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Bacillus amyloliquefaciens exopolysaccharide for inhibiting growth of enterotoxin-producing escherichia coli

A technology for dissolving starch spores and exopolysaccharides, which is applied in the field of bioengineering and can solve the problems of different inhibitory activities and small yields.

Active Publication Date: 2019-10-25
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, their inhibitory activities vary due to the diversity of monosaccharide composition and polysaccharide structure
However, the exopolysaccharide production of most lactic acid bacteria is very small, less than 1g / L, so it has not attracted the interest of commercial development
In contrast, Bacillus sp. has been found to produce higher exopolysaccharides, but unfortunately it has not been shown to act as an anti-adhesion agent for ETEC

Method used

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  • Bacillus amyloliquefaciens exopolysaccharide for inhibiting growth of enterotoxin-producing escherichia coli
  • Bacillus amyloliquefaciens exopolysaccharide for inhibiting growth of enterotoxin-producing escherichia coli
  • Bacillus amyloliquefaciens exopolysaccharide for inhibiting growth of enterotoxin-producing escherichia coli

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The Bacillus amyloliquefaciens of the present invention is selected from the intestinal tract of healthy piglets.

[0036] (1) Bacillus amyloliquefaciens with the strain preservation number is activated and expanded on the LB slope;

[0037] (2) Inoculate the expanded strain obtained in step (1) into the fermentation medium, culture at 37° C. at 200 r / min for 48 to 72 hours, and obtain a fermentation broth containing exopolysaccharide of Bacillus amyloliquefaciens;

[0038] (3) centrifuging the liquid fermentation broth prepared in step (2), and collecting the supernatant;

[0039] (4) Concentrate the supernatant obtained in step (3) by ultrafiltration to 1 / 2 of the original volume to obtain a concentrated solution, then add three times the volume of absolute ethanol to the concentrated solution, and after alcohol precipitation, centrifuge, collect sediment;

[0040] (5) dissolve the precipitate collected in step (4) with water, add 1 / 4 volume of seveage reagent to re...

Embodiment 2

[0048] Artificially simulated gastrointestinal juice needs to be freshly prepared: use PBS to prepare pepsin with a concentration of 3g / L and pH 3.0 and trypsin with a pH of 1g / L and pH 8.0 respectively, and filter the prepared solutions with 0.22μm filter membranes to obtain simulated gastric juice and Simulated intestinal fluid.

[0049] Resuspend the spores of Bacillus amyloliquefaciens JN4 in saline, 9 The final concentration of CFU / mL was added to simulated gastric juice (pH 3.0), incubated at 37°C for 3 hours, and the number of viable bacteria was detected every hour. After 3 hours, take 1ml of the culture solution in the simulated gastric juice (pH 3.0) and add it to 9ml of the simulated intestinal juice (pH 8.0), mix well, incubate at 37°C for 8 hours, and measure the number of viable bacteria every 2 hours.

[0050] The results showed that Bacillus amyloliquefaciens JN4 could survive for 3 hours in artificial simulated gastric juice (pH 3.0), and the survival rate wa...

Embodiment 3

[0052] Take the spores of Bacillus amyloliquefaciens JN4 in the logarithmic growth phase at a final concentration of 1×10 6 The inoculum amount of CFU / mL was respectively inserted into the fermentation medium (preparation method is the same as in Example 1) with NaCl mass fraction of 0%, 2%, 4%, 6%, 7%, 8%, and 9%, and fully mixed Uniform, measure the OD of the bacterial solution after culturing at 37°C for 24 hours 600 value.

[0053] The results showed that the survival rate of Bacillus amyloliquefaciens was more than 85% when the concentration of NaCl was 9%, which indicated that Bacillus amyloliquefaciens JN4 had strong tolerance to NaCl.

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Abstract

The invention discloses bacillus amyloliquefaciens exopolysaccharide for inhibiting growth of enterotoxin-producing escherichia coli and belongs to the technical field of bioengineering. The molecularweight of the exopolysaccharide is 8005 Da, and the exopolysaccharide is prepared from fructose and glucose and is a levan with a special structure, and the molecular structure of the exopolysaccharide has 7 specific repeating units. Each repeating unit contains 7 fructosyls, uses 6 beta-(2,6)-fructosyl as a framework, and uses one beta-(1,2)-fructosyl as a branched chain. The bacillus amyloliquefaciens exopolysaccharide EPS-JN4 has the effect of inhibiting the growth of enterotoxigenic escherichia coli, has the lowest inhibitory concentration of 5 * 10-7 mg polysaccharide / CFU escherichia coli and has good potential of replacing antibiotics to treat diarrhea caused by escherichia coli.

Description

technical field [0001] The invention relates to an exopolysaccharide of bacillus amyloliquefaciens capable of inhibiting the growth of enterotoxigenic Escherichia coli, and belongs to the technical field of bioengineering. Background technique [0002] For weaned piglets, about 80% of acute diarrhea is considered to be caused by enterotoxigenic Escherichia coli (ETEC), and its mortality rate exceeds 10%. Currently, the conventional treatment is the addition of antibiotics, yet antibiotic therapy is the main cause of genetic progression of drug-resistant strains. In addition, it can lead to an imbalance of intestinal flora and a disordered immune system, which can lead to weight loss and poor growth of surviving piglets, eventually causing huge economic losses. Therefore, finding a safe and effective alternative to antibiotics is of great significance to animal husbandry. [0003] Some sugar compounds, such as galactooligosaccharides from plants and lactic acid bacteria ext...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12P19/04C08B37/00A61K31/715A61P1/12A61P1/00C12R1/07
CPCA61K31/715A61P1/00A61P1/12C08B37/006C12P19/04C12N1/205C12R2001/07Y02A50/30
Inventor 蔡国林陆健李晓敏
Owner JIANGNAN UNIV
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