Effect and mechanisms of thermal sterilization methods on the in vitro phenolic bioaccessibility of rose tea with milk.

Rose polyphenols, key functional components in roses, require adequate bioaccessibility for their health benefits, subject to influence by food components and processing. Investigating the impact of various thermal sterilization methods on the bioaccessibility of rose polyphenols in rose tea with milk and the underlying mechanisms, our findings indicated a significant increase in bioaccessibility following treatment at 85 °C/30 min. Conversely, 121 °C/15 min treatment decreased bioaccessibility. Examining the interaction between ß-casein in milk and rose polyphenols under different sterilization conditions, SEM and particle size analysis revealed binding, with fluorescence spectroscopy indicating non-covalent bonds. Binding forces followed the order 121 °C > 85 °C > 25 °C. Notably, at 85 °C, non-covalent binding improved polyphenol bioaccessibility, while the intensified binding at 121 °C decreased it. SDS-PAGE and amino acid analysis confirmed no covalent bond. This study establishes a theoretical basis for selecting thermal sterilization temperatures for milk-flower combinations, considering polyphenol bioaccessibility.

A Ni-NTA-based red fluorescence probe for protein labelling in live cells.

The great success of a His6-Ni-nitrilotriaceate (Ni-NTA) system in protein purification has inspired scientists to develop novel Ni-NTA based fluoresent probes for imaging of proteins in live cells. Despite a great deal of effort being made, only very few such probes can cross the cell membrane to label intracellular proteins. Herein, a new red fluorescence probe (Ni-NTA-AB) consisting of a Ni-NTA moiety, BODIPY and an arylazide was designed and synthesized to selectively label His6-tagged proteins. Guided by the Ni-NTA, the probe selectively binds to proteins with a genetically fused His6-tag exemplified by a DNA repair protein, xerodermapigmentosum group A (XPA122), leading to ca. 26-fold fluorescence enhancement. Ni-NTA-AB could enter into live bacterial cells to label intracellular His6-tagged proteins with the assistance of Tween 80 and also to quantify His6-tagged proteins in SDS-PAGE upon photoactivation of arylazide with a wide linear range (25-1000 ng). This study may provide a new horizon for the multiple-channel tracking and labelling of His6-tagged proteins.

Rapid labeling of intracellular His-tagged proteins in living cells.

Small molecule-based fluorescent probes have been used for real-time visualization of live cells and tracking of various cellular events with minimal perturbation on the cells being investigated. Given the wide utility of the (histidine)6-Ni(2+)-nitrilotriacetate (Ni-NTA) system in protein purification, there is significant interest in fluorescent Ni(2+)-NTA-based probes. Unfortunately, previous Ni-NTA-based probes suffer from poor membrane permeability and cannot label intracellular proteins. Here, we report the design and synthesis of, to our knowledge, the first membrane-permeable fluorescent probe Ni-NTA-AC via conjugation of NTA with fluorophore and arylazide followed by coordination with Ni(2+) ions. The probe, driven by Ni(2+)-NTA, binds specifically to His-tags genetically fused to proteins and subsequently forms a covalent bond upon photoactivation of the arylazide, leading to a 13-fold fluorescence enhancement. The arylazide is indispensable not only for fluorescence enhancement, but also for strengthening the binding between the probe and proteins. Significantly, the Ni-NTA-AC probe can rapidly enter different types of cells, even plant tissues, to target His-tagged proteins. Using this probe, we visualized the subcellular localization of a DNA repair protein, Xeroderma pigmentosum group A (XPA122), which is known to be mainly enriched in the nucleus. We also demonstrated that the probe can image a genetically engineered His-tagged protein in plant tissues. This study thus offers a new opportunity for in situ visualization of large libraries of His-tagged proteins in various prokaryotic and eukaryotic cells.