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1.
Phys Chem Chem Phys ; 22(4): 2142-2156, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31912070

RESUMO

There are several important phenomena in chemistry, biology, and physics where molecules (or parts of a molecule) having charges of the same sign come closer together and become stable. DNA condensation, RNA folding, colloid-colloid interactions are some of the examples of this kind. In the current work, we have investigated how ß-lactoglobulin, a protein found in milk, in spite of carrying +13 charge, favors the homodimer formation in the presence of salt. We have focussed on calculating the protein-protein binding free energy in the presence of salt and identifying the thermodynamic and microscopic mechanism of the process. Estimation of binding free energy of this salt-dependent process is done by combining molecular dynamics simulation with statistical mechanical theory of three-dimensional reference interaction site model (3D-RISM). Binding free energy is evaluated from the chemical potential of the solutes as opposed to potential of mean force calculation, which gives only a constrained free energy. Our calculated values semi-quantitatively match with the experimental results. By examining the different components of binding free energy, we have found that the role of salt ions (especially of Cl-) is to shift the equilibrium towards the dimer. Non-polar (Lennard-Jones) interactions between the monomers is also favorable to the binding free energy. However, water slightly disfavors the dimer formation. For the microscopic mechanism, heterogeneous of both Na+ and Cl- near the charged residues at the binding interface and change of this charge distribution on dimer formation contribute to the stability. A fine-tuning of enthalpic and entropic effects of salt ions is found to operate at different salt concentrations. Both thermodynamic and microscopic mechanism of dimer formation gives detailed insight into the complex electrostatics of charged protein-protein binding.


Assuntos
Lactoglobulinas/química , Modelos Moleculares , Simulação de Dinâmica Molecular , Sais/química , Dimerização , Lactoglobulinas/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína
2.
Food Chem ; 302: 125349, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31442700

RESUMO

Knowledge about the critical interfacial concentration of a protein supports our understanding of the kinetic stability of an emulsion. Its determination is currently limited to either invasive or indirect methods. The aim of our study was the determination of the critical interfacial concentration of whey protein ß-lactoglobulin at oil/water-interfaces through fluorescence and pendant drop analysis and the comparison to an in situ Fourier-transform-infrared-spectroscopy (FTIR) method. Exponentially decreasing interfacial tension with increasing ß-lactoglobulin content (0.10-1.00 wt%) in pendant drop analysis could partly be confirmed by fluorescence spectra. A critical interfacial concentration of 0.20-0.31 wt% ß-lactoglobulin (1.80-2.69 mg/m2) in oil/water (5/95)-emulsions was determined via FTIR, analyzing the Amide I/Amide II peak intensity ratio. This was confirmed by the increasing formation of intermolecular ß-sheets, revealed by second derivative spectra. With this FTIR method we expand current options to investigate the interfacial behavior of food proteins by determination of secondary structure elements.


Assuntos
Lactoglobulinas/química , Óleos/química , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Emulsões/química , Cinética , Estrutura Secundária de Proteína , Dodecilsulfato de Sódio/química , Tensão Superficial , Água/química
3.
Food Chem ; 304: 125442, 2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-31491714

RESUMO

In this study, the effects of moderate electric fields during thermal denaturation of ß-lactoglobulin were examined through an in situ circular dichroism approach, complemented by intrinsic extrinsic fluorescence analysis. Results have shown that the effects of electric fields in protein unfolding were linearly dependent on the applied electric field intensity (V/cm) and increased by the use of low electric frequencies - i.e. 50 to 200 Hz. These electric effects caused significant changes on ß-lactoglobulin melting temperature, unfolded conformation and subsequent intermolecular interactions, revealed by the increase of surface hydrophobicity (ANS affinity) and higher conservation of retinol binding. The obtained data provides a clear evidence that moderate electric fields contribute to distinct folding/unfolding of ß-lactoglobulin, resulting in structural modifications. These findings are relevant for (bio)-technological applications involving electric fields processing, bringing new insights for the development of innovative strategies to control protein function and tune production of functional protein systems.


Assuntos
Campos Eletromagnéticos , Lactoglobulinas/química , Desdobramento de Proteína , Temperatura Ambiente , Dicroísmo Circular , Interações Hidrofóbicas e Hidrofílicas , Lactoglobulinas/metabolismo , Conformação Proteica
4.
Food Chem ; 308: 125596, 2020 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-31648097

RESUMO

The protective mechanism of glycerol on ß-lactoglobulin were studied in 0-60% glycerol solutions by experimental and molecular simulation approaches. Results showed that the stability of ß-lactoglobulin increased with glycerol concentration, with little secondary structure changes induced by glycerol. The tertiary structure altered slightly with glycerol concentration, resulting in a stronger near UV circular dichroism signal and intrinsic tryptophan fluorescence quenching, indicating aromatic side chains closer to hydrophobic microenvironment. The Rg of ß-lactoglobulin increased with glycerol concentration without dimer dissociation, due to expansion of the quaternary structures. Moreover, the flexibility (RMSF) of ß-lactoglobulin decreased by glycerol. Distance between areas enclosing Asp33 and Arg40 from separate chains did not increase, suggesting possibly reinforced electrostatic attractions. In conclusion, the stabilization of ß-lactoglobulin in glycerol solution is probably the comprehensive results of the decreased molecular flexibility, the strengthened hydrophobic interaction in individual chain, and the possibly reinforced electrostatic attractions between two chains of ß-lactoglobulin.


Assuntos
Glicerol/química , Lactoglobulinas/química , Dicroísmo Circular , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
5.
J Sci Food Agric ; 100(2): 607-613, 2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-31591730

RESUMO

BACKGROUND: α-Dicarbonyl compounds are widely generated in the Maillard reaction, caramelization and oil oxidation during heat treatment. These compounds can readily react with lysine and arginine residues of a protein, whereas the influence of these compounds on protein structure and quality has seldom been revealed. This study compared influence of glycation by glucose and α-dicarbonyl compounds on amyloid-like aggregation of ß-lactoglobulin (ß-LG), both fibrillation kinetics and conformation of aggregates were studied. RESULTS: Compared with glycation by glucose, the glycation by α-dicarbonyl compounds resulted in faster reduction of free amino group, sulfydryl group, and the relative content of ß-sheet secondary structure, according to the ultraviolet (UV) spectra or circular dichroism (CD) spectra results. Based on the analysis of fibrillation kinetics using thioflavin T (ThT) binding assay, the glycation by α-dicarbonyls were more efficient in suppressing the growth of fibrillar aggregates. In addition, glycation by α-dicarbonyl resulted in amorphous oligomers, which were compared with the amyloid-like aggregates in control and glucose-glycated samples, based on the transmission electron microscopy (TEM) observation. CONCLUSIONS: Glycation by α-dicarbonyl compounds induced larger decline in the ß-sheet structure of ß-LG than glycation by glucose, and thus largely suppressed the amyloid-like aggregation of ß-LG and changed the morphology of aggregates. © 2019 Society of Chemical Industry.


Assuntos
Amiloide/química , Lactoglobulinas/química , Animais , Bovinos , Dicroísmo Circular , Glucose/química , Glicosilação , Temperatura Alta , Agregados Proteicos , Estrutura Secundária de Proteína
6.
J Agric Food Chem ; 67(45): 12511-12519, 2019 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-31626537

RESUMO

Self-assembled and cross-linked hybrid hydrogels for entrapment and delivery of hydrophilic and hydrophobic bioactive compounds were developed based on N-acetyl-l-cysteine (NAC)- or l-cysteine (CYS)-functionalized chitosan-ß-lactoglobulin nanoparticles (NPs). In both the systems, amphiphilic protein ß-lactoglobulin (ß-lg) was self-assembled by using glutaraldehyde for affinity binding with egg white-derived peptides (EWDP) and curcumin and then coated with NAC- or CYS-functionalized chitosan (CS) by electrostatic interaction. The resulting NPs were characterized in terms of size, polydispersity, and surface charge by dynamic light scattering. Results corroborated pH-sensitive properties of NAC-CS-ß-lg NPs and CYS-CS-ß-lg NPs with the particle size as small as 118 and 48 nm, respectively. The two kinds of NPs also showed excellent entrapment of EWDP and curcumin with the entrapment efficiency (EE) of EWDP and curcumin ranging from 51 to 89% and 42 to 57% in NAC-CS-ß-lg NPs, as well as 50-81% and 41-57% in CYS-CS-ß-lg NPs under different pH values. Fourier transform infrared and molecular docking studies provided support for the interaction mechanism of NAC/CYS-CS with ß-lg as well as the NPs with EWDP and curcumin. Strikingly, the in vitro release kinetics of EWDP and curcumin exhibited the controlled and sustained release properties up to 58 and 70 h from the NPs, respectively. Note that the permeability of QIGLF (pentapeptide, isolated from EWDP) and curcumin passing through Caco-2 cell monolayers were all improved after the entrapment in the NPs. This work offers promising methods for effective entrapment and oral delivery of both hydrophilic and hydrophobic bioactive compounds.


Assuntos
Acetilcisteína/química , Quitosana/química , Curcumina/química , Cisteína/química , Sistemas de Liberação de Medicamentos/métodos , Lactoglobulinas/química , Células CACO-2 , Curcumina/metabolismo , Portadores de Fármacos/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Simulação de Acoplamento Molecular , Nanopartículas/química , Tamanho da Partícula , Peptídeos/química
7.
J Agric Food Chem ; 67(42): 11741-11751, 2019 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-31566971

RESUMO

Dietary phenolic compounds display strong antioxidant capabilities but face limited practical applications as a result of their poor biocompatibility (high immune resistance). Some food proteins possess mild antioxidant capabilities but are often not sufficient to maintain a reactive oxidative species balance. In this study, we overcome these barriers by covalently conjugating a natural phenolic antioxidant, gentisic acid (GA), onto an antioxidant protein, ß-lactoglobulin (ßLG). Upon optimization of conjugation conditions, we confirm the formation of ßLG-GA conjugates with mass spectrometry, Fourier transform infrared spectroscopy, and ultraviolet-visible absorption. Surface charge analysis revealed a saturation molar ratio of 150:1 (GA/ßLG), while far-ultraviolet circular dichroism revealed substantial changes in the protein secondary structure upon conjugation. The antioxidant capability of resultant conjugates was probed by monitoring the decay of 1,1-diphenyl-2-picrylhydrazyl radical content via time-resolved electron paramagnetic resonance spectroscopy, which suggested two possible pathways to scavenge radicals, i.e., the antioxidant GA on the protein surface and the protein conformational change that exposes more antioxidant amino acids. To our best knowledge, this work is the first report on the fabrication of a dual-effect antioxidant biopolymer using a nature-inspired template via covalent linking with the antioxidant mechanism probed. Our findings are essential for opening a new route to design functional materials with enhanced antioxidant activity and biocompatibility.


Assuntos
Antioxidantes/química , Gentisatos/química , Lactoglobulinas/química , Animais , Bovinos , Dicroísmo Circular , Espectrometria de Massas , Espectroscopia de Infravermelho com Transformada de Fourier
8.
J Agric Food Chem ; 67(43): 12054-12060, 2019 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-31560529

RESUMO

The purpose of current research is to design and acquire novel biological macromolecule materials with enhanced functional properties. Chitosan-ferulic acid binary conjugate (CFC) was synthesized based on the carbodiimide-mediated coupling reaction, and then ß-lactoglobulin-ferulic acid-chitosan ternary conjugate (BFCC) was fabricated by laccase induction. Furthermore, the impact of laccase concentration on the formation mechanism of BFCC was investigated by the analyses of reaction group content, ultraviolet-visible (UV-vis) absorption, circular dichroism (CD), and fluorescence spectroscopy. Results showed that hetero- and homo-conjugates between CFC and ß-lactoglobulin (ß-LG) were achievable at the low concentration (≤4 U/mL) and high concentration (≥6 U/mL) of laccase, respectively. The CD spectrum indicated that the interaction with CFC made ß-LG more disorderly. Functional evaluation results revealed that the antioxidant activity and thermal stability of BFCC were improved compared with ß-LG. The knowledge obtained in the present study provided an effective method to acquire innovative biological macromolecule materials with desirable functional characteristics.


Assuntos
Quitosana/química , Ácidos Cumáricos/química , Lacase/química , Lactoglobulinas/química , Animais , Biocatálise , Bovinos , Dicroísmo Circular , Proteínas Fúngicas/química , Conformação Proteica , Trametes/enzimologia
9.
Food Chem ; 301: 125298, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31387044

RESUMO

A synthetic scenario for functionalization of ß-lactoglobulin (ßLg) with polymeric units containing caffeic acid (ßLg-polyCA) was developed; and all intermediates and final products were structurally confirmed using nuclear magnetic resonance spectroscopy, matrix assisted laser desorption ionization time-of-flight mass spectrometry, and physico-chemically characterized using differential scanning calorimetry and circular dichroism. The antioxidant properties and emulsion stability of ßLg, ßLg-CA conjugate and ßLg-polyCA based systems containing high percentage of fish oil (50%) were evaluated; and ßLg-polyCA presented the highest antioxidant and free radical-scavenging activity based on DPPH, ABTS and HS scavenging assays (92.4, 87.92 and 67.35% respectively). Thiobarbituric acid (TBARS) test demonstrated that compared to native ßLg, ßLg-polyCA afford up 4-5 fold of inhibition of oxidative rancidity and displayed drastic secondary structure changes. Compared to native ßLg based emulsions, ßLg-polyCA had larger oil droplet sizes, stronger negative zeta potentials (-69.9 mv), narrower size distributions (PDI: 0.22) and less creaming index.


Assuntos
Antioxidantes/farmacologia , Emulsões/química , Óleos de Peixe/química , Lactoglobulinas/química , Fenóis/química , Antioxidantes/química , Ácidos Cafeicos/química , Varredura Diferencial de Calorimetria , Dicroísmo Circular , Difusão Dinâmica da Luz , Peroxidação de Lipídeos , Espectroscopia de Ressonância Magnética , Oxirredução , Polimerização , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
10.
Chem Commun (Camb) ; 55(74): 11143-11146, 2019 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-31463510

RESUMO

We develop a membrane technology based on amyloid fibrils to remove aluminium from water and minimize its exposure to humans. We study aluminium adsorption by amyloid fibrils by evaluating the binding isotherms, the thermodynamics and the effects of different parameters. Amyloid-based membranes demonstrate outstanding removal efficiencies beyond 98%.


Assuntos
Alumínio/metabolismo , Proteínas Amiloidogênicas/metabolismo , Lactoglobulinas/metabolismo , Membranas Artificiais , Poluentes Químicos da Água/metabolismo , Purificação da Água/métodos , Adsorção , Alumínio/química , Proteínas Amiloidogênicas/química , Bebidas , Concentração de Íons de Hidrogênio , Lactoglobulinas/química , Ligação Proteica , Temperatura Ambiente , Termodinâmica , Águas Residuárias/química , Poluentes Químicos da Água/química
11.
J Agric Food Chem ; 67(37): 10412-10422, 2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31464443

RESUMO

In this study, aggregation of fucoxanthin (FX) and its effects on binding and delivery properties of whey proteins were explored. Initially, the H- and J-aggregates of FX were successfully prepared by adjusting the water/ethanol ratio and water-dripping rate. The transition from J- to H-aggregates was observed over the standing time. Then, the molecular arrangement of FX H-aggregates was analyzed using the point-dipole approximation model and molecular dynamics, showing that their intermolecular distance and angle were about 5.0-6.7 Å and -35° to 35°, respectively. The transformation of J- to H-aggregates was also observed during molecular dynamics, with a shortened intermolecular distance, a reduced solvent accessible surface area, an enhanced interaction force, and a narrowed dihedral angle. Further, the interactions of whey proteins with different forms of FX were investigated, indicating that both ß-lactoglobulin and whey protein isolates could form complexes with the monomers, H-aggregates, and J-aggregates of FX. In terms of affinity, whey proteins bound FX monomers more strongly than aggregates. Furthermore, the complexes comprising whey proteins and monomeric FX had better delivery capabilities than aggregated FX, manifested in encapsulation efficiency, physical stability, and bioaccessibility.


Assuntos
Proteínas do Soro do Leite/química , Xantofilas/química , Concentração de Íons de Hidrogênio , Lactoglobulinas/química , Simulação de Dinâmica Molecular , Agregados Proteicos , Ligação Proteica
12.
Soft Matter ; 15(31): 6362-6368, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31298681

RESUMO

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.


Assuntos
Biopolímeros/química , Lactoglobulinas/química , Pectinas/química , Adsorção , Cinética , Transição de Fase , Reologia , Eletricidade Estática , Viscosidade
13.
J Agric Food Chem ; 67(34): 9601-9610, 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31334648

RESUMO

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.


Assuntos
Emulsificantes/química , Lactoglobulinas/química , Lecitinas/química , Polissorbatos/química , Sacarose/análogos & derivados , Emulsões/química , Géis/química , Temperatura Alta , Agregados Proteicos , Sacarose/química , Viscosidade
14.
Food Chem ; 298: 125024, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31261007

RESUMO

We investigated the allergenicity, digestibility and functional properties of whey protein isolate (WPI) after covalent conjugation with chlorogenic acid (CHA). The covalent conjugation of CHA may cause an unfolded protein structure. The WPI-CHA conjugate showed lower IgE binding capacity but higher intestinal digestibility than unmodified WPI. Furthermore, after digestion, the IgE binding capacity of ß-lactoglobulin and α-lactoalbumin was lower in the digested WPI-CHA conjugate than digested WPI. Moreover, the solubility, emulsifying activity, foaming properties and antioxidant capacity of WPI were enhanced by covalent conjugation of CHA. Covalent conjugation with CHA might reduce the allergenicity in vitro of WPI by improving the functional properties of the protein.


Assuntos
Alérgenos/imunologia , Proteínas do Soro do Leite/química , Proteínas do Soro do Leite/imunologia , Alérgenos/química , Ácido Clorogênico/química , Digestão , Emulsões/química , Lactoglobulinas/química , Solubilidade
15.
Molecules ; 24(11)2019 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-31181617

RESUMO

The compound 3,3'-diindolylmethane (DIM) has a broad spectrum of anticancer activities. However, low stability and bioavailability limit its application. Elucidating interactions between DIM and ß-lactoglobulin (ß-LG) may be useful for fabricating whey protein-based protecting systems. Interaction with DIM increased the diameter and absolute zeta potential value of ß-LG. UV-absorption spectra suggested that there was a complex of DIM and ß-LG. ß-LG showed enhanced fluorescence intensity by complexing with DIM with a binding constant of 6.7 × 105 M-1. Upon interaction with DIM, ß-LG was decreased in secondary structure content of helix and turn while increased in ß-sheet and unordered. FT-IR spectra and molecular docking results indicated the roles of hydrophobic interaction and hydrogen bond for the formation of DIM and ß-LG nanocomplexes. Data suggested that ß-LG may be a good vehicle for making a protein-based DIM protection and delivery system due to the tight binding of DIM to ß-LG.


Assuntos
Anticarcinógenos/metabolismo , Indóis/metabolismo , Lactoglobulinas/química , Lactoglobulinas/metabolismo , Anticarcinógenos/química , Estabilidade de Medicamentos , Interações Hidrofóbicas e Hidrofílicas , Indóis/química , Modelos Biológicos , Simulação de Acoplamento Molecular , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Espectroscopia de Infravermelho com Transformada de Fourier , Biologia de Sistemas , Proteínas do Soro do Leite/química , Proteínas do Soro do Leite/metabolismo
16.
Food Funct ; 10(6): 3626-3636, 2019 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-31162493

RESUMO

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.


Assuntos
Lactoglobulinas/farmacologia , Leite/química , Selênio/farmacologia , Animais , Apoptose/efeitos dos fármacos , Caspase 8/genética , Caspase 8/metabolismo , Bovinos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Lactoglobulinas/química , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Selênio/química
17.
Int J Biol Macromol ; 136: 804-812, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31228500

RESUMO

To investigate the interaction mechanism between bovine protein ß-lactoglobulin (ß-LG) and theaflavin (TA), chlorogenic acid (CA) and delphinidin-3-O-glucoside (D3G), multi-spectrometry analytical methods and molecular modeling were applied. Fluorescence experiments proved that polyphenols strongly quenched the intrinsic fluorescence of ß-LG mainly through static quenching and the main interaction force was hydrophobic interaction. Moreover, Fourier transform infrared (FTIR) and circular dichroism (CD) indicated that polyphenols changed ß-LG secondary and tertiary structure. Enzyme-linked immunosorbent assay and molecular modeling study manifested that complex of ß-LG with polyphenols could significantly reduce the IgE-binding capacity of ß-LG due to the polyphenol binding site directly obscures the IgE linear epitopes. In conclusion, polyphenols had impact on the structure and potential functionality of ß-LG, which would be valuable in dairy processing industry and food nutrition security.


Assuntos
Lactoglobulinas/metabolismo , Polifenóis/metabolismo , Animais , Bovinos , Lactoglobulinas/química , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica , Termodinâmica
18.
Int J Pharm ; 567: 118470, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31252148

RESUMO

Powders containing one of four model proteins (myoglobin, bovine serum albumin, lysozyme, ß-lactoglobulin) were formulated with either sucrose, trehalose, or mannitol and dried using lyophilization or spray-drying. The powders were characterized using solid-state Fourier transform infrared spectroscopy (ssFTIR), solid-state fluorescence spectroscopy, differential scanning calorimetry (DSC) and solid-state hydrogen/deuterium exchange mass spectrometry (ssHDX-MS). ssFTIR and fluorescence spectroscopy identified minor structural differences among powders with different excipients and drying methods for some proteins. Using ssHDX-MS, differences in protein structure were observed among protein formulations containing sucrose or trehalose and mannitol, and/or with varying processing conditions, including proteins like ß-lactoglobulin, for which standard characterization techniques showed no differences. Proteins processed by spray-drying typically showed greater heterogeneity by ssHDX-MS than those lyophilized; these differences were not detected by ssFTIR or solid-state fluorescence spectroscopy. The ssHDX-MS metrics were better correlated with protein physical instability measured by size-exclusion chromatography in 90-day stability studies (40 °C, 33% RH) than with the results of DSC, ssFTIR, or fluorescence spectroscopy. Thus, ssHDX-MS detected subtle changes in conformation and/or matrix interactions for these proteins that were correlated with storage stability, suggesting that the method can be used to design robust solid-state pharmaceutical protein products more rapidly.


Assuntos
Dessecação/métodos , Lactoglobulinas/química , Muramidase/química , Mioglobina/química , Soroalbumina Bovina/química , Excipientes/química , Liofilização , Conformação Proteica , Estabilidade Proteica
19.
J Agric Food Chem ; 67(27): 7775-7782, 2019 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-31088053

RESUMO

Analyzing an in vitro gastroduodenal digest of whey proteins by high-performance liquid chromatography (HPLC) coupled to high-resolution/high-sensitivity tandem mass spectrometry (MS/MS), we sought to evaluate if state-of-art peptidomics provide comprehensive peptide coverage of food "digestomes". A multitude of small-sized peptides derived from both α-lactalbumin and ß-lactoglobulin as well as disulfide cross-linked hetero-oligomers remained unassigned, even when the digests were compared before and after S-S reduction. The precipitation with 12% trichloroacetic acid demonstrated the occurrence of large-sized polypeptides that escaped the bioinformatic identification. The analysis of a HPLC-MS/MS run with different proteomic search engines generated dissimilar peptide subsets, thus emphasizing the demand of refined searching algorithms. Although the MS/MS fragmentation of monocharged ions with exclusion of non-peptide-interfering compounds enlarged the inventory of short peptides, the overall picture of the "digestome" was still incomplete. These findings raise relevant implications for the identification of possible food-derived bioactive peptides or allergenic determinants.


Assuntos
Digestão , Duodeno/metabolismo , Mucosa Gástrica/metabolismo , Peptídeos/análise , Proteínas do Soro do Leite/química , Proteínas do Soro do Leite/metabolismo , Alérgenos/análise , Cromatografia Líquida de Alta Pressão/métodos , Dissulfetos/química , Análise de Alimentos/métodos , Lactalbumina/química , Lactalbumina/metabolismo , Lactoglobulinas/química , Lactoglobulinas/metabolismo , Proteólise , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos
20.
Food Chem ; 289: 223-231, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-30955606

RESUMO

Although the connection between protein oxidation, amyloid aggregation and diseases such as Alzheimer's is well known there is no information on such effects during preparation of beta-lactoglobulin fibrils. Different morphologies of amyloid aggregates of beta-lactoglobulin were prepared by incubation at pH 2 or pH 3.5 for up to 72 h. After 5 h, amyloid aggregates at pH 2 formed typical fibrils, which consisted of peptides. At pH 3.5, the amyloid aggregates were worm-like and consisted of intact protein. After 72 h, the building blocks at both pH values changed towards smaller peptides. The apparent tyrosine oxidation reached a maximum after 5 h at both pH values, whereas N-formylkynurenine and carbonyls increased continuously during 72 h. In case amyloid structures are used as edible material, the health related effects caused by protein oxidation needs to be considered.


Assuntos
Lactoglobulinas/química , Agregados Proteicos , Amiloide/metabolismo , Animais , Bovinos , Cromatografia em Gel , Concentração de Íons de Hidrogênio , Lactoglobulinas/metabolismo , Oxirredução , Peptídeos/análise , Temperatura Ambiente , Tirosina/química
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