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Lactic acid bacteria engineering bacteria for improving survival capacity in acid stress environment

A technology for viability, lactic acid bacteria

Active Publication Date: 2019-04-23
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the addition of alkaline substances often leads to the accumulation of by-products, and the salts formed in the by-products will once again lead to a hypertonic environment for cells, resulting in osmotic stress, which will affect the growth and metabolism of bacteria again

Method used

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  • Lactic acid bacteria engineering bacteria for improving survival capacity in acid stress environment
  • Lactic acid bacteria engineering bacteria for improving survival capacity in acid stress environment
  • Lactic acid bacteria engineering bacteria for improving survival capacity in acid stress environment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment 1: Construction of recombinant bacterial strain

[0054] Specific steps are as follows:

[0055](1) Obtain the ctrA gene sequence shown in SEQ ID NO.1 from the NCBI database (the ctrA gene is the gene encoding the amino acid permease CtrA, and the amino acid permease CtrA is a protein used to transport amino acids on the membrane), The bglF gene sequence as shown in SEQ ID NO.3 (bglF gene is the gene of encoding β-glucoside-specific PTS system IIABC component BglF, and β-glucoside-specific PTS system IIABC component BglF is used for transporting β on the membrane -glucoside), the oppB gene sequence shown in SEQID NO.4 (the oppB gene is the gene encoding the peptide transport system permease OppB, and the peptide transport system permease OppB is the protein used to transport oligopeptides on the membrane ), according to the gene sequence design primers shown in Table 1 respectively;

[0056] (2) Using the genome of L. lactis NZ9000 as a template, respective...

Embodiment 2

[0064] Embodiment 2: the growth performance test of recombinant bacterial strain

[0065] Specific steps are as follows:

[0066] (1) The bacterial strain L lactis (Vector) (control) containing only the blank plasmid pNZ8148 (control) and the bacterial strains L lactis (CtrA), L lactis (BglF) and L lactis (OppB) obtained in Example 1 were inoculated respectively in a mixture of 10 μg / mL of chloramphenicol in GM17 liquid medium for activation, and placed in a 30°C incubator for static culture overnight;

[0067] (2) Transfer the above-obtained seed solution to fresh chloramphenicol (10 μg / mL) GM17 liquid medium with an inoculum size of 2%, and culture it statically at 30° C.;

[0068] (3) During the culturing process, samples were taken at regular intervals to measure the OD value at a wavelength of 600nm;

[0069] (4) Cultivate to OD 600 Add 10ng / mL Nisin at 0.4 to induce the expression of the transporter, with time as the abscissa, OD 600 The value is the ordinate, and ...

Embodiment 3

[0071] Example 3: Tolerance test of recombinant strains under lactic acid stress conditions

[0072] Specific steps are as follows:

[0073] The bacterial strain L lactis (Vector) (control) containing only the blank plasmid pNZ8148 (control) and the bacterial strain L lactis (CtrA) obtained in Example 1 were respectively induced and cultured in GM17 medium at 30° C. for 6 h, and the cells were collected by centrifugation and subjected to 0.85 % normal saline was washed twice and then resuspended in fresh pH 4.0 (adjusted by lactic acid) GM17 (containing chloramphenicol 10 μg / mL) equal to the volume of the collected bacterial solution, and stressed for 1.5h, 2.5h and 3h respectively ; the bacterial suspension after the stress was washed twice and then resuspended in equal-volume physiological saline, and 10 μL of the resuspension was taken, diluted with different gradients and planted on the GM17 chloramphenicol flat plate to determine the number of viable bacteria and the surv...

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Abstract

The invention discloses lactic acid bacteria engineering bacteria for improving the survival capacity in the acid stress environment, and belongs to the technical field of gene engineering and microorganism engineering. A gene of coded amino acid permease CtrA is used as a target gene, lactic acid bacteria are used as an expression host, and the lactic acid bacteria engineering bacteria capable ofbeing widely applied to preparation of food, medicine, feed and chemicals are successfully established. The survival capacity of the lactic acid bacteria engineering bacteria in the acid stress environment is remarkably improved, and the survival capacity is improved by 35.7 times compared with contrast strains.

Description

technical field [0001] The invention relates to a lactic acid bacteria engineering bacterium with improved viability in an acid stress environment, and belongs to the technical fields of genetic engineering and microbial engineering. Background technique [0002] Lactic acid bacteria are the general term for a class of non-spore and Gram-positive bacteria that ferment glucose and the main metabolic end product is lactic acid, including Lactobacillus, Weissella, and Carnobacterium, etc., which can promote the digestion and absorption of nutrients, reduce Serum cholesterol, regulating the balance of intestinal flora, activating the body's immune system, improving food flavor and preventing cancer and other physiological functions also have important applications in food, medicine, feed, fine chemicals and other important industrial fields closely related to human life value. [0003] However, in the process of industrial fermentation production of lactic acid bacteria and the...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/74C12N15/31C12R1/01
CPCC07K14/195C12N15/746
Inventor 张娟杨佩珊刘为佳朱政明陈坚堵国成
Owner JIANGNAN UNIV
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