CN105219657A - Rainbow conk liquid fermenting high polysaccharide bacterial strain and selection thereof - Google Patents
Rainbow conk liquid fermenting high polysaccharide bacterial strain and selection thereof Download PDFInfo
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Abstract
The invention discloses a kind of rainbow conk liquid fermenting high polysaccharide bacterial strain and selection thereof, does is bacterial strain deposit number? CGMCC? No.? 10903; Classification And Nomenclature: rainbow conk, Latin name: <i>Coriolus</iGr eatT.GreaT.GT<i>? versicolor</i>.Mutagenesis of the present invention goes out the rainbow conk mutagenic strain of a strain prepared from coriolus versicolor mycelium polysaccharide yield higher than former starting strain, and the output that improve krestin can create good economic benefit.
Description
Technical field
The present invention relates to a kind of rainbow conk liquid fermenting high polysaccharide bacterial strain and selection thereof.
Background technology
Intracellular Polysaccharide of Poly-stictus Versicolor PS-K (Coriolusversicolorintracellu-larpolysaccharides) is the associated proteins polysaccharide belonging to extraction purification the mycelia of fungi rainbow conk or cultivation from Basidiomycetes polyporaceae rainbow conk, it is good biological response modifiers, there is the multiple pharmacological effect such as Tumor suppression, immunity moderation, reduction Radiotherapy chemotherapy toxic side effects, and have anti-HIV activity.Due to the resource-constrained of field rainbow conk, adopting solid fermentation cultured mycelia to extract, polysaccharide cost is high, cycle length, unstable product quality, yield poorly, and Drug Administration of China requires that changing to liquid culture prepared from coriolus versicolor mycelium by solid culture extracts intracellular polyse recently, so not enough in order to make up these, adapt to extensive intensive manufacture.Krestin mainly obtains from the mycelium extraction of liquid fermenting, the research that current rainbow conk liquid fermenting improves polysaccharide yield is mostly that the research in the condition and krestin extraction process of rainbow conk liquid fermenting is a lot, and pass through induction mutation of bacterium, obtain specific strain to improve the content of tunning krestin, almost do not report, output is high, biological character is excellent, the good rainbow conk bacterial classification of the production traits is the prerequisite ensureing that rainbow conk is produced and rainbow conk industry development is good, the most output of rainbow conk bacterial classification be separated from field is not high, need through domestication and continuous seed selection, to improve output and the production traits of rainbow conk, therefore, the research of the seed selection aspect of bacterial classification just seems particularly important.The efficiency of spontaneons screening is lower, and takes the method effect of artificially breeding better, and rainbow conk, as a kind of macro fungi, has the feature of himself, and the election effects of its novel bacterial be unable to do without the research of the Breeding of Edible Mushroom aspect.
Excellent medicinal fungi engineering bacteria is the key improving medicinal fungi liquid fermenting level.At present, natural selection, selection by mutation and cross-breeding are mainly contained to the breeding technique that medicinal fungi adopts.Except utilizing the physics and chemistry behavior such as ultraviolet, laser and chemical mutagen because usually filtering out except excellent medicinal fungi, in order to improve validity and the purpose of medicinal fungi breeding, researchist more and more payes attention to utilizing Protoplast Fusion Technique, gene recombination technology etc. to build novel medicinal fungi fermentation.Protoplast fusion is also known as cytogamy, this technology is not by the impact of the affinity factor, in the kind that can realize medicinal fungi between different varieties, plant between and effective mixing of full-length genome between belonging in cell, between the edge kind far away making genetic distance larger, intergeneric hybridization becomes possibility.But the widespread use of this technology is have impact on due to heterozygote standard of perfection also existing technical difficulty.
In the strain improvement of edible mushrooms, usually many difficult problems are had, the yields screening cycle of such as novel bacterial is long, workload is large, the efficiency of strain improvement is not high, adopt traditional method to carry out edible fungus species seed selection, the difficulty screening the high yield new strains that a strain output is high, biological character is excellent, the production traits is excellent is larger.Therefore, a kind of new breeding technique and technique efficiently is urgently introduced in edible fungus species election effects field.
At present in Microbial Breeding, utilize ionic fluid to carry out selection by mutation to receive and pursue, ionic fluid develops very rapid because of the mutagenic mechanism of its uniqueness and biological effect as a kind of new mutation source in selection by mutation, ion implantationly cause DNA bond rupture, cause a large amount of acceptor atom to be shifted, recombinate, form new molecular structure and gene, produce abundant transgenation, thus improve mutation rate.Compared with classic mutagenesis source, ion implantationly can obtain higher mutation rate, wider mutation spectrum with less physiological damage, and have equipment simple, easy to use, with low cost, to human body and the advantage such as environmentally friendly.In recent years, there is report to be used in the mutagenesis of antibiotic strain by Mutation technique induced by ion beam, and succeed.The people such as Xu Liqing, Zhang Yinfen adopt ion beam implantation to be applied to the mutagenesis of the anti-strain producing strains of ribostamycin, kantlex producing strains and kantlex, result shows that titer of antibodies unit is significantly improved, with production bacterial strain-gentamicin and the kantlex producing strains of N+ process, the superior strain selected for the production of, increase rate is respectively more than about 20% and 12.5%, and economic benefit is obvious.Within 2006, people such as Zhao Shu glad grade adopts ion beam implantation to carry out mutagenic and breeding to a saccharomycete TQ601, successfully obtains a strain and produces the low bacterial strain TQ105 bacterium of higher alcohols, reduce 34.15% than starting strain.Ion beam mutation technology is used for the seed selection work of bacterial strain, and effect is better than traditional induced-mutation technique.
Few for rainbow conk breeding of new variety report at present, yet there are no the relevant report about utilizing ion beam mutation mutagenic and breeding high yield krestin fermentation strain both at home and abroad, this project implementation will fill up the blank of this respect.Introduce the seed selection that Mutation technique induced by ion beam applies to high yield krestin fermentation strain, play its advantage, greatly improve the success ratio of mutagenic and breeding, rainbow conk liquid fermenting strain selection is set up a new high efficiency method, and new road is opened up in the development for fungi fermentation technology.
Summary of the invention
The object of the present invention is to provide a kind of rainbow conk liquid fermenting high polysaccharide bacterial strain and selection thereof.
Technical solution of the present invention is:
A kind of rainbow conk liquid fermenting high polysaccharide bacterial strain, is characterized in that: deposit number is CGMCCNo.10903; Classification And Nomenclature: rainbow conk, Latin name: Coriolusversicolor.
Described rainbow conk liquid fermenting high polysaccharide bacterial strain, fermentation condition is: fermenter volume 100L, liquid amount 60L, mixing speed 150r/win, air flow 1:0.5-1, and pressure differential method is inoculated, and inoculum size is cultivate under 8%, 27 ~ 28 DEG C of conditions; Add 0.1% soya-bean oil as defoamer; The fermentation termination time is 60 hours; Fermention medium: often liter of fermented liquid Semen Maydis powder 30g, soybean cake powder 15g, peptone 3g, glucose 30g, carbon-nitrogen ratio is 19:1.
A selection for rainbow conk liquid fermenting high polysaccharide bacterial strain, is characterized in that: comprise the following steps:
(1) select multiple different rainbow conk to produce bacteria strain, carry out the liquid fermenting test of rainbow conk, select the high yield starting strain of the highest rainbow conk bacterial strain of prepared from coriolus versicolor mycelium polysaccharide yield as mutagenesis;
(2) starting strain utilizes protoplastis to prepare technology, carries out the preparation of rainbow conk protoplastis;
(3) protoplastis of rainbow conk starting strain is as low energy N
+the material of ion implantation mutagenesis: by the preparation of rainbow conk protoplastis, and make rainbow conk protoplastis reach the suspension of 106/ml, draws 0.1ml and coats in blank culture dish, air-dry, carry out low energy N
+ion implantation mutagenesis;
(4) N
+ion implantation mutagenesis: carry out N+ ion implantation mutagenesis to the material that step (3) is coated in culture dish, then uses 1ml0.6mol/L sucrose homeo-osmosis agent wash-out, and absorption 0.1ml elutriant is coated on regeneration plating medium and cultivated;
(5) screening of mutagenic strain: by the 0.6mol/L sucrose homeo-osmosis agent wash-out of the flat board after ion beam mutation, coat on regeneration culture medium flat board, after bacterium colony grows up to, select mycelia sturdy dense, grow mycelia faster and carry out liquid fermenting test, mycelium polysaccharides is measured, select the rainbow conk bacterial strain that mycelium polysaccharides content is higher than starting strain, screening and culturing is carried out to it, finally obtain the rainbow conk new strains of a strain liquid fermenting high polysaccharide;
(6) stabilization characteristics of genetics checking: by bacterial strain continuous passage 20 generation after above-mentioned mutagenesis screening, then carry out liquid fermenting test, stable yield, proterties are unconverted to be finally defined as the production of upper new mutagenesis rainbow conk liquid fermenting engineering strain.
Described N
+ion implanting conditions is Implantation Energy is 20KeV, and implantation dosage is 9 × 1014ions/cm
2, target chamber vacuum tightness is 10-3pa, injects, be spaced apart 15s with 5s pulsed.
Regeneration culture medium is: maltose 5 grams, glucose 10 grams, yeast powder 4 grams, 0.6mol/L N.F,USP MANNITOL, and 5.5 grams, agar, pH nature, it is 1000mL that distilled water is settled to cumulative volume.
Advantage of the present invention and positively effect are:
1, the breeding of the edible fungus species such as rainbow conk adopts the means such as cross-breeding and UV mutation to carry out usually in the past, the shortcomings such as efficiency of inducing mutation is low, easily cause negative sudden change, inherited character is unstable, and the mutagenic and breeding by adopting the edible mushrooms protoplastiss such as ion implantation technique mutagenesis rainbow conk to carry out bacterial strain, improve the efficiency of mutagenesis and the probability of gain mutant, and the rainbow conk bacterial strain after mutagenesis is not easily degenerated.Adopt mutagenesis of the present invention to go out the rainbow conk mutagenic strain of a strain prepared from coriolus versicolor mycelium polysaccharide yield higher than former starting strain, the output that improve krestin can create good economic benefit.
2, the present invention is by the research to the best optimal culture condition of the rainbow conk new strains of high polysaccharide and optimal liquid fermentation parameter, with tunning polysaccharide for index, the best fermentation parameter screening applicable high polysaccharide new strains is carried out from the nutrition, pH, fermentation time etc. of substratum, from shake-flask culture to the different fermentations parameter of factory's fermentative production, improve the yield of factorial praluction krestin, for factorial praluction krestin provides ripe technique.
3, the rainbow conk new strains of a strain liquid fermenting high polysaccharide is obtained by low energy N+ implantation mutagenesis, make fermentation krestin output higher than high yield starting strain 50% to the research of new strains optimal medium and liquid parameter, often liter of liquid fermenting amount mycelium polysaccharides reaches 2.26 grams.
Below in conjunction with embodiment, present invention process is described further.
The method of low energy N+ implantation mutagenic and breeding disease-resistant high yield rainbow conk bacterial strain and the bacterial strain of institute's seed selection, deposit number is CGMCCNo.10903; Classification And Nomenclature: rainbow conk, Latin name: Coriolusversicolor, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date on 06 11st, 2015.
Embodiment
The processing method of mutagenesis screening rainbow conk liquid fermenting high polysaccharide new strains, the steps include:
1, high yield prepared from coriolus versicolor mycelium polysaccharide starting strain screening
From rainbow conk bacterial classification SYZ1, SYZ2, SYZ3, SYZ4, SYZ5, SYZ6, SYZ7, SYZ8 of domestic introduction different varieties.Bacterial strain is respectively from the Chinese Academy of Agricultural Sciences, and northeast, and the bacterial strain of this institute preservation, be inoculated on PDA slant medium, and 28 DEG C of lucifuges are cultivated and activated for 7 days.By the inoculation of activation in the central authorities that PDA culture medium culturing ware is housed, the time of record rainbow conk mycelium germination, measure its speed of growth, observe the form (mycelia thickness, dense) of mycelia.Then the test of rainbow conk liquid fermenting is carried out, quantitatively to be seeded to by punching in liquid nutrient medium (culture medium prescription see under), 150 revs/min of rotary shaker, 28 DEG C of lucifuges cultivate 7 days, 4000 revs/min centrifugal 5 minutes, collect mycelium, dry to constant weight, measure its quality and obtain rainbow conk liquid fermenting biomass, carry out prepared from coriolus versicolor mycelium polysaccharide simultaneously and measure for 65 DEG C.Using prepared from coriolus versicolor mycelium polysaccharide yield soprano as krestin high yield starting strain.Test finally to be determined using SYZ2 from a strain rainbow conk bacterial strain of the Chinese Academy of Agricultural Sciences as the high yield starting strain of mutagenesis.(see table 1)
Producing Strain mycelium polysaccharide starting strain screening (table 1)
2, low energy N
+ion implantation mutagenesis
(1) sample pre-treatments: starting strain SYZ2 cultivates the mycelia of 5 days, with 2% lywallzyme enzymolysis 5 hours at 30 DEG C, filters enzymolysis solutions with 8 layers of lens wiping paper, with homeo-osmosis agent 8ml gradation washing and filtering altogether, gets filtered liquid.Centrifugal 10 minutes of 4000r/min, removes supernatant liquor, with homeo-osmosis agent 8ml centrifuge washing, obtains the protoplastis of purifying.Protoplastis homeo-osmosis agent 10ml is diluted use, makes protoplast suspension, count with blood counting chamber.Refining protoplast suspension is diluted to 10
6individual/ml.Getting 0.1ml coats in culture dish, and sterile wind is air-dry, and it is ion implantation to carry out low energy N+ immediately.
(2) ion implanting conditions: Implantation Energy is 20KeV, implantation dosage is 9.00 × 10
14ionscm
-2target chamber vacuum tightness is 10
-3pa, injects with 5s pulsed, is spaced apart 15s.(the best ion injection condition that this condition filters out through research in province's science and technology support project " using the multi-stage cross coupling method seed selection rainbow conk disease-resistant high yield new strains such as induced mutations breeding " for my research team)
3, the screening of prepared from coriolus versicolor mycelium polysaccharide superior strain
(1) primary dcreening operation: by the 0.6mol/L sucrose homeo-osmosis agent wash-out of the flat board after ion beam mutation, getting 0.1ml coats on regeneration culture medium flat board, after bacterium colony grows up to, select the bacterial strain having obvious antagonism between mycelium, pick out altogether 96 strains.Then proceed on PDA culture medium flat plate and carry out antagonistic effect with starting strain SYZ2, filtering out 5 strains altogether has obvious antagonistic strain.5 strain bacterial strains are SYZ203, SYZ209, SYZ225, SYZ261, SYZ285; (2) multiple sieve: then by the bacterial strain rainbow conk liquid fermenting test after primary dcreening operation, select mycelium polysaccharides output higher than the bacterial strain of control strain as the high polysaccharide bacterial strain of mutagenesis, the rainbow conk new strains SYZ209 finally obtaining a plant height product mycelium polysaccharides is numbered SYZ9, exceed 35.9% than starting strain SYZ2, finally carry out isozyme electrophoresis test and determine that SYZ9 is high yield mycelia polysaccharide new strains.(see table 2)
Table 2:5 strain dissociant with to impinging upon PD solution culture fermentation information slip
4, genetic stability checking
L, the bacterial strain obtained is carried out 20 times go down to posterity after mutagenesis screening, and then carry out rainbow conk experiment in cultivation, compare with the first-generation, select mycelium polysaccharides stable yield (namely each generation bacterial strain output between there is not significant difference, statistical analysis, variance p>O.05), proterties is unconverted as final rainbow conk mutagenicity high-yield bacterial strain.Finishing screen chooses a strain disease-resistant high yield rainbow conk mutagenic strain, higher by 35.9% than the former high yield producing strain that sets out, and stabilization characteristics of genetics, deposit number is CGMCCNo.10903.
5, the research of the best optimal culture condition of the rainbow conk new strains of high polysaccharide and optimal liquid fermentation parameter.
With tunning mycelium polysaccharides for index, the best fermentation parameter screening applicable high polysaccharide new strains is carried out from the nutrition, PH, fermentation time etc. of substratum, from shake-flask culture to the different fermentations parameter of factory's fermentative production, improve the yield of factorial praluction krestin, for factorial praluction krestin provides ripe technique.
1. the screening of high polysaccharide optimal medium formula
(determination of the suitableeest carbon source, the determination of the suitableeest nitrogenous source, carbon-nitrogen ratio (C/N) measure the impact of fermenting, pH)
Study a lot at the research researcher of the suitableeest carbon source of rainbow conk liquid fermenting, nitrogenous source, carbon-nitrogen ratio (C/N), and work out the carbon source of rainbow conk liquid fermenting the best, nitrogenous source, carbon-nitrogen ratio (C/N), this research team uses the liquid fermentation medium formula worked out in edible medicinal fungus liquid fermenting for many years as basic fermention medium (glucose 20g, Semen Maydis powder 20g, soybean cake powder 10g, KH
2pO
410g, MgSO
45g, VB
l100mg, H
2o1000ml) screen about 20 with C/N in the research method of rainbow conk liquid fermenting in conjunction with more domestic researchers, carry out research screening with tunning mycelium production, polysaccharide for index and draw one group of rainbow conk new strains optimal medium being applicable to high polysaccharide.Optimal medium is: (glucose 30g, Semen Maydis powder 30g, soybean cake powder 15g, peptone 3 grams, KH
2pO
410g, MgSO
45g, VB
l100mg, H
2o1000ml) in table 3.
Table 3:SYZ9 liquid fermenting inoculum size 8%, 140 revs/min of rotary shaker 28 DEG C of lucifuges cultivate 6 days
Optimal medium is: glucose 30g, Semen Maydis powder 30g, soybean cake powder 15g, peptone 3 grams, KH
2pO
410g, MgSO
45g, VB
l100mg, H
2o1000ml, C:N are 19:1.
2. from laboratory to the research of the different parameters change factorial praluction process
(with biomass, polysaccharide yield, the shortest fermentation time for factorial praluction parameter)
Laboratory shake flask liquid fermenting is tested
Laboratory shake flask liquid fermenting is tested: new strains SYZ9 take optimal medium as formula, culture temperature 28 DEG C, shaking speed 150 revs/min, inoculum size 8%.The test of shaking flask liquid fermenting finds when pH value is from low to high rising and then declining, and is exactly best fermentation time point when reaching the highest.In whole fermenting process, reducing sugar is in reduction, does not substantially have too large change to reducing sugar during fermentation ends.Laboratory shake flask liquid fermenting test Best Times is 6 days, and fermentation end products, prepared from coriolus versicolor mycelium dry weight 28.42g, mycelium polysaccharides content 8.6%, often liter of fermented product can produce prepared from coriolus versicolor mycelium polysaccharide 2.41g.
The fermentation test of batch production 100L fermentor liquid
Note: due to the existence of W-Gum when fermentation does not complete, when using anthrone-sulphuric acid method to detect, polysaccharide numerical value is higher.
Batch production 100 liters of liquid fermentation tank tests: new strains SYZ9 take optimal medium as formula, fermentor tank (ventilation adds stirring) volume 100L, liquid amount 60L, mixing speed 150r/win, air flow 1:0.5-1, pressure differential method is inoculated, inoculum size is cultivate under 8%, 27 ~ 28 DEG C of conditions.Add 0.1% soya-bean oil as defoamer.Whole fermenting process finds to test with laboratory shake flask liquid fermenting to have consistence, 60 hours ferment tank time is best, fermentation end products, prepared from coriolus versicolor mycelium dry weight 26.58g, mycelium polysaccharides content 8.5%, often liter of fermented product can produce prepared from coriolus versicolor mycelium polysaccharide 2.26g.
6, by low energy N
+inject the rainbow conk new strains that mutagenesis obtains a strain liquid fermenting high polysaccharide, make fermentation krestin output higher than high yield starting strain 50% to the research of new strains optimal medium and liquid parameter, often liter of liquid fermenting amount mycelium polysaccharides reaches 2.26 grams.
Polysaccharide detection method: anthrone-sulphuric acid method measures
Reducing sugar detection method: DNS method measures
Culture medium prescription:
Regeneration culture medium: RM4 regeneration culture medium: maltose 5 grams, glucose 10 grams, yeast powder 4 grams, 0.6mol/L N.F,USP MANNITOL, 5.5 grams, agar, PH nature, it is 1000mL that distilled water is settled to cumulative volume.
PD liquid nutrient medium: potato 200g liquor, glucose 20g, KH
2pO
410g, MgS ()
45g, H
2o1000m1.
Basic fermention medium: glucose 20g, Semen Maydis powder 20g, soybean cake powder 10g, KH
2pO
410g, MgSO
45g, VB
l100mg, H
2o1000ml.
Claims (5)
1. a rainbow conk liquid fermenting high polysaccharide bacterial strain, is characterized in that: deposit number is CGMCCNo.10903; Classification And Nomenclature: rainbow conk, Latin name: Coriolusversicolor.
2. rainbow conk liquid fermenting high polysaccharide bacterial strain according to claim 1, is characterized in that: fermentation condition is: fermenter volume 100L, liquid amount 60L, mixing speed 150r/win, air flow 1:0.5-1, pressure differential method is inoculated, inoculum size is cultivate under 8%, 27 ~ 28 DEG C of conditions; Add 0.1% soya-bean oil as defoamer; The fermentation termination time is 60 hours; Fermention medium: often liter of fermented liquid Semen Maydis powder 30g, soybean cake powder 15g, peptone 3g, glucose 30g, carbon-nitrogen ratio is 19:1.
3. a selection for rainbow conk liquid fermenting high polysaccharide bacterial strain according to claim 1, is characterized in that: comprise the following steps:
(1) select multiple different rainbow conk to produce bacteria strain, carry out the liquid fermenting test of rainbow conk, select the high yield starting strain of the highest rainbow conk bacterial strain of prepared from coriolus versicolor mycelium polysaccharide yield as mutagenesis;
(2) starting strain utilizes protoplastis to prepare technology, carries out the preparation of rainbow conk protoplastis;
(3) protoplastis of rainbow conk starting strain is as low energy N
+the material of ion implantation mutagenesis: by the preparation of rainbow conk protoplastis, and make rainbow conk protoplastis reach the suspension of 106/ml, draws 0.1ml and coats in blank culture dish, air-dry, carry out low energy N
+ion implantation mutagenesis;
(4) N
+ion implantation mutagenesis: carry out N+ ion implantation mutagenesis to the material that step (3) is coated in culture dish, then uses 1mlO.6mol/L sucrose homeo-osmosis agent wash-out, and absorption 0.1ml elutriant is coated on regeneration plating medium and cultivated;
(5) screening of mutagenic strain: by the O.6mol/L sucrose homeo-osmosis agent wash-out of the flat board after ion beam mutation, coat on regeneration culture medium flat board, after bacterium colony grows up to, select mycelia sturdy dense, grow mycelia faster and carry out liquid fermenting test, mycelium polysaccharides is measured, select the rainbow conk bacterial strain that mycelium polysaccharides content is higher than starting strain, screening and culturing is carried out to it, finally obtain the rainbow conk new strains of a strain liquid fermenting high polysaccharide;
(6) stabilization characteristics of genetics checking: by bacterial strain continuous passage 20 generation after above-mentioned mutagenesis screening, then carry out liquid fermenting test, stable yield, proterties are unconverted to be finally defined as the production of upper new mutagenesis rainbow conk liquid fermenting engineering strain.
4. the selection of rainbow conk liquid fermenting high polysaccharide bacterial strain according to claim 3, is characterized in that: described N
+ion implanting conditions is Implantation Energy is 20KeV, and implantation dosage is 9 × 1014ions/cm
2, target chamber vacuum tightness is 10-3pa, injects, be spaced apart l5s with 5s pulsed.
5. the selection of rainbow conk liquid fermenting high polysaccharide bacterial strain according to claim 3, is characterized in that: regeneration culture medium is: maltose 5 grams, glucose 10 grams, yeast powder 4 grams, 0.6mol/L N.F,USP MANNITOL, 5.5 grams, agar, pH nature, it is l000mL that distilled water is settled to cumulative volume.
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