A kind of preparation method of Bacillus subtilis C3 and anti-Listeria monocytogenes bacteriocin

A technology of Bacillus subtilis and Listeria monocytogenes, which is applied in the field of microbiology, can solve problems such as no related reports, and achieve the effect of simple extraction process, wide sources and efficient purification method

Active Publication Date: 2015-07-29
BEIJING UNIV OF AGRI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there is no relevant report on the preparation method of Bacillus subtilis bacteriocin against Listeria monocytogenes in domestic invention patents and literature

Method used

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  • A kind of preparation method of Bacillus subtilis C3 and anti-Listeria monocytogenes bacteriocin
  • A kind of preparation method of Bacillus subtilis C3 and anti-Listeria monocytogenes bacteriocin
  • A kind of preparation method of Bacillus subtilis C3 and anti-Listeria monocytogenes bacteriocin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1, the preparation of Bacillus subtilis C3 bacteriocin crude extract

[0026] 1. Preparation of Bacillus subtilis C3 fermentation broth

[0027] (1) Preparation of seed solution

[0028] Seed medium formula (W / V, mass-volume ratio concentration): glucose 1%, peptone 1%, NaCl 1%, KH 2 PO 4 0.15%, MgS0 4 ·7H 2 O0.15%, pH natural;

[0029] Seed culture conditions: The activated Bacillus subtilis was transferred into the seed medium at 1% inoculum amount, the culture temperature was 37°C, the liquid volume was 75mL / 250mL, and the shaker was shaken at 150r / min for 18h, and the number of viable bacteria was about 10. 9 CFU / mL.

[0030] (2) Preparation of fermentation broth

[0031] Fermentation medium formula (W / V, mass-volume ratio concentration): glucose 1%, peptone 1.5%, yeast extract powder 1.5%, KH 2 PO 4 0.1%, MgSO 4 ·7H 2 O0.15%, NaCl0.5%, pH7.0;

[0032] Fermentation conditions: The seed liquid was transferred into the fermentation medium at 2% ...

Embodiment 2

[0036] Example 2. Bacteriostatic properties and physicochemical properties of the crude extract of Bacillus subtilis C3 bacteriocin

[0037] During the experiment, the antibacterial and physicochemical properties of the crude extract of C3 bacteriocin were determined by the tube-plate method.

[0038] Tube-dish method: first dilute the indicator strain to 10 7 CFU / mL, after mixing with the corresponding solid medium that has been heated and melted, pour about 15 mL into a petri dish. After solidification, gently place a sterile Oxford cup. Take 100 μL of Bacillus subtilis C3 crude extract and add it to the Oxford cup and culture at 37 °C. 12h, the inhibition zone was observed, and the diameter of the inhibition zone was measured with a vernier caliper, and the reading was accurate to 0.01 mm.

[0039] 1. Antibacterial properties of crude extract of Bacillus subtilis C3 bacteriocin

[0040] The bacteriostatic effect of table 1 Bacillus subtilis C3 bacteriocin crude extract

...

Embodiment 3

[0062] Embodiment 3, Bacillus subtilis C3 bacteriocin purification method

[0063] 1. Purification of the crude extract of Bacillus subtilis C3 bacteriocin

[0064] (1) Step-by-step elution and purification by anion exchange chromatography

[0065] The crude extract of Bacillus subtilis C3 bacteriocin was filtered and sterilized with a 0.2 μm sterile filter, and was eluted and purified by DEAE-Sepharose Fast Flow anion exchange chromatography step by step. The column size was 10 mm × 200 mm; buffer: A Solution 0.025mol / L pH7.5Tric-HCl, solution B contains 0.4mol / L NaCl in 0.025mol / L pH7.5Tric-HCl; step-by-step elution steps: 50min 100% solution A→90min 70% solution A+30% solution B →40min50%A solution+50%B solution→40min100%B solution; the flow rate is 1mL / min, the sample volume is 5mL, the automatic collector is 5mL / tube, and the tube plate method is used to detect the effluent of the collected eluate for Listeria monocytogenes. Antibacterial activity, the test showed that ...

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Abstract

The name of the invention is a bacillus subtilis C3 and its anti-Listeria monocytogenes bacteriocin preparation method. The bacteriocin, used as a natural antiseptic, is suitable for low temperature meat and meat product and fruit and vegetable fresh-keeping, and can be used to raise safety of food. According to the invention, Bacillus subtilis C3 CGMCC No.9713 is utilized by ammonium sulfate precipitation, anion exchange chromatography and semi-preparative reversed phase liquid chromatography for fine purification. Thus, titer of the bacillus subtilis C3 bacteriocin is raised by 32 times, and purity is raised by 42.80 times. The bacteriocin has a very strong bacteriostatic activity of Listeria monocytogenes and good heat and pH stability, can be degraded by protease K, and is a natural safe biological preservative. The bacteriocin provided by the invention has advantages of wide sources of raw materials for production, simple extraction technology and efficient and stable purification method. The preparation method provided by the invention is suitable for industrial production.

Description

technical field [0001] The invention relates to a preparation method of Bacillus subtilis C3 and an anti-Listeria monocytogenes bacteriocin in the field of microorganisms. As a natural preservative, the bacteriocin is suitable for keeping meat and meat products, fruits and vegetables fresh at low temperature, and can improve the safety of food. Background technique [0002] In the process of processing, storage, transportation, etc., food is easily contaminated by harmful microorganisms, leading to spoilage or food poisoning. The method of adding preservatives to inhibit the growth of microorganisms has gradually become an important means of food preservation. Most traditional meat products are kept fresh by chemical preservatives, which have certain safety hazards. Improper use will have certain side effects. Long-term excessive intake will cause certain damage to consumers' bodies and lead to environmental pollution. Therefore, the development of efficient, safe and envir...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C07K14/32C07K1/36C07K1/30C07K1/20C07K1/18C12R1/125
Inventor 谢远红刘慧张红星杨永青熊利霞高秀芝马晓丹
Owner BEIJING UNIV OF AGRI
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