Design of micro- and nanostructures from β-lactoglobulin under selected environmental conditions

The 19th Gums & Stabilisers for the Food Industry Conference: Hydrocolloid multifunctionalityBovine -lactoglobulin (-Lg) is a globular protein and the major component of whey proteins (ca. 50 % of its protein content). Besides the high nutritional value, the biological properties and resistance to proteolytic degradation in the stomach, its gelation capacity is particularly important allowing the formation of bio-based micro- and nanostructures (e.g. particles and hydrogels). -Lg when heated above a critical temperature (i.e. denaturation temperature: 76 ºC) undergoes conformational changes followed by subsequent protein-protein interactions. The order and rates of aggregation is highly dependent on the temperature, pH and protein concentration and can result in the formation of micro- and nanostructures with different properties and morphologies. The understanding of the kinetics of aggregation and of the combined effect of such environmental conditions is essential to design protein structures with the desired functionalities and applications. The objective of the present work was to understand the heat-induced aggregation of -Lg, affected by combined environmental conditions (various pH, heating temperature and protein concentrations) that lead to the formation of -Lg food-grade micro- and nanostructures. In this study, -Lg at various concentration (5, 10 and 15 mg·mL-1) was solubilized in 25 mM of sodium phosphate buffer at different pH values (3, 4, 6 and 7) and heated at different temperatures (60, 70 and 80 ºC) below and above the denaturation temperature of -Lg. Afterwards, the effect of aforementioned conditions on the -Lg micro- and nanostructures formation was evaluated in terms of their particle size and polydispersity index (PDI) by dynamic light scattering. -Lg nanostructures showed particle sizes below 50 nm when formed at pH 3 and 7 for -Lg concentrations of 5, 10 and 15 mg·mL-1 and heating temperatures of 60, 70 and 80 ºC, however displayed high PDI values ( 0.5). When the temperature of heating increased above the denaturation temperature of -Lg (i.e. 80 °C), the PDI values of the structures at pH 6 showed the lowest values ( 0.2), independent of the -Lg concentration used. At pH 4, it was possible to obtain structures at microscale (i.e. 3 µm) independent of the -Lg concentration and heating temperature of 70 and 80 °C. At this pH, which is relatively close to the isoelectric point of -Lg (i.e. 5.2), the net charge of proteins is ca. zero, so the protein structures tend to aggregate, thus showing higher size values. Therefore, protein aggregation mechanisms appear to be controlled by the environmental conditions applied; therefore, an understanding of the quantitative effect of these conditions is crucial for rational design of protein structures at micro or nanoscale with tailor-made functionalities.SFRH/BPD/80766/2011, Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq,Brasil) and to the Fundação para a Ciência e Tecnologia (FCT,Portugal), respectively. This study was supported by FCT under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte 2020-Programa Operacional Regional do Norteinfo:eu-repo/semantics/publishedVersio

Characterization of β-Lactoglobulin nano-hydrogels formed upon heat treatment and under selective pH range conditions

Bovine β-lactoglobulin (β-lg) is a globular protein from milk that has considerable potential as a functional ingredient in food, cosmetic and pharmaceutical applications. It is the major component (i.e. 50 wt%) and the primary gelling agent of whey proteins. β-Lg is known to be stable at low pH and highly resistant to proteolytic degradation in the stomach. Other property includes its ability to act as an encapsulating agent. The objective of this work was to understand the kinetics of aggregation during heat treatment under narrow pH range conditions that leads to the formation of food-grade β-lg nano-hydrogels and to propose a model for the mechanism of aggregation. In this study, aqueous dispersions of β-lg were generated and the formation of stable β-lg nano aggregates were evaluated after heating under different temperatures (i.e. 75, 80 and 85ºC), holding periods (i.e. 5, 10, 15 and 20 min), and pH values (i.e. 4, 5, 6 and 7), with particular emphasis around pH 6.0. The protein aggregates formed were characterized as to their stability (turbidity), morphology, size, surface charge and content in accessible thiol groups. Stable dispersions of β-lg nano-hydrogels were obtained at pH 6, corresponding to an aggregation yield of about 65, 77 and 92% at 75, 80 and 85ºC, respectively, during a holding period of 20 min. Nano-hydrogels at pH 6 were characterized by a particle size between 160 and 250 nm and low polydispersity (<0.2). Their ζ-potential was +20 or −40 mV, depending on the pH values. Nano-hydrogels obtained at pH 5 displayed a lower content of accessible thiol groups as compared to those obtained at pH above or below this value. Between pH 5 and 6, large sedimenting protein particulates were obtained, whereas soluble aggregates were formed at pH <5 or ≥6

Design of β-lactoglobulin micro- and nanostructures by controlling gelation through physical variables

β-lactoglobulin (β-Lg) is the major protein fraction of bovine whey serum and its principal gelling agent. Its gelation capacity enables conformational changes associated with protein-protein interactions that allow the design of structures with different properties and morphologies. Thus, the aim of this work was to successfully use β-Lg, purified from a commercial whey protein isolate, to develop food-grade micro- (with diameters between 200 and 300 nm) and nano- (with diameters ≤ 100 nm) structures. For this purpose, the phenomena involved in β-Lg gelation were studied under combined effects of concentrations (from 5 to 15 mg mL−1), heating temperature (from 60 to 80 °C) and heating time (from 5 to 25 min) for pH values of 3, 4, 6 and 7. The effects of such conditions on β-Lg structures were evaluated and the protein was fully characterized in terms of size, polydispersity index (PDI) and surface charge (by dynamic light scattering – DLS), morphology (by transmission electron microscopy - TEM) and conformational structure (circular dichroism, intrinsic and extrinsic fluorescence). Results have shown that β-Lg nanostructures were formed at pH 3 (with diameters between 12.1 and 22.3 nm) and at 7 (with diameters between 8.9 and 35.3 nm). At pH 4 structures were obtained at macroscale (i.e., ≥ 6 μm) for all β-Lg concentrations when heated at 70 and 80 °C, independent of the time of heating. For pH 6, it was possible to obtain β-Lg structures either at micro- (245.0 – 266.4 nm) or nanoscale (≤ 100 nm) with the lowest polydispersity (PDI) values (≤ 0.25), in accordance with TEM analyses, for heating at 80 °C for 15 min. Intrinsic and extrinsic fluorescence data and far-UV circular dichroism spectra measurements revealed conformational changes on β-Lg structure that support these evidences. A strict control of the physical and environmental conditions is crucial for developing β-Lg structures with the desired characteristics, thus calling for the understanding of the mechanisms of protein aggregation and intermolecular interaction when designing β-Lg structures with novel functionalities.Lívia de Souza Simões gratefully acknowledges her grant to CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brasil) from Brazil. Oscar L. Ramos gratefully acknowledges the Fundação para a Ciência e Tecnologia (FCT, Portugal) for his fellowship (SFRH/BPD/80766/2011). The authors also would like to acknowledge Ana I. Bourbon, from the International Iberian Nanotechnology Laboratory, for assistance in native polyacrylamide gel electrophoresis. This study was supported by the FCT under the scope of the strategic funding of UID/BIO/04469 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte, Portugal.info:eu-repo/semantics/publishedVersio

Structural characterization of heat-induced β-lactoglobulin nanohydrogels under the effects of selected physical conditions

The 19th Gums & Stabilisers for the Food Industry Conference: Hydrocolloid multifunctionalityβ-Lactoglobulin (β-Lg) is the major protein fraction in bovine whey serum (ca. 50% of its protein content). It is a bio-based and a Generally Recognized As Safe (GRAS) material, with a high nutritional value, that can be used to encapsulate nutraceuticals essentially due to its gelation capacity, which allows the formation of nanohydrogels. Furthermore, β-Lg displays a high binding capacity, under specific environmental conditions and it is resistant to proteolytic degradation in the stomach. These features make of β-Lg an excellent bio-based material to be used as carrier of nutraceuticals. The objective of this work was to understand the impact of different conditions (β-Lg concentration and heating times) in the physical properties of β-Lg nanohydrogels. In this study, β-Lg at various concentration (5, 10 and 15 mg·mL-1 ), were solubilized in 25 mM of sodium phosphate buffer (at pH 6) and heated at 80 °C for several holding time periods (5, 15 and 25 min). The protein structures formed were then characterized in terms of their stability, morphology, polydispersity index, size and surface charge. Stable nanohydrogels were obtained at pH 6, when treated at 80 ºC for heating periods longer than 15 min, characterized by a low polydispersity (< 0.2). At these conditions, nanohydrogels showed increasing particle size values, ranging from 50 nm to 110 nm, and surface charge from -15 mV to -20 mV, as β-Lg concentration increased. Nanohydrogels prepared at the same conditions, but treated for shorter periods of time, showed to be unstable, characterized by higher polydispersity (≥ 0.5) and surface charges of - 7 mV, independently of the β-Lg concentration used. The results obtained in this study represent a significant contribute to enrich the knowledge about the impact of several environmental conditions on β-Lg nanohydrogel characteristics and thus in the desired properties intended for their final application.Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI - 01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte 2020 - Programa Operacional Regional do Norteinfo:eu-repo/semantics/publishedVersio

Production of whey protein hydrogels through application of electric fields

Whey protein based gelling systems may present several functional roles in food formulations by enhancing textural properties (e.g. mouthfeel), acting as stabilizing agents or by being used as carrier of bioactive substances (e.g. nutraceuticals). The rates and pathways for the production of a protein gel system are controlled by heating conditions, protein concentration, pH, ionic strength, and solvent medium. The combination of heat and electric treatment has the potential to interfere with unfolding and aggregation of whey proteins and thus with protein-protein interactions. The general objective of this study was to evaluate and understand the effects of electric fields (EF) during thermal aggregation of liquid dispersions of whey protein isolate (WPI) and hydrogels made thereof. The main goal of this study was to induce thermal aggregation of a liquid dispersion of WPI into a three-dimensional network, a so called hydrogel, through combined application of instantaneous heating and EF. Nanostructures and nano-scale phenomena of the initial steps of whey protein aggregation were assessed by nano-tracking analysis and dynamic light scattering techniques. To assess the effects of EF on macroscopic properties of the produced hydrogels, rheological measurements were performed under steady shear flow. This study shows that EF interferes with inter- and intra-molecular protein interactions producing a marked reduction in whey protein aggregation. This particle size reduction can be controlled by the intensity of electrical treatment applied (e.g. EF treatments of 0, 3 and 10 V/cm have determined particle sizes of 156.9, 141.1 and 117.7 nm, respectively). The rheological measurements show that apparent viscosity of the produced hydrogels is also affected by the presence and magnitude of the EF applied. In conclusion, EF induces changes from a nanometer to micrometer range offering a great potential to the development of GRAS (generally recognized as safe) engineered nanotechnology-based delivery systems for food applications

Improving the properties of heat-induced whey protein hydrogels produced under the effects of moderate electric fields

Research on whey utilization is now being largely focused on exploiting their physiochemical and bioactive properties. Whey protein isolate (WPI) is among the most importante whey-derived ingredients and consists of products purified to a high protein content (>90%). The denaturation and aggregation kinetic behavior of WPI upon heating is of particular relevance; when properly engineered and controlled, it results in the production of novel nano-structures with many potential uses in Food formulations (i.e. enhancement of textural properties, action as stabilizing agents and delivery of biologically active substances. Combination of heat and moderate electric fields (MEF) treatment interferes with unfolding and aggregation pathways of whey biopolymer

Structural characterization of heat-induced whey protein hydrogels produced under the effects of moderate electric fields

Whey protein based gelling systems may present several functional roles in food formulations by enhancing textural properties (e.g. mouthfeel), acting as stabilizing agents or by being used as carrier of biologically active substances (e.g. pharmaceuticals). The rates and pathways for the production of a protein gel system are controlled by heating conditions, protein concentration, pH, ionic strength, and solvent medium. The combination of heat and electric treatment has the potential to interfere with unfolding and aggregation of whey proteins and thus with protein-protein interactions. The general objective of this study was to evaluate and understand the effects of electric fields (EF) on properties of liquid dispersions of whey protein and hydrogels made thereof. The results show that use of EF in combination with heating treatments (at 90 ºC for 20 minutes) modifies the rheological behavior of the obtained hydrogels, which presented nearly identical values for G‘ and G‘‘ or alternatively higher G‘‘ than G’. Through dynamic light scattering and size measurements it was possible to observe that EF treatment applied around critical particulate gel formation conditions (pH near isolectric point and high ionic strength medium) resulted in a whey gel less prone to formation of large aggregates and protein sedimentation. Structural characterization was assessed by dissolving the formed gels in various reducing and non-reducing buffers. Results showed that the hydrogel formed under an EF was almost completely solubilized in water (> 60 %) and largely soluble in NaCl 6 M (> 30%), SDS or urea (> 45 %) buffers, without using a reducing agent thus showing the importance of non-covalent and electrostatic interactions in maintaining the gel structure. As conclusion, the presence of an EF during heating favor whey proteins’ water binding properties resulting in a more stable dispersions of β-lactoglobulin aggregates with different mechanical and microstructual features

Development and characterization of β-lactoglobulin nanohydrogels for bioactive compound delivery

Whey proteins produced during bovine cheese manufacture, or membrane fractionation of bovine milk, account for increasingly valuable food ingredients – owing to their nutritionally-balanced composition in amino acid residues, coupled with their functional properties (e.g. solubility, gel-forming, emulsifying and foaming features). Whey protein-based gelling systems may indeed play several functional roles in food formulation by enhancing textural properties, acting as stabilizing agents or by being used as carrier of bioactive substances (e.g. nutraceuticals) [1]. The rates and pathways for manufacture of a protein gel system are controlled by heating conditions, protein concentration pH and ionic strength. The combination of different treatments can potentially interfere with unfolding and aggregation of whey proteins, and thus with protein-protein interactions. β-Lactoglobulin (β-Lg) is the major protein fraction of bovine whey serum (i.e. 50 wt%), and a primary gelling agent; it is stable at low pH and highly resistant to proteolytic degradation in the stomach, further to its appropriateness as encapsulating agent [2]. The objective of this work was to understand the kinetics of aggregation during heat treatment, under narrow pH ranges, leading to formation of edible β-Lg nanohydrogels, as well as to rationalize the contribution and importance of covalent and noncovalent interactions to maintain structure thereof. Furthermore, the ability of said nanohydrogels to encapsulate bioactive compounds (e.g. vitamin B2) was also assessed. Aqueous dispersions of β-Lg (10 mg/mL) were accordingly produced, and formation of stable nanostructures was ascertained after heating at 80 ºC for 20 min, under different pH values (i.e. 4, 5, 6 and 7); particular emphasis was placed on pH 6.0. The protein aggregates formed were characterized for stability, role of covalent or non-covalent bonds, morphology, size, surface charge and content in accessible thiol groups. Stable dispersions of β-Lg nanohydrogels were obtained at pH 6, corresponding to aggregation yield of ca. 77% at 80 ºC, for a holding period of 20 min. Such structures were characterized by particle size between 100 and 150 nm, low degree of polydispersity (<0.2), and association efficiency of vitamin B2 above 85%. Their ζ-potential varied from +20 to −40 mV, depending on pH. Nanohydrogels obtained at pH 5 displayed a lower content of accessible thiol groups than those obtained at pH values above or below. For pH between 5 and 6, large settling protein particulates were obtained, whereas soluble aggregates were formed at pH values off that range

Development and characterization of bioactive β-lactoglobulin nano-hydrogels for food applications

Whey proteins produced during bovine cheese manufacture, or membrane fractionation of bovine milk account for increasingly valuable food ingredient – owing to their nutritionally- balanced composition in amino acid residues coupled with their functional properties (e.g. solubility, and gel-forming, emulsifying and foaming features)1. β-Lactoglobulin (β-Lg) is the major fraction of bovine whey proteins (i.e. 50 wt%), and a primary gelling agent; it is stable at low pH and highly resistant to proteolytic degradation in the stomach, besides its ability to act as encapsulating agent2

Assessing the in vitro digestion of lactoferrin-curcumin nanoparticles using the realistic gastric model

Nanosized delivery systems have been the subject of research and discussion in the scientific community due to their unique properties and functionality. However, studies reporting the behaviour of nanodelivery systems under dynamic in vitro digestion conditions are still very scarce. To address this gap, this study aims to assess the dynamic in vitro gastric digestion of lactoferrin/curcumin nanoparticles in the realistic gastric model (RGM). For this purpose, the INFOGEST standard semi-dynamic digestion protocol was used. The nanosystems were characterized in terms of hydrodynamic size, size distribution, polydispersity index (PdI), and zeta potential using dynamic light scattering (DLS), before and during the digestion process. Confocal laser scanning microscopy (CLSM) was also used to examine particle aggregation. In addition, the release of curcumin was evaluated spectroscopically and the intrinsic fluorescence of lactoferrin was measured throughout the digestion process. The protein hydrolysis was also determined by UV-VIS-SWNIR spectroscopy to estimate, in real-time, the presence of free NH2 groups during gastric digestion. It was possible to observe that lactoferrin/curcumin nanoparticles were destabilized during the dynamic digestion process. It was also possible to conclude that low sample volumes can pose a major challenge in the application of dynamic in vitro digestion models.info:eu-repo/semantics/publishedVersio