CN101225431A - Rapid screening method for dihydroxypropanone high-yield fungus - Google Patents
Rapid screening method for dihydroxypropanone high-yield fungus Download PDFInfo
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- CN101225431A CN101225431A CNA2008100595172A CN200810059517A CN101225431A CN 101225431 A CN101225431 A CN 101225431A CN A2008100595172 A CNA2008100595172 A CN A2008100595172A CN 200810059517 A CN200810059517 A CN 200810059517A CN 101225431 A CN101225431 A CN 101225431A
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- 238000000034 method Methods 0.000 title claims abstract description 26
- 238000012216 screening Methods 0.000 title claims abstract description 19
- UOQFZGVGGMHGEE-UHFFFAOYSA-N 1,1-dihydroxypropan-2-one Chemical compound CC(=O)C(O)O UOQFZGVGGMHGEE-UHFFFAOYSA-N 0.000 title claims description 12
- 241000233866 Fungi Species 0.000 title claims description 12
- 239000007788 liquid Substances 0.000 claims abstract description 32
- 239000012029 Fehling's reagent Substances 0.000 claims abstract description 27
- 238000000855 fermentation Methods 0.000 claims abstract description 9
- 230000004151 fermentation Effects 0.000 claims abstract description 9
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 78
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 48
- 235000011187 glycerol Nutrition 0.000 claims description 39
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 34
- 239000000843 powder Substances 0.000 claims description 34
- 230000001580 bacterial effect Effects 0.000 claims description 31
- 241000894006 Bacteria Species 0.000 claims description 28
- 229920001817 Agar Polymers 0.000 claims description 23
- 239000008272 agar Substances 0.000 claims description 23
- 239000002904 solvent Substances 0.000 claims description 22
- 239000006228 supernatant Substances 0.000 claims description 13
- 239000002689 soil Substances 0.000 claims description 11
- 239000001888 Peptone Substances 0.000 claims description 10
- 108010080698 Peptones Proteins 0.000 claims description 10
- 235000019319 peptone Nutrition 0.000 claims description 10
- 239000002504 physiological saline solution Substances 0.000 claims description 9
- RXKJFZQQPQGTFL-UHFFFAOYSA-N dihydroxyacetone Chemical compound OCC(=O)CO RXKJFZQQPQGTFL-UHFFFAOYSA-N 0.000 abstract description 27
- 238000012360 testing method Methods 0.000 abstract description 6
- 230000000813 microbial effect Effects 0.000 abstract description 3
- 229940120503 dihydroxyacetone Drugs 0.000 abstract 4
- 239000012530 fluid Substances 0.000 abstract 2
- 239000001963 growth medium Substances 0.000 abstract 2
- 230000003247 decreasing effect Effects 0.000 abstract 1
- 125000001475 halogen functional group Chemical group 0.000 abstract 1
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- 230000010355 oscillation Effects 0.000 abstract 1
- 230000000007 visual effect Effects 0.000 abstract 1
- 244000005700 microbiome Species 0.000 description 16
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 15
- 238000001816 cooling Methods 0.000 description 12
- 238000002360 preparation method Methods 0.000 description 12
- 239000000243 solution Substances 0.000 description 9
- 239000007787 solid Substances 0.000 description 8
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 238000002156 mixing Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- LJCNRYVRMXRIQR-OLXYHTOASA-L potassium sodium L-tartrate Chemical compound [Na+].[K+].[O-]C(=O)[C@H](O)[C@@H](O)C([O-])=O LJCNRYVRMXRIQR-OLXYHTOASA-L 0.000 description 5
- 235000011006 sodium potassium tartrate Nutrition 0.000 description 5
- 241000589232 Gluconobacter oxydans Species 0.000 description 4
- 244000208060 Lawsonia inermis Species 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 238000010438 heat treatment Methods 0.000 description 4
- 238000011081 inoculation Methods 0.000 description 4
- 230000001954 sterilising effect Effects 0.000 description 4
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- 239000000126 substance Substances 0.000 description 4
- 241000589220 Acetobacter Species 0.000 description 3
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- 238000000926 separation method Methods 0.000 description 3
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- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 241000228212 Aspergillus Species 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- 241000194108 Bacillus licheniformis Species 0.000 description 2
- 244000063299 Bacillus subtilis Species 0.000 description 2
- 241000588807 Bordetella Species 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- 241001508813 Clavispora lusitaniae Species 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 101000892220 Geobacillus thermodenitrificans (strain NG80-2) Long-chain-alcohol dehydrogenase 1 Proteins 0.000 description 2
- 241000589236 Gluconobacter Species 0.000 description 2
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- 241000235527 Rhizopus Species 0.000 description 2
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- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
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- WGTYBPLFGIVFAS-UHFFFAOYSA-M tetramethylammonium hydroxide Chemical compound [OH-].C[N+](C)(C)C WGTYBPLFGIVFAS-UHFFFAOYSA-M 0.000 description 2
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
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- DHRLEVQXOMLTIM-UHFFFAOYSA-N phosphoric acid;trioxomolybdenum Chemical compound O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.OP(O)(O)=O DHRLEVQXOMLTIM-UHFFFAOYSA-N 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a fast screening method for dihydroxyacetone high-yielding strain, which is characterized in that a waiting colony is inoculated in the culture medium of perforated distribution plate until a single colony is grown; the fehling reagent is added to test; if a brown-red halo spot is shown around the single colony, the single colony is a positive strain; the positive strain is fetched to be inoculate to the inclined culture medium, and cultivated at 28 to 37 DEG C in 1 to 2 days; the inclined strain of the positive strain is obtained; the inclined strain is inoculated in the fermentation medium, and the zymotic fluid is cultivated by oscillation; the content of DNA in the zymotic liquid is determined; the DNA content of the zymotic fluid obtained by fermentation is higher than the DNA content of the 2 g/L positive strain, which is dihydroxyacetone high-producing strain. The fast screening method for dihydroxyacetone high-yielding strain has the advantages of simple operation, visual and convenient judgment to result, fast and high-flux screening of microbial strain well producing dihydroxyacetone, significantly decreased amount of labor, and increased labor efficiency.
Description
(1) technical field
The present invention relates to a kind of rapid screening method of dihydroxypropanone high-yield fungus, belong to industrial microorganism engineering field.
(2) background technology
Otan is called 1 again, and (1,3-Dihydroxyacetone) (hereinafter to be referred as DHA) is the most single ketose to the 3-otan, has the white powder crystallization of sweet taste, organic solvent such as soluble in water, ethanol, acetone and ether.The molecular weight of this chemical substance is 90.08.
DHA is of many uses, market capacity is big, the research of producing otan with bio-transformation glycerine has great importance, and microbe transformation method production DHA compares with chemical synthesis has many advantages: advantages such as specificity is strong, reaction conditions is gentle, substrate utilization ratio is high, transformation efficiency is high, production technique is simple.
Abroad be the existing a lot of patents of research and the bibliographical information of otan with the microbial method glycerine converting, if used being used to produces the microbial host Gluconobacter (Gluconobacter) (US 5770411) and genus acetobacter (Acetobacter) (US 4076589) microorganism of glycerol dehydrogenase, especially the report of gluconobacter oxydans (Gluconobacter oxydans) and weak oxide acetobacter (Acebobacter Suboxydans) is in the majority, studied the Bacillus subtilus (Bacillus subtilis FERM P-10524) (JP 2286089) of bacillus (Bacillus) in addition, Micremonospora (Micromonospora) (JP 62210994), Pseudomonas (Pseudomonas), serratia (Serratia), restrain mould uncle Bordetella (Klebsiella), the Irving program Bordetella (Erwinia) of planting, Aspergillus (Aspergillus), Penicillium (Penicillium), Rhizopus (Rhizopus) (US 4576913).
Although it is more that glycerine converting is produced the microorganism of otan, the high yield bacterium that really is used to produce DHA is also fewer, and the report of the rapid screening aspect of the microorganism that relevant glycerine converting is an otan is blank especially.
(3) summary of the invention
The object of the invention is to provide a kind of screening method of dihydroxypropanone high-yield fungus fast, easily.
The technical solution used in the present invention is:
The screening method of dihydroxypropanone high-yield fungus, described method comprises following sequential steps:
(1) get bacterial strain to be screened, be seeded in the porous plate substratum, be cultured under 28～37 ℃ and grow single bacterium colony, add fehling reagent, place 10～60min down for 25～35 ℃, single periphery of bacterial colonies manifests the red-brown milky spot, positive bacterial strain; Described porous plate substratum is composed as follows: glycerine 20～50g/L, and yeast powder 2～5g/L, agar 18～20g/L, solvent are water, pH5.0～7.0;
(2) get positive strain in addition and be seeded to slant medium, cultivated 1～2 day down, obtain the slant strains of positive strain for 28～37 ℃; Described slant medium is composed as follows: glycerine 20～50g/L, and yeast powder 2～5g/L, agar 18～20g/L, solvent are water, pH5.0～7.0;
(3) slant strains with positive strain is seeded to fermention medium, 28～37 ℃, 100～200rpm shaking culture, 24～60h obtain fermented liquid, measure DHA content in the fermented liquid, DHA content is higher than the positive strain of 2g/L in the fermented liquid that fermentation obtains, and is dihydroxypropanone high-yield fungus; Described fermention medium is composed as follows: glycerine 10～50g/L, yeast powder 2～20g/L, peptone 1～20g/L, NaH
2PO
42H
2O 1～5g/L, CaCO
31～4g/L, solvent are water, pH4.0～7.0.
The present invention can directly apply to the seed selection of known bacterial strain, also can be applicable to screen the unknown microorganism in the soil.When being applied to screen in the soil sample microorganism, method is as follows: get soil sample to be measured, pulverize, add stroke-physiological saline solution, vibration is got supernatant liquor and is coated the plate substratum, cultivated 1～2 day for 28～37 ℃, single bacterium colony of acquisition screens according to step (1)～(3) step as bacterial strain to be screened; Described plate substratum is composed as follows: glycerine 20～50g/L, and yeast powder 2～5g/L, agar 18～20g/L, solvent are water, pH5.0～7.0.
The porous plate substratum that the present invention relates to, slant medium are the identical solid mediums of moiety scope with the plate substratum.Prepare as follows: heating condition down in the 1000mL water adding agar 18～20g make it to dissolve, add glycerine 20～50g and yeast powder 2～5g again, 121 ℃ of sterilization 20min, when being cooled to 60 ℃, in gnotobasis, substratum is joined in each aperture of porous plate, obtain the porous plate substratum through cooling; In gnotobasis, substratum is poured in the aseptic test tube, obtained slant medium through cooling; In gnotobasis substratum is poured in the aseptic plate, cooling obtains the plate substratum.
Described fermention medium is prepared as follows, adds glycerine 10～50g, yeast powder 2～20g, peptone 1～20g, NaH in the 1000mL water
2PO
42H
2O 1～5g transfers pH to 4.0～7.0 with 0.1mol/L HCl or NaOH, adds CaCO at last
31～4g, 121 ℃ of sterilization 20min, cooling obtains fermention medium.
Fehling reagent is a qualitative experiment reagent, and its consumption is unrestricted, and those skilled in the art can be as required, add according to general knowledge, and usually, when operating in porous plate, every hole is added 0.1～0.2mL fehling reagent and got final product.
In the described step (1), cultivate in each parallel inoculation at least 2 hole of bacterial strain to be measured, cultivation obtains single bacterium colony, wherein at least 1 hole is added fehling reagent and is carried out color reaction, the single bacterium colony that obtains in the hole of not adding fehling reagent of the bacterial strain correspondence to be measured that is positive is used for next step operation, can save the operating time like this, and can realize batch operation.
Concrete, described method is as follows:
(1) get soil sample to be measured, pulverize, add stroke-physiological saline solution, vibration is got supernatant liquor and is coated the plate substratum, cultivates 1～2 day for 28～37 ℃, and single bacterium colony of acquisition is as bacterial strain to be screened; Described plate substratum is composed as follows: glycerine 20～50g/L, and yeast powder 2～5g/L, agar 18～20g/L, solvent are water, pH5.0～7.0;
(2) under the aseptic condition, get bacterial strain to be screened, parallel 2 holes that are seeded to the porous plate substratum, be cultured under 28～37 ℃ and grow single bacterium colony, add fehling reagent 0.1～0.2mL therein in 1 hole, place 10～60min down for 25～35 ℃, single periphery of bacterial colonies manifests the red-brown milky spot, positive bacterial strain, the corresponding single bacterium colony that does not add the positive strain that obtains in the hole of fehling reagent is used for next step operation; Described porous plate is cultivated and is formed with the plate substratum;
(3) get positive strain and be seeded to slant medium, cultivated 1～2 day down, obtain the slant strains of positive strain for 28～37 ℃; Described slant medium is formed with the plate substratum;
(4) slant strains with positive strain is seeded to fermention medium, 28～37 ℃, 100～200rpm shaking culture, 24～60h obtain fermented liquid, measure DHA content in the fermented liquid, DHA content is higher than the positive strain of 2g/L in the fermented liquid that fermentation obtains, and is dihydroxypropanone high-yield fungus; Described fermention medium is composed as follows: glycerine 10～50g/L, yeast powder 2～20g/L, peptone 1～20g/L, NaH
2PO
42H
2O 1～5g/L, CaCO
31～4g/L, solvent are water, pH4.0～7.0.
Preferably, described method is as follows:
A) get soil sample to be measured, pulverize, add stroke-physiological saline solution, vibration is got supernatant liquor and is coated the plate substratum, cultivates 1 day for 28 ℃, and single bacterium colony of acquisition is as bacterial strain to be screened; Described plate substratum is composed as follows: glycerine 20g/L, and yeast powder 2g/L, agar 18g/L, solvent are water, pH5.0;
B) under the aseptic condition, get bacterial strain to be screened, in parallel 2 holes that are seeded to the porous plate substratum, be cultured under 28 ℃ and grow single bacterium colony, add fehling reagent 0.1mL therein in 1 hole, place 60min down for 25 ℃, single periphery of bacterial colonies manifests the red-brown milky spot, positive bacterial strain, the corresponding single bacterium colony that does not add the positive strain that obtains in the hole of fehling reagent is used for next step operation; Described porous plate substratum is composed as follows: glycerine 20g/L, and yeast powder 2g/L, agar 18g/L, solvent are water, pH5.0;
C) get positive strain and be seeded to slant medium, cultivated 1 day down, obtain the slant strains of positive strain for 28 ℃; Described slant medium composed as follows: glycerine 20g/L, yeast powder 2g/L, agar 18g/L, solvent are water, pH5.0;
D) slant strains with positive strain is seeded to fermention medium, 30 ℃, 200rpm shaking culture 24h obtain fermented liquid, measure DHA content in the fermented liquid, DHA content is higher than the positive strain of 2g/L in the fermented liquid that fermentation obtains, and is dihydroxypropanone high-yield fungus; Described fermention medium is composed as follows: glycerine 10g/L, yeast powder 2g/L, peptone 1g/L, NaH
2PO
42H
2O 1g/L, CaCO
31g/L, solvent are water, pH5.0.
Fehling reagent comprises two kinds of solution of A, B, A solution is that mass concentration is the copper-bath of 0.044g/mL, B solution is that mass concentration is respectively the Seignette salt of 0.258g/mL and the sodium hydroxide mixing solutions of 0.142g/mL, during use, gets and uses immediately after equivalent A, B liquid mix.
Described fehling reagent can be prepared as follows:
A liquid: use 34.6g CuSO
45H
2O is dissolved in the 200mL water, adds the dense H of 0.5mL again
2SO
4, behind the mixing, be diluted with water to 500mL; B liquid: with 173g Seignette salt (KNaC
4H
4O
64H
2O) and the 71gNaOH solid be dissolved in the 400mL water, redilution becomes 500mL solution, during use, gets and uses immediately after equivalent A, B liquid mix.
Among the present invention, the analytical procedure of DHA can adopt gas-chromatography and thin-layer chromatography method to carry out:
DHA thin-layer chromatography method: G type silica-gel plate is adopted in experiment, and (20mm * 10mm), developping agent: chloroform-methanol-distilled water mixed solvent (volume ratio is 80: 19: 1), developer consists of: phospho-molybdic acid 3g, methyl alcohol 45mL, distilled water 45mL and vitriol oil 10mL.Concrete operations step: the silica gel thin sheet behind the point sample is placed on downwards in the chromatography cylinder that developping agent is housed with reference line launches 10～15min, diffuse to apart from 1～2cm place, thin plate top until developping agent.Take out thin plate, dry up, stifling 2～5min in the iodine cylinder, baking oven is put in colour developing, toasts about 10min down at 110 ℃, can present the DHA spot, adopts the thin layer chromatography scanner of CAMMA to measure DHA content.
DHA gas chromatography analysis method: used instrument: Varian varian cp-3800 gas chromatograph, band flame ionization ditector; PY-2020iD type cracker (Frontier LaboratoriesLtd.Japan).Agents useful for same: TMAH (25% aqueous solution), otan (chromatographically pure), glycerine, anhydrous methanol (analytical pure), distilled water.Chromatographic condition: Ultra ALLOY-5 Stainless Steel Capillary tubing string (30m * 0.25mm * 0.25 μ m); 400 ℃ of vaporization temperatures; 280 ℃ of detector temperatures; 50 ℃ of beginnings of column temperature rise to 60 ℃ with 1 ℃/min, rise to 280 ℃ with 10 ℃/min again; Splitting ratio 30: 1; Carrier gas, nitrogen; Post flow 1mL/min.Application of sample: 0.2 μ L fermentation supernatant and 0.4 μ LTMAH (25% aqueous solution).
Principle of the present invention: with glycerine is substrate, utilizes the glycerol dehydrogenase enzymic catalytic reaction of microorganisms to produce 1, the 3-otan, the product otan can with fehling reagent generation color reaction, generate henna precipitation.Based on this colour developing principle, designed quick, the high-throughout screening model of product otan microorganism.Practice shows that this model in the test of bacterial screening, received good effect.By this model, screened the microorganism strains of several strains energy high yield otans.
Beneficial effect of the present invention is: simple to operate, result judges intuitive and convenient, and the microorganism strains that energy is quick, high flux screening produces otan significantly reduces labor capacity, raises labour efficiency.
(4) embodiment
The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:
Embodiment 1:
(1) preparation of fehling reagent:
A liquid 34.6gCuSO
45H
2O is dissolved in the 200mL water, adds the dense H of 0.5mL again
2SO
4, behind the mixing, be diluted with water to 500mL,
B liquid 173g Seignette salt (KNaC
4H
4O
64H
2O) and 71g NaOH solid be dissolved in the 400mL water, redilution becomes 500mL solution,
In the time of use, get and use immediately after equivalent A, B liquid mix;
(2) preparation of substratum:
Plate substratum, porous plate substratum prepare with slant medium: described plate substratum, porous plate substratum and slant medium are the identical solid mediums of moiety, its component is as follows: glycerine 20g, yeast powder 2g, agar 18g, water 1000mL, pH value 5.0.Preparation as follows: heating condition down in the 1000mL water adding agar 18g make it to dissolve, add glycerine 20g and yeast powder 2g again, 121 ℃ of sterilization 20min, when being cooled to 60 ℃, in sterile environment, substratum is joined in each aperture of 96 aseptic hole porous plates, cooling obtains the porous plate substratum then, and is stand-by; In sterile environment substratum is poured in the aseptic plate, cooling obtains the plate substratum, and is stand-by; Pour substratum into aseptic test tube in sterile environment, cooling obtains slant medium, and is stand-by; The fermention medium preparation: fermention medium is composed as follows: glycerine 10g, yeast powder 2g, peptone 10g, NaH
2PO
42H
2O 1g, CaCO
31g, water 1000mL, pH value 5.0;
(3) soil sample that will screen grinds, and adds stroke-physiological saline solution, fully vibration, and it is stand-by to get supernatant liquor;
(4) separation and Culture:
The supernatant of step (3) preparation is coated on the plate substratum, cultivated 1 day at 28 ℃, select single bacterium colony one by one, parallelly be inoculated into 2 same porous plate substratum (porous plate X, porous plate Y) goes up in the corresponding mutually aperture, porous plate is placed on 28 ℃ cultivated 1 day, will grow single bacterium colony in the aperture;
(5) preliminary screening:
With the fehling reagent of newly preparing, in the aperture of porous plate X, add fehling reagent 0.1mL, place 60min down at 25 ℃, visual inspection, if the positive reaction of henna milky spot occurs, as positive control, on porous plate Y, select positive single bacterium colony then in the corresponding aperture with the otan standard substance in periphery of bacterial colonies, be inoculated into slant medium, cultivated 1 day down for 28 ℃;
(6) multiple sieve:
In fermention medium, shaking culture 24h on 30 ℃, the constant temperature shaking table of 200rpm measures DHA content with cultured inclined-plane inoculation, and DHA content is higher than the microorganism that otan is produced in the conduct of 2g/L.
In this screening process, screen the microorganism strains that otan is produced in 2 strains, be preserved in Chinese typical culture collection center (being called for short CCTCC) on October 18th, 2005:
Clavispora lusitaniae yeast (Clavispora lusitaniae), deposit number is CCTCC No.M205116;
Pichia membranaefaciens (Pichia membranifaciens), deposit number is CCCTCC No.M205117.
Embodiment 2:
(1) preparation of fehling reagent:
A liquid: use 34.6gCuSO
45H
2O is dissolved in the 200mL water, adds the dense H of 0.5mL again
2SO
4, behind the mixing, be diluted with water to 500mL;
B liquid: with 173g Seignette salt (KNaC
4H
4O
64H
2O) and 71g NaOH solid be dissolved in the 400mL water, redilution becomes 500mL solution;
In the time of use, get and use immediately after equivalent A, B liquid mix;
(2) preparation of substratum:
Plate substratum, porous plate substratum prepare with slant medium: described plate substratum, porous plate substratum and slant medium are the identical solid mediums of moiety, its component is as follows: glycerine 30g, yeast powder 3g, agar 20g, water 1000mL, pH value 6.0; Preparation as follows: heating condition down in the 1000mL water adding agar 20g make it to dissolve, add glycerine 30g and yeast powder 3g again, 121 ℃ of sterilization 20min, when being cooled to 60 ℃, in sterile environment, substratum is joined in each aperture of 96 aseptic hole porous plates, cooling obtains the porous plate substratum then, and is stand-by; In gnotobasis substratum is poured in the aseptic plate, cooling obtains the plate substratum then, and is stand-by; In sterile environment substratum is poured in the aseptic test tube, cooling obtains slant medium then, and is stand-by;
The fermention medium preparation: fermention medium is composed as follows: glycerine: 50g, yeast powder: 20g, peptone: 1g, NaH
2PO
42H
2O:5g, CaCO
3: 4g, water 1000mL, pH value 5.0,
(3) soil sample that will screen grinds, and adds stroke-physiological saline solution, fully vibration, and it is stand-by to get supernatant liquor;
(4) separation and Culture:
The supernatant of step (3) preparation is coated on the plate substratum, cultivated 1 day at 37 ℃, select single bacterium colony one by one, parallelly be inoculated into 2 same porous plate substratum (porous plate X, porous plate Y) goes up in the corresponding mutually aperture, porous plate is placed on 37 ℃ cultivated 1 day, will grow single bacterium colony in the aperture;
(5) preliminary screening:
With the fehling reagent of newly preparing, in the aperture of porous plate X, add fehling reagent 0.2mL, place 30min down at 30 ℃, visual inspection, if the positive reaction of henna milky spot occurs, as positive control, on porous plate Y, select positive single bacterium colony then in the corresponding aperture with the otan standard substance in periphery of bacterial colonies, be inoculated into slant medium, cultivated 1 day down for 28 ℃;
(6) multiple sieve:
In fermention medium, shaking culture 36h on 30 ℃, the constant temperature shaking table of 200rpm measures DHA content with cultured inclined-plane inoculation, and DHA content is higher than the microorganism that otan is produced in the conduct of 2g/L.
In this screening process, screen 1 strain and produce the microorganism strains of otan, be preserved in Chinese typical culture collection center (being called for short CCTCC) on August 29th, 2006: Bacillus licheniformis B-05571 (Bacillus licheniformis B-05571), preserving number CCTCC No.M206082.
Embodiment 3:
(1) preparation of fehling reagent:
A liquid 34.6g CuSO
45H
2O is dissolved in the 200mL water, adds the dense H of 0.5mL again
2SO
4, behind the mixing, be diluted with water to 500mL,
B liquid 173g Seignette salt (KNaC
4H
4O
64H
2O) and 71g NaOH solid be dissolved in the 400mL water, redilution becomes 500mL solution,
In the time of use, get and use immediately after equivalent A, B liquid mix;
(2) preparation of substratum:
Plate substratum, porous plate substratum prepare with slant medium: described plate substratum, porous plate substratum and slant medium are the identical solid mediums of moiety, its component is as follows: glycerine 50, yeast powder 5, agar 20, water 1000mL, pH value 7.0, preparation as follows: heating condition down adds agar 20g in the 1000mL water to be made it to dissolve, and adds glycerine 50g and yeast powder 5g again, 121 ℃ of 20min that sterilize, when being cooled to 60 ℃
In sterile environment substratum is joined in each aperture of 384 aseptic hole porous plates, to obtain the porous plate substratum stand-by in cooling then;
With freshly prepared substratum, pour in the aseptic plate in gnotobasis, cooling obtains the plate substratum, and is stand-by;
Pour freshly prepared substratum into aseptic test tube in sterile environment, it is stand-by to prepare slant medium.
The fermention medium preparation: fermention medium is composed as follows: glycerine: 50g, yeast powder: 5g, peptone: 20g, NaH
2PO
42H
2O:1g, CaCO3:4g, water 1000mL, pH value 7.0, the soil sample that (3) will screen grinds, and adds stroke-physiological saline solution, fully vibration, it is stand-by to get supernatant liquor;
(4) separation and Culture:
The supernatant of step (3) preparation is coated on the plate substratum, cultivated 2 days at 30 ℃, select single bacterium colony one by one, parallelly be inoculated into 2 same porous plate substratum (porous plate X, porous plate Y) goes up in the corresponding mutually aperture, porous plate is placed on 30 ℃ cultivated 1 day, will grow single bacterium colony in the aperture;
(5) preliminary screening:
With the fehling reagent of newly preparing, in the aperture of porous plate X, add fehling reagent 0.1mL, place 20min down at 35 ℃, visual inspection, if the positive reaction of henna milky spot occurs, as positive control, on porous plate Y, select positive single bacterium colony then in the corresponding aperture with the otan standard substance in periphery of bacterial colonies, be inoculated into slant medium, cultivated 1 day down for 30 ℃;
(6) multiple sieve:
In fermention medium, shaking culture 48h on 30 ℃, the constant temperature shaking table of 100rpm measures DHA content with cultured inclined-plane inoculation, and DHA content is higher than the microorganism that otan is produced in the conduct of 2g/L.
In this screening process, screen the microorganism strains that otan is produced in 1 strain: gluconobacter oxydans (Gluconobacter oxydans).
Claims (4)
1. the rapid screening method of dihydroxypropanone high-yield fungus, described method comprises following sequential steps:
(1) get bacterial strain to be screened, be seeded in the porous plate substratum, be cultured under 28～37 ℃ and grow single bacterium colony, add fehling reagent, place 10～60min down for 25～35 ℃, single periphery of bacterial colonies manifests the red-brown milky spot, positive bacterial strain; Described porous plate substratum is composed as follows: glycerine 20～50g/L, and yeast powder 2～5g/L, agar 18～20g/L, solvent are water, pH5.0～7.0;
(2) get positive strain in addition and be seeded to slant medium, cultivated 1～2 day down, obtain the slant strains of positive strain for 28～37 ℃; Described slant medium composed as follows: glycerine 20～50g/L, yeast powder 2～5g/L, agar 18～20g/L, solvent are water, pH5.0～7.0;
(3) slant strains with positive strain is seeded to fermention medium, 28～37 ℃, 100～200rpm shaking culture, 24～60h obtain fermented liquid, measure DHA content in the fermented liquid, DHA content is higher than the positive strain of 2g/L in the fermented liquid that fermentation obtains, and is dihydroxypropanone high-yield fungus; Described fermention medium is composed as follows: glycerine 10～50g/L, yeast powder 2～20g/L, peptone 1～20g/L, NaH
2PO
42H
2O 1～5g/L, CaCO
3, 1～4g/L, solvent are water, pH4.0～7.0.
2. the method for claim 1, it is characterized in that described method is: get soil sample to be measured, pulverize, add stroke-physiological saline solution, vibration is got supernatant liquor and is coated the plate substratum, cultivates 1～2 day for 28～37 ℃, the single bacterium colony that obtains screens according to step (1)～(3) step as bacterial strain to be screened; Described plate substratum is composed as follows: glycerine 20～50g/L, and yeast powder 2～5g/L, agar 18～20g/L, solvent are water, pH5.0～7.0.
3. the method for claim 1 is characterized in that described method is as follows:
(1) get soil sample to be measured, pulverize, add stroke-physiological saline solution, vibration is got supernatant liquor and is coated the plate substratum, cultivates 1～2 day for 28～37 ℃, and single bacterium colony of acquisition is as bacterial strain to be screened; Described plate substratum is composed as follows: glycerine 20～50g/L, and yeast powder 2～5g/L, agar 18～20g/L, solvent are water, pH5.0～7.0;
(2) under the aseptic condition, get bacterial strain to be screened, in parallel 2 holes that are seeded to the porous plate substratum, be cultured under 28～37 ℃ and grow single bacterium colony, add fehling reagent 0.1～0.2mL therein in 1 hole, place 10～60min down for 25～35 ℃, single periphery of bacterial colonies manifests the red-brown milky spot, positive bacterial strain, the corresponding single bacterium colony that does not add the positive strain that obtains in the hole of fehling reagent is used for next step operation; Described porous plate substratum is composed as follows: glycerine 20～50g/L, and yeast powder 2～5g/L, agar 18～20g/L, solvent are water, pH5.0～7.0;
(3) get positive strain and be seeded to slant medium, cultivated 1～2 day down, obtain the slant strains of positive strain for 28～37 ℃; Described slant medium composed as follows: glycerine 20～50g/L, yeast powder 2～5g/L, agar 18～20g/L, solvent are water, pH5.0～7.0;
(4) slant strains with positive strain is seeded to fermention medium, 28～37 ℃, 100～200rpm shaking culture, 24～60h obtain fermented liquid, measure DHA content in the fermented liquid, DHA content is higher than the positive strain of 2g/L in the fermented liquid that fermentation obtains, and is dihydroxypropanone high-yield fungus; Described fermention medium is composed as follows: glycerine 10～50g/L, yeast powder 2～20g/L, peptone 1～20g/L, NaH
2PO
42H
2O 1～5g/L, CaCO
31～4g/L, solvent are water, pH4.0～7.0.
4. method as claimed in claim 3 is characterized in that described method is as follows:
(1) get soil sample to be measured, pulverize, add stroke-physiological saline solution, vibration is got supernatant liquor and is coated the plate substratum, cultivates 1 day for 28 ℃, and single bacterium colony of acquisition is as bacterial strain to be screened; Described plate substratum is composed as follows: glycerine 20g/L, and yeast powder 2g/L, agar 18g/L, solvent are water, pH5.0;
(2) under the aseptic condition, get bacterial strain to be screened, in parallel 2 holes that are seeded to the porous plate substratum, be cultured under 28 ℃ and grow single bacterium colony, add fehling reagent 0.1mL therein in 1 hole, place 60min down for 25 ℃, single periphery of bacterial colonies manifests the red-brown milky spot, positive bacterial strain, the corresponding single bacterium colony that does not add the positive strain that obtains in the hole of fehling reagent is used for next step operation; Described porous plate substratum is composed as follows: glycerine 20g/L, and yeast powder 2g/L, agar 18g/L, solvent are water, pH5.0;
(3) get positive strain and be seeded to slant medium, cultivated 1 day down, obtain the slant strains of positive strain for 28 ℃; Described slant medium composed as follows: glycerine 20g/L, yeast powder 2g/L, agar 18g/L, solvent are water, pH5.0;
(4) slant strains with positive strain is seeded to fermention medium, 30 ℃, 200rpm shaking culture 24h obtain fermented liquid, measure DHA content in the fermented liquid, DHA content is higher than the positive strain of 2g/L in the fermented liquid that fermentation obtains, and is dihydroxypropanone high-yield fungus; Described fermention medium is composed as follows: glycerine 10g/L, yeast powder 2g/L, peptone 1g/L, NaH
2PO
42H
2O 1g/L, CaCO
31g/L, solvent are water, pH5.0.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101591681B (en) * | 2009-04-13 | 2011-11-23 | 浙江工业大学 | Method for producing dihydroxyacetone through microbial transformation |
| CN102492732A (en) * | 2011-11-24 | 2012-06-13 | 厦门大学 | Preparation method of dihydroxyacetone by transforming glycerol |
| CN105695517A (en) * | 2016-04-06 | 2016-06-22 | 常州市阿曼特化工有限公司 | Synthesis method of dihydroxyacetone |
| CN108486209A (en) * | 2018-03-22 | 2018-09-04 | 浙江工业大学 | A kind of method of high-flux fast screening high yield R-2- (4- hydroxyphenoxies) propionic acid bacterial strain |
| CN108663361A (en) * | 2018-05-22 | 2018-10-16 | 福州大学 | A kind of method of biomass in quick measurement liquid state fermentation liquid |
| CN112831537A (en) * | 2021-01-24 | 2021-05-25 | 广东石油化工学院 | Preparation method of bio-based end-capped polyether |
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2008
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101591681B (en) * | 2009-04-13 | 2011-11-23 | 浙江工业大学 | Method for producing dihydroxyacetone through microbial transformation |
| CN102492732A (en) * | 2011-11-24 | 2012-06-13 | 厦门大学 | Preparation method of dihydroxyacetone by transforming glycerol |
| CN102492732B (en) * | 2011-11-24 | 2014-09-03 | 厦门大学 | Preparation method of dihydroxyacetone by transforming glycerol |
| CN105695517A (en) * | 2016-04-06 | 2016-06-22 | 常州市阿曼特化工有限公司 | Synthesis method of dihydroxyacetone |
| CN108486209A (en) * | 2018-03-22 | 2018-09-04 | 浙江工业大学 | A kind of method of high-flux fast screening high yield R-2- (4- hydroxyphenoxies) propionic acid bacterial strain |
| CN108486209B (en) * | 2018-03-22 | 2022-03-18 | 浙江工业大学 | Method for rapidly screening (R) -2- (4-hydroxyphenoxy) propionic acid producing strains in high flux |
| CN108663361A (en) * | 2018-05-22 | 2018-10-16 | 福州大学 | A kind of method of biomass in quick measurement liquid state fermentation liquid |
| CN108663361B (en) * | 2018-05-22 | 2020-12-25 | 福州大学 | Method for rapidly determining biomass in liquid fermentation broth |
| CN112831537A (en) * | 2021-01-24 | 2021-05-25 | 广东石油化工学院 | Preparation method of bio-based end-capped polyether |
| CN112831537B (en) * | 2021-01-24 | 2022-11-15 | 广东石油化工学院 | Preparation method of bio-based end-capped polyether |
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