Method for improving endurance capacity of lactic acid bacteria under acidic conditions
An acid condition, lactic acid bacteria technology, applied in the field of genetic engineering and microbial engineering, can solve the problems of osmotic stress, accumulation of by-products, influence on bacterial growth and metabolism, etc., to achieve improved tolerance, less workload, and high success rate Effect
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Embodiment 1
[0050] Embodiment 1: Construction of recombinant bacterial strain
[0051] Specific steps are as follows:
[0052] (1) Obtain the glnP gene sequence as shown in SEQ ID NO.1 from the NCBI database (glnP gene is the gene of encoding glutamine ABC transporter GlnP, and glutamine ABC transporter GlnP is used for transporting gluten on the membrane. amino acid), the glnQ gene sequence shown in SEQ ID NO.2 (glnQ is the gene encoding the glutamine transport ATP binding protein GlnQ, and the glutamine transport ATP binding protein GlnQ is used to transport glutamate on the membrane and aspartic acid), the oppB gene sequence shown in SEQID NO.3 (oppB is the gene encoding the peptide transport system permease OppB, and the peptide transport system permease OppB is used to transport oligopeptides on the membrane protein), the malF gene sequence shown in SEQ ID NO.4 (malF is the gene encoding the maltose ABC transporter permease MalF, and the maltose ABC transporter permease MalF is the ...
Embodiment 2
[0060] Embodiment 2: the growth performance test of recombinant bacterial strain
[0061] Specific steps are as follows:
[0062] (1) The bacterial strain L lactis NZ9000 (pNZ8148) (control) containing only the blank plasmid pNZ8148 and the bacterial strain L lactis (GlnP), L lactis (GlnQ), L lactis (OppB) and L lactis (MalF) obtained in Example 1 They were respectively inoculated in GM17 liquid medium supplemented with 10 μg / mL chloramphenicol for activation, and placed in a 30°C incubator for static culture overnight;
[0063] (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.;
[0064] (3) During the culturing process, samples were taken at regular intervals to measure the OD value at a wavelength of 600nm;
[0065] (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 t...
Embodiment 3
[0067] Example 3: Tolerance test of recombinant strains under lactic acid stress conditions
[0068] Specific steps are as follows:
[0069] The bacterial strain L lactis (Vector) (control) containing only the blank plasmid pNZ8148 and the bacterial strains L lactis (GlnP) and L lactis (GlnQ) obtained in Example 1 were respectively induced and cultured in GM17 medium at 30° C. for 6 h, The cells were collected by centrifugation, washed twice with 0.85% normal saline, and then resuspended in fresh GM17 (containing 10 μg / mL of chloramphenicol) at pH 4.0 (adjusted by lactic acid) equal to the volume of the collected bacterial solution, respectively. 1.5h, 2.5h, and 3h; wash the stressed bacterial suspension twice and resuspend it in an equal volume of normal saline, take 10 μL of the resuspension, dilute different gradients and inoculate it on a GM17 chloramphenicol plate to determine the viable bacteria number and survival rate (results such as Figure 6-7 shown);
[0070] Am...
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