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Method for preparing bacillus subtilis lipopeptid biosurfactant

A Bacillus subtilis, biological surface technology, which is applied in the field of producing lipopeptide biosurfactant Surfactin, can solve the problems of unreachable commercial use and low yield, and achieve the effects of efficient production and simple fermentation process operation.

Inactive Publication Date: 2010-09-22
DALIAN BITEOMICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the low yield, which cannot be used commercially, many inventors are committed to increasing the yield of Surfactin

Method used

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  • Method for preparing bacillus subtilis lipopeptid biosurfactant
  • Method for preparing bacillus subtilis lipopeptid biosurfactant
  • Method for preparing bacillus subtilis lipopeptid biosurfactant

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Experimental program
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Embodiment 1

[0028] Take the strains BIT09S1, BIT09S2 and BIT09A2 preserved on the slant, inoculate them into 25mL / 250mL LB medium and cultivate them at 37°C for 36h; the obtained seed solution is inoculated into fermentation medium according to 5% inoculation amount: maltose (6.7%), soybean powder (4%), K 2 HPO 4 (0.5%), MgSO 4 ·7H 2 O (0.05%), CaCl 2 2H 2 O (0.018%), FeSO 4 ·7H 2 O(25ppm), MnCl 2 4H 2 O (22ppm), yeast powder (0.1%), water, pH 7.0, loading volume 100mL / 250mL, culture at 37°C for 72h. After the cultivation, take 40mL of the fermentation broth and centrifuge at 8000r / min for 10min to remove the bacteria. The obtained supernatant is used for the measurement of the surface tension. Acid precipitation. Dry at 60°C for 24 hours, take it out and add 0.6mL methanol to dissolve it, sonicate for 1 hour, take the supernatant and save it for HPLC analysis. The mensuration of surface tension first records the density of fermented liquid supernatant, measures the surface tens...

Embodiment 2

[0033] Take the strains preserved on the slant: BIT09S1, BIT09S2 and BIT09A2, inoculate them in 25mL / 250mL LB medium respectively, and cultivate them at 200rpm at 37°C for 36h. NH 4 NO 3 (0.2%), K 2 HPO 4 ·3H 2 O (0.3%), NaH 2 PO 4 2H 2 O (1%), MgSO 4 ·7H 2 O (0.02%), MnCl 2 4H 2 O (2ppm), yeast powder (0.02%), water, pH 7.2, cultured at 150rpm at 28°C for 72h. After the cultivation, take 30mL of the fermentation broth and centrifuge at 8000r / min for 10min to remove the bacteria. The obtained supernatant is used for the measurement of surface tension. After the measurement, adjust the pH to 2.0 with 6mol / L HCl, put it in the refrigerator at 4°C overnight, and centrifuge to obtain the acid precipitate. .Dry at 60°C for 24 hours, take it out and add 0.6mL of methanol to dissolve it, sonicate for 1 hour, take the supernatant and save it for HPLC analysis. The analysis of surface tension measures the density of fermented liquid supernatant earlier, then measures the su...

Embodiment 3

[0038] Get the bacterial strain BIT09S1 that slant preserves, inoculate in the LB culture medium of loading capacity 25mL / 250mL, cultivate 36h at 37 ℃ of 200rpm, gained seed liquid is inoculated in fermentation medium by the inoculum of 5%: guar gum gel-breaking liquid (20g guar gum Dr. Glue Enzyme biological enzyme breaker), 0.5% NH 4 h 2 PO 4 , 0.3% (NH 4 ) 2 HPO 4 , K 2 HPO 4 ·3H 2 O (0.3%), MgSO 4 ·7H 2 O (0.02%), MnCl 2 4H 2O (2ppm), yeast extract (0.02%), water, pH 7.2, cultured at 37°C for 72h. After the cultivation, take 25 mL of the fermented liquid and centrifuge at 8000r / min for 10 min to remove the bacteria. The obtained supernatant is used for the measurement of the surface tension. Acid precipitation. Dry at 60°C for 24 hours, take it out and add 0.6mL methanol to dissolve it, sonicate for 1 hour, take the supernatant and save it for HPLC analysis. The measurement of surface tension first measures the density of the supernatant of the fermentation ...

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Abstract

The invention relates to a method for preparing a bacillus subtilis lipopeptid biosurfactant, which is characterized in that the adopted bacterial strain is efficient lipopeptid producing strain bacillus subtilis BIT09S1,BIT09S2 and BIT09A2, and the lipopeptid biosurfactant is prepared by taking cheap molasses, pulse flour, konjac gum finemeal, guar gum and gum breaking liquid of modified guar gum as carbon sources for the culture medium; adding other nitrogen sources, phosphorus sources, inorganic salt components and nutrient elements into the culture medium; and carrying out fermentation, separation and purification. The raw materials used for producing lipopeptid by the method are easily obtained and can be enormously purchased. The crude extract of the obtained lipopeptid biosurfactant Surfactin can be used in various drilling and production processes such as oil-gas field fracturing, acidizing, de-plugging, controlling and water shut-off, oily water treatment, environment renovation and the like, and the biosurfactant has wide prospect.

Description

technical field [0001] The invention relates to a biosurfactant and Bacillus subtilis, in particular to a method for producing lipopeptide biosurfactin Surfactin by using Bacillus subtilis. Background technique [0002] Biosurfactants are secondary metabolites produced by microorganisms under certain conditions with both hydrophilic and hydrophobic structures. In addition to having the same or similar physical and chemical properties as chemically synthesized surfactants, biosurfactants also have complex structures, strong specificity, low toxicity, biodegradability, and environmental friendliness, and can be fermented with cheap agricultural and sideline products. Some biosurfactants also have pharmacological effects and physiological activities such as antibacterial, antiviral, and antitumor. Biosurfactants have been widely used in the fields of petroleum, food, cosmetics and medicine. [0003] In 1968, Arima et al. discovered that a lipopeptide surfactant produced by Ba...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12P21/02C12R1/125
Inventor 梅晓丹刘向阳叶伟孙强陈朋王薇粱莹江磊磊靳祥史兴来
Owner DALIAN BITEOMICS INC
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