The flowering process has been reported to play crucial roles in improving the flavor and efficacy of fermented tea. Hippophae rhamnoides leaves containing many beneficial ingredients are a suitable plant source for tea processing. In this study, we isolated a β-glucosidase-producing fungus Eurotium amstelodami BSX001 from the fermented tea and used Hippophae rhamnoides leaves (HRL) as a substrate to explore the detailed process of bioconversion of some important functional factors. The results show that the contents of total phenolic compounds and flavonoids increased significantly after seven days, especially flavonoid aglycones (e.g., quercetin, kaempferol, and isorhamnetin). Such compounds greatly enhance the antioxidative activity of fermented products. Metabolic analysis of the standard compounds (rutin, quercetin-3-glucoside, kaempferol-3-glucoside, quercetin, isorhamnetin-3-glucoside, isorhamnetin, and kaempferol) further confirmed the effective biotransformation by E. amstelodami. Mechanisms of the bioconversion could be involved in deglycosylation, dihydroxylation, and O-methylation. Our findings expand the understanding of tea fermentation process and provide further guidance for the fermented tea industry.

Deep eutectic solvents (DES), as a green alternative to traditional organic solvents in biocatalysis, not only activate proteins but even increase the efficiency of enzymatic reactions. Here, DES were used in a combinatorial enzyme-catalyzed system containing β-glucosidase BGLAt and β-galactosidase BGALAo to produce deglycosylated ginsenosides (De-g) from ginseng extracts (GE). The results showed that DES prepared with betaine and ethylene glycol (molar ratio, 1:2) could significantly stimulate the activity of the combinatorial enzymes as well as improve the acid resistance and temperature stability. The DES-based combinatorial enzyme-catalyzed system could convert 5 g of GE into 1.24 g of De-g (F₁, F₂, 20 (S)-PPT, and CK) at 24 h, which was 1.1 times that of the buffer sample. As confirmed by the spectral data, the changes in the conformations of the combinatorial enzymes were more favorable for the binding reaction with the substrates. Moreover, the constructed DES-based aqueous two-phase system enabled the recovery of substantial amounts of DES and De-g from the top phase. These results demonstrated that DES shows great application as a reaction solvent for the scale-up production of De-g and provide insights for the green extraction of natural products.

This study aimed to investigate the alkaloid secondary metabolites of Aspergillus amstelodami BSX001, a fungus isolated from Anhua dark tea, and to improve the extraction yield of the active ingredients by optimizing the extraction process. The structural characterization of the compounds was investigated using mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy. The antioxidant activity of echinulin-related alkaloids was evaluated by determining the total reducing power and DPPH radical scavenging capacity. The extraction process of the compound with optimum activity was optimized by a single-factor test and response surface methodology (RSM) combined with Box–Behnken design (BBD). The optimized result was validated. Finally, a new alkaloid 8-hydroxyechinulin (1), and four known alkaloids, variecolorin G (2), echinulin (3), neoechinulin A (4), and eurocristatine (5), were isolated. Echinulin-related compounds 1, 3, and 4 possessed certain antioxidant activities, with IC₅₀ values of 0.587 mg/mL, 1.628 mg/mL, and 0.219 mg/mL, respectively, against DPPH radicals. Their total reducing power at a concentration of 0.5 mg/mL was 0.29 mmol/L, 0.17 mmol/L, and 4.25 mmol/L. The extraction process of neoechinulin A was optimized with the optimum extraction parameters of 72.76% methanol volume fraction, 25 mL/g solid–liquid ratio, and 50.8 °C soaking temperature. Under these conditions, the extraction yield of neoechinulin A was up to 1.500 mg/g.

Trichlorfon (TCF), a widely used organophosphorus pesticide for crop protection, poses severe dermal toxicity risks as it readily penetrates the epidermal barrier and metabolizes into the more toxic dichlorvos (DDVP), triggering neurotoxicity, oxidative stress and inflammatory damage in exposed individuals. As a promising stoichiometric bioscavenger for pesticide detoxification, natural acetylcholinesterase (AChE) is still plagued by bottlenecks in thermal stability and transdermal delivery efficiency. To address these issues, this study employed a previously engineered high-activity and thermostable AChE variant (CpA-M5), evaluated its detoxification efficacy and mechanisms both in vitro and in vivo, and further developed a CpA-M5-loaded dissolvable microneedle system (CpA-M5-MN) for targeted dermal detoxification. In vitro assays demonstrated that CpA-M5 effectively restored endogenous AChE activity, scavenged ROS, reduced inflammation and alleviated apoptosis in TCF-exposed cells. Enzyme kinetics and molecular interaction analyses confirmed irreversible binding between CpA-M5 and TCF/DDVP, with DDVP showing higher binding affinity (K_D = 9.23 × 10⁻⁶ M, Ki = 15.56 μM) and complex stability (K_a = 2450.191 M⁻¹, ΔG = −39.37 ± 1.48 kcal/mol). In vivo, the CpA-M5-loaded microneedle system achieved targeted transdermal delivery, effectively overcoming the limitations of low permeability and rapid degradation of free enzymes. Pathological and metabolomic analyses demonstrated that CpA-M5-MN reduced skin residual TCF/DDVP levels, restored local oxidative-inflammatory homeostasis, and regulated glutathione and amino acid metabolic pathways, thereby achieving synergistic detoxification and metabolic repair effects. Collectively, this work presents a promising targeted detoxification platform for pesticide-induced dermal injury, and provides new insights into the clinical translation of engineered enzyme-based bioscavengers.