CN104450834A - Method of increasing yield of spinosad by improving fermentation condition of saccharopolyspora spinosa based on metabonomics - Google Patents
Method of increasing yield of spinosad by improving fermentation condition of saccharopolyspora spinosa based on metabonomics Download PDFInfo
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- CN104450834A CN104450834A CN201410736930.3A CN201410736930A CN104450834A CN 104450834 A CN104450834 A CN 104450834A CN 201410736930 A CN201410736930 A CN 201410736930A CN 104450834 A CN104450834 A CN 104450834A
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Abstract
The invention discloses a method of increasing the yield of spinosad by improving the fermentation condition of saccharopolyspora spinosa based on metabonomics. The method comprises the following steps: (1) determining intracellular metabolite of the saccharopolyspora spinosa; (2) analyzing the capability of the saccharopolyspora spinosa of producing the spinosad; (3) carrying out PLS analysis; and (4) carrying out process analysis. According to the method disclosed by the invention, the metabolite change law in the process of producing the spinosad by virtue of fermentation of the saccharopolyspora spinosa is revealed by utilizing the metabonomics means and combining multivariate statistics, and a metabolic change mechanism related to production of the spinosad is further explained; by analyzing the spinosad production capability and the metabolic level of the saccharopolyspora spinosa in different growth environments, the metabolin related to the production of the spinosad is found, thus providing a direction for optimizing the culture process of the saccharopolyspora spinosa and increasing the fermentation yield of the spinosad. The invention also provides new thinking and method for study in other culture technological processes of producing macrolides microorganisms.
Description
Technical field
The invention belongs to field of biological pesticide, be specifically related to a kind of improvement based on metabolism group and sting saccharopolyspora strain fermentation condition to improve the method for producing pleocidin output.
Background technology
Pleocidin has another name called Spinosyn or polyoxin, being a kind of by stinging saccharopolyspora strain macrolide antibiotics through secondary metabolism generation under aerobic fermentation condition, mainly comprising A83543A (accounting for 85%-90%) and A83543D (accounting for 10%-15%) two kinds of compounds.Pleocidin is as a kind of high-efficiency broad spectrum Macrolide insecticide active substance, due to its there is mode of action uniqueness, insecticidal spectrum is wide, the transformation period is short, insecticidal activity is high, without resistance, residual low, low in the pollution of the environment and mechanism of action to the physico-chemical property of some uniquenesses such as person poultry harmless, biological characteristics and novelty, be called as the green novel biopesticide of wide spectrum of a new generation.
Pleocidin is used widely as green novel environmentally-friendly biological agricultural chemicals, and lot of domestic and foreign mechanism and researchist have carried out the research of thorn saccharopolyspora strain being produced to pleocidin, and its route of synthesis is illustrated gradually.However, research domestic at present only rests on laboratory stage, and unrealized industrialization.Research shows, improves the raising that dissolved oxygen contributes to pleocidin output in fermenting process, but but still fuzzy to its associated metabolic mechanism, and this also becomes the stumbling-block of pleocidin suitability for industrialized production to a certain extent.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, provide a kind of and improve thorn saccharopolyspora strain fermentation condition to improve the method for pleocidin output based on metabolism group.
Technical scheme of the present invention is summarized as follows:
Improve thorn saccharopolyspora strain fermentation condition to improve the method for pleocidin output based on metabolism group, comprise the following steps:
(1) metabolite in thorn saccharopolyspora strain born of the same parents is measured:
1. cell harvesting and cancellation:
In substratum 1, fermentation culture is carried out to thorn saccharopolyspora strain, in logarithmic phase and the stationary phase of the fermentation of thorn saccharopolyspora strain, each period at least one point in time sampling 5-10ml, each point in time sampling 3-5 part, institute's sample thief is mixed with 18-20mL cancellation liquid and places 5-10min afterwards, at 3000-5000rpm, 4 DEG C, centrifugal 3-5min, abandons supernatant liquor; With the medium component on 4 DEG C of distilled water wash removing cells; 3000-5000rpm, 4 DEG C, centrifugal 3-5min, abandons supernatant liquor, retains thalline; Repetitive scrubbing 2-4 time; Described cancellation liquid to be volumetric concentration be 60% methanol aqueous solution; Described substratum 1 is: glucose 72g/L, corn steep liquor 12g/L, Sodium Glutamate 2.4g/L, bean powder 18g/L, and surplus is water, adjusts pH=7.5, sterilizing;
2. endocellular metabolism thing is extracted:
Get the cell that 1. step obtain and add liquid nitrogen grinding in mortar, take the good cell of 40-50mg liquid nitrogen grinding and be placed in 1.5mL centrifuge tube, add the extracting solution that 0.5-1mL temperature is-30 DEG C, vortex vortex mixer fully mixes rear frozen-thawed and obtains freeze thawing liquid for 2-4 time; By freeze thawing liquid at 4 DEG C, 3000-5000rpm, centrifugal 3-5min, get supernatant liquor and deserve to be called clear liquid a in another centrifuge tube; Lower confluent monolayer cells adds 0.5-1mL again, temperature is the extracting solution of-30 DEG C, after vortex vortex mixer fully mixes, and 4 DEG C, 8000-10000rpm, centrifugal 3-5min, get supernatant liquor and deserve to be called clear liquid b, supernatant liquor a and b is mixed to obtain supernatant liquor c, 8000-10000rpm, 4 DEG C, centrifugal 3-5min, get 260 μ L supernatant liquor c and go to 1.5mL centrifuge tube, adding 50uL concentration is 0.3mgmL
-1succsinic acid-2,2,3,3-d
4the aqueous solution, after vortex vortex mixer fully mixes, under-40 DEG C of conditions, vacuum lyophilization obtains sample, preserves in Ultralow Temperature Freezer; Described extracting solution is volumetric concentration is 50% methanol aqueous solution;
3. metabolite derivatize
In the sample that 2. step obtains, add 50 μ L concentration is 20mgmL
-1the pyridine solution of methoxyl group ammonium salt hydrochlorate, in 40 DEG C of water-baths, react 70-100min, add the trimethyl silicon based trifluoroacetamide of 80 μ L N-methyl-N-, in 40 DEG C of water-bath 20-60min, put into numbered GC sample injection bottle, room temperature leaves standstill 2h;
4. GC-MS detects
Adopt GC-MS to step 3. gained sample carry out quantitative and qualitative analysis process, condition is as follows:
Chromatographic column: silicon chromatographic column, HP-5MS, 30m × 0.25mm, 0.25 μm;
Sample size: 1 μ L; ;
Splitting ratio is 1:10;
Carrier gas: high-purity helium, purity: 99.9995%;
Injector temperature: 280 DEG C;
GC – MS interface temperature: 280 DEG C;
Helium flow velocity: constant voltage, 91KPa;
Heating schedule: 70 DEG C keep 2min, with 5 DEG C of min
-1speed be raised to 290 DEG C, and keep 3min at 290 DEG C, then cool to 30 DEG C;
Ionization mode: electron impact ionization;
Ion(ic)current: 40 μ A;
Ionization voltage: 70eV;
Source temperature: 250 DEG C;
Sweep limit: 50-800m/z;
Sweep velocity: 2scans
-1;
Thus obtain the metabolite quantitative and qualitative analysis data of thorn saccharopolyspora strain;
(2) produce pleocidin ability to thorn saccharopolyspora strain to analyze:
1. cell harvesting and cancellation:
In substratum 2, fermentation culture is carried out to thorn saccharopolyspora strain, respectively at logarithmic phase and at least one point in time sampling 5-10ml of each phase of stationary phase of the fermentation of thorn saccharopolyspora strain, each point in time sampling 3-5 part, institute's sample thief is mixed with 18-20mL cancellation liquid and places 5-10min afterwards, at 3000-5000rpm, 4 DEG C, centrifugal 3-5min, abandons supernatant liquor; With the medium component on 4 DEG C of distilled water wash removing cells; 3000-5000rpm, 4 DEG C, centrifugal 3-5min, abandons supernatant liquor, retains thalline; Repetitive scrubbing 2-4 time; Described cancellation liquid to be volumetric concentration be 60% methanol aqueous solution; Described substratum 2 is: n-dodecane 2-5ml/L, glucose 72g/L, corn steep liquor 12g/L, Sodium Glutamate 2.4g/L, bean powder 18g/L, and surplus is water, adjusts pH=7.5, sterilizing;
2. endocellular metabolism thing is extracted:
Get the cell that 1. step obtain and add liquid nitrogen grinding in mortar, take the good cell of 40-50mg liquid nitrogen grinding and be placed in 1.5mL centrifuge tube, add the extracting solution that 0.5-1mL temperature is-30 DEG C, vortex vortex mixer fully mixes rear frozen-thawed and obtains freeze thawing liquid for 2-4 time; By freeze thawing liquid at 4 DEG C, 3000-5000rpm, centrifugal 3-5min, get supernatant liquor d in another centrifuge tube; Lower confluent monolayer cells adds the extracting solution that 0.5-1mL temperature is-30 DEG C again, after vortex vortex mixer fully mixes, 4 DEG C, 8000-10000rpm, centrifugal 3-5min, get supernatant liquor e, supernatant liquor d and e is mixed, 8000-10000rpm, 4 DEG C, centrifugal 3-5min, get 260 μ L supernatant liquor f and go to 1.5mL centrifuge tube, adding 50uL concentration is 0.3mgmL
-1succsinic acid-2,2,3,3-d
4the aqueous solution, after vortex vortex mixer fully mixes, under-40 DEG C of conditions, vacuum lyophilization obtains sample, preserves in Ultralow Temperature Freezer; Described extracting solution is volumetric concentration is 50% methanol aqueous solution;
3. metabolite derivatize
In the sample that 2. step obtains, add 50 μ L concentration is 20mgmL
-1the pyridine solution of methoxyl group ammonium salt hydrochlorate, in 40 DEG C of water-baths, react 70-100min, add the trimethyl silicon based trifluoroacetamide of 80 μ L N-methyl-N-, in 40 DEG C of water-bath 20-60min, put into numbered GC sample injection bottle, room temperature leaves standstill 2h;
4. GC-MS detects
Adopt GC-MS to step 3. gained sample carry out quantitative and qualitative analysis process, condition is as follows:
Chromatographic column: silicon chromatographic column, HP-5MS, 30m × 0.25mm, 0.25 μm;
Sample size: 1 μ L; ;
Splitting ratio is 1:10;
Carrier gas: high-purity helium, purity: 99.9995%;
Injector temperature: 280 DEG C;
GC – MS interface temperature: 280 DEG C;
Helium flow velocity: constant voltage, 91KPa;
Heating schedule: 70 DEG C keep 2min, with 5 DEG C of min
-1speed be raised to 290 DEG C, and keep 3min at 290 DEG C, then cool to 30 DEG C;
Ionization mode: electron impact ionization;
Ion(ic)current: 40 μ A;
Ionization voltage: 70eV;
Source temperature: 250 DEG C;
Sweep limit: 50-800m/z;
Sweep velocity: 2scans
-1;
Thus obtain the metabolite quantitative and qualitative analysis data of thorn saccharopolyspora strain;
5. born of the same parents' intracellular metabolite thing qualitative and quantitative analysis
Mzmine is used to carry out spectrum unscrambling and peak alignment to the data of GC-MS, mass spectra peak is deconvoluted by AMDIS storehouse, peak width is at more than 2.0s, and signal to noise ratio higher than 10 peak be just considered to the peak of metabolite, by mass spectrometric detection peak and business-like mass spectral database NIST database are compared, detected metabolite is identified, by built-in program, automatic integration is carried out and manual correction to the peak of each metabolite in total ion current figure, thus obtain the data of internal standard substance and each metabolite peak area, then, by peak area quantitative in the unit mass color atlas of the characteristic ion of often kind of metabolite divided by the internal standard substance succsinic acid-2 on same spectrogram, 2, 3, 3-d
4peak area be able to stdn, and calculate the relative content of each metabolite, the relative abundance of often kind of metabolite can be obtained,
(3) PLS analyzes
Use centralization and sized method to carry out pre-treatment to the metabolite relative content data obtained in (1) and (2), then use SIMCA-P 11.5 Demo software to carry out partial least squares discriminant analysis to the data obtained;
(4) process analysis
The content of metabolite markers step (3) obtained makes chart according to culture condition difference, observe and analyze these metabolites change rule, and then find to produce at thorn saccharopolyspora strain the metabolite and associated metabolic network that play a crucial role in pleocidin process, thus for providing optimal anchor direction by the thorn saccharopolyspora strain culture condition improved for the purpose of pleocidin output.
Advantage of the present invention:
(1) utilize metabolism group means and disclose the metabolite Changing Pattern in thorn saccharopolyspora strain fermentation product pleocidin process in conjunction with multivariate statistics, relevant metabotic change mechanism produced by explaination and pleocidin further.
(2) by analyzing the product pleocidin ability of thorn saccharopolyspora strain under Different growing environment and metaboilic level, find the metabolite relevant to producing pleocidin, thus be the optimization of its culture process, the raising provider of pleocidin fermentation yield to.
(3) present method also can be other researchs of producing macrolide microorganism culturing technological process and provides new thinking and countermeasure.
Accompanying drawing explanation
Fig. 1 for glucose and pleocidin under thorn saccharopolyspora strain different fermentations condition over time ("+" represents the n-dodecane adding 2ml/L);
Fig. 2 for biomass and dissolved oxygen under thorn saccharopolyspora strain different fermentations condition over time ("+" represents the n-dodecane adding 2ml/L);
Fig. 3 is the PLS analysis shot chart that thorn saccharopolyspora strain produces pleocidin ability and the change of corresponding metaboilic level under different culture condition;
Fig. 4 is the PLS analysis VIP figure that thorn saccharopolyspora strain produces pleocidin ability and the change of corresponding metaboilic level under different culture condition;
Fig. 5 is that thorn saccharopolyspora strain synthesizes relevant main metabolic metabolite variation tendency under different culture condition to pleocidin.
Fig. 6 be in fermention medium 1, add different concns n-dodecane on the impact of pleocidin output.
Fig. 7 be in fermention medium 1, add 0.05g/L amino acid on the impact of pleocidin output.
Embodiment
Succsinic acid-2,2,3,3-d
4(buying company: SIGMA-ALORICH, production code member 293075|CAS 14493-42-6|Aldrich)
Below in conjunction with specific embodiment, the present invention is further illustrated:
Embodiment 1
Improve thorn saccharopolyspora strain fermentation condition to improve the method for pleocidin output based on metabolism group, comprise the following steps:
(1) metabolite in thorn saccharopolyspora strain born of the same parents is measured:
1. cell harvesting and cancellation:
In substratum 1, fermentation culture is carried out to thorn saccharopolyspora strain, at logarithmic phase 62h, 86h, 110h and stationary phase 134h, 158h and 182h six time points of the fermentation of thorn saccharopolyspora strain, each point in time sampling 3 parts, every increment savors fermented liquid 5ml, institute's sample thief is mixed with 18mL cancellation liquid and places 5min afterwards, at 3000rpm, 4 DEG C, centrifugal 3min, abandons supernatant liquor; With the medium component on 4 DEG C of distilled water wash removing cells; 3000rpm, 4 DEG C, centrifugal 3min, abandons supernatant liquor, retains thalline; Repetitive scrubbing 3 times;
Cancellation liquid to be volumetric concentration be 60% methanol aqueous solution;
Substratum 1 is: glucose 72g/L, corn steep liquor 12g/L, Sodium Glutamate 2.4g/L, bean powder 18g/L, and surplus is water, adjusts pH=7.5, sterilizing;
2. endocellular metabolism thing is extracted:
Get the cell that 1. step obtain and add liquid nitrogen grinding in mortar, take the good cell of 40mg liquid nitrogen grinding and be placed in 1.5mL centrifuge tube, add the extracting solution that 0.5mL temperature is-30 DEG C, vortex vortex mixer fully mixes rear frozen-thawed and obtains freeze thawing liquid 3 times; By freeze thawing liquid at 4 DEG C, 3000rpm, centrifugal 3min, get supernatant liquor and deserve to be called clear liquid a in another centrifuge tube; Lower confluent monolayer cells adds 0.5mL again, temperature is the extracting solution of-30 DEG C, after vortex vortex mixer fully mixes, and 4 DEG C, 8000rpm, centrifugal 3min, get supernatant liquor and deserve to be called clear liquid b, supernatant liquor a and b is mixed to obtain supernatant liquor c, 8000rpm, 4 DEG C, centrifugal 3min, get 260 μ L supernatant liquor c and go to 1.5mL centrifuge tube, adding 50uL concentration is 0.3mgmL
-1succsinic acid-2,2,3,3-d
4the aqueous solution, after vortex vortex mixer fully mixes, under-40 DEG C of conditions, vacuum lyophilization obtains sample, preserves in Ultralow Temperature Freezer;
Described extracting solution is volumetric concentration is 50% methanol aqueous solution;
3. metabolite derivatize
In the sample that 2. step obtains, add 50 μ L concentration is 20mgmL
-1the pyridine solution of methoxyl group ammonium salt hydrochlorate, in 40 DEG C of water-baths, react 70min, add the trimethyl silicon based trifluoroacetamide of 80 μ L N-methyl-N-, in 40 DEG C of water-bath 20min, put into numbered GC sample injection bottle, room temperature leaves standstill 2h;
4. GC-MS detects
Adopt GC-MS to step 3. gained sample carry out quantitative and qualitative analysis process, condition is as follows:
Chromatographic column: silicon chromatographic column, HP-5MS, 30m × 0.25mm, 0.25 μm;
Sample size: 1 μ L; ;
Splitting ratio is 1:10;
Carrier gas: high-purity helium, purity: 99.9995%;
Injector temperature: 280 DEG C;
GC – MS interface temperature: 280 DEG C;
Helium flow velocity: constant voltage, 91KPa;
Heating schedule: 70 DEG C keep 2min, with 5 DEG C of min
-1speed be raised to 290 DEG C, and keep 3min at 290 DEG C, then cool to 30 DEG C;
Ionization mode: electron impact ionization;
Ion(ic)current: 40 μ A;
Ionization voltage: 70eV;
Source temperature: 250 DEG C;
Sweep limit: 50-800m/z;
Sweep velocity: 2scans
-1;
Thus obtain the metabolite quantitative and qualitative analysis data of thorn saccharopolyspora strain;
(2) produce pleocidin ability to thorn saccharopolyspora strain to analyze:
1. cell harvesting and cancellation:
In substratum 2, fermentation culture is carried out to thorn saccharopolyspora strain, at logarithmic phase 62h, 86h, 110h and stationary phase 134h, 158h and 182h six time points of the fermentation of thorn saccharopolyspora strain, each point in time sampling 3 parts, every increment savors fermented liquid 5ml, institute's sample thief is mixed with 18mL cancellation liquid and places 5min afterwards, at 3000rpm, 4 DEG C, centrifugal 3min, abandons supernatant liquor; With the medium component on 4 DEG C of distilled water wash removing cells; 3000rpm, 4 DEG C, centrifugal 3min, abandons supernatant liquor, retains thalline; Repetitive scrubbing 3 times;
Cancellation liquid to be volumetric concentration be 60% methanol aqueous solution;
Substratum 2 is: n-dodecane 2ml/L, glucose 72g/L, corn steep liquor 12g/L, Sodium Glutamate 2.4g/L, bean powder 18g/L, and surplus is water, adjusts pH=7.5, sterilizing;
2. endocellular metabolism thing is extracted:
Get the cell that 1. step obtain and add liquid nitrogen grinding in mortar, take the good cell of 40mg liquid nitrogen grinding and be placed in 1.5mL centrifuge tube, add the extracting solution that 0.5mL temperature is-30 DEG C, vortex vortex mixer fully mixes rear frozen-thawed and obtains freeze thawing liquid 3 times; By freeze thawing liquid at 4 DEG C, 3000rpm, centrifugal 3min, get supernatant liquor and deserve to be called clear liquid d in another centrifuge tube; Lower confluent monolayer cells adds 0.5mL again, temperature is the extracting solution of-30 DEG C, after vortex vortex mixer fully mixes, and 4 DEG C, 8000rpm, centrifugal 3min, get supernatant liquor and deserve to be called clear liquid e, supernatant liquor d and e is mixed to obtain supernatant liquor f, 8000rpm, 4 DEG C, centrifugal 3min, get 260 μ L supernatant liquor f and go to 1.5mL centrifuge tube, adding 50uL concentration is 0.3mgmL
-1succsinic acid-2,2,3,3-d
4the aqueous solution, after vortex vortex mixer fully mixes, under-40 DEG C of conditions, vacuum lyophilization obtains sample, preserves in Ultralow Temperature Freezer; Described extracting solution is volumetric concentration is 50% methanol aqueous solution;
3. metabolite derivatize
In the sample that 2. step obtains, add 50 μ L concentration is 20mgmL
-1the pyridine solution of methoxyl group ammonium salt hydrochlorate, in 40 DEG C of water-baths, react 70min, add the trimethyl silicon based trifluoroacetamide of 80 μ L N-methyl-N-, in 40 DEG C of water-bath 20min, put into numbered GC sample injection bottle, room temperature leaves standstill 2h;
4. GC-MS detects
Adopt GC-MS to step 3. gained sample carry out quantitative and qualitative analysis process, condition is as follows:
Chromatographic column: silicon chromatographic column, HP-5MS, 30m × 0.25mm, 0.25 μm;
Sample size: 1 μ L; ;
Splitting ratio is 1:10;
Carrier gas: high-purity helium, purity: 99.9995%;
Injector temperature: 280 DEG C;
GC – MS interface temperature: 280 DEG C;
Helium flow velocity: constant voltage, 91KPa;
Heating schedule: 70 DEG C keep 2min, with 5 DEG C of min
-1speed be raised to 290 DEG C, and keep 3min at 290 DEG C, then cool to 30 DEG C;
Ionization mode: electron impact ionization;
Ion(ic)current: 40 μ A;
Ionization voltage: 70eV;
Source temperature: 250 DEG C;
Sweep limit: 50-800m/z;
Sweep velocity: 2scans
-1;
Thus obtain the metabolite quantitative and qualitative analysis data of thorn saccharopolyspora strain;
5. born of the same parents' intracellular metabolite thing qualitative and quantitative analysis
Mzmine is used to carry out spectrum unscrambling and peak alignment to the data of GC-MS, mass spectra peak is deconvoluted by AMDIS storehouse, peak width is at more than 2.0s, and signal to noise ratio higher than 10 peak be just considered to the peak of metabolite, by mass spectrometric detection peak and business-like mass spectral database NIST database are compared, detected metabolite is identified, by built-in program, automatic integration is carried out and manual correction to the peak of each metabolite in total ion current figure, thus obtain the data of internal standard substance and each metabolite peak area, then, by peak area quantitative in the unit mass color atlas of the characteristic ion of often kind of metabolite divided by the internal standard substance succsinic acid-2 on same spectrogram, 2, 3, 3-d
4peak area be able to stdn, and calculate the relative content of each metabolite, the relative abundance of often kind of metabolite can be obtained,
(3) PLS analyzes
Use centralization and sized method to carry out pre-treatment to the metabolite relative content data obtained in (1) and (2), then use SIMCA-P 11.5 Demo software to carry out partial least squares discriminant analysis to the data obtained;
(4) process analysis
The content of metabolite markers step (3) obtained makes chart according to culture condition difference, observe and analyze these metabolites change rule, and then find to produce at thorn saccharopolyspora strain the metabolite and associated metabolic network that play a crucial role in pleocidin process, thus for providing optimal anchor direction by the thorn saccharopolyspora strain culture condition improved for the purpose of pleocidin output.
As shown in Figure 1, after with the addition of n-dodecane, the dissolved oxygen level of fermented liquid entirety is improved, and makes dissolved oxygen substantially can maintain more than 25%, and under the same operating conditions, dissolved oxygen when not adding n-dodecane in fermented liquid is on the low side, is minimumly even down to less than 10%.Under the good condition of dissolved oxygen condition, thalli growth is vigorous, and thalli growth speed obviously increases.After adding n-dodecane as shown in Figure 2, glucose consumption rate increases, and pleocidin synthesized in a large number in stationary phase, and the ultimate capacity of pleocidin reaches 381.58mg/L, exceeds 57.8% than control group.
To sting the metaboilic level of saccharopolyspora strain for independent variable(s) matrix under the culture medium culturing condition of adding n-dodecane, pleocidin ability is produced for Y matrix with the thorn saccharopolyspora strain of same sample, PLS analysis is carried out to data and obtains Fig. 3, as seen from Figure 3, add n-dodecane, on the metabolism of thorn saccharopolyspora strain product pleocidin, there is remarkably influenced, and Fig. 4 gives this PLS VIP figure analyzed, the potential metabolite relevant to producing pleocidin ability can be found by VIP figure, as seen from the figure, these metabolites relate to central carbon metabolism (lactic acid, citric acid, oxysuccinic acid, Cori's eater Cori, succsinic acid, α-ketoglutaric acid), phosphopentose pathway (maltonic acid), amino acid metabolism (ALANINE, oxy-acetyl-Serine), polyalcohols (inositol).
The impact of n-dodecane on the metabolism of thorn saccharopolyspora strain product pleocidin is added for understanding further, Fig. 5 is made in the metabolic markers change obtained by Fig. 4, shown in Fig. 5, metabolic markers relates generally to three major types, and organic acid is as lactic acid, oxysuccinic acid, α-ketoglutaric acid, fumaric acid, citric acid, succsinic acid; Amino acid metabolism is as L-Ala, leucine, α-amino-isovaleric acid, proline(Pro), L-glutamic acid, Methionin, aspartic acid, Threonine etc.; Pleocidin precursor as; Cori's eater Cori, L-rhamnosyl.Add n-dodecane, lactic acid metabolism level obviously declines, and illustrates that in born of the same parents, aerobic metabolism is vigorous, indicate cell free fermentation liquid dissolved oxygen level to increase, dissolved oxygen level improves, and pleocidin precursor species metabolite flux is increased, and represents that dissolved oxygen increases the synthesis promoting pleocidin.Amino acids metabolism synthesis flux increases, and amino acid is the fundamental unit of protein synthesis, illustrates that dissolved oxygen increases to promote biomass synthesis, and biomass increases, and also can promote the synthesis of pleocidin.
By upper, substratum dissolved oxygen level produces pleocidin ability to thorn saccharopolyspora strain material impact, in conjunction with associated metabolic change and product pleocidin capability analysis, find to improve dissolved oxygen, make aminoacids content and pleocidin precursor Cori's eater Cori in thorn saccharopolyspora strain born of the same parents, the content of L-rhamnosyl significantly improves, and then pleocidin fermentation yield is improved.Following optimisation strategy can be proposed accordingly and improve pleocidin fermentation yield:
1. optimize n-dodecane addition and improve dissolved oxygen further;
2. during the fermentation, in substratum, add amino acid or rhamnosyl raising pleocidin output
By figure six, the n-dodecane adding 0.2%-2% all can promote the synthesis of pleocidin, but its promoter action is not increase along with the increase adding concentration.When to add the concentration of n-dodecane be 0.5%, the output of pleocidin reaches maximum value, exceeds 57.8% than control group, continues to increase the concentration promoter action of n-dodecane and not obvious.When adding concentration and reaching 4%, can produce restraining effect to the synthesis of pleocidin on the contrary, this may be because n-dodecane excessive concentration inhibits thalli growth.
By figure seven, L-Ala, α-amino-isovaleric acid, leucine, aspartic acid all can promote the synthesis of pleocidin, and wherein L-Ala metabolism mainly produces acetyl-CoA, and the output of pleocidin is comparatively contrasted raising 22%; α-amino-isovaleric acid is converted mainly into succinyl-coenzyme A, is further converted to methylmalonyl CoA and makes output increased 21%; Leucine and aspartic acid are that pleocidin output improves 0.8% and 1.2% respectively.Other amino acid, except proline(Pro), have no significant effect pleocidin output, and the interpolation of proline(Pro) may have restraining effect to thorn saccharopolyspora strain, and pleocidin output is declined to some extent.
To sum up, based on the method for metabolism group, improve thorn saccharopolyspora strain culture process and all achieve beneficial effect to improve pleocidin output, the output of pleocidin is had raising in various degree.
Embodiment 2
Improve thorn saccharopolyspora strain fermentation condition to improve the method for pleocidin output based on metabolism group, comprise the following steps:
(1) metabolite in thorn saccharopolyspora strain born of the same parents is measured:
1. cell harvesting and cancellation:
In substratum 1, fermentation culture is carried out to thorn saccharopolyspora strain, at logarithmic phase 62h, 86h, 110h and stationary phase 134h, 158h and 182h six time points of the fermentation of thorn saccharopolyspora strain, each point in time sampling 4 parts, every increment savors fermented liquid 8ml, institute's sample thief is mixed with 19mL cancellation liquid and places 8min afterwards, at 4000rpm, 4 DEG C, centrifugal 3min, abandons supernatant liquor; With the medium component on 4 DEG C of distilled water wash removing cells; 4000rpm, 4 DEG C, centrifugal 4min, abandons supernatant liquor, retains thalline; Repetitive scrubbing 2 times;
Cancellation liquid is with embodiment 1, and substratum 1 is with embodiment 1;
2. endocellular metabolism thing is extracted:
Get the cell that 1. step obtain and add liquid nitrogen grinding in mortar, take the good cell of 45mg liquid nitrogen grinding and be placed in 1.5mL centrifuge tube, add the extracting solution that 0.8mL temperature is-30 DEG C, vortex vortex mixer fully mixes rear frozen-thawed and obtains freeze thawing liquid 2 times; By freeze thawing liquid at 4 DEG C, 4000rpm, centrifugal 4min, get supernatant liquor and deserve to be called clear liquid a in another centrifuge tube; Lower confluent monolayer cells adds 0.8mL again, temperature is the extracting solution of-30 DEG C, after vortex vortex mixer fully mixes, and 4 DEG C, 9000rpm, centrifugal 4min, get supernatant liquor and deserve to be called clear liquid b, supernatant liquor a and b is mixed to obtain supernatant liquor c, 9000rpm, 4 DEG C, centrifugal 4min, get 260 μ L supernatant liquor c and go to 1.5mL centrifuge tube, adding 50uL concentration is 0.3mgmL
-1succsinic acid-2,2,3,3-d
4the aqueous solution, after vortex vortex mixer fully mixes, under-40 DEG C of conditions, vacuum lyophilization obtains sample, preserves in Ultralow Temperature Freezer;
Extracting solution is with embodiment 1;
3. metabolite derivatize
In the sample that 2. step obtains, add 50 μ L concentration is 20mgmL
-1the pyridine solution of methoxyl group ammonium salt hydrochlorate, in 40 DEG C of water-baths, react 80min, add the trimethyl silicon based trifluoroacetamide of 80 μ L N-methyl-N-, in 40 DEG C of water-bath 40min, put into numbered GC sample injection bottle, room temperature leaves standstill 2h;
4. GC-MS detects
GC-MS is adopted to carry out quantitative and qualitative analysis process to sample, with embodiment 1 step (1) 4. GC-MS detection;
(2) produce pleocidin ability to thorn saccharopolyspora strain to analyze:
1. cell harvesting and cancellation:
In substratum 2, fermentation culture is carried out to thorn saccharopolyspora strain, at logarithmic phase 62h, 86h, 110h and stationary phase 134h, 158h and 182h six time points of the fermentation of thorn saccharopolyspora strain, each point in time sampling 4 parts, every increment savors fermented liquid 8ml, institute's sample thief is mixed with 18mL cancellation liquid and places 8min afterwards, at 4000rpm, 4 DEG C, centrifugal 4min, abandons supernatant liquor; With the medium component on 4 DEG C of distilled water wash removing cells; 4000rpm, 4 DEG C, centrifugal 4min, abandons supernatant liquor, retains thalline; Repetitive scrubbing 2 times;
Cancellation liquid to be volumetric concentration be 60% methanol aqueous solution;
Substratum 2 is: n-dodecane 3ml/L, glucose 72g/L, corn steep liquor 12g/L, Sodium Glutamate 2.4g/L, bean powder 18g/L, and surplus is water, adjusts pH=7.5, sterilizing;
2. endocellular metabolism thing is extracted:
Get the cell that 1. step obtain and add liquid nitrogen grinding in mortar, take the good cell of 45mg liquid nitrogen grinding and be placed in 1.5mL centrifuge tube, add the extracting solution that 0.8mL temperature is-30 DEG C, vortex vortex mixer fully mixes rear frozen-thawed and obtains freeze thawing liquid 2 times; By freeze thawing liquid at 4 DEG C, 4000rpm, centrifugal 4min, get supernatant liquor d in another centrifuge tube; Lower confluent monolayer cells adds 0.8mL again, temperature is the extracting solution of-30 DEG C, after vortex vortex mixer fully mixes, and 4 DEG C, 9000rpm, centrifugal 4min, get supernatant liquor e, supernatant liquor d and e is mixed, 9000rpm, 4 DEG C, centrifugal 4min, get 260 μ L supernatant liquor f and go to 1.5mL centrifuge tube, adding 50uL concentration is 0.3mgmL
-1succsinic acid-2,2,3,3-d
4the aqueous solution, after vortex vortex mixer fully mixes, under-40 DEG C of conditions, vacuum lyophilization obtains sample, preserves in Ultralow Temperature Freezer; Described extracting solution is volumetric concentration is 50% methanol aqueous solution;
Extracting solution is volumetric concentration is 50% methanol aqueous solution;
3. metabolite derivatize
In the sample that 2. step obtains, add 50 μ L concentration is 20mgmL
-1the pyridine solution of methoxyl group ammonium salt hydrochlorate, in 40 DEG C of water-baths, react 90min, add the trimethyl silicon based trifluoroacetamide of 80 μ L N-methyl-N-, in 40 DEG C of water-bath 40min, put into numbered GC sample injection bottle, room temperature leaves standstill 2h;
4. GC-MS detects
GC-MS is adopted to carry out quantitative and qualitative analysis process to sample, with embodiment 1 step (2) 4.;
5. born of the same parents' intracellular metabolite thing qualitative and quantitative analysis
With embodiment 1 step (2) 5.;
(3) PLS analyzes
With embodiment 1 step (3);
(4) process analysis
The content of metabolite markers step (3) obtained makes chart according to culture condition difference, observe and analyze these metabolites change rule, and then find to produce at thorn saccharopolyspora strain the metabolite and associated metabolic network that play a crucial role in pleocidin process, thus for providing optimal anchor direction by the thorn saccharopolyspora strain culture condition improved for the purpose of pleocidin output.
Prove by experiment, result is similar to embodiment 1.
Embodiment 3
Improve thorn saccharopolyspora strain fermentation condition to improve the method for pleocidin output based on metabolism group, comprise the following steps:
(1) metabolite in thorn saccharopolyspora strain born of the same parents is measured:
1. cell harvesting and cancellation:
In substratum 1, fermentation culture is carried out to thorn saccharopolyspora strain, at logarithmic phase 62h, 86h, 110h and stationary phase 134h, 158h and 182h six time points of the fermentation of thorn saccharopolyspora strain, each point in time sampling 5 parts, every increment savors fermented liquid 10ml, institute's sample thief is mixed with 20mL cancellation liquid and places 10min afterwards, at 5000rpm, 4 DEG C, centrifugal 5min, abandons supernatant liquor; With the medium component on 4 DEG C of distilled water wash removing cells; 5000rpm, 4 DEG C, centrifugal 5min, abandons supernatant liquor, retains thalline; Repetitive scrubbing 4 times;
Cancellation liquid to be volumetric concentration be 60% methanol aqueous solution;
Substratum 1 is: glucose 72g/L, corn steep liquor 12g/L, Sodium Glutamate 2.4g/L, bean powder 18g/L, and surplus is water, adjusts pH=7.5, sterilizing;
2. endocellular metabolism thing is extracted:
Get the cell that 1. step obtain and add liquid nitrogen grinding in mortar, take the good cell of 50mg liquid nitrogen grinding and be placed in 1.5mL centrifuge tube, add the extracting solution that 1mL temperature is-30 DEG C, vortex vortex mixer fully mixes rear frozen-thawed and obtains freeze thawing liquid 4 times; By freeze thawing liquid at 4 DEG C, 5000rpm, centrifugal 5min, get supernatant liquor and deserve to be called clear liquid a in another centrifuge tube; Lower confluent monolayer cells adds the extracting solution that 1mL temperature is-30 DEG C again, after vortex vortex mixer fully mixes, 4 DEG C, 10000rpm, centrifugal 5min, get supernatant liquor and deserve to be called clear liquid b, supernatant liquor a and b is mixed to obtain supernatant liquor c, 10000rpm, 4 DEG C, centrifugal 5min, get 260 μ L supernatant liquor c and go to 1.5mL centrifuge tube, adding 50uL concentration is 0.3mgmL
-1succsinic acid-2,2,3,3-d
4the aqueous solution, after vortex vortex mixer fully mixes, under-40 DEG C of conditions, vacuum lyophilization obtains sample, preserves in Ultralow Temperature Freezer;
Described extracting solution is volumetric concentration is 50% methanol aqueous solution;
3. metabolite derivatize
In the sample that 2. step obtains, add 50 μ L concentration is 20mgmL
-1the pyridine solution of methoxyl group ammonium salt hydrochlorate, in 40 DEG C of water-baths, react 100min, add the trimethyl silicon based trifluoroacetamide of 80 μ L N-methyl-N-, in 40 DEG C of water-bath 60min, put into numbered GC sample injection bottle, room temperature leaves standstill 2h;
4. GC-MS detects
GC-MS is adopted to carry out quantitative and qualitative analysis process to sample, with embodiment 1 step (1) 4. GC-MS detection;
(2) produce pleocidin ability to thorn saccharopolyspora strain to analyze:
1. cell harvesting and cancellation:
In substratum 2, fermentation culture is carried out to thorn saccharopolyspora strain, at logarithmic phase 62h, 86h, 110h and stationary phase 134h, 158h and 182h six time points of the fermentation of thorn saccharopolyspora strain, each point in time sampling 5 parts, every increment savors fermented liquid 10ml, institute's sample thief is mixed with 20mL cancellation liquid and places 10min afterwards, at 5000rpm, 4 DEG C, centrifugal 5min, abandons supernatant liquor; With the medium component on 4 DEG C of distilled water wash removing cells; 5000rpm, 4 DEG C, centrifugal 5min, abandons supernatant liquor, retains thalline; Repetitive scrubbing 4 times; Cancellation liquid,
Substratum 2 is: n-dodecane 5ml/L, glucose 72g/L, corn steep liquor 12g/L, Sodium Glutamate 2.4g/L, bean powder 18g/L, and surplus is water, adjusts pH=7.5, sterilizing;
2. endocellular metabolism thing is extracted:
Get the cell that 1. step obtain and add liquid nitrogen grinding in mortar, take the good cell of 50mg liquid nitrogen grinding and be placed in 1.5mL centrifuge tube, add the extracting solution that 1mL temperature is-30 DEG C, vortex vortex mixer fully mixes rear frozen-thawed and obtains freeze thawing liquid 4 times; By freeze thawing liquid at 4 DEG C, 5000rpm, centrifugal 5min, get supernatant liquor and deserve to be called clear liquid d in another centrifuge tube; Lower confluent monolayer cells adds 1mL again, temperature is the extracting solution of-30 DEG C, after vortex vortex mixer fully mixes, and 4 DEG C, 10000rpm, centrifugal 5min, get supernatant liquor and deserve to be called clear liquid e, supernatant liquor d and e is mixed to obtain supernatant liquor f, 10000rpm, 4 DEG C, centrifugal 5min, get 260 μ L supernatant liquor f and go to 1.5mL centrifuge tube, adding 50uL concentration is 0.3mgmL
-1succsinic acid-2,2,3,3-d
4the aqueous solution, after vortex vortex mixer fully mixes, under-40 DEG C of conditions, vacuum lyophilization obtains sample, preserves in Ultralow Temperature Freezer; Described extracting solution is volumetric concentration is 50% methanol aqueous solution;
3. metabolite derivatize
In the sample that 2. step obtains, add 50 μ L concentration is 20mgmL
-1the pyridine solution of methoxyl group ammonium salt hydrochlorate, in 40 DEG C of water-baths, react 100min, add the trimethyl silicon based trifluoroacetamide of 80 μ L N-methyl-N-, in 40 DEG C of water-bath 60min, put into numbered GC sample injection bottle, room temperature leaves standstill 2h;
4. GC-MS detects
GC-MS is adopted to carry out quantitative and qualitative analysis process to sample, with embodiment 1 step (2) 4.;
5. born of the same parents' intracellular metabolite thing qualitative and quantitative analysis
With embodiment 1 step (2) 5.;
(3) PLS analyzes
With embodiment 1 step (3);
(4) process analysis
The content of metabolite markers step (3) obtained makes chart according to culture condition difference, observe and analyze these metabolites change rule, and then find to produce at thorn saccharopolyspora strain the metabolite and associated metabolic network that play a crucial role in pleocidin process, thus for providing optimal anchor direction by the thorn saccharopolyspora strain culture condition improved for the purpose of pleocidin output.
Prove by experiment, result is similar to embodiment 1.
Claims (1)
1. improve thorn saccharopolyspora strain fermentation condition to improve the method for pleocidin output based on metabolism group, it is characterized in that comprising the following steps:
(1) metabolite in thorn saccharopolyspora strain born of the same parents is measured:
1. cell harvesting and cancellation:
In substratum 1, fermentation culture is carried out to thorn saccharopolyspora strain, in logarithmic phase and the stationary phase of the fermentation of thorn saccharopolyspora strain, each period at least one point in time sampling 5-10ml, each point in time sampling 3-5 part, institute's sample thief is mixed with 18-20mL cancellation liquid and places 5-10min afterwards, at 3000-5000rpm, 4 DEG C, centrifugal 3-5min, abandons supernatant liquor; With the medium component on 4 DEG C of distilled water wash removing cells; 3000-5000rpm, 4 DEG C, centrifugal 3-5min, abandons supernatant liquor, retains thalline; Repetitive scrubbing 2-4 time; Described cancellation liquid to be volumetric concentration be 60% methanol aqueous solution; Described substratum 1 is: glucose 72g/L, corn steep liquor 12g/L, Sodium Glutamate 2.4g/L, bean powder 18g/L, and surplus is water, adjusts pH=7.5, sterilizing;
2. endocellular metabolism thing is extracted:
Get the cell that 1. step obtain and add liquid nitrogen grinding in mortar, take the good cell of 40-50mg liquid nitrogen grinding and be placed in 1.5mL centrifuge tube, add the extracting solution that 0.5-1mL temperature is-30 DEG C, vortex vortex mixer fully mixes rear frozen-thawed and obtains freeze thawing liquid for 2-4 time; By freeze thawing liquid at 4 DEG C, 3000-5000rpm, centrifugal 3-5min, get supernatant liquor and deserve to be called clear liquid a in another centrifuge tube; Lower confluent monolayer cells adds 0.5-1mL again, temperature is the extracting solution of-30 DEG C, after vortex vortex mixer fully mixes, and 4 DEG C, 8000-10000rpm, centrifugal 3-5min, get supernatant liquor and deserve to be called clear liquid b, supernatant liquor a and b is mixed to obtain supernatant liquor c, 8000-10000rpm, 4 DEG C, centrifugal 3-5min, get 260 μ L supernatant liquor c and go to 1.5mL centrifuge tube, adding 50uL concentration is 0.3mgmL
-1succsinic acid-2,2,3,3-d
4the aqueous solution, after vortex vortex mixer fully mixes, under-40 DEG C of conditions, vacuum lyophilization obtains sample, preserves in Ultralow Temperature Freezer; Described extracting solution is volumetric concentration is 50% methanol aqueous solution;
3. metabolite derivatize
In the sample that 2. step obtains, add 50 μ L concentration is 20mgmL
-1the pyridine solution of methoxyl group ammonium salt hydrochlorate, in 40 DEG C of water-baths, react 70-100min, add the trimethyl silicon based trifluoroacetamide of 80 μ L N-methyl-N-, in 40 DEG C of water-bath 20-60min, put into numbered GC sample injection bottle, room temperature leaves standstill 2h;
4. GC-MS detects
Adopt GC-MS to step 3. gained sample carry out quantitative and qualitative analysis process, condition is as follows:
Chromatographic column: silicon chromatographic column, HP-5MS, 30m × 0.25mm, 0.25 μm;
Sample size: 1 μ L; ;
Splitting ratio is 1:10;
Carrier gas: high-purity helium, purity: 99.9995%;
Injector temperature: 280 DEG C;
GC – MS interface temperature: 280 DEG C;
Helium flow velocity: constant voltage, 91KPa;
Heating schedule: 70 DEG C keep 2min, with 5 DEG C of min
-1speed be raised to 290 DEG C, and keep 3min at 290 DEG C, then cool to 30 DEG C;
Ionization mode: electron impact ionization;
Ion(ic)current: 40 μ A;
Ionization voltage: 70eV;
Source temperature: 250 DEG C;
Sweep limit: 50-800m/z;
Sweep velocity: 2scans
-1;
Thus obtain the metabolite quantitative and qualitative analysis data of thorn saccharopolyspora strain;
(2) produce pleocidin ability to thorn saccharopolyspora strain to analyze:
1. cell harvesting and cancellation:
In substratum 2, fermentation culture is carried out to thorn saccharopolyspora strain, respectively at logarithmic phase and at least one point in time sampling 5-10ml of each phase of stationary phase of the fermentation of thorn saccharopolyspora strain, each point in time sampling 3-5 part, institute's sample thief is mixed with 18-20mL cancellation liquid and places 5-10min afterwards, at 3000-5000rpm, 4 DEG C, centrifugal 3-5min, abandons supernatant liquor; With the medium component on 4 DEG C of distilled water wash removing cells; 3000-5000rpm, 4 DEG C, centrifugal 3-5min, abandons supernatant liquor, retains thalline; Repetitive scrubbing 2-4 time; Described cancellation liquid to be volumetric concentration be 60% methanol aqueous solution; Described substratum 2 is: n-dodecane 2-5ml/L, glucose 72g/L, corn steep liquor 12g/L, Sodium Glutamate 2.4g/L, bean powder 18g/L, and surplus is water, adjusts pH=7.5, sterilizing;
2. endocellular metabolism thing is extracted:
Get the cell that 1. step obtain and add liquid nitrogen grinding in mortar, take the good cell of 40-50mg liquid nitrogen grinding and be placed in 1.5mL centrifuge tube, add the extracting solution that 0.5-1mL temperature is-30 DEG C, vortex vortex mixer fully mixes rear frozen-thawed and obtains freeze thawing liquid for 2-4 time; By freeze thawing liquid at 4 DEG C, 3000-5000rpm, centrifugal 3-5min, get supernatant liquor d in another centrifuge tube; Lower confluent monolayer cells adds the extracting solution that 0.5-1mL temperature is-30 DEG C again, after vortex vortex mixer fully mixes, 4 DEG C, 8000-10000rpm, centrifugal 3-5min, get supernatant liquor e, supernatant liquor d and e is mixed, 8000-10000rpm, 4 DEG C, centrifugal 3-5min, get 260 μ L supernatant liquor f and go to 1.5mL centrifuge tube, adding 50uL concentration is 0.3mgmL
-1succsinic acid-2,2,3,3-d
4the aqueous solution, after vortex vortex mixer fully mixes, under-40 DEG C of conditions, vacuum lyophilization obtains sample, preserves in Ultralow Temperature Freezer; Described extracting solution is volumetric concentration is 50% methanol aqueous solution;
3. metabolite derivatize
In the sample that 2. step obtains, add 50 μ L concentration is 20mgmL
-1the pyridine solution of methoxyl group ammonium salt hydrochlorate, in 40 DEG C of water-baths, react 70-100min, add the trimethyl silicon based trifluoroacetamide of 80 μ L N-methyl-N-, in 40 DEG C of water-bath 20-60min, put into numbered GC sample injection bottle, room temperature leaves standstill 2h;
4. GC-MS detects
Adopt GC-MS to step 3. gained sample carry out quantitative and qualitative analysis process, condition is as follows:
Chromatographic column: silicon chromatographic column, HP-5MS, 30m × 0.25mm, 0.25 μm;
Sample size: 1 μ L; ;
Splitting ratio is 1:10;
Carrier gas: high-purity helium, purity: 99.9995%;
Injector temperature: 280 DEG C;
GC – MS interface temperature: 280 DEG C;
Helium flow velocity: constant voltage, 91KPa;
Heating schedule: 70 DEG C keep 2min, with 5 DEG C of min
-1speed be raised to 290 DEG C, and keep 3min at 290 DEG C, then cool to 30 DEG C;
Ionization mode: electron impact ionization;
Ion(ic)current: 40 μ A;
Ionization voltage: 70eV;
Source temperature: 250 DEG C;
Sweep limit: 50-800m/z;
Sweep velocity: 2scans
-1;
Thus obtain the metabolite quantitative and qualitative analysis data of thorn saccharopolyspora strain;
5. born of the same parents' intracellular metabolite thing qualitative and quantitative analysis
Mzmine is used to carry out spectrum unscrambling and peak alignment to the data of GC-MS, mass spectra peak is deconvoluted by AMDIS storehouse, peak width is at more than 2.0s, and signal to noise ratio higher than 10 peak be just considered to the peak of metabolite, by mass spectrometric detection peak and business-like mass spectral database NIST database are compared, detected metabolite is identified, by built-in program, automatic integration is carried out and manual correction to the peak of each metabolite in total ion current figure, thus obtain the data of internal standard substance and each metabolite peak area, then, by peak area quantitative in the unit mass color atlas of the characteristic ion of often kind of metabolite divided by the internal standard substance succsinic acid-2 on same spectrogram, 2, 3, 3-d
4peak area be able to stdn, and calculate the relative content of each metabolite, the relative abundance of often kind of metabolite can be obtained,
(3) PLS analyzes
Use centralization and sized method to carry out pre-treatment to the metabolite relative content data obtained in (1) and (2), then use SIMCA-P 11.5Demo software to carry out partial least squares discriminant analysis to the data obtained;
(4) process analysis
The content of metabolite markers step (3) obtained makes chart according to culture condition difference, observe and analyze these metabolites change rule, and then find to produce at thorn saccharopolyspora strain the metabolite and associated metabolic network that play a crucial role in pleocidin process, thus for providing optimal anchor direction by the thorn saccharopolyspora strain culture condition improved for the purpose of pleocidin output.
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