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Bacillus subtilis DK-03 and application thereof

A technology of Bacillus subtilis and DK-03, which is applied in the field of microorganisms, can solve the problems of limited research reports on the application of Bacillus subtilis, and achieve the effects of improving the balance of intestinal flora, promoting digestion and absorption, and improving growth performance

Pending Publication Date: 2022-07-29
LIAOCHENG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the research reports on the application of Bacillus subtilis in donkey foal feed are relatively limited, and the specific application dosage and the effect on the growth performance of donkey foals need further research

Method used

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  • Bacillus subtilis DK-03 and application thereof
  • Bacillus subtilis DK-03 and application thereof
  • Bacillus subtilis DK-03 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Screening of Bacillus subtilis

[0033] The Bacillus screening method of the present invention comprises the following steps:

[0034] Collected from the healthy intestinal contents, fresh feces and the surrounding soil of the donkey farms in Yucheng and Qiping, Liaocheng City, after being heated in a water bath at 85°C for 10 minutes, diluted to 10 -7 , take 10 -5 and 10 -7 100 μL of each diluted bacterial solution was injected and inoculated onto a nutrient agar plate, and cultured in an incubator at 37 °C for 48 h. Pick a single colony from the plate, gram-stain it, select the gram-positive bacteria, and purify and cultivate the single bacteria until there are no impurities observed under the microscope. The purified strains were stored in an ultra-low temperature freezer at -80°C with a volume fraction of 30% glycerol.

[0035] Through the above method, a colony was isolated from the intestinal contents of a donkey with a flat, dry, dull surface, opaqu...

Embodiment 2

[0036] Example 2 Sequencing and identification of Bacillus subtilis

[0037] Using forward primer P1: 5'-AGAGTTTGATCCTGGCTCAG-3', reverse primer P2: 5'-GGTTACCTTGTTACGACTT-3', the primers were synthesized by Shanghai Lifei Biotechnology Co., Ltd. (Beijing), and 16SrDNA was amplified. PCR reaction system: 2x PCR mix 30 μL, primers 2.5 μL each, bacterial solution 2 μL, ddH 2 O 23 μL. The PCR program was: 95°C for 5 min; 95°C for 30 s, 55°C for 30 s, 72°C for 90 s, a total of 30 cycles; the last extension at 72°C for 10 min, the PCR amplification product was incubated in 1.0% agarose Electrophoresis on a gel (containing SYBR® Green I at a concentration of 1 / 10,000) for 30 minutes, observe under UV light, and confirm that the PCR product of the product is successful and meets the requirements, and then send the PCR product to Shanghai Lifey Biotechnology Co., Ltd. (Beijing) for sequence determination. The sequencing results were subjected to homology analysis in NCBI. Through ...

Embodiment 3

[0038] Example 3 Safety analysis of Bacillus subtilis

[0039] A single colony of Bacillus subtilis DK-03 was seeded on a blood agar plate, and placed in a 37°C incubator for 24-48 hours to observe whether there were haemolysis zones and the type of haemolysis zones.

[0040] Colony hemolysis has the following three situations:

[0041] (1) Alpha hemolysis: also known as grass green hemolysis, the narrow (1-2 mm), grass green hemolysis ring formed around the colony is caused by methemoglobin;

[0042](2) β hemolysis: also known as complete hemolysis, a completely clear and transparent hemolysis ring is formed around the colony, which is caused by the complete lysis of red blood cells by hemolysin produced by bacteria;

[0043] (3) γ-hemolysis: that is, no hemolysis, no change in the medium around the colony, and no lysis or defect of red blood cells.

[0044] After 48 hours, the isolated Bacillus subtilis DK-03 showed γ-type hemolysis on the hemolytic plate, that is, no hemo...

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Abstract

The invention provides bacillus subtilis DK-03 and application thereof. The bacillus subtilis DK-03 is preserved in the China Center for Type Culture Collection on January 5, 2021, and the preservation number of the bacillus subtilis DK-03 is CCTCC (China Center for Type Culture Collection) No. M2021018. The bacillus subtilis DK-03 disclosed by the invention has relatively good enzyme production performance and antibacterial performance, is high in safety, and can be used as a fungicide or a feed additive for improving the intestinal flora balance of the donkey foals, improving the organism immune function of the donkey foals, promoting the digestion and absorption of the donkey foals on nutrient substances in feed, improving the growth performance of the donkey foals and improving the survival rate of the donkey foals. The economic benefits of the donkey farm are favorably improved.

Description

technical field [0001] The invention belongs to the technical field of microorganisms, and in particular relates to a Bacillus subtilis DK-03 and its application. Background technique [0002] The donkey industry is a characteristic animal husbandry in my country, and the healthy breeding of donkey foals is a prerequisite for the development of the donkey industry. In the donkey foal stage, the intestinal microbes of the donkey have low diversity and abundance, single function, insufficient secretion of endogenous digestive enzymes such as intestinal lipase and amylase, and are affected by weaning stress, Escherichia coli and other pathogenic microorganisms. , prone to problems such as intestinal flora imbalance, indigestion, and decreased production performance. Studies have found that lactic acid bacteria preparations can antagonize pathogenic microorganisms, regulate the balance of intestinal flora, promote the digestion and absorption of nutrients, and improve immune fu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20A23K10/18A23K50/20A61K35/742A61P1/00A61P37/04A61P31/04C12R1/125
CPCC12N1/20A23K10/18A23K50/20A61K35/742A61P1/00A61P37/04A61P31/04
Inventor 李艳王长法马青山王永晖刘桂芹
Owner LIAOCHENG UNIV
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