Antimicrobial protein product of bacillus amyloliquefaciens NCPSJ7 and preparation method of antimicrobial protein product

A technology of amylolytic spores and antibacterial proteins, which is applied in the preparation method of peptides, chemical instruments and methods, food preservation, etc., can solve problems such as no further research, achieve good application prospects, improve purity, and reduce costs

Active Publication Date: 2014-07-30
INST OF AGRO FOOD SCI & TECH SHANDONG ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The literature does not further study its various formulations

Method used

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  • Antimicrobial protein product of bacillus amyloliquefaciens NCPSJ7 and preparation method of antimicrobial protein product
  • Antimicrobial protein product of bacillus amyloliquefaciens NCPSJ7 and preparation method of antimicrobial protein product
  • Antimicrobial protein product of bacillus amyloliquefaciens NCPSJ7 and preparation method of antimicrobial protein product

Examples

Experimental program
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Effect test

Embodiment 1

[0032] Example 1: Preparation of crude antibacterial protein extract

[0033] For the culture and fermentation conditions of Bacillus amyloliquefaciens NCPSJ7, see Example 2 of Chinese Patent Application 201310112580.9 (Name of Invention: Broad-spectrum Antibacterial Bacillus amyloliquefaciens strain and its application). Finally, it was cultured in shake flask for 6 days to obtain fermentation broth.

[0034] Centrifuge 10L fermentation broth at 4℃, 13000rpm for 20 minutes, collect the fermentation supernatant, slowly add solid ammonium sulfate to 60% saturation while stirring, let stand overnight at 4℃, centrifuge at 13000rpm for 20 minutes, collect the precipitate, add 50mM pH7 .0 phosphate buffer 1L reconstituted, and centrifuged at 13000rpm for 20 minutes, collect the supernatant, which is the crude antibacterial protein extract;

[0035] Place 1L of the crude antibacterial protein extract in the ultrafiltration device, filter with 5000Da ultrafiltration membrane at 4℃, when the...

Embodiment 2

[0036] Example 2: DEAE Sepharose Fast Flow Anion Exchange Column Chromatography of Antibacterial Protein

[0037] The crude antimicrobial protein extract was loaded at natural flow rate, and eluted 1 column volume with 50mM pH7.0 phosphate buffer, and then 0.25M NaCl solution (prepared with 50mM pH7.0 phosphate buffer) was used for elution 1- 2 column volumes to remove impurities; then 4-5 column volumes of 0.25-0.6M NaCl (prepared with 50mM pH 7.0 phosphate buffer) solution gradient elution were used to obtain two elution peaks (0.25- 0.4M NaCl elution peak, 0.4-0.6M NaCl elution peak). The elution speed is 1.0ml / min, the detection wavelength is 280nm; the elution peak of 50mM pH7.0 phosphate buffer, the elution peak of 0.25M NaCl and the two elution peaks of 0.25-0.6M NaCl are collected. The chromatogram is shown in figure 1 , Using Oxford cup method to detect the antibacterial activity of each elution peak against the pathogen of watermelon fusarium wilt, see figure 2 .

[003...

Embodiment 3

[0039] Example 3: Preparation of antibacterial protein spray powder

[0040] Put the above-mentioned antibacterial protein sample in an ultrafiltration device, filter with 5000Da ultrafiltration membrane at 4°C, add 50mM pH6.5-8.0 phosphate buffer to the original volume of the intercepted part, repeat 3 times, collect the intercepted part, It is the antibacterial protein sample after desalination and concentration;

[0041] The crude antibacterial protein extract sample is spray-dried at an inlet temperature of 130°C, an outlet temperature of 65°C, and a material flow rate of 3.3 mL / min, and the spray dried powder is collected, which is an antibacterial protein product. After testing, its main components are: 24kDa and 36kDa two antibacterial proteins, the total content of these two antibacterial proteins accounted for more than 65% of the mass of the spray dried powder.

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Abstract

The invention discloses an antimicrobial protein product of bacillus amyloliquefaciens NCPSJ7 and a preparation method of the antimicrobial protein product. The preparation method comprises the steps of centrifuging a bacillus amyloliquefaciens NCPSJ7 fermentation liquid, and collecting a supernatant liquid; carrying out ammonium sulfate precipitation, ultrafiltration, desalination and concentration on the fermented supernatant liquid; then, carrying out anion exchange column chromatography, ultrafiltration, desalination and concentration; next, carrying out spray drying to obtain the antimicrobial protein product. The antimicrobial protein product prepared by using the method mainly contains the two antimicrobial proteins: 24kDa and 36kDa, and the total content of the two antimicrobial proteins accounts for more than 60% of the mass of spray dried powder. The antimicrobial protein product is favorable in reconstitution properties, strong in water solubility and favorable in antimicrobial activity and has a certain industrial application prospect so as to be applied to the fields such as fruit and vegetable preservation and fresh keeping, food additives, fish medicines, veterinary medicines and the like.

Description

Technical field [0001] The invention belongs to the technical field of separation, purification and preparation of microbial fermentation broth, and particularly relates to a Bacillus amyloliquefaciens antibacterial protein product and a preparation method thereof. Background technique [0002] Plant diseases are a global problem, causing huge economic losses. Statistics from the Food and Agriculture Organization of the United Nations (FAO) show that the average annual loss caused by plant diseases is about 10%-15% of the total output, of which plant diseases caused by fungi account for about 80%. At present, chemical fungicides are mainly used to control plant diseases. Although chemical fungicides can effectively reduce plant diseases, chemical fungicides also cause negative effects. Chemical residues threaten the health of humans and animals and endanger the environment. Therefore, research and development of new methods that can replace chemical fungicides to control plant ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/32C07K1/36C07K1/34C07K1/30C07K1/18A23B7/154
CPCA23B7/154C07K14/32
Inventor 裘纪莹陈相艳陈蕾蕾祝清俊王未名刘孝永周庆新张翔
Owner INST OF AGRO FOOD SCI & TECH SHANDONG ACAD OF AGRI SCI
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