Endogenous Fluorescence Research Articles

Elucidation of the binding behavior between tetracycline and bovine casein by multi-spectroscopic and molecular simulation methods

Bovine casein was used as a model protein to explore the binding behavior of food matrix with residual antibiotics. The interaction mechanism between casein and tetracycline (TC) was characterized by fluorescence quenching and isothermal titration calorimetry. Through forming the non-fluorescent ground-state complex, TC effectively quenched the endogenous fluorescence of casein. The formed casein-TC complex was maintained by hydrogen bonding, electrostatic and hydrophobic interactions (ΔH < 0, ΔS > 0). Circular dichroism and infrared spectroscopic studies indicated that the complexation of TC did not affect the secondary structure of casein. Molecular docking further theoretically validated the main interaction forces and deciphered the amino acid residues surrounding the antibiotic in the casein binding pocket. Interestingly, 1H NMR results demonstrated the π-π stacking interaction formed between PHE-189 and the benzene ring of TC, which was consistent with the docking findings. Besides, saturation transfer difference (STD) NMR revealed that the binding of TC engages its –N(CH3)2 group. This study sheds new light on the transition of residual antibiotics from the free to the bound state in foodstuffs.

Quantitative Endogenous Fluorescence Analysis Discriminates Glioblastoma Tumor through Visible and NIR Excitation

Nowadays, the ability to diagnose brain tumors intraoperatively and delineate their margins, as accurately as possible, is of primordial importance during surgery. However, the exact tumor boundaries and targets are difficult to find due to the similar visual appearances especially at the margins, leading in many cases to poor surgical outcomes and a high risk of recurrences. To solve this dogma, our group tried to determine different tissue optical signatures such as optical parameters and endogenous fluorescence. For that, we conducted a comparative study to differentiate between healthy and tumorous tissues under one- and two-photon excitations on optical properties to explore several optical parameters. In addition, the study of endogenous fluorescence can successfully help with the discrimination between tissue types using one- and two-photon excitations. This works suggests that the multimodal analysis of optical signatures of tissues could be a reliable technique that can be used to differentiate between healthy and tumorous tissues. This can help in the future with the implementation of such techniques in vivo during surgery to help the surgeon with the decision that needs to be taken as for tissue resection.

Mechanism of interaction between urolithin A and α-glucosidase: Analysis by inhibition kinetics, fluorescence spectroscopy, isothermal titration calorimetry and molecular docking

In this study, the mechanism of interaction between urolithin A, a gut metabolite of ellagitannins, and α-glucosidase was characterized by inhibition kinetics, fluorescence spectroscopy, isothermal titration calorimetry, and molecular docking. Urolithin A exhibited potential reversible inhibitory activity for α-glucosidase in an uncompetitive manner with an IC50 value of (28.03 ± 0.59) μM. The results of fluorescence titration and isothermal titration calorimetry analysis showed that urolithin A statically quenched the endogenous fluorescence of α-glucosidase, which was a spontaneous exothermic process, mainly driven by hydrogen bond and/or van der Waals force. Synchronous fluorescence spectroscopy revealed that urolithin A increased the polarity of tryptophan microenvironment and the hydrophobicity of tyrosine microenvironment. The combination of urolithin A with acarbose, a competitive α-glucosidase inhibitor, showed an additive inhibitory effect. Furthermore, molecular docking showed that urolithin A formed hydrogen bonds with key residues Arg269, Thr273, and His258, which are the sites outside the active center of α-glucosidase. These findings could highlight the value of ellagitannin rich diet as an antihyperglycemic food in the prevention of postprandial hyperglycemia, and indicate that urolithin A may be a promising lead compound of α-glucosidase inhibitor.

Effect of pH-shifting and sonication-assisted treatment on properties and stability of vegetable oil-based whipped cream stabilized by kidney bean protein aggregates

Whipped cream stabilized by kidney bean protein aggregates (KPAgs) was constructed, and the effects of pH-shifting and sonication-assisted treatment on the structure of KPAgs and the properties and stability of the resulting whipped cream were invFestigated. The relationship between the properties and stability of whipped cream and the structure of KPAgs was analyzed. The visual appearance, ThT fluorescence, TEM, endogenous fluorescence and FT-IR results showed that KPAgs prepared at pH 2.0 had better gel properties than those at pH 4.5 and 7.0, because the former produced fibril aggregates while the latter two mainly produced particle aggregates. The CLSM results of the emulsion showed that the strength of the protein gel structure decreased with increasing pH during preparation, and gradually failed to form a continuous phase. Kidney bean protein was more suitable than soybean protein for preparing fibril protein aggregates suitable for whipped cream. The protein gel three-dimensional network structure of the kidney bean protein after sonication-assisted treatment was more compact because more KPAgs were generated. Protein fibril aggregates had a higher aspect ratio and structural flexibility than particle aggregates, and were easier to arrange on the interface to form an interface layer, thereby avoiding the aggregation of air bubbles and thus having better whipped cream structural stability.

Abstract 6512: Label-free autofluorescence imaging reveals different metabolic responses to adverse growth conditions between normal and breast cancer cells lines

Abstract Cancer cells are metabolically distinct from normal cells, and can adapt differently to adverse growth conditions. We sought to compare normal MCF10A and Ras-transformed MCF10AT cells under various conditions (regular growth media, doxorubicin-treatment, and glucose-deprived media) by imaging endogenous cellular FAD and NADH fluorescence and comparing it to proliferation rates (measured by MTS assay) and chromatin structure (measured by Hoescht staining). Under normal growth conditions, MCF10AT cells displayed similar levels of FAD and higher levels of mitochondrial NADH than MCF10A cells. Elevated NADH in MCF10AT cells seems to reflect the preference for anaerobic glycolysis rather than oxidative phosphorylation (the so-called Warburg effect). Our MTS data showed that doxorubicin treatment did not significantly affect MCF10AT cell proliferation but led to a modest reduction in cell proliferation in the normal cell line. Likewise, in MCF10AT cells, doxorubicin treatment led to an increase in FAD and a decrease in NADH. Conversely, in MCF10A cells, doxorubicin led to a decrease in FAD levels and an increase in mitochondrial NADH. These results suggest that the cancer line may be switching to oxidative phosphorylation to resist doxorubicin-induced toxicity, while the normal cells reduce oxidative phosphorylation with growth inhibition. In contrast to the doxorubicin response, glucose deprivation produced significant growth inhibition and cell death in MCF10AT cells while having a modest growth-inhibitory effect on the normal line. Interestingly, low glucose decreased FAD and mitochondrial NADH in MCF10AT cells, suggesting that both aerobic and anaerobic respiration was reduced and consistent with the marked reduction in observed cell viability. In contrast, MCF10A cells showed a slight decrease in FAD and an increase in NADH, suggesting a decrease in oxidative phosphorylation consistent with the modest growth inhibition observed. Finally, MCF10ATs displayed higher levels of Hoechst staining than MCF10A cells, consistent with a higher proportion of condensed chromatin observed in cancer cells. In addition, we found a larger increase in Hoechst staining in MCF10AT cells in response to both doxorubicin and low glucose, as compared to MCF10A cells. This data suggests that the chromatin state of the two lines is also differentially affected by these treatments. In conclusion, label-free measurement of autofluorescence of cellular FAD and mitochondrial NADH reveals the different metabolic and chromatin responses to adverse growth conditions between cancer and non-cancer cells. These differences are associated with the different proliferation rates and may have important clinical implications for cancer risk stratification. Citation Format: Katherine Junkins, Angel Perez Martinez, Margaret Rodgers, Shelley A. Phelan, Min Xu. Label-free autofluorescence imaging reveals different metabolic responses to adverse growth conditions between normal and breast cancer cells lines. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6512.

NAD(P)H binding configurations revealed by time-resolved fluorescence and two-photon absorption

NADH and NADPH play key roles in the regulation of metabolism. Their endogenous fluorescence is sensitive to enzyme binding, allowing changes in cellular metabolic state to be determined using fluorescence lifetime imaging microscopy (FLIM). However, to fully uncover the underlying biochemistry, the relationships between their fluorescence and binding dynamics require greater understanding. Here we accomplish this through time- and polarization-resolved fluorescence and polarized two-photon absorption measurements. Two lifetimes result from binding of both NADH to lactate dehydrogenase and NADPH to isocitrate dehydrogenase. The composite fluorescence anisotropy indicates the shorter (1.3–1.6 ns) decay component to be accompanied by local motion of the nicotinamide ring, pointing to attachment solely via the adenine moiety. For the longer lifetime (3.2–4.4 ns), the nicotinamide conformational freedom is found to be fully restricted. As full and partial nicotinamide binding are recognized steps in dehydrogenase catalysis, our results unify photophysical, structural, and functional aspects of NADH and NADPH binding and clarify the biochemical processes that underlie their contrasting intracellular lifetimes.

Changes in Texture Characteristics and Special Requirements of Sichuan-Style Braised Beef for Industrial Production: Based on the Changes in Protein and Lipid of Beef

This study aimed to investigate the optimal stewing time (0, 30, 60, 90, 120, and 150 min) for industrialized preparation of Sichuan-style braised beef with different demands. With prolonged stewing time, the hardness and chewiness of the braised beef initially increased and then decreased (p < 0.05), whereas springiness and cohesiveness gradually decreased. The moisture content of braised beef and the endogenous fluorescence intensity of braised beef protein significantly decreased (p < 0.05). However, the thiobarbituric acid reaction substances (TBARS) value and protein carbonyl content of braised beef greatly increased (p < 0.05). During the stewing process, the texture properties of Sichuan-style braised beef were affected by the moisture content, oxidation of proteins and lipids, and integrity of the muscle fibers. Considering texture traits, when Sichuan-style pre-braised beef bought by consumers is stewed with other ingredients for about 30 min, its corresponding stewing time is 60 min in industrialized production processes. This process parameter can not only save energy consumption for practical production, but also improve the hardness value of the as-obtained Sichuan-style pre-braised beef, which is conducive to transportation through refraining from cracking of pre-braised beef pieces. When consumers only use simple heating to eat the Sichuan-style pre-braised beef product, stewing times of 120 or 150 min can be considered in industrialized production processes. This work provided a theoretical reference for the industrialized and standardized production of different types of prepared Sichuan-style braised beef.

Encapsulation of docosahexaenoic acid (DHA) using self-assembling food-derived proteins for efficient biological functions

Docosahexaenoic acid (DHA; 22n-6) possesses multiple biological functions, including antioxidant activity and ameliorating hypertriglyceridemia. However, the application of DHA has been limited due to poor aqueous solubility and susceptible to oxidation. Here, ovalbumin (O), myosin (M), 7S soy globulin (S), and β-lactoglobulin (β), hydrolyzed by chymotrypsin, self-assembled into micelles, respectively. Adding incremental DHA to micelles caused endogenous fluorescence quenching of O, M, S, and β micelles, implying successful incorporation of DHA into hydrophobic cores of micelles (O (DHA), M (DHA), S (DHA), and β (DHA)). The results showed that micelles provided spatial stability and improved solubility, and stability against thermal and ultraviolet (UV) light for DHA. The uptake of DHA from M (DHA), β (DHA), O (DHA), and S (DHA) was 3.27-, 3.91-, 2.7-, and 3.95-fold higher, respectively, than that of DHA by Caco-2 cells. Encapsulation in micelles increased DHA aqueous solubility and uptake, which enhanced cellular endogenous antioxidant defense. Meanwhile, increased uptake of DHA was verified by HepG2 cells, and O, M, S, and β micelles were proven to increase DHA uptake to reduce lipid deposition. Our findings strongly support the possibility that O, M, S, and β micelles can be regarded as a carrier for loading DHA.

Intraoperative Optical Diagnostics of Uterine Microcirculation during Myomectomy

Previous studies have shown that intraoperative optical diagnostics can be used to solve fundamental and applied problems in gynecology. Our article presents results from experimental in vivo intraoperative measurements of endogenous fluorescence and blood perfusion in various sections of the uterus, myometrium, endometrium, myomas, and pseudocapsule in female patients undergoing myomectomy. We found that myomas have different blood supply and fluorescence level at wavelength 365 nm depending on their growth intensity and size, most likely due to collagen accumulation in the extracellular matrix and changes in the vascular architecture. The possibility of laser Doppler flowmetry to assess endometrial perfusion is also demonstrated for investigating the effect of myomas of different types on endometrial tissue blood supply. The results obtained can be used to provide a better understanding of pathological processes in uterine leiomyoma, thus helping physicians to choose treatment strategies.

The inhibitory effect and mechanism of small molecules on acetic anhydride-induced BSA acetylation and aggregation

Protein acetylation is a significant post-translational modification, and hyperacetylation results in amyloid aggregation, which is closely related to neurodegenerative diseases (Alzheimer's disease, Huntington's disease, and so on). Therefore, it is significant to inhibit the hyperacetylation of proteins and their induced aggregation. In the present study, we aimed to explore the anti-acetylation and anti-amyloid properties of five small molecules (gallic acid, menadione, resveratrol, apigenin, and quercetin) in the process of acetic anhydride-induced protein hyperacetylation and its aggregation. Optical detection methods, such as SDS-PAGE, inverted fluorescence microscopy, and endogenous fluorescence spectroscopy, were used to investigate the effects of small molecules on protein acetylation, aggregation, and structure. In addition, fluorescence quenching and molecular docking techniques were used to explore the relationship between small molecules and acetylation. The results showed that gallic acid (200 μM), menadione (100 μM), quercetin (40 μM), resveratrol (5 μM), and apigenin (20 μM) (unmodified rates were 61.12 %, 67.76 %, 65.11 %, 62.66 %, and 67.81 %, respectively) had strong inhibitory effects on acetylation, and there was no significant difference (P < 0.05). In addition, gallic acid (200 μM), menadione (100 μM), and resveratrol (5 μM) (inhibition rates of 29.89 %, 26.53 %, and 26.09 %, respectively) had more substantial inhibitory effects on protein aggregation, indicating that the five small molecules could inhibit acetic anhydride-induced hyperacetylation and protein aggregation. The underlying mechanism might be that it could inhibit hyperacetylation and resist amyloid aggregation by interacting with proteins to occupy acetylation sites. Collectively, our findings showed that gallic acid, menadione, and resveratrol could potentially prevent and treat neurodegenerative diseases, such as Alzheimer's disease, by inhibiting acetylation and acetylation-induced aggregation.

Protease-Activatable Porphyrin Molecular Beacon for Osteoarthritis Management.

Despite the substantial burden posed by osteoarthritis (OA) globally, difficult challenges remain in achieving early OA diagnosis and adopting effective disease-modifying treatments. In this study, we use a biomolecular approach to address these limitations by creating an inherently theranostic molecular beacon whose imaging and therapeutic capabilities are activated by early pathological changes in OA. This platform comprised (1) a peptide linker substrate for metalloproteinase-13 (MMP-13), a pathological protease upregulated in OA, which was conjugated to (2) a porphyrin moiety with inherent multimodal imaging, photodynamic therapy, and drug delivery capabilities, and (3) a quencher that silences the porphyrin's endogenous fluorescence and photoreactivity when the beacon is intact. In diseased OA tissue with upregulated MMP-13 expression, this porphyrin molecular beacon (PPMMP13B) was expected to undergo sequence-specific cleavage, yielding porphyrin fragments with restored fluorescence and photoreactivity that could, respectively, be used as a readout of MMP-13 activity within the joint for early OA imaging and disease-targeted photodynamic therapy. This study focused on the synthesis and characterization of PPMMP13B, followed by a proof-of-concept evaluation of its OA imaging and drug delivery potential. In solution, PPMMP13B demonstrated 90% photoactivity quenching in its intact form and robust MMP-13 activation, yielding a 13-fold increase in fluorescence post-cleavage. In vitro, PPMMP13B was readily uptaken and activated in an MMP-13 cell expression-dependent manner in primary OA synoviocytes without exuding significant cytotoxicity. This translated into effective intra-articular cartilage (to a 50 μm depth) and synovial uptake and activation of PPMMP13B in a destabilization of the medial meniscus OA mouse model, yielding strong fluorescence contrast (7-fold higher signal than background) at the diseased joint site. These results provide the foundation for further exploration of porphyrin molecular beacons for image-guided OA disease stratification, effective articular delivery of disease-modify agents, and OA photodynamic therapy.

Binding characteristics of Bruton’s tyrosine kinase inhibitor ibrutinib with bovine serum albumin: multi-spectroscopic combined with molecular simulation

Ibrutinib, as an oral Bruton’s tyrosine kinase inhibitor, has highly potent, covalent irreversible, and other characteristics. In this work, the binding characteristics of ibrutinib with bovine serum albumin were investigated using multi-spectroscopic techniques and molecular simulations. The findings demonstrated that ibrutinib could quench the endogenous fluorescence of bovine serum albumin by static quenching mode, meanwhile bovine serum albumin could also quench the fluorescence of ibrutinib. In the binding process of ibrutinib with bovine serum albumin, ibrutinib caused a bathochromic shift in the absorption band of bovine serum albumin, further indicating that ibrutinib and bovine serum albumin formed a ground state ibrutinib and bovine serum albumin complex. It is confirmed that ibrutinib has a strong affinity to bovine serum albumin due to the binding constant of more 104 M−1. Experimental findings showed that ibrutinib resulted in the decrease in the hydrophobicity or the enhancement in the polarity of the surroundings around Tyr and Trp residues of bovine serum albumin and diminishing in α-helix and β-sheet content of bovine serum albumin. The findings from site-competition experiments confirmed that the binding site of ibrutinib onto bovine serum albumin was the same as that of ibuprofen, that is, ibrutinib bound to the site II' site of bovine serum albumin, which was verified by molecular docking. The findings from thermodynamic analysis revealed that ibrutinib spontaneously bound onto bovine serum albumin through an enthalpy-driven and the driving forces included hydrogen bonding and van der Waals forces being the dominating forces. The findings from molecular dynamics simulation confirmed that some residues such as ARG-208, ALA-209, ALA-212, LEU-326, and LYS-350 made major contributions in ibrutinib and bovine serum albumin complexation.

Study on the Structure, Function, and Interface Characteristics of Soybean Protein Isolate by Industrial Phosphorylation

The impacts of industrial phosphorylation on the structural changes, microstructure, functional, and rheological features of soybean protein isolate (SPI) were spotlighted. The findings implied that the spatial structure and functional features of the SPI changed significantly after treatment with the two phosphates. Sodium hexametaphosphate (SHMP) promoted aggregation of SPI with a larger particle size; sodium tripolyphosphate (STP) modified SPI with smaller particle size. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) results showed insignificant alterations in the structure of SPI subunits. Fourier transform infrared (FTIR) and endogenous fluorescence noted a decline in α-helix quantity, an amplification in β-fold quantity, and an increase in protein stretching and disorder, indicating that phosphorylation treatment fluctuated the spatial structure of the SPI. Functional characterization studies showed that the solubility and emulsion properties of the SPI increased to varying degrees after phosphorylation, with a maximum solubility of 94.64% for SHMP-SPI and 97.09% for STP-SPI. Emulsifying activity index (EAI) and emulsifying steadiness index (ESI) results for STP-SPI were better than those for SHMP-SPI. Rheological results showed that the modulus of G' and G″ increased and the emulsion exhibited significant elastic behavior. This affords a theoretical core for expanding the industrial production applications of soybean isolates in the food and various industries.

Time-specific ultrasonic treatment of litchi thaumatin-like protein inhibits inflammatory response in RAW264.7 macrophages via NF-κB and MAPK transduction pathways

The pro-inflammation activity of litchi thaumatin-like protein (LcTLP) led to be responsible for the occurrence of adverse reactions after excessive consumption of litchi. This study aimed to characterize the changes in the structure and inflammatory activity of LcTLP induced by ultrasound treatment. Significant molecular structure of LcTLP changes occured at 15 min ultrasound treatment, and then tended to recover with subsequent treatment. Secondary structure (α-helices decreased from 17.3% to 6.3%), tertiary structure (the maximum endogenous fluorescence intensity decreased), and microstructure (mean hydrodynamic diameter reduced from 4 μm to 50 nm) of the LcTLP treated for 15 min (LT15) were significantly affected, which led to the inflammatory epitope of LcTLP (domain II and V-cleft) unfolded. In vitro, LT15 had a significant anti-inflammatory response, which inhibited NO production and had the best effect at 50 ng/mL in RAW264.7 macrophages (73.24%). Moreover, proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) secretion and mRNA expression levels were also significantly lower compared with untreated LcTLP (p < 0.05). Western blot further confirmed that the expressions of IκB-α, p65, p38, ERK and JNK reduced markedly (p < 0.05), which indicated LT15 inhibited the inflammatory response through NF-κB and MAPK transduction pathways. Overall, it can be hypothesized that LT15 exposed to low frequency ultrasonic fields have a direct effect on the protein surface structure and thus on the entry of LT15 into cells, making 15-minute ultrasound treatment potentially useful in reducing the pro-inflammatory properties of litchi or related liquid products.

New Insights into the Inhibition of Hesperetin on Polyphenol Oxidase: Inhibitory Kinetics, Binding Characteristics, Conformational Change and Computational Simulation.

The inhibitory activity of hesperetin on polyphenol oxidase (PPO) and their interaction characteristics were investigated using multiple spectroscopic methods and computational simulation. Hesperetin, a mixed inhibitor, reversibly inhibited PPO activity, and its half-maximum inhibitory concentration (IC50) values on monophenolase and diphenolase were 80.8 ± 1.4 μM and 776.0 ± 15.5 μM, respectively. Multivariate curve resolution-alternate least squares (MCR-ALS) analysis suggested PPO interacted with hesperetin and formed PPO-hesperetin complex. Hesperetin statically quenched PPO's endogenous fluorescence, and hydrophobic interactions mainly drove their binding. Hesperetin affected the polarity of the microenvironment around the Trp residues in PPO, but had no effect on that around Tyr residues. Circular dichroism (CD) results showed that hesperetin increased α-helix content and decreased β-fold and random coil contents, thus tightening PPO's structure. Molecular docking showed that hesperetin entered the hydrophobic cavity of PPO, bound near the dinuclear copper active center, interacted with Val283, Phe264, His85, Asn260, Val248, and His263 via hydrophobic interactions, formed hydrogen bonds with Met280, His89, and His259 residues and also interacted with Phe292, His61, Phe90, Glu256, His244, Asn260, Phe264, and Gly281 via van der Waals forces. The molecular dynamics simulation results also demonstrated that the addition of hesperetin reduced the stability and hydrophobicity of PPO and increased PPO's structural denseness. Thus, the inhibition of hesperetin on PPO may be because hesperetin bound near the active center of PPO, interacted with the surrounding residues, occupied the binding site for substrate, and induced the changes in PPO's secondary structure, thus inhibiting the catalytic activity of PPO. This study may provide novel views for the inhibition of hesperetin on PPO and theoretical guidance for developing flavonoids as new and efficient PPO inhibitors.

Characterizing the binding interactions between virgin/aged microplastics and catalase in vitro

Microplastics (MPs) undergo physical, chemical, and biological aging in the environment, leading to changes in their physicochemical properties, affecting migration characteristics and toxicity. Oxidative stress effects induced by MPs in vivo have been extensively studied, but the toxicity difference between virgin and aged MPs and the interactions between antioxidant enzymes and MPs in vitro have not been reported yet. This study investigated the structural and functional changes of catalase (CAT) induced by virgin and aged PVC-MPs. It was shown that light irradiation aged the PVC-MPs, and the aging mechanism was photooxidation, resulting in a rough surface and appearing holes and pits. Because of the changes in physicochemical properties, aged MPs had more binding sites than virgin MPs. Fluorescence and synchronous fluorescence spectra results suggested that MPs quenched the endogenous fluorescence of CAT and interacted with tryptophane and tyrosine residues. The virgin MPs had no significant effect on the skeleton of CAT, while the skeleton and the polypeptide chains of CAT became loosened and unfolded after binding with the aged MPs. Moreover, the interactions of CAT with virgin/aged MPs increased the α-helix and decreased the β-sheet contents, destroyed the solvent shell, and resulted in a dispersion of CAT. Due to the large size, MPs cannot enter the interior of CAT and have no effects on the heme groups and activity of CAT. The interaction mechanism between MPs and CAT may be that MPs adsorb CAT to form the protein corona, and aged MPs had more binding sites. This study is the first comprehensive investigation of the effect of aging on the interaction between MPs and biomacromolecules and highlights the potential negative effects of MPs on antioxidant enzymes.

Structure and emulsifying properties of rice bran protein alkylated using an electrochemical reactor

To replace the application of reducing agents for protein alkylation, electrochemical reduction techniques have been considered. In this study, a custom-made electrochemical reactor was used to alkylate rice bran protein (RBP). The structure, morphology, and emulsification properties of RBP were investigated under different voltages. When treated at 35 V, the content of the α-helix and β-sheet of RBP decreased at first and then increased, whereas the content of the β-turn and random coil increased steadily. The CH3 group of RBP was exposed and S-S decreased. The endogenous fluorescence spectral curve exhibited a redshift. The free sulfhydryl (-SH) content increased. The average particle size of the modified RBP decreased by 69.35 %, and its zeta potential decreased to −21.8 mV. Atomic force microscopy (AFM) revealed that the treated protein particles dispersed more evenly and their roughness (Rq) decreased. The contact angle, water holding capacity (WHC), fat holding capacity (FHC), and solubility were enhanced. The emulsification capacity increased to 65.82 m2/g and emulsification stability increased to 36.34 min. These results demonstrated that the RBP was alkylated by the electrochemical reactor and the modified RBP showed improved emulsification properties compared to the untreated RBP.

Fluorescence-Activating and Absorption-Shifting Nanoprobes for Anaerobic Tracking of Gut Microbiota Derived Vesicles.

Outer membrane vesicles (OMVs) are crucial for bacterial intercellular communication and the crosstalk between the gut microbiota and its host. Methods capable of visualizing gut microbiota derived OMVs would be of great significance but have been rarely reported. Here, nanoprobes carrying a fluorescence-activating and absorption-shifting tag are prepared by combining genetic engineering and antibiotic-boosted vesicle formation and release. Benefiting from their natural structure and molecular oxygen-independent emission, the resulting nanovesicles can be applied as endogenous fluorescence probes to anaerobically track gut microbiota associated OMVs. These nanoprobes show flexibility in on-demand fluorescence turn-on/off and reversibly switchable emission bands for intelligent and dual-color imaging. With these special characteristics, the behaviors of microbiota OMVs to not only inhibit specific pathogenic strains through membrane fusion but also repair the intestinal barrier via entering intestinal epithelia and promoting the expressions of tight junctions are tracked and identified in the gut. Based on these discoveries, OMVs are disclosed to be able to remit inflammation in a murine model of colitis following transplantation to the intestine by oral delivery. This work provides an approach to visualize the dynamics of the gut microbiota and disclose potential targets for disease intervention.

Effects of Packaging Materials on Structural and Simulated Digestive Characteristics of Walnut Protein during Accelerated Storage.

Walnuts are rich in fat and proteins that become oxidized during the processing and storage conditions of their kernels. In this study, the effect of three packaging materials (e.g., polyethylene sealed packaging, polyamide/polyethylene vacuum packaging, and polyethylene terephthalate/aluminum foil/polyethylene vacuum packaging) were investigated on the oxidation, structural and digestive properties of walnut kernel proteins. Results showed that the amino acid content gradually decreased and carbonyl derivatives and dityrosine were formed during storage. The protein molecule structure became disordered as the α-helix decreased and the random coil increased. The endogenous fluorescence intensity decreased and the maximum fluorescence value was blue-shifted. After 15 days of storage, surface hydrophobicity decreased, while SDS-PAGE and HPLC indicated the formation of large protein aggregates, leading to a reduction in solubility. By simulating gastrointestinal digestion, we found that oxidation adversely affected the digestive properties of walnut protein isolate and protein digestibility was best for polyethylene terephthalate/aluminum foil/polyethylene vacuum packaging. The degree of protein oxidation in walnuts increased during storage, which showed that except for fat oxidation, the effect of protein oxidation on quality should be considered. The results of the study provided new ideas and methods for walnut quality control.

The structure, antioxidant activity, and stability of fish gelatin/chitooligosaccharide nanoparticles loaded with apple polyphenols.

Apple polyphenols (APs) with multiple biological effects have attracted extensive attention due to their broad opportunities for application. However, the use of APs is hampered by their instability in the face of environmental changes. Designing efficient carriers to improve the bioavailability of APs is the key to solving these problems. In this study, gelatin-chitooligosaccharide nanoparticles produced by the Maillard reaction (GCM) were fabricated to encapsulate AP, and the structure, antioxidant activity, and stability of the GMM-AP nanoparticle system were evaluated. The results of endogenous fluorescence spectrum, Fourier-transform infrared (FTIR) spectroscopy, X-ray diffraction, and simultaneous thermal analysis confirmed structural changes and interactions between GCM and AP. Combination with GCM did not adversely affect the antioxidant properties of AP, and the GCM-AP nanoparticles possessed superior temperature and storage stability. In comparison with fish gelatin-apple polyphenol nanoparticles, the GCM-AP nanoparticles were more stable at a wider pH range, and were more resistant to the electrostatic shielding effect of NaCl. After simulating gastric digestion, the particle size and polydispersity index (PDI) of GCM-AP nanoparticles were almost unchanged. The findings suggest that GCM nanoparticles loaded with AP could be used as good carriers with good antioxidant activity and stability. This study therefore provides a theoretical foundation for the development and industrial application of food functional factors. © 2023 Society of Chemical Industry.