Bacillus subtilis C3 and its anti-Listeria monocytogenes bacteriocin preparation method

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: 2013-09-25
BEIJING UNIV OF AGRI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no relevant report on the preparation method of Bacillus subtilis

Method used

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  • Bacillus subtilis C3 and its anti-Listeria monocytogenes bacteriocin preparation method
  • Bacillus subtilis C3 and its anti-Listeria monocytogenes bacteriocin preparation method
  • Bacillus subtilis C3 and its anti-Listeria monocytogenes bacteriocin preparation method

Examples

Experimental program
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Example Embodiment

[0025] Example 1. Preparation of crude extract of Bacillus subtilis C3 bacteriocin

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

[0027] (1) Preparation of seed liquid

[0028] Seed medium formula (W / V, mass volume ratio concentration): glucose 1%, peptone 1%, NaCl1%, KH 2 PO 4 0.15%, MgS0 4 ·7H 2 O0.15%, natural pH;

[0029] Seed culture conditions: transfer the activated Bacillus subtilis into the seed culture medium according to the 1% inoculum, the culture temperature is 37℃, the liquid volume is 75mL / 250mL, the shaking culture is 150r / min for 18h, the number of viable bacteria is 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 1.5%, KH 2 PO 4 0.1%, MgSO 4 ·7H 2 O0.15%, NaCl0.5%, pH7.0;

[0032] Fermentation conditions: transfer the seed liquid into the fermentation medium according to the 2% inoculum, the fermentation temperature...

Example Embodiment

[0036] Example 2: Antibacterial properties and physical and chemical properties of crude extract of Bacillus subtilis C3 bacteriocin

[0037] During the test, the tube-disc method was used to determine the antibacterial properties and physical and chemical properties of the crude extract of C3 bacteriocin.

[0038] Tube dish method: first dilute the indicator strain to 10 7 CFU / mL, after mixing with the corresponding solid medium that has been melted by heating, pour about 15mL into a petri dish, gently place a sterile Oxford cup after solidification, and take 100μL of Bacillus subtilis C3 crude extract into the Oxford cup and cultivate at 37℃ At 12h, observe the inhibition zone and use a vernier caliper to determine the diameter of the inhibition zone, and the reading is accurate to 0.01mm.

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

[0040] Table 1 Antibacterial effect of crude extract of Bacillus subtilis C3 bacteriocin

[0041]

[0042...

Example Embodiment

[0062] Example 3: Purification method of Bacillus subtilis C3 bacteriocin

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

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

[0065] The crude extract of Bacillus subtilis C3 bacteriocin was filtered and sterilized with a 0.2μm sterile filter, and was eluted and purified step by step by DEAE-Sepharose Fast Flow anion exchange chromatography. The column size was 10mm×200mm; buffer: A Solution 0.025mol / L pH7.5Tric-HCl, solution B contains 0.025mol / L pH7.5Tric-HCl of 0.4mol / L NaCl; 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-disc method is used to detect and collect the eluate against Listeria monocytogenes. Antibacterial activity. Tests show that the eluate at 95~105min h...

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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 anti-Listeria monocytogenes bacteriocin in the field of microorganisms. As a natural preservative, the bacteriocin is suitable for keeping low-temperature meat and meat products, fruits and vegetables fresh, and can improve food safety. Background technique [0002] During the process of processing, storage, transportation, etc., food is easily contaminated by harmful microorganisms, resulting in spoilage or food poisoning. The method of adding preservatives to inhibit the growth of microorganisms has gradually become an important means of food preservation. Traditional meat products are mostly preserved 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 environment...

Claims

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Application Information

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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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