Unraveling the effects of low protein-phenol binding affinity on the structural properties of beta-lactoglobulin.

Non-covalent interactions of phenolics with proteins cannot always be readily identified, often leading to contradictory results described in the literature. This results in uncertainties as to what extent phenolics can be added to protein solutions (for example for bioactivity studies) without affecting the protein structure. Here, we clarify which tea phenolics (epigallocatechin gallate (EGCG), epicatechin and gallic acid) interact with the whey protein ß-lactoglobulin by combining various state-of-the-art-methods. STD-NMR revealed that all rings of EGCG can interact with native ß-lactoglobulin, indicating multidentate binding, as confirmed by the small angle X-ray scattering experiments. For epicatechin, unspecific interactions were found only at higher protein:epicatechin molar ratios and only with 1H NMR shift perturbation and FTIR. For gallic acid, none of the methods found evidence for an interaction with ß-lactoglobulin. Thus, gallic acid and epicatechin can be added to native BLG, for example as antioxidants without causing modification within wide concentration ranges.

The Assembly Mechanism and Mesoscale Architecture of Protein-Polysaccharide Complexes Formed at the Solid-liquid Interface.

Protein-polysaccharide composite materials have generated much interest due to their potential use in medical science and biotechnology. A comprehensive understanding of the assembly mechanism and the mesoscale architecture is needed for fabricating protein-polysaccharide composite materials with desired properties. In this study, complex assemblies were built on silica surfaces through a layer-by-layer (LbL) approach using bovine beta-lactoglobulin variant A (ßLgA) and pectin as model protein and polysaccharide, respectively. We demonstrated the combined use of quartz crystal microbalance with dissipation monitoring (QCM-D) and neutron reflectometry (NR) for elucidating the assembly mechanism as well as the internal architecture of the protein-polysaccharide complexes formed at the solid-liquid interface. Our results show that ßLgA and pectin interacted with each other and formed a cohesive matrix structure at the interface consisting of intertwined pectin chains that were cross-linked by ßLgA-rich domains. Although the complexes were fabricated in an LbL fashion, the complexes appeared to be relatively homogeneous with ßLgA and pectin molecules spatially distributed within the matrix structure. Our results also demonstrate that the density of ßLgA-pectin complex assemblies increased with both the overall and local charge density of pectin molecules. Therefore, the physical properties of the protein-polysaccharide matrix structure, including density and level of hydration, can be tuned by using polysaccharides with varying charge patterns, thus promoting the development of composite materials with desired properties.

Micronutritional supplementation with a holoBLG-based FSMP (food for special medical purposes)-lozenge alleviates allergic symptoms in BALB/c mice: Imitating the protective farm effect.

BACKGROUND: Previously, the protective farm effect was imitated using the whey protein beta-lactoglobulin (BLG) that is spiked with iron-flavonoid complexes. Here, we formulated for clinical translation a lozenge as food for special medical purposes (FSMP) using catechin-iron complexes as ligands for BLG. The lozenge was tested in vitro and in a therapeutical BALB/c mice model. METHODS: Binding of iron-catechin into BLG was confirmed by spectroscopy and docking calculations. Serum IgE binding of children allergic or tolerating milk was assessed to loaded (holo-) versus empty (apo-) BLG and for human mast cell degranulation. BLG and Bet v 1 double-sensitized mice were orally treated with the holoBLG or placebo lozenge, and immunologically analysed after systemic allergen challenge. Human PBMCs of pollen allergic subjects were flow cytometrically assessed after stimulation with apoBLG or holoBLG using catechin-iron complexes as ligands. RESULTS: One major IgE and T cell epitope were masked by catechin-iron complexes, which impaired IgE binding of milk-allergic children and degranulation of mast cells. In mice, only supplementation with the holoBLG lozenge reduced clinical reactivity to BLG and Bet v 1, promoted Tregs, and suppressed antigen presentation. In allergic subjects, stimulation of PBMCs with holoBLG led to a significant increase of intracellular iron in circulating CD14+ cells with significantly lower expression of HLADR and CD86 compared to their stimulation with apoBLG. CONCLUSION: The FSMP lozenge targeted antigen presenting cells and dampened immune activation in human immune cells and allergic mice in an antigen-non-specific manner, thereby conferring immune resilience against allergic symptoms.

Milk Proteins-Their Biological Activities and Use in Cosmetics and Dermatology.

Milk and colostrum have high biological potential, and due to their natural origin and non-toxicity, they have many uses in cosmetics and dermatology. Research is ongoing on their potential application in other fields of medicine, but there are still few results; most of the published ones are included in this review. These natural products are especially rich in proteins, such as casein, ß-lactoglobulin, α-lactalbumin, lactoferrin, immunoglobulins, lactoperoxidase, lysozyme, and growth factors, and possess various antibacterial, antifungal, antiviral, anticancer, antioxidant, immunomodulatory properties, etc. This review describes the physico-chemical properties of milk and colostrum proteins and the natural functions they perform in the body and compares their composition between animal species (cows, goats, and sheep). The milk- and colostrum-based products can be used in dietary supplementation and for performing immunomodulatory functions; they can enhance the effects of certain drugs and can have a lethal effect on pathogenic microorganisms. Milk products are widely used in the treatment of dermatological diseases for promoting the healing of chronic wounds, hastening tissue regeneration, and the treatment of acne vulgaris or plaque psoriasis. They are also increasingly regarded as active ingredients that can improve the condition of the skin by reducing the number of acne lesions and blackheads, regulating sebum secretion, ameliorating inflammatory changes as well as bestowing a range of moisturizing, protective, toning, smoothing, anti-irritation, whitening, soothing, and antiaging effects.

Bovine ß-lactoglobulin peptides as novel carriers for flavonoids extracted with supercritical fluids from yellow onion skins.

Our study describes in detail the binding mechanism between the main flavonoids that were extracted from onion skins by supercritical CO2 and peptides from whey proteins, from the perspective of obtaining multifunctional ingredients, with health-promoting benefits. The supercritical CO2 extract had 202.31 ± 11.56 mg quercetin equivalents/g DW as the major flavonoid and antioxidant activity of 404.93±1.39 mM Trolox/g DW. The experiments on thermolysin-derived peptides fluorescence quenching by flavonoids extract allowed estimating the binding parameters, in terms of binding constants, and the number of binding sites. The thermodynamic analysis indicated that the main forces involved in complex formation were hydrogen bonds and van der Waals interactions. Molecular docking tests indicated that peptide fluorescence quenching upon gradual addition of onion skin extract might be due to flavonoids binding by Val15 -Ser21 . All 7 to 14 amino acids long peptides appeared to have affinity toward quercetin-3,4'-O-diglucoside and quercetin-4'-O-monoglucoside. The study is important as a potential solution for reuse of valuable resources, underutilized, such as whey peptides and yellow onion skins flavonoids for efficient microencapsulation, as a holistic approach to deliver healthy and nutritious food. PRACTICAL APPLICATION: A growing interest was noticed in the last years in investigating the interactions between proteins and different biologically active compounds, such as to provide knowledge for efficient development of new food, pharmaceutical, and cosmetic products. Recent studies suggest that flavonoid-protein complexes may be designed to improve the functional performance of the flavonoids. The results obtained in this study bring certain benefits in terms of exploiting the bioactive potential of both flavonoids and bioactive peptides, for developing of formulas with improved functional properties.

Mechanism of Selenium Nanoparticles Inhibiting Advanced Glycation End Products.

Selenium nanoparticles (SeNPs) have been applied in fields of nanobiosensors, environment, nanomedicine, etc. as a result of their excellent characteristics. Early studies had shown that SeNPs have certain inhibition ability against glycation, but the inhibition mechanism, especially for the influence of SeNPs on the reaction activity of glycation sites, remains unclear. The aim of the presented research was to reveal the effects of SeNPs on the ß-lactoglobulin (ß-Lg)/d-ribose glycation system at the molecular level and explore the possible inhibitory mechanism of SeNPs on the formation of advanced glycation end products (AGEs) by analyzing the glycation sites via high-performance liquid chromatography (HPLC)-Orbitrap-tandem mass spectrometry (MS/MS). Changes in contents of AGE formation and free amino acid contents had indicated that SeNPs could significantly slow the glycation process, thus attenuating the formation of AGEs. HPLC-Orbitrap-MS/MS analysis revealed that, at 6, 12, and 24 h, the number of glycation sites of glycated ß-Lg decreased from 7, 7, and 9 to 5, 5, and 6 after the intervention of SeNPs, respectively. The glycation extent of each glycation site was controlled, and the dual-glycation ability of K8, K14, K47, K91, and K101 was changed. All of these results confirmed that SeNPs could indeed slow the process of protein glycation at the molecular level. This may be the reason for SeNPs reducing the formation of AGEs during glycation. Therefore, this study shed light on the insight of how SeNPs reduce the formation of AGEs.

Beta-lactoglobulin-nutrition allergen and nanotransporter of different nature ligands therapy with therapeutic action.

ß-lactoglobulin is one of the nutrition allergens present in the milk of many mammals, with the exception of human. This protein belongs to the family of lipocalins, consisting of nine antiparallel ß-strands (ß-A to ß-I) and one α-helix. This structure allows it to serve as a nanotransporter of various nature ligands in a pH dependent manner, which allows us to confidently consider it as a reliable carrier of drugs directly into the intestine, bypassing the destructive acidic environment of the stomach. Based on the latest data, this review describes the currently known methods of reducing the allergenicity of beta-lactoglobulin, as well as the mechanisms and methods of forming complexes of this protein with ligands, which emphasizes its importance and versatility and explains the growing interest in studying its properties in recent decades, and also opens up prospects for its practical application in medicine and pharmaceuticals.

The heteropolysaccharide of Mangifera indica fruit: Isolation, chemical profile, complexation with ß-lactoglobulin and antioxidant activity.

A 91 kDa heteropolysaccharide (F2) was isolated from Mangifera indica fruit via extraction with H2O, purification by C2H5OH, starch removal and ion exchange chromatography. This polymer was made up mostly of Ara, Gal, Glc, Rha, Xyl, and GalA in a 37: 29: 9:3:2:19 molar proportion. It inherited a small backbone containing GalpA and Rhap units substituted with very large side chains containing differently linked Ara and Gal units plus esterified gallic acid (GA) residue. Several enzymes generated oligosaccharides including (i) Ara2-10Ac6-22, (ii) Gal1-8Ac5-26 and (iii) GA1Gal1Ac7 were characterized. This polysaccharide, which showed dose dependent antioxidant activity, exhibited synergism with gallic acid, and formed a complex (K = 1.2 × 106 M-1) with ß-lactoglobulin. Accordingly, H2O treatment produces a polysaccharide with desired biochemical properties; this could be effective in designing innovative functional food with flexible makeup.

In vitro study of bioaccessibility, antioxidant, and α-glucosidase inhibitory effect of pelargonidin-3-O-glucoside after interacting with beta-lactoglobulin and chitosan/pectin.

Polysaccharides and fruit extracts are applied in dairy products to enhance their nutritional property, but the effects of such formulations on the functions and biological activities are yet to be explored. Therefore, this study was aimed at evaluating the effect of interactions among milk protein (beta-lactoglobulin; BLG), polysaccharides (pectin, P; chitosan, CH), and anthocyanin (pelargonidin-3-O-glucoside; P3G) in improving the bioavailability and biological activity of P3G. After gastrointestinal digestion (GID), the content of free P3G in different model solutions were as follows: P3G-alone (73.59 µg/mL), P3G-P (66.59 µg/mL), P3G-CH (36.72 µg/mL), P3G-BLG (64.92 µg/mL), P3G-P-BLG (64.92 µg/mL), and P3G-CH-BLG (39.61 µg/mL). Less amount of free P3G in model solutions indicated increased complex formation of P3G with protein and/or polysaccharides during GID. These complexes resulted in protection and progressive release of P3G in the gastrointestinal tract. Chitosan exhibited more protection to P3G compared with P and BLG. In addition, α-glucosidase inhibitory activity and ROS scavenging activities of conjugated-P3G samples were potentially augmented after GID. However, the presence of polysaccharides and protein in the model solutions did not show any negative effect on the biological activity of P3G. Thus, pure P3G can be used as a nutritional ingredient in dairy industries.

Transient measurement and structure analysis of protein-polysaccharide multilayers at fluid interfaces.

The formation of electrostatic protein-polysaccharide multilayers has attracted attention for the design of fluid interfaces with enhanced stability and functionality. However, current techniques are often limited to measuring final multilayer properties. We present an interfacial shear rheology setup with simultaneous subphase exchange, allowing the transient measurement of biopolymer multilayers by their viscoelasticity. The successive and simultaneous adsorption of ß-lactoglobulin (ß-lg) and low-methoxyl pectin were investigated at the n-dodecane/water interface at pH 4. The successive injection of pectin increased the viscoelasticity of an adsorbed ß-lg layer by electrostatic complexation. On the other hand, simultaneous adsorption impeded adsorption kinetics and interfacial layer strength due to complexation in the bulk phase prior to adsorption. Neutron reflectometry at the air-water interface confirmed the formation of an initial ß-lg layer and electrostatic complexation of a secondary pectin layer, which desorbed upon pH-induced charge inversion. The layer formed by simultaneous adsorption mainly consisted of ß-lg. We conclude that protein-polysaccharide complexes show limited surface activity and result in a lower effective protein concentration available for adsorption.

Properties of ß-Lactoglobulin Aggregates and Gels as Affected by Ternary Emulsifier Mixtures of Tween 20, Lecithin, and Sucrose Palmitate.

The influence of sucrose palmitate, Tween 20, and lecithin on the properties of heat-induced aggregates and cold-set gels of ß-lactoglobulin was studied based on an experimental mixture design with a fixed total emulsifier concentration. Emulsifiers were added to the protein solution before heating. Aggregate size and absolute values of ζ potential increased with the addition of emulsifiers, among which lecithin had the most pronounced effect. The water retention of the aggregates correlated positively with the aggregate size. Gels had reduced fracture stress and strains with increasing sucrose palmitate and decreasing Tween 20 contents. The fracture properties correlated with the ζ potentials of the aggregates, and larger aggregates led to gels with higher water-holding capacities. The emulsifiers hence influenced the gel properties indirectly via the aggregate properties. The impact of emulsifiers on food structures should therefore be considered when a food product is designed.

Antitumor activity of selenium modification of the bovine milk component ß-Lg (Se-ß-Lg) on H22 cells.

In this study, the apoptosis induction and antitumor activity of a novel complex, seleno-ß-lactoglobulin (Se-ß-Lg), on H22 cells were explored. In in vitro experiments, the MTT assay showed that Se-ß-Lg was cytotoxic to H22 cells in a concentration- and time-dependent manner and displayed few proliferation inhibition effects on normal liver L02 cells. Annexin V-FITC/PI and PI staining assays showed that Se-ß-Lg induced apoptosis changes of H22 cells from early to late apoptosis and led to S phase cell cycle arrest. Western blot and Z-VAD-FMK inhibitor assays showed that Se-ß-Lg triggered the Fas/FasL-mediated caspase 8-dependent extrinsic death receptor pathway in H22 cells. In in vivo experiments, Se-ß-Lg effectively repressed the growth of transplanted H22 solid tumors in a dose-dependent manner and exhibited few toxic effects on the host animals. H&E and PI staining of tumor tissues showed that Se-ß-Lg caused the occurrence of typical apoptosis morphology features and dose-dependently increased the proportion of apoptosis peaks (Sub-G1 peak) in H22 solid tumors. These results suggest that Se-ß-Lg has the capacity to induce H22 tumor cell apoptosis in vitro and in vivo and support that Se-ß-Lg can be applied as a functional complex in food.

How do biological stimuli modulate conformational changes of biomedical thermoresponsive polymer?

Continuing efforts to develop stimuli-responsive polymers (SRPs) as novel smart materials/biomaterials are anticipated to upgrade the quality life of humans. The details of the molecular, physico chemical and biophysical interactions between SRPs and proteins are not fully understood. Indeed, protein - polymer interactions play a major role in a wide range of biomedical/biomaterial applications. In this regard, we have demonstrated the influence of proteins (ß-lactoglobulin (BLG) and stem bromelain (BM) as biological stimuli) on the phase transition behavior of biomedical thermoresponsive poly(N-isopropylacrylamide) (PNIPAM). In order to predict these, we have used a set of biophysical techniques to unveil the influence of biological stimuli on the phase transition behavior of PNIPAM. Absorption spectroscopy, steady-state fluorescence spectroscopy, Fourier transform infrared (FTIR) spectroscopy, field emission scanning electron microscopy (FESEM) were operated at room temperature to examine the changes in absorbance, fluorescence intensity, molecular interactions and surface morphologies, respectively. Furthermore, temperature dependent fluorescence spectroscopy and dynamic light scattering (DLS) studies were also performed to analyze conformational changes, agglomeration behavior, particle size, coil to globule transition and phase behavior. The significant variations obtained in the phase transition temperature values, conformational changes and agglomeration behavior clearly reflects the different molecular interplay induced in presence of biological stimuli. The results demonstrated that the added proteins act as biological stimuli via preferential interactions between the amide group of the polymer and water molecules. The present study can be useful for the design and development of the next generation smart responsive materials/biomaterials.

Identification of angiotensin converting enzyme and dipeptidyl peptidase-IV inhibitory peptides derived from oilseed proteins using two integrated bioinformatic approaches.

Angiotensin-converting enzyme (ACE) and dipeptidyl peptidase-IV (DPP-IV) play critical roles in the development of hypertension and type 2 diabetes, respectively. Inhibiting ACE and DPP-IV activity using peptides has become part of new therapeutic strategies for supporting medicinal treatment of both diseases. In this study, oilseed proteins, including soybean, flaxseed, rapeseed, sunflower and sesame are evaluated for the possibility of generating ACE and DPP-IV inhibitory peptides using different integrated bioinformatic approaches (UniProt knowledgebase, ProtParam, BLAST, BIOPEP, PeptideRanker, Pepsite2 and ToxinPred), and three bovine proteins (ß-lactoglobulin, ß-casein and κ-casein) as comparisons. Compared with bovine proteins, the potency indices of ACE and DPP-IV inhibitory peptides, calculated using the BIOPEP database, suggest that oilseed proteins may be considered as good precursors of ACE inhibitory peptides but generate a relative lower yield of DPP-IV inhibitory peptides following subtilisin, pepsin (pH = 1.3) or pepsin (pH > 2) hydrolysis. Average scores aligned using PeptideRanker confirmed oilseed proteins as significant potential sources of bioactive peptides: over 105 peptides scored over 0.8. Pepsite2 predicted that these peptides would largely bind via Gln281, His353, Lys511, His513, Tyr520 and Tyr523 of ACE to inhibit the enzyme, while Trp629 would be the predominant binding site of peptides in reducing DPP-IV activity. All peptides were capable of inhibiting ACE and DPP-IV whilst 65 of these 105 peptides are not currently recorded in BIOPEP database. In conclusion, our in silico study demonstrates that oilseed proteins could be considered as good precursors of ACE and DPP-IV inhibitory peptides as well as so far unexplored peptides that potentially have roles in ACE and DPP-IV inhibition and beyond.

Oral administration of lipid oil-in-water emulsions performed with synthetic or protein-type emulsifiers differentially affects post-prandial triacylglycerolemia in rats.

In this study, we compared the impact of administration of size-calibrated lipid emulsions prepared with either synthetic or natural emulsifiers on the post-absorptive plasma triacylglycerol responses in rats. We did this using four types of size-calibrated (10 µm diameter) and metastable (3 days) emulsions with 20% of an oleic acid-rich sunflower oil and 1% of either synthetic emulsifiers (Tween 80 or sodium 2-stearoyl-lactylate) or two proteins (ß-lactoglobulin or sodium caseinate). An oral fat tolerance test was performed in fasted rats by oral administration of each of these formulations in continuous or emulsified forms. Kinetic parameters (AUC0-inf., AUC0-6h, Cmax, Tmax, and T1/2) for the description of the plasma triacylglycerol responses were calculated. AUC0-6h and AUC0-inf. calculated for the protein groups were significantly lower than those of the control and the synthetic groups. These lower values were associated with significant decreases in the Cmax, exacerbated by the emulsion form and with marked decreases in the Tmax as compared to the control group. T1/2 values were differentially affected by the lipid administration forms and by the nature of the emulsifiers. As compared with the control group, T1/2 was largely increased in the sodium stearoyl-2-lactylate group, but on the contrary, largely lowered in the casein group. We concluded that the use of proteins as natural emulsifiers in lipid emulsions decreased the magnitude of post-prandial triacylglycerolemia for the same amount of ingested lipids, when the emulsion size is controlled for. Proteins could be a promising alternative to the widespread use of synthetic emulsifiers in the food industry.

Nanomechanics of Pectin-Linked ß-Lactoglobulin Nanofibril Bundles.

Nanofibrils of ß-lactoglobulin can be assembled into bundles by site-specific noncovalent cross-linking with high-methoxyl pectin (Hettiarachchi et al. Soft Matter 2016, 12, 756). Here we characterized the nanomechanical properties of bundles using atomic force microscopy and force spectroscopy. Bundles had Gaussian cross sections and a mean height of 17.4 ± 1.4 nm. Persistence lengths were calculated using image analysis with the mean-squared end-to-end model. The relationship between the persistence length and the thickness had exponents of 1.69-2.30, which is consistent with previous reports for other fibril types. In force spectroscopy experiments, the bundles stretched in a qualitatively different manner to fibrils, and some of the force curves were consistent with peeling fibrils away from bundles. The flexibility of pectin-linked nanofibril bundles is likely to be tunable by modulating the stiffness and length of fibrils and the ratio of pectin to fibrils, giving rise to a wide range of structures and functionalities.

Structure-affinity relationship of the interaction between phenolic acids and their derivatives and ß-lactoglobulin and effect on antioxidant activity.

In this study, 71 phenolic acids and their derivatives were used to investigate the structure-affinity relationship of ß-lactoglobulin binding, and the effect of this interaction on antioxidant activity. Based on a fluorescence quenching method, an improved mathematical model was adopted to calculate the binding constants, with a correction for the inner-filter effect. Hydroxylation at the 3-position increased the affinity of the phenolic acids for ß-lactoglobulin, while hydroxylation at the 2- or 4-positions had a negative effect. Complete methylation of all hydroxy groups, except at the 3-position, enhanced the binding affinity. Replacing the hydroxy groups with methyl groups at the 2-position also had a positive effect. Hydrogen bonding was one of the binding forces for the interaction. The antioxidant activity of phenolic acid-ß-lactoglobulin complexes was higher than that of phenolic acids alone. These findings provide an understanding of the structure-activity relationship of the interaction between ß-lactoglobulin and phenolic acids.

Development, physicochemical characterization and cytotoxicity of selenium nanoparticles stabilized by beta-lactoglobulin.

Novel Selenium nanoparticles (SeNPs) were developed using beta-lactoglobulin (Blg) as a stabilizer in redox systems of selenite and ascorbic acid in this study. Particle size, morphology, stability, and in vitro biological activity of synthesized Blg stabilized selenium nanoparticles (Blg-SeNPs) were characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), ultraviolet-visible spectrophotometry (UV/Vis), and cell toxicity assays, respectively. Stabilizing mechanisms of Blg-SeNPs were investigated by Fourier-transform infrared spectroscopy (FTIR) and protein fluorescence probe. The results revealed that the Blg-SeNPs were spherical with mean particle size of 36.8±4.1nm. They were stable in acidic or neutral to basic solutions (pH 2.5-3.5 or 6.5-8.5) at 4°C for 30days as a result of electrostatic repulsions. FTIR results showed that functional groups of NH2 and OH on Blg molecules were responsible for binding with SeNPs. Furthermore, decreases in protein surface hydrophobicity indicated that possible binding happened between Se and the hydrophobic domains of Blg. The cell toxicity of Blg-SeNPs was significantly lower than that of sodium selenite on both cancerous and non-cancerous cells. This study provides a facile and green method for chemically synthesizing stable SeNPs which are suitable for further evaluation in medicinal applications.

Amyloid fibril systems reduce, stabilize and deliver bioavailable nanosized iron.

Iron-deficiency anaemia (IDA) is a major global public health problem. A sustainable and cost-effective strategy to reduce IDA is iron fortification of foods, but the most bioavailable fortificants cause adverse organoleptic changes in foods. Iron nanoparticles are a promising solution in food matrices, although their tendency to oxidize and rapidly aggregate in solution severely limits their use in fortification. Amyloid fibrils are protein aggregates initially known for their association with neurodegenerative disorders, but recently described in the context of biological functions in living organisms and emerging as unique biomaterial building blocks. Here, we show an original application for these protein fibrils as efficient carriers for iron fortification. We use biodegradable amyloid fibrils from ß-lactoglobulin, an inexpensive milk protein with natural reducing effects, as anti-oxidizing nanocarriers and colloidal stabilizers for iron nanoparticles. The resulting hybrid material forms a stable protein-iron colloidal dispersion that undergoes rapid dissolution and releases iron ions during acidic and enzymatic in vitro digestion. Importantly, this hybrid shows high in vivo iron bioavailability, equivalent to ferrous sulfate in haemoglobin-repletion and stable-isotope studies in rats, but with reduced organoleptic changes in foods. Feeding the rats with these hybrid materials did not result in abnormal iron accumulation in any organs, or changes in whole blood glutathione concentrations, inferring their primary safety. Therefore, these iron-amyloid fibril hybrids emerge as novel, highly effective delivery systems for iron in both solid and liquid matrices.

Inhibition of Snake Venom Metalloproteinase by ß-Lactoglobulin Peptide from Buffalo (Bubalus bubalis) Colostrum.

Bioactive peptide research has experienced considerable therapeutic interest owing to varied physiological functions, efficacy in excretion, and tolerability of peptides. Colostrum is a rich natural source of bioactive peptides with many properties elucidated such as anti-thrombotic, anti-hypertensive, opioid, immunomodulatory, etc. In this study, a variant peptide derived from ß-lactoglobulin from buffalo colostrum was evaluated for the anti-ophidian property by targeting snake venom metalloproteinases. These are responsible for rapid local tissue damages that develop after snakebite such as edema, hemorrhage, myonecrosis, and extracellular matrix degradation. The peptide identified by LC-MS/MS effectively neutralized hemorrhagic activity of the Echis carinatus venom in a dose-dependent manner. Histological examinations revealed that the peptide mitigated basement membrane degradation and accumulation of inflammatory leucocytes at the venom-injected site. Inhibition of proteolytic activity was evidenced in both casein and gelatin zymograms. Also, inhibition of fibrinolytic and fibrinogenolytic activities was seen. The UV-visible spectral study implicated Zn2+ chelation, which was further confirmed by molecular docking and dynamic studies by assessing molecular interactions, thus implicating the probable mechanism for inhibition of venom-induced proteolytic and hemorrhagic activities. The present investigation establishes newer vista for the BLG-col peptide with anti-ophidian efficacy as a promising candidate for therapeutic interventions.