CN1259414C - High proportion psendomonas syringae production strain and its fermentation method to produce psendomonas syringae - Google Patents
High proportion psendomonas syringae production strain and its fermentation method to produce psendomonas syringae Download PDFInfo
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Abstract
The present invention provides a novel strain for fermenting and producing high-component crown bacterin, namely a pseudomonas syringae Y-2018 strain, and a preservation number is CGMCC NO. 1105. The strain has no need to be cultivated at low temperature, and overcomes the weakness that a wild strain can accumulate the crown bacterin at low temperature. The present invention also provides a method for industrially producing the crown bacterin, which comprises the steps that the pseudomonas syringae Y-2018 strain is prepared from bevel strains through seed culture in a shake flask, aeration and fermentation to obtain fermentation liquid which comprises the crown bacterin; the fermentation liquid is concentrated, extracted, crystallized and recrystallized to obtain crystalline powder and noncrystallizable mother liquid. The method can use an organic nitrogen culture medium to fermentation, and organic nitrogen not only can be used for adding bacterin quantity, but also can synthesize a large amount of crown bacterin. Consequently, the method overcomes the defects that the wild strain can not resist the organic nitrogen, and the accumulation of the crown bacterin is inhibited by the organic nitrogen.
Description
Technical field
The present invention relates to the field of microorganism and fermentative production compound thereof.Specifically, relate to the new bacterial strain that is used for the fermentative production psendomonas syringae, and utilize this strain fermentation to produce the method for psendomonas syringae.
Background technology
Psendomonas syringae is the Chinese that renames, the nineties in last century, China phytopathologist claimed once that it was the hat toxin, it is now know that psendomonas syringae is except there being the toxin one side to plant, also having prior one side is that plant is had high physiologically active, and it is more suitable therefore to be called psendomonas syringae.English coronatine by name.Ichikara in 1977 etc. find the deep red red pathogenic mutation of pseudomonas syringae (Psendomonas syingae pv.alropurea), can cause the Lolium multiflorum spot that fades, the reason that causes this spot that fades is due to a kind of compound of producing of this bacterium, Ichihara is with this compound called after coronatine, this is because the old name of this bacterium is called Ps.corofaciens var.aropurpurea, gets the prefix of its kind name and forms.Also had many people in the similar scab of different plants, to isolate pathogenic bacteria afterwards, also produced coronatine.
Since nineteen ninety-five, Mittal etc. find psendomonas syringae and dormin and jasmonic, especially with jasmonic similar physiological function are arranged, but its activity almost is 100-1000 times of jasmonic, the cotton test that the psendomonas syringae of producing with us is done is an example, and psendomonas syringae is made into 1ppm-10
-6The solution soaking cotton seeds of ppm, the main root length of mensuration seed germination, hypocotyl length, lateral root number, percentage of germination etc. find 10 after 9 days
-2The concentration of ppm still has restraining effect, only 10
-4The concentration of ppm just shows and promotes the growth effect.Pertinent data shows that psendomonas syringae also makes the amount of the anticarcinogen-taxol in the Ramulus et folium taxi cuspidatae improve 10 times, and it is better than jasmonic as herbicidal activity, might become the leading compound of new herbicides.Other and the similar function of jasmonic as promoting phytosynthesis ethene, promote and remove dormancy, organ differentiation, adventive root formation, blade and fruit abscission, induce flower formation or the like that activity all is higher than jasmonic.Because psendomonas syringae has efficiently the plant growth regulating function, to person poultry harmless, free from environmental pollution, be expected to replace the preparation of the same type of present chemosynthesis, be the green product of significant from now on.
The structure of psendomonas syringae such as Fig. 1, it has a hat bacterium acid (coronafacine acid) that contains an amino acid whose hat alkanoic acid (coronamine acid) and a polyketone structure to form, and two components connect with amido linkage.An activity is also had in independent hat bacterium acid, but does not have psendomonas syringae active high.
Though psendomonas syringae is had some scattered researchs, but there is not the psendomonas syringae product in the world, this be since the amount that the wild strain of occurring in nature produces psendomonas syringae seldom, totally 200 milligrams of the psendomonas syringae that the former Di Min in city of Hokkaido, Japan university etc. obtains from 250 liters of fermented liquids, hat bacterium acid and hat alkanoic acids, wherein psendomonas syringae has only 60 milligrams.Therefore the former Di Min in city thinks that psendomonas syringae output is so low, can not pass through fermentative Production.The method of (1998) chemosynthesis such as the former Di Min in city is successful, probably need the reaction of 15 steps, but expense is very high, is unsuitable for suitability for industrialized production.
The present invention adopts nature screening, and means such as induced mutations have obtained can the ferment microorganism strains of generation high component psendomonas syringae of a strain, utilizes this bacterial strain can the high-load psendomonas syringae of fermentative production, has realized the suitability for industrialized production of psendomonas syringae.
Summary of the invention
The invention provides new the fermenting of a strain and produce the bacterial strain of high component psendomonas syringae, i.e. pseudomonas syringae (Psendomonas syringae) Y-2018 bacterial strain.Pseudomonas syringae Y-2018 is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on March 5th, 2004, and preserving number is CGMCC NO.1105.
The present invention also provides a kind of fermentation and extracting method of suitability for industrialized production psendomonas syringae, this method comprises cultivates pseudomonas syringae Y-2018 bacterial strain through slant strains, shake-flask seed is cultivated, and fermentation culture obtains to contain the fermented liquid of psendomonas syringae, through concentrated, solvent extraction, decolouring, concentrated extract, obtain extracting solution through silica gel column chromatography (or without silica gel column chromatography) again, make its crystallization, recrystallization obtains crystalline flour and non crystallized mother liquor.
One. strain selection
Fs:
At first from the wild pseudomonas of 27 strains, screen, obtain a strain and produce 011 higher bacterial strain of psendomonas syringae, improve 39% No. 156 bacterial strains than starting strain output through the mutagenic obtained strain of ultraviolet ray repeatedly etc.
Subordinate phase:
Will be than 156 bacterial strains of high yield through mutagenesis such as nitrosoguanidines, do not need to obtain 343 and 422 bacterial strains through low temperature fermentation.The mutant strain that obtains can be cultivated down at 26 ℃.Because low temperature is cultivated, need to use a large amount of cold water coolings, and mutant strain has overcome the weakness that wild strain must low temperature could accumulate psendomonas syringae, makes normal fermentation process become possibility, saves a large amount of waters and power consumption.
Phase III:
To not need cryogenic 343 bacterial strains again through repeatedly mutagenesis repeatedly such as ultraviolet ray and nitrosoguanidines, and obtain a strain and neither needed low temperature 774 bacterial strains of ability organonitrogen again.Because use organonitrogen can make thalli growth good, thus synthetic more psendomonas syringae, but the not anti-organonitrogen of wild strain, no matter supply organonitrogen early stage, or the middle and later periods supply, all can suppress the accumulation of psendomonas syringae.Overcome this defective by mutagenesis, the bacterium amount is increased, also made the psendomonas syringae accumulation become possibility.And then by the mutagenesis repeatedly of nitrosoguanidine and laser etc., obtain the Y-2018 bacterial strain, original production is brought up to more than the 40ug/mL less than 10ug/mL.
Pseudomonas syringae Y-2018 bacterial strain has nothing different with wild bacterium on bacterium colony and thalli morphology, bacterium colony is thin oyster white, and is little and smooth, presenting yellow-green fluorescence under the rayed; Thalline is a dialister bacterium, the blunt circle in two ends.Not existing together of Y-2018 and wild bacterium is that this mutant strain has changed the requirement of ecological condition and makes the output of present psendomonas syringae improve 4.3 times.
Two. the fermentative production of psendomonas syringae
1. the preparation of slant strains
Get the Y-2018 inoculation to slant medium, cultivated 2 days for 26～28 ℃.
The slant medium component is: glucose 1g, and beef extract 0.3g, peptone 0.5g, agar 1.5g, water 100mL, pH7.4 sterilized 30 minutes for 121 ℃.
2. culture of seed liquid
Make the bottled 100mL seed culture medium of 500mL taper, medium component is: white sugar 1-2g, corn steep liquor 2g, ammonium sulfate 0.1g, yeast extract paste 0.1g, dipotassium hydrogen phosphate 0.15g, sal epsom 0.08g, water 100mL, pH7.4,121 sterilizations 30 minutes.
Insert slant strains, 26～28 ℃ of shaking tables were cultivated 24 hours, and revolution shaking table speed is 160r/min.
The enlarged culturing of seed liquor, for example 3000mL Erlenmeyer flask or seeding tank can be undertaken by similar approach.Seeding tank need add the bubble enemy of 0.03-0.09%.
3. fermentation culture
Successively used two kinds of fermention mediums:
A substratum (not containing organonitrogen):
White sugar 1.5-5.0g, ammonium chloride 0.1-0.3g (or ammonium sulfate 0.12-0.36g), saltpetre 0.01-0.05g, dipotassium hydrogen phosphate 0.1-0.5g, potassium primary phosphate 0.10-0.40g, water 100mL, pH7.0-7.6.
No. two substratum (containing organonitrogen):
White sugar 1.5-4.5g, corn steep liquor 2-5g, soybean cake powder 0.6-1.5g, ammonium sulfate 0.04-0.2g (or ammonium chloride 0.03-0.18g), saltpetre 0.01-0.3g, dipotassium hydrogen phosphate 0.05-0.2g, water 100mL, pH7.0-7.4.
Seed liquor is inserted respectively in above two kinds of fermention mediums with 2% inoculum size.Enlarged culturing comprises fermentor cultivation, can be undertaken by similar approach, and fermentor cultivation need add 0.03-0.09% bubble enemy.No matter shaking table or fermentor tank, fermentation period were about 96 hours.Two kinds of substratum all can be 26～28 ℃ and 18 ℃ of cultivations, and 18 ℃ are suitable for wild bacteria strain, and 26～28 ℃ are suitable for mutant strain.The high pressure liquid chromatography external standard method is all used in fermenting process and end, converses their output according to the psendomonas syringae peak area.
4. psendomonas syringae extracts:
Near exhausting, psendomonas syringae has accumulated the highest, is called maturing fermentation liquid for the cultivation of fermented liquid through about 96 hours, nutritive ingredient, should finish fermentation and extract.Psendomonas syringae extract mainly comprise acidifying, macroporous resin adsorption, wash-out, concentrate eluant, solvent extraction, extraction liquid decolouring, concentrate, column chromatography, extracting solution condensing crystal etc.
Maturing fermentation liquid can be used sulfuric acid, hydrochloric acid, phosphoric acid etc. are regulated fermented liquid to pH5-2, filtration sterilization body (or the do not remove thalline) macroporous resin adsorption of flowing through post then, remove large quantity of moisture, mineral substance, residual sugar, protein and most of pigment, with 75-80% acetone wash-out by the psendomonas syringae of resin absorption, after reclaiming acetone, the concentrated elutriant acid adjustment more than 20 times is to pH5-2, carry out solvent (ethyl acetate, butylacetate etc.) extraction, the extraction liquid decolouring, reclaim solvent and become spissated extraction liquid, behind the column chromatography or post is not analysed and is allowed its spontaneous nucleation (can carry out recrystallization in case of necessity), obtain crystalline flour and non crystallized mother liquor, measure psendomonas syringae content in crystalline flour purity and the mother liquor with high performance liquid chromatography.
Beneficial effect of the present invention:
The mutant strain that the present invention obtains has higher psendomonas syringae output, and this bacterial strain does not need low temperature to cultivate, overcome the weakness that wild strain must low temperature could accumulate psendomonas syringae, saved a large amount of waters and the power consumption that have been used to lower the temperature when low temperature is cultivated, made the normal fermentation process become possibility.In addition, mutant strain can use the organonitrogen substratum to ferment, and use organonitrogen that the bacterium amount is increased, and can synthesize more psendomonas syringae, but the not anti-organonitrogen of wild strain, organonitrogen suppresses the accumulation of psendomonas syringae.
Description of drawings
Fig. 1 psendomonas syringae structure.Be respectively from left to right: psendomonas syringae, hat alkanoic acid and the acid of hat bacterium.
The outer mapping of Fig. 2 liquid spectrum is produced method mode chart (the peak area figure that Fig. 2-1 draws for the standard specimen concentration known, Fig. 2-2 surveys peak area figure for sample and ask concentration from standard specimen).
The gas spectrogram of Fig. 3 psendomonas syringae (upward be standard, be sample down).
The mass spectrum of Fig. 4 psendomonas syringae (left side is a standard, and the right side is a sample).
The nucleus magnetic resonance figure of Fig. 5 psendomonas syringae (the critical function group that psendomonas syringae is described conforms to chemical structural drawing).
Fig. 6 potato block method (middle for handling, about be contrast).
The big bean seedlings method of Fig. 7 (Fig. 7-1 left side is short in the contrast seedling on the right side for handling seedling, and Fig. 7-2 handles on a left side seedling and macula lutea occurs).
Embodiment
Embodiment one
Get Y-2018 bacterial strain and starting strain 011 slant strains, insert on the slant medium, 26～28 ℃ cultivate 2 days stand-by.Slant medium is: glucose 1g, and beef extract 0.3g, peptone 0.5g, agar 1.5g, water 100mL, pH7.4 sterilized 30 minutes for 121 ℃.
Make 2 bottles of the bottled 100ml seed culture mediums of 500mL taper, a bottle graft is gone into the Y-2018 bacterial strain, and a bottle graft is gone into 011,26 ℃ of shaking table cultivation of starting strain 24 hours, and revolution shaking table speed is 160r/min, and it is stand-by that shake-flask seed is grown good back.Seed culture medium is: white sugar 1g, corn steep liquor 2g, ammonium sulfate 0.1g, yeast extract paste 0.1g, dipotassium hydrogen phosphate 0.15g, sal epsom 0.08g, water 100mL, pH7.4,121 sterilizations 30 minutes.
Make two kinds of fermention mediums, the one, do not contain No. 1 substratum of organonitrogen, filling a prescription is: white sugar 1.5g, ammonium chloride 0.1g, saltpetre 0.03g, dipotassium hydrogen phosphate 0.36g, potassium primary phosphate 0.40g, water 100mL, pH7.0-7.6.
The 2nd, contain No. 2 substratum of organonitrogen, filling a prescription is: white sugar 2.5g, corn steep liquor 3g, soybean cake powder 0.6g, ammonium sulfate 0.2g, saltpetre 0.05g, dipotassium hydrogen phosphate 0.2g, water 100mL, pH7.0-7.4.
Two kinds of fermention mediums each 12 bottles (the bottled 100mL of 500mL taper), every kind long good seed culture fluid, inoculum size by 2% inserts two kinds of fermention mediums, be the Y-2018 bacterial strain connect No. 16 bottles, connect No. 26 bottles, 011 bacterial strain connect No. 16 bottles, No. 26 bottles, two kinds of fermention medium branches are put 26 ℃ and 18 ℃ of shaking tables then, under identical rotating speed, cultivated 96 hours, and took out and to use their output (see figure 2) of high performance liquid chromatography external standard method respectively, 3 bottles of multiple average results such as following table (unit is ug/mL):
| Bacterial strain number | 26℃ | 18℃ | ||
| No. 1 substratum | No. 2 substratum | No. 1 substratum | No. 2 substratum | |
| Y-2018 | 38.77 | 43.81 | 38.94 | 44.3 |
| 011 | 0.57 | 0.22 | 10.18 | 4.36 |
Broken away from must be 18 ℃ envrionment conditions for mutant strain Y-2018 as can be seen from the above table, and on the substratum that organonitrogen exists, improve than the substratum that does not have organonitrogen is existing, show the potentiality that continue raising are arranged, starting strain 011 necessary low temperature, and the organonitrogen substratum will greatly reduce the accumulation of psendomonas syringae.
Embodiment two
This prescription of testing used substratum is identical with embodiment one.
The Y-2018 bacterial strain is inserted on the slant medium at the bacterial classification that 4 ℃ of refrigerators are preserved, cultivated 48 hours, and made its activation for 28 ℃.Then connect two collariums in the seed culture medium of the bottled 100mL of 500mL taper, inoculate 3 bottles, 26 ℃ of shaking tables were cultivated 24 hours.After seed is grown well, insert in the fermention medium (No. 2 substratum) of the bottled 100mL of 500mL taper, inoculum size 2%, totally 100 bottles, 28 ℃ of shaking tables were cultivated 4 days, merged 100 bottles and got the 10L fermented liquid, measured the psendomonas syringae peak area with high performance liquid chromatography, obtaining psendomonas syringae concentration as embodiment 1 from standard specimen is 36ug/mL, and 10L psendomonas syringae total amount is 360mg.Fermented liquid is transferred to pH3 to be filtered, flow through macroporous resin (the Yangzhou pharmaceutical factory of 400mL volume then, model YPR-II) post, psendomonas syringae is adsorbed on the resin, water cleans the impurity in the resin gap, use 75-80% acetone wash-out then, reclaim acetone, the about 300mL of the aqueous solution that stays transfers pH 3 back 400mL ethyl acetate extractions, activated carbon decolorizing, reclaim ethyl acetate, stay spissated extraction liquid 10mL, admix silica gel, oven dry, install on the silicagel column, use the trichloromethane methanol-eluted fractions, collection contains that part of solvent of psendomonas syringae, boils off organic solvent and obtains extracting solution, because the psendomonas syringae amount is few, impurity is many, can't crystallization, have only the 1.7mL mother liquor, get the 0.1mL mother liquor and be diluted to 100mL with methyl alcohol, high performance liquid chromatography records peak area, obtaining psendomonas syringae concentration as embodiment 1 identical way is 168ug/mL, and the 10L fermented liquid obtains psendomonas syringae 168mg, and yield is 47%.
Embodiment three
Press the seed culture method of embodiment two, obtain 3 bottles in the seed (the bottled 100mL of 500mL taper) of logarithmic growth, make 3 bottles of the bottled 1000mL seed culture mediums of 3000mL taper then, respectively 100mL grown good seed and insert, 26 ℃ of shaking tables cultivate 18-20 hour stand-by.Then make the fermention medium (i.e. No. 2 substratum) of fermentor tank, and add 0.03% bubble enemy, the 200L fermentor tank prepares the 150L fermentation culture, be cooled to 28 ℃ through sterilization, the 1000mL that inserts 3 bottles of 3000mL dresses grows good seed, cultivates 4 days for 26 ℃, and ventilation 0-16 hour is 0.8v.v.m, 17-80 hour is 1.2v.v.m, and 81-96 hour is 0.9v.v.m.Maturing fermentation liquid is through high-performance liquid chromatogram determination, and obtaining psendomonas syringae content with embodiment 1 method is 41.42ug/mL, and the 150L total content is 6.213g.Fermented liquid is transferred pH3, and through the absorption of 7.0kg macroporous resin (factory, model are with embodiment 2) post, 14L80% acetone wash-out gets elutriant 15L, so just makes fermented liquid concentrate 10 times.Reclaim acetone then, stay and contain the about 3L of the psendomonas syringae aqueous solution, transfer pH3 after, ethyl acetate extraction 3 times, obtain extraction liquid 3.6L, use the 30g activated carbon decolorizing, reclaim ethyl acetate again, obtain the about 50mL of brown concentrated extract, volatilization at normal temperatures makes its crystallization, isolates mother liquor.Coarse crystallization makes its crystallization again with acetic acid ethyl dissolution, evaporation again, obtain crystalline flour 1.54g, crystalline flour is through online detection of gas chromatography/mass spectrometry and magnetic resonance detection, molecular weight conform to fully with chemical structure (seeing Fig. 3,4,5), and to record relative purity be 94%, 1.54g pure psendomonas syringae is 1.45g in the crystalline flour, accounts for 23.3% of total amount.Stay non crystallized mother liquor 18mL, get 500 times of 0.1mL dilutions, it is 187.6ug/mL that high performance liquid chromatography is measured concentration, extrapolates to contain psendomonas syringae 1.69g in the 18mL mother liquor, accounts for total amount 27.2%, psendomonas syringae addition in crystalline flour and the mother liquor, and yield is 50.5%.
Embodiment four
Biological assay is one of qualitative detection of psendomonas syringae, also is crystallization and mother liquor are confirmed again.
One. the potato block method:
Psendomonas syringae is mixed with 10
-15-10
-21Solution, be applied to 1cm
3The charlotte piece on, will scribble the charlotte piece of various concentration, be placed in the incubator that 25 ℃ of dark preserve moisture 2-3 days, take out and check, scribbling 10
-18-10
-21On the potato block of concentration psendomonas syringae, can find that there is outgrowth irregular projection on the surface, only not be coated with the charlotte piece of clear water and concentration greater than 10 and be not coated with psendomonas syringae
-18The charlotte piece, then still maintain the original state or rot, show that psendomonas syringae concentration has restraining effect when big, only be diluted to behind the finite concentration just tool stimulating growth effect (see figure 6).
Two. big bean seedlings method
Planting soybean in the flowerpot in advance, it is long to 10cm left and right sides Gao Shi to treat that soybean emerges, and will make to be higher than 10
-6The psendomonas syringae of concentration is applied on the leaf that sassafras hinders slightly, or from vein injection psendomonas syringae solution, allow the bean seedlings continued growth, can find to be coated with or to inject the bean seedlings of psendomonas syringae solution after one week, growth seriously is obstructed, the bean seedlings comparison is not obviously downgraded according to (not being coated with the seedling of psendomonas syringae solution), also occurs macula lutea on the blade face.The proof psendomonas syringae has the growth of inhibition effect (see figure 7).
Claims (2)
1. a strain has the microorganism strains that is used to produce high component psendomonas syringae that adapts to comparatively high temps and the fermentation of anti-organonitrogen, and it is pseudomonas syringae (Psendomonas syringae) Y-2018 CGMCC NO.1105 bacterial strain.
2. the described microorganism strains of claim 1 is produced the method for psendomonas syringae, described bacterial strain is cultivated through slant strains, shake-flask seed is cultivated and aerobic fermentation, acquisition contains the fermented liquid of psendomonas syringae, obtain extracting solution through concentrated, extraction, chromatography, make its crystallization, recrystallization obtains crystalline flour and non crystallized mother liquor; Wherein fermention medium is for containing the organonitrogen substratum, and filling a prescription is: white sugar 1.5-4.5g, corn steep liquor 2-5g, soybean cake powder 0.6-1.5g, ammonium sulfate 0.04-0.2g or ammonium chloride 0.03-0.18g, saltpetre 0.01-0.3g, dipotassium hydrogen phosphate 0.05-0.2g, water 100mL, pH7.0-7.4.
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| CN1316897C (en) * | 2005-03-21 | 2007-05-23 | 中国农业大学 | Method of raising taxinol content in tazus chinensis |
| CN101033457B (en) * | 2007-02-16 | 2010-12-15 | 江西农业大学 | Method of producing coronatine by burkholderiacepacia and fermentation culture medium thereof |
| CN101948764B (en) * | 2010-05-13 | 2012-07-18 | 江苏科技大学 | Pseudomonas syringae pv.mori bacterial strain for producing coronatine and method for producing coronatine by fermentation thereof |
| CN102239876B (en) * | 2011-05-18 | 2013-09-04 | 中国农业大学 | Application of coronatine as novel cotton defoliant |
| CN102229543B (en) * | 2011-06-07 | 2013-12-04 | 中国农业大学 | Extraction method for purified coronatine (COR) from fermentation liquor |
| CN102250975A (en) * | 2011-06-21 | 2011-11-23 | 江苏科技大学 | Brown sugar culture medium for producing coronatine by fermenting |
| CN102422787B (en) * | 2011-09-14 | 2013-06-05 | 江苏科技大学 | Method for increasing content of secondary metabolite 1-deoxynojirimycin of mulberry tree |
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