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Screening and application of probiotic bacillus licheniformis of high-yield compound enzyme

The technology of Bacillus licheniformis and compound enzyme, which is applied in the field of agricultural microbiology, can solve the problems of being susceptible to the influence of internal and external environment, high cost of enzyme preparation preparation, poor growth-promoting effect, etc., and achieves fast growth and reproduction speed and obvious bacteriostatic effect. , the effect of strong resistance

Active Publication Date: 2018-09-28
湖北华大瑞尔科技有限公司
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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 improve the poor growth-promoting effect of a single probiotic, the disadvantages of high enzyme preparation cost, unstable properties, and easy to be affected by internal and external environments, and provide a screening and application of a high-yield compound enzyme probiotic Bacillus licheniformis

Method used

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  • Screening and application of probiotic bacillus licheniformis of high-yield compound enzyme
  • Screening and application of probiotic bacillus licheniformis of high-yield compound enzyme
  • Screening and application of probiotic bacillus licheniformis of high-yield compound enzyme

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Isolation and identification of strains

[0032] 1. Separation of strains

[0033] Isolate samples were taken from 90-day-old healthy broiler manure from a chicken farm in Wuhan, Hubei Province. After the sample is collected, use a sterile cotton swab to pick up a small amount of feces in a 7mL EP tube filled with 3mL sterile water. After 10 minutes in a water bath at 80°C, spread the bacterial solution on the LB solid medium and select the rough surface The dirty white colonies with pleats and irregular edges are purely cultured. The pure cultures are morphologically observed by Gram stain for preliminary screening. The bacteria are long rod-shaped, single, paired or chain-like gram-positive bacilli. After 20 hours, spores were formed, producing near-mesophytic ellipsoid spores with slightly enlarged cysts. The strain of the invention was named Bacillus licheniformis HDRaBLp, and it was subcultured and preserved. The colony characteristics of this strain on sol...

Embodiment 2

[0061] Example 2: Verification of antibacterial performance of strains

[0062] When preparing a suspension of indicator bacteria (enterpathogenic E. coli, Staphylococcus aureus, Clostridium weichii), use an inoculation needle to pick a small amount of indicator bacteria from the slope and inoculate them into the conventional nutrient broth respectively. Incubate at ℃ for 24h, adjust the concentration of the bacterial suspension to about 10 9 CFU / mL.

[0063] Using the method of plate diffusion (Lyver et al., 1998; Lin Dong et al., 2001), dip a small amount of Bacillus licheniformis with an inoculating rod and spot them in the center of the poured nutrient agar plate. Incubate at 37°C for 24 hours, then buckle the plate upside down. Cover the petri dish with a thin layer of chloroform and fumigate for about 30 minutes to kill the living cells and allow the colony to be fixed on the surface of the plate, then remove the petri dish cover and let the residual chloroform in the plate c...

Embodiment 3

[0066] Example 3: Stress resistance and growth characteristics of Bacillus licheniformis strain HDRaBLp

[0067] 1. Tolerance test

[0068] (1) Bile salt tolerance test

[0069] After the Bacillus licheniformis strain HDRaBLp (isolated from 90-day-old healthy broiler manure of a chicken farm in Wuhan, Hubei Province in December 2013) was activated for 2 generations, 1 mL was inoculated into 9 mL containing 0%, 0.15%, and 0.30% , 0.50% bile salt in conventional LB liquid culture medium, culture for 12h at 37°C, dilute to a suitable multiple, take 0.1mL and apply to solid LB plate, observe the growth and count, repeat each treatment 3 times. The results show that HDRaBLp can tolerate 0.15%, 0.30%, and 0.50% bile salt concentration (see Table 4). The concentration of bile in the digestive tract fluctuates in the range of 0.03% to 0.3%. Therefore, the Bacillus licheniformis strain HDRaBLp can tolerate the environment of intestinal bile salts.

[0070] Table 4 Tolerance of Bacillus liche...

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Abstract

The invention belongs to the technical field of agricultural microorganism application, in particular to the screening and application of a probiotic bacillus licheniformis of a high-yield compound enzyme. The bacillus licheniformis has obvious bacteriostatic effect on common livestock intestinal pathogenic bacteria such as escherichia coli, staphylococcus aureus and clostridium welchii. The screening and application of the probiotic bacillus licheniformis of the high-yield compound enzyme comprises the isolation and identification of bacillus licheniformis strains, the study of biological characteristics and the application as livestock feed additives. According to the screening and application of the probiotic bacillus licheniformis of high-yield compound enzyme, the strain of the invention produces cellulase, amylase and proteinase, is resistant to low pH value of gastric acid and high osmotic pressure of intestinal bile salt, has the characteristics of fast growth, high stress resistance, high bacteriostasis and high enzyme production ability, and can be used for preparing livestock feed microbial additives.

Description

Technical field [0001] The invention belongs to the technical field of agricultural microbiology, and specifically relates to the screening and application of a probiotic Bacillus licheniformis with high-yield complex enzymes. The bacillus licheniformis is effective against common intestinal pathogens such as Escherichia coli and golden yellow. Staphylococcus and Clostridium wischii have obvious antibacterial effects. The invention includes the isolation and identification of Bacillus licheniformis strains, the study of biological characteristics and their application as livestock and poultry feed additives. Background technique [0002] The use of antibiotic feed additives was once considered to be one of the greatest biotechnologies in animal husbandry production in the 20th century. It played a huge role in preventing and treating animal diseases, improving the production efficiency of livestock and poultry, and meeting the needs of human animal products. However, with the pa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20A23K10/18C12R1/10
CPCA23K10/18C12N1/205C12R2001/10
Inventor 王喜亮李越章恺伦金卉肖运才周祖涛石德时金秀娥
Owner 湖北华大瑞尔科技有限公司
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