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Acid-stress-resisting recombinant lactobacillus

A technology for recombining lactic acid bacteria and lactic acid bacteria, applied in the field of bioengineering, can solve problems such as low efficiency, weakening existing metabolic pathways, and large workload.

Active Publication Date: 2018-06-01
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The method for improving the acid stress tolerance of lactic acid bacteria mainly contains at present: (1) mutagenesis breeding, this method has the characteristics such as easy, type is various, but workload is big, efficient is its main shortcoming; (2) biochemical engineering strategy, It has been reported that exogenous aspartic acid has been used 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 strategies, currently using metabolic engineering strategies to improve the environmental stress tolerance of lactic acid bacteria The methods mainly include constructing new metabolic pathways, expanding existing metabolic pathways and weakening existing metabolic pathways.
The above methods either have cost issues or low success rates

Method used

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Construction of embodiment 1 recombinant strain

[0025] The gene sequence of greAB as shown in SEQ ID NO.2 was obtained from L.lactis NZ9000 of the NCBI database, and cloned into the expression plasmid pNZ8148 of Lactococcus lactis to obtain the recombinant plasmid pNZ8148 / GreAB, which was then electroporated into In the host strain L. lactis NZ9000, a recombinant strain L. lactis NZ9000 (pNZ8148 / GreAB) was obtained.

[0026] details as follows:

[0027] According to the gene sequence of GreAB, primers greAB-F and greAB-R (Table 1) shown in SEQ ID NO.3 and SEQ ID NO.4 were designed respectively, and the genome of L.lactis NZ9000 was used as template PCR amplification to obtain SEQ ID NO. The gene fragment shown in ID NO.2. The PCR product and the vector pNZ8148 were double-digested with NcoI and HindIII, respectively, and the digested products were purified and ligated. The ligation product was transformed into Escherichia coli MC1061 (commercial strain) competent, ...

Embodiment 2

[0031] The growth performance test of embodiment 2 overexpression GreAB bacterial strain

[0032] To investigate the growth of bacterial strains when GreAB was overexpressed, the bacterial strains L.lactis NZ9000 (pNZ8148 / GreAB) and L.lactis NZ9000 (pNZ8148) (control) were inoculated in GM17 liquid medium ( 1 mL) for activation, and placed in a 30°C incubator for static culture overnight. The seed solution was then transferred to fresh chloramphenicol (10 μg / mL) GM17 liquid medium with a 2% inoculation amount, and cultured statically at 30°C. Samples were taken every 2 hours, and the OD value at a wavelength of 600nm was measured. Grow to OD 600 At 0.4, 10ng / mL nisin was added to induce the expression of GreAB protein. Taking time as the abscissa, OD 600 The value is the vertical axis, and the growth curve is drawn.

[0033] The result is as figure 2 shown. The growth performance test analysis showed that the biomass of the recombinant strain was not significantly diff...

Embodiment 3

[0034] Tolerance test under the acid stress condition of embodiment 3

[0035] For the analysis of the tolerance of the investigated strains to acid, the survival rates of the recombinant strains and the control strains at pH 4.0 were measured.

[0036] The specific operation method is as follows: the strain was induced and cultured for 6 hours, the cells were collected by centrifugation, washed twice with 0.85% normal saline, and then resuspended in an equal volume of fresh GM17 (containing 10 μg / mL chloramphenicol) at pH 4.0 (adjusted by lactic acid). , coercion at different times. After the stress, the bacterial suspension was washed twice and then resuspended in an equal volume of normal saline. 10 μL of the resuspension was taken, diluted with different gradients, and planted on a GM17 chloramphenicol plate to determine the number of viable bacteria and the survival rate.

[0037] According to the analysis of the tolerance test, the survival rate of the recombinant strai...

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Abstract

The invention discloses acid-stress-resisting recombinant lactobacillus and belongs to the technical field of biological engineering. A GreAB gene obtained from lactococcus lactis (L.lactis NZ9000) isexcessively expressed in the lactococcus lactis (L.lactis NZ9000) to obtain recombinant lactococcus lactis (L.lactis NZ9000) (pNZ8148 / GreAB) with the remarkably improved acid-stress-resisting capability. The survival rate of a recombinant strain is 10.4 times as much as that of a control group after the recombinant strain is stressed for 3h under the condition that the pH (Potential of Hydrogen)is 4.0. The invention further provides a method for improving the acid stress resistance; the method has good industrial application value.

Description

technical field [0001] The invention relates to a recombinant lactic acid bacteria resistant to acid stress, which belongs to the technical field of bioengineering. Background technique [0002] When lactic acid bacteria are used for industrial production, during the fermentation process, along with the metabolic growth of the bacteria, acidic substances are also produced and accumulated, causing the cells to face severe acid stress. In order to maintain the stability of fermentation production and improve production efficiency, the industry usually maintains the pH in a stable range by adding exogenous neutralizers during the fermentation process. For example, the pH value of the fermentation environment is controlled by adding alkaline substances (ammonia or NaOH). However, the addition of alkaline substances often leads to the accumulation of by-products. The salts formed in the by-products will again cause the cells to be in a hypertonic environment, resulting in osmot...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C07K14/195C12R1/46
CPCC07K14/195
Inventor 张娟陈坚堵国成朱政明
Owner JIANGNAN UNIV
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