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Method for purifying nisin through preparative high performance liquid chromatography

A technology of nisin and high-performance liquid chromatography, which is applied in peptide preparation methods, chemical instruments and methods, organic chemistry, etc., can solve the problems that the purification process needs to be further developed, it is not suitable for industrial production, and the process flow is complicated. Achieve the effect of improving purification effect, fast separation speed, simple and efficient process

Active Publication Date: 2021-06-01
宁波博睿瀚达生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The Chinese patent with publication number CN105755074A discloses a method for extracting Nisin. Although the activity is improved, its technological process is complicated and requires four steps of chromatographic separation. The preparation period is long, the efficiency is low, and the cost is high. Suitable for industrial production
[0004] Therefore, ensuring activity and stability under high-purity conditions is still a difficult problem for the preparation of high-purity nisin, and the green and efficient purification process still needs to be further developed

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  • Method for purifying nisin through preparative high performance liquid chromatography
  • Method for purifying nisin through preparative high performance liquid chromatography
  • Method for purifying nisin through preparative high performance liquid chromatography

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Embodiment 1

[0042] This embodiment provides a method for purifying nisin by preparative high performance liquid chromatography, comprising the following steps:

[0043] (1) Salting out: at room temperature, in the nisin fermented liquid, add the sodium chloride solution that concentration is 20wt%, after stirring and dissolving, leave standstill for 2 hours, centrifuge, discard the supernatant, collect the precipitate, Redissolve with a sodium acetate-acetic acid buffer solution with a concentration of 50mmol / L and a pH value of 3.6 to obtain a Nisin crude product solution;

[0044] (2) Reversed-phase chromatographic separation: take the Nisin crude product solution and inject it with a flow rate of 300mL / min, first use the equilibrium liquid I of 5 times the column volume to the phenyl-bonded silica gel chromatographic column (diameter 100mm × length 1000mm, particle diameter 5 μm, Pore ​​diameter of 120 angstroms) for column balance, and then eluted with eluent I, when the baseline rise...

Embodiment 2

[0050] This embodiment provides a method for purifying nisin by preparative high performance liquid chromatography, comprising the following steps:

[0051] (1) Salting out: at room temperature, in the nisin fermented liquid, add the sodium chloride solution that concentration is 20wt%, after stirring and dissolving, leave standstill for 2 hours, centrifuge, discard the supernatant, collect the precipitate, Redissolve with a sodium citrate-citric acid buffer solution with a concentration of 50mmol / L and a pH value of 3.6 to obtain a crude Nisin solution;

[0052] (2) Reversed-phase chromatographic separation: take the Nisin crude product solution and inject it with a flow rate of 300mL / min, first use the equilibrium liquid I of 5 times the column volume to the phenyl-bonded silica gel chromatographic column (diameter 100mm × length 1000mm, particle diameter 5 μm, Pore ​​diameter of 120 angstroms) for column balance, and then eluted with eluent I, when the baseline rises, start...

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Abstract

The invention belongs to the technical field of biological medicine separation, and particularly relates to a method for purifying nisin through preparative high performance liquid chromatography. The method for purifying nisin comprises the following steps: taking a Nisin crude product, firstly carrying out hydrophobic chromatography by taking phenyl bonded silica gel as a stationary phase, then carrying out weak cation chromatography by taking carboxyl bonded silica gel as a stationary phase, and separating to obtain high-purity Nisin. Hydrophobic chromatography and weak cation chromatography are respectively carried out on the salted-out crude pure nisin liquid, the high-purity Nisin is prepared only through three steps, the purity of the nisin is improved, the inactivation rate of polypeptide is reduced, and the total activity recovery rate of the nisin can reach 75% or above. The two chromatographic methods are combined to improve the purification effect of the nisin, the selectivity is good, the resolution ratio is high, the separation speed is high, the used chromatographic column does not need to be regenerated by strong acid and strong alkali and is green and environment-friendly, the service life of the chromatographic column is long, and therefore the industrial production cost can be reduced.

Description

technical field [0001] The invention relates to the technical field of separation of biomedicine, in particular to a method for purifying nisin by preparative high performance liquid chromatography. Background technique [0002] Nisin (Nisin, Nisin), also known as nisin, is a polypeptide compound synthesized and secreted by Streptococcus nisin during the metabolic process. It consists of 34 amino acid residues, and its molecular terminal has amino and carboxyl groups. The terminal is isoleucine, the carboxyl terminal is lysine, and the molecular weight is about 3500Da. Streptococcus lactis has stable and high-efficiency antibacterial activity, can be absorbed and utilized by the human body, will not change the normal flora in the human intestinal tract, and will not produce drug resistance or cross-resistance like other antibiotics. Nisin can effectively inhibit a variety of Gram-positive bacteria that cause food spoilage. It is a good product for food preservation and anti...

Claims

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Application Information

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IPC IPC(8): C07K14/315C07K1/36C07K1/30C07K1/20C07K1/18
CPCC07K14/315
Inventor 朱文瑾李浛君陈平李浛民
Owner 宁波博睿瀚达生物科技有限公司