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A high-yielding immune polysaccharide and bacteriocin-producing Bacillus subtilis strain and its application

A Bacillus subtilis, exopolysaccharide technology, applied in bacteria, applications, antibacterial drugs, etc., can solve the problems of not many exopolysaccharides, limited interest in research and development, large-scale application, low cost, etc., and achieve application prospects Broad, excellent bacteriocin-producing ability, good immunomodulatory effect

Active Publication Date: 2022-07-05
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although there are many studies on exopolysaccharides produced by Bacillus subtilis, there are not many exopolysaccharides used in industrial production, and there are rare reports on the application of exopolysaccharides in animal husbandry.
Mainly due to the scarcity of research, it may also be due to the low cost required by animal husbandry, which largely limits the interest in its research and development and large-scale application

Method used

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  • A high-yielding immune polysaccharide and bacteriocin-producing Bacillus subtilis strain and its application
  • A high-yielding immune polysaccharide and bacteriocin-producing Bacillus subtilis strain and its application
  • A high-yielding immune polysaccharide and bacteriocin-producing Bacillus subtilis strain and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Isolation and identification of Bacillus subtilis H-4

[0035] 1. Sample processing

[0036] Take an appropriate amount of frozen healthy poultry intestinal content samples in a Bacillus-enriched liquid medium, put an appropriate amount of glass beads into the conical flask, activate at 32°C and 180r / min for 24-48h, and place the bacterial suspension in a Boil for 15 minutes in a water bath at 85°C to kill other bacteria that cannot form spores. Dilute the treated bacterial suspension with sterilized normal saline or sterilized enriched medium for 10-fold gradient dilution, and draw 10 5 -10 7 100uL of diluents with different concentrations were evenly spread on BPY solid medium, and cultured at 32°C for 24-48h. Do three repetitions for each set of gradients.

[0037] Spore enrichment medium: tryptone 1%, yeast extract powder 0.3%, starch 0.3%, manganese sulfate 0.01%, potassium dihydrogen phosphate 1.5%, disodium hydrogen phosphate 2%, natural pH, 121 ° C...

Embodiment 2

[0077] Example 2 Molecular weight determination and partial sequence identification of bacteriocins produced by Bacillus subtilis H-4

[0078] 1. Separation and purification of antibacterial substances produced by H-4

[0079] Take 100ml of H-4 fermentation broth and centrifuge it at 12,000g for 15 minutes to remove most of the bacterial cells, and then filter the centrifuged supernatant with a 0.22 μm filter to remove the remaining bacterial cells. The supernatant at this time is cell-free Supernatant (cell-freesupernatant, CFS).

[0080]The packing material for anion exchange chromatography is Q-Sepharose Fast Flow, and the size of the column is 1.6cm×25cm. After packing, use 15mM ammonium acetate-ammonia buffer at pH 8.0 to equilibrate for 2 column volumes. The concentrated protein in the previous step was loaded and eluted with 1 mol / L NaCl prepared in buffer at a flow rate of 2 ml / min. The eluate was collected by tube, and the antibacterial activity was determined. Com...

Embodiment 3

[0102] Example 3 Bacteriostatic properties or antibacterial spectrum of Bacillus subtilis

[0103] 1. Preparation of fermentation strains

[0104] After activation, Bacillus subtilis H-4 was inoculated into LB liquid medium and cultivated for 12 h as seed liquid. According to the inoculum of 4%, it was inoculated into BPY medium and fermented for 24h.

[0105] BPY medium formula: beef extract 0.5%, yeast extract 0.5%, tryptone 1%, sodium chloride 0.5%, glucose 0.5%, sucrose 0.5%.

[0106] 2. Indicator bacteria plate preparation

[0107] In this experiment, 19 strains of pathogenic bacteria in the laboratory (see Table 6) were selected for bacteriostatic test. After activating the indicator bacteria, pick a single colony and inoculate it in the liquid medium by streaking on a plate. 10 CFU / mL, then diluted to 10 with sterile normal saline 6-7 CFU / mL for use, take a sterile petri dish, add 15-20 mL of LB medium (agar content 2%), and place it on a horizontal surface to soli...

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Abstract

The invention provides a pleiotropic Bacillus subtilis with high production of immune polysaccharide and bacteriocin and application thereof. The preservation number of Bacillus subtilis provided by the present invention is CGMCC NO.20463, the bacteria has excellent ability to produce extracellular immune polysaccharide, and the yield of extracellular polysaccharide is as high as 8203.64 mg / L measured by purifying the fermentation liquid after fermentation by the conventional method, And the bacteria has excellent bacteriocin-producing ability and endotoxin-digesting ability, can effectively inhibit Salmonella, Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Sarcinus luteus, Streptococcus, Clostridium perfringens. pathogenic or harmful bacteria. The Bacillus subtilis of the present invention can be used as a food additive or a feed additive, has good immune regulation, antibacterial, antiviral, antiinflammatory and antioxidative abilities, and has broad application prospects in disease resistance and disease prevention and replacement of livestock and poultry antibiotics.

Description

technical field [0001] The invention relates to the technical field of microorganisms and fermentation, in particular to the application of a Bacillus subtilis high-yielding extracellular polysaccharide and a bacterial agent thereof. Background technique [0002] Microbial exopolysaccharide (EPS) is a secondary metabolite that is secreted to the outside of the cell and separated from the body in the process of growth and metabolism. It is ubiquitous and has a wide range of biological functions. For example, starch and glycogen are energy sources for plants and animals, and cellulose and chitin are involved in the skeleton of animals and plants. There are two types of exopolysaccharides, homopolysaccharides and heteropolysaccharides. Homopolysaccharides contain only one monosaccharide, while heteropolysaccharides are composed of repeating units ranging in size from disaccharides to heptasaccharides. Bacterial exopolysaccharides were first discovered from wine in the mid-19th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20A23K10/18A23K50/75A23L33/135A61K35/742A61K8/99A61Q19/00A61P37/02A61P31/04A61P31/12A61P39/06C12P19/04C12P21/00C12R1/125
CPCC12N1/20A23K10/18A23K50/75A23L33/135A61K35/742A61K8/99A61Q19/00A61P37/02A61P31/04A61P31/12A61P39/06C12P19/04C12P21/00A61K2800/10C12R2001/125C12N1/205
Inventor 张日俊姚蒙蒙胡聪李仲玄黄燕斯大勇王俊永
Owner CHINA AGRI UNIV
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