CN105483191A - Preparation method of high- mildew-activity-resistance antimicrobial lipopeptide V7-surfactin - Google Patents
Preparation method of high- mildew-activity-resistance antimicrobial lipopeptide V7-surfactin Download PDFInfo
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- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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Abstract
Provided is a preparation method of high-mildew-activity-resistance antimicrobial lipopeptide V7-surfactin. Bacillus natto serves as the bacterium strain, and high-mildew-activity-resistance surfactin with the condensed structural formula of Cyclo(C14BOHFA-E1-I/L2-I/L3-V4-D5-I/L6-V7), the molecular weight of 1008 Da and shorter form of V7-surfactin is extracted through a solid fermenting method. The method is simple in process step, the product is easy to separate and purify, and the obtained V7-surfactin has broad-spectrum resistance to various mildews and has higher safety than surfactin of other bacterium sources. The method is used for preparing a culture medium with V7-surfactin yield of 800-1100 mg/L and purity of 85-99%.
Description
Technical field
The present invention relates to a kind of high anti-mildew active antibacterial lipopeptid V
7the preparation method of-surfactin, especially relates to one with bacillus natto (Bacillusnatto) for bacterial classification, and employing solid state fermentation prepares the active V of high anti-mildew
7its skeleton symbol of-surfactin(is: Cyclo(C
14bOHFA-E
1-I/L
2-I/L
3-V
4-D
5-I/L
6-V
7), molecular weight is 1008Da) method.
Background technology
At present, common on market anti-mildew mushroom medicine is main mainly with chemicals.Often there is the problems such as anti-mildew activity is low, drug effect is not lasting in natural anti-mildew product and biological products, is difficult to drop into commercial applications.The drug resistance problems that life-time service chemicals causes mould to produce and chemicals are to the toxic side effect of organism and environment, and making to seek and develop efficient, green new bio source anti-mildew active substance becomes inevitable.
Antibacterial lipopeptid Surfactin is the bacterial origin cyclic lipopeptide produced by genus bacillus, also known as tensio-active agent or surfactant peptides.Natural surfactin is the mixture of A, B, C, D tetra-kinds of hypotypes produced by subtilis (Bacillussubtilis) mostly.[skeleton symbol is the ring texture that they are normally made up of the beta-hydroxy fatty acid being 13-16 containing 7 amino acid whose depsipeptides and carbonatoms: Cyclo (CnBOHFA-E
1-L
2-L
3-V
4-D
5-L
6-L
7), n gets 13-16, is called for short L
7-surfactin].Because having the several functions such as antibacterial and mouldproof, anti-inflammatory, inhibition tumor cell propagation, surfactin achieves extensive concern in multiple fields such as food, medicine, makeup, has great Research Significance and using value.And the surfactin reported in most literature data is 7 amino acids is common surfactin types of leucine (L), and 7 amino acids are the V of α-amino-isovaleric acid (V)
7-surfactin is rare relevant report.
Bacillus natto (B.natto) is called for short Bacillus natto again, owing to deriving from traditional fermented food natto, more credible in security.In classification, Bacillus natto is considered to subtilis subspecies (B.subtilissubsp.natto), but its institute's antibacterial lipopeptid that produces is in kind with quantitatively all there is significant difference with subtilis.About the research of Bacillus natto, focused mostly in the past generation have on the Nattokinase of thrombus dissolving function.Up to now, there are no utilizing fermenting bacillus natto to produce the active V of high anti-mildew
7the pertinent literature report of-surfactin.
Summary of the invention
The technical problem to be solved in the present invention is, overcomes the deficiencies in the prior art, provides a kind of high anti-mildew active antibacterial lipopeptid V
7the preparation method of-surfactin take bacillus natto as bacterial classification, produces high anti-mildew active antibacterial lipopeptid V with solid state fermentation
7[skeleton symbol is-surfactin: Cyclo(C
14bOHFA-E
1-I/L
2-I/L
3-V
4-D
5-I/L
6-V
7)].
Described bacillus natto (B.natto) is separated from commercially available natto or Bacillus natto bacterium powder, and separation method is: get natto 2-3 grain or bacillus natto bacterium powder 0.5-1g under aseptic condition, put into the tubes Sterile water of 10mL, and fully concussion to water slightly becomes muddy.By the fundamental operation that microbial strains line is separated, pick the water after feculence one ring with aseptic inoculation ring, sectional streak on beef extract-peptone flat board.Then flat-plate inverted is placed in 35-37 DEG C of constant incubator and cultivates 48h, flat board grows white, and edge is irregular, and matter is glued, and can form the bacterium colony of elongated wire drawing, be B.natto when provoking.Microscopy guarantees that the single B.natto colony inoculation of picking is on beef extract-peptone inclined-plane, and 35-37 DEG C of constant temperature culture 60-72h, obtains bacillus natto (B.natto) slant strains not containing after miscellaneous bacteria.
The high anti-mildew active antibacterial lipopeptid V of the present invention
7the preparation method of-surfactin, comprises the following steps:
(1) preparation (prior art) of bacillus natto (B.natto) seed liquor: prepare seed culture medium according to the formula of extractum carnis 2-4g, peptone 8-12g, NaCl5-8g, water 1L, the initial pH value of substratum is regulated to be 6.8-7.0 with HCl or NaOH that mass concentration is 5-10%, 121 DEG C of sterilizing 20-30min, cooling; By the bacillus natto slant strains after the activation of transfering loop picking, by the cooled seed culture medium of inoculum size access sterilizing of 2-3 ring bacterial classification/50mL substratum; 24-30h is cultivated in 32-35 DEG C, 150-180rpm concussion; Obtain bacillus natto (B.natto) seed liquor;
(2) fermention medium is prepared according to following formula: glucose 20-40g, yeast extract powder 1-5g, soy peptone 4-6g, Na
2hPO
41-2g, KH
2pO
40.5-1.5g, L-Ala (Ala) 0.2-0.6g, L-glutamic acid (Glu) 0.2-0.8g, aspartic acid (Asp) 0.2-0.8g, α-amino-isovaleric acid (Val) 0.2-4.0g, agar powder 15-20g, water 1L; PH6.3-6.8,121 DEG C of sterilizing 20-30min, obtain fermention medium;
(3) inoculate, ferment: step (2) gained fermention medium is cooled to 45-50 DEG C, and aseptically poured into by substratum in aseptic culture dish, substratum gauge control is at 2-3mm; Leave standstill 5-10min, after culture medium solidifying, according to inoculum size access step (1) the gained bacillus natto seed liquor of 0.02-0.08mL seed liquor/9cm diameter Petri dishes, and with aseptic spreading rod, seed liquor is even in media surface coating; Be placed in 28-35 DEG C again, relative humidity 70-85% condition bottom fermentation cultivates 48-96h;
The inoculum size of described 0.02-0.08mL seed liquor/9cm diameter Petri dishes, its implication is: when the culture dish diameter used is for 9cm, the inoculum size of seed liquor is 0.02-0.08mL; When the culture dish diameter used is not 9cm, according to the area of culture dish, the corresponding inoculum size determining seed liquor; As follows;
Step (4) and step (5) are V
7the purification step of-surfactin:
(4) V
7volumetric concentration is that the methyl alcohol of 90-100% sprays into culture surface according to the amount of 4-5mL methyl alcohol/9cm diameter Petri dishes by the extraction of-surfactin: after fermentation ends, lixiviate 3-5min under normal temperature; Repeat leaching step 2-3 time, merge each vat liquor; Vat liquor is concentrated into the 1/10-1/5 of original volume through rotary evaporation, obtains extraction concentrated solution;
(5) V
7the preparation of-surfactin: step (4) gained is extracted concentrated solution and cross 0.22 μm or 0.45 μm of filter membrane or through Solid-Phase Extraction pre-treatment (Solid-Phase Extraction preferably adopts C18 solid-phase extraction column), then be separated with preparation HPLC.HPLC condition is: C18 or the C8 preparative column of particle diameter 2.7-5 μm, UV-detector, and determined wavelength is 210 or 215nm.Mobile phase A is water, and B is acetonitrile or methyl alcohol or both mixed solutions, and gradient is 5-95%, elution time 20-60min.Collect the eluate of corresponding peak value, rotary evaporation removes organic solvent, and under 40-50 DEG C of condition, low temperature is dried to constant weight, obtains white solid, is V
7-surfactin.
Adopt the preparation method of the present invention, gained V
7-surfactin product yield is 800-1100mg/L substratum, and purity is 85-99%.
Adopt the V of gained of the present invention
7-surfactin carries out anti-mildew active testing to multiple moulds such as aspergillus niger (A.niger), flavus (A.flavus), Aspergillus fumigatus (A.fumigatus), head molds (Rhizopussp.).Anti-mildew active testing result shows: V
7-surfactin all has stronger restraining effect to multiple moulds such as aspergillus niger (A.niger), flavus (A.flavus), Aspergillus fumigatus (A.fumigatus), head molds (Rhizopussp.).
Carry out also finding in the separation and purification research process of sample at employing HPLC, using water as mobile phase A, methyl alcohol and acetonitrile mixture carry out the gradient elution of product as Mobile phase B time, V
7-surfactin is obviously comparatively Zao than other surfactin homologous components to be eluted (see accompanying drawing 1), thus is easy to separate with other surfactin homologues, is convenient to the V that obtained purity is higher
7-surfactin monomer.
The solid state fermentation of the present invention is adopted to produce V
7-surfactin, directly can obtain surfactin crude extract in the lixiviate of culture surface spraying methyl alcohol after fermentation ends, and processing step is simple, and in gained crude extract, the interference substances content such as pigment is low, is beneficial to later stage separation and purification.
The present invention bacterial classification B.natto used is the production bacterium of traditional zymotic bean product natto.As a kind of leavened food, the natto containing viable bacteria can quantity-unlimiting human consumption.Therefore, have higher security with the surfactin that fermenting bacillus natto is produced, its range of application is wider.
Accompanying drawing explanation
Fig. 1 is the HPLC figure of the B.natto solid fermentation product of the present invention;
Wherein, the corresponding peak value of 42min is V
7the corresponding peak value of-surfactin, 50-53.5min is respectively the L of C atomicity 13-15
7-surfactin.
HPLC condition is: Shimadazu20AHPLC system, 2.7 μm of HalosC184.6mm*150mm chromatographic columns, mobile phase A is the ultrapure water containing 0.04%TFA, Mobile phase B is be the mixed solution of 5:1 containing the acetonitrile of 0.04%TFA and methyl alcohol volume ratio, flow velocity is 1mL/min, and gradient is 5%B-95%B (40min).
Fig. 2 is that Bacillus natto produces V
7the second order ms of-surfactin and three grades of mass spectrums;
Wherein, 1008
[M+H]for Cyclo (C
14bOHFA-E
1-I/L
2-I/L
3-V
4-D
5-I/L
6-V
7), resolve as follows:
1008(b
8)-V
7-909(b
7)-I/L
6-796(b
6)-D
5-681(b
5)-V
4-582(b
4)-I/L
3-469(b
3)-I/L
2-356(b
2:HO-C
14BOHFA-E
1-CO
+)
1008(y
8)-C
14BOHFA-782(y
7)-E
1-653(y
6)-I/L
2-540(y
5)-I/L
3-427(y
4)-V
4-328(y
3)-D
5-213(y
2:H
2N-I/L
6-V
7-CO
+)
Specific charge 671 is H
3n
+-I/L
2-I/L
3-V
4-D
5-I/L
6-V
7-COOH fragment:
671(b
6)-(H-V
7-OH)-554(b
5)-I/L
6-441(b
4)-D
5-326(b
3)-V
4-227(b
2:H
2N-I/L
2-I/L
3-CO
+)
671(y
6)-I/L
2-558(y
5)-I/L
3-445(y
4)-V
4-346(y
3)-D
5-231(y
2:H
3N
+-I/L
6-V
7-COOH)。
Embodiment
Below in conjunction with embodiment, the invention will be further described.
In each embodiment; described bacillus natto (B.natto) is separated from commercially available natto or Bacillus natto bacterium powder; separation method is: get natto 2 or bacillus natto bacterium powder 0.5g under aseptic condition, put into the tubes Sterile water of 10mL, and fully concussion to water slightly becomes muddy.By the fundamental operation that microbial strains line is separated, pick the water after feculence one ring with aseptic inoculation ring, sectional streak on beef extract-peptone flat board.Then flat-plate inverted is placed in 35-37 DEG C of constant incubator and cultivates 48h, flat board grows white, and edge is irregular, and matter is glued, and can form the bacterium colony of elongated wire drawing, be B.natto when provoking.Microscopy guarantees that the single B.natto colony inoculation of picking is on beef extract-peptone inclined-plane, and 35-37 DEG C of constant temperature culture 72h, obtains bacillus natto (B.natto) slant strains not containing after miscellaneous bacteria.
Embodiment 1
The present embodiment comprises the following steps:
(1) preparation of B.natto seed liquor: take extractum carnis 3g, peptone 8g, NaCl5g be dissolved in 1L water, medium pH to 6.8 is regulated with HCl or NaOH of mass concentration 5%, by the dispensed loading amount of 50mL/250mL triangular flask, seed culture medium is distributed in 250mL triangular flask, sealed membrane seals, 121 DEG C of sterilizing 20min, cooling; By the bacillus natto slant strains after the activation of transfering loop picking, in the seed culture medium cooled after every bottle of sterilizing, access the B.natto slant strains after 2 ring activation.32 DEG C, 30h is cultivated in 150rpm concussion; Obtain bacillus natto (B.natto) seed liquor;
(2) fermention medium is prepared according to following formula: glucose 20g, yeast extract powder 1g, soy peptone 4g, Na
2hPO
41g, KH
2pO
40.5g, Ala0.6g, Glu0.2g, Asp0.2g, Val0.2g, agar powder 20g, water 1L, pH6.8,121 DEG C of sterilizing 20min, obtain fermention medium;
(3) inoculate, ferment: when step (2) gained fermention medium is cooled to 50 DEG C, aseptically poured into by substratum in aseptic culture dish, substratum gauge control is at 3mm; Leave standstill 10min, after culture medium solidifying, according to inoculum size access step (1) the gained B.natto seed liquor of 0.05mL seed liquor/9cm diameter Petri dishes, with aseptic spreading rod, seed liquor is even in media surface coating; Be placed in 30 DEG C again, relative humidity 70% condition bottom fermentation cultivates 72h.
Step (4) and step (5) are V
7the purification step of-surfactin:
(4) V
7volumetric concentration is that the methyl alcohol of 100% sprays into culture surface according to the amount of 5mL methyl alcohol/9cm diameter Petri dishes by the extraction of-surfactin: after fermentation ends, normal temperature lixiviate 5min; Repeat leaching step 3 times, merge each vat liquor; Vat liquor is concentrated into 1/5 of original volume through rotary evaporation, obtains extraction concentrated solution;
(5) V
7the preparation of-surfactin: step (4) gained is extracted concentrated solution and crosses 0.22 μm of filter membrane, gained filtrate is separated preparation through preparation HPLC.HPLC condition is: the C18 preparative column of particle diameter 3 μm, UV-detector, and determined wavelength is 215nm.Mobile phase A is water, and B is acetonitrile: methyl alcohol=5:1(volume ratio) mixed solution, gradient is 5-95%, elution time 40min.Collect the eluate that organic phase concentration is the interval corresponding peak value of 70%-80%, rotary evaporation removes organic solvent, and under 40 DEG C of conditions, low temperature is dried to constant weight, obtains white solid, is V
7-surfactin.
In the present embodiment, gained V
7-surfactin product yield is 1100mg/L substratum, and purity is 90%.
Embodiment 2
The present embodiment comprises the following steps:
(1) preparation of B.natto seed liquor: prepare seed culture medium according to the formula of extractum carnis 3g, peptone 10g, NaCl5g, water 1L, the initial pH of substratum is regulated to be 6.9 with HCl or NaOH of mass concentration 5%, by the dispensed loading amount of 50mL/250mL triangular flask, seed culture medium is distributed in 250mL triangular flask, sealed membrane seals, 121 DEG C of sterilizing 20min.By the B.natto slant strains after the activation of transfering loop picking, by the cooled seed culture medium of inoculum size access sterilizing of 3 ring bacterial classification/50mL substratum.33 DEG C, 27h is cultivated in 160rpm concussion; Obtain bacillus natto (B.natto) seed liquor;
(2) fermention medium is prepared according to following formula: glucose 30g, yeast extract powder 3g, soy peptone 5g, Na
2hPO
41.5g, KH
2pO
41.0g, Ala0.3g, Glu0.5g, Asp0.5g, Val1.0g, agar powder 18g, water 1L, pH6.4,121 DEG C of sterilizing 20min, obtain fermention medium;
(3) inoculate, ferment: step (2) gained fermention medium is cooled to 50 DEG C, aseptically substratum is poured in aseptic culture dish, substratum thickness 2.5mm; Leave standstill 5min, after culture medium solidifying, according to inoculum size access step (1) the gained B.natto seed liquor of 0.03mL seed liquor/9cm diameter Petri dishes, with aseptic spreading rod, seed liquor is even in media surface coating; Be placed in 30 DEG C again, relative humidity 80% condition bottom fermentation cultivates 60h.
Step (4) and step (5) are V
7the purification step of-surfactin:
(4) V
7volumetric concentration is that the methyl alcohol of 95% sprays into culture surface according to the amount of 4mL methyl alcohol/9cm diameter Petri dishes by the extraction of-surfactin: after fermentation ends, lixiviate 4min under normal temperature; Repeat leaching step 3 times, merge each vat liquor; Vat liquor is concentrated into 1/5 of original volume through rotary evaporation, obtains extraction concentrated solution;
(5) V
7the preparation of-surfactin: step (4) gained is extracted concentrated solution and crosses 0.45 μm of ultra-filtration membrane, gained filtrate is separated through preparation HPLC.HPLC condition is: the C18 preparative column of particle diameter 5 μm, UV-detector, and determined wavelength is 210nm.Moving phase is A is water, and B is acetonitrile, and gradient is 5-95%, elution time 60min.Collect the eluate that organic phase concentration is the interval corresponding peak value of 75%-80%, rotary evaporation removes organic solvent, and 50 DEG C of cryodrying samples, to constant weight, obtain white solid, are V
7-surfactin.
V in the present embodiment
7-surfactin product yield is 1000mg/L substratum, and purity is 85%.
Embodiment 3
The present embodiment comprises the following steps:
(1) preparation of B.natto seed liquor: prepare seed culture medium according to extractum carnis 3g, peptone 12g, NaCl5g, water 1L formula, regulate the initial pH of substratum to be 7.0 with HCl or NaOH that mass concentration is 10%.By the dispensed loading amount of 50mL/250mL triangular flask, seed culture medium is distributed into 250mL triangular flask, sealed membrane seals, 121 DEG C of sterilizing 20min, cooling; Enter in every bottle of cooled seed culture medium of sterilizing with B.natto slant strains 3 articulating after the activation of transfering loop picking; 35 DEG C, 30h is cultivated in 180rpm concussion; Obtain bacillus natto (B.natto) seed liquor;
(2) fermention medium is prepared according to following formula: glucose 40g, yeast extract powder 5g, soy peptone 6g, Na
2hPO
42g, KH
2pO
41.5g, Ala0.2g, Glu0.8g, Asp0.8g, Val4.0g, agar powder 20g, water 1L, pH6.3-6.8,121 DEG C of sterilizing 20min, obtain fermention medium;
(3) inoculate, ferment: step (2) gained fermention medium is cooled to 50 DEG C, and aseptically poured into by substratum in aseptic culture dish, substratum gauge control is at 2mm; Leave standstill 10min, after culture medium solidifying, according to inoculum size access step (1) the gained B.natto seed liquor of 0.02mL seed liquor/9cm diameter Petri dishes, and with aseptic spreading rod, seed liquor is even in media surface coating; Be placed in 32 DEG C again, relative humidity 85% condition bottom fermentation cultivates 66h;
Step (4) and step (5) are V
7the purification step of-surfactin:
(4) V
7volumetric concentration is that the methyl alcohol of 100% sprays into culture surface according to the amount of 5mL methyl alcohol/9cm diameter Petri dishes by the extraction of-surfactin: after fermentation ends, normal temperature lixiviate 3min; Repeat leaching step 2 times, merge vat liquor; Vat liquor is concentrated into 1/5 of original volume through rotary evaporation, obtains lixiviate concentrated solution;
(5) V
7the preparation of-surfactin: in the set-up procedure that adds water (4) gained concentrated solution, methanol content is 10%, through WatersC18 Solid-Phase Extraction column extracting, 100% methanol-eluted fractions adsorptive, gained elutriant is separated through preparation HPLC.HPLC condition is: the C18 preparative column of particle diameter 3 μm, UV-detector, and determined wavelength is 215nm.Moving phase is A is water, and B is acetonitrile: methyl alcohol=5:1(volume ratio) mixed solution, gradient is 5-95%, time 40min.Collect the eluate that organic phase concentration is the interval corresponding peak value of 70%-75%, rotary evaporation removes organic solvent, and 45 DEG C of cryodrying samples, to constant weight, obtain white solid, are V
7-surfactin.
The present embodiment gained V
7-surfactin product yield is 1100mg/L substratum, and purity is 99%.
Embodiment 4
Application Example
Adopt the V of embodiment of the present invention gained
7-surfactin carries out anti-mildew active testing to multiple moulds such as aspergillus niger (A.niger), flavus (A.flavus), Aspergillus fumigatus (A.fumigatus), head molds (Rhizopussp.).Anti-mildew active testing result shows: V
7-surfactin all has stronger restraining effect to multiple moulds such as aspergillus niger (A.niger), flavus (A.flavus), Aspergillus fumigatus (A.fumigatus), head molds (Rhizopussp.).V
7-surfactin and L
7-surfactin to the minimal inhibitory concentration (MIC) of several mould in table 1.MIC measuring method is with reference to the method for the people such as DanielMania, KaiHilpert, SergeRuden.（DanielMania,KaiHilpert,SergeRuden,etal.ScreeningforAntifungalPeptidesandTheirModesofActioninAspergillusnidulans.AppliedandEnvironmentalMicroiology,2010,11:7102-7108.）
From table 1, V
7the MIC value of-surfactin to several tested mould compares L
7the respective value of-surfactin all has reduction in various degree, and decline multiple is between 0.9-2.5.
(note: V used
7-surfactin adopts the application's method to obtain, and purity is 95wt%; L
7-surfactin is same purity commercial standard; MIC value tests the mean+SD recorded three times.）
Carry out also finding in the separation and purification research process of sample at employing HPLC, using water as mobile phase A, methyl alcohol and acetonitrile mixture carry out the gradient elution of product as Mobile phase B time, V
7-surfactin is obviously comparatively Zao than other surfactin homologous components to be eluted (see accompanying drawing 1), thus is easy to separate with other surfactin homologues, is convenient to the V that obtained purity is higher
7-surfactin monomer.
Claims (10)
1. one kind high anti-mildew active antibacterial lipopeptid V
7the preparation method of-surfactin, is characterized in that, take bacillus natto as bacterial classification, produces high anti-mildew active antibacterial lipopeptid V with solid state fermentation
7-surfactin.
2. high anti-mildew active antibacterial lipopeptid V according to claim 1
7the preparation method of-surfactin, is characterized in that, the fermention medium of described bacillus natto is: glucose 20-40g, yeast extract powder 1-5g, soy peptone 4-6g, Na
2hPO
41-2g, KH
2pO
40.5-1.5g, L-Ala 0.2-0.6g, L-glutamic acid 0.2-0.8g, aspartic acid 0.2-0.8g, α-amino-isovaleric acid 0.2-4.0g, agar powder 15-20g, water 1L; PH6.3-6.8,121 DEG C of sterilizing 20-30min.
3. high anti-mildew active antibacterial lipopeptid V according to claim 1 and 2
7the preparation method of-surfactin, is characterized in that, comprises the following steps:
(1) preparation of bacillus natto seed liquor;
(2) fermention medium is prepared according to following formula: glucose 20-40g, yeast extract powder 1-5g, soy peptone 4-6g, Na
2hPO
41-2g, KH
2pO
40.5-1.5g, L-Ala 0.2-0.6g, L-glutamic acid 0.2-0.8g, aspartic acid 0.2-0.8g, α-amino-isovaleric acid 0.2-4.0g, agar powder 15-20g, water 1L; PH6.3-6.8,121 DEG C of sterilizing 20-30min, obtain fermention medium;
(3) inoculate, ferment: step (2) gained fermention medium is cooled to 45-50 DEG C, and aseptically poured into by substratum in aseptic culture dish, substratum gauge control is at 2-3mm; Leave standstill 5-10min, after culture medium solidifying, according to inoculum size access step (1) the gained bacillus natto seed liquor of 0.02-0.08mL seed liquor/9cm diameter Petri dishes, and with aseptic spreading rod, seed liquor is even in media surface coating; Be placed in 28-35 DEG C again, relative humidity 70-85% condition bottom fermentation cultivates 48-96h.
4. high anti-mildew active antibacterial lipopeptid V according to claim 1 and 2
7the preparation method of-surfactin, is characterized in that, V
7the purification step of-surfactin:
(4) V
7volumetric concentration is that the methyl alcohol of 90-100% sprays into culture surface according to the amount of 4-5mL methyl alcohol/9cm diameter Petri dishes by the extraction of-surfactin: after fermentation ends, lixiviate 3-5min under normal temperature; Repeat leaching step 2-3 time, merge each vat liquor; Vat liquor is concentrated into the 1/10-1/5 of original volume through rotary evaporation, obtains extraction concentrated solution;
(5) V
7the preparation of-surfactin: step (4) gained is extracted concentrated solution and cross 0.22 μm or 0.45 μm of filter membrane or through Solid-Phase Extraction pre-treatment, then be separated with preparation HPLC.
5. high anti-mildew active antibacterial lipopeptid V according to claim 4
7the preparation method of-surfactin, is characterized in that, HPLC condition is: C18 or the C8 preparative column of particle diameter 2.7-5 μm, UV-detector, and determined wavelength is 210 or 215nm; Mobile phase A is water, and B is acetonitrile or methyl alcohol or both mixed solutions, and gradient is 5-95%, elution time 20-60min; Collect the eluate of corresponding peak value, rotary evaporation removes organic solvent, and under 40-50 DEG C of condition, low temperature is dried to constant weight, obtains white solid, is V
7-surfactin.
6. high anti-mildew active antibacterial lipopeptid V according to claim 3
7the preparation method of-surfactin, is characterized in that, V
7the purification step of-surfactin:
(4) V
7volumetric concentration is that the methyl alcohol of 90-100% sprays into culture surface according to the amount of 4-5mL methyl alcohol/9cm diameter Petri dishes by the extraction of-surfactin: after fermentation ends, lixiviate 3-5min under normal temperature; Repeat leaching step 2-3 time, merge each vat liquor; Vat liquor is concentrated into the 1/10-1/5 of original volume through rotary evaporation, obtains extraction concentrated solution;
(5) V
7the preparation of-surfactin: step (4) gained is extracted concentrated solution and cross 0.22 μm or 0.45 μm of filter membrane or through Solid-Phase Extraction pre-treatment, then be separated with preparation HPLC.
7. high anti-mildew active antibacterial lipopeptid V according to claim 6
7the preparation method of-surfactin, is characterized in that, HPLC condition is: C18 or the C8 preparative column of particle diameter 2.7-5 μm, UV-detector, and determined wavelength is 210 or 215nm; Mobile phase A is water, and B is acetonitrile or methyl alcohol or both mixed solutions, and gradient is 5-95%, elution time 20-60min; Collect the eluate of corresponding peak value, rotary evaporation removes organic solvent, and under 40-50 DEG C of condition, low temperature is dried to constant weight, obtains white solid, is V
7-surfactin.
8. high anti-mildew active antibacterial lipopeptid V according to claim 4
7the preparation method of-surfactin, is characterized in that, in step (5), Solid-Phase Extraction adopts C18 solid-phase extraction column.
9. high anti-mildew active antibacterial lipopeptid V according to claim 5
7the preparation method of-surfactin, is characterized in that, in step (5), Solid-Phase Extraction adopts C18 solid-phase extraction column.
10. high anti-mildew active antibacterial lipopeptid V according to claim 6
7the preparation method of-surfactin, is characterized in that, in step (5), Solid-Phase Extraction adopts C18 solid-phase extraction column.
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| CN102321182A (en) * | 2011-09-22 | 2012-01-18 | 广东海洋大学 | Composite antibiosis peptide and working method thereof that bacillus natto NT-6 bacterial strain is produced |
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| Title |
|---|
| CAO XIAO-HONG ET AL: "Evaluation of a lipopeptide biosurfactant from Bacillus Natto TK-1 as a potential source of anti-adhesive,antimicrobial and antitumor activities", 《BRAZILIAN JOURNAL OF MICROBIOLOGY》 * |
| 王东: "纳豆菌抗菌脂肽固态发酵工艺优化及其在对虾保鲜上的应用", 《中国优秀硕士学位论文全文数据库工程科技Ⅰ辑》 * |
| 钱朝东: "天目类芽孢杆菌新种所产脂肽类抗生素体内外抗菌活性及抗菌机理研究", 《中国优秀博士论文全文数据库基础科学辑》 * |
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