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Multi-effect bacillus subtilis for high yield of immune polysaccharide and bacteriocin and application

A Bacillus subtilis, exopolysaccharide technology, applied in the application, bacteria, antibacterial drugs and other directions, can solve the problems of low cost, few exopolysaccharides, limited interest in research and development and large-scale application, etc., to achieve good immunity The effect of regulation, broad application prospect and excellent bacteriocin-producing ability

Active Publication Date: 2020-11-20
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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  • Multi-effect bacillus subtilis for high yield of immune polysaccharide and bacteriocin and application
  • Multi-effect bacillus subtilis for high yield of immune polysaccharide and bacteriocin and application
  • Multi-effect bacillus subtilis for high yield of immune polysaccharide and bacteriocin and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Isolation and Identification of Bacillus subtilis (Bacillus subtilis) H-4

[0035] 1. Sample processing

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

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

Embodiment 2

[0077] Example 2 Molecular Weight Determination and Partial Sequence Identification of Bacteriocin 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 at 12000g for 15 minutes to remove most of the bacteria, then filter the centrifuged supernatant with a 0.22μm filter membrane to remove the remaining bacteria, the supernatant at this time is cell-free Supernatant (cell-free supernatant, CFS).

[0080]The anion-exchange chromatography filler is Q-Sepharose Fast Flow, and the size of the chromatographic column used is 1.6cm×25cm. After loading the filler, first use 15mM ammonium acetate-ammonia buffer solution with a pH of 8.0 to balance 2 column volumes. The concentrated protein in the previous step was loaded and eluted with 1mol / L NaCl prepared in buffer at a flow rate of 2ml / min. Collect the eluate by tube and measure the antibacterial activity. T...

Embodiment 3

[0102] Bacteriostatic properties or bacteriostatic spectrum of embodiment 3 Bacillus subtilis

[0103] 1. Preparation of fermented strain bacterial liquid

[0104] The activated Bacillus subtilis H-4 was inoculated in LB liquid medium and cultured for 12 hours as seed solution. According to the inoculum amount of 4%, it was inoculated into BPY medium and fermented for 24 hours for later use.

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

[0106] 2. Preparation of indicator bacteria plate

[0107] In this test, 19 strains of pathogenic bacteria in the laboratory (see Table 6) were selected for antibacterial test. After activating the indicator bacteria, pick a single colony and inoculate it in the liquid medium by streaking on a plate. After culturing for 24 hours at 37°C with a rotation speed of 200r / min, the concentration reaches about 10 10 CFU / mL, then diluted to 10 with sterile ...

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Abstract

The invention provides multi-effect bacillus subtilis for high yield of immune polysaccharide and bacteriocin and application. The preservation number of the bacillus subtilis provided by the invention is CGMCC NO.20463. The strain has excellent extracellular immune polysaccharide production capability; after fermentation by a conventional method, fermentation liquor is purified, and the yield ofthe exopolysaccharide is as high as 8203.64 mg / L; and the strain has excellent bacteriocin production capacity and endotoxin digestion capacity, and can effectively inhibit pathogenic or harmful bacteria such as salmonella, escherichia coli, staphylococcus aureus, listeria monocytogenes, sarcina lutea, streptococcus, clostridium perfringens and the like. The bacillus subtilis disclosed by the invention can be used as a food additive or a feed additive, has good immunoregulation, antibacterial, antiviral, anti-inflammatory and antioxidant capacities, and has a wide application prospect in the aspects of disease resistance, disease prevention and livestock and poultry antibiotic substitution.

Description

technical field [0001] The invention relates to the technical field of microorganisms and fermentation, in particular to the application of a strain of Bacillus subtilis with high exopolysaccharide production and bacterial agents thereof. Background technique [0002] Microbial exopolysaccharide (exopolysaccharide, EPS) is a secondary metabolite that is secreted outside the cell and separated from the body during growth and metabolism. It is a high molecular polymer composed of more than 10 aldose or ketose with glycosidic bonds. It is found in nature It is ubiquitous and has a wide range of biological functions. For example, starch and glycogen are the energy sources of plants and animals, and cellulose and chitin are involved in the skeleton formation of animals and plants. Exopolysaccharides are of two types, homopolysaccharides containing only one type of monosaccharide, and heteropolysaccharides, which are composed of repeating units ranging in size from disaccharides t...

Claims

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Application Information

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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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