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Bacillus subtilis producing lipase and inhibiting Vibrio splendidus and use method thereof

A technology of Bacillus subtilis and lipase, applied in the field of biology, can solve the problems of no reported pathogenic bacteria and lipase-secreting strains, and achieve the effects of avoiding economic losses, increasing survival rate, and huge economic benefits

Active Publication Date: 2018-12-07
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report that can inhibit the main pathogenic bacteria and the strains that secrete lipase at the same time.

Method used

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  • Bacillus subtilis producing lipase and inhibiting Vibrio splendidus and use method thereof
  • Bacillus subtilis producing lipase and inhibiting Vibrio splendidus and use method thereof
  • Bacillus subtilis producing lipase and inhibiting Vibrio splendidus and use method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Embodiment 1: Isolation and screening of bacterial strain

[0022] 1.1 Isolation of beneficial bacteria

[0023] (1) Primary screening:

[0024] The lipase-producing strains were initially screened by bromocresol purple oil assimilation plate color circle method. Prepare oil assimilation plate medium, adjust pH to 6.5, add 0.04% bromocresol purple indicator and emulsified olive oil after sterilization. Use an inoculation loop to scrape a small amount of bacteria and plant them on the plate, place it upside down at 28 for 3 days and then observe the size of the discoloration circle. The discoloration circle indicates that the bacteria has the ability to secrete lipase. The larger the discoloration circle, the stronger the enzyme production ability .

[0025] The primary screening medium is an oil assimilation plate medium, and the formula is as follows (%, w / w):

[0026] Emulsified olive oil 12, potassium dihydrogen phosphate 0.1, magnesium sulfate 0.05, iron sulfate...

Embodiment 2

[0032] Embodiment 2: identification of strain

[0033] The total DNA of the bacterial liquid was extracted and used as a PCR template for the bacterial liquid, and PCR amplification was performed with 27F and 1492R primers. PCR reaction system (10μl): cDNA 1μl, 10×Buffer 1μl, 2.5mM dNTP 0.8μl, 10μM forward and reverse primers 0.5μl each, 0.05μl rTaq enzyme, 2H2O 6.15μl.

[0034] PCR primers:

[0035]

[0036] The PCR reaction conditions are:

[0037]

[0038] After the whole reaction was finished, the reaction products were detected by 1.0% agarose gel electrophoresis. The target band was cut back under the gel imaging system, and the DNA was recovered and purified according to the instructions of the Shanghai Sangong SanPrep column kit. Recovered DNA - stored at 20°C or used for transformation link. The recovered product was connected to the PMD18-T cloning vector, and connected overnight at 16°C to obtain the target strain gene sequence PMD18-T recombinant vector. ...

Embodiment 3

[0045] Embodiment 3: bacterial strain activation condition

[0046] The Bacillus subtilis strain is inoculated into LB medium, and cultured at a temperature of 20-28°C for 8-10 hours to obtain activated Bacillus subtilis. The formula of the LB medium is: peptone 10g / L, yeast extract 5g / L, NaCl 10g / L.

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Abstract

The invention provides a strain of Bacillus subtilis ZF003 (Bacillus subtilis s), which is preserved in China General Microbiological Culture Collection Center and is assigned the accession number ofCGMCC No.15645, preservation date being Apr 26th, 2018. The invention also discloses an application of the Bacillus subtilis, wherein the Bacillus subtilis is applied to cultivation of stichopus japonicas, comprising two processes: (1) fermenting a stichopus japonicas feed with the Bacillus subtilis; (2) spreading a microbial liquid of the Bacillus subtilis in a breeding pool of the stichopus japonicas. The strain, on one hand, can secrete the lipase extracellularly, so that fat in the feed is decomposed by the lipase; and on the other hand, the strain can inhibit the main pathogenic bacteria,Vibrio splendidus, of skin ulcer syndrome of the stichopus japonicas.

Description

technical field [0001] The invention belongs to the field of biology and relates to a bacillus subtilis, in particular to a bacillus that produces lipase and inhibits Vibrio and its use method. Background technique [0002] With the rapid development of the aquaculture industry, the scale of high-density intensive farming is increasing day by day, and the economic loss of the aquaculture industry due to the deterioration of the environment of aquaculture and the harm of diseases has become more and more obvious. The traditional solution has been antibiotics; the use of antibiotics is known to lead to problems with resistant strains and lingering in the body. Probiotics are living microorganisms that can have beneficial effects on animals within a certain concentration range. Probiotics play a growth-promoting role in animals by enhancing the animal's inhibitory effect on harmful intestinal microbial flora, or by enhancing non-specific immune function to prevent disease. Th...

Claims

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Application Information

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IPC IPC(8): C12N1/20A23K10/18A23K50/80C12R1/125
CPCA23K10/18A23K50/80C12N1/20C12N1/205C12R2001/125
Inventor 朱伟冯政夫杨少辉董文欣
Owner QINGDAO AGRI UNIV
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