Evaluation of ginsenoside bioconversion of lactic acid bacteria isolated from kimchi.
ABSTRACT: BACKGROUND:Panax ginseng is a physiologically active plant widely used in traditional medicine that is characterized by the presence of ginsenosides. Rb1, a major ginsenoside, is used as the starting material for producing ginsenoside derivatives with enhanced pharmaceutical potentials through chemical, enzymatic, or microbial transformation. METHODS:To investigate the bioconversion of ginsenoside Rb1, we prepared kimchi originated bacterial strains Leuconostoc mensenteroides WiKim19, Pediococcus pentosaceus WiKim20, Lactobacillus brevis WiKim47, Leuconostoc lactis WiKim48, and Lactobacillus sakei WiKim49 and analyzed bioconversion products using LC-MS/MS mass spectrometer. RESULTS:L. mesenteroides WiKim19 and Pediococcus pentosaceus WiKim20 converted ginsenoside Rb1 into the ginsenoside Rg3 approximately five times more than Lactobacillus brevis WiKim47, Leuconostoc lactis WiKim48, and Lactobacillus sakei WiKim49. L mesenteroides WIKim19 showed positive correlation with ?-glucosidase activity and higher transformation ability of ginsenoside Rb1 into Rg3 than the other strains whereas, P. pentosaceus WiKim20 showed an elevated production of Rb3 even with lack of ?-glucosidase activity but have the highest acidity among the five lactic acid bacteria (LAB). CONCLUSION:Ginsenoside Rg5 concentration of five LABs have ranged from ?2.6 ?g/mL to 6.5 ?g/mL and increased in accordance with the incubation periods. Our results indicate that the enzymatic activity along with acidic condition contribute to the production of minor ginsenoside from lactic acid bacteria.
Project description:Previous studies using traditional biochemical identification methods to study the ecology of commercial sauerkraut fermentations revealed that four species of lactic acid bacteria, Leuconostoc mesenteroides, Lactobacillus plantarum, Pediococcus pentosaceus, and Lactobacillus brevis, were the primary microorganisms in these fermentations. In this study, 686 isolates were collected from four commercial fermentations and analyzed by DNA fingerprinting. The results indicate that the species of lactic acid bacteria present in sauerkraut fermentations are more diverse than previously reported and include Leuconostoc citreum, Leuconostoc argentinum, Lactobacillus paraplantarum, Lactobacillus coryniformis, and Weissella sp. The newly identified species Leuconostoc fallax was also found. Unexpectedly, only two isolates of P. pentosaceus and 15 isolates of L. brevis were recovered during this study. A better understanding of the microbiota may aid in the development of low-salt fermentations, which may have altered microflora and altered sensory characteristics.
Project description:Rushan cheese, an essential part of the Bai culture, has been produced and consumed for centuries by the Bai people living mostly in Yunnan province of China, however, studies on the naturally occurring microbial communities of Rushan cheese are lacking. In this study, we applied high throughput sequencing technique to analyze the microbial compositions of Rushan cheese samples from three different geographical origins (i.e., Weishan, Eryuan, and Jianchuan). The microbiota in Weishan, Eryuan and Jianchuan Rushan cheese samples were distinct in terms of taxonomic composition and abundance. Linear discriminant analysis (LDA) of effect size (LEfSe) analysis found the characteristic taxonomic species in Weishan Rushan cheese samples were Lactobacillus pentosus, Lactobacillus crustorum, Lactobacillus brevis, Leuconostoc mesenteroides, and Pediococcus pentosaceus; the representing taxonomic species in Eryuan Rushan cheese samples were Lactobacillus kefiranofaciens, Lactococcus lactis, Acetobacter pasteurianus and Moraxella osloensis; by comparison, Acinetobacter was enriched in Jianchuan Rushan cheese samples. Characterization of the microbial diversity in Rushan cheese samples from different geographical origins will contribute to the understanding of microorganisms responsible for the Rushan cheese fermentation, and enable us to develop bioresources derived from Rushan cheese in the future.
Project description:Kimchi fermentation depends on diverse lactic acid bacteria, which convert raw materials into numerous metabolites that contribute to the taste of food. Amino acids and saccharides are important primary metabolites. Arginine is nearly exhausted during kimchi fermentation, whereas the concentrations of other amino acids are reported not to increase or decrease dramatically. These phenomena could imply that arginine is an important nutritional component among the amino acids during kimchi fermentation. In this study, we investigated the arginine-catabolism pathway of seven lactic acid bacteria isolated from kimchi and evaluated the products of arginine catabolism (citrulline and ornithine) associated with the bacteria. The arginine content dramatically decreased in cultures of Lactobacillus brevis and Weissella confusa from 300 ?g/mL of arginine to 0.14 ± 0.19 and 1.3 ± 0.01 ?g/mL, respectively, after 6 h of cultivation. Citrulline and ornithine production by L. brevis and W. confusa showed a pattern that was consistent with arginine catabolism. Interestingly, Pediococcus pentosaceus, Lactobacillus plantarum, Leuconostoc mesenteroides, and Leuconostoc lactis did not show increased citrulline levels after arginine was added. The ornithine contents were higher in all bacteria except for L. lactis after adding arginine to the culture. These results were consistent with the absence of the arginine deiminase gene among the lactic acid bacteria. Arginine consumption and ornithine production were monitored and compared with lactic acid bacteria by metagenomics analysis, which showed that the increment of ornithine production correlated positively with lactic acid bacteria growth.
Project description:<h4>Background</h4>Genomes of gram-positive bacteria encode many putative cell-surface proteins, of which the majority has no known function. From the rapidly increasing number of available genome sequences it has become apparent that many cell-surface proteins are conserved, and frequently encoded in gene clusters or operons, suggesting common functions, and interactions of multiple components.<h4>Results</h4>A novel gene cluster encoding exclusively cell-surface proteins was identified, which is conserved in a subgroup of gram-positive bacteria. Each gene cluster generally has one copy of four new gene families called cscA, cscB, cscC and cscD. Clusters encoding these cell-surface proteins were found only in complete genomes of Lactobacillus plantarum, Lactobacillus sakei, Enterococcus faecalis, Listeria innocua, Listeria monocytogenes, Lactococcus lactis ssp lactis and Bacillus cereus and in incomplete genomes of L. lactis ssp cremoris, Lactobacillus casei, Enterococcus faecium, Pediococcus pentosaceus, Lactobacillius brevis, Oenococcus oeni, Leuconostoc mesenteroides, and Bacillus thuringiensis. These genes are neither present in the genomes of streptococci, staphylococci and clostridia, nor in the Lactobacillus acidophilus group, suggesting a niche-specific distribution, possibly relating to association with plants. All encoded proteins have a signal peptide for secretion by the Sec-dependent pathway, while some have cell-surface anchors, novel WxL domains, and putative domains for sugar binding and degradation. Transcriptome analysis in L. plantarum shows that the cscA-D genes are co-expressed, supporting their operon organization. Many gene clusters are significantly up-regulated in a glucose-grown, ccpA-mutant derivative of L. plantarum, suggesting catabolite control. This is supported by the presence of predicted CRE-sites upstream or inside the up-regulated cscA-D gene clusters.<h4>Conclusion</h4>We propose that the CscA, CscB, CscC and CscD proteins form cell-surface protein complexes and play a role in carbon source acquisition. Primary occurrence in plant-associated gram-positive bacteria suggests a possible role in degradation and utilization of plant oligo- or poly-saccharides.
Project description:Changes in the ecology of the various lactic acid bacteria (LAB) species, which are involved in traditional fermented sausages, were investigated in the light of the use of different breeds of pork, each of which was raised in two different environments and processed using two different technologies. The semi-quantitative molecular method was applied in order to understand how the different species alternate over time, as well as their concentration ratios. A significant increase in LAB over the first days of fermentation characterized the trials where the starter culture wasn't added (T), reaching values of 107-108 cfu g-1. On the other hand, in the trials in which sausages were produced with starter addition, LAB counts had a less significant incremental jump from about 106 cfu g-1 (concentration of the inoculum) to 108 cfu g-1. Lactobacillus sakei and Lb. curvatus were detected as the prevalent population in all the observed fermentations. Pediococcus pentosaceus, Lb. casei, Leuconostoc mesenteroides, Lactococcus garviae, and Lb. graminis also appeared, but their concentration ratios varied depending on the diverse experimental settings. The results of cluster analysis showed that a plant- and breed-specific LAB ecology exists. In addition, it was also observed that the breeding system can influence the presence of certain LAB species.
Project description:Reactive oxygen species (ROS), such as hydroxyl and superoxide anion radicals, are highly reactive molecules derived from the metabolism of oxygen. ROS play positive roles in cell physiology, but they may also damage cell membranes and DNA, inducing oxidation that causes membrane lipid peroxidation and decreases membrane fluidity. Soymilk yogurt, which is soymilk fermented using lactic acid bacteria (LAB), is an excellent food item with numerous functional substances with antioxidant effects. In this study, the antioxidative activities of soymilk yogurt were investigated. Sixteen of the 26 tested LAB strains solidified soymilk. In antioxidant capacity tests for bacterial cells, Leuconostoc mesenteroides MYU 60 and Pediococcus pentosaceus MYU 759 showed the highest values in the oxygen radical antioxidant capacity (ORAC) and hydroxyl radical antioxidant capacity (HORAC) tests, respectively. The supernatant of soymilk yogurt made with Lactobacillus gasseri MYU 1 showed the highest ORAC and HORAC values. L. mesenteroides MYU 60, Lactobacillus plantarum MYU 74, Lactobacillus reuteri MYU 220, and P. pentosaceus MYU 759 showed significantly high N-acetylcysteine equivalent values compared with the control in a total ROS reducing assay (p<0.05). These strains were selected, and a comet assay was performed, which exhibited decreased values in all selected strains compared with the control, indicating DNA protection. An acidic exopolysaccharide produced by P. pentosaceus MYU 759 showed high antioxidant capacity. The antioxidant substances produced by LAB fermentation may be exopolysaccharides, antioxidant peptides, and isoflavone aglycones. Soymilk yogurt can be used as a functional food useful for various diseases related to oxidation.
Project description:Ginsenoside Rb1is the main component in ginsenosides. It is a protopanaxadiol-type ginsenoside that has a dammarane-type triterpenoid as an aglycone. In this study, ginsenoside Rb1 was transformed into gypenoside XVII, ginsenoside Rd, ginsenoside F2 and compound K by glycosidase from Leuconostoc mesenteroides DC102. The optimum time for the conversion was about 72 h at a constant pH of 6.0 to 8.0 and the optimum temperature was about 30?. Under optimal conditions, ginsenoside Rb1 was decomposed and converted into compound K by 72 h post-reaction (99%). The enzymatic reaction was analyzed by highperformance liquid chromatography, suggesting the transformation pathway: ginsenoside Rb1? gypenoside XVII and ginsenoside Rd?ginsenoside F2?compound K.
Project description:Lactic acid bacteria (LAB) are responsible for olfactory changes in wine during malolactic fermentation (MLF). A side characteristic of MLF is the release of grape derived aroma compounds from their glycosylated precursors by β-glycosidase activities of these bacteria. Apart from Oenococcus oeni, which is regarded as the most promising species for MLF, glycosidic activities have also been observed in wine related members of the genera Lactobacillus and Pediococcus. Nevertheless, information on the involved enzymes including their potential use in winemaking is limited. In this study we report that β-glucosidases with similar protein sequences can be identified in the genomes of Lactobacillus brevis, O. oeni and Leuconostoc mesenteroides. TTG serves as start codon for the glucosidase gene of O. oeni. The β-glucosidase of O. oeni ATCC BAA-1163 was expressed in E. coli and partially characterized. The enzyme displayed characteristics similar to β-glucosidases isolated from L. brevis and L. mesenteroides. A pH optimum between 5.0 and 5.5, and a K(m) of 0.17 mmol L(-1 )pNP-β-D-glucopyranoside were determined. A glycosyltransferase activity was observed in the presence of ethanol. The enzyme from O. oeni was capable to hydrolyze glycosides extracted from Muskat wine. This study also contains a report on glycosidase activities of several LAB species including Oenococcus kitaharae.
Project description:A total of 161 low-G+C-content gram-positive bacteria isolated from whole-crop paddy rice silage were classified and subjected to phenotypic and genetic analyses. Based on morphological and biochemical characters, these presumptive lactic acid bacterium (LAB) isolates were divided into 10 groups that included members of the genera Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, and WEISSELLA: Analysis of the 16S ribosomal DNA (rDNA) was used to confirm the presence of the predominant groups indicated by phenotypic analysis and to determine the phylogenetic affiliation of representative strains. The virtually complete 16S rRNA gene was PCR amplified and sequenced. The sequences from the various LAB isolates showed high degrees of similarity to those of the GenBank reference strains (between 98.7 and 99.8%). Phylogenetic trees based on the 16S rDNA sequence displayed high consistency, with nodes supported by high bootstrap values. With the exception of one species, the genetic data was in agreement with the phenotypic identification. The prevalent LAB, predominantly homofermentative (66%), consisted of Lactobacillus plantarum (24%), Lactococcus lactis (22%), Leuconostoc pseudomesenteroides (20%), Pediococcus acidilactici (11%), Lactobacillus brevis (11%), Enterococcus faecalis (7%), Weissella kimchii (3%), and Pediococcus pentosaceus (2%). The present study, the first to fully document rice-associated LAB, showed a very diverse community of LAB with a relatively high number of species involved in the fermentation process of paddy rice silage. The comprehensive 16S rDNA-based approach to describing LAB community structure was valuable in revealing the large diversity of bacteria inhabiting paddy rice silage and enabling the future design of appropriate inoculants aimed at improving its fermentation quality.
Project description:BACKGROUND:Several studies have reported that ginsenoside Rg3(S) is effective in treating metastatic diseases, obesity, and various cancers, however, its presence in white ginseng cannot be estimated, and only a limited amount is present in red ginseng. Therefore, the use of recombinant glycosidases from a Generally Recognized As Safe (GRAS) host strain is a promising approach to enhance production of Rg3(S), which may improve nutritional activity, human health, and quality of life. METHOD:Lactobacillus ginsenosidimutans EMML 3041T, which was isolated from Korean fermented pickle (kimchi), presents ginsenoside-converting abilities. The strain was used to enrich the production of Rg3(S) by fermenting protopanaxadiol (PPD)-mix-type major ginsenosides (Rb1, Rb2, Rc, and Rd) in four different types of food-grade media (1, MRS; 2, Basel Food-Grade medium; 3, Basel Food-Grade medium-I, and 4, Basel Food-Grade medium-II). Due to its tendency to produce Rg3(S), the presence of glycoside hydrolase in Lactobacillus ginsenosidimutans was proposed, the whole genome was sequenced, and the probable glycoside hydrolase gene for ginsenoside conversion was cloned. RESULTS:The L. ginsenosidimutans EMML 3041T strain was whole genome sequenced to identify the target genes. After genome sequencing, 12 sets of glycoside hydrolases were identified, of which seven sets (?,?-glucosidase and ?,?-galactosidase) were cloned in Escherichia coli BL21 (DE3) using the pGEX4T-1 vector system. Among the sets of clones, only one clone (BglL.gin-952) showed ginsenoside-transforming abilities. The recombinant BglL.gin-952 comprised 952 amino acid residues and belonged to glycoside hydrolase family 3. The enzyme exhibited optimal activity at 55 °C and a pH of 7.5 and showed a promising conversion ability of major ginsenoside Rb1?Rd?Rg3(S). The recombinant enzyme (GST-BglL.gin-952) was used to mass produce Rg3(S) from major ginsenoside Rb1. Scale-up of production using 50 g of Rb1 resulted in 30 g of Rg3(S) with 74.3% chromatography purity. CONCLUSION:Our preliminary data demonstrated that this enzyme would be beneficial in the preparation of pharmacologically active minor ginsenoside Rg3(S) in the functional food and pharmaceutical industries.