In the current study, the probiotic potential of approximately 250 strains of lactic acid bacteria (LAB) isolated from piglet fecal samples were investigated; among them Lactobacillus plantarum strain JDFM LP11, which possesses significant probiotic potential, with enhanced acid/bile tolerance, atta...
In the current study, the probiotic potential of approximately 250 strains of lactic acid bacteria (LAB) isolated from piglet fecal samples were investigated; among them Lactobacillus plantarum strain JDFM LP11, which possesses significant probiotic potential, with enhanced acid/bile tolerance, attachment to porcine intestinal epithelial cells (IPEC-J2), and antimicrobial activity. The genetic characteristics of strain JDFM LP11 were explored by performing whole genome sequencing (WGS) using a PacBio system. The circular draft genome have a total length of 3,206,883 bp and a total of 3,021 coding sequences were identified. Phylogenetically, three genes, possibly related to survival and metabolic activity in the porcine host, were identified. These genes encode p60, lichenan permease IIC component, and protein TsgA, which are a putative endopeptidase, a component of the phosphotransferase system (PTS), and a major facilitator in the gut environment, respectively. Our findings suggest that understanding the functional and genetic characteristics of L. plantarum strain JDFM LP11, with its candidate genes for gut health, could provide new opportunities and insights into applications in the animal food and feed additive industries.
In the current study, the probiotic potential of approximately 250 strains of lactic acid bacteria (LAB) isolated from piglet fecal samples were investigated; among them Lactobacillus plantarum strain JDFM LP11, which possesses significant probiotic potential, with enhanced acid/bile tolerance, attachment to porcine intestinal epithelial cells (IPEC-J2), and antimicrobial activity. The genetic characteristics of strain JDFM LP11 were explored by performing whole genome sequencing (WGS) using a PacBio system. The circular draft genome have a total length of 3,206,883 bp and a total of 3,021 coding sequences were identified. Phylogenetically, three genes, possibly related to survival and metabolic activity in the porcine host, were identified. These genes encode p60, lichenan permease IIC component, and protein TsgA, which are a putative endopeptidase, a component of the phosphotransferase system (PTS), and a major facilitator in the gut environment, respectively. Our findings suggest that understanding the functional and genetic characteristics of L. plantarum strain JDFM LP11, with its candidate genes for gut health, could provide new opportunities and insights into applications in the animal food and feed additive industries.
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제안 방법
All isolated LAB were maintained at –80℃ in MRS broth containing glycerol solution for further studies.
An 8–12 kb library was prepared following the Pacific Biosciences manual; sequencing procedure and filtration/de-novo assembly of raw sequence data was carried out on the PacBio RS II system (Pacific Biosciences, Menlo Park, CA, USA) using C4 chemistry on single molecule, real-time cells with a 120 min sequence capture protocol according to the PacBio Hierarchical Genome Assembly Process using the Quiver software package.
Assembled contigs, including short lengths (< 20,000 bps) and low coverage (< 50×) were sorted for further experiments.
Following exposure of C. elegans to individual probiotic candidates on NGM plates containing nystatin (5 μg/mL) for five days, ten worms were picked at random, washed twice in M9 buffer, and placed on brain heart infusion (BHI) plates containing both kanamycin (100 μg/mL) and streptomycin (100 μg/mL).
coli OP50 as described previously (Brenner, 1974). In order to evaluate whether the probiotic candidates had an effect on host lifespan, nematode lifespan was measured according to previously described methods (Aballay et al., 2000; Garigan et al., 2002) with modifications. Groups of twenty worms were exposed to each probiotic candidate on NGM plates and the numbers of live worms were scored every day.
4B). Next, we conducted an ANI, comparative tree analysis of the strain JDFM LP11 compared with genome sequences of the same species. The ANI trees were constructed from the eight available complete genome sequences in the Ensemble bacteria database (Fig.
Groups of twenty worms were exposed to each probiotic candidate on NGM plates and the numbers of live worms were scored every day. To correctly determine scores, worms were transferred to fresh plates every three days. A worm was regarded as dead when it failed to respond to a gentle touch.
대상 데이터
Amplified products were purified for sequencing using a Gel Extraction Kit (Intron, Korea). The sequences of the final products were analyzed using an ABI 377 automated DNA sequencer (Perkin Elmer, USA). Sequence homologies were examined by comparing the obtained sequences with those in DNA databases (http://www.
5). Three strains, namely WCFS1, UCMA3037, and ZJ316, grouped together with JDFM LP11. Subsequently, dN/dS analysis in a branch-site model was performed to identify the phylogenetically featured genes in the JDFM LP11 strain.
The inhibitory effects of the lactobacilli on intestinal pathogens was measured using an overlay method. Two representative porcine intestinal pathogens including pathogenic E. coli and Salmonella Typhimurium were used for the test. Bacteria were grown in Luria-Bertani (LB) broth (Difco, USA) at 37℃ for 18 h.
데이터처리
Nematode survival was analyzed using the Kaplan-Meier method and differences were determined using the log-rank test (STATA6; STATA, College Station, TX, USA). Student's t-test was performed to determine statistical differences in CFU counts for determining bacterial counts.
Student's t-test was performed to determine statistical differences in CFU counts for determining bacterial counts.
이론/모형
The inhibitory effects of the lactobacilli on intestinal pathogens was measured using an overlay method. Two representative porcine intestinal pathogens including pathogenic E.
성능/효과
4, the predicted ORFs were grouped by COG functional and EggNOG subsystem categorizations. First, COG functional categorization showed that 1,472 ORFs (48.72% of the COG assigned ORFs) belonged to five major COG functional categories including amino acid transport and metabolism, carbohydrate transport and metabolism, translation, ribosomal structure and biogenesis, recombination, replication, repair, and general function prediction (Fig. 4A). In addition, EggNOG categorization mainly classified ORFs into those responsible for amino acid transport and metabolism, carbohydrate transport and metabolism, cell wall/membrane/envelope biogenesis, and replication/recombination/repair as well as transcription (55.
Unfortunately, there is no information for TsgA in the literature as a transporter in the genus Lactobacillus. In conclusion, we suggest that the featured genes may positively influence the colonization and survival of L. plantarum JDFM LP11 and its interaction with commensal microbiota or pathogens in the gut.
plantarum strain JDFM LP11, providing genomic evidence for the observed functional activities. The results of this combined analysis demonstrate that the newly identified L. plantarum strain JDFM LP11 hold promise for use as probiotic functional food additives.
Subsequently, dN/dS analysis in a branch-site model was performed to identify the phylogenetically featured genes in the JDFM LP11 strain. Three phylogenetically featured genes and their amino acid sequence changes were identified in this study (Table 1 and 2); putative endopeptidase p60, lichenan permease IIC component, and protein TsgA, which are an endopeptidase, a component of the phosphotransferase system (PTS), and a major facilitator in the gut environment, respectively. It has been established that proteolytic enzymes are inhibited in vivo by protein inhibitors produced by symbiotic bacteria in our gut to protect themselves from digestive peptidases (Garcia-Ferrer et al.
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