Bacteriocin with antibacterial activity as well as production method and application thereof

A production method and technology for bacteriostatic activity, which are applied in the preparation methods of peptides, microorganism-based methods, biochemical equipment and methods, etc., to achieve the effect of stable properties

Inactive Publication Date: 2021-04-06
OCEAN UNIV OF CHINA +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Bacillus flexus is a Gram-positive bacterium. It has been reported in the literature that it has good applications in degrading organic nitrogen, catechol, promoting biogas fermentation, and producing β-amylase, but no inhibitory effect has been found. Related reports on bacterial activity

Method used

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  • Bacteriocin with antibacterial activity as well as production method and application thereof
  • Bacteriocin with antibacterial activity as well as production method and application thereof
  • Bacteriocin with antibacterial activity as well as production method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] The preparation of embodiment 1 campycin fermented liquid

[0037] (1) Basal medium LBM: yeast powder 1-15g / L, sodium chloride 1-60g / L, tryptone 1-30g / L, glucose 1-50g / L, BG11 10-30g / L.

[0038] (2) Seed liquid preparation: pick R29-2 single colonies from the inclined surface of the test tube to the first-level seed medium, culture for 16-20 h, then transfer to the second-level seed medium, and culture at 32°C, 120rpm for 16-20h to logarithmic growth period.

[0039] (3) Liquid submerged fermentation: according to (1) preparation of 20L LBM culture is based on 50L fermenter, the inoculum size is 1%, during the fermentation process, sampling is carried out every 2h to monitor the OD of the fermented liquid 600 , pH, dissolved oxygen (DO) and antibacterial activity changes (see figure 1 ), to be OD 600 The value tends to be stable, that is, the lower tank of the fermentation is stopped to obtain the bacteriocin fermentation liquid.

Embodiment 2

[0040] The extraction and purification of embodiment 2 campycin

[0041] (1) Screening of macroporous resins: 10 kinds of macroporous resins with different adsorption effects were selected, and D3520, XR925, D101, XR901, XR926M, XR925CSB, AB-8, XR929C, XAD-1, D4006 were used respectively. After pretreatment Add the same amount of sterile fermentation supernatant, static adsorption for 2h. Remove the unadsorbed fermentation broth supernatant, add an equal amount of ethanol solution to elute. Collect ethanol eluate and detect bacteriostasis activity, screen and obtain effective adsorption resin accordingly and be: D4006 macroporous resin, D3520 macroporous resin, preferably, best adsorption resin is D4006 type macroporous resin (see figure 2 ).

[0042] (2) Rough extraction of bacteriocin: get the bacteriocin-containing fermented liquid and collect the supernatant after centrifuging at 10000rpm after leaving the tank, choose D4006 type macroporous resin, and carry out prelimi...

Embodiment 3

[0048] The identification method of embodiment 3 campycin

[0049] (1) By performing Tricine-SDS-PAGE and antibacterial experiments on the crude extract, the sample was diluted 1:1 (V / V) with tricine buffer, and heated at 40°C for 30-60min. After adding the sample, electrophoresis was performed at a constant voltage of 30V for 1 hour, followed by electrophoresis at a constant voltage of 150V for 2 hours, and a low molecular weight protein standard was used as a control. After electrophoresis, after Coomassie brilliant blue staining and decolorization, there is a blue band on the swimming lane of the bacteriocin sample corresponding to the standard product between 5.8-7.8kDa, which also confirms the protein properties of campycin (see Figure 6 ).

[0050] (2) Utilize matrix-assisted laser desorption ionization time-of-flight mass spectrometry to measure the molecular weight and identify its structure of the separated and purified campycin, wherein, the mass spectrometry analy...

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Abstract

The invention relates to bacteriocin with antibacterial activity. The bacteriocin is prepared by fermenting Bacillus flexus, the molecular weight of the bacteriocin is 6094 Da, and the bacteriocin is stable under acid-base conditions, can tolerate high temperature, is not sensitive to trypsin, pepsin and other digestive enzymes, and has very strong prevention and antagonism effects on gram-positive pathogenic bacteria such as staphylococcus aureus, streptococcus agalactiae and clostridium perfringens, and has a wide application prospect in the fields of medical treatment, food, feed, cultivation, planting and the like.

Description

technical field [0001] The present invention relates to bacteriocins, and relates to the field of microbial fermentation, in particular to a campycin produced by deep-sea-derived Campylobacter R29-2, which can effectively antagonize Staphylococcus aureus, Streptococcus agalactiae and Enterococcus faecalis , Clostridium perfringens and other pathogenic bacteria. Background technique [0002] Since Fleming's discovery of penicillin in the late 1920s, antibiotics have revolutionized medicine, saving the lives of millions of patients each year and playing a key role in preventing infectious disease and performing surgical procedures. However, in recent years, the misuse and abuse of antibiotics for humans and animals has greatly accelerated the formation of microbial resistance, and research on new antibacterial substances has attracted much attention. [0003] Bacteriocins are a class of peptides with antibacterial activity that are synthesized by ribosomes and need to be acti...

Claims

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Application Information

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IPC IPC(8): C07K14/32C07K1/36C07K1/30C07K1/16C12P21/00A61K38/16A61P31/04C12N1/20C12R1/07
CPCC07K14/32C12P21/00A61P31/04C12N1/20A61K38/00
Inventor 何增国乔晓妮孙晓雯汤伟唐涛张军
Owner OCEAN UNIV OF CHINA
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