A kind of lactic acid bacteria engineering bacteria with improved acid stress resistance

An acid stress, engineering bacteria technology, applied in the field of genetic engineering and microbial engineering, can solve the problems affecting the growth and metabolism of bacteria, accumulation and weakening of by-products, etc., to improve amino acid stress resistance, improve acid stress resistance, lactic acid The effect of increasing the resistance of

Active Publication Date: 2020-11-06
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] 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
[0008] At present, the method that improves the acid stress resistance such as lactic acid, acetic acid of lactic acid bacteria then mainly contains: (1) mutagenesis breeding, this method has characteristics such as easy, various types, but workload is big, efficient is its main shortcoming; (2) ) biochemical engineering strategy, it has been reported that exogenous aspartic acid has been added to improve the acid stress tolerance of lactic acid bacteria, but the use of this method has caused an increase in production costs; (3) metabolic engineering strategy, currently using metabolic engineering Strategies to improve the environmental stress of lactic acid bacteria mainly include constructing new metabolic pathways, expanding existing metabolic pathways and weakening existing metabolic pathways. However, this method has the problems of high cost and low success rate.

Method used

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  • A kind of lactic acid bacteria engineering bacteria with improved acid stress resistance
  • A kind of lactic acid bacteria engineering bacteria with improved acid stress resistance
  • A kind of lactic acid bacteria engineering bacteria with improved acid stress resistance

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 purC gene sequence shown in SEQ ID NO.1 from the NCBI database (the gene encoding phosphoribosylaminoimidazole-succinamide synthetase PurC, participates in the IMP biosynthetic pathway in purine metabolism, itself is A part of purine metabolism, regulation of purine metabolism can alleviate the intrusion of abiotic stress to a certain extent), design primers as shown in Table 1 respectively according to gene sequence;

[0056] (2) Using the genome of L.lactis NZ9000 as a template, respectively use the primers in Table 1 to obtain the gene fragment shown in SEQ ID NO.1 by PCR amplification;

[0057] (3) The PCR product and the carrier pNZ8148 were double-digested with the restriction endonucleases in Table 1, and the digested products were purified and ligated;

[0058] (4) Transform the ligation product into Escherichia coli MC1061 (commercialized...

Embodiment 2

[0062] Embodiment 2: the growth performance test of recombinant bacterial strain

[0063] Specific steps are as follows:

[0064] (1) The bacterial strain L lactis NZ9000 (pNZ8148) (control) and the bacterial strain L lactis NZ9000 (pNZ8148 / purC) obtained in Example 1 were respectively inoculated in GM17 liquid medium added with 10 μg / mL chloramphenicol for activation. Incubate overnight in a 30°C incubator;

[0065] (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.;

[0066] (3) During the culturing process, samples were taken at regular intervals to measure the OD value at a wavelength of 600nm;

[0067] (4) Cultivate to OD 600 Add 10ng / mL nisin at 0.4 to induce the expression of the transporter, take time as the abscissa, OD 600 The value is the ordinate, and the growth curve is drawn (the drawn growth curve is as follows figure 2 shown).

[0068] The ...

Embodiment 3

[0069] Example 3: Tolerance test of recombinant strains under acid stress conditions

[0070] Specific steps are as follows:

[0071] The bacterial strain L lactis NZ9000 (pNZ8148) (control) and the bacterial strain L lactis NZ9000 (pNZ8148 / purC) obtained in Example 1 were respectively induced and cultured for 6 hours, the cells were collected by centrifugation, washed twice with 0.85% normal saline, and resuspended in an equal volume of In fresh GM17 (containing 10 μg / mL of chloramphenicol) at pH 4.0 (adjusted by lactic acid), stress for different times; wash the stressed bacterial suspension twice and resuspend in an equal volume of normal saline, take 10 μL for resuspension solution, dilute different gradient points and plant on the GM17 chloramphenicol plate to measure the number of viable bacteria and the survival rate (the results are as follows: image 3 shown);

[0072] Survival rate = (N / N 0 )×100%;

[0073] Among them, N 0 is the number of viable colonies on the...

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Abstract

The invention discloses lactic acid bacteria engineering bacteria with improved acid stress resistance, and belongs to the genetic engineering and microbial engineering technical fields. The lactic acid bacteria engineering bacteria which can be widely applied in preparation of food, medicines, feeds and chemicals are successfully constructed by taking a gene encoding phosphoribosylaminimidazole-succinimide synthetase PurC as a target gene and lactic acid bacteria as an expression host. The acid stress resistance of the lactic acid bacteria engineering bacteria is significantly improved, and the maximum acid stress resistance is improved by 83.2 times than that of wild strains.

Description

technical field [0001] The invention relates to a lactic acid bacteria engineering bacterium with improved acid stress resistance, belonging to the technical fields of genetic engineering and microbial engineering. Background technique [0002] Lactic acid bacteria is a general term for a group of bacteria that can produce large amounts of lactic acid from fermentable carbohydrates. These bacteria are widely distributed in nature and have rich species diversity. They are not only ideal materials for studying classification, biochemistry, genetics, molecular biology and genetic engineering, and have important academic value in theory, but also have important industrial fields closely related to human life, such as food, medicine, feed, and fine chemicals. It also has important application value. [0003] However, in the process of industrial fermentation production of lactic acid bacteria and their function as probiotics in the human gastrointestinal system, it is inevitabl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/74C12R1/01
CPCC12N9/93C12N15/746C12Y603/02006
Inventor 张娟杨佩珊刘为佳陈坚堵国成
Owner JIANGNAN UNIV
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