Preparation method of (Lactobacillus planetarium subsp. plantarum)Zhang-LL and its listeria monocytogene-resistant bacteriocin

A technology of Lactobacillus plantarum and bacteriocin, applied in the field of microorganisms, can solve the problem that there is no literature and report on the preparation method of Lactobacillus plantarum plant subsp.

Active Publication Date: 2015-04-22
BEIJING BEINONG HONGZE BIOTECH CO LTD
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Problems solved by technology

Although there are literatures and patents about the preparation method of Lactobacillus plantarum bacteriocin in China at

Method used

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  • Preparation method of (Lactobacillus planetarium subsp. plantarum)Zhang-LL and its listeria monocytogene-resistant bacteriocin
  • Preparation method of (Lactobacillus planetarium subsp. plantarum)Zhang-LL and its listeria monocytogene-resistant bacteriocin
  • Preparation method of (Lactobacillus planetarium subsp. plantarum)Zhang-LL and its listeria monocytogene-resistant bacteriocin

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Experimental program
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Effect test

Embodiment 1

[0029] Embodiment 1, screening and identification of lactic acid bacteria strain Zhang-LL with bacteriostatic activity

[0030] 1. Screening of lactic acid bacteria strain Zhang-LL with antibacterial activity

[0031] Take 1 g of the ground bacon product and put it into 9 mL of sterile saline, shake it fully for 20 minutes to make a suspension, and dilute it 10 times to 10 -4 ~10 -7 Smear each dilution sample suspension on the MRS selective medium containing 0.4% bromocresol purple, culture at 37°C for 24 hours, pick yellow colonies for Gram staining, and transfer to MRS In the liquid medium, the bacteria were enriched for 2 to 3 generations at 37°C, and the fermentation supernatant was collected by centrifugation, and the Oxford cup method was used to judge whether the strain had antibacterial activity by observing the size of the inhibition zone.

[0032] The determination of antibacterial activity adopts the Oxford cup method: first dilute the indicator bacterial strain t...

Embodiment 2

[0057] Embodiment 2, the extraction of Lactobacillus plantarum subspecies Zhang-LL bacteriocin

[0058] 1. The fermentation process conditions of bacteriocin

[0059] The medium components (w / v) for cultivating Lactobacillus plantarum subspecies Zhang-LL producing bacteriocin: tryptone 1%, beef extract 1%, yeast extract powder 0.5%, triammonium citrate 0.2%, glucose %, Tween-800.1%, sodium acetate 0.5%, dipotassium hydrogen phosphate 0.2%, magnesium sulfate 0.05%, manganese sulfate 0.025%, pH 6.5-7.0, sterilized at 121°C for 20min.

[0060] Fermentation process conditions for producing bacteriocin: fermentation temperature 37° C., fermentation time 20 h, initial pH of the medium 6.5, inoculum size 1%.

[0061] 2. Extraction of bacteriocin in fermentation broth

[0062] (1) Adsorption of bacteriocins to producing bacterial cells The adsorption characteristics of bacteriocins to producing bacterial cells were measured by test. The lactic acid bacteria fermentation broth obtain...

Embodiment 3

[0072] Embodiment 3, Lactobacillus plantarum subspecies Zhang-LL bacteriocin purification method

[0073]1. Purification method of Lactobacillus plantarum subspecies Zhang-LL bacteriocin

[0074] (1) Preliminary purification of bacteriocin by cation exchange chromatography. The crude bacteriocin extracted sample was redissolved in sterile distilled water, filtered through a 0.22 μm sterile filter membrane, and then purified by SP Sepharose Fast Flow cation exchange chromatography. Analysis column size is 10mm×100mm; loading buffer: 20mmol / L pH3.4 citric acid-sodium citrate buffer; eluent: 0.8mol / L NaCl citric acid-sodium citrate buffer; elution conditions : Isocratic elution with 20% eluent for 20 minutes, linear gradient elution with 20% to 100% eluent for 100 minutes; flow rate: 1mL / min, sample volume: 1mL, automatic collector 5mL / tube, and absorbance at 280nm There is an eluate with an absorption peak and antibacterial activity; put the collected solution with antibacteria...

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Abstract

The invention relates to a preparation method of (Lactobacillus planetarium subsp.plantarum)Zhang-LL and its listeria monocytogene-resistant bacteriocin. The preparation method is suitable for preservation and fresh-keeping of meat and meat products, milk and milk products, fruits and vegetable, and instant foods. The (Lactobacillus planetarium subsp.plantarum)Zhang-LL (CGMCC No.6936) is selected from bacon on the Fujian farmer's market. A bacteriocin production broth is obtained by fermentation of a Zhang-LL strain, and the Zhang-LL strain bacteriocin is extracted and purified by a pH-dependent adsorption-desorption method, a cation exchange chromatography and a reversed phase high-performance liquid chromatography so that titer is improved by 32 times and purity is improved by 36.65 times. The bacteriocin can inhibit a plurality of food-borne pathogenic bacteria such as listeria monocytogenes, has high bacteriostatic activity, good heat, acid and base stability, can be degraded by human protease and is a natural and safe biological preservative. The preparation method has the advantages of simple processes, stability, high efficiency, source convenience, low cost and industrial production feasibility.

Description

technical field [0001] The invention relates to a plant subspecies of Lactobacillus plantarum in the field of microorganisms and a preparation method thereof for producing anti-Listeria monocytogenes bacteriocin. The bacteriocin is used as a natural preservative and is suitable for meat and meat products, milk and dairy products, fruits and vegetables, Improving food safety in ready-to-eat foods. Background technique [0002] In the process of processing, storage, transportation, etc., food is easily polluted by harmful microorganisms, resulting in spoilage or food poisoning. Adding preservatives to inhibit the growth of microorganisms is an important means to prevent food spoilage. Studies have shown that some chemical preservatives are carcinogenic, teratogenic and cause food poisoning, which have a greater impact on human health, and their application is increasingly restricted by many countries. Biological preservatives are a new class of natural preservatives discovere...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12P21/00C07K14/335C07K1/36C07K1/34C07K1/18C12R1/25
CPCC07K14/335C12N1/205C12R2001/25
Inventor 张红星刘慧谢远红郝艳芳金君华段慧霞熊利霞高秀芝
Owner BEIJING BEINONG HONGZE BIOTECH CO LTD
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