Bacillus flexus R29-2 from deep sea, micro-ecological preparation and application thereof

A technology of Bacillus flexus and microecological preparations, applied in the field of microorganisms, to achieve the effects of increasing survival rate, improving disease resistance, and reducing feed-to-meat ratio

Active Publication Date: 2020-04-14
OCEAN UNIV OF CHINA +3
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] 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 antibacterial effect has been found. Related reports on the activity, the present invention isolated a strain of Bacillus flexus from the deep sea, which has the activity of inhibiting Gram-positive bacteria such as Staphylococcus aureus, Streptococcus agalactiae, Enterococcus faecalis, and Bacillus cereus, and was verified by hemolysis experiments Non-hemolytic, good application in broiler and tilapia farming

Method used

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  • Bacillus flexus R29-2 from deep sea, micro-ecological preparation and application thereof
  • Bacillus flexus R29-2 from deep sea, micro-ecological preparation and application thereof
  • Bacillus flexus R29-2 from deep sea, micro-ecological preparation and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1, identification of bacterial strain R29-2

[0026] (1) Morphological identification

[0027] Bacterial colonies are beige opaque on the LB plate, with smooth edges, wet and flat; after Gram staining, the bacterial cells are purple short rods under an optical microscope (see figure 1 ).

[0028] (2) Hemolytic

[0029] Pick a colony of Bacillus flexus R29-2, spot it on a blood agar plate, and observe after culturing at 37°C for 48 hours, no hemolysis.

[0030] (3) Strain identification

[0031] 16S rRNA gene amplification and sequencing: R29-2 was inoculated into LB broth and cultured overnight at 37°C, using the bacterial liquid as a template, the bacterial 16S rRNA gene sequence was amplified using bacterial universal primers 27F and 1492R. The PCR amplification system is 50 μL, 1 μL of bacterial liquid as a template, 1 μL of upstream and downstream primers, 25 μL of 2×Mix, ddH 2 O 22 μL. PCR reaction program: pre-denaturation at 95°C for 5 minutes; d...

Embodiment 2

[0035] Embodiment 2, the antibacterial activity assay of bacillus flexus R29-2

[0036] For the determination of the antibacterial activity of Bacillus curvedus R29-2, Escherichia coli K88, Aeromonas hydrophila T2W-1, Klebsiella pneumoniae THM1 were selected as Gram-negative indicator bacteria; Gram-positive indicator bacteria Select Staphylococcus aureus JP, Streptococcus agalactiae Y405, Enterococcus faecium2#XB, Bacillus cereus 3③.

[0037] Pick R29-2 single colony on LBH plate, culture at 28°C for 24 hours, take 100 μl of indicator bacteria solution in 10ml LB or MRS medium (containing 0.8% agar), mix well and pour it on the cultured R29-2 single colony On the colony plate, observe the diameter of the inhibition zone after culturing at 37°C for 12 hours.

[0038] Its antibacterial effect is shown in Table 1 below: the bacterium has antagonistic effects on a variety of Gram-positive bacteria, including Staphylococcus aureus, Streptococcus agalactiae, Enterococcus faecalis ...

Embodiment 3

[0041] Embodiment 3, the liquid submerged aerated fermentation condition exploration of Bacillus flexus R29-2

[0042] (1) Preparation of seed liquid: Pick a single colony from the LBH agar medium on the inclined plane of the test tube of Bacillus flexus R29-2, inoculate it in a test tube of LBH liquid medium, and cultivate it at 32°C and 120rpm for 16-20h to obtain the first-grade seeds liquid; the primary seed liquid was inoculated in LBH liquid medium at a concentration of 2%, cultivated at 32°C and 120rpm until the logarithmic growth phase, and the concentration was 10 7 -10 8 cfu / ml of secondary seed solution.

[0043] (2) Liquid submerged fermentation: preparation for culture is based on a 50L fermenter with an inoculum size of 1-2%, and a glucose solution is separately sterilized, and glucose is added as a carbon source during the fermentation process. Adjust the dissolved oxygen conditions in the fermenter by setting the rotation speed and ventilation, and monitor th...

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Abstract

The invention relates to a bacillus flexus strain. The preservation name of the strain is bacillus flexus R29-2, the preservation unit is China General Microbiological Culture Collection Center, and the preservation number is CGMCC No.18731. The strain is derived from the west Pacific Caroline seamount, and is a circular beige smooth opaque bacterial colony in an LB agar culture medium. The strainis in a short rod shape under a microscope, and gram staining is positive. The strain has a good antibacterial effect on gram-positive bacteria such as staphylococcus aureus, streptococcus agalactiae, enterococcus faecalis and bacillus cereus, and can effectively increase the survival rate of poultry, reduce the feed conversion ratio and improve the disease resistance of aquaculture animals whenbeing prepared into a micro-ecological preparation and serving as a feed additive in poultry breeding and aquaculture.

Description

technical field [0001] The invention relates to the technical field of microorganisms, in particular to a deep-sea source of Bacillus flexus R29-2, which can effectively inhibit Gram-positive bacteria such as Staphylococcus aureus, Streptococcus agalactiae, Enterococcus faecalis, and Bacillus cereus , can significantly increase the survival rate of poultry, reduce the ratio of feed to meat, and can effectively improve the disease resistance of aquaculture animals. Background technique [0002] In recent years, the misuse and abuse of antibiotics for humans and animals has greatly accelerated the formation of microbial resistance. As the time limit for a complete ban on antibiotics in the breeding industry in 2020 is approaching, it is imminent to find safe and efficient alternative antibiotic products. There are a large number of microbial flora in the intestinal tract of animals. When using antibacterial drugs to kill harmful bacteria in animals, it will also kill the norma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20A23K10/18A23K50/80A23K50/75C12R1/07
CPCA23K10/18A23K50/75A23K50/80C12N1/20C12N1/205C12R2001/07
Inventor 何增国乔晓妮汤伟孙晓雯唐涛张军许向阳
Owner OCEAN UNIV OF CHINA
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