Nanoencapsulation of quercetin into bio-based nanostructures obtained from assembling of α-lactalbumin and lysozyme

Nanotechnology possesses an intrinsic potential to produce new food ingredients and innovative products, with considerable benefits to human health. This can be attained via development of innovative structures for application in functional foods. In recent years, consumption of foods providing health benefits has risen chiefly as a result of significant investments from the food industry and widening consumer awareness in this field. Polyphenols constitute one such functional ingredient: it entails a large group of plant metabolites with a large spectrum of recognized biological activities in humans. Quercetin is, in particular, one of the most representative compounds of the flavonoid family; it has been assigned a wide range of health benefits, including anti-inflammatory agent, cancer prevention, DNA protection agent, antioxidant and cardio-protective agent. However, its bioavailability is low, so limited biological effects may be noticed arising from its poor solubility, gastrointestinal instability and low uptake rate through the gastrointestinal tract. A possible solution to overcome such limitations is nanoencapsulation of quercetin. Therefore, our study was aimed at encapsulating quercetin into bio-based nanostructures obtained from assembling of α-lactalbumin (α-La) and lysozyme (Lys), as promoted by heating at 75 oC for 15 min, at pH 11; evaluation of their association efficiency was performed. Such nanostructures were prepared via solubilization of 2 mg mL-1 of Lys and α-La powders in water, at a molar ratio of 1:0.54, and were extensively characterized by dynamic light scattering (for particle size, polydispersity and zeta potential) and transmission electron microscopy (for microstructure and morphology). Quercetin has been successfully encapsulated into protein nanostructures above 50% efficiency. These nanostructures exhibited spherical morphology, with average size below 100 nm and zeta potential around -35 mV. Our results suggest that quercetin encapsulated in such proteinaceous nanostructures may be used for manufacture of functional foods

Design of bio-based supramolecular structures through self-assembly of α-lactalbumin and lysozyme

Bovine α-lactalbumin (α-La) and lysozyme (Lys), two globular proteins with highly homologous tertiary structures and opposite isoelectric points, were used to produce bio-based supramolecular structures under various pH values (3, 7 and 11), temperatures (25, 50 and 75 °C) and times (15, 25 and 35 min) of heating. Isothermal titration calorimetry experiments showed protein interactions and demonstrated that structures were obtained from the mixture of α-La/Lys in molar ratio of 0.546. Structures were characterized in terms of morphology by transmission electron microscopy (TEM) and dynamic light scattering (DLS), conformational structure by circular dichroism and intrinsic fluorescence spectroscopy and stability by DLS. Results have shown that protein conformational structure and intermolecular interactions are controlled by the physicochemical conditions applied. The increase of heating temperature led to a significant decrease in size and polydispersity (PDI) of α-La–Lys supramolecular structures, while the increase of heating time, particularly at temperatures above 50 °C, promoted a significant increase in size and PDI. At pH 7 supramolecular structures were obtained at microscale – confirmed by optical microscopy – displaying also a high PDI (i.e. > 0.4). The minimum size and PDI (61 ± 2.3 nm and 0.14 ± 0.03, respectively) were produced at pH 11 for a heating treatment of 75 °C for 15 min, thus suggesting that these conditions could be considered as critical for supramolecular structure formation. Its size and morphology were confirmed by TEM showing a well-defined spherical form. Structures at these conditions showed to be stable at least for 30 or 90 days, when stored at 25 or 4 °C, respectively. Hence, α-La–Lys supramolecular structures showed properties that indicate that they are a promising delivery system for food and pharmaceutical applications.CNPq and CAPES, and their support to FAPEMIG and CNPEM-LNBio (Centro Nacional de Pesquisa em Energia e Materiais-Laboratório Nacional de Biociências) both from Brazil. Fundação para a Ciência e a Tecnologia. The authors thank the FCT Strategic Project of UID/BIO/04469/2013 unit, COMPETE 2020 (POCI-01-0145-FEDER-006684), the project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462), and the project “BioInd - Biotechnology and Bioengineering for improved Industrial and Agro-Food processes”, REF. NORTE-07-0124-FEDER-000028 Co-funded by the Programa Operacional Regional do Norte (ON.2 – O Novo Norte), QREN, FEDER

Design of nanostructures, obtained from assembling of α-lactalbumin and lysozyme upon heat treatment and selective environmental conditions

Protein assembly into supramolecular structures (e.g. aggregates, fibrils and nanotubes) is a widespread phenomenon in biological sciences. Nowadays, it is well documented that the amphiphilic properties of proteins is a driving force to their self-assembling into innovative micro- and nanostructures of high interest in the food and pharmaceutical fields. Formation of such structures is strongly dependent on physicochemical conditions and protein conformation. In this study, bio-based nanostructures were produced from assembly of hen egg white lysozyme (Lys) and bovine α-lactalbumin (α-La) – i.e. two homologous globular proteins with opposite charge), under various processing conditions: heating treatment (55 °C and 75 °C), holding time (25 and 35 min) and pH (3 and 11). The nano-scale structures prepared by solubilization of 2 mg mL-1 of Lys and α-La powders in water, at a molar ratio of 1:0.54, were characterized via dynamic light scattering (in terms of particle size, polydispersity and zeta potential), and further analyzed by transmission electron microscopy (TEM). Smaller sized particles (75 nm) and low podydispersity values (0.24) were produced at pH 11 after heating at 75 °C for 25 min, whereas at pH 3 (and similar conditions) the average mean particle size was ca. 402 nm with polydispersity of 0.45. The nanostructure stability was also assessed; higher stability was obtained at pH 11 than 3, with zeta potentials of -35 and +27 mV, respectively, by 60 d. The nanostructure entities prepared at pH 11 were shown by TEM to possess a well defined spherical shape. Protein assembly mechanisms and intermolecular interactions involved appear to be controlled by the environmental conditions applied; therefore, an understanding of the quantitative effects of these conditions are crucial for rational design of new protein assemblies with tailor-made functionalities