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A method for separating and purifying lipopeptide surfactin family compounds

A technology for separation and purification of lipopeptides, which is applied to the preparation methods of peptides, chemical instruments and methods, compositions of drilling holes, etc., can solve the problems of cumbersome complexity, high cost, low purity, etc. Low, high purity effect

Active Publication Date: 2021-09-28
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There are many problems in the whole technological process. For example, the problems in the crude extraction process include: (1) After acid precipitation, the protein will also precipitate out together with the surfactin family compounds, and many small molecular impurities will be brought in, resulting in organic The purity after solvent extraction is not high, and the literature reports are generally between 50% and 70%
(2) The surfactin product obtained after organic solvent extraction is dissolved in organic solvent, which is not conducive to the processing and storage of subsequent products
The problems in the fine extraction process include: many unit operations, tedious and complicated, high cost, disadvantages that are not conducive to industrial application, and excessive dependence on expensive organic solvents, such as methanol, isopropanol, n-butanol, ethyl acetate Esters, etc., and these organic solvents are usually expensive and toxic, which not only increases the cost, but also pollutes the environment, and is not suitable for the use of products such as food and medicine

Method used

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  • A method for separating and purifying lipopeptide surfactin family compounds
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  • A method for separating and purifying lipopeptide surfactin family compounds

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1 prepares fermented liquid

[0035] The fermentation strain is Bacillus amyloliquefaciens MT45, which is isolated from Moutai Daqu in China and stored in the China General Microorganism Culture Collection and Management Center. Patent pending.

[0036] The composition of LB medium is: yeast powder 5.0g / L, tryptone 10.0g / L, NaCl 10.0g / L.

[0037] Optimal medium composition: sucrose 65g / L, ammonium nitrate 8g / L, peptone 3.73g / L, potassium dihydrogen phosphate 4.08g / L, disodium hydrogen phosphate 10g / L, magnesium sulfate 0.096g / L, calcium chloride 7μM , ferrous sulfate 4 μM, EDTA 4 μM.

[0038] Bacillus amyloliquefaciens MT45 was activated in LB medium, 37°C, 200rpm, cultured for 12h, then inoculated into a 250mL flask containing 50mL optimized medium according to the inoculum size of 2%, and cultured at 30°C, 200rpm for 72h. The fermentation broth was collected, and the supernatant was collected after centrifugation to prepare for the separation and extract...

Embodiment 2

[0039] Embodiment 2 detects the content of protein in the fermented liquid

[0040] First establish a standard curve, prepare bovine serum albumin (BSA) to the concentration of 0, 0.0625, 0.125, 0.25, 0.5, 0.75, 1g / L, add Coomassie Brilliant Blue G-250 solution and measure different bovine serum albumin at 595nm The absorbance of the solution.

[0041]After reacting the fermentation broth with Coomassie Brilliant Blue G-250, measure the absorbance at 595nm, establish a standard curve and calculate the protein content in the fermentation broth. like figure 1 As shown, the standard curve is established as: y=2.1295x-0.0275, R 2 = 0.9949. It was detected that the protein content in the initial fermentation liquid was 0.129g / L.

Embodiment 3

[0042] Embodiment 3 calculates recovery rate and purity, protein removal rate of surfactin

[0043] Recovery rate:

[0044] purity:

[0045] Protein removal rate:

[0046] Calculation of the content of surfactin: Quantitative analysis of the concentration of surfactin in the culture medium was carried out by ultra-high performance liquid chromatography (UPLC). Using Waters BEH C 18 Chromatographic column (100mm×2.1mm, 1.7μm particle) for liquid phase gradient separation; mobile phase A is 0.1% formic acid aqueous solution, mobile phase B is HPLC grade methanol; elution gradient: 0.1min, 70% B; 0.1–2.0min , 70% B; 2.0–8.0 min, 70%–100% B; 8.0–10 min, 100% B; 10.1 min, 70% B, 10.1–13 min 70% B; the flow rate was 0.3 mL / min. The UV detection wavelength is 205nm. Configure methanol standard solutions of surfactin with different concentration gradients, and make a surfactin standard curve according to the changes in peak area and concentration. Then, according to the in...

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Abstract

The invention discloses a method for separating and purifying lipopeptide surfactin family compounds, which belongs to the technical field of fermentation engineering. Invented the method of using ethanol to remove protein and then using acid precipitation and pickling to remove small molecular impurities, and obtained high-purity surfactin family compounds. Among them, the purity of surfactin can reach 94.2%, and the recovery rate can reach 96.3%, and the recovery rate of lichenin can reach 95.0%, and the purity can reach 92.6%. The obtained product is a milky white powder with high purity and no further refining is required. A series of methods for separating and purifying lipopeptide surfactin family compounds adopted in the present invention are simple in operation, environmentally friendly, low in cost, high in product purity and recovery rate, and can be effectively applied to actual industrial production.

Description

technical field [0001] The invention relates to a method for separating and purifying lipopeptide surfactin family compounds, belonging to the technical field of fermentation engineering. Background technique [0002] Since 1949, more than 30 Bacillus lipopeptides with different structures have been discovered successively. These lipopeptide compounds have unique and highly similar structures, and are all cyclic heptapeptide compounds synthesized by the non-ribosome peptide pathway (Non-Ribosome Peptide Synthases, NRPS). According to the different amino acid sequences and ring-forming methods in the polypeptide, Bacillus lipopeptides are divided into four families: surfactin, fengycin, iturin, and kurstakin, and each The family contains a variety of structural analogs. Among them, the surfactin family can be further divided into surfactin and lichenin. Surfactin is mainly synthesized by Bacillus subtilis and Bacillus amyloliquefaciens, while lichenin is mainly synthesized ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/64C07K1/04C09K8/584C09K8/60
CPCC07K7/64C09K8/584C09K8/602
Inventor 吴群杨娜熊钊徐岩
Owner JIANGNAN UNIV
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