CN103725728B - The preparation method of a kind of Bacillamide compound and Bacillamide precursor - Google Patents
The preparation method of a kind of Bacillamide compound and Bacillamide precursor Download PDFInfo
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Abstract
The invention discloses a kind of method of a large amount of production Bacillamide compound and Bacillamide precursor, comprising: actinomycetes are inoculated in fermention medium, cultivate 5 ~ 10 days; Be inoculated in by bacillus mycoides in fermention medium, symbiosis culture 10 ~ 20 days, obtains Dual culture fermention medium, after being separated, obtain Bacillamide compound and Bacillamide precursor.The inventive method, require low to substratum, simple to operate, industrial production cost is low, and tunning is simple, active constituent content is high, foreign matter content is few, not only reduces cost, and can also produce in a large number and obtain Bacillamide compound and Bacillamide precursor, have very high using value, industrial production feasibility is large.
Description
Technical field
The present invention relates to the field that microbial culture prepares compound, be specifically related to the preparation method of a kind of a large amount of production Bacillamide compound and Bacillamide precursor.
Background technology
The red tide not only havoc marine eco-environment, marine fishery and oceanic resources, and the various toxin that red tide brings, as research of diarrhetic shellfish poisons, Toxin-Domoic Acid, paralytic shellfish poisoning (PSP) etc., the health of the serious harm mankind.To the improvement of algae reproduction in red tide and aquaculture water, propose many methods both at home and abroad, physical method, biological method and chemical process can be divided into.Physical method generally uses the throwing out sedimentation algae of modified clay, but this method is cured the symptoms, not the disease, and algae also can amount reproduction.Biological method utilizes complex microorganism preparations, suppresses the growth of algae, and this method effect is strong, can have certain application in aquaculture, but the seashore that can contact for the mankind and lake, the input of a large amount of pathogenic microorganisms, and infeasible.Chemical process has and uses the algicide of copper ions, and the chemical herbicide of a few species, but large quantity research shows, cupric ion and existing chemical algicide, to the effect as all toxic in fish and people of non-targeted biology, effective but dangerous.In chemical process, also have the another kind of natural algicide coming from the Nature, this type of algicide inactivation of algae is strong, and toxic side effect is little, be well suited for the control being applied to algal bloom, but the natural algicide that at present algae killing effect that finds of investigator is strong is few.The mixture of Bacillamide compound and Bacillamide precursor is the natural algicide that a kind of algae killing effect is strong.
Bacillamide compound mainly comprises Bacillamide A, Bacillamide B and Bacillamide C, and its structural formula is as follows:
Bacillamide precursor, i.e. N-Acetriptine, its structural formula is as follows:
They are from bacillus marinus or actinomycetes, be separated the indoles alkaloid containing thiazole obtained, research finds that Bacillamide composition has good inhibition to red tide algae, if Bacillamide A, B, C are to half effect concentration (Effect concentration, the EC of Skeletonema Costatum
50,72h) be respectively 0.011mg/L, 15.59mg/L and 34.70mg/L, to the EC of Gymnodinium breve
50,72hbe respectively 112mg/L, 234.56mg/L and 57.13mg/L, different Bacillamide compounds is different to different algae inhibit activities, and therefore, as algicide, the mixture of Bacillamide compound and Bacillamide precursor is better than monomeric compound.Separately there are some researches show, the mixture of Bacillamide compound and Bacillamide precursor to other bacterium, fungi unrestraint effect, and to human cell's nontoxicity, is a class very promising natural product in red-tide control.
But it is considerably less that regrettably, conventional fermentation process is separated the Bacillamide compounds amount obtained.Publication number is CN101948891A(application number is 201010278110.6) Chinese patent disclose a kind of substratum utilizing fermentation atrophy genus bacillus to prepare Bcallamide C, be specially 3.64g/L W-Gum, 6.0g/L Tryptones, 6.29g/L calcium carbonate, 4.0g/L soybean protein, 0.10g/L halfcystine, 0.02g/L tryptophane, solvent is artificial seawater, the maximum production that its fermentation produces Bacillamide C is 22.197mg/L, the method substratum used is nutritious, cost is high, but can only obtain a Bacillamide compound.Its tunning is analyzed (Fig. 1 of this Figure of description) at the HPLC of 220nm, and the peak of arrow indication is Bacillamide C, as seen from the figure, although the output increased of Bacillamide C, impurity peaks is also many.Therefore, a kind of method that simultaneously can obtain the mixture of Bacillamide compound and Bacillamide precursor with low cost in a large number, will have very high using value.
Microorganism can express many important secondary metabolites, but in its natural state, the amount extremely pettiness that secondary metabolite is expressed, only when environmental change or rival occur, the amount of expression just can increase.Dual culture is as a kind of mode increasing microbial secondary meta-bolites and express, and its application also has many problems, such as, does not have hormesis between bacterial strain, or has the effect that stimulates meta-bolites to express but co-culture system is unstable, poor repeatability etc.A foundation for stable co-culture system, can not only increase the expression of active secondary metabolites, and greatly can improve the transformation efficiency of medium nutrient content, reduces production cost, improves resource utilization.
Summary of the invention
The present invention is directed to the application demand of Bacillamide compound and Bacillamide precursor and existing methodical deficiency, provide a kind of method of a large amount of production Bacillamide compound and Bacillamide precursor.
A preparation method for Bacillamide compound and Bacillamide precursor, comprises the following steps:
1) actinomycetes are inoculated in fermention medium, cultivate 5 ~ 10 days;
2) be inoculated in by bacillus mycoides in the fermention medium in step 1), symbiosis culture 10 ~ 20 days, obtains Dual culture fermention medium, after being separated, obtain Bacillamide compound and Bacillamide precursor.
For meeting the stable growth of bacterium in physical environment, as preferably, step 1) and step 2) in, described cultivation is quiescent culture.Under quiescent culture, the Dual culture of actinomycetes and bacillus mycoides can produce Bacillamide composition in a large number.Namely quiescent culture is adopted in the present invention.
In step 1), described fermention medium, for can make actinomycetes and the well-grown substratum of bacillus mycoides simultaneously, described fermention medium can adopt prior art, as ISP2(international streptomyces project medium2) substratum or Gao Shi substratum.As preferably, in fermention medium 1L, the concrete formula of described fermention medium is:
By acid-base buffer, this fermention medium is regulated pH to 7-8.This fermention medium nutrition is lower, and nitrogenous source is less, can make actinomycetes and bacillus mycoides well-grown simultaneously, and mutually promote generation Bacillamide compound and Bacillamide precursor.
In step 1), described actinomycetes, prioritizing selection streptomycete, the streptomyces nodocus (Streptomyces nodosus1A0101684) that Beijing North Na Chuanlian Bioteknologisk Institut specifically can be selected commercially available, variant streptomycete (Streptomyces variabilis CGMCC4.798) or streptomycete (Streptomyces sp.MCC1A03740).
Step 2) in, the symbiosis stage is that the bacillus mycoides of inoculation utilizes residue in environment to nourish and grow, breeds, induces actinomycetes to produce the stage of secondary metabolite.The described symbiosis stage terminates, and mark is that bacillus mycoides is dead, thalline melts and actinomycetes produce aerial hyphae.After the symbiosis stage terminates, in substratum, liquid clarification, is covered with aerial hyphae, is then separated by culture medium flat plate method of scoring.
The bacillus mycoides (Bacillus mycoides CGMCC1.197) that described bacillus mycoides specifically can select Beijing North Na Chuanlian Bioteknologisk Institut commercially available.The bacillus mycoides of inoculation will be cultivated 18 ~ 24 hours at 100 ~ 200 revs/min of shaking tables.
Step 2) in, the inoculation volume of described bacillus mycoides is the 0.1%-5% of fermention medium volume.More preferably 0.1%-0.75%.
Step 2) in, described separation comprises: be extracted with ethyl acetate by Dual culture fermention medium, and concentrated, obtain ethyl acetate extract, ethyl acetate extract is the mixture containing Bacillamide compound and Bacillamide precursor.
The mixture of Bacillamide compound and Bacillamide precursor can adopt reversed phase chromatography separation method or normal-phase chromatography partition method to obtain Bacillamide compound and Bacillamide precursor.Namely described ethyl acetate extract adopts reversed phase chromatography separation method or normal-phase chromatography partition method to obtain Bacillamide compound and Bacillamide precursor.
The filler that described reversed phase chromatography separation method adopts is octadecyl silane, and the moving phase of employing is methanol-water.
Described reversed phase chromatography separation method detailed process is: first remove large polar impurity with the methanol aqueous solution wash-out formed by volume ratio 20 ~ 30:80 ~ 70 first alcohol and water, obtain Bacillamide precursor with the methanol aqueous solution wash-out that volume ratio 35 ~ 45:65 ~ 55 first alcohol and water is formed again, then obtain Bacillamide compound with the methanol aqueous solution wash-out that volume ratio 50 ~ 70:50 ~ 30 first alcohol and water is formed.Bacillamide compound is obtained with the methanol aqueous solution wash-out that volume ratio 50 ~ 70:50 ~ 30 first alcohol and water is formed, specifically comprise: obtain Bacillamide compd B and C with the methanol aqueous solution wash-out that volume ratio 50:50 first alcohol and water is formed, obtain Bacillamide compd A with the methanol aqueous solution wash-out that volume ratio 60:40 first alcohol and water is formed.
The filler that described normal-phase chromatography partition method adopts is silica gel, and the moving phase of employing is methylene chloride-methanol.
Described normal-phase chromatography partition method detailed process is: first remove little polar impurity with by the methylene dichloride of volume ratio 100:1 and the eluant solution of the formation of methanol, then obtains Bacillamide compound and Bacillamide precursor with the methylene dichloride of volume ratio 80 ~ 25:1 and the eluant solution of the formation of methanol.Bacillamide compound and Bacillamide precursor is obtained with the methylene dichloride of volume ratio 80 ~ 25:1 and the eluant solution of the formation of methanol, specifically comprise: first obtain Bacillamide compd A with the eluant solution of volume ratio 80:1 methylene dichloride and the formation of methanol, obtain Bacillamide precursor and Bacillamide compd B (in chronological sequence eluting) with the eluant solution of volume ratio 50:1 methylene dichloride and the formation of methanol again, finally obtain Bacillamide Compound C with the eluant solution of volume ratio 25:1 methylene dichloride and the formation of methanol.
Described Bacillamide compound comprises Bacillamide A, Bacillamide B and Bacillamide C, and its structural formula is as follows:
Described Bacillamide precursor, i.e. N-Acetriptine, its structural formula is as follows:
The preparation method of Bacillamide compound provided by the invention and Bacillamide precursor, based on microorganism in extraneous environmental change or there is rival to deposit in case, the principle that the expression originally expressing the active secondary metabolites of denier can increase greatly.But different strains Dual culture, as a kind of method stimulating microorganism great expression secondary metabolite, also exists a lot of problem, the effect of such as co-culture system increase secondary metabolite expression, poor repeatability, culturing process do not have evaluation index etc.And the preparation method of Bacillamide compound provided by the invention and Bacillamide precursor, effectively preparation Bacillamide compound and Bacillamide precursor in the co-culture system of expressing with a kind of good stability, repeatability is high, suitability is strong enhancing secondary metabolite.
Compared with prior art, the invention has the beneficial effects as follows:
One, the preparation method of Bacillamide compound of the present invention and Bacillamide precursor, a large amount of Bacillamide compounds and Bacillamide precursor can be produced, require low to substratum, simple to operate, industrial production cost is low, and tunning is simple, active constituent content is high, foreign matter content is few, compare the method that traditional zymotic atrophy genus bacillus prepares Bcallamide C, not only reduce cost, and can also produce in a large number and obtain Bacillamide compound and Bacillamide precursor, there is very high using value, industrial production feasibility is large.
Two, the Bacillamide compound prepared of the present invention, contains Bacillamide A, Bacillamide B and Bacillamide C.Because different Bacillamide compounds is different to different algae inhibit activities, activity and adaptability that therefore Bacillamide composition is killing algae aspect will be better than single Bacillamide compound, therefore the present invention is very large as the application prospect of algicide.
Three, the Bacillamide precursor prepared of the present invention, i.e. N-Acetriptine, can be used as the raw material of Bacillamide biology, chemosynthesis, and also can be used as the synthesis material of melatonin N-ethanoyl 5-methoxytryptamine, using value is high.
Four, the preparation method of Bacillamide compound of the present invention and Bacillamide precursor, select first alcohol and water to be eluting solvent system, solvent price is relatively cheap, and preparation method is simple, be applicable to very much a large amount of preparations of Bacillamide compound and Bacillamide precursor, application prospect is large.
Accompanying drawing explanation
Fig. 1 is the high-efficient liquid phase chromatogram of the ethyl acetate extract of A group preparation in embodiment 1;
Fig. 2 is the high-efficient liquid phase chromatogram of the ethyl acetate extract of B group preparation in embodiment 1;
Fig. 3 is the high-efficient liquid phase chromatogram of the ethyl acetate extract of C group preparation in embodiment 1;
Fig. 4 is the high-efficient liquid phase chromatogram of the ethyl acetate extract of D group preparation in embodiment 1;
Fig. 5 is the high-efficient liquid phase chromatogram of ethyl acetate extract prepared by comparative example 1;
Fig. 6 is the high-efficient liquid phase chromatogram of ethyl acetate extract prepared by comparative example 2;
Fig. 7 is the high-efficient liquid phase chromatogram of ethyl acetate extract prepared by embodiment 2;
Fig. 8 is the high-efficient liquid phase chromatogram of the Bacillamide compound of embodiment 3 preparation and the mixture of Bacillamide precursor;
Fig. 9 is the high-efficient liquid phase chromatogram of ethyl acetate extract prepared by embodiment 4;
Figure 10 is the high-efficient liquid phase chromatogram of ethyl acetate extract prepared by embodiment 5.
Embodiment
Microorganism (the streptomyces nodocus Streptomyces nodosus1A0101684 related in the present invention, bacillus mycoides Bacillus mycoides CGMCC1.197, variant streptomycete Streptomyces variabilis CGMCC4.798, streptomycete Streptomyces sp.MCC1A03740) all purchased from Beijing North Na Chuanlian Bioteknologisk Institut.
Embodiment 1
1) by actinomycetes (streptomyces nodocus Streptomyces nodosus1A0101684, Beijing North Na Chuanlian Bioteknologisk Institut) be inoculated in 500mL Erlenmeyer flask, every bottle contains 200mL fermention medium (in fermention medium 1L, yeast extract 5g, glycerine 4g, Zulkovsky starch 1g, surplus is seawater, by acid-base buffer, this fermention medium is regulated pH to 7.5) in, divide ABCD tetra-groups, wherein A group and B group quiescent culture 6 days at 28 DEG C, C group and D group quiescent culture 10 days at 28 DEG C;
2) bacillus mycoides (the Bacillus mycoides CGMCC1.197 of 24 hours will be cultivated through 150 revs/min of shaking tables, from Beijing North Na Chuanlian Bioteknologisk Institut) be inoculated in step 1) and grown in actinomycetic fermention medium, wherein, A group and C group inoculation volume are 400 μ L, B group and D group inoculation volume are 1000 μ L, at 28 DEG C, symbiosis quiescent culture 15 days, obtains Dual culture fermention medium;
3) the Dual culture fermention medium of each group of gained is utilized extraction into ethyl acetate, be concentrated into dry, obtain ethyl acetate extract, the ethyl acetate extract efficient liquid phase chromatographic analysis that gained is respectively organized, filler is octadecyl silane, take methanol-water as moving phase, flow velocity 0.6mL/min, elution program is: 0-5min, 5%(volume fraction) methyl alcohol; 5-100min, 5%-100%(volume fraction) methyl alcohol; 100-120min, 100%(volume fraction) methyl alcohol carries out gradient elution, and determined wavelength is 220nm.The high-efficient liquid phase chromatogram of the ethyl acetate extract of A group as shown in Figure 1, the high-efficient liquid phase chromatogram of the ethyl acetate extract of B group as shown in Figure 2, as shown in Figure 3, the high-efficient liquid phase chromatogram of the ethyl acetate extract of D group as shown in Figure 4 for the high-efficient liquid phase chromatogram of the ethyl acetate extract of C group.From Fig. 1, Fig. 2, Fig. 3 and Fig. 4, under different fermentations condition, fermented ingredient good stability.
After testing, with 200mL fermention medium for benchmark, in the ethyl acetate extract that A group obtains, the ultimate production of Bacillamide compound and Bacillamide precursor is 31mg/L.In the ethyl acetate extract that B group obtains, the ultimate production of Bacillamide compound and Bacillamide precursor is 38mg/L.
In the ethyl acetate extract that C group obtains, the ultimate production of Bacillamide compound and Bacillamide precursor is 36mg/L.
In the ethyl acetate extract that D group obtains, the ultimate production of Bacillamide compound and Bacillamide precursor is 40mg/L.
Embodiment 2
1) by actinomycetes (streptomyces nodocus Streptomyces nodosus1A0101684, Beijing North Na Chuanlian Bioteknologisk Institut) be inoculated in 500mL Erlenmeyer flask, every bottle contains 250mL fermention medium (in fermention medium 1L, yeast extract 5g, glycerine 5g, surplus is seawater, by acid-base buffer, this fermention medium is regulated pH to 7.5) in, quiescent culture 7 days at 28 DEG C;
2) bacillus mycoides (the Bacillus mycoides CGMCC1.197 of 24 hours will be cultivated through 140 revs/min of shaking tables, from Beijing North Na Chuanlian Bioteknologisk Institut) be inoculated in step 1) and grown in actinomycetic substratum, inoculum is long-pending is respectively 1000 μ L, at 28 DEG C, symbiosis quiescent culture 15 days, obtains Dual culture fermention medium;
3) the Dual culture fermention medium of each group of gained is utilized extraction into ethyl acetate, be concentrated into dry, obtain ethyl acetate extract.
Be benchmark with fermention medium, in the ethyl acetate extract obtained, the ultimate production of Bacillamide compound and Bacillamide precursor is 36mg/L.
Comparative example 1
1) by actinomycetes (streptomyces nodocus Streptomyces nodosus1A0101684, Beijing North Na Chuanlian Bioteknologisk Institut) be inoculated in 500mL Erlenmeyer flask, every bottle contains 250mL fermention medium (in fermention medium 1L, yeast extract 5g, glycerine 4g, surplus is seawater, by acid-base buffer, this fermention medium is regulated pH to 7.5) in, quiescent culture 7 days at 28 DEG C;
2) quiescent culture 15 days at 28 DEG C, obtains the fermention medium of streptomyces nodocus single culture;
3) fermention medium of streptomyces nodocus single culture is utilized extraction into ethyl acetate, be concentrated into dry, obtain ethyl acetate extract.
Be benchmark with fermention medium, in the ethyl acetate extract obtained, the ultimate production of Bacillamide compound and Bacillamide precursor is 0mg/L.
Comparative example 2
1) select in 500mL Erlenmeyer flask, every bottle containing 250mL fermention medium (in fermention medium 1L, yeast extract 5g, glycerine 4g, surplus is seawater, by acid-base buffer, this fermention medium is regulated pH to 7.5) in, at 28 DEG C, leave standstill 7 days;
2) bacillus mycoides (the Bacillus mycoides CGMCC1.197 of 24 hours will be cultivated through 140 revs/min of shaking tables, from Beijing North Na Chuanlian Bioteknologisk Institut) be inoculated in step 1) fermention medium, inoculum is long-pending is respectively 1000 μ L, at 28 DEG C, quiescent culture 15 days, obtains the fermention medium of bacillus mycoides single culture;
3) fermention medium of bacillus mycoides single culture is utilized extraction into ethyl acetate, be concentrated into dry, obtain ethyl acetate extract.
Be benchmark with fermention medium, in the ethyl acetate extract obtained, the ultimate production of Bacillamide compound and Bacillamide precursor is 0mg/L.
By ethyl acetate extract efficient liquid phase chromatographic analysis prepared by comparative example 1, comparative example 2 and embodiment 2, filler is octadecyl silane, take methanol-water as moving phase, flow velocity 0.6mL/min, elution program is: 0-10min, 40%(volume fraction) methyl alcohol; 10-40min, 40-70%(volume fraction) methanol elution gradient, 40-55min, 70-100%(volume fraction) methanol elution gradient, determined wavelength is 220nm.As shown in Figure 5, as shown in Figure 6, the high-efficient liquid phase chromatogram of ethyl acetate extract prepared by embodiment 2 as shown in Figure 7 for the high-efficient liquid phase chromatogram of ethyl acetate extract prepared by comparative example 2 for the high-efficient liquid phase chromatogram of ethyl acetate extract prepared by comparative example 1.According to Fig. 5, Fig. 6 and Fig. 7, result shows, adopts the relative single culture of co-culture method greatly can improve the output of secondary metabolic product, produces many new chromatographic peaks.
Embodiment 3
Ethyl acetate extract embodiment 2 obtained adopts reversed phase chromatography separation method to obtain Bacillamide compound and Bacillamide precursor.The filler that reversed phase chromatography separation method adopts is octadecyl silane, and the moving phase of employing is methanol-water.This reversed phase chromatography separation method detailed process is: first remove large polar impurity with the methanol aqueous solution wash-out formed by volume ratio 30:70 first alcohol and water, Bacillamide precursor is obtained again with the methanol aqueous solution wash-out that volume ratio 40:60 first alcohol and water is formed, then Bacillamide compd B is obtained and C(in chronological sequence elutes with the methanol aqueous solution wash-out that volume ratio 50:50 first alcohol and water is formed), the methanol aqueous solution wash-out that 60:40 first alcohol and water is formed obtains Bacillamide compd A.
Take fermention medium as benchmark, the output of the output of Bacillamide precursor to be the output of 20mg/L, Bacillamide compd A be 8mg/L, Bacillamide compd B is the output of 7.5mg/L, Bacillamide Compound C is 0.5mg/L.
The methanol aqueous solution wash-out formed via volume ratio 30:70 first alcohol and water removes the mixture of the Bacillamide compound after large polar impurity and Bacillamide precursor through efficient liquid phase chromatographic analysis, high-efficient liquid phase chromatogram as shown in Figure 8, syncaryon mr qualification structure, result shows, adopt co-culture method of the present invention, the main compound of generation is Bacillamide compound and Bacillamide precursor.
The nucleus magnetic resonance appraising datum of Bacillamide compound is as shown in table 1, and the nucleus magnetic resonance appraising datum of Bacillamide precursor is as shown in table 2.
Table 1
Table 2
From the nuclear-magnetism appraising datum of table 1, Bacillamide compound mainly comprises Bacillamide A(1), Bacillamide B(2) and Bacillamide C(3).
From the nuclear-magnetism appraising datum of table 2, Bacillamide precursor is N-Acetriptine, and its structural formula is as follows:
Ethyl acetate extract embodiment 2 obtained adopts normal-phase chromatography partition method to obtain Bacillamide compound and Bacillamide precursor.The filler that positive phase chromatography adopts is silica gel, and the moving phase of employing is methylene chloride-methanol, wash-out ratio (i.e. the volume ratio of methylene dichloride and the methyl alcohol) difference of employing: 100:1,80:1,50:1,25:1.The compound of wash-out gained passes through liquid-phase chromatographic analysis, gained qualification result is: methylene chloride-methanol 100:1 wash-out is little polar impurity compound, methylene chloride-methanol 80:1 wash-out obtains Bacillamide compd A, methylene chloride-methanol 50:1 wash-out obtains Bacillamide precursor and Bacillamide compd B (in chronological sequence eluting), and methylene chloride-methanol 25:1 wash-out obtains Bacillamide Compound C.
Embodiment 4
1) by actinomycetes (variant streptomycete Streptomyces variabilis CGMCC4.798, Beijing North Na Chuanlian Bioteknologisk Institut) be inoculated in 500mL Erlenmeyer flask, every bottle containing 200mL fermention medium (ISP2, international streptomyces project medium2) in, quiescent culture 10 days at 28 DEG C;
2) bacillus mycoides (the Bacillus mycoides CGMCC1.197 of 24 hours will be cultivated through 120 revs/min of shaking tables, from Beijing North Na Chuanlian Bioteknologisk Institut) be inoculated in step 1) and grown in actinomycetic fermention medium, inoculation volume is 200 μ L, 28 DEG C of symbiosis quiescent culture 20 days, obtain Dual culture fermention medium;
3) the Dual culture fermention medium of gained is utilized extraction into ethyl acetate, be concentrated into dry, obtain ethyl acetate extract, by the ethyl acetate extract efficient liquid phase chromatographic analysis of gained, filler is octadecyl silane, take methanol-water as moving phase, flow velocity 0.6mL/min, elution program is: 0-10min, 40%(volume fraction) methyl alcohol; 10-40min, 40-70%(volume fraction) methanol elution gradient, 40-55min, 70-100%(volume fraction) methanol elution gradient, determined wavelength is 220nm.The high-efficient liquid phase chromatogram of ethyl acetate extract prepared by embodiment 4 as shown in Figure 9.Result shows, carry out Dual culture to variant streptomycete and bacillus mycoides, product is mainly the mixture of Bacillamide compound and Bacillamide precursor, and foreign matter content is few.
Be benchmark with fermention medium, in the ethyl acetate extract obtained, the ultimate production of Bacillamide compound and Bacillamide precursor is 34mg/L.
Embodiment 5
1) be inoculated in 500mL Erlenmeyer flask by actinomycetes (streptomycete Streptomyces sp.MCC1A03740, Beijing North Na Chuanlian Bioteknologisk Institut), every bottle contains 200mL Gao Shi substratum, quiescent culture 7 days at 28 DEG C;
2) bacillus mycoides (the Bacillus mycoides CGMCC1.197 of 24 hours will be cultivated through 180 revs/min of shaking tables, from Beijing North Na Chuanlian Bioteknologisk Institut) be inoculated in the actinomycetic fermention medium of growth of step 1), inoculation volume is 1500 μ L, at 28 DEG C, symbiosis quiescent culture 20 days, obtains Dual culture fermention medium;
3) the Dual culture fermention medium of gained is utilized extraction into ethyl acetate, be concentrated into dry, obtain ethyl acetate extract, by the ethyl acetate extract efficient liquid phase chromatographic analysis of gained, filler is octadecyl silane, take methanol-water as moving phase, flow velocity 0.8mL/min, elution program is: 0-70min, 30-100%(volume fraction) methanol elution gradient, determined wavelength is 220nm.The high-efficient liquid phase chromatogram of ethyl acetate extract prepared by embodiment 5 as shown in Figure 10.Ethyl acetate extract (condition analysis with the same) comparison of gained in gained color atlas and comparative example 1 and comparative example 2, compound identification result as shown in Figure 10, these strain actinomycetes and bacillus mycoides Dual culture, the product of gained is mainly the mixture of Bacillamide compound and Bacillamide precursor.
Be benchmark with fermention medium, in the ethyl acetate extract obtained, the ultimate production of Bacillamide compound and Bacillamide precursor is 52mg/L.
Claims (7)
1. a preparation method for Bacillamide compound and Bacillamide precursor, is characterized in that, comprises the following steps:
1) actinomycetes are inoculated in fermention medium, cultivate 5 ~ 10 days;
Described actinomycetes are commercially available streptomyces nodocus Streptomyces nodosus 1A0101684, variant streptomycete Streptomyces variabilis CGMCC4.798 or the streptomycete Streptomyces sp.MCC1A03740 of Beijing North Na Chuanlian Bioteknologisk Institut;
In fermention medium 1L, the concrete formula of described fermention medium is:
By acid-base buffer, this fermention medium is regulated pH to 7-8;
2) bacillus mycoides is inoculated in step 1) in fermention medium in, symbiosis culture 10 ~ 20 days, obtains Dual culture fermention medium, through be separated after obtain Bacillamide compound and Bacillamide precursor;
Described bacillus mycoides is the commercially available bacillus mycoides Bacillus mycoides CGMCC1.197 of Beijing North Na Chuanlian Bioteknologisk Institut;
Step 1) and step 2) in, described cultivation is quiescent culture.
2. the preparation method of Bacillamide compound according to claim 1 and Bacillamide precursor, is characterized in that, step 2) in, the bacillus mycoides of inoculation will be cultivated 18 ~ 24 hours at 100 ~ 200 revs/min of shaking tables.
3. the preparation method of Bacillamide compound according to claim 1 and Bacillamide precursor, is characterized in that, step 2) in, the inoculation volume of described bacillus mycoides is the 0.1%-5% of fermention medium volume.
4. the preparation method of Bacillamide compound according to claim 1 and Bacillamide precursor, it is characterized in that, step 2) in, described separation comprises: be extracted with ethyl acetate by Dual culture fermention medium, concentrated, obtain ethyl acetate extract, ethyl acetate extract is the mixture containing Bacillamide compound and Bacillamide precursor.
5. the preparation method of Bacillamide compound according to claim 4 and Bacillamide precursor, it is characterized in that, described separation also comprises: adopted by ethyl acetate extract reversed phase chromatography separation method or normal-phase chromatography partition method to obtain Bacillamide compound and Bacillamide precursor.
6. the preparation method of Bacillamide compound according to claim 5 and Bacillamide precursor, is characterized in that, the filler that described reversed phase chromatography separation method adopts is octadecyl silane, and the moving phase of employing is methanol-water;
The filler that described normal-phase chromatography partition method adopts is silica gel, and the moving phase of employing is methylene chloride-methanol.
7. the Bacillamide compound according to claim 5 or 6 and the preparation method of Bacillamide precursor, it is characterized in that, described reversed phase chromatography separation method detailed process is: first remove large polar impurity with the methanol aqueous solution wash-out formed by volume ratio 20 ~ 30:80 ~ 70 first alcohol and water, obtain Bacillamide precursor with the methanol aqueous solution wash-out that volume ratio 35 ~ 45:65 ~ 55 first alcohol and water is formed again, then obtain Bacillamide compound with the methanol aqueous solution wash-out that volume ratio 50 ~ 70:50 ~ 30 first alcohol and water is formed;
Described normal-phase chromatography partition method detailed process is: first remove little polar impurity with by the methylene dichloride of volume ratio 100:1 and the eluant solution of the formation of methanol, then obtains Bacillamide compound and Bacillamide precursor with the methylene dichloride of volume ratio 80 ~ 25:1 and the eluant solution of the formation of methanol.
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| CN111850066A (en) * | 2020-07-16 | 2020-10-30 | 上海交通大学 | Precursor-guided preparation method of Bacillamide halogenated analogue |
| CN114958706A (en) * | 2022-04-11 | 2022-08-30 | 中国科学院海洋研究所 | Method for improving capacity of bacillus for removing red tide algae cells |
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