Method for quickly and efficiently screening nisin high-yield strain

A technology of nisin and high-yield strains, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve problems such as difficult industrial application, restricted application, low reliability, etc., to improve efficiency and workload The effect of reducing and increasing the probability

Inactive Publication Date: 2017-04-26
昆山博青生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the original method has a large workload, small scope, and other factors, which restrict its application in the large-scale screening of Lactococcus lactis lactis streptococcus strains with high Nisin production
[0006] Chinese patent, ZL.2011100251152, discloses a screening method for Aspergillus niger with high glucosidase production, however, the technology provided by this patent has low reliability and is difficult to apply industrially

Method used

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  • Method for quickly and efficiently screening nisin high-yield strain
  • Method for quickly and efficiently screening nisin high-yield strain
  • Method for quickly and efficiently screening nisin high-yield strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] (1) primary screening; Lactococcus lactis was subjected to ultraviolet mutagenesis for 30s to obtain Lactococcus lactis subsp. lactis, as screening starting strains, the mutagenized Lactococcus lactis was added to a sodium chloride solution of 0.75% by weight to obtain Bacterial suspension containing Lactococcus lactis;

[0060] a. Preparation of Lactococcus lactis fermented metabolite liquid, the mutagenized Lactococcus lactis was cultured in a lactic acid bacteria medium for 6hh, and then the lactic acid with a weight concentration of 10% was used to adjust pH 1.5 and let stand for 5min , Centrifuge at 6000rpm for 5min, pass through a filter membrane with a 0.1μm pore size, filter the filtrate after residual bacterial cells, and obtain Lactococcus lactis fermentation metabolite;

[0061] Lactic acid bacteria culture medium, including the following components by weight:

[0062] K 2 HPO 4 0.2%

[0063] Sodium acetate 0.3%

[0064] M g SO 4 7H 2 0 0.2%

[0065...

Embodiment 2

[0093] (1) primary screening; with Lactococcus lactis, sodium nitrite mutagenesis 1min, obtain Lactococcus lactis subsp. lactis, as screening starting bacterial strain, will contain the Lactococcus lactis after mutagenesis and add the sodium chloride solution of weight concentration 0.9% , to obtain a bacterial suspension containing Lactococcus lactis;

[0094] a. Preparation of Lactococcus lactis fermented metabolite, the mutagenized Lactococcus lactis was cultured in lactic acid bacteria medium for 48h, then lactic acid was adjusted to pH 3.0, left standing for 30min, centrifuged at 12000rpm for 30min, passed through 22 μm Filter membrane with pore size, filter the filtrate after residual bacterial cells, and obtain Lactococcus lactis fermentation metabolite;

[0095] Lactic acid bacteria culture medium, the following components by weight:

[0096] K 2 HPO 4 1%

[0097] Sodium acetate 0.8%

[0098] M g SO 4 7H 2 0 0.02%

[0099] Ammonia citrate 0.2%

[0100] Sucro...

Embodiment 3

[0127] Compared with the existing method, the method of the present invention has higher work efficiency, obviously reduces the workload and improves the screening probability. The purpose is stronger, and the probability of obtaining the target mutant strain is higher.

[0128] Table 1 Comparison of existing screening method and screening preliminary screening efficiency of the present invention

[0129]

[0130] It can be seen from the results that taking the Nisin resistance plate containing 48000U / mL as an example, the existing initial screening plate, 100uL bacterial suspension can finally obtain 430 potential strains per plate. The titer was determined, and finally the high-yielding strains were screened according to the comparison of the titer. However, in the method of the present invention, 17 potential strains can be obtained for each plate from the same bacterial suspension, and only the 17 strains need to be fermented to determine the titer, the workload is gre...

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Abstract

The invention provides a method for quickly and efficiently screening a nisin high-yield strain. The method is characterized in that induced mutant lactococcus lactis servers as a starting strain, and low pH-Nisin resistance plate serves as a strain culture carrier for preliminary screening. The method is simple, and required agents are common; by adoption of the low pH resistance plate, screening efficiency is improved greatly through double indexes, a screening range is widened, possibility of screening out of the high-yield mutant strain is increased, and the grown-out strain is more representative; cell growth metabolite is treated to directly prepare a screening culture medium to screen a substrate tolerant strain, a substrate retroinhibition effect of cells is improved, cell growth and metabolism performances are improved, and the obtained strain is superior. By a re-screening step, all strains growing on the preliminary screening plate can be rescreened by one step, screening omission is avoided, and screening workload can be relieved effectively.

Description

technical field [0001] The invention relates to a screening method for high nisin producing strains. Background technique [0002] Nisin, a small peptide with strong bactericidal effect synthesized and secreted by certain Lactococcus Lactis during the metabolic process, also known as lactococcin or nisin. [0003] Nisin can inhibit the growth and reproduction of most Gram-positive bacteria and their spores, such as Staphylococcus, Streptococcus, Lactococcus, Clostridium and Bacillus, especially Staphylococcus aureus, Hemolytic streptococcus and botulinum have obvious inhibitory effect. Therefore, Nisin can significantly extend the preservation time of food or shorten the time required for sterilization, and has a good effect on the preservation and preservation of dairy products, meat products, canned foods and vegetable protein foods. [0004] At present, one of the main problems in Nisin research is that the Nisin-producing ability of Lactococcus lactis obtained from nat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/01C12R1/46
CPCC12N15/01
Inventor 叶贵子黄青山
Owner 昆山博青生物科技有限公司
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