CN103477994B - Bacterial strain used for producing ganoderma lucidum polysaccharides by complete feed liquid fermentation of rice bran and wheat bran - Google Patents
Bacterial strain used for producing ganoderma lucidum polysaccharides by complete feed liquid fermentation of rice bran and wheat bran Download PDFInfo
- Publication number
- CN103477994B CN103477994B CN201310274915.7A CN201310274915A CN103477994B CN 103477994 B CN103477994 B CN 103477994B CN 201310274915 A CN201310274915 A CN 201310274915A CN 103477994 B CN103477994 B CN 103477994B
- Authority
- CN
- China
- Prior art keywords
- strain
- rice bran
- wheat bran
- cctcc
- ganoderma lucidum
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the field of microbial application technology and food biotechnology, and discloses a ganoderma lucidum strain used for producing polysaccharides by complete feed liquid fermentation of rice bran and wheat bran. Ganoderma lucidum JSU6161314 is stored in China Center for Type Culture Collection (CCTCC) on 25th, June, 2013, preservation number is CCTCC M2013287, and the bacterial strain is named as Ganoderma lucidum CCTCC M2013287. According to a method disclosed in a drawing of the invention, the novel bacterial strain, which is capable of realizing high yield of ganoderma lucidum polysaccharides, is obtained by modifying ganoderma lucidum CFCC6043. The novel bacterial strain is used for rapid fermentation in a rice bran and wheat bran complete feed liquid culture medium and high yield of ganoderma lucidum polysaccharides. After fermentation, hypha dry weight and hypha polysaccharide yield increase 23.0% and 45% respectively compared with that of the original strain.
Description
Technical field
The present invention relates to food microorganisms applied technical field, relate in particular to a strain and be adapted at the also new mutagenic strain of the ganoderma strain capable of high yield ganoderan of growth in rice bran and the composite liquefied substratum of wheat bran complete feed.
Background technology
Since the people such as Chihara (reference: Chihara G, MaedaY, Hamuro J, et al.Inhibition of mouse sarcoma180by polysaccharides from Lentinus edodes (Berk.) sing[J] .Nature, 1969, 222 (5194): 687-688.) published thesis on " Nature " in 1969, since announcement lentinan has anti-tumor activity, have been found that the medicinal fungi with anti-tumor activity reaches kind more than 200, a wherein part or edible fungus, as Grifola frondosa, Hericium erinaceus (Bull. Ex Fr.) Pers., glossy ganoderma etc.In mycelium, sporophore, sclerotium or the spore of these edible mushroomss, can produce various active such as amino acid, protein, VITAMIN, polysaccharide, glycoside, flavonoid and microbiotic composition or Rich in Trace Element selenium, zinc etc., have the body immunity of raising, antitumor, strengthen liver function, the multiple efficacies such as anti-oxidant.The existing edible mushroom health-care food in domestic market is if ganoderma lucidum capsule, Grifola frondosa capsule and capsule of Chinese caterpillar fungus etc. are in fast sale.New Zealand, Japan, the U.S. etc. all produce and have similar product in the world.Zelanian " GanoPoly " series product are formed by the polysaccharide composite chitosan of the extraction such as glossy ganoderma, rainbow conk, are mainly used in improving the aspect such as immunizing power, auxiliary antineoplaston.With regard to ganoderan, research for it shows: ganoderan has good raising immunizing power, the curative effects such as auxiliary antineoplaston, for example Huang, the test of pesticide effectiveness that Seto etc. extract ganoderan from glossy ganoderma thalline shows, ganoderan has immunoregulation effect and antitumor action, and (reference is shown in 1. Huang S.Q., Ning Z.X.Extraction of polysaccharide from Ganoderma lucidum and its immune enhancement activity[J] .Int J Biol Macromol, 2010, 47 (3): 336～341, 2. Seto S.W., Lam T.Y, Tam H.L., et al.Novel hypoglycemic effects of Ganoderma lueidum water-extract in obese/diabetic (db/+db) mice[J] .Phytomedicine, 2009, (16): 426～436, 3. Hikino H, Konno C, Mirin Y, et al.Isolation and hypoglycemic activity of ganoderans A and B, glycans of Ganoderma lucidum fruit bodies[J].Planta?Med，1985，(12)：339～340)
Glossy ganoderma (Ganoderma luccidum) another name sesame grass, auspicious grass, it is Mycophyta, Basidiomycotina, Hymenomycetes, Aphyllophorales, glossy ganoderma Cordycepps, Ganoderma fungi, is mainly distributed in the torrid zone and the subtropical zone in Asia, Australia, Africa and America, and minority is also distributed in Temperate Region in China, only there are 4 kinds of Ganoderma in the Europe that is located in temperate zone, the Northern Hemisphere, and about 5 kinds of North America.China extends across the torrid zone to cool temperature zone, and Ganodermataceae kind is many and distribute extensively, is one of country that world's Ganoderma fungal species diversity is the abundantest.Traditionally, the obtain manner of glossy ganoderma is mainly artificial culture or field acquisition, is used as medicine with sporophore or spore powder.But the Ganoderma Lucidum cycle is long, production efficiency is low, labour intensity is large, is subject to again the restriction such as season, environment, subjects to disease and pest, and quality and output are unstable, and field acquisition glossy ganoderma limited amount is difficult to meet large production requirement.Edible fungus liquid submerged fermentation technology, has obvious advantage than traditional sporophore cultivation.For High-efficient Production ganoderan, researchist attaches great importance to the research of the liquid cultivating method to promoting suitability for industrialized production, has obtained many achievements in research.Chu Xiaoyan, Zeng Xi and so on Ganoderma liquid culture conditions were studied, the main content of such studies involving the production strain, medium composition and fermentation parameters, will reduce production costs and increase the yield of polysaccharides as a research focus.Wherein most process using are if starch, potato, glucose etc. are as medium component, even if use processing of farm products byproduct as rice bran or wheat bran, also be to set it as submember, be still taking grain class raw material, glucose or other raw materials as main Carbon and nitrogen sources.
China is large agricultural country, and agricultural byproducts source is abundant.Rice bran, wheat bran are as the by product of paddy and wheat processing, and not only its nutritive ingredient is abundant, and cheap.The material such as rich in starch, Mierocrystalline cellulose in rice bran, and wheat bran is rich in the material such as albumen, Mierocrystalline cellulose.Theoretically, rice bran and wheat bran are compound has possessed the glossy ganoderma needed Carbon and nitrogen sources material of growing.Under the effect of glossy ganoderma self cellulase and other enzymes, glossy ganoderma can change into rice bran and wheat bran the nutritive substance of self and grow, and produces ganoderan.Therefore, use the cheap agricultural byproducts such as rice bran, wheat bran, the required nutritive substances of Ganoderma lucidum submerged fermentation such as complete place of glucose, produce the ganodermal drop with auxiliary oncotherapy, high-valued application to low side raw material is significant, has high economic worth.
According to inventor herein Liu Wei people seminar years of researches experience, some edible mushroom strainses, not add on the rice bran of glucose or other grain class raw materials or wheat bran liquid culture medium upgrowth situation very desirable, need to be done special processing if induction mutation of bacterium is to obtain good growth.For example, Liu Weimin instructs several Master degree candidates to carry out deep research to edible mushrooms Grifola frondosa liquid state fermentation rice bran or wheat bran, obtaining some achievements, formed as Publication about Document: (1) Yang Suohua. Grifola frondosa ferment rice bran is prepared polysaccharide [D]. master thesis. Zhenjiang: the .2006 of Jiangsu University; (2) Gu Huimin. Grifola frondosa is liquid research [D] of cultivating product polysaccharide and enrichment organoselenium in rice bran substratum. master thesis. Zhenjiang: the .2009 of Jiangsu University; (3) to open and build. Physical mutagenesis Grifola frondosa liquid fermenting rice bran wheat bran produces the research [D] of polysaccharide. master thesis. Zhenjiang: Jiangsu University, 2010; (4) Guo Chunmei. the induction mutation of bacterium of Grifola frondosa, liquid fermenting rice bran wheat bran produce polysaccharide and rich selenium research [D]. master thesis. Zhenjiang: Jiangsu University, 2011; (5) Li Yongzhuan. transform rice bran and the Grifola frondosa strain mutagenesis of wheat bran high polysaccharide and the fermentation test [D] of existing bacterium. master thesis. Zhenjiang: Jiangsu University, 2012; (6) Liu Weimin, Zhang Jian, Guo Chunmei, etc. for the Grifola frondosa strain [P] of rice bran and wheat bran compound material production polysaccharide, 10579078.5,2010; (7) Liu Weimin, Zhang Jian, Guo Chunmei, etc. use rice bran wheat bran compound material and Grifola frondosa mutagenic strain to produce the method [P] of polysaccharide, 1010579048.4,2010; (7) Liu Weimin, Guo Chunmei, Zhang Jian, etc. for the bacterial strain [P] of ferment rice bran and wheat bran extracting solution production grifolan, 10150888.3,2011 (applications); (8) Li Yanan. the mutagenesis of Grifola frondosa bacterial classification and fermentation and product property research [D]. master thesis. Zhenjiang: Jiangsu University, 2013 (this paper publication date is in June, 2013, identical days with the application submission of this patent); (9) Zhao Li. the research [D] of Grifola frondosa liquid state fermentation enrichment manganese in rice bran wheat bran complex medium. master thesis. Zhenjiang: Jiangsu University, 2012; (10) Liu Lili. high value transforms Ganoderma lucidum submerged fermentation and the induction mutation of bacterium [D] of rice bran wheat bran. master thesis.Zhenjiang: Jiangsu University, 2012; (11) Guo Tianlong. the full rice bran wheat bran research of the high-valued conversion of Ganderma lucidum strain mutagenesis and liquid state fermentation [D]. master thesis. Zhenjiang: Jiangsu University, 2013 (this paper publication date is in June, 2013, identical days with the application submission of this patent).
From above-mentioned reference, the inventor herein Liu Wei people study around efficient high-valued this special topic of rare edible fungi polysaccharide that is converted into of rice bran wheat bran always, realize multiple innovation and creation imaginations, and propose to carry out rice bran wheat bran complete feed liquid fermenting with glossy ganoderma, the Research Thinking that efficient high-valued conversion rice bran wheat bran is ganoderan, to original strain mutagenic treatment in addition, (be negative mutagenesis instructing Liu Lili mutagenesis testing unsuccessful, see the analysis of Liu Lili Master's thesis p46 his-and-hers watches 5.2, the Master's thesis of Liu Lili all adopts rice bran wheat bran to cross leaching juice and makes substratum, rice bran wheat bran complete feed liquid culture medium of the present invention) basis on, summing up experience, instruct again Guo Tianlong to carry out systematic research, obtain positive mutagenic strain, and mutagenic strain liquid state fermentation rice bran wheat bran complete feed newly proposed, the method that efficient high-valued conversion rice bran wheat bran is ganoderan, the content of another patent of invention that has formed patent of the present invention and simultaneously declare.The Master's thesis publication date of Guo Tianlong is in June, 2013, submits to days identical with the application of this patent, does not affect novelty of the present invention.
One of key issue of the present invention is for must obtain the new bacterial strain of a kind of glossy ganoderma, and this new bacterial strain can effectively be grown on rice bran wheat bran complete feed liquid culture medium and Efficient Conversion rice bran wheat bran complete feed is ganoderan.The foundation of new bacterial strain and new bacterial strain all need innovation to the mode of newly utilizing of rice bran wheat bran, being suitable for the rice bran of new ganoderma strain capable and the wheat bran composition proportioning of liquid nutrient medium and the processing condition of liquid state fermentation all needs recent studies on to draw, have not been reported, therefore the present invention has uniqueness, innovation and practicality, possesses the essential characteristic of patent of invention.
The physics selection of microorganism mainly contains ultraviolet mutagenesis, microwave irradiation, ion beam mutagenesis etc. at present.For mutagenesis screening goes out to be adapted to grow on full rice bran wheat bran complex medium and produces the ganoderma strain capable of polysaccharide, the present invention adopts simple new approaches of physical mutagenesis, carry out ultraviolet mutagenesis and microwave irradiation test simultaneously, the site scope that expands tested glossy ganoderma starting strain sudden change, improves the possibility that obtains gain mutant bacterial strain.What mutagenesis of the present invention obtained be adapted at grows on full rice bran wheat bran complex medium and the bacterial strain that produces ganoderan obtains for invention first.
Summary of the invention
The present invention to use soil and reduces the production cycle in order to reduce, reduce production costs, by low side raw material high-valued trans-utilization in addition, finally can reduce ganoderan product price, benefit masses, the byproduct rice bran of the consideration large agricultural-food paddy of employing and wheat processing and wheat bran, as substratum, no longer add other carbon sources as glucose and nitrogenous source, carry out the liquid fermenting of glossy ganoderma, produce ganoderan.Realize this object, need mutagenesis screening to go out the ganoderma strain capable of suitable growth on rice bran wheat bran complete feed liquid culture medium.Therefore, the present invention is by by the method for ultraviolet, microwave irradiation, and taking high polysaccharide as index, the directed rice bran wheat bran that transforms be basic, selects high yield, ganoderma strain capable cheaply.By the foundation of the screening methods such as a series of stability, heredity, ensure the good character of glossy ganoderma, for further utilizing on a large scale the ganoderan of rice bran and wheat bran production high value cheaply to lay the foundation.
The technical solution used in the present invention is as follows:
The present invention is set out by the glossy ganoderma CFCC6043 (Ganoderma lucidum CFCC6043) of China Forest microbial strains preservation administrative center (CFCC), by the mutagenesis screening of ultraviolet and microwave, a kind of new glossy ganoderma mutagenic strain glossy ganoderma JSU6161314 (Ganoderma lucidum JSU6161314) is provided, can on the rice bran wheat bran complete feed liquid culture medium that does not add other Carbon and nitrogen sources, there is high growth rates and high polysaccharide yield; This glossy ganoderma JSU6161314 has been deposited in Wuhan, China Wuhan University Chinese Typical Representative culture collection center (CCTCC) on June 25th, 2013, preservation strain is numbered CCTCC M2013287, and name is called glossy ganoderma CCTCC M2013287 (Latin is called Ganoderma lucidum CCTCC M2013287).
In one aspect of the invention, provide the purposes of above-mentioned glossy ganoderma CCTCC M2013287, produce ganoderan for ferment rice bran wheat bran complete feed liquid culture medium.
Beneficial effect of the present invention
The present invention adopts simple physical mutagenesis technology ultraviolet and microwave irradiation technology seed selection glossy ganoderma superior strain, by laboratory, existing glossy ganoderma CFCC6043 sets out, carry out the parallel mutagenesis of ultraviolet and microwave, with growth velocity, mycelia dry weight and exocellular polysaccharide are that index is screened, the final glossy ganoderma CCTCC M2013287 that obtains, in the rice bran wheat bran complete feed liquid culture medium that does not add other Carbon and nitrogen sources, produce glossy ganoderma and produce polysaccharide, output is higher compared with original strain glossy ganoderma CFCC6043, while fermentation by tank on microwave irradiation bacterial strain obtained strains glossy ganoderma CCTCC M2013287, mycelia dry weight and mycelia polysaccharide productive rate output are 6.3g/100mL substratum and 0.203mg/100mL substratum, starting strain glossy ganoderma CFCC6043 mycelia dry weight and mycelia polysaccharide output are 5.12g/100mL substratum and 0.14mg/100mL substratum, the mycelia dry weight of mutagenic strain glossy ganoderma CCTCC M2013287 fermentation and mycelia dry weight and the mycelia polysaccharide output of mycelia polysaccharide productivity ratio starting strain glossy ganoderma CFCC6043 have increased respectively 23.0% and 45%.Prove that through the antagonistic effect of glossy ganoderma CCTCC M2013287 and starting strain glossy ganoderma CFCC6043 bacterial strain and starting strain after mutagenesis are different in hereditary property, when screening, mutagenic strain glossy ganoderma CCTCC M2013287 growth characteristics are obviously faster than starting strain glossy ganoderma CFCC6043, thereby can judge that mutagenic strain is as desirable new bacterial strain.This new bacterial strain glossy ganoderma CCTCC M2013287 obtains for pioneering, belongs to a kind of new Microbial resources, has uniqueness, novelty.Taking new bacterial strain glossy ganoderma CCTCC M2013287 as basis, form the method for new ferment rice bran wheat bran complete feed liquid culture medium, the method has changed in the past rice bran wheat bran and has crossed and get juice after leaching juice or cellulose treatment and make the low shortcoming of rice bran wheat bran utilization ratio, efficiently utilize cheap rice bran wheat bran, can reduce production costs, reduce resource consumption, increase output.Described mutagenic fungi glossy ganoderma CCTCC M2013287 is under identical rice bran wheat bran usage quantity condition, the mycelia dry weight of mutagenic strain glossy ganoderma CCTCC M2013287 fermentation and mycelia dry weight and the mycelia polysaccharide output of mycelia polysaccharide productivity ratio starting strain glossy ganoderma CFCC6043 have increased respectively 23.0% and 45%, obvious effect of increasing production.The present invention has produced the beneficial effect of technical progress.
The finished product are the ganoderan with the effect such as strengthening immunity, auxiliary oncotherapy, can become the useful product of popular health care.By high-valued low side raw material trans-utilization, cost and resource are saved.Therefore, the present invention has good social benefit.
End product ganoderan at present on market price higher, be high-valued product, the present invention has good economic worth.
To sum up, the present invention has possessed uniqueness, creativeness and the practicality of patent of invention, has produced useful technology, society and economical effectiveness.
Brief description of the drawings
Fig. 1 is the schema of the parallel mutagenic breeding method of bacterial strain ultraviolet microwave of the present invention;
Fig. 2 is uv irradiating effect, wherein note: the left side is for irradiating 0s, and the right is for irradiating 90s;
Fig. 3 is microwave irradiation effect, wherein note: the left side is for irradiating 0s, and the right is for irradiating 10s;
Fig. 4 is strain growth state, wherein note: be followed successively by from left to right ultraviolet No. 9, glossy ganoderma CCTCC M2013287, starting strain;
Fig. 5 is the comparison of strain liquid seed morphology, wherein note: be followed successively by from left to right starting strain, No. 9, ultraviolet, glossy ganoderma CCTCC M2013287;
Fig. 6 is the antagonism figure of mutagenic fungi glossy ganoderma CCTCC M2013287 and starting strain glossy ganoderma CFCC6043, wherein note: the left side is original strain glossy ganoderma CFCC6043, and the right is mutagenic fungi glossy ganoderma CCTCC M2013287.
Embodiment
The present invention is the flow process shown in accompanying drawing 1 to specifications, provide ultraviolet and microwave irradiation seed selection on the rice bran wheat bran complex medium that does not add other Carbon and nitrogen sources, to have the method for the ganoderma strain capable of high growth rates and high polysaccharide yield, described method comprises the following steps:
Getting the existing glossy ganoderma in laboratory is starting strain;
Ganoderma strain capable is inoculated on solid slant culture base and is normally cultivated;
After yeast culture, physiological saline wash-out makes spore suspension;
Spore suspension obtained above is irradiated under ultraviolet lamp and microwave radiation is carried out after mutagenesis, unglazed dark cultivation, primary screening goes out very fast, the more stable bacterial strain of growth velocity;
On full rice bran, wheat bran liquid fermentation medium, ferment, the bacterial strain of postsearch screening high growth rates and high polysaccharide yield;
Carry out stability and heredity analysis and identification;
In one embodiment, solid slant culture base used is potato 200g/L, glucose 20g/L, peptone 5g/L, potassium primary phosphate 1.5g/L, magnesium sulfate 0.75g/L, vitamins B
110mg/L, agar 20g/L, pH nature.
In one embodiment, described constant temperature is 28 DEG C.
In one embodiment, described ultraviolet mutagenesis adopts ruddiness secretly to operate, and the ultraviolet lamp tube using two power as 30W, as the light source of ultraviolet mutagenesis, apart from 20cm, irradiates 30s.
In one embodiment, described unglazed dark cultivation is unglazed cultivation 2-3 days at 28 DEG C.
In one embodiment, described unglazed dark cultivation used medium is rice bran, wheat bran solid plate substratum: rice bran 20g/L, wheat bran 30g/L, potassium primary phosphate 1.5g/L, magnesium sulfate 0.75g/L, vitamins B
110mg/L, agar 20g/L, pH nature.
In one embodiment, described screening method is plate diameter assay method.
In one embodiment, described microwave irradiation adopts ruddiness secretly to operate, microwave power 700W, pulse-repetition 2450Hz, mutation time 20s.
In one embodiment, described primary screening step is: from ultraviolet mutagenesis, microwave irradiation is unglazed dark culture plate, the well-grown single bacterium colony of picking is seeded to respectively in new rice bran wheat bran solid plate substratum, pick out 10 strain fast growths and dense mutagenic fungi, to its cultivation of going down to posterity, therefrom select that 3 strains are fast with respect to starting strain growth, shapeliness, bacterial strain that stability is high.
In one embodiment, described Secondary Fermentation screening step is: the definite high growth rates variant of first secondary growth, as the object of postsearch screening, screens the bacterial strain of definitive variation strain good character stably express thereby ferment together with starting strain.First bacterium and starting strain are carried out to shake flask fermentation test, continuously fermented for 5 generations, determine object mutagenic fungi by index.
In one embodiment, described shaking flask is 250mL Erlenmeyer flask.
In one embodiment, described rice bran, wheat bran liquid fermentation medium are: rice bran 20g/L, wheat bran 30g/L (it is complete utilization that rice bran, wheat bran poach 4h do not get juice), potassium primary phosphate 1.5g/L, magnesium sulfate 0.75g/L, vitamins B
110mg/L, pH nature.
In one embodiment, described index is exocellular polysaccharide, mycelia (mixing a small amount of raw material) dry weight and mycelia polysaccharide.
The Analysis and Identification method that transforms the glossy ganoderma mutant strain of rice bran and wheat bran compound material in the present invention, described method comprises the following steps:
Colonial morphology comparison;
Stability analysis;
In one embodiment, described is colonial morphology comparison, to on inclined-plane, set out ganoderma strain capable and JSU61613 inoculation to solid slant culture base, each 6 repetitions, in 28 DEG C of constant incubators, cultivate 7 days, observe once every day, observes the speed of growth, color, colony shape, smell etc. that comprise bacterium colony.
In one embodiment, described stability analysis is for investigating stability taking mycelial growth rate as index, finishing screen is selected to preferably dissociant of 10 strains, go down to posterity through 5 times, select optimum JSU6161314 bacterial strain, this strain dissociant was gone down to posterity for 3 generations again, observe the variation of mycelial growth rate, thereby determine stability, and contrast with starting strain.
In one embodiment, described heredity analysis is antagonism analysis.
Embodiment 1 glossy ganoderma ultraviolet ray-microwave irradiation
1. the preparation of spore suspension
Glossy ganoderma is seeded in to 9cm and has gone out after cultivating 4d on the solid medium flat board of bacterium and rinse by 10mL stroke-physiological saline solution, with the writing brush brush nourishing body back and forth of sterilizing, collect washing lotion and get final product.
2. the selection of ultraviolet mutagenesis effect curve and ultraviolet mutagenesis dosage
Get 20 9cm plates, every ware is equipped with respectively freshly prepd spore suspension 5mL.Every two is one group, is divided into 10 groups.Irradiate respectively 0,5,10,15,20,25,30,45,60,150s (90s radiation response is shown in accompanying drawing 2), then every ware is got 1mL and is suitably coated with after dilution dull and stereotyped.Dull and stereotyped 28 DEG C of thermostat containers cultivations that are placed on dark black out with black cloth parcel.After flat board grows bacterium colony, enumeration.Every group of mean value using two ware colony numbers is as the colony number of this group.Calculate mutagenesis lethality rate, select ultraviolet mutagenesis dosage taking mutagenesis lethality rate as index.
In formula, A is bacterium colony regeneration number after mutagenesis; B is colony number before mutagenesis.
3. ultraviolet mutagenesis
Open the about 20min of ultraviolet preheating, cut-off footpath 9cm sterile petri dish 2 is overlapped, and adds respectively the above-mentioned spore suspension 5mL adjusting, and put on electromagnetic shaker, open plate lid, is 20cm in distance, under the ultraviolet lamp that power is 30W, irradiates respectively by the best irradiation dose of above-mentioned selection.Cover ware lid, close ultraviolet lamp.When dose meter, from uncapping, only add a cover.First drive electromagnetic shaker switch, then the irradiation of uncapping, make cell in spore suspension accept to irradiate even etc.
4. microwave irradiation
The spore suspension that absorption makes, injects the smooth plate in bottom, and the suspension amount of each plate is 10mL, adjustment microwave power is 700W, pulse-repetition is 2450Hz, according to the different treatment times, spore suspension is carried out to radiation treatment (10s radiation response is shown in accompanying drawing 3).Draw spore suspension 0.3mL, coating rice bran, wheat bran solid plate substratum, be then placed in 28 DEG C of thermostat containers and cultivate 3d.Live bacterial count, calculates lethality rate.10 batches of continually mutagenizes, select on rice bran bran mass can grow first out, healthy and strong, pure bacterial strain.
4. the screening of mutagenic strain
Get the bacteria suspension 0.2mL of uv irradiating, microwave radiation, be coated with rod with aseptic glass and fill equably rice bran, wheat bran solid plate media surface, every batch is coated with 5 plates, 10 batches of mutagenesis, single bacterium colony of regeneration is seeded to respectively in new plate, pick out 10 strain fast growths and dense mutagenic fungi, to its cultivation of going down to posterity, therefrom select that 3 strains are fast with respect to starting strain growth, shapeliness, bacterial strain that stability is high.
Above-mentioned glossy ganoderma mutagenic fungi is carried out to shake flask fermentation test, mix dry weight as index taking exocellular polysaccharide, mycelia, filter out object mutagenic fungi.
Table 1 is selected bacterial strain and is respectively mixed dry weight for fermented hypha
Table 2 is selected the each generation fermentation of bacterial strain exocellular polysaccharide
From table 1 and 2, can find out that the mycelia of dissociant JSU-10 (being glossy ganoderma JSU6161314) first-generation fermentation and polysaccharide yield are all higher than starting strain, but in the s-generation and the third generation, there is certain variation in its proterties, and glossy ganoderma JSU6161314 is relatively stable, its mycelia mixing dry weight and exocellular polysaccharide are all higher than starting strain, its third generation mycelia mixes dry weight and exopolysaccharides has improved respectively 1.8% and 5.9% in 100mL shake-flask culture, glossy ganoderma JSU6161314 is deposited in Chinese Typical Representative culture collection center (CCTCC) on June 25th, 2013, preservation strain is numbered CCTCC M2013287, name is called glossy ganoderma CCTCC M2013287 (Latin is called Ganoderma lucidum CCTCC M2013287).
Test the analysis of a dissociant
1, biomorph Epidemiological Analysis
Ganderma lucidum strain on inclined-plane is inoculated on solid slant culture base, 6 repetitions, in 28 DEG C of constant incubators, cultivate 7 days, observe once every day, and the results such as the speed of growth of observable bacterium colony, color, colony shape, smell are: the glossy ganoderma CCTCC M2013287 speed of growth is very fast, dull and stereotyped covering in 5 days, the dull and stereotyped growth velocity of starting strain is 0.83mm/h, the dull and stereotyped growth velocity of glossy ganoderma CCTCC M2013287 is 0.94mm/h, has improved 13.25%, sees accompanying drawing 4.Glossy ganoderma CCTCC M2013287 bacteria colony white, is just fine hair shape, rear change flocculence, and felted in a measure, bacterium colony is thick.Reverse side is slightly micro-yellow, has slight milk fragrance.The form of liquid one, two generations seed is shown in accompanying drawing 5.
2, stability analysis
Investigate stability taking mycelial growth rate as index, finishing screen is selected to preferably dissociant of 10 strains, go down to posterity through 5 times, select optimum glossy ganoderma CCTCC M2013287 bacterial strain, this strain dissociant was gone down to posterity for 3 generations again, observe the variation of mycelial growth rate, and contrast with starting strain, thereby determine stability, then 3 generations of going down to posterity the results are shown in Table 3.
Table 3 is selected bacterial strain respectively for the speed of growth
As can be seen from Table 3, the speed of growth of mutagenic strain JSU-10 (being glossy ganoderma JSU6161314) is higher than starting strain, its speed of growth is degenerated not clearly, go down to posterity through 8 generations, still have the speed of growth of 0.911mm/h, the character variation that glossy ganoderma produces after ultraviolet, microwave irradiation as can be seen here can heredity.
3. heredity analysis
Antagonism is analyzed
Dissociant and starting strain are seeded on PDA flat board simultaneously, and two bacterial strains are at a distance of certain distance, are placed in the incubator of 28 DEG C and cultivate, and observe the antagonism between bacterium colony after 4d.
Hypha of edible fungus not of the same race is in growing, and mycelia mutually restriction the other side's growth spreads, and produces antagonism, forms antagonism line at intersection, Here it is antagonism.Antagonism is not only in the existence not of the same race of edible mushrooms, and of the same race but between the discrepant bacterial strain of hereditary property also exist, between bacterial strain, the power of antagonistic action has reflected the size of genetic diversity between bacterial strain, therefore antagonism not only can be for judging the sibship between bacterial strain, and can be for breeding, whether qualification bacterial strain produces variation.This test is connected to dull and stereotyped upper bacterium by starting strain and dissociant and has produced certain antagonism, there is certain variation in the growthhabit that can find out the right dissociant glossy ganoderma CCTCC M2013287 from Figure of description 6, its mycelial growth densification, and form antagonism line with left side starting strain, illustrated that starting strain and dissociant have produced genetic diversity.
Test two mutagenic strain glossy ganoderma CCTCC M2013287 and the comparison of starting strain glossy ganoderma CFCC6043 output
Mutagenic strain and starting strain through screening carry out ferment tank, relatively leavening property.
Ganoderma strain capable adopts respectively mutagenic strain glossy ganoderma CCTCC M2013287 and starting strain glossy ganoderma CFCC6043.Fermentor tank sample-loading amount is 80% of fermentor tank volume, culture temperature is 28 DEG C, ventilation 1:0.8v/v/mim, stirring velocity 60r/min, tank gauge pressure 0.05MPa, inoculum size 8%, incubation time 5d, fermention medium is rice bran 50g/L, wheat bran 30g/L, potassium primary phosphate 1.5g/L, magnesium sulfate 0.75g/L, pH nature.Weighting method is measured mycelia dry weight, and phenol sulfuric acid process is measured mycelia polysaccharide.The mycelia mixture of liquid culture gained, after centrifugation, then is used to distilled water wash 3 times, and the nutrient solution being sticked to remove mycelium surface, puts into air dry oven, under 60 DEG C of conditions, is dried to constant weight, mixes dry weight through weighing and obtain mycelia.In the mycelia of drying, add certain volume distilled water, after grinding, lixiviate 3h in 100 DEG C of water-baths, quantitatively to a constant volume, with phenolsulfuric acid method mensuration mycelia mixing polysaccharide content.Test-results is: (1) mutagenic strain glossy ganoderma CCTCC M2013287 and starting strain glossy ganoderma CFCC6043 ferment in the same terms and substratum, mycelia dry weight is respectively 63.0g/L and 51.2g/L, and the mycelia dry weight of mutagenic strain glossy ganoderma CCTCC M2013287 has improved 23% compared with starting strain.(2) glossy ganoderma CCTCC M2013287 and a bacterial strain ferment in the same terms and substratum, and both are respectively 2.03g/L and 1.40g/L mycelia polysaccharide, and the mycelia polysaccharide of glossy ganoderma CCTCC M2013287 has improved 45% compared with starting strain.Fermentation test shows, glossy ganoderma CCTCC M2013287 compares with starting strain glossy ganoderma CFCC6043, leavening property and produce mycelia polysaccharide performance good variation has occurred.
Claims (1)
1. for the bacterial strain of rice bran and wheat bran complete feed liquid state fermentation production ganoderan, it is characterized in that being preserved in Chinese Typical Representative culture collection center, the Wuhan glossy ganoderma CCTCC M2013287 (Ganoderma lucidum CCTCC M2013287) of (being called for short CCTCC).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310274915.7A CN103477994B (en) | 2013-06-28 | 2013-06-28 | Bacterial strain used for producing ganoderma lucidum polysaccharides by complete feed liquid fermentation of rice bran and wheat bran |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310274915.7A CN103477994B (en) | 2013-06-28 | 2013-06-28 | Bacterial strain used for producing ganoderma lucidum polysaccharides by complete feed liquid fermentation of rice bran and wheat bran |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103477994A CN103477994A (en) | 2014-01-01 |
| CN103477994B true CN103477994B (en) | 2014-12-17 |
Family
ID=49818865
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310274915.7A Expired - Fee Related CN103477994B (en) | 2013-06-28 | 2013-06-28 | Bacterial strain used for producing ganoderma lucidum polysaccharides by complete feed liquid fermentation of rice bran and wheat bran |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103477994B (en) |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105586266B (en) * | 2015-12-25 | 2019-03-05 | 江苏大学 | A method of producing selenium-rich Chinese caterpillar fungus bacterial filament raw material |
| CN105586267B (en) * | 2015-12-25 | 2018-11-06 | 江苏大学 | Produce the ganoderma lucidum mutagenic strain of ganoderma lucidum mycelium |
| CN105754865B (en) * | 2015-12-25 | 2019-04-30 | 江苏大学 | Utilize the method for mutagenic strain production ganoderma lucidum mycelium raw material |
| CN105586270B (en) * | 2015-12-25 | 2018-10-09 | 江苏大学 | The production method of Se-rich lucid ganoderma mycelia raw material |
| CN109943488B (en) * | 2019-02-19 | 2022-05-03 | 中国科学院合肥物质科学研究院 | Ganoderma lucidum polysaccharide high-yield strain RWHBW-1 and application thereof |
| CN110283861B (en) * | 2019-07-05 | 2023-05-02 | 江苏大学 | Method for producing ganoderan by using ganoderma lucidum strains generated by mutagenesis |
| CN110438010A (en) * | 2019-07-05 | 2019-11-12 | 江苏大学 | One plant of ganoderma strain obtained by ultraviolet mutagenesis |
| CN115474504B (en) * | 2021-05-30 | 2023-07-25 | 北京京诚生物科技有限公司 | New strain ganoderma lucidum and industrial application thereof |
| CN115053752A (en) * | 2022-06-30 | 2022-09-16 | 安徽金寨仙芝灵生物科技有限公司 | Novel ganoderma lucidum strain cultivation method |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1283629A (en) * | 1999-08-05 | 2001-02-14 | 武汉大学 | Antineoplastic gancoderma mycelium-glucoprotein composition and its application and preparing process |
| CN101455274A (en) * | 2008-12-29 | 2009-06-17 | 江南大学 | Method for producing feedstuff containing ganoderma lucidum active ingredient and feedstuff using ganoderma lucidum liquid fermentation liquid |
| TW201028477A (en) * | 2009-01-22 | 2010-08-01 | qiu-lan Song | Method of fermenting with cycle-packed reactor to produce ganoderma lucidum polysaccharide |
| CN101838673A (en) * | 2010-05-18 | 2010-09-22 | 湛江三角威力神酿酒集团有限公司 | Ganoderma lucidum polysaccharide liquid fermentation preparation method using rice wine vinasse as raw material |
| CN102643884A (en) * | 2012-05-04 | 2012-08-22 | 苏州百趣食品有限公司 | Method for producing polysaccharide by utilizing fermentation of ganoderma |
| CN102919513A (en) * | 2012-11-07 | 2013-02-13 | 南开大学 | Method for producing feed by using fungi to ferment Chinese medicine residue |
-
2013
- 2013-06-28 CN CN201310274915.7A patent/CN103477994B/en not_active Expired - Fee Related
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1283629A (en) * | 1999-08-05 | 2001-02-14 | 武汉大学 | Antineoplastic gancoderma mycelium-glucoprotein composition and its application and preparing process |
| CN101455274A (en) * | 2008-12-29 | 2009-06-17 | 江南大学 | Method for producing feedstuff containing ganoderma lucidum active ingredient and feedstuff using ganoderma lucidum liquid fermentation liquid |
| TW201028477A (en) * | 2009-01-22 | 2010-08-01 | qiu-lan Song | Method of fermenting with cycle-packed reactor to produce ganoderma lucidum polysaccharide |
| CN101838673A (en) * | 2010-05-18 | 2010-09-22 | 湛江三角威力神酿酒集团有限公司 | Ganoderma lucidum polysaccharide liquid fermentation preparation method using rice wine vinasse as raw material |
| CN102643884A (en) * | 2012-05-04 | 2012-08-22 | 苏州百趣食品有限公司 | Method for producing polysaccharide by utilizing fermentation of ganoderma |
| CN102919513A (en) * | 2012-11-07 | 2013-02-13 | 南开大学 | Method for producing feed by using fungi to ferment Chinese medicine residue |
Non-Patent Citations (3)
| Title |
|---|
| Relationship between mycelium morphology and extracellular polysaccharide production of medicinal mushroom Ganoderma lucidum in submerged culture;zhongyan ding,et al.;《Journal of medicinal plants research》;20120530;第6卷(第14期);第2868-2874页 * |
| 灵芝米糠发酵液制作保健饮料的工艺研究;顾颖娟等;《食品科学》;20091231;第30卷(第4期);第295-298页 * |
| 高值转化米糠麸皮的灵芝液体发酵及菌种诱变;刘丽丽;《江苏大学硕士学位论文》;20090903;第16页倒数第1-2段，第17页第5段，第18页第4-6段，第47页第1-4段，第59页倒数第1段，第60页第1段 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103477994A (en) | 2014-01-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103477994B (en) | Bacterial strain used for producing ganoderma lucidum polysaccharides by complete feed liquid fermentation of rice bran and wheat bran | |
| CN1358413A (en) | Method for preparing mushroom mycelium and use | |
| CN103343152B (en) | The method of polysaccharide produced by a kind of Grifola frondosa mutagenic fungi liquid state fermentation rice bran wheat bran complete feed | |
| CN102119631B (en) | Grifola frondosa strain for producing polysaccharide with composite raw material of rice bran and wheat bran | |
| CN103396973B (en) | Method for producing mycoprotein feed through microbial fermentation of kitchen wastes | |
| CN104145719A (en) | Cordyceps sinensis mycelium fermentation production method | |
| CN109355204A (en) | A kind of method of fermenting and producing cordyceps sinensis mycelium powder | |
| CN109251951A (en) | A kind of method that semicontinuous Liquid Culture efficiently produces ganoderma lucidum polysaccharide | |
| CN109076882A (en) | A kind of mycelial cultural method of selenium-enriched edible mushroom and its application | |
| CN109337895A (en) | A kind of production method of good quality and high output selenium-enriched hericium erinaceus strain | |
| CN102816806A (en) | Production method of grifolan selenium compound | |
| CN103416313B (en) | Grifola frondosa strain produced through rice bran and wheat bran complete feed liquid fermentation | |
| CN102080113A (en) | Method for producing polysaccharide by rice husk bran composite raw material and grifola frondosa mutant strain | |
| CN102925527A (en) | Method for mixing and fermenting flammulina velutipes and lucid ganoderma | |
| CN104651294B (en) | The fermentation medium and its preparation and application of suitable Metarhizium anisopliae production spore | |
| CN103421861B (en) | The method of Cordyceps Polysaccharide produced by a kind of liquid state fermentation rice bran wheat bran complete feed | |
| CN103555786B (en) | A kind of glossy ganoderma mutagenic fungi liquid state fermentation rice bran wheat bran complete feed is produced the method for polysaccharide | |
| CN102816701A (en) | Strain used for fermenting rice bran and wheat bran extracts for producing grifolan | |
| CN105586269B (en) | The method for producing maitake mushroom mycelia raw material using mutagenesis Grifola frondosa strain | |
| CN103849575B (en) | A kind of production method of single cell protein | |
| CN105586267B (en) | Produce the ganoderma lucidum mutagenic strain of ganoderma lucidum mycelium | |
| CN105754865B (en) | Utilize the method for mutagenic strain production ganoderma lucidum mycelium raw material | |
| CN1225172C (en) | Batch production process of biological herbicide | |
| CN1173029C (en) | Fermentation process of producing agaric and its culture medium | |
| CN110527702A (en) | Utilize the method for mutagenic obtained Inonotus obliquus bacterial strain production Fuscoporia obliqua polysaccharide |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB03 | Change of inventor or designer information | ||
| CB03 | Change of inventor or designer information |
Inventor after: Liu Weimin Inventor after: Zhang Xiaofei Inventor after: Shen Guodong Inventor after: Guo Tianlong Inventor after: Zhou Wenbiao Inventor after: Zhao Jiewen Inventor after: Ma Haile Inventor before: Liu Weimin Inventor before: Guo Tianlong Inventor before: Shen Guodong Inventor before: Zhang Xiaofei Inventor before: Zhou Wenbiao Inventor before: Zhao Jiewen Inventor before: Ma Haile |
|
| COR | Change of bibliographic data |
Free format text: CORRECT: INVENTOR; FROM: LIU WEIMIN GUO TIANLONG SHEN GUODONG ZHANG XIAOFEI ZHOU WENBIAO ZHAO JIEWEN MA HAILE TO: LIU WEIMIN ZHANG XIAOFEI SHEN GUODONG GUO TIANLONG ZHOU WENBIAO ZHAO JIEWEN MA HAILE |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20141217 Termination date: 20190628 |