Antibiotic Resistance in Acetic Acid Bacteria Originating from Vinegar.

Acetic acid bacteria (AAB) are major contributors to the production of fermented vinegar, offering various cultural, culinary, and health benefits. Although the residual unpasteurized AAB after vinegar production are not pathogens, these are necessary and require safety evaluations, including antibiotic resistance, before use as a starter. In this research, we investigated the antibiotic resistance profiles of 26 AAB strains, including various species of Komagataeibacter and Acetobacter, against 10 different antibiotics using the E-test method. All strains exhibited resistance to aztreonam and clindamycin. Komagataeibacter species demonstrated a 50% resistance rate to ciprofloxacin, analogous to Acetobacter species, but showed twice the resistance rates to chloramphenicol and erythromycin. Genomic analysis of K. saccharivorans CV1 identified intrinsic resistance mechanisms, such as multidrug efflux pumps, thereby enhancing our understanding of antibiotic resistance in acetic acid-producing bacteria. These findings enhance understanding of antibiotic resistance in AAB for food safety and new antimicrobial strategies, suggesting the need for standardized testing methods and molecular genetic study.

Health Benefits of Antioxidant Bioactive Compounds in Ginger (Zingiber officinale) Leaves by Network Pharmacology Analysis Combined with Experimental Validation.

Network pharmacology is an ideal tool to explore the effects of therapeutic components derived from plants on human metabolic diseases that are linked to inflammation. This study investigated the antioxidant effects of ginger leaves (GLs) and predicted targets for antioxidant activity. Quantitative and free radical scavenging analyses were performed to detect the main bioactive compounds of GLs and evaluate their antioxidant activities. Chemical diversity and network pharmacology approaches were used to predict key antioxidant components of GLs and their molecular targets. Nine major bioactive compounds of GLs were quantified using an internal standard method, and the antioxidant activity was evaluated using the DPPH and ABTS free radical scavenging methods. We first built the compound-gene-pathways and protein-protein interaction networks of GLs-related antioxidant targets and then conducted gene ontology and Kyoto Encyclopedia of Gene and Genome (KEGG) pathway enrichment analyses. Molecular docking results show that astragalin, a compound isolated from GLs, had the highest level of connectivity in the compound-target network and was involved in inflammation-related biosynthesis by directly impacting cytokine gene expression and PTGS2 inhibition markers. These findings not only suggest that the compounds isolated from GLs can be developed as potential antioxidants, but also demonstrate the applicability of network pharmacology to assess the potential of foods for disease treatment.

Mechanistic manifold in a hemoprotein-catalyzed cyclopropanation reaction with diazoketone.

Hemoproteins have recently emerged as promising biocatalysts for new-to-nature carbene transfer reactions. However, mechanistic understanding of the interplay between productive and unproductive pathways in these processes is limited. Using spectroscopic, structural, and computational methods, we investigate the mechanism of a myoglobin-catalyzed cyclopropanation reaction with diazoketones. These studies shed light on the nature and kinetics of key catalytic steps in this reaction, including the formation of an early heme-bound diazo complex intermediate, the rate-determining nature of carbene formation, and the cyclopropanation mechanism. Our analyses further reveal the existence of a complex mechanistic manifold for this reaction that includes a competing pathway resulting in the formation of an N-bound carbene adduct of the heme cofactor, which was isolated and characterized by X-ray crystallography, UV-Vis, and Mössbauer spectroscopy. This species can regenerate the active biocatalyst, constituting a non-productive, yet non-destructive detour from the main catalytic cycle. These findings offer a valuable framework for both mechanistic analysis and design of hemoprotein-catalyzed carbene transfer reactions.

The Cactus (Opuntia ficus-indica) Cladodes and Callus Extracts: A Study Combined with LC-MS Metabolic Profiling, In-Silico, and In-Vitro Analyses.

Opuntia ficus-indica (OF) phytochemicals have received considerable attention because of their health benefits. However, the structure-activity relationship between saponin and flavonoid antioxidant compounds among secondary metabolites has rarely been reported. In a molecular docking study, selected compounds from both Opuntia ficus-indica callus (OFC) and OF ethanol extract were found to be involved in Toll-like receptor 4 and mitogen-activated protein kinase (MAPK) signaling pathways. High affinity was specific for MAPK, and it was proposed to inhibit the oxidative and inflammatory responses with poricoic acid H (-8.3 Kcal/mol) and rutin (-9.0 Kcal/mol). The pro-inflammatory cytokine factors at a concentration of 200 µg/mL were LPS-stimulated TNF-α (OFC 72.33 ng/mL, OF 66.78 ng/mL) and IL-1ß (OFC 49.10 pg/mL, OF 34.45 pg/mL), both of which significantly decreased OF (p < 0.01, p < 0.001). Taken together, increased NO, PGE2, and pro-inflammatory cytokines were significantly decreased in a dose-dependent manner in cells pretreated with OFC and the OF extract (p < 0.05). These findings suggest that OFC and OF have important potential as natural antioxidant, anti-inflammatory agents in health-promoting foods and medicine.

Enantioselective Synthesis of α-Trifluoromethyl Amines via Biocatalytic N-H Bond Insertion with Acceptor-Acceptor Carbene Donors.

The biocatalytic toolbox has recently been expanded to include enzyme-catalyzed carbene transfer reactions not occurring in Nature. Herein, we report the development of a biocatalytic strategy for the synthesis of enantioenriched α-trifluoromethyl amines through an asymmetric N-H carbene insertion reaction catalyzed by engineered variants of cytochrome c552 from Hydrogenobacter thermophilus. Using a combination of protein and substrate engineering, this metalloprotein scaffold was redesigned to enable the synthesis of chiral α-trifluoromethyl amino esters with up to >99% yield and 95:5 er using benzyl 2-diazotrifluoropropanoate as the carbene donor. When the diazo reagent was varied, the enantioselectivity of the enzyme could be inverted to produce the opposite enantiomers of these products with up to 99.5:0.5 er. This methodology is applicable to a broad range of aryl amine substrates, and it can be leveraged to obtain chemoenzymatic access to enantioenriched ß-trifluoromethyl-ß-amino alcohols and halides. Computational analyses provide insights into the interplay of protein- and reagent-mediated control on the enantioselectivity of this reaction. This work introduces the first example of a biocatalytic N-H carbenoid insertion with an acceptor-acceptor carbene donor, and it offers a biocatalytic solution for the enantioselective synthesis of α-trifluoromethylated amines as valuable synthons for medicinal chemistry and the synthesis of bioactive molecules.

Effects of High-Pressure, Hydrothermal, and Enzyme-Assisted Treatment on the Taste and Flavor Profile of Water-Soluble Ginger (Zingiber officinale) Extract.

Ginger, a plant widely consumed worldwide, is used as a spice or to enhance the flavor of foods. In this study, the taste characteristics (gingerol, shogaol, and amino acid) of extracts treated with various solubilizing methods were objectively compared. In addition, an E-nose confirmed the flavor pattern combined with principal component analysis (PCA) between each extract gas chromatogram-tandem mass spectrometry was performed to compare and analyze volatile compounds between extraction methods. As a result, high-pressure enzyme-assisted extraction (HPE) and hydrothermal enzyme-assisted extraction (HWE) treatment effectively improved the extraction yield of ginger and the contents of gingerol and shogaol and removed the bitter taste. In addition, radar charts of both E-nose and PCA provided the distribution of flavor substances in HPE and HWE products of ginger. After enzyme-assisted treatment, a strong fruity and piquant flavor was noted. In conclusion, it is suggested that ginger extract of enzyme-assisted treatment has increased flavor compounds and can be an excellent food material.

Optimization of pectinase-assisted extraction condition of mulberry (Morus alba L.) fruit using response surface methodology and its effect on anthocyanin synthesis pathway-related metabolites.

Mulberry (Morus alba L.) fruit (MF) is a rich source of functional compounds, such as anthocyanin. However, during solvent extraction, these compounds are not fully dispersed into the substrate, leading to incomplete extraction. Moreover, raw MF rapidly ripens and deteriorates after harvesting; hence, innovative methods to process MF are needed. Here, a pectinase-assisted extraction method is developed to liberate polyphenols and anthocyanins from cell wall matrices in MF. We optimized the procedure to maximize water solubility index (WSI), total phenolic (TP) content, and total anthocyanin (TA) content using a central composite design to perform a response surface methodology (RSM) analysis. The optimal conditions predicted by the RSM were a 1:5 w/v material/water ratio with 3.5% pectinase (v/w) and 1.5% citric acid (w/w) for 113 min at 50°C. Under these conditions, the WSI, TP, and TA were significantly higher compared with those in the untreated control. The results well matched (within 5% differences) with the predicted RSM values. Furthermore, metabolite analysis revealed that the levels of cyanidin-3-O-glucoside, delphinidin hexoside, and quercetin were higher in pectinase-assisted MF extraction compared with the untreated control. This work demonstrated that pectinase-assisted extraction using citric acid could be an efficient technique to enhance the value of MF and its potential applications in the food industry. PRACTICAL APPLICATION: A pectinase-assisted extraction method was optimized to enhance the WSI, TP, and TA yields from MF extracts. The optimal conditions were predicted to be 1:5 w/v material/water ratio, 3.5% pectinase (v/w), and 1.5% CA (w/w) with a 113 min reaction time at 50°C. Under these conditions, WSI, TP, and TA were significantly increased compared with the untreated control. These results suggested the potential of mulberry plants for use in the food industry via the development of a simple, efficient process to extract functional compounds from MF.

A Diverse Library of Chiral Cyclopropane Scaffolds via Chemoenzymatic Assembly and Diversification of Cyclopropyl Ketones.

Chiral cyclopropane rings are key pharmacophores in pharmaceuticals and bioactive natural products, making libraries of these building blocks a valuable resource for drug discovery and development campaigns. Here, we report the development of a chemoenzymatic strategy for the stereoselective assembly and structural diversification of cyclopropyl ketones, a highly versatile yet underexploited class of functionalized cyclopropanes. An engineered variant of sperm whale myoglobin is shown to enable the highly diastereo- and enantioselective construction of these molecules via olefin cyclopropanation in the presence of a diazoketone carbene donor reagent. This biocatalyst offers a remarkably broad substrate scope, catalyzing this reaction with high stereoselectivity across a variety of vinylarene substrates as well as a range of different α-aryl and α-alkyl diazoketone derivatives. Chemical transformation of these enzymatic products enables further diversification of these molecules to yield a collection of structurally diverse cyclopropane-containing scaffolds in enantiopure form, including core motifs found in drugs and natural products as well as novel structures. This work illustrates the power of combining abiological biocatalysis with chemoenzymatic synthesis for generating collections of optically active scaffolds of high value for medicinal chemistry and drug discovery.

Response Surface Methodology for Optimization of Process Parameters and Antioxidant Properties of Mulberry (Morus alba L.) Leaves by Extrusion.

Mulberry (Morus alba L.) leaves (MLs), originally used to feed silkworms, have recently been recognized as a food ingredient containing health-beneficial, bioactive compounds. In this study, the extrusion process was applied for the enhancement of the amount of extractable flavonoids from MLs. Extrusion conditions were optimized by water solubility index, total phenolic content, and total flavonoid content (TF) using response surface methodology, and antioxidative stress activities were evaluated in macrophage cells. According to the significance of regression coefficients of TF, the optimal extrusion parameters were set as barrel temperature of 114 °C, moisture feed content of 20%, and screw speed of 232 rpm. Under these conditions, the TF of extruded ML reached to 0.91% and improved by 63% compared with raw ML. Fifteen flavonoids were analyzed using ultra-high-performance liquid chromatograph coupled with photodiode array detection and quadrupole time-of-flight mass spectrometry (UPLC-PDA-QTOF/MS), and the extrusion resulted in increases in quercetin-3-gentiobioside, quercetin-3,7-di-O-glucoside, kaempferol-3,7-di-O-glucoside, rutin, isoquercitrin, and moragrol C. Besides, regarding antioxidative activity, extruded ML water extract inhibited the production of H2O2-induced reactive oxygen species and attenuated nuclear morphology alterations in macrophage cells. The findings of this study should be useful in food processing design to improve the extractable functional compounds in MLs.

Optimal conditions for the encapsulation of Weissella cibaria JW15 using alginate and chicory root and evaluation of capsule stability in a simulated gastrointestinal system.

The delivery of active probiotic cells in capsules can reduce probiotic cell loss induced by detrimental external factors during digestion. In this study, we determined the optimal conditions for the encapsulation of Weissella cibaria JW15 (JW15) within calcium and polyethylene glycol (PEG)-alginate with chicory root extract powder (CREP). JW15 was encapsulated as the core material (109 cells/mL, 2 mL/min), and a solution containing a mixture of 1.5% sodium alginate and 1% CREP was extruded into a receiving bath with 0.1 M calcium chloride (CaCl2 ) and 0.05% PEG. Capsule morphology and size were measured using optical microscopy. The optimal air pressure and frequency vibration for capsules containing alginate only (Al) were 200 mbar and 200 Hz, respectively and 100 mbar and 350 Hz for capsules containing alginate with CREP (Ch), respectively. The voltage for both capsules types was fixed at 1.35 kV. Then, the capsules were incubated in a simulated gastrointestinal (GI) system for 6 hr at 37 °C. The addition of PEG in a CaCl2 hardening solution led to degradation of the Ch capsule (Ch-PEG) and the release of cells into the small intestine vessel in the simulated GI system. By contrast, the cells were trapped within the Al capsules. Based on these data, effective encapsulation using alginate with CREP and PEG can enable JW15 to be released at a targeted anatomical site of activity within the GI system, thereby, enhancing the efficacy of probiotic cells. These protective effects can be leveraged during the development of probiotic products. PRACTICAL APPLICATION: Weissella cibaria JW15 (109 cells/mL) was encapsulated in biodegradable and biocompatible capsules, prepared by mixing 1.5% alginate with 1% chicory root extract powder (CREP) in 0.1 M CaCl2 and 0.05% PEG using an encapsulator. The optimal processing parameters were as follows: pressure, 100 mbar; vibration frequency, 350 Hz; voltage, 1.35 kV; and core flow rate, 2 mL/min. When the resulting capsules were subjected to a simulated gastrointestinal system for 6 hr, the cells were released into the small intestine, and up to 95% cell viability was preserved. These results suggest that capsules made from alginate with CREP and formulated using calcium and PEG are a promising delivery system for probiotic cells.

Enhancement of viability, acid, and bile tolerance and accelerated stability in lyophilized Weissella cibaria JW15 with protective agents.

Dietary supplementation with lactic acid bacteria to maintain or improve intestinal health is advocated. Weissella spp. are present in different fermented vegetable-based foods like kimchi, as well as in the normal gastrointestinal (GI) tract of humans. Weissella cibaria strains have been proposed as potential probiotics. Freeze-drying is a promising treatment method for these strains for industrial applications and to increase the accessibility of their health-promoting benefits. Moreover, probiotic strains need to be able to survive in the host GI tract, and acid and bile are both environmental stressors that can reduce strain survival. Therefore, this study evaluated the effect of the combination of protective agents on the acid and bile resistance of W. cibaria JW15 after freeze-drying. A protective agent combination with a 1:1 ratio of 5 g + 5 g/100 ml w/v soy flour + yeast extract (SFY) retained nearly 100% viability after freeze-drying and was resistant to artificial bile acids. Remarkably, skim milk + soy flour (SSF) was resistant to an acidic solution, and the viability of W. cibaria JW15 in artificial gastric acid was enhanced when treated with this mixture. Furthermore, SFY and SSF were found to maintain high numbers of viable cells with a low specific rate of cell death (k) after storage at 50°C, 60°C, and 70°C. These results support an effective probiotic formulation system with a high number of viable cells, and its protective effects can be leveraged in the development of probiotic products with health benefits.