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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 ; 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
3.
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
4.
J Dairy Sci ; 102(9): 7863-7873, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31326163

RESUMO

The effect of the contents of casein (CN) and whey protein fractions on curd yield (CY) and composition was estimated using 964 individual milk samples. Contents of αS1-CN, αS2-CN, ß-CN, γ-CN, glycosylated κ-CN (Gκ-CN), unglycosylated κ-CN, ß-LG, and α-LA of individual milk samples were measured using reversed-phase HPLC. Curd yield and curd composition were measured by model micro-cheese curd making using 25 mL of milk. Dry matter CY (DMCY) was positively associated with all casein fractions but especially with αS1-CN and ß-CN. Curd moisture decreased at increasing ß-CN content and increased at increasing γ-CN and Gκ-CN content. Due to their associations with moisture, Gκ-CN and ß-CN were the fractions with the greatest effect on raw CY, which decreased by 0.66% per 1-standard deviation (SD) increase in the content of ß-CN and increased by 0.62% per 1-SD increase in the content of Gκ-CN. The effects due to variation in percentages of the casein fractions in total casein were less marked than those exerted by contents. A 1-SD increase in ß-CN percentage in casein (+3.8% in casein) exerted a slightly negative effect on DMCY (ß = -0.05%). Conversely, increasing amounts of αS1-CN percentage were associated with a small increase in DMCY. Hence, results suggest that, at constant casein and whey protein contents in milk, the DMCY depends to a limited extent on the variation in the αS1-CN:ß-CN ratio. κ-Casein percentage did not affect DMCY, indicating that the positive relationship detected between the content of κ-CN and DMCY can be attributed to the increase in total casein resulting from the increased amount of κ-CN and not to variation in κ-CN relative content. However, milk with increased Gκ-CN percentage in κ-CN also shows increased raw CY and produces curds with increased moisture content. Curd yield increased at increasing content and relative proportion of ß-LG in whey protein, but this is attributable to an improved capacity of the curd to retain water. Results obtained in this study support the hypothesis that, besides variation in total casein and whey protein contents, variation in protein composition might affect the cheese-making ability of milk, but this requires further studies.


Assuntos
Caseínas/química , Queijo/análise , Leite/química , Proteínas do Soro do Leite/química , Animais , Glicosilação , Lactoglobulinas/metabolismo , Água/análise
5.
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
6.
Nutrients ; 11(6)2019 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-31242665

RESUMO

The effect of glycation and aggregation of thermally processed ß-lactoglobulin (BLG) on binding to sRAGE and specific immunoglobulin E (sIgE) from cow milk allergic (CMA) patients were investigated. BLG was heated under dry conditions (water activity < 0.7) and wet conditions (in phosphate buffer at pH 7.4) at low temperature (<73 °C) and high temperatures (>90 °C) in the presence or absence of the milk sugar lactose. Nε-(carboxymethyl)-l-lysine (CML) western blot and glycation staining were used to directly identify glycation structures on the protein fractions on SDS-PAGE. Western blot was used to specify sRAGE and sIgE binding fractions. sRAGE binding was highest under wet-heated BLG independent of the presence of the milk sugar lactose. Under wet heating, high-molecular-weight aggregates were most potent and did not require the presence of CML to generate sRAGE binding ligands. In the dry system, sRAGE binding was observed only in the presence of lactose. sIgE binding affinity showed large individual differences and revealed four binding profiles. Dependent on the individual, sIgE binding decreased or increased by wet heating independent of the presence of lactose. Dry heating required the presence of lactose to show increased binding to aggregates in most individuals. This study highlights an important role of heating condition-dependent protein aggregation and glycation in changing the immunogenicity and antigenicity of cow's milk BLG.


Assuntos
Epitopos , Produtos Finais de Glicação Avançada/metabolismo , Temperatura Alta , Imunoglobulina E/metabolismo , Lactoglobulinas/metabolismo , Lisina/análogos & derivados , Hipersensibilidade a Leite/metabolismo , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Água/química , Produtos Finais de Glicação Avançada/imunologia , Imunoglobulina E/imunologia , Lactoglobulinas/imunologia , Lactose/química , Ligantes , Lisina/imunologia , Lisina/metabolismo , Hipersensibilidade a Leite/imunologia , Agregados Proteicos , Ligação Proteica , Conformação Proteica , Receptor para Produtos Finais de Glicação Avançada/imunologia
7.
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
8.
Int J Mol Sci ; 20(10)2019 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-31130632

RESUMO

Neuromelanin (NM) is a dark brown pigment found in dopaminergic neurons of the substantia nigra (SN) and in norepinephrinergic neurons of the locus coeruleus (LC). Although NM is thought to be involved in the etiology of Parkinson's disease (PD) because its content decreases in neurodegenerative diseases such as PD, details are still unknown. In this study, we characterized the biosynthetic pathway of the oxidation of dopamine (DA) by tyrosinase in the presence of thiol peptides and proteins using spectroscopic and high-performance liquid chromatography (HPLC) methods and we assessed the binding of DA via cysteine residues in proteins by oxidation catalyzed by redox-active metal ions. To examine whether the protein-bound DA conjugates exhibit pro-oxidant activities, we measured the depletion of glutathione (GSH) with the concomitant production of hydrogen peroxide. The results suggest that the fate of protein-bound DA conjugates depends on the structural features of the proteins and that DA-protein conjugates produced in the brain possess pro-oxidant activities, which may cause neurodegeneration due to the generation of reactive oxygen species (ROS) and the depletion of antioxidants.


Assuntos
Dopamina/metabolismo , Estresse Oxidativo , Doença de Parkinson/metabolismo , Proteínas/metabolismo , Agaricales/enzimologia , Animais , Bovinos , Glutationa/metabolismo , Humanos , Lactoglobulinas/metabolismo , Melaninas/metabolismo , Monofenol Mono-Oxigenase/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Soroalbumina Bovina/metabolismo
9.
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
10.
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
11.
Talanta ; 199: 116-123, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30952234

RESUMO

In this study, we present the use of microreactors packed with immobilized trypsin particles for the rapid and efficient bottom-up analysis of proteins by on-line immobilized enzyme microreactor capillary electrophoresis mass spectrometry (IMER-CE-MS). The results obtained digesting ß-lactoglogulin (ß-LG) off-line with free trypsin in solution and with immobilized trypsin particles were taken as a reference for the optimization of the on-line protein digestion. Under the optimized conditions, on-line digestion, separation and characterization of the protein digests were possible in less than 30 min. The limit of detection for complete sequence coverage was around 10 µg mL-1 (~500 µM) of ß-LG, the repeatability was comparable to the off-line digestion methods and the microreactor could be reused until thirty times. The good performance of IMER-CE-MS was also demonstrated for several other proteins as α-casein (α-CSN), ß-casein (ß-CSN), and κ-casein (κ-CSN), as well as for a complex protein mixture (an Escherichia coli whole cell lysate).


Assuntos
Caseínas/metabolismo , Enzimas Imobilizadas/metabolismo , Lactoglobulinas/metabolismo , Tripsina/metabolismo , Caseínas/química , Eletroforese Capilar , Enzimas Imobilizadas/química , Humanos , Lactoglobulinas/química , Espectrometria de Massas , Tripsina/química
12.
J Biosci Bioeng ; 128(2): 156-161, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30862433

RESUMO

Food processing technology such as protein hydrolysis using proteases has been receiving a lot of attention, and it is important to accurately understand the cleavage specificity of each protease for selecting a protease suited to aims. Although numerous methods have been reported to reveal the substrate specificity of proteases, there is no method to evaluate simply, quickly, reasonably, and accurately. This study set out to devise Pep-MS assay, a novel assay system that can be used to comprehensively clarify positions at which proteases cleave, by combining a mass spectrometer and a photo-cleavable peptide array. First, we evaluated peptide array corresponding to the primary sequences of αS1-casein, αS2-casein and ß-casein with trypsin to verify the accuracy of the Pep-MS assay. The evaluation of cleavage positions by the trypsin protease reagent using the Pep-MS assay resulted in a matching rate of about 96.8% to rational cleavage positions. Next, we confirmed the cleavage positions in αS2-casein or ß-lactoglobulin by an industrial bacterial protease from Bacillus subtilis at some protease reaction temperatures or reaction times. The Pep-MS assay clarified the differences in the cleavage patterns due to the reaction temperature, and the change in the cleavage strength with the reaction time. Pep-MS assay is a promising method for evaluating the substrate specificity of proteases, which will be useful to find effective production conditions for functional peptide from foods and effective hydrolysis conditions for decreasing allergen of food proteins.


Assuntos
Caseínas/metabolismo , Lactoglobulinas/metabolismo , Espectrometria de Massas , Processos Fotoquímicos , Análise Serial de Proteínas , Tripsina/metabolismo , Bacillus subtilis/enzimologia , Manipulação de Alimentos , Hidrólise , Cinética , Proteólise , Especificidade por Substrato , Temperatura Ambiente
13.
Food Chem ; 288: 276-282, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-30902293

RESUMO

A methylglyoxal (MG)-ß-lactoglobulin (bLG) model was established to simulate reheating conditions (60-100 °C) to investigate the modification effect that α-dicarbonyl compounds had on protein structure and on the digestibility of milk protein. The results showed that bLG can be modified by MG, and the modification degree increased with the increase in reheating temperature. The reacted lysine and arginine as well as the generated protein-bound NƐ-carboxymethyllysine and NƐ-carboxyethyllysine in the modified bLG also increased with temperature. The high-resolution mass spectrometry results revealed that the modification site is at the lysine and arginine residue of bLG. Additionally, nine types of modifications were detected, and NƐ-carboxyethyllysine was the dominant modification product. The in vitro digestibility of MG-modified bLG clearly decreased with the increase in reheating temperature. This result was consistent with the degree of structural modification and could be explained by the specific action sites (lysine and arginine) of the digestive enzyme, which were modified by MG.


Assuntos
Laticínios/análise , Lactoglobulinas/metabolismo , Aldeído Pirúvico/química , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Lactoglobulinas/química , Lisina/análogos & derivados , Lisina/química , Pepsina A/metabolismo , Espectrometria de Massas em Tandem , Temperatura Ambiente
14.
Food Chem ; 288: 306-314, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-30902298

RESUMO

Health benefits are routinely attributed to whey proteins, their hydrolysates and peptides based on in vitro chemical and cellular assays. The objective of this study was to track the fate of whey proteins through the upper gastrointestinal tract, their uptake across the intestinal barrier and then assess the physiological impact to downstream target cells. Simulated gastrointestinal digestion (SGID) released a selection of whey peptides some of which were transported across a Caco-2/HT-29 intestinal barrier, inhibited free radical formation in muscle and liver cells. In addition, SGID of ß-lactoglobulin resulted in the highest concentration of free amino acids (176 nM) arriving on the basolateral side of the co-culture with notable levels of branched chain and sulphur-containing amino acids. In vitro results indicate that consumption of whey proteins will deliver bioactive peptides to target cells.


Assuntos
Estresse Oxidativo/efeitos dos fármacos , Peptídeos/farmacologia , Proteínas do Soro do Leite/metabolismo , Sequência de Aminoácidos , Animais , Transporte Biológico , Células CACO-2 , Bovinos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Trato Gastrointestinal/metabolismo , Células HT29 , Humanos , Lactoglobulinas/metabolismo , Leite/metabolismo , Peptídeos/isolamento & purificação , Peptídeos/metabolismo
15.
Food Chem ; 287: 313-323, 2019 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-30857705

RESUMO

The objective of this work was to study molecular binding of safranal to whey proteins by taking advantage of headspace solid-phase microextraction combined with gas chromatography (HS-SPME/GC), fluorescence and circular dichroism (CD) spectroscopies, and docking studies. The results of HS-SPME/GC indicated that bovine serum albumin (BSA) had the highest affinity toward safranal, with binding constant of 3.196 × 103 M-1. Also, binding strength was reduced in the order of α-lactalbumin (α-Lact), whey protein isolate (WPI), and ß-lactoglobulin (ß-Lg). Although there was a good agreement between results of HS-SPME/GC and fluorescence spectroscopy regarding the safranal binding site on whey proteins, the order of their binding affinity toward safranal was not consistent for both techniques. According to docking studies, conformational alterations in secondary and tertiary structures of whey proteins induced by safranal association resulted from hydrophobic interactions and hydrogen bonds.


Assuntos
Cromatografia Gasosa/métodos , Cicloexenos/metabolismo , Microextração em Fase Sólida/métodos , Terpenos/metabolismo , Proteínas do Soro do Leite/química , Proteínas do Soro do Leite/metabolismo , Sítios de Ligação , Dicroísmo Circular , Cicloexenos/química , Ligações de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Lactalbumina/química , Lactalbumina/metabolismo , Lactoglobulinas/química , Lactoglobulinas/metabolismo , Simulação de Acoplamento Molecular , Estrutura Secundária de Proteína , Soroalbumina Bovina/química , Soroalbumina Bovina/metabolismo , Espectrometria de Fluorescência , Terpenos/química
16.
Anal Chim Acta ; 1052: 163-169, 2019 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-30685035

RESUMO

Immunochemical detection of food allergens is usually based on the use of polyclonal or monoclonal immunoglobulins G (IgG) antibodies. However, due to differences in epitopes recognition between IgG and IgE, an epitope modification during food processing can potentially alter allergenicity and detection in such different way. For that reason, the use of traditional immunological methods to anticipate or study the protein allergenicity is not recommended. The objective of this work was to develop a standardized competitive ELISA, based on recombinant IgE antibodies to ß-lactoglobulin (ß-lg), to reveal and to measure changes in ß-lg immunoreactivity provoked by technological processes in foods containing this milk protein. As a result, a sensitive immunochemical method (Limit of Detection, LOD < 0.2 µg mL-1) has been developed for ß-lg recognition. Also, this technique has been confirmed to detect modifications in ß-lg-IgE binding after food processing (thermal treatment) in a similar way to those results obtained with allergic patients in previous works. Consequently, data obtained with this assay could be used as a preliminary test to predict in vitro ß-lg allergenic behavior in allergenicity studies in processed foods.


Assuntos
Alérgenos/análise , Ensaio de Imunoadsorção Enzimática/métodos , Manipulação de Alimentos , Imunoglobulina E/metabolismo , Lactoglobulinas/análise , Leite/química , Alérgenos/metabolismo , Animais , Bovinos , Lactoglobulinas/metabolismo , Limite de Detecção , Temperatura Ambiente
17.
Langmuir ; 35(2): 446-452, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30565468

RESUMO

In presence of calcium ions, ß-lactoglobulin (BLG) unfolds and subsequently aggregates after heating. This process has important pharmaceutical and agroalimentary applications. Nowadays, the molecular mechanism of unfolding and BLG aggregation, and the role of calcium in the mechanism, is poorly understood. Actually, in most studies, data have been acquired at room temperature, after heating and after aggregation, which makes it difficult to establish a clear causal-temporal relation between calcium binding, heat, and aggregation. Thus, the goal of the present study is to get accurate, nanoscale data about the molecular events leading to BLG unfolding and calcium-dependent aggregation. The molecular transformation of BLG during heating has been investigated, using the NMR pulse field gradient technique, operating in a high field (900 MHz). Thanks to this technique, the molecular conformation of newly formed unfolded BLG molecules can be distinguished in a large pool of native ones. The present work shows that BLG at neutral pH at 65 °C displays fast, cooperative-like unfolding, in which no long-lived intermediary state (as a molten globule one) is detected, before aggregation. These data also indicate that calcium ions bind unfolded BLG in specific sites which might be a necessary feature to form the aggregate. Finally, these data also provide an NMR-based methodology to monitor the rate of protein unfolding using NMR.


Assuntos
Lactoglobulinas/metabolismo , Agregados Proteicos , Animais , Cálcio/metabolismo , Bovinos , Calefação , Temperatura Alta , Lactoglobulinas/química , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Desdobramento de Proteína
18.
Colloids Surf B Biointerfaces ; 174: 493-500, 2019 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-30497011

RESUMO

During the last decade a special interest has been focused on studying the relationship between the composition and structure of emulsions and the extent of lipolysis, driven by the necessity of modulate lipid digestion to decrease or delay fats absorption or increase healthy fat nutrients bioavailability. Because bile salts (BS) play a crucial role in lipids metabolism, understanding how typical food emulsifiers affect the structures of BS under duodenal conditions, can aid to further understand how to control lipids digestion. In the present work the BS-binding capacity of three emulsifiers (Lecithin, Tween 80 and ß-lactoglobulin) was studied under duodenal conditions. The combination of several techniques (DLS, TEM, ζ-potential and conductivity) allowed the characterization of molecular assemblies resulting from the interactions, as modulated by the relative amounts of BS and emulsifiers in solution.


Assuntos
Ácidos e Sais Biliares/metabolismo , Emulsificantes/metabolismo , Secreções Intestinais/metabolismo , Intestinos/fisiologia , Lactoglobulinas/metabolismo , Lecitinas/metabolismo , Digestão , Alimentos , Humanos , Técnicas In Vitro
19.
Int J Biol Macromol ; 125: 596-604, 2019 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-30528992

RESUMO

Silver nanoparticles (SNPs) have been increasingly used in medicines and biomaterials as a drug carriers and diagnostic or therapeutic material due to their smaller size, large surface area and cell penetration ability. Here we report the preparation of SNPs of diameter 10 ±â€¯3 nm by using silver nitrate and sodium borohydride and the interaction of synthesized SNPs with our model protein ß-lactoglobulin (ß-lg) in 10 mM phosphate buffer at pH 7.5 after thermal exposure at 75 °C. Heat exposed ß-lg forms amyloidal fibrillar aggregates whereas this protein aggregates adopt rod-like shape instead of fibrillar structure in presence of SNP under the same conditions. Size of the synthesized SNPs is confirmed by UV-Visible spectroscopy, SEM and TEM. Interactions and subsequent formation of molecular assembly of heat stressed ß-lg with SNP were investigated using Th-T assay and ANS binding assay, DLS, RLS, CD, FT-IR, SEM, TEM. Docking study parallely also support the experimental findings.


Assuntos
Lactoglobulinas/metabolismo , Nanopartículas Metálicas/administração & dosagem , Agregados Proteicos/efeitos dos fármacos , Prata/administração & dosagem , Amiloide/metabolismo , Temperatura Alta , Interações Hidrofóbicas e Hidrofílicas , Polimorfismo de Nucleotídeo Único/fisiologia
20.
Biosensors (Basel) ; 8(4)2018 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-30445706

RESUMO

In this article, we introduce the use of a carboxy-functionalized waterborne carbon nanotube ink for the fabrication of an amperometric biosensor aimed at the quantification of ß-lactoglobulin. Detection of this protein from cow's milk was performed by a sandwich immunoassay onto printed carbon nanotube electrodes. The electrodes were printed using a carbon nanotube ink modified with polystyrene beads containing a high amount of carboxylic groups for protein immobilization. This strategy showed enhanced sensing performance compared to the use of oxidative treatments for the functionalization of electrodes. These electrodes showed an excellent electrochemical behavior, and proteins could be immobilized on their surface via the carbodiimide reaction. These antibody-immobilized carbon nanotube electrodes allowed for the detection of ß-lactoglobulin in sub-ppm concentrations.


Assuntos
Técnicas Biossensoriais/instrumentação , Lactoglobulinas/metabolismo , Nanotubos de Carbono/normas , Animais , Bovinos , Tinta , Leite
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