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Bacillus subtilis LFB112 as well as bacteriocin produced by same and application thereof

A technology of Bacillus subtilis and LFB112, applied in the field of new strains of Bacillus subtilis and bacteriocin, can solve the problems of limited bacteriostatic spectrum, lack of credible evidence, and no production test data.

Active Publication Date: 2013-05-22
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the application research of bacteriocins has the following deficiencies: 1. The bacteriocins found have a limited antibacterial spectrum, and can only inhibit some Gram-positive pathogenic bacteria. Pathogens and fungi have no inhibitory power, so they have great limitations in practical application; 2. The problem of the inhibition of bacteriocins on clinical drug-resistant bacteria has not been solved
Antibiotic resistance is the biggest problem facing the current clinical treatment. According to reports, as many as 18,000 patients die from super-resistant bacteria "MRSA" (Methicillin-resistant Staphylococcus aureus) in the United States every year. The bacteriocins of bacteria have practical significance; 3. The relevant research only stays at the laboratory level, and does not give specific production test or clinical use data, so there is a lack of credible evidence in practical application

Method used

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  • Bacillus subtilis LFB112 as well as bacteriocin produced by same and application thereof
  • Bacillus subtilis LFB112 as well as bacteriocin produced by same and application thereof
  • Bacillus subtilis LFB112 as well as bacteriocin produced by same and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Antibacterial Activity Analysis of Bacillus subtilis LFB112

[0037] Antibacterial activity analysis method: first pour 15ml NA (nutrient agar) solid medium (composition is: 10 grams of peptone, 5 grams of beef extract, 5 grams of sodium chloride, 15 grams of agar, 1000 ml of distilled water, pH7. 0) Make a nutrient agar plate. Then, mix the pathogenic indicator bacteria Staphylococcus aureus CVCC1885 (China Veterinary Microbial Strain Collection Center) cultured overnight with NA (Nutritional Soft Agar) medium (containing 0.75% agar, w / v), and make it into The concentration of indicator bacteria was diluted by 10 6 About cfu / ml, take 5ml and pour it into the prepared nutrient agar plate. Place the Oxford cup gently on the plate, add 100 μl of centrifuged supernatant to the Oxford cup and let it stand at 4°C for 2 hours, then culture at 37°C for 18 hours to observe the appearance of the inhibition zone. Determination of the antibacterial activity unit of ba...

Embodiment 2

[0046] Example 2 Preparation of bacteriocin

[0047] 1. Fermentation culture

[0048] The best medium for producing bacteriocin by Bacillus subtilis LFB112 fermentation is the improved BPY medium, and its ingredients are: 20 grams of peptone, 10 grams of glucose, 10 grams of yeast extract, 10 grams of beef extract, 3 grams of salt, 2.5 dipotassium hydrogen phosphate gram, distilled water 1000ml, pH value 7.2, at 37 ℃, 120 rpm shaker fermentation for 24 hours.

[0049] 2. Separation of crude bacteriocin

[0050] Centrifuge the fermentation broth at 10,000 rpm for 15 minutes, then take the supernatant, slowly add ammonium sulfate solid powder to the supernatant for salting out, stir while adding, until the saturation reaches 80%, and then incubate at 4°C Let stand for 12 hours, then centrifuge at 12,000 rpm at 4°C for 30 minutes, discard the supernatant to obtain the crude precipitated bacteriocin.

[0051] 3. Purification

[0052] A certain volume of 20 mM phosphate buffer ...

Embodiment 3

[0054] The bacteriostatic spectrum of embodiment 3 bacteriocin

[0055] Test strains:

[0056] Escherichia coli: Escherichia coli CVCC195, Escherichia coli CVCC249, Escherichia coli CVCC247, Escherichia coli CVCC8099, Escherichia coli CVCC245, Escherichia coli CVCC232, Escherichia coli CVCC1498, Escherichia coli CVCC1158, drug-resistant Escherichia coli F015

[0057] Salmonella: Salmonella CVCC79301, Salmonella C79-52, Salmonella typhimurium CVCC541, drug-resistant Salmonella E103

[0058] Staphylococcus aureus: Staphylococcus aureus CVCC1885, Staphylococcus aureus CVCC6538, Staphylococcus aureus 1882,

[0059] Enterococcus: Enterococcus faecalis 1.130, Enterococcus faecalis 1.595, Enterococcus faecalis 1.2025.

[0060] Pseudomonas: Pseudomonas aeruginosa CVCC2087, Pseudomonas aeruginosa CVCC2088.

[0061] Micrococcus luteus: Micrococcus luteus 1600

[0062] Listeria: Listeria monocytogenes 54002, Listeria monocytogenes 1599

[0063] Streptococcus: Streptococcus lactis CI...

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Abstract

The invention provides bacillus subtilis LFB112 with the collection number of CGMCC No.2996. The bacterial strain has relatively high antibacterial activity and has antibacterial effect on various animal pathogenic bacteria and food pathogenic bacteria. The invention also provides bacteriocin produced by the bacterial strain, wherein the bacteriocin is relatively stable against heat and acid and has a broad-spectrum antibacterial effect without inhibiting beneficial bacteria. Experiments indicate that the bacterial strain LFB112 provided by the invention and the bacteriocin produced by the same can be used for improving the clinical symptoms of chicken and increasing the growth speed and feed price, and can be used in biological veterinary medicaments or feed additives.

Description

technical field [0001] The invention relates to microorganism application technology, in particular to a new strain of bacillus subtilis, the bacteriocin produced by it and its application. Background technique [0002] Since the discovery of penicillin by Fleming in England in 1929, antibiotics have made great contributions to human disease control and animal health care. However, in recent years, the use of antibiotic resistance caused by the abuse of antibiotics has attracted more and more attention and anxiety. In the aquaculture industry, with the large-scale use of antibiotics in feed, their side effects have become increasingly prominent, such as causing animal intestinal flora imbalance, and more and more drug-resistant strains and even superbugs "MRSA" (formazan-resistant bacteria) have appeared clinically. Staphylococcus aureus), and drug residues in animal products. The emergence of these problems not only affects the healthy development of the aquaculture indust...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20A23K1/16C12P1/04C12R1/125A23K10/18
Inventor 张日俊谢建华李桂冠
Owner CHINA AGRI UNIV
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