Hydrolysis improves the inhibition efficacy of bovine lactoferrin against infection by SARS-CoV-2 pseudovirus.

The entry of SARS-CoV-2 into host cells may involve the spike protein cleavage by cathepsin L (CTSL). Certain food proteins such as lactoferrin (Lf) inhibit CTSL. The current study investigated the impact of hydrolysis (0-180 min) by proteinase K on electrophoretic pattern, secondary structure, cathepsin inhibitory and SARS-CoV-2 pseudovirus infectivity inhibitory of bovine Lf. Gel electrophoresis indicated that hydrolysis cut Lf molecules to half lobes (∼40 kDa) and produced peptides ≤18 kDa. Approximation of the secondary structural features through analysis of the second-derivative amide I band collected by infra-red spectroscopy suggested a correlative-causative relationship between cathepsin inhibition and the content of helix-unordered structures in Lf hydrolysate. The half maximal inhibitory concentration (IC50) of Lf hydrolysed for 90 min (H90) against CTSL was about 100 times smaller than that of the Lf hydrolysed for 0 min (H0). H90 had also double activity against SARS-CoV-2 pseudo-types infectivity compared with H0.

Encapsulation of DHA oil with heat-denatured whey protein in Pickering emulsion improves its bioaccessibility.

This study compared the bioaccessibility of docosahexaenoic acid (DHA) provided encapsulated or unencapsulated within a food matrix. DHA oil was composed of DHA-enriched triacylglycerols prepared as Pickering emulsion by encapsulation with heat-denatured whey protein isolate particles and then incorporated into homogenized liquid egg to get omelets. The effect of encapsulation was analyzed by using a static in vitro digestion model of the adult, which digestive fluid enzymes have also been characterized by proteomics. First, the size of lipid droplets was shown to be smaller and uniformly dispersed in omelets with encapsulated-DHA oil compared to non-encapsulated-DHA oil. Distribution of droplets was more regular with encapsulated-DHA oil as well. As a consequence, we showed that encapsulating DHA oil promoted the hydrolysis by pancreatic lipase during the intestinal phase. A larger proportion of DHA enriched-triacylglycerols was hydrolyzed after two hours of digestion, leading to a greater release in free DHA. Thus, only 32% of DHA remained esterified in the triacylglycerols with encapsulated-DHA oil, compared to 43% with non-encapsulated-DHA oil. The DHA in free form ultimately represented 52% of the total DHA with encapsulated-DHA oil, compared to 40% with non-encapsulated-DHA oil. Finally, our results showed that as much DHA was released after one hour of intestinal digestion when the DHA oil was encapsulated as after two hours when the DHA oil was not encapsulated. Therefore, DHA bioaccessibility was significantly improved by encapsulation of DHA oil in omelets.

Emulsion gels loaded with pancreatic lipase: Preparation from spontaneously made emulsions and assessment of the rheological, microscopic and cargo release properties.

Emulsion gels are solidified emulsions, which can be used for delivery of both hydrophilic and lipophilic substances. In this research, at first fish oil-in-water (O/W; 10% w/w) emulsions were prepared through the spontaneous emulsification technique. As emulsifier, a blend of the small-molecule surfactant tween 80, and either low-acyl (LaG) or high-acyl (HaG) gellan was used. For making fully stable (100% stability index) emulsions, a 10-fold higher concentration of LaG than HaG in the emulsion aqueous phase was required. The difference in gellan concentration resulted in a bigger mean drop size, as well as lesser consistency coefficient and yield stress for HaG emulsion than LaG emulsion. Subsequently, the fully stable HaG and LaG emulsions were gelled by CaCl2 addition. LaG emulsion gel was self-supporting and had a dense microstructure (as observed by electron microscopy), whereas HaG emulsion gel was not self-supporting. Loading lipase into the emulsions before ionotropic gelation did not lead to unacceptable acid values for fish oil during the emulsion gels storage. When the lipase-loaded fish O/W emulsion gels were immersed in an acid solution which imitated the gastric fluid (yet without digestive enzymes) oil droplets flocculated (as observed by confocal microscopy). The acid immersion also increased the dynamic moduli of the gels. Lipase was not released into the surrounding acid solution from LaG emulsion gel. A subsequent immersion within an alkaline solution imitating the small intestine fluid (yet without digestive enzymes) reduced the dynamic moduli of both kinds of emulsion gels. The alkaline immersion also caused extensive crack propagation in LaG emulsion gel network, which was found associated with diminished value of tan δ (G''/G') as an index of gel energy dissipation. Lipase was released form LaG emulsion gel into the alkaline solution, however, it took a remarkable period of time to begin.

Covalent ß-lactoglobulin-maltodextrin amyloid fibril conjugate prepared by the Maillard reaction.

The surface modification of ß-lactoglobulin amyloid fibrils (AFs) was investigated by performing the Maillard reaction with the free anomeric carbon of the maltodextrin in water at pH 9.0 and 90 °C. The bonding of maltodextrin to fibrils was confirmed by determining the free amino group content and the presence of final products from the Maillard reaction. The secondary structure of AFs was preserved as observed by circular dichroism analysis. Atomic force microscopy evidenced that prolonged heat treatment caused hydrolysis of the attached polysaccharide and consequently lowered the height of the fibrils from 8.0 nm (after 1 h) to 6.0 nm (after 24 h), which led to the reduction of hydrophilicity of resulting conjugate. Increasing the reaction time, however, resulted in the improvement of colloidal stability and decrease in turbidity ascribed to the increment of glycation degree, as well as, a decrease in the isoelectric point of the protein-based supramolecular object.

Food proteins are a potential resource for mining cathepsin L inhibitory drugs to combat SARS-CoV-2.

The entry of SARS-CoV-2 into host cells proceeds by a proteolysis process, which involves the lysosomal peptidase cathepsin L. Inhibition of cathepsin L is therefore considered an effective method to decrease the virus internalization. Analysis from the perspective of structure-functionality elucidates that cathepsin L inhibitory proteins/peptides found in food share specific features: multiple disulfide crosslinks (buried in protein core), lack or low contents of (small) α-helices, and high surface hydrophobicity. Lactoferrin can inhibit cathepsin L, but not cathepsins B and H. This selective inhibition might be useful in fine targeting of cathepsin L. Molecular docking indicated that only the carboxyl-terminal lobe of lactoferrin interacts with cathepsin L and that the active site cleft of cathepsin L is heavily superposed by lactoferrin. A controlled proteolysis process might yield lactoferrin-derived peptides that strongly inhibit cathepsin L.

Gelatin as texture modifier and porogen in egg white hydrogel.

In the present study, composite egg white/gelatin hydrogels were produced and their porosity was increased through the subsequent removal of gelatin by leaching into water. The composite gel with 0.5% gelatin showed a higher degree of swelling than did the control gelatin-free sample after 60 min of immersion in an aqueous medium which was ascribed to the formation of capillary channels due to gelatin leaching. The composite gel containing 0.3% gelatin showed the highest water-holding capacity and firmness indices among all samples. Gel porosity decreased with increasing gelatin content. However, after gelatin depletion, higher concentrations of gelatin yielded hydrogels with higher porosity, as confirmed by scanning electron microscopy. Based on Fourier transform infra-red spectroscopy, it was concluded that the count of hydrogen bonds decreased after gelatin depletion. X-ray diffraction analysis indicated that intermolecular interaction between gelatin and egg white proteins had taken place in the amorphous phase.

Structural Assessment and Catalytic Oxidation Activity of Hydrophobized Whey Proteins.

Chemical modification of whey proteins allows manipulation of their characteristics, such as surface charge and hydrophobicity. Herein, we report the influence of hydrophobization accomplished by a preacetylation stage and a subsequent combined acetylation-heating process on some characteristics of whey proteins. Hydrophobization extensively (≥90%) acetylated the available free amino groups of whey proteins. The produced protein particles were nanoscaled (75 nm) and had a significantly low isoelectric point (3.70). Hydrophobization increased the ß-sheet content of whey proteins and significantly decreased the solvent exposure of tyrosine residues. It also conferred a less compact tertiary structure to the proteins and decreased the extent of disulfide-bond formation by heating. The mobility of α-lactalbumin in nonreducing electrophoresis gel increased as a consequence of hydrophobization. Then, the ability of whey proteins to catalyze hydroquinone autoxidation was examined, and it was found that the activity decreased as a result of hydrophobization. The catalytic activity of the proteins was associated with the free-amino-group content, which determined the extent of cation-π attractive interactions; ζ-potential, which determined the extent of anion-π repulsive interactions; and π-stacking between hydrophobic residues and the electron cloud of the quinone ring.

Characterization of Carboxylated Cellulose Nanocrytals Isolated through Catalyst-Assisted H2O2 Oxidation in a One-Step Procedure.

A green and facile method was designed to isolate a type of cellulose nanocrystal (CNC) with carboxylated surfaces from native cellulose materials. Because isolation and modification processes of cellulosic particles are generally performed separately using harmful chemicals and multiple steps, the one-pot approach employed in this work is interesting from both an economical and ecological point of view. The reaction is carried out by adding hydrogen peroxide as an oxidant and copper(II) sulfate as a catalyst in acidic medium under mild thermal conditions. The charge content of the carboxylated CNC is about 1.0 mmol g-1, measured by a conductometric titration. Fourier transform infrared spectroscopy also proved the presence of carboxyl groups on the CNC particles. Atomic force microscopy along with optical polarized microscopy readily showed a rod shape morphology for the cellulosic particles. An average length of 263 nm and width of 23 nm were estimated by transmission electron microscopy. Dynamic laser scattering on carboxylated CNC suspensions by adding salt confirmed that nanoparticles are electrostatically stable. Carboxylated CNCs were furthermore characterized by solid carbon-13 nuclear magnetic resonance and X-ray spectroscopy.

Interface-related attributes of the Maillard reaction-born glycoproteins.

Interfacial behavior of proteins which is a chief parameter to their emulsifying and foaming properties can be tailored through the Maillard reaction. The reaction can increase protein solubility at isoelectric point and ought to be controlled for example by high pressure processing to suppress melanoidins formation. Branched and long saccharides bring considerable steric hindrance which is associated with their site of conjugation to proteins. Conjugation with high molecular weight polysaccharides (such as 440 kDa dextran) may indeed increase the thickness of globular proteins interfacial film up to approximately 25 nm. However, an overly long saccharide can shield protein charge and slow down the electrophoretic mobility of conjugate. Maillard conjugation may decrease the diffusion rate of proteins to interface, allowing further unfolding at interface. As well, it can increase desorption iteration of proteins from interface. In addition to tempering proteins adsorption to interface, Maillard conjugation influences the rheology of protein membranes. Oligosaccharides (especially at higher glycation degrees) decrease the elastic modulus and Huggins constant of protein film; whereas, monosaccharides yield a more elastic interface. Accordingly, glycation of random coil proteins has been exploited to stiffen the corresponding interfacial membrane. Partial hydrolysis of proteins accompanied with anti-solvent-triggered nanoparticulation either before or after conjugation is a feasible way to enhance their emulsifying activity.

Bioactive whey peptide particles: An emerging class of nutraceutical carriers.

Whey-based diets have been linked with prolonged life expectancy and improved physical performance. These observations based on numerous clinical and simulated studies are attributed to diverse biological activities of whey peptides. Recently, bioactive whey peptides were exploited for enveloping nutraceuticals and drugs in view of fabricating capsules that the carrier matrix is also bioactive. Some of the most considered bioactivities of whey peptides including antihypertension, antioxidant, anti-obesity, anti-diabetes, and hypocholesterolemic properties with corresponding underlying mechanisms are briefly discussed. Then, we overview the supramolecular and gelation-prompted encapsulation of nutraceuticals with whey proteins, followed by summarizing recent developments in utilization of synthetic peptides for gene and drug delivery. Finally, particulation of bioactive whey peptides are communicated. Whey peptides may exert both biologically beneficial and technologically appreciated activities. Two procedures including desolvation and internal gelation have been so far employed for bioactive peptides particulation. Crosslinking is a prerequisite to confer acid-induced cold-set gelation to bioactive peptides. It also increases peptides Fe3+-reducing power. Surface activity of a population of peptides in whey protein hydrolysate may result in co-adsorption of the peptides together with small molecule surfactants onto oil-water interface, leading to modulated interfacial architecture and particle morphology.

Functional and in vitro gastric digestibility of the whey protein hydrogel loaded with nanostructured lipid carriers and gelled via citric acid-mediated crosslinking.

Nanostructured lipid carriers (NLCs) with mean size of 347nm were fabricated and added into a heat-denatured whey protein solution. The subsequent crosslinking of proteins by citric acid or CaCl2 resulted in the formation of cold-set hydrogels. Fourier transform infrared spectroscopy (FTIR) proposed formation of more hydrogen bonds in gel due to NLC loading or citric acid-mediated gelation. It was also found based on FITR spectroscopy that citric acid crosslinking disordered whey proteins. Scanning electron microscopy (SEM) imaging showed a non-porous and finely meshed microstructure for the crosslinked gels compared to non-crosslinked counterparts. Crosslinking also increased the firmness and water-holding capacity of gels. In pepsin-free fluid, a strong correlation existed between reduction in gel swellability and digestibility over periods up to 60min due to NLC loading and citric acid gelation. However, in peptic fluid, NLC loading and citric acid crosslinking brought about much higher decrease in digestibility than swellability.

Effect of salts and nonionic surfactants on thermal characteristics of egg white proteins.

Effect of salts (Sodium chloride (NaCl), Sodium sulfate (Na2SO4), Ammonium sulfate ((NH4)2SO4), and nonionic surfactants (glycerol, tween20, tween80) on thermal properties of egg white proteins as a whole were investigated. Egg white solutions with additive (0, 0.5 and 1%) were collected after 0, 1 and 2min heat treatment. Physico-chemical properties of egg white proteins were evaluated by measuring heat coagulation time, solubility and turbidity of solution. Adding glycerol caused the most significant decrease in turbidity and increase in heat coagulation time and solubility of egg white, although Sodium Chloride had the least positive impact on physico-chemical properties of egg white under heat treatment. The Fourier transform infrared (FT-IR) spectroscopy analysis of heat treated egg white proteins as a whole with additives demonstrated changes in secondary protein structure, which are presented regarding the shape, intensity and position of FT-IR band. Meanwhile, it showed a good correlation with the physico-chemical properties consequences. Generally, the effect of nonionic surfactants were more noticeable than that of salts in preventing of egg white proteins aggregation under heat treatment. By improving thermal stability of egg white proteins, its usage in thermal processing industry can be evolved.

Structure of starch aerogel as affected by crosslinking and feasibility assessment of the aerogel for an anti-fungal volatile release.

Starch suspensions were crosslinked with trisodium citrate for either 0 or 17h, gelled and then freeze-dried to corresponding aerogels. The aerogel from the 17h-crosslinked suspension was loaded with the antifungal compound, trans-2-hexenal, and coated with the surfactant, sorbitan monooleate. Aerogel hardness was increased by the citrate-mediated crosslinking, whereas its adhesiveness decreased. Starch gelation decreased the crystallinity index (CrI) from 59% to ≈23%; however, the pre-gelation crosslinking resulted in a higher CrI value (i.e. ≈38%) for the aerogel. The voids at the internal microstructure of the 17h-crosslinked aerogel were more uniform and coating with surfactant closed the surface openings. The latter accordingly resulted in a more sustained release of the volatile, trans-2-hexenal, from the crosslinked starch aerogel and led to slower lethality of Aspergillus parasiticus cells inoculated on pistachio nuts compared with the non-coated condition.

Textural and cargo release attributes of trisodium citrate cross-linked starch hydrogel.

An alkaline starch suspension was charged with citric acid and incubated for different durations (0, 8.5 or 17h). The suspension was then supplemented with caffeine and gelatinized to fabricate hydrogels which were subsequently stored for varying periods (0, 24 or 48h). Charging of the well-dissolved alkaline starch suspension with citric acid decreased at first both the flow index and consistency coefficient (K); however, starch cross-linking over time by the generated trisodium citrate increased the K value. The latter also inhibited gel syneresis and increased its water-holding capacity. Trisodium citrate did not nonetheless influence the gel hardness except for the sample incubated for maximum duration and stored for the longest period. The amount of the caffeine released from hydrogel decreased by citrate cross-linking and was higher at neutral pH than pH 2.0. Fourier-transform infra-red spectroscopy suggested that caffeine was enclosed within the gel network via non-covalent interactions.

Fast Protein Liquid Chromatography.

Fast protein liquid chromatography (FPLC) is a form of high-performance chromatography that takes advantage of the high resolution made possible by small-diameter stationary phases. It was originally developed for proteins and features high loading capacity, biocompatible aqueous buffer systems, fast flow rates, and availability of stationary phases in most common chromatography modes (e.g., ion exchange, gel filtration, reversed phase, and affinity). The system makes reproducible separation possible by incorporating a high level of automation including autosamplers, gradient program control, and peak collection. In addition to proteins, the method is applicable to other kinds of biological samples including oligonucleotides and plasmids. The most common type of FPLC experiment is anion exchange of proteins. This chapter describes such an experiment carried out using an ÄKTA FPLC explorer system (Amersham Pharmacia Biotech, Sweden).

Cold-set hydrogels made of whey protein nanofibrils with different divalent cations.

Whey protein nanofibrils are gaining interest to fabricate cold-set hydrogels due to their ability to gel at lower concentrations than parent proteins. In the present research, fibrillated protein solution was gelled with three different divalent cation salts including CaCl2, MnCl2 and ZnCl2 and the textural and functional characteristics of the resulting hydrogel samples were studied. Atomic force microscopy indicated that the flexible micron-scaled fibrils with nanometric thickness (up to 8.0nm) that formed at pH 2.0 underwent breaking in length upon post-formation pH rise to 7.5. Whilst heat-denatured protein solution failed to form self-supporting gel at pH 7.5, fibrillated protein solution gelled by all three types of cations. Fibrillation increased the protein solution consistency coefficient (K) much more than heat denaturation. It was suggested based on Fourier-transform infra-red (FT-IR) spectra that some hydrogen bonds were disrupted by fibrillation. Zn(2+)-induced gel was firmer, had a higher water holding capacity and a more compact microstructure, as well, required a higher compressive stress to fracture than its counterparts. Nonetheless, the Mn(2+)- and Ca(2+)-induced gels disintegrated to a much lesser extent in both pepsin-free and pepsin-present simulated gastric juice than Zn(2+)-induced sample. Chitosan coating approximately halved the simulated degradability of all gel samples.

One-pot nanoparticulation of potentially bioactive peptides and gallic acid encapsulation.

Whey protein isolate was hydrolyzed to an in vitro antioxidative hydrolysate, followed by transglutaminase-induced cross-linking and microemulsification in an oil phase. The obtained microemulsion was then dispersed in a gallic acid-rich model wastewater which caused gallic acid transportation into internal nanodroplets. Whey peptides were consequently gelled, yielding nanoparticles. Electrophoresis showed that ß-lactoglobulin and low molecular weight peptides were cross-linked by transglutaminase. Protein hydrolysis and subsequent enzymatic cross-linking increased the ζ-potential value. Microscopic investigation indicated that most particles were non-spherical. Non-cross-linked and cross-linked peptides underwent a form of heat-triggered self-assembly in the dry state, while nanoparticles did not show such behavior. Peptide crystallites size was increased by cross-linking and acid-induced particle formation. The latter also caused a reduction in intensity of C-H stretching and C-N bending peaks in infra-red spectrum. Gallic acid release from particles to simulated gastrointestinal fluids was through diffusion from swollen particles, and reached almost 70% release.

Microwave-assisted isomerisation of lactose to lactulose and Maillard conjugation of lactulose and lactose with whey proteins and peptides.

Lactose was isomerised to lactulose by microwave heating and purified by a methanolic procedure to a product with approximately 72% lactulose content. Afterwards, lactose and the lactulose-rich product (PLu) were conjugated with either whey protein isolate (WPI) or its antioxidant hydrolysate (WPH) through microwaving. Lactose had a higher Maillard reactivity than PLu, and WPH was more reactive than WPI. The browning intensity of WPI-sugar systems was however higher than that of WPH-sugar pairs. Atomic force microscopy showed larger (up to ≈103 nm) particles for WPI-PLu conjugates compared to WPH-PLu counterparts (up to ≈39 nm). The Maillard conjugation progressively increased the radical-scavenging activity of WPI/WPH-sugar pairs with increasing conjugation time and improved the foaming properties of WPI and WPH. The WPI/WPH-sugar conjugates showed higher solubility and emulsification index than unreacted counterpart pairs. For native WPI, ß-lactoglobulin was not degraded by in vitro gastric digestion, whereas for WPH-PLu conjugates degraded completely.

One-Pot Procedure for Recovery of Gallic Acid from Wastewater and Encapsulation within Protein Particles.

A whey protein isolate solution was heat-denatured and treated with the enzyme transglutaminase, which cross-linked ≈26% of the amino groups and increased the magnitude of the ζ-potential value. The protein solution was microemulsified, and then the resulting water-in-oil microemulsion was dispersed within a gallic acid-rich model wastewater. Gallic acid extraction by the outlined microemulsion liquid membrane (MLM) from the exterior aqueous phase (wastewater) and accumulation within the internal aqueous nanodroplets induced protein cold-set gelation and resulted in the formation of gallic acid-enveloping nanoparticles. Measurements with a strain-controlled rheometer indicated a progressive increase in the MLM viscosity during gallic acid recovery corresponding to particle formation. The mean hydrodynamic size of the nanoparticles made from the heat-denatured and preheated enzymatically cross-linked proteins was 137 and 122 nm, respectively. The enzymatic cross-linking of whey proteins led to a higher gallic acid recovery yield and increased the glass transition enthalpy and temperature. A similar impact on glass transition indices was observed by the gallic acid-induced nanoparticulation of proteins. Scanning electron microscopy showed the existence of numerous jammed/fused nanoparticles. It was suggested on the basis of the results of Fourier transform infrared spectroscopy that the in situ nanoparticulation of proteins shifted the C-N stretching and C-H bending peaks to higher wavenumbers. X-ray diffraction results proposed a decreased ß-sheet content for proteins because of the acid-induced particulation. The nanoparticles made from the enzymatically cross-linked protein were more stable against the in vitro gastrointestinal digestion and retained almost 19% of the entrapped gallic acid after 300 min sequential gastric and intestinal digestions.