Pathway and Production Differences in Branched-Chain Hydroxy Acids as Bioactive Metabolites in Limosilactobacillus fermentum, Ligilactobacillus salivarius, and Latilactobacillus sakei.

Branched-chain hydroxy acids (BCHAs) as bioactive metabolites of Lactobacillaceae include 2-hydroxy isovaleric acid (HIVA), 2-hydroxy isocaproic acid (HICA), and 2-hydroxy-3-methyl isovaleric acid (HMVA). Combining targeted and untargeted metabolomics, this study elucidates differences in extracellular BCHA production in Limosilactobacillus fermentum, Ligilactobacillus salivarius, and Latilactobacillus sakei alongside comparing comprehensive metabolic changes. Through targeted metabolomics, BCHA production among 38 strains exhibited strain specificity, except for L. sakei, which showed significantly lower BCHA production. Explaining the lower production in L. sakei, which lacks the branched-chain amino acid (BCAA)-utilizing pathway, comparison of BCHA production by precursor reaction revealed that the pathway utilizing BCAAs is more dominant than the pathway utilizing pyruvate. Expanding upon the targeted approach, untargeted metabolomics revealed the effects of the reaction compound on other metabolic pathways besides BCHAs. Metabolism alterations induced by BCAA reactions varied among species. Significant differences were observed in glycine, serine, and threonine metabolism, pyruvate metabolism, butanoate metabolism, and galactose metabolism (p < 0.05). These results emphasize the importance of the synergy between fermentation strains and substrates in influencing nutritional components of fermented foods. By uncovering novel aspects of BCAA metabolism pathways, this study could inform the selection of fermentation strains and support the targeted production of BCHAs.

Synergistic Antioxidant and Anti-Inflammatory Activities of Kale Juice Fermented with Limosilactobacills reuteri EFEL6901 or Limosilactobacills fermentum EFEL6800.

This study investigates the synergistic impact of fermenting kale juice with Limosilactobacillus strains on its antioxidant and anti-inflammatory properties. Kale's rich nutrient profile, especially its flavonoids, offers potential health benefits. Probiotic lactic acid bacteria are employed in kale fermentation to enhance nutrient bioavailability and generate bioactive compounds. Kale juices fermented with L. reuteri EFEL6901 or L. fermentum EFEL6800 exhibited superior microbial growth. Free sugars and amino acids were converted to alcohols and organic acids, affecting the organoleptic and health-related properties of the product. In addition, fermentation increased quercetin and kaempferol content, indicating improved availability. Furthermore, the fermented juice exhibited notable antioxidant activity and suppressed nitric oxide (NO) production, revealing anti-inflammatory potential. Gene expression analysis confirmed reduced pro-inflammatory markers such as iNOS, COX-2, IL-6, and IL-1ß and elevated anti-inflammatory cytokines, including IL-10. This research highlights the promising potential of fermented kale juice, enriched with Limosilactobacillus strains, as a functional food with combined antioxidant and anti-inflammatory benefits.

Strain-specific metabolomic diversity of Lactiplantibacillus plantarum under aerobic and anaerobic conditions.

The chemotaxonomic diversity of 20 Lactiplantibacillus plantarum strains was investigated using non-targeted metabolite profiling under different culture conditions. Multivariate and metabolic pathway analyses based on GC-MS and LC-MS/MS datasets showed that amino acid metabolism, especially 2-hydroxy acids, was enriched under aerobic conditions (AE), whereas fatty acid & sugar metabolism was increased under anaerobic conditions (AN). Based on the metabolite profiles, L. plantarum strains were clustered into three main groups (A, B, and C). Overall, 79 and 83 significantly discriminant metabolites were characterized as chemical markers of AE and AN growth conditions, respectively. Notably, alcohols were more abundant in group A whereas amino acids, peptides, purines, and pyrimidines were significantly higher in group C. 2-hydroxy acids and oxylipins biosynthesized through amino acid and fatty acid metabolism, respectively, were more abundant in groups A and B. Furthermore, we observed a strong correlation between the chemical diversity of L. plantarum groups and their antioxidant activity from metabolite extracts. We propose a non-targeted metabolomic workflow to comprehensively characterize the chemodiversity of L. plantarum strain under different culture conditions, which may help reveal specific biomarkers of individual strains depending on the culture conditions.

Phenotypic and genomic analyses of bacteriocin-producing probiotic Enterococcus faecium EFEL8600 isolated from Korean soy-meju.

Enterococcus faecium is a prevalent species found in fermented soybean products, known for its contributions to flavor development and inhibition of pathogenic microorganisms during fermentation. This study aims to provide comprehensive phenotypic and genomic evidence supporting the probiotic characteristics of E. faecium EFEL8600, a bacteriocin-producing strain isolated from Korean soy-meju. Phenotypic analysis revealed that EFEL8600 produced a peptide with inhibitory activity against Listeria monocytogenes, estimated to be 4.6 kDa, corresponding to the size of enterocins P or Q. Furthermore, EFEL8600 exhibited probiotic traits, such as resilience in gastrointestinal conditions, antioxidant and anti-inflammatory activities, and protection of the intestinal barrier. Safety assessments demonstrated no hemolytic and bile salt deconjugation activities. Genomic analysis revealed the presence of several genes associated with probiotic characteristics and bacteriocin production, while few deleterious genes with a low likelihood of expression or transferring were detected. Overall, this study highlights E. faecium EFEL8600 as a potent anti-listeria probiotic strain suitable for use as a starter culture in soymilk fermentation, providing potential health benefits to consumers.

A qPCR Method to Assay Endonuclease Activity of Cas9-sgRNA Ribonucleoprotein Complexes.

The CRISPR-Cas system has emerged as the most efficient genome editing technique for a wide range of cells. Delivery of the Cas9-sgRNA ribonucleoprotein complex (Cas9 RNP) has gained popularity. The objective of this study was to develop a quantitative polymerase chain reaction (qPCR)-based assay to quantify the double-strand break reaction mediated by Cas9 RNP. To accomplish this, the dextransucrase gene (dsr) from Leuconostoc citreum was selected as the target DNA. The Cas9 protein was produced using recombinant Escherichia coli BL21, and two sgRNAs were synthesized through in vitro transcription to facilitate binding with the dsr gene. Under optimized in vitro conditions, the 2.6 kb dsr DNA was specifically cleaved into 1.1 and 1.5 kb fragments by both Cas9-sgRNA365 and Cas9-sgRNA433. By monitoring changes in dsr concentration using qPCR, the endonuclease activities of the two Cas9 RNPs were measured, and their efficiencies were compared. Specifically, the specific activities of dsr365RNP and dsr433RNP were 28.74 and 34.48 (unit/µg RNP), respectively. The versatility of this method was also verified using different target genes, uracil phosphoribosyl transferase (upp) gene, of Bifidobacterium bifidum and specific sgRNAs. The assay method was also utilized to determine the impact of high electrical field on Cas9 RNP activity during an efficient electroporation process. Overall, the results demonstrated that the qPCR-based method is an effective tool for measuring the endonuclease activity of Cas9 RNP.

Integrated genome-based assessment of safety and probiotic characteristics of Lactiplantibacillus plantarum PMO 08 isolated from kimchi.

Lactiplantibacillus plantarum PMO 08 has been used as a probiotic starter culture for plant-based fermented beverages, with various health-promoting effects such as cholesterol-lowering and anti-inflammatory activities. This study aimed to analyze the genome sequence of Lp. plantarum PMO 08 and identify its safety and probiotic characteristics at the genomic level. For this, complete genome sequencing was conducted to investigate the genes associated with risk and probiotic characteristics by using Pacbio combined with Illumina HiSeq. This bacterial strain has one circular chromosome of 3,247,789 bp with 44.5% G + C content and two plasmids of 50,296 bp with 39.0% G + C content and 19,592 bp with 40.5% G + C content. Orthologous average nucleotide identity analysis showed that PMO 08 belongs to the Lp. plantarum group with 99.14% similarity to Lp. plantarum WCFS1. No deleterious genes were determined in the virulence factor analysis, and no hemolysin activity or secondary bile salt synthesis were detected in vitro test. In the case of antibiotic resistance analysis, PMO 08 was resistant to ampicillin in vitro test, but these genes were not transferable. In addition, the strain showed same carbohydrate utilization with Lp. plantarum WCFS1, except for mannopyranoside, which only our strain can metabolize. The strain also harbors a gene for inositol monophosphatase family protein related with phytate hydrolysis and have several genes for metabolizing various carbohydrate which were rich in plant environment. Furthermore, in probiotic characteristics several genes involved in phenotypes such as acid/bile tolerance, adhesion ability, and oxidative stress response were detected in genome analysis. This study demonstrates that Lp. plantarum PMO 08 harbors several probiotic-related genes (with no deleterious genes) and is a suitable probiotic starter for plant-based fermentation.

Probiotic potential of Tetragenococcus halophilus EFEL7002 isolated from Korean soy Meju.

BACKGROUND: Probiotic starters can improve the flavor profile, texture, and health-promoting properties of fermented foods. Tetragenococcus halophilus is a halophilic lactic acid bacterium that is a candidate starter for high-salt fermented foods. However, the species is known to produce biogenic amines, which are associated with neurotoxicity. Here, we report a probiotic starter strain of T. halophilus, EFEL7002, that is suitable for use in high-salt fermentation. RESULTS: EFEL7002 was isolated from Korean meju (fermented soybean) and identified as T. halophilus, with 99.85% similarity. The strain is safe for use in food as it is a non-hemolytic and non-biogenic amine producer. EFEL7002 is tolerant to gastrointestinal conditions and can adhere to Caco-2 cells. This strain showed antioxidant, anti-inflammatory, and protective effects against the human gut epithelial barrier. EFEL7002 grew well in media containing 0-18% NaCl showing maximum cell densities in 6% or 12% NaCl. CONCLUSIONS: T. halophilus EFEL7002 can be used as a health-promoting probiotic starter culture for various salty fermented foods.

Co-packaging of opposing neurotransmitters in individual synaptic vesicles in the central nervous system.

Many mammalian neurons release multiple neurotransmitters to activate diverse classes of postsynaptic ionotropic receptors. Entopeduncular nucleus somatostatin (EP Sst+) projection neurons to the lateral habenula (LHb) release both glutamate and GABA, but it is unclear whether these are packaged into the same or segregated pools of synaptic vesicles. Here, we describe a method combining electrophysiology, spatially patterned optogenetics, and computational modeling designed to analyze the mechanism of glutamate/GABA co-release in mouse brain. We find that the properties of postsynaptic currents elicited in LHb neurons by optogenetically activating EP Sst+ terminals are only consistent with co-packaging of glutamate/GABA into individual vesicles. Furthermore, presynaptic neuromodulators that weaken EP Sst+ to LHb synapses maintain the co-packaging of glutamate/GABA while reducing vesicular release probability. Our approach is applicable to the study of multi-transmitter neurons throughout the brain, and our results constrain the mechanisms of neuromodulation and synaptic integration in LHb.

Analytical approaches to examine gamma-aminobutyric acid and glutamate vesicular co-packaging.

Multi-transmitter neurons, i.e., those that release more than one type of neurotransmitter, have been found in many organisms and brain areas. Given the peculiar biology of these cells, as well as the potential for diverse effects of each of the transmitters released, new tools, and approaches are necessary to parse the mechanisms and functions of synaptic co-transmission. Recently, we and others have studied neurons that project to the lateral habenula and release both gamma-aminobutyric acid (GABA) and glutamate, in some cases by packaging both transmitters in the same synaptic vesicles. Here, we discuss the main challenges with current electrophysiological approaches to studying the mechanisms of glutamate/GABA co-release, a novel statistical analysis that can identify co-packaging of neurotransmitters versus release from separate vesicle, and the implications of glutamate/GABA co-release for synapse function and plasticity.

Dynamics and Entropy of Cyclohexane Rings Control pH-Responsive Reactivity.

Activation entropy (ΔS ‡) is not normally considered the main factor in determining the reactivity of unimolecular reactions. Here, we report that the intramolecular degradation of six-membered ring compounds is mainly determined by the ΔS ‡, which is strongly influenced by the ring-flipping motion and substituent geometry. Starting from the unique difference between the pH-dependent degradation kinetics of geometric isomers of 1,2-cyclohexanecarboxylic acid amide (1,2-CHCAA), where only the cis isomer can readily degrade under weakly acidic conditions (pH < 5.5), we found that the difference originated from the large difference in ΔS ‡ of 16.02 cal·mol-1·K-1. While cis-1,2-CHCAA maintains a preference for the classical chair cyclohexane conformation, trans-1,2-CHCAA shows dynamic interconversion between the chair and twisted boat conformations, which was supported by both MD simulations and VT-NMR analysis. Steric repulsion between the bulky 1,2-substituents of the trans isomer is one of the main reasons for the reduced energy barrier between ring conformations that facilitates dynamic ring inversion motions. Consequently, the more dynamic trans isomer exhibits much a larger loss in entropy during the activation process due to the prepositioning of the reactant than the cis isomer, and the pH-dependent degradation of the trans isomer is effectively suppressed. When the ring inversion motion is inhibited by an additional methyl substituent on the cyclohexane ring, the pH degradability can be dramatically enhanced for even the trans isomer. This study shows a unique example in which spatial arrangement and dynamic properties can strongly influence molecular reactivity in unimolecular reactions, and it will be helpful for the future design of a reactive structure depending on dynamic conformational changes.