The Construction of the Lactobacillus Fermentum MG 2CRS Mutant and Its Changes on Probiotic Effects

Abstract: As is known to all, lactic acid bacteria have been granted the GRAS (generally regarded as safe) status and widely used in various fields because of its potential probiotic effect. Lactic acid bacteria are not only used for a variety of dairy starter cultures, but also produce enzymes, antimicrobial peptides and used as food additives, even it can be developed for viable vaccine to produce antibodies or antigens.In the present paper, Lactobacillus fermentum was studied for the general survey and elimination of endogenous plasmid, and then established a strain free of endogenous plasmid which solved the problem of plasmid incompatibility and conducted a basis of importing foreign plasmid DNA. PCR method were used to clone a fragment gene of Two-component regulatory gene RR and then this gene was cloned into Knockout vector plasmid pRV300, thus novel knockout vector pRV-RR was constructed. Electro transformation method was used to transfered pRV-RR into L. fermentum and destructed the integrity of the RR gene. 16S rDNA gene sequencing and Southern blot method were used to identify mutants.Finally, the physiological and biochemical experiments and probiotic property experiments including sugar fermentation test, acid and bile salt tolerance test and adhesion and colonization test were characterized for the L. fermentum mutations. The results are showed as follows:1. Six strains of Lactobacillus isolated from wild bovine milk were subjected to extract and inspect the endogenous plasmid DNA through three different methods. The result showed that all six strains contained different sizes of endogenous plasmid DNA. The modified Anderson-Mckay method was the best, followed by the liquid nitrogen freezing method and the Alkaline lysis method was the worst. Approximately 23 kb of endogenous plasmid DNA of L. fermentum can completely eliminated by SDS- heterothermic method.2. 400bp of two-component regulatory gene RR (partial gene) of L. fermentum were exactly cloned by PCR. And then through AT cloning and sequencing the homology of RR compared with known sequence in Genbank is 100%. Its nucleotide homology in comparison with different strains of lactic acid bacteria was over 70%, which means close genetic relationship. While in comparison with pathogenic bacteria strains was less than 50%, which means distant genetic relationship. But the homology of amino acids between various strains was surprisingly similar except some regions. EcoRI/HindIII double macrorestriction was used to identify pRV-RR and its accuracy. The mutant was obtained by electrotransformed (voltage 1.5kv, time 2.0ms) the knockout vector pRV-RR into L. fermentum. Result of 16S rDNA gene sequence comparison showed that the homology of the mutant is 100% to wild-type L. fermentum, suggesting they are identical. The mutant was identified by southern blot indicated four quite strong signals, while the wild-type strain appeared two strong signals, suggesting that RR gene was knocked out successfully.3. Physiological and biochemical tests and probiotic characteristics of L. fermentum mutant (Lf -mutantfor short) and wild-type L. fermentum (Lf-wild type for short) were compared with each other. The sugar fermentation test showed that the difference was slight. Fermentation capability of fructose and sucrose of the Lf -mutantdecreased obviously, while fermentation capability of other kinds of sugers did not changed. Salt tolerance test showed that the Lf -mutantbegan to be restrained in 2.0%NaCl but Lf-wild type and inside plasmid eliminated Lf were not restrained, and the Lf -mutantwas significantly in 4%NaCl but Lf-wild type and plasmid eliminated Lf were still not. Only 6%NaCl can restrain the growing of all three. Acid tolerance test showed that: the mutant Lf, Lf-wild type and plasmid eliminated Lf grew well at pH4.5. At pH3.0, the Lf -mutantwas restrained, yet wild Lf and plasmid eliminated Lf still grew well. Bile tolerance test showed that before 12 h , the tolerance of the Lf -mutantwas the same as Lf-wild type in MRS culture which contained different concentrations of bile salt, such as 2.0﹪, 1.8﹪,1.0﹪and 0.3﹪, but after 12 h culturing, the growing of the Lf -mutantwas obviously higher than the Lf-wild type, especially in the concentration of 2.0﹪and 1.8﹪. Mouse gastrointestinal adherence test showed that the live counts bacteria from faeces of the Lf -mutant were significantly higher than Lf-wild type while live counts from gastrointestinal contents and intestinal mucosa were on the contrary.This study established a basis on the analysis of probiotic mechanism of L. fermentum at molecular level…
Key words: L. fermentum; mutant; 2CRS; RR gene; probiotics

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