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Bacillus subtilis shou003, anti-vibrio protein and preparation method and applications of bacillus subtilis shou003 and anti-vibrio protein

A Bacillus subtilis, anti-Vibrio protein technology, applied in the field of microorganisms, can solve the problems of increasing control difficulty and endangering human health, etc.

Inactive Publication Date: 2014-06-04
SHANGHAI OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a Bacillus subtilis shou003, aiming to solve the problems of bacterial drug resistance caused by existing antibiotics to prevent and treat vibriosis, increase the difficulty of prevention and endanger human health, etc.

Method used

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  • Bacillus subtilis shou003, anti-vibrio protein and preparation method and applications of bacillus subtilis shou003 and anti-vibrio protein
  • Bacillus subtilis shou003, anti-vibrio protein and preparation method and applications of bacillus subtilis shou003 and anti-vibrio protein
  • Bacillus subtilis shou003, anti-vibrio protein and preparation method and applications of bacillus subtilis shou003 and anti-vibrio protein

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Experimental program
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Effect test

Embodiment 1

[0046] The separation of embodiment 1 bacillus subtilis shou003

[0047] The Bacillus subtilis shou003 (Bacillus subtilis) described in the present invention is obtained by separating from the intestinal tract of healthy large yellow croaker, and the separation method is as follows:

[0048] First, use 75% alcohol to disinfect the body surface; dissect under aseptic conditions, take out the intestine, wipe the outer wall of the intestine with 75% alcohol cotton ball, and rinse repeatedly with sterile normal saline. When preparing the total intestinal bacterial suspension (including the intestinal wall and intestinal contents), the foregut, midgut, and hindgut were ligated with sterile cotton thread, and the tissues with a length of about 0.5 cm were mixed together as samples. After weighting, add sterilized physiological saline in a ratio of 1:10 (w / v) and mix, grind thoroughly with a sterilized homogenizer, and set the ground sample to 10 -1 , and then sterilized saline for ...

Embodiment 2

[0049]The fermentation culture of embodiment 2 bacillus subtilis shou003

[0050] (1) Take the Bacillus subtilis shou003 strain and inoculate it into the activation medium, culture it on a shaker at 37°C for 18-20 hours, centrifuge it at 8000rpm / min for 5min, wash the precipitate with sterile water and prepare a concentration of 10 7 CFU / ml bacterial liquid; the preparation process of the activated medium is as follows: 9g of peptone, 2g of beef extract powder, 4g of sodium chloride, 1000ml of distilled water, heated and dissolved, divided into flasks, and sterilized at 121°C for 15min.

[0051] (2) Inoculate the bacterial liquid into the fermentation medium with an inoculation amount of 1% of the volume of the fermentation medium, and culture it on a shaking table at 4-40°C and pH 4 for 10-16 hours to obtain a Bacillus subtilis shou003 fermentation liquid The preparation process of the fermentation medium is as follows: maltose 1g, beef extract 1g, CaCl 20.2g, add distilled ...

Embodiment 3

[0052] Example 3 Tolerance of Bacillus subtilis shou003

[0053] Use an inoculation loop to pick up the slant strain of Bacillus subtilis shou003 and inoculate it into the activated medium, culture it on a shaker at 37°C for 18-20 hours, centrifuge at 8000rpm / min for 5min, discard the supernatant, wash it with sterile water for 3 times, and finally wash it with sterile water. Water was used to make bacterial liquid, and the concentration of bacterial liquid was adjusted to 10 7 CFU / ml for tolerance tests to temperature, NaCl, gastric juice, intestinal juice, and bile salts.

[0054] (1) Temperature resistance

[0055] Take 12 crude test tubes and divide them into 4 groups, each group has 3 replicates in parallel, each of which is added with 10ml of fermentation medium, after sterilization, Bacillus subtilis shou003 is inoculated into 12 test tubes with an inoculum amount of 1%, and then inoculated in 70 After treatment at ℃, 80℃, 90℃ and 100℃ for 30 minutes, after incubating...

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Abstract

The invention provides bacillus subtilis (Bacillussubtilis) shou003, an anti-vibrio protein and a preparation method and applications of the bacillus subtilis shou003 and the anti-vibrio protein. The preparation method of the anti-vibrio protein comprises the following steps: screening out bacillus subtilis shou003 (preserved in China Center for Type Culture Collection with a number of CCTCC No.: M2013571 on November 13, 2013) from intestinal tract of a healthy large yellow croaker; and then extracting the anti-vibrio protein from the fermentation broth of bacillus subtilis shou003, wherein the amino acid sequence of the anti-vibrio protein is shown as SEQ ID No.: 1. The bacillus subtilis shou003 and the anti-vibrio protein show good effects on inhibiting aquatic pathogenic bacteria, in particular pathogenic vibrio; in addition, the bacillus subtilis shou003 shows outstanding tolerance to temperature, NaCl, gastric juice, intestinal juice and cholate and can be widely applied to the prevention of germs during aquaculture; on that basis, the optimal fermentation culture method of the bacillus subtilis shou003, and a preparation method of the anti-vibrio protein are provided.

Description

technical field [0001] The invention belongs to the technical field of microbes, and in particular relates to a bacillus subtilis shou003, an antivibrio protein and a preparation method and application thereof. Background technique [0002] Vibriosis is caused by bacteria of the genus Vibrio. It is the most prevalent and harmful bacterial disease in aquatic animals such as fish, shrimp, crab and shellfish all over the world. It has caused great harm to the aquaculture industry. The economic loss is immeasurable. [0003] At present, the main method used to control vibriosis in the aquaculture industry is antibiotic prevention and control, because antibiotics are convenient to use and easy to operate, and they are effective faster and have better curative effect. However, due to the long-term application and abuse of chemical drugs such as antibiotics, many bacteria have gradually developed resistance to them, which has increased the difficulty of disease prevention, and som...

Claims

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Application Information

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IPC IPC(8): C12N1/20A01N63/00A01P1/00C12P21/02C07K14/32C07K1/34C12R1/125
Inventor 张庆华郭婧王娟封永辉宋增福陈彪张永华
Owner SHANGHAI OCEAN UNIV
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