Search Results (3,940)

N-linked glycans (N-glycans) are complex carbohydrate structures covalently attached to the asparagine residues of glycoproteins and play a substantial role in protein folding, stability and biological activity. One of the major challenges in glycomics is the enzymatic release of intact N-glycans from native glycoproteins, as conventional deglycosylation approaches often require prior protein denaturation and exhibit limited activity toward structurally diverse substrates. In this study, we recombinantly produced EndoBI-1, an endo-β-N-acetylglucosaminidase derived from Bifidobacterium longum subsp. infantis ATCC 15697, and investigated its N-glycan release activity on two structurally distinct glycoprotein substrates: bovine lactoferrin (bLF), an animal-derived glycoprotein, and soy protein, a plant-derived glycoprotein. EndoBI-1 was produced using an in vivo cloning approach and purified to homogeneity, as validated by SDS-PAGE analysis. The released N-glycan profiles of each substrate were characterized by HILIC-FLD-QTOF-MS/MS analysis, which showed different glycan compositions depending on the origin: complex-type and sialylated N-glycans were the major structures in the bLF derived fractions, while the fractions derived from soy protein mainly contained high-mannose-type structures. Together, these results demonstrate the capacity of EndoBI-1 to release structurally intact N-glycans from both animal- and plant-derived glycoproteins under native, non-denaturing conditions, establishing this enzyme as a biocatalytic tool for the preparative-scale production of bioactive N-glycans from diverse protein sources. Full article

Poly[(R)-3-hydroxybutyrate] [P(3HB)] is a biological polyester synthesized by microorganisms and can be used as a biodegradable plastic. The higher the molecular weight of P(3HB), the stronger it can be processed into films and fibers. Therefore, high-molecular-weight P(3HB) can be used as high-performance plastics for engineering and specialty applications. The P(3HB) biosynthetic operon consists of three genes, canonically arranged as phaC-phaA-phaB (phaCAB). This study specifically rearranges the phaCAB operon to the phaBCA order to investigate P(3HB) production and its molecular weight from glucose and/or xylose, which can be derived from wood hydrolysates, using Escherichia coli XL1-Blue as a host. By rearranging the gene order to phaBCA, the expression balance of PHA synthase (PhaC, encoded by phaC) and 3HB monomer-supplying enzymes (PhaA and PhaB, encoded by phaA and phaB, respectively) changed, enabling the host to produce P(3HB) with a higher molecular weight than that with the canonical operon. This effect was significant when xylose was used as the carbon source or when a nutritionally rich medium was used. However, the amount of P(3HB) produced by the phaCAB-expressing strain was consistently better than that by the phaBCA-expressing strain. This study establishes that high-molecular-weight P(3HB) can be produced through an integrated approach of operon engineering and wood-derived sugar bioprocessing, offering a sustainable production route for high-performance biodegradable plastics from non-food biomass. Full article

The dramatic rise in global aquaculture has brought renewed focus on filamentous fungi impacting fish health and aquafeeds. The fungal genus Aspergillus has a Janus-like role in aquaculture. On the pathogenic side, opportunistic pathogens like A. flavus, A. niger and A. fumigatus cause outbreaks of aspergillosis, with granulomatous lesions and significant mortality, especially in fish with impaired immunity and/or poor environmental conditions. Toxigenic strains of A. flavus and A. parasiticus also contaminate aquafeeds with aflatoxins, some of the most toxic natural hepatotoxins and carcinogens, which have been shown to affect growth, immunity and aflatoxin residues in edible fish fillets. On the positive side, non-toxigenic strains of A. niger, A. oryzae and A. awamori are increasingly used as fungal probiotics, solid-state fermenters to upgrade plant protein feed supplements, and as sources of industrially useful hydrolytic enzymes, secondary metabolites and antifungal compounds. Synthesizing evidence from over 90 peer-reviewed studies, this narrative review elucidates how species, strain, and rearing context jointly determine whether Aspergillus behaves as a pathogen or as a functional additive. We highlight knowledge gaps, offer an interpretative model and suggest practical strategies to limit risks and leverage the biotechnological uses of Aspergillus for sustainable aquaculture. The novelty of this review lies in integrating, within a single finfish-focused framework, the three usually separate faces of Aspergillus—pathogen, aflatoxin producer, and functional feed additive—and in translating this dual nature into practical, strain-level guidance for aquaculture. Full article

Cow’s milk allergy (CMA) is the most common food allergy in infants. Goat milk exhibits relatively low allergenicity and is widely regarded as a potential substitute for cow milk. However, the high sequence homology between cow and goat milk proteins may trigger cross-reactivity, significantly restricting the practical application of goat milk. This study aims to disrupt cross-linear epitopes of cow and goat milk proteins through enzymatic hydrolysis. We prepare hypoallergenic goat milk protein hydrolysates and systematically evaluate their desensitization effects. Bioinformatics methods were employed to predict the B-cell linear epitopes of six major allergens (α_S1-casein, α_S2-casein, β-casein, κ-casein, α-lactalbumin, and β-lactoglobulin) from cow, goat, and sheep milk, and sequence homology was analyzed through protein-protein Basic Local Alignment Search Tool (BLASTP). The results showed that the sequence similarity of homologous allergens among the three milk sources all exceeded 30%. Subsequently, six proteases (protamex, alcalase, pepsin, trypsin, papain and bromelain) were used to hydrolyze whole goat milk protein. Indirect enzyme-linked immunosorbent assay (ELISA) results indicated that all six hydrolysates significantly reduced immunoreactivity with antibodies against the five major cow’s milk allergens. Among them, alcalase exhibited significant efficacy against all five allergens; papain and protamex were particularly effective against β-casein; trypsin showed pronounced efficacy against α-lactalbumin and β-lactoglobulin; and bromelain and pepsin also significantly reduced immunoreactivity against certain allergens. Mass spectrometry revealed the peptide composition and epitope coverage of the hydrolysates: bromelain yielded two overlapping peptides (FAWPQY, LKDLKDY), protamex one (LAMAAS), Alcalase three (PPQSVLS, IPIQYVLS, LPYPYY), trypsin two (YLGYLEQL, AMAASDISLL), papain three (NEINQFYQK, FQSEEQQQTEDELQDK, AMAASDISL); and no matching peptide was detected for pepsin. These results confirmed at the molecular level that enzymatic hydrolysis can effectively disrupt cross-reactive epitopes. Full article

Marine-associated polar fungi are promising sources of structurally diverse natural products. To investigate polyketide metabolites from the Antarctic moss-derived fungus Penicillium sp. OUCMDZ-4014, HSQC NMR combined with DeepSAT analysis was used to prioritize fractions enriched in cyclic and aromatic polyketides, and chromane-like metabolites. Chromatographic separation yielded ten resorcylic acid lactone-derived aromatic polyketides, including RAL macrolides, ring-opened esters, and isocoumarins, among which compounds 4–7 were new. Their planar structures were established by HRESIMS and 1D/2D NMR analyses. Their chemical structures including configurations were established by HRESIMS, 1D/2D NMR, ECD calculations, 13C NMR calculations, and DP4+ analysis. Isocoumarins 7–10 displayed diverse levels of α-glucosidase inhibition, with compound 9 being the most active (IC₅₀ = 47.0 ± 3.83 μM). Kinetic analysis indicated noncompetitive inhibition by 9 and mixed-type inhibition by 10. Genome mining revealed a putative res biosynthetic gene cluster containing HR-PKS, NR-PKS, and tailoring-enzyme genes. Compound 1 underwent nonenzymatic conversion into 9 under culture-medium conditions, while trace conversion was also observed during concentration, revealing a chemical link between the RAL macrolide and isocoumarin scaffolds, and highlighting the contribution of post-biosynthetic chemical transformation to fungal polyketide diversification. Full article

Seventy-five thermophilic fungi capable of growing at 45 °C were isolated from sugarcane bagasse samples and cultivated under solid-state fermentation (SSF). Two isolates showing the highest xylanase and cellulase production were selected and identified as Aspergillus fumigatus 5S2 and Aspergillus sp. 1.6C. Water hyacinth (WH) and sugarcane bagasse (SCB) were evaluated as lignocellulosic supports and carbon sources for enzyme production under SSF. Eight culture conditions were evaluated to identify the most suitable condition for enzyme production. Results revealed that substrate recalcitrance was a major limiting factor for enzyme production and that thermochemical pretreatment significantly improved substrate utilization. The highest enzyme activities were achieved on pretreated WH (PWH) supplemented with NaNO₃ and mineral salts. A. fumigatus 5S2 reached 810 U/g dry matter (DM) for xylanase and 95 U/g DM for cellulase, whereas Aspergillus sp. 1.6C reached 649 U/g DM of xylanase and 33 U/g DM of cellulase under the corresponding optimal culture conditions. PWH was mixed with SCB to improve the physical properties of the SSF support, including aeration, porosity, and water retention. The highest enzyme production levels were obtained using 1:1 and 1:0.5 (w/w) mixtures of PWH and SCB for A. fumigatus 5S2 and Aspergillus sp. 1.6C, respectively. SSF column bioreactors packed with these mixtures produced similar enzyme titers at 30 °C and 40 °C, confirming the thermotolerant nature of both strains. Full article

The search for sustainable alternatives to fish oil (FO) in aquafeeds is imperative for the long-term sustainability of aquaculture. This study evaluated the effects of partially replacing FO with oils derived from Hermetia illucens (RH) and Musca domestica (RY) larvae at 10%, 30%, and 50% levels on growth, lipid metabolism, hepatic antioxidant capacity, and gut microbiota in juvenile Cyprinus carpio. Results showed distinct source- and dose-dependent responses. RY produced a consistent dose-dependent trend in growth parameters, whereas RH exhibited a biphasic pattern, with growth impairment at 30% replacement and recovery at 50%. Both oils significantly suppressed hepatic fatty acid synthase (FAS) activity, yet this suppression was accompanied by dose-dependent up-regulation of fas expression, particularly at high RH and RY levels, indicating complex post-transcriptional regulation. RH at higher replacement levels reduced digestive enzyme activities and antioxidant capacity, while higher RY caused minimal disruption. Both oils reshaped gut microbiota toward oil-specific community structures. Two-way ANOVA revealed significant synergistic interactions between oil source and replacement level for most serum biochemical and hepatic antioxidant parameters (ηp² > 0.90). These findings demonstrate that RY is a tolerable alternative across a wide replacement range, though all RY groups exhibited lower growth than the control, 30% is identified as the maximum tolerable level; whereas RH requires precise dosing at 10% to avoid the 30% impairment threshold. Full article

Agricultural and horticultural by-products represent an underexploited source of valuable bioactive compounds that can contribute to the development of more sustainable production systems. This study investigated the chemical composition and biological activities of the essential oil obtained from the horticultural byproducts of Artemisia dracunculus. The waste aerial biomass was subjected to steam distillation, and the essential oil was characterized by gas chromatography–mass spectrometry. Its antioxidant, α-amylase and α-glucosidase inhibitory, phytotoxic, antibacterial, and antibiofilm activities were subsequently evaluated. The essential oil was characterized as an estragole-rich chemotype (70.17%), with trans-β-ocimene and cis-β-ocimene as other main constituents. The essential oil showed moderate antioxidant activity and measurable enzyme inhibitory activity and, despite showing limited effects on seed germination, it significantly inhibited radical elongation in selected plant species. Furthermore, it demonstrated excellent antibiofilm activity, significantly reducing the metabolic activity of mature cells of bacteria associated with biofilm formation: Listeria monocytogenes, Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae and Staphylococcus aureus. The results demonstrate how horticultural by-products from A. dracunculus maintain a chemical profile comparable to conventional plant material and represent a valuable source of bioactive compounds with promising potential for sustainable agri-food applications. Full article

Background/Objectives: Dittrichia viscosa (L.) W. Greuter (Asteraceae) is a Mediterranean medicinal plant whose polar phenolic fraction remains insufficiently characterized. This study aimed to characterize the phenolic composition and evaluate the antioxidant, antimicrobial, and mechanistic bioactivity of aqueous and methanolic leaf extracts collected from the Rmilate Forest in Tangier, northern Morocco. Methods: Extracts were characterized by ICP-AES for elemental content and by HPLC-PDA-ESI-MS for phenolic annotation, and total phenolic, flavonoid, and tannin contents were quantified. Antioxidant capacity was assessed using DPPH, ABTS, FRAP, and ORAC assays. Antibacterial activity was evaluated against four reference strains, with time–kill kinetics and membrane integrity assays used to probe the mechanism of action, and antifungal activity was tested against dermatophytic and phytopathogenic fungi. Molecular docking against four bacterial, fungal, and antioxidant enzyme targets and ProTox-3 toxicity prediction were also performed. Results: ICP-AES revealed high concentrations of calcium (12,476.75 mg/kg) and potassium (29,699 mg/kg). The methanolic extract showed higher total phenolic (55.05 mg GAE/g), flavonoid (84.18 mg QE/g), and tannin (262.1 mg TAE/g) contents and superior antioxidant activity (DPPH IC₅₀: 0.090 mg/mL). Twelve phenolic compounds were tentatively annotated, including caffeic acid, caffeoylquinic acid derivatives, and quercetin/kaempferol glycosides. Antibacterial activity (MIC: 0.25–1 mg/mL) was consistent with cell envelope disruption, and up to 88% mycelial inhibition was achieved against Trichophyton rubrum. Docking ranked kaempferol-3-O-pentoside (−10.3 kcal/mol) among the top-scoring ligands, and the top compounds were assigned to toxicity class 5. Conclusions:D. viscosa is a promising source of bioactive phenolics for pharmaceutical and agrochemical development. Full article

This critical perspective review first classifies glyphosate-sensing platforms and then evaluates their analytical applicability to agricultural runoff. Platforms are divided at the primary level into optical and electrochemical transduction, because these families measure different physical signals and have different sources of matrix interference. They are then grouped by the process that produces selectivity or signal change: direct interaction or metal coordination, affinity recognition by aptamers, antibodies, or molecularly imprinted polymers, catalytic modulation by enzymes or nanozymes, and separation or preconcentration before detection. This hierarchy distinguishes recognition chemistry from transduction method and device configuration. The review next defines four intended analytical applications—trace surveillance, runoff event screening, spill triage, and laboratory-adjacent confirmation—and compares them in terms of matrix, target concentration range, sample preparation, reporting metrics, and quality control requirements. Glyphosate occurs in dissolved and particle-associated forms, degrades mainly to AMPA, and coexists with phosphate, glufosinate, divalent cations, natural organic matter, and suspended sediment. Consequently, the lowest reported LOD is rarely the sole criterion for selecting a method. Matrix-matched calibration, spike recovery, selectivity, response time, storage stability, reader requirements, and invalid result rules determine whether an assay is suitable for a specified analytical application. The most defensible near-term approach combines matrix-specific sample preparation, platform-specific controls, and LC-MS/MS confirmation when results are regulatory, contested, or close to a decision threshold. Full article

Background/Objectives: Flavonoids are a diverse group of dietary polyphenolic compounds associated with a range of important biological activities. However, their low systemic bioavailability and extensive metabolism raise questions about how they exert their purported benefits. Methods: A comprehensive survey of literature published over the past 10 years was conducted, focusing on human, animal, and in vitro studies addressing the metabolic fate of major flavonoid subclasses. Results: Original research articles (n = 159) were selected following predetermined inclusion criteria. Data from diverse experimental models—including intestinal and hepatic systems—demonstrate that flavonoids undergo substantial transformation by Phase I and Phase II enzymes, alongside extensive biotransformation by the gut microbiota. In this critical narrative review we summarize dietary sources, bioavailability patterns, metabolic pathways, and analytical strategies, emphasizing advances enabled by high-resolution mass spectrometry. Recent findings indicate that enterohepatic recirculation and the biological activities of circulating and tissue-associated metabolites contribute significantly to the observed health effects, despite limited levels of parent compounds. Conclusions: Flavonoids exhibit limited oral bioavailability and undergo complex metabolic processing, yet growing evidence indicates that their metabolites play key roles in mediating beneficial effects. Understanding these metabolic pathways is essential for interpreting epidemiological associations and designing more effective intervention studies. Full article

Eruca vesicaria subsp. sativa (rocket) sprouts are a rich source of bioactive compounds used in cosmetics. Phytochemical profiling (LC-PDA-MS/MS) of 5-day-old sprouts identified 26 metabolites, mainly glucosinolates (e.g., glucoraphanin and glucoerucin) and flavonol glycosides of quercetin and kaempferol. The extracts exhibited strong antioxidant activity in DPPH^•, ABTS^•+, and ferric-reducing assays and moderate, dose-dependent inhibition of skin-degrading enzymes: tyrosinase, elastase, and collagenase, suggesting anti-wrinkle and skin-brightening effects. Cytotoxicity assays on human BJ skin fibroblasts demonstrated that the rocket sprout extracts were non-cytotoxic at concentrations up to 125 µg/mL, with low-dose treatments enhancing the cell viability. Hemocompatibility tests confirmed safety, with <2% hemolysis observed. The incorporation of these compounds into model emulsions yielded stable formulations over 90 days. These findings support the novelty of rocket sprout extracts as functional/nutricosmetic ingredients with relevance to both food and skin health. Full article

Dietary protein is essential for poultry performance, influencing egg production, growth, immunity, and physiological functions. However, the high cost and environmental impact of soybean meal has encouraged the development of reduced crude protein (CP) diets supplemented with crystalline amino acids (AAs), coupled with the reduced cost and improved availability of synthetic AA. This strategy enables precise AA balancing, improves nitrogen utilization, and reduces nitrogen excretion and ammonia emissions. Among these strategies, supplementation with arginine (Arg), an indispensable AA in chickens, is particularly important due to its critical roles in protein synthesis, reproductive function, and eggshell formation. In addition, functional Arg precursors, such as guanidinoacetic acid (GAA) and citrulline (Cit), can further enhance Arg availability while supporting energy metabolism and hormonal regulation. Despite these benefits, excessive protein reduction without adequate AA balance can impair laying performance, particularly in aged hens. Furthermore, nutrient digestibility remains a key limitation in reduced protein (RP) diets, as non-starch polysaccharides present in cereal-based feeds can reduce nutrient availability. The inclusion of exogenous enzymes, such as xylanase and β-glucanase, can improve digestibility, while phytase supplementation further enhances nutrient utilization by increasing phosphorus availability and mitigating the anti-nutritional effects of phytate; however, responses are often inconsistent. Another critical factor is maintaining an optimal energy-to-protein ratio, as imbalances may lead to increased fat deposition and reduced productive performance. While supplementation of key limiting AAs (lysine, methionine, and threonine) supports protein reduction, other AAs, such as Arg, isoleucine, and valine, may become limiting in RP diets. Hence, this review explores developments toward RP diets by highlighting three key strategies: enzyme supplementation to enhance nutrient digestibility, inclusion of functional Arg sources to improve performance and bone quality, and optimization of the energy–protein ratio in RP diets through precise AA formulation, with the overall aim to improve the sustainability of the poultry industry. Full article

The gut microbiota actively metabolizes steroid hormones, but the mechanisms of these transformations—particularly the sequence of enzymatic reactions and the source of hydrogen atoms—remain poorly understood. Conventional analytical approaches are hampered by the complex fecal matrix, isomeric metabolites, and the lack of authentic reference standards. Here we present a strategy based on parallel incubation of steroids with pooled human gut microbiota in H₂O and D₂O, followed by HPLC-HRMS, with the aim of determining the sequence of reductive steps and tracking the incorporation of atoms into steroid metabolites. Using a pooled fecal inoculum from multiple donors (n = 18) and three steroid substrates, we demonstrate that the characteristic mass shift (1.0063 Da per deuterium atom) enables detection of metabolites and distinguishes multi-step enzymatic reactions in the microbial community without recombinant enzymes and authentic standards. Progesterone and 19-hydroxy-4-androstene-3,17-dione underwent sequential two-step reduction incorporating up to three deuterium atoms, while 17α-hydroxypregnenolone followed a three-step pathway incorporating up to four deuterium atoms, consistent with localization to the A-ring. This workflow provides a practical tool for investigating gut microbial steroid metabolism in the context of human physiology and disease, with potential applications in monitoring microbial activity in endocrine disorders, inflammatory bowel disease, and neuropsychiatric conditions. Full article

The skin barrier is essential for maintaining epidermal homeostasis and preventing transepidermal water loss (TEWL). This study investigated the ability of a Hyssopus officinalis cell culture hydroethanolic extract to support epidermal barrier function by modulating ceramide-related enzymes and structural proteins. Hydroethanolic extracts from cell cultures grown under light and dark conditions were characterized by high-resolution mass spectrometry. Dark-grown cultures accumulated higher levels of rosmarinic acid, hydroxytyrosol glycosides, and polymethoxyflavones than light-grown cultures and intact plants. In keratinocytes, the extract from dark-grown cells (HoEx) at 0.002% w/v exhibited significant antioxidant activity (20% ROS reduction) and increased β-glucocerebrosidase (GBA) activity (25%) as well as the expression of β-glucocerebrosidase (44%), serine palmitoyltransferase (34%), involucrin (17%), and filaggrin (44%) compared with control. In human skin explants, 0.5% w/w HoEx increased ceramide content (36%) and total epidermal lipid levels (99%) relative to control. In a clinical study involving 10 volunteers with mechanically stressed skin, 0.5% w/w HoEx increased GBA activity by 30% and reduced TEWL by 15%, whereas placebo treatment led to a 55% decrease in GBA activity and a 25% increase in TEWL. Collectively, these findings indicate that HoEx modulates multiple epidermal pathways involved in lipid processing and differentiation under in vitro, ex vivo, and stressed in vivo conditions. While the study was not designed to establish mechanistic causality, the coordinated modulation of ceramide-related enzymes, structural proteins, and TEWL suggests that HoEx may support epidermal barrier function within the limits of the tested models. HoEx therefore represents a standardized source of bioactive compounds with potential relevance for dermocosmetic applications. Full article