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Method for screening lactic acid bacteria capable of producing bacteriocin from plant source materials

A bacteriocin-producing, plant-derived technology, applied in microorganism-based methods, biochemical equipment and methods, bacteria, etc., can solve problems such as loss of activity, inability to screen out bacteriocin-producing strains, and inability to completely exclude the influence of small molecules.

Inactive Publication Date: 2011-11-23
ANHUI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since lactic acid bacteria can produce antibacterial substances such as various organic acids and hydrogen peroxide, as well as small molecule antibacterial substances such as diacetyl during the fermentation process, traditional conventional methods cannot completely eliminate the influence of small molecules. , and the accuracy of the experimental results is not high, there are often some false positive results
In addition, a considerable portion of bacteriocins are acid-dependent bacteriocins. If the pH of the fermentation supernatant is adjusted to 5.5-6.0, these acid-dependent bacteriocins will lose their activity, making it impossible to screen out these bacteriocin-producing strains

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] (1) Processing of plant source materials

[0041] Cut the leaves and stems of purslane into small pieces, put them into test tubes, add a small amount of sterile water, compact them, seal them with plastic bags and kraft paper, enrich at 25°C for 15 days, and obtain the enrichment solution;

[0042] (2) Isolation and purification of lactic acid bacteria

[0043] A. Under sterile conditions, use a sampler to draw 0.1mL enrichment solution from the bottom of the test tube, and perform gradient dilution to obtain 10 -4 、10 -5 、10 -6 、10 -7 gradient dilution;

[0044] B. Take 0.1mL of the above-mentioned dilutions of different gradients, add them to the MRS solid plate respectively, and evenly spread the bacterial suspension on the surface of the plate with a triangular spatula;

[0045] C. Under anaerobic conditions at 28°C, culture the above plate for 48 hours;

[0046] D. According to the difference in colony morphology, use an inoculation loop to pick various typi...

Embodiment 2

[0064] (1) Processing of plant source materials

[0065] Cut Chinese cabbage leaves into small pieces, put them into test tubes, add a small amount of sterile water, compact them, seal them with plastic bags and kraft paper, and enrich at 25°C for 15 days to obtain the enrichment solution;

[0066] (2) Isolation and purification of lactic acid bacteria

[0067] A. Under sterile conditions, use a sampler to draw 0.1mL enrichment solution from the bottom of the test tube, and perform gradient dilution to obtain 10 -4 、10 -5 、10 -6 、10 -7 gradient dilution;

[0068] B. Take 0.1mL of the above-mentioned dilutions of different gradients, add them to the MRS solid plate respectively, and use a triangular spatula to evenly spread the bacterial suspension on the surface of the plate;

[0069] C. Under anaerobic conditions at 28°C, culture the above plate for 48 hours;

[0070] D. According to the difference in colony morphology, pick various typical colonies with an inoculation ...

Embodiment 3

[0088] (1) Processing of plant source materials

[0089] Put the millet into a test tube, add a small amount of sterile water, compact it, seal it with a plastic bag and kraft paper, and enrich it at 25°C for 15 days to obtain the enrichment solution;

[0090] (2) Isolation and purification of lactic acid bacteria

[0091] A. Under sterile conditions, use a sampler to draw 0.1mL of bacterial suspension from the bottom of the test tube, and perform gradient dilution to obtain 10 -4 、10 -5 、10 -6 、10 -7 gradient dilution;

[0092] B. Take 0.1mL of the above-mentioned dilutions of different gradients, add them to the MRS solid plate respectively, and use a triangular spatula to evenly spread the bacterial suspension on the surface of the plate;

[0093] C. Under anaerobic conditions at 28°C, place the above-prepared plate in culture for 48 hours;

[0094] D. According to the difference in colony morphology, pick various typical colonies with an inoculation loop, draw four z...

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Abstract

The invention relates to a method for screening lactic acid bacteria capable of bacteriocin from plant source materials. The method comprises the following steps: treating the plant source materials; separating and screening out the lactic acid bacteria; screening a lactic acid bacterium strain capable of producing antibiotic substances by using a method of fermenting a supernatant fluid by use of the strain to carry out an antibiotic test; eliminating interference of small molecules by using dialyzate of the supernatant fluid to carry out the antibiotic test, and screening out the lactic acid bacteria capable of producing the bacteriocin; and performing an antibiotic test by using a protein crude extract which is precipitated by use of ammonium sulfate, and performing a crude extract enzymolysis test by using various proteases to prove that the lactic acid bacterium strain capable of producing the bacterocin can be effectively screened by utilizing the method. In the method, the method for separating and purifying the lactic acid bacteria capable of producing the bacteriocin is improved, the fermented supernatant fluid is dialyzed by a dialysis bag with smaller cut-off molecular weight, so that the inhibition effect of small molecular antibiotic substances in the fermented supernatant fluid on indicator bacteria can be eliminated, and the activity of the bacteriocin can be guaranteed. By utilizing the method, the experimental steps are reduced, and the screening efficiency is improved.

Description

technical field [0001] The invention relates to a method for screening and obtaining bacteriocin-producing lactic acid bacteria strains from plant source materials. Background technique [0002] Bacteriocins are a class of proteins or polypeptides synthesized by bacteria through ribosomes, and have attracted widespread attention because of their antibacterial activity. Compared with chemical preservatives and antibiotics, bacteriocin, as a protein substance, has strong antibacterial activity and good stability, can be enzymatically hydrolyzed without remaining in the human body, is not easy to cause drug resistance, and has high safety It can be widely used in the fields of food natural preservatives and medicine. Many lactic acid bacteria are bacteriocin-producing bacteria, and Nisin, a bacteriocin produced by Lactococcus lactis, has been approved by FAO for use in food preservation. Narrow, poor solubility and low stability under neutral conditions, thus greatly limiting...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12R1/225C12R1/25
Inventor 张明陈一然蔡庆霞陈晓琳祁克宗
Owner ANHUI AGRICULTURAL UNIVERSITY
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