Effect of potential probiotic Leuconostoc mesenteroides FB111 in prevention of cholesterol absorption by modulating NPC1L1/PPARα/SREBP-2 pathways in epithelial Caco-2 cells.

Mustard kimchi consumption reduces cholesterol levels in rats. To identify lactic acid bacteria (LAB) in kimchi which exert this effect, 20 LAB isolates were evaluated for cholesterol reduction in an in vitro screen. The FB111 strain showed the highest cholesterol-lowering activity and was identified as Leuconostoc mesenteroides. This strain was characterized as a potential probiotic through sequential analyses for resistance to gastrointestinal digestion and bile salts, and adhesion to Caco-2 cells. The Caco-2 cells treated with L. mesenteroides FB111 (6-8 log CFU/mL) showed toxicological effect. The reduction of cholesterol uptake in these cells was inhibited by 48.6% compared to the control and significantly higher than that of the Lactobacillus rhamnosus GG (LGG) strain-treated group after 2-h incubation. The levels of NPC1L1, ABCG5, ABCG8, SREBP-1, SREBP-2, and PPARα gene expression were determined by reverse transcription-quantitative polymerase chain reaction analysis. The L. mesenteroides FB111 and LGG inhibited the mRNA expression of NPC1L1 (P < 0.05), whereas the expression of PPARα was increased. Moreover, the FB111 strain also inhibited the expression of SREBP-2 mRNA. Overall, we found that L. mesenteroides FB111 has efficient cholesterol-lowering effects and might be useful as a probiotic in the food industry.

Genetic diversity analysis of Leuconostoc mesenteroides from Korean vegetables and food products by multilocus sequence typing.

In the present study, 35 Leuconostoc mesenteroides strains isolated from vegetables and food products from South Korea were studied by multilocus sequence typing (MLST) of seven housekeeping genes (atpA, groEL, gyrB, pheS, pyrG, rpoA, and uvrC). The fragment sizes of the seven amplified housekeeping genes ranged in length from 366 to 1414 bp. Sequence analysis indicated 27 different sequence types (STs) with 25 of them being represented by a single strain indicating high genetic diversity, whereas the remaining 2 were characterized by five strains each. In total, 220 polymorphic nucleotide sites were detected among seven housekeeping genes. The phylogenetic analysis based on the STs of the seven loci indicated that the 35 strains belonged to two major groups, A (28 strains) and B (7 strains). Split decomposition analysis showed that intraspecies recombination played a role in generating diversity among strains. The minimum spanning tree showed that the evolution of the STs was not correlated with food source. This study signifies that the multilocus sequence typing is a valuable tool to access the genetic diversity among L. mesenteroides strains from South Korea and can be used further to monitor the evolutionary changes.

A proposal of Leuconostoc mesenteroides subsp. jonggajibkimchii subsp. nov. and reclassification of Leuconostoc mesenteroides subsp. suionicum (Gu et al., 2012) as Leuconostoc suionicum sp. nov. based on complete genome sequences.

The type strains of four subspecies of Leuconostocmesenteroides, L. mesenteroidessubsp. mesenteroides, L. mesenteroidessubsp. cremoris, L. mesenteroidessubsp. dextranicum and L. mesenteroidessubsp. suionicum, and strain DRC1506T, used as a starter culture for commercial kimchi production in Korea, were phylogenetically analyzed on the basis of their complete genome sequences. Although the type strains of the four L. mesenteroides subspecies and strain DRC1506T shared very high 16S rRNA gene sequence similarities (>99.72 %), the results of analysis of average nucleotide identity (ANI), in silico DNA-DNA hybridization (DDH) and core-genome-based relatedness indicated that they could form five different phylogenetic lineages. The type strains of L. mesenteroidessubsp. mesenteroides, L. mesenteroidessubsp. cremoris and L. mesenteroidessubsp. dextranicum and DRC1506T shared higher ANI and in silico DDH values than the thresholds (95-96 % and 70 %, respectively) generally accepted for different species delineation, whereas the type strain of L. mesenteroidessubsp. suionicum (DSM 20241T) shared lower ANI (<94.1 %) and in silico DDH values (<57.0 %) with the other four L. mesenteroides lineage strains, indicating that DSM 20241T couldn be reclassified as representing a different species. Here, we report that DRC1506T represents a novel subspecies within the species Leuconostoc mesenteroides, for which the name Leuconostoc mesenteroidessubsp. jonggajibkimchii subsp. nov. is proposed. The type strain is DRC1506T (=KCCM 43249T=JCM 31787T). In addition, L. mesenteroidessubsp. suionicum is also reclassified as Leuconostoc suionicum. sp. nov. (type strain DSM 20241T=ATCC 9135T=LMG 8159T=NCIMB 6992T).

Isolation and Characterization of Kimchi Starters Leuconostoc mesenteroides PBio03 and Leuconostoc mesenteroides PBio104 for Manufacture of Commercial Kimchi.

This study was focused on developing and obtaining a kimchi starter for use in commercial kimchi production. Kimchi varieties made with selected starters are of high quality, have high levels of mannitol, and extended shelf life. The starters were screened for properties such as mannitol production, low gas/acid production, and acid resistance. Finally, kimchi fermentation testing was performed using selected LAB starters. Kimchi samples were prepared with lactic acid bacteria (LAB) starters, including Leuconostoc mesenteroides PBio03 and Leuconostoc mesenteroides PBio104. The LAB starters are isolated from kimchi and can grow under pH 3.0 and low temperature conditions of 5°C. Four kimchi samples were fermented and stored for 28 days at 5°C. The kimchi samples made with starters (PBio03 and PBio104) had better quality (production of mannitol and maintenance of heterofermentative LAB dominance) than the non-starter kimchi samples. In the starter kimchi, Leu. mesenteroides was the dominant LAB, comprising 80% and 70% of total LAB counts at 7 and 21 days, respectively. Mannitol content of the kimchi with Leu. mesenteroides PBio03 was 1,423 ± 19.1 mg/ 100 g at 28 days, which was higher than that of the non-starter kimchi sample (1,027 ± 12.2 mg/100 g). These results show the possibility of producing kimchi with improved qualities using Leu. mesenteroides PBio03 and PBio104 as starters.

Analysis of rpoB polymorphism and PCR-based approaches for the identification of Leuconostoc mesenteroides at the species and subspecies level.

Leuconostoc mesenteroides includes the subsp. cremoris, subsp. dextranicum, subsp. mesenteroides and subsp. jonggajibkimchii, but the identification at the subspecies level using current phenotypic and/or genotypic methods is still difficult. In this study, a polyphasic approach based on the analysis of rpoB gene polymorphism, Multiplex-PCR and phenotypic tests was optimised and used to identify a collection of Leuc. mesenteroides strains at the species and subspecies levels. The annotation of published Leuc. mesenteroides genomes was also revised. A polymorphic region of rpoB gene was effective in separating Leuc. mesenteroides strains at the species (rpoB-species-specific-PCR) and subspecies (phylogenetic comparison) levels. Multiplex-PCR discriminated the subsp. mesenteroides from subsp. cremoris, but strains of uncertain attribution were found among subsp. dextranicum and subsp. jonggajibkimchii. Most of phenotypic features were not suitable for subspecies discrimination. Our assays may provide a rapid and reliable identification of subsp. mesenteroides and subsp. cremoris strains in fermented foods. The discrimination of subsp. dextranicum and subsp. jonggajibkimchii suffered from several limitations (e.g. low number of available strains and genomes, phenotypic profile close to subsp. mesenteroides, discrepancy between genotypic and phenotypic traits) and further investigations are needed to clarify their delineation and taxonomical position.

Composition of lactic acid bacteria during spontaneous curly kale (Brassica oleracea var. sabellica) fermentation.

The present work is the first report on spontaneous fermentation of curly kale and characteristics of autochthonous lactic acid bacteria (LAB). Our results indicate that curly kale fermentation is the new possibility of the technological use of this vegetable. Bacteria representing ten different species were isolated from three phases of curly kale fermentation and identified by MALDI-TOF mass spectrometry and 16S rRNA gene sequencing. Among them, four species were identified as Lactobacillus spp. (Lb. plantarum 332, Lb. paraplantarum G2114, Lb. brevis R413, Lb. curvatus 154), two as Weissella spp. (W. hellenica 152, W. cibaria G44), two as Pediococcus spp. (P. pentosaceus 45AN, P. acidilactici 2211), one as Leuconostoc mesenteroides 153, and one as Lactococcus lactis 37BN. The functional properties of isolates, i.e. acid, NaCl and bile salt tolerance, enzyme activities, adhesion to hydrocarbons, and antibiotic resistance, were examined. Among the tested strains, Lb. plantarum 332, Lb. paraplantarum G2114, P. pentosaceus 2211, and Lb. brevis R413 exhibited the best hydrophobicity value and high tolerance to bile salts, NaCl, and low pH.

Emergence of Leuconostoc mesenteroides as a causative agent of oozing in carrots stored under non-ventilated conditions.

Long-term storage and transport of post-harvest carrots (Daucus carota L.) require a low-temperature, high-relative-humidity environment, usually with low ventilation. Following long-term storage, a slimy exudate (oozing) often appears on the carrots, leading to severe spoilage. We characterized the environmental conditions leading to these symptoms and identified the causative agent. Simulation of non-ventilated storage conditions revealed accumulation of CO2 (to 80%) and ethanol (to 1000 ppm); then, a transparent exudate appeared on the carrot surface which, upon ventilation, developed into tissue browning and soft rot. Peels from oozing carrots contained over 10-fold the total bacterial counts of healthy carrots. The total peel microbiome was determined by 16S rDNA sequencing. During oozing stage, the surface of carrots incubated in a CO2 -rich (98%) environment harboured a bacterial population dominated by Lactobacillales and Enterobacteriales, differing markedly from those incubated in air. Three prevalent bacterial isolates from the oozing carrots were identified as Pantoea agglomerans, Rahnella aquatilis and Leuconostoc mesenteroides. Inoculation of carrot discs with L. mesenteroides, but not the others, induced oozing under high CO2 , suggesting that this bacterium is responsible for oozing of stored carrots. These findings should enable development of approaches to preventing carrot spoilage during long-term storage.

Pan-genomic and transcriptomic analyses of Leuconostoc mesenteroides provide insights into its genomic and metabolic features and roles in kimchi fermentation.

The genomic and metabolic features of Leuconostoc (Leu) mesenteroides were investigated through pan-genomic and transcriptomic analyses. Relatedness analysis of 17 Leu. mesenteroides strains available in GenBank based on 16S rRNA gene sequence, average nucleotide identity, in silico DNA-DNA hybridization, molecular phenotype, and core-genome indicated that Leu. mesenteroides has been separated into different phylogenetic lineages. Pan-genome of Leu. mesenteroides strains, consisting of 999 genes in core-genome, 1,432 genes in accessory-genome, and 754 genes in unique genome, and their COG and KEGG analyses showed that Leu. mesenteroides harbors strain-specifically diverse metabolisms, probably representing high evolutionary genome changes. The reconstruction of fermentative metabolic pathways for Leu. mesenteroides strains showed that Leu. mesenteroides produces various metabolites such as lactate, ethanol, acetate, CO2, mannitol, diacetyl, acetoin, and 2,3-butanediol through an obligate heterolactic fermentation from various carbohydrates. Fermentative metabolic features of Leu. mesenteroides during kimchi fermentation were investigated through transcriptional analyses for the KEGG pathways and reconstructed metabolic pathways of Leu. mesenteroides using kimchi metatranscriptomic data. This was the first study to investigate the genomic and metabolic features of Leu. mesenteroides through pan-genomic and metatranscriptomic analyses, and may provide insights into its genomic and metabolic features and a better understanding of kimchi fermentations by Leu. mesenteroides.

Lactic acid bacteria involved in cocoa beans fermentation from Ivory Coast: Species diversity and citrate lyase production.

Microbial fermentation is an indispensable process for high quality chocolate from cocoa bean raw material. lactic acid bacteria (LAB) are among the major microorganisms responsible for cocoa fermentation but their exact role remains to be elucidated. In this study, we analyzed the diversity of LAB in six cocoa producing regions of Ivory Coast. Ribosomal 16S gene sequence analysis showed that Lactobacillus plantarum and Leuconostoc mesenteroides are the dominant LAB species in these six regions. In addition, other species were identified as the minor microbial population, namely Lactobacillus curieae, Enterococcus faecium, Fructobacillus pseudoficulneus, Lactobacillus casei, Weissella paramesenteroides and Weissella cibaria. However, in each region, the LAB microbial population was composed of a restricted number of species (maximum 5 species), which varied between the different regions. LAB implication in the breakdown of citric acid was investigated as a fundamental property for a successful cocoa fermentation process. High citrate lyase producer strains were characterized by rapid citric acid consumption, as revealed by a 4-fold decrease in citric acid concentration in the growth medium within 12h, concomitant with an increase in acetic acid and lactic acid concentration. The production of citrate lyase was strongly dependent on environmental conditions, with optimum production at acidic pH (pH<5), and moderate temperature (30-40°C), which corresponds to conditions prevailing in the early stage of natural cocoa fermentation. This study reveals that one of the major roles of LAB in the cocoa fermentation process involves the breakdown of citric acid during the early stage of cocoa fermentation through the activity of citrate lyase.

Effect of potential probiotic Leuconostoc mesenteroides FB111 in prevention of cholesterol absorption by modulating NPC1L1/PPARalpha/SREBP-2 pathways in epithelial Caco-2 cells

Mustard kimchi consumption reduces cholesterol levels in rats. To identify lactic acid bacteria (LAB) in kimchi which exert this effect, 20 LAB isolates were evaluated for cholesterol reduction in an in vitro screen. The FB111 strain showed the highest cholesterol-lowering activity and was identified as Leuconostoc mesenteroides. This strain was characterized as a potential probiotic through sequential analyses for resistance to gastrointestinal digestion and bile salts, and adhesion to Caco-2 cells. The Caco-2 cells treated with L. mesenteroides FB111 (6-8 log CFU/mL) showed toxicological effect. The reduction of cholesterol uptake in these cells was inhibited by 48.6% compared to the control and significantly higher than that of the Lactobacillus rhamnosus GG (LGG) strain-treated group after 2-h incubation. The levels of NPC1L1, ABCG5, ABCG8, SREBP-1, SREBP-2, and PPARalpha gene expression were determined by reverse transcription-quantitative polymerase chain reaction analysis. The L. mesenteroides FB111 and LGG inhibited the mRNA expression of NPC1L1 (P < 0.05), whereas the expression of PPARalpha was increased. Moreover, the FB111 strain also inhibited the expression of SREBP-2 mRNA. Overall, we found that L. mesenteroides FB111 has efficient cholesterol-lowering effects and might be useful as a probiotic in the food industry

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