A kind of anti-acid stress component and its application

A resistance, amino acid technology, applied in the field of bioengineering, can solve problems such as low success rate, low efficiency, and increased production cost

Active Publication Date: 2019-11-08
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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  • A kind of anti-acid stress component and its application
  • A kind of anti-acid stress component and its application
  • A kind of anti-acid stress component and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Construction of embodiment 1 recombinant strain

[0031] Obtain the gene sequence of recT-A76 as shown in SEQ ID NO.3 from L.lactis A76 of NCBI database, and clone it on the expression plasmid pNZ8148 of Lactococcus lactis to obtain recombinant plasmid pNZ8148 / RecT-A76, and then It was electroporated into the host strain L.lactis NZ9000 to obtain the recombinant strain L.lactis NZ9000 (pNZ8148 / RecT-A76); the recT-MG1655 shown in SEQ ID NO.4 was obtained from E.coli MG1655 in the NCBI database. The gene sequence was cloned into the expression plasmid pNZ8148 to obtain the recombinant plasmid pNZ8148 / RecT-MG1655, which was then electrotransformed into the host strain L. lactis NZ9000 to obtain the recombinant strain L. lactis NZ9000 (pNZ8148 / RecT-MG1655).

[0032] details as follows:

[0033]According to the gene sequence of recT, primers recT-A76-BF, recT-A76-BR, recT-MG1655- BF, recT-MG1655-BR (Table 1), using the genomes of L.lactis A76 and E.coli MG1655 as templates...

Embodiment 2

[0037] The growth performance test of embodiment 2 overexpression RecT protein strain

[0038] To investigate the growth of the strains when the RecT protein was overexpressed, the strains L.lactis NZ9000 (pNZ8148 / RecT-A76), L.lactis NZ9000 (pNZ8148 / RecT-MG1655) and L.lactis NZ9000 (pNZ8148) (control) were inoculated Activation was carried out in GM17 liquid medium (1 mL) added with 10 μg / mL chloramphenicol, 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 RecT protein. Taking time as the abscissa, OD 600 The value is the vertical axis, and the growth curve is drawn.

[0039] The result is as figure 2 shown. The growth performance test ana...

Embodiment 3

[0040] Tolerance test under the acid stress condition of embodiment 3

[0041] 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.

[0042] 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.

[0043] According to the analysis of the tolerance test, the survival rates of the recombinant stra...

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Abstract

The invention discloses an anti-acid-stress component and application thereof, belonging to the field of bioengineering technology. According to the invention, two recombinant Lactococcus lactis strains with substantially improved resistance to acid stress, i.e., L. lactis NZ9000 (pNZ8148 / RecT-A76) and L. lactis NZ9000 (pNZ8148 / RecT-MG1655), are obtained by overexpression of recT genes, originated from Lactococcus lactis L. lactis A76 and Escherichia coli E. coli MG1655, in Lactococcus lactis NZ9000, respectively. After the two recombinant Lactococcus lactis strains suffer from stress in an environment with a pH value of 4.0 for 3h, the survival rates of the two strains are 10.5 times and 1.4 times the survival rates of control strains, respectively; and after the two strains suffer from stress caused by 20% (v / v) ethanol for 6 h, the survival rates of the two strains are 3.7 times and 2.1 times the survival rates of control strains, respectively. The invention also provides a method for improving resistance to acid stress. The method has good industrial application value.

Description

technical field [0001] The invention relates to an anti-acid stress component and its application, belonging 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 osmotic stress, whi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C07K14/245C07K14/315A23L33/135A23K10/16C12R1/46
CPCA23K10/16A23L33/135C07K14/245C07K14/315
Inventor 张娟陈坚堵国成吉晓梅朱政明
Owner JIANGNAN UNIV
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