidum new strains obtained reach through examining using mark for liquid state fermentation Quasi- wheat bran and raw rice bran complete feed culture medium as main component, produces the fermentation of ganoderma lucidum mycelium raw material and fermentation liquid Object；

In one aspect of the invention, the fermentation material of ganoderma lucidum mycelium raw material and fermentation liquid is produced by ultrasonic wave added Extraction, freeze concentration, citric acid extraction displacement heavy metal ion, alcohol precipitation alcohol remove the miscellaneous ganoderma lucidum polysaccharide product for obtaining qualification；

In one aspect of the invention, wheat bran is to execute the former Ministry of Agriculture to eat feed wheat bran standard NY/ using standard T3218-2018, additional Heavy Metals lead, arsenic, mercury, cadmium, chromium level of pollution not more than 3mg/kg, wheat planting mistake Applying pesticides meet agrotechnical specification in journey, do harm in wheat growth without serious head blight, harvesting season causes 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 physical mutagenesis ultraviolet mutagenesis technology One plant of ganoderma lucidum new strains of breeding, original strain are ganoderma lucidum JSU LIU17, the bacterial strain saved from other seminars, this institute, After be named as ganoderma lucidum JSU LIUYU0.Thus original strain sets out, and ultraviolet mutagenesis is carried out, with growth rate, pure dry mycelial weight It is that index is screened with mycelia polysaccharide, final to obtain ganoderma lucidum new strains, laboratory number is JSULIUYU18, preservation mechanism Deposit number is CCTCC NO:M 2018908.The ganoderma lucidum new strains use mark in the raw material that meets that other carbon sources and nitrogen source are not added Ganoderma lucidum mycelium raw material is produced in the liquid culture medium of quasi- wheat bran rice bran complete feed, production capacity is higher compared with original strain, pure Mycelial yield is improved, and the polysaccharide in mycelia polysaccharide and fermentation liquid is also accordingly improved.

When shake flask fermentation, a kind of wheat bran rice bran complete feed, the dry weight concentrations of pure mycelia, mycelia used in gained ganoderma lucidum new strains are thick Polysaccharide concentration, crude extracellular polysaccharide concentration and total Thick many candies concentration respectively reach 2.184g/100mL, 113.746mg/100mL, 310.88mg/100mL and 424.626mg/100mL, the polysaccharide mycelia polysaccharide that every hundred mL culture medium obtains after separating impurity elimination, Exocellular polysaccharide and total starches product are respectively 93.271mg, 242.486mg and 335.757mg.And original ganoderma strain Ganoderma lucidum JSU LIU17 dry weight concentrations of pure mycelia, mycelia Thick many candies concentration, extracellular thick under the same conditions Polysaccharide concentration and total Thick many candies concentration be respectively 1.908g/100mL, 95.988mg/100mL, 267.079mg/100mL and 363.067mg/100mL, polysaccharide every hundred mL culture medium obtains after separating impurity elimination mycelia polysaccharide, exocellular polysaccharide and total starches Product is respectively 81.59mg, 202.98mg, 284.57mg.The dry mycelial weight concentration of new strains increases by 14.4%, every hundred mL culture The mycelia polysaccharide weight that base obtains increases 14.3%, and exocellular polysaccharide weight increases 19.5%, and total starches weight increases 18.0%, illustrate the ability that new strains obtained by mutagenesis have stronger ferment rice bran and wheat bran, the fermenting property of bacterial strain has occurred Significant positive variation.Final products total starches are the polysaccharide separated after impurity elimination, are clean products, product different from the past is only It is calculated with mycelia polysaccharide, also achieves the novelty of conspicuousness progress.

The present invention is finally obtained one plant of ganoderma lucidum new strains, deposit number is by Uv-induced screening research repeatedly CCTCC NO:M 2018908, the new strains be not added other carbon sources and nitrogen source only contain meet it is complete using the wheat bran rice bran of standard In the liquid culture medium of material the speed of growth faster, the ability of fermentation wheat bran and rice bran it is stronger so that the bacterium in specified fermentation volume Silk amount and mycelia polysaccharide yield are higher, and the fermentation character of new strains changes compared with original strain, embody the only of distinctness Characteristic.Based on the ganoderma lucidum new strains, the method for foring new fermentation wheat bran rice bran complete feed liquid culture medium, this method will Wheat bran and rice bran complete feed are subject to liquid state fermentation application, and have comprehensively been more than original strain on capacity and output, it is seen that this The ganoderma lucidum new strains in invention achieve " novelty for obtaining substantive distinguishing features outstanding and marked improvement ".The invention patent Cheap wheat bran rice bran is efficiently utilized, production cost can be reduced, reduces resource consumption, hence it is evident that increases yield, has practical Property.

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 ganoderma lucidum new strains obtained.

Fig. 2 is the antagonism figure of ganoderma lucidum new strains and original starting strain, and note: the left side is the original starting strain of ganoderma lucidum in figure, The right is ganoderma lucidum new strains.

Fig. 3 is the chadogram of ganoderma lucidum new strains.

Specific embodiment

The present invention attached process shown in FIG. 1 to specifications, provides through ultraviolet mutagenesis, breeding is in wheat bran rice bran complete feed On liquid culture medium the speed of growth faster, the method for the higher ganoderma lucidum new strains of polysaccharide yield, the method includes the following steps:

Taking the original strain Ganoderma lucidum JSU LIU17 of laboratory preservation 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 the screening training of rice bran wheat bran It supports on base

It is protected from light culture, 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 examination

It tests, secondary screening selects the bacterial strain that growth rate is very fast, more stable；

Shake flask fermentation is carried out with rice bran 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 28 DEG C.

In one embodiment, the ultraviolet mutagenesis is secretly operated using feux rouges, in the ultraviolet lamp for being 30W apart from power 30s is irradiated under 20cm respectively.

In one embodiment, described to be protected from light culture to be protected from light culture 7 days at 28 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, wheat bran, rice bran Complete feed uses, and wheat bran rice bran has to comply with using 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 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 wheat bran solid plate culture medium, picks out growth 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 ganoderma lucidum new strains and original starting strain that secondary screening is selected carry out shake flask fermentation test, continuously ferment for 5 generations, determine target by index Mutagenic fungi.

In one embodiment, the ganoderma lucidum mutagenesis new strains Ganoderma lucidum is No. 2 bacterial strain, multiple Sieving the growth rate on the 5th generation plate is 1.18cm/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 2.0g/100mL, wheat bran 3.0g/100mL, potassium dihydrogen phosphate 0.15g/100mL, magnesium sulfate 0.075g/100mL, pH are natural.

In one embodiment, the index is pure dry mycelial weight, mycelia polysaccharide weight and the hair of 100mL fermentation volume Zymotic fluid exocellular polysaccharide weight.

In one embodiment, inhereditary feature is characterized with antimicrobial experiment, result is as shown in Fig. 2, and antagonism line is bright Aobvious, the ganoderma lucidum 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 shape.

In one embodiment, ganoderma lucidum new strains, deposit number CCTCC NO:M 2018908, Jing Shenggong bioengineering (Shanghai) limited liability company DNA sequencing is determined as one plant of ganoderma lucidum new strains.

The ganoderma lucidum new strains are deposited in the Chinese Typical Representative positioned at Wuhan, China Wuhan University on December 19th, 2018 Culture collection (CCTCC), deposit number be CCTCC NO:M 2018908, it is proposed that classification naming be ganoderma strain JSU LIUYU18 or latin name are Ganoderma lucidum JSU LIUYU18, and ganoderma lucidum new strains of the present invention are equal Refer to this bacterial strain (see Fig. 3).

In one embodiment, the obtained ganoderma lucidum mutagenesis new strains that screen are obtained using wheat bran rice bran fermentation medium Fermentation results are as follows: the exocellular polysaccharide concentration in the dry weight concentrations of pure mycelia, mycelia polysaccharide concentration and fermentation liquid respectively reaches 2.184g/100mL, 113.746mg/100mL and 310.88mg/100mL, polysaccharide every hundred mL culture medium after separating impurity elimination obtain To mycelia polysaccharide, exocellular polysaccharide and total starches product be respectively 93.271mg, 242.486mg and 335.757mg.

In one embodiment, the more original starting strain of the dry mycelial weight concentration of ganoderma lucidum mutagenesis new strains increases 14.4%, mycelia polysaccharide concentration increases 14.3%, and exocellular polysaccharide increases 19.5%, and total starches increase 18.0%, explanation New strains obtained by mutagenesis have the ability of stronger ferment rice bran and wheat bran, and the fermenting property of bacterial strain has occurred significant positive Variation.

In one embodiment, the Ganoderma lucidum mycelium that obtains after fermentation is by centrifuge separation, multiple frequency ultrasonic assisted extraction, cold Freeze the method that concentration, heavy metal ion is replaced in citric acid extraction, alcohol precipitation alcohol is washed, is freeze-dried and obtains qualified Ganoderma lucidum mycelium 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 ganoderma lucidum exocellular polysaccharide.

In one embodiment, qualified Ganoderma lucidum mycelium polysaccharide and qualified ganoderma lucidum exocellular polysaccharide sum of the two are ganoderma lucidum Total starches, referred to as ganoderma lucidum polysaccharide.

In one embodiment, the mycotoxin aflatoxin B1 of ganoderma lucidum polysaccharide, B2 and deoxynivalenol bacterium alkene Limitation, heavy metal lead, arsenic, mercury, cadmium, the limitation of chromium and pesticide carbendazim, avermectin, the residue limits process of butachlor of alcohol Measurement not higher than Limited Doses as defined in GB2761-2017, GB2762-2017, GB2763-2016 or is not detected.

Embodiment 1

The ganoderma lucidum new strains that ganoderma strain is obtained using the present invention, Ganoderma lucidum CCTCC NO:M 2018908.Rice bran usage amount is 2.0g/100mL culture medium, and the usage amount of wheat bran is 3.0g/100mL culture medium, biphosphate Potassium 0.15g/100mL, epsom salt 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%, 28 DEG C of cultivation temperature, revolving speed 120r/min, training Support time 7d.Implement below by step (1)~(4): (1) using the resulting mycelium containing a small amount of rice bran wheat bran of Liquid Culture After the separation of 20 mesh metallic sieves, and it is washed twice with water；(2) gained wet mycelium multiple frequency ultrasonic auxiliary warm water is carried out to extract, Water temperature is no more than 60 DEG C, filters to take clear liquid, chilled concentration, citric acid extraction displacement heavy metal ion, alcohol precipitation, centrifugal filtration, Alcohol washes, is freeze-dried and obtains qualified mycelia polysaccharide product；(3) gained fermentation liquid is through filtering, freeze concentration, citric acid extraction displacement Heavy metal ion, alcohol precipitation, centrifugal filtration, alcohol wash, is freeze-dried and obtains qualified exocellular polysaccharide product；(4) fermentation gained Thick many candies Polyoses content is measured using Phenol sulfuric acid procedure, result is dry weight concentrations, the mycelia Thick many candies concentration, crude extracellular polysaccharide of pure mycelia Concentration and total Thick many candies concentration respectively reach 2.184g/100mL, 113.746mg/100mL, 310.88mg/100mL and 424.626mg/100mL, polysaccharide every hundred mL culture medium obtains after separating impurity elimination mycelia polysaccharide, exocellular polysaccharide and total starches Product is respectively 93.271mg, 242.486mg and 335.757mg；(5) through detecting, the mycotoxin aspergillus flavus poison of ganoderma lucidum polysaccharide The limitation of plain B1, B2 and deoxynivalenol, heavy metal lead, arsenic, mercury, cadmium, the limitation of chromium and pesticide carbendazim, AVM hereinafter The residue limits process measurement of rhzomorph, butachlor is provided not higher than GB2761-2017, GB2762-2017, GB2763-2016 Limited Doses or be not detected.

Claims (1)

1. one plant by ultraviolet mutagenesis obtain ganoderma strain, deposit number be CCTCC NO:M 2018908, it is proposed that classification It is named as ganoderma strain JSU LIUYU18, latin name is Ganoderma lucidum JSU LIUYU18.