Disaggregation of Islet Amyloid Polypeptide Fibrils as a Potential Anti-Fibrillation Mechanism of Tetrapeptide TNGQ.

Islet amyloid polypeptide (IAPP) fibrillation has been commonly associated with the exacerbation of type 2 diabetes prognosis. Consequently, inhibition of IAPP fibrillation to minimize ß-cell cytotoxicity is an important approach towards ß-cell preservation and type 2 diabetes management. In this study, we identified three tetrapeptides, TNGQ, MANT, and YMSV, that inhibited IAPP fibrillation. Using thioflavin T (ThT) fluorescence assay, circular dichroism (CD) spectroscopy, dynamic light scattering (DLS), and molecular docking, we evaluated the potential anti-fibrillation mechanism of the tetrapeptides. ThT fluorescence kinetics and microscopy as well as transmission electron microscopy showed that TNGQ was the most effective inhibitor based on the absence of normal IAPP fibrillar morphology. CD spectroscopy showed that TNGQ maintained the α-helical conformation of monomeric IAPP, while DLS confirmed the presence of varying fibrillation species. Molecular docking showed that TNGQ and MANT interact with monomeric IAPP mainly by hydrogen bonding and electrostatic interaction, with TNGQ binding at IAPP surface compared to YMSV, which had the highest docking score, but interact mainly through hydrophobic interaction in IAPP core. The highly polar TNGQ was the most active and appeared to inhibit IAPP fibrillation by disaggregation of preformed IAPP fibrils. These findings indicate the potential of TNGQ in the development of peptide-based anti-fibrillation and antidiabetic nutraceuticals.

Curcumin-Induced Stabilization of Protein-Based Nano-Delivery Vehicles Reduces Disruption of Zwitterionic Giant Unilamellar Vesicles.

Curcumin-loaded native and succinylated pea protein nanoparticles, as well as zwitterionic giant unilamellar vesicles were used in this study as model bioactive compound loaded-nanoparticles and biomembranes, respectively, to assess bio-nano interactions. Curcumin-loaded native protein-chitosan and succinylated protein-chitosan complexes, as well as native protein-chitosan and succinylated protein-chitosan hollow, induced leakage of the calcein encapsulated in the giant unilamellar vesicles. The leakage was more pronounced with hollow protein-chitosan complexes. However, curcumin-loaded native protein and curcumin-loaded succinylated protein nanoparticles induced calcein fluorescence quenching. Dynamic light scattering measurements showed that the interaction of curcumin-loaded native protein, curcumin-loaded succinylated protein, native protein-chitosan, and succinylated protein-chitosan complexes with the giant unilamellar vesicles caused a major reduction in the size of the lipid vesicles. Confocal and widefield fluorescence microscopy showed rupturing of the unilamellar vesicles after treatment with native pea protein-chitosan and succinylated pea protein-chitosan complexes. The nature of interaction between the curcumin-loaded protein nanoparticles and the biomembranes, at the bio-nano interface, is influenced by the encapsulated curcumin. Findings from this study showed that, as the protein plays a crucial role in stabilizing the bioactive compound from chemical and photodegradation, the encapsulated nutraceutical stabilizes the protein nanoparticle to reduce its interaction with biomembranes.

Defatted Wheat Germ Protein-Derived Peptides Showed Multiple Biological Activities from the Stomach to Small Intestine: In Silico and In Vitro Approaches.

This study aimed to test the hypothesis that bioactive peptides can exert multiple bioactivities at different sites in the gastrointestinal tract. Our previous research identified 33 gastric-resistant peptides derived from wheat germ with potential antiadhesive activity against Helicobacter pylori in the stomach. In this work, in silico digestion of these peptides with trypsin, thermolysin, and chymotrypsin produced 67 peptide fragments. Molecular docking was conducted to predict their ACE and DPP-IV inhibitory activities in the small intestine. Three peptides (VPIPNPSGDR, VPY, and AR) were selected and synthesized for in vitro validation. Their generation in the gastrointestinal tract was verified via in vitro digestion, followed by mass spectrometry analysis. The IC50 values for ACE inhibition were 199.5 µM (VPIPNPSGDR), 316.3 µM (VPY), and 446.7 µM (AR). For DPP-IV inhibition, their IC50 values were 0.5, 1.6, and 4.0 mM, respectively. This research pioneers new directions in the emerging field of multifunctional peptides, providing scientific evidence to support the utilization of wheat germ as value-added food ingredients.

Molecular Interaction of Pea Glutelin and Lipophilic Bioactive Compounds: Structure-Binding Relationship and Nano-/Microcomplexation.

This study investigated the impact of ionic strength and lipophilicity of bioactive compounds on their interaction with the alkaline soluble pea glutelin fraction (ASF) using the fluorescence quenching technique. A Stern-Volmer quenching constant, KD, of 8.9 ± 0.10, 5.3 ± 0.06, 4.0 ± 0.01, 1.1 ± 0.00, 0.9 ± 0.02, and 0.1 ± 0.00 (×104 M-1) was observed for curcumin-ASF (CuASF), astaxanthin-ASF (AsASF), cholecalciferol-ASF (ChASF), ß-carotene-ASF (ßCaASF), coenzyme Q10-ASF (Q10ASF), and ß-sitosterol-ASF (ßSiASF) complexes, respectively. An increase in ionic strength did not significantly change KD, the effective quenching constant K, and the bimolecular quenching rate constant KQ. However, it changed the mode of interaction of the ASF with cholecalciferol, ß-carotene, coenzyme Q10, and ß-sitosterol from static to static-dynamic quenching. Transmission electron microscopy showed that the morphology formed with protein (spherical nanocomplexes, microaggregates, or fiber-like particles) differed among the compounds. The favorable binding of CuASF, AsASF, ChASF, and ßCaASF complexes provides stable matrices for formulating protein-based delivery systems for lipophilic nutraceuticals.

Inhibition of Islet Amyloid Polypeptide Fibrillation by Structurally Diverse Phenolic Compounds and Fibril Disaggregation Potential of Rutin and Quercetin.

The influence of 12 food-derived phenolic compounds on islet amyloid polypeptide (IAPP) fibrillation was investigated. Results from thioflavin T assay demonstrated that gallic acid, caffeic acid, and rutin and its aglycone, quercetin, inhibited IAPP fibrillation at 1:0.5, 1:1, and 1:2 IAPP-phenolic molar ratios. Circular dichroism and dynamic light scattering at the 1:1 IAPP-phenolic ratio confirmed the inhibition of fibril formation. Rutin and quercetin increased the lag time by 90 and 6%, and the relative α-helix content by 63 and 48%, respectively. Gallic acid decreased the elongation rate by 30%, whereas caffeic acid decreased the maximum fluorescence intensity by 65%. Furthermore, fluorescence microscopy and transmission electron microscopy (TEM) showed IAPP fibril morphologies indicative of fibrillation reduction by the compounds. Molecular docking and TEM showed that rutin and quercetin disaggregated preformed IAPP fibrils potentially through fibrillar-monomeric equilibrium shifts. These findings demonstrate important structural features of phenolic compounds for disaggregating IAPP fibrils or inhibiting their formation.

In vitro inhibition of acetylcholinesterase activity by yellow field pea (Pisum sativum) protein-derived peptides as revealed by kinetics and molecular docking.

Compounds with structural similarities to the neurotransmitter (acetylcholine) are mostly used to inhibit the activity of acetylcholinesterase (AChE) in Alzheimer's disease (AD) therapy. However, the existing drugs only alleviate symptoms of moderate to mild conditions and come with side effects; hence, the search is still on for potent and safer options. In this study, High performance liquid chromatography (HPLC) fractionations of AChE-inhibitory pea protein hydrolysates obtained from alcalase, flavourzyme and pepsin digestions were carried out followed by sequence identification of the most active fractions using mass spectrometry. Subsequently, 20 novel peptide sequences identified from the active fractions were synthesized and five peptides, QSQS, LQHNA, SQSRS, ETRSQ, PQDER (IC50 = 1.53 - 1.61 µg/mL) were selected and analyzed for ability to change AChE protein conformation (fluorescence emission and circular dichroism), kinetics of enzyme inhibition, and enzyme-ligand binding configurations using molecular docking. The kinetics studies revealed different inhibition modes by the peptides with relatively low (<0.02 mM and <0.1 mM) inhibition constant and Michaelis constant, respectively, while maximum velocity was reduced. Conformational changes were confirmed by losses in fluorescence intensity and reduced α-helix content of AChE after interactions with different peptides. Molecular docking revealed binding of the peptides to both the catalytic anionic site and the peripheral anionic site. The five analyzed peptides all contained glutamine (Q) but sequences with Q in the penultimate N-terminal position (LQHNA, SQSRS, and PQDER) had stronger binding affinity. Results from the different analysis in this study confirm that the peptides obtained from enzymatic digestion of pea protein possess the potential to be used as novel AChE-inhibitory agents in AD management.

Lentil Protein and Tannic Acid Interaction Limits in Vitro Peptic Hydrolysis and Alters Peptidomic Profiles of the Proteins.

In this study, the nature of lentil protein-tannic acid (LPTA) interaction and its effect on in vitro pepsin digestion were investigated. LPTA mixtures containing 1% w/v LP and 0.001-0.5% TA were prepared and characterized in terms of particle size, thermal properties, and secondary and tertiary structures. A 20-fold increase in particle size was observed in LPTA0.5% compared to LP control (without TA), indicating aggregation. Static quenching of tryptophan residues within the protein hydrophobic folds was observed. Increasing TA levels also enhanced protein thermal stability. Over 50% reduction in free amino groups of LPTA 0.5%, relative to LP, was observed after pepsin digestion. Cleavage specificity of pepsin and peptidomic profile of LP were modified by the presence of TA in LPTA 0.5%. This study showed that 0.5% w/v TA induced protein aggregation and reduced LP digestibility by hindering the accessibility of pepsin to the protein network, thus modifying the profile of released peptides.

Peptide-Mucin Binding and Biosimilar Mucus-Permeating Properties.

This study aimed to understand the role of the mucus layer (a biological hydrogel) in the transport mechanisms of peptides. Using established in vitro models, the mucin-binding activity and mucus-permeating property of peptides were determined. Uncharged peptides with relatively high hydrophilicity, including MANT, TNGQ, and PASL, as well as cationic peptides, including KIPAVF and KMPV, possessed strong mucin-binding activity. Contrarily, uncharged peptides with high hydrophobicity index, including YMSV and QIGLF, exhibited weak mucin-binding activity. Only TNGQ, which has high Boman index and hydrophilicity, showed a high biosimilar mucus-permeating property with a permeability of 96 ± 30% after 60 min. TNGQ showed the potential for high bioavailability due to the high mucin-binding and biosimilar mucus-permeating activities.

Effects of sonication on the in vitro digestibility and structural properties of buckwheat protein isolates.

The present work investigated the effects of sonication at different amplitudes and durations on the in vitro digestibility of buckwheat protein isolates (BPIs). The conformation, particle size and microstructures of the BPIs were also studied to explicate the possible mechanisms of the sonication-induced changes. The results showed that sonication conditions of 20 kHz, pulsed on-time 10 s, off-time 5 s, amplitude of 60% and duration of 10 min (SA6T10) improved the digestibility of BPIs from 41.4% (control) to 58.2%. The tertiary structure analysis showed that sonication exposed the hydrophobic core buried inside the protein molecules and broke the intramolecular crosslinks, based on the increase in the surface hydrophobicity and intrinsic fluorescence and the decrease in the disulphide content. The secondary structure analysis showed that SA6T10 decreased the content of ß-turn and ß-sheet by 40.9% and 22.4%, respectively, and increased the content of anti-parallel ß-sheet, random coil, and α-helix by 40.9%, 30.6%, and 25.5%, respectively. The particle size of the control BPIs (427.7 ± 76.7 nm) increased to 2130.8 ± 356.2 nm in the SA6T10 sonicated sample with a corresponding decrease in the polydispersity index from 0.97 ± 0.04 to 0.51 ± 0.13. Moreover, scanning electron microscopy indicated that sonication broke the macroparticles into smaller fragments and changed the surface state of the proteins. Taken together, sonication has proven to be a promising approach for improving the digestibility of buckwheat proteins, which can be explored as a source of plant-based alternative protein for food applications.

Utilization of insect proteins to formulate nutraceutical delivery systems: Encapsulation and release of curcumin using mealworm protein-chitosan nano-complexes.

The potential of using insect proteins to encapsulate and protect hydrophobic nutraceuticals within biopolymer nano-complexes was examined. Insect proteins were used to form nanoparticles that were uncoated or coated with chitosan. Initially, the nature of the curcumin-mealworm protein interaction was investigated. Curcumin mainly interacted with the hydrophobic core of the insect protein nanoparticles through hydrophobic forces. About one curcumin molecule bound per protein molecule in both the absence and presence of chitosan. The binding constants (K) were 1.1 × 104 M-1 and 0.7 × 104 M-1 for curcumin loaded in the uncoated and coated nanoparticles, respectively. Differential scanning calorimetry showed increased thermal stability of the proteins after interaction with curcumin or chitosan. Encapsulation efficiency of the curcumin within the biopolymer nano-complexes was 30-47% depending on the system. Transmission electron microscopy and dynamic light scattering analysis showed that the biopolymer nano-complexes were spherical and relatively small (d = 143-178 nm). FTIR suggests that curcumin was stabilized more effectively in the coated nano-complexes, due to non-covalent intermolecular interactions. Curcumin release under oral, gastric, and intestinal conditions showed that over 90% of the nutraceutical was released after exposure to model gastrointestinal conditions. The findings demonstrate the potential of using insect proteins for fabricating colloidal delivery systems for water-insoluble nutraceuticals.

Peptide-Mineral Complexes: Understanding Their Chemical Interactions, Bioavailability, and Potential Application in Mitigating Micronutrient Deficiency.

Iron, zinc, and calcium are essential micronutrients that play vital biological roles to maintain human health. Thus, their deficiencies are a public health concern worldwide. Mitigation of these deficiencies involves micronutrient fortification of staple foods, a strategy that can alter the physical and sensory properties of foods. Peptide-mineral complexes have been identified as promising alternatives for mineral-fortified functional foods or mineral supplements. This review outlines some of the methods used in the determination of the mineral chelating activities of food protein-derived peptides and the approaches for the preparation, purification and identification of mineral-binding peptides. The structure-activity relationship of mineral-binding peptides and the potential use of peptide-mineral complexes as functional food ingredients to mitigate micronutrient deficiency are discussed in relation to their chemical interactions, solubility, gastrointestinal digestion, absorption, and bioavailability. Finally, insights on the current challenges and future research directions in this area are provided.