Formation and characterization of soy protein nanoparticles by controlled partial enzymatic hydrolysis

Highlights

• Soy protein nanoparticles (SPNPs) were fabricated by controlled partial enzymatic hydrolysis.

• SPNPs were spherical and uniformly distributed with mean particle size between 80–170 nm.

• SPNPs were dominantly maintained by hydrophobic interactions, disulfide bonds and hydrogen bonds.

• The ratio of α-helix to β-sheet in SPNPs was in a narrow range of around 45%.

• SPNPs showed favorable anti-oxidative activity.

Abstract

In the present study, the potential of enzymatic hydrolysis as an efficient approach for the fabrication of soy protein nanoparticles (SPNPs) was investigated. Three types of enzymes including Flavorzyme, Alcalase and Protamex were selected to hydrolyze soy protein isolate (SPI) to different degrees (DH, 3%, 7% and 11%). Both the enzymes used and the DH obtained were vital for nanoparticle formation. The fabricated SPNPs were spherical and showed homogeneous size distributions with z-average size between 80 and 170 nm. Mechanisms involved in the formation of SPNPs were discussed by evaluating changes in SDS-PAGE patterns, secondary structures and interactive forces maintaining particle structure. Results suggested that the main subunits of 7S and 11S were retained in SPNPs, where hydrophobic interactions mainly dominated their structure formation, along with hydrogen bonds and disulfide bonds stabilizing the external and internal structure of nanoparticles, respectively. Secondary structure analysis implied that the ratio of α-helix to β-sheet in SPNPs was in a narrow range of around 45%, and a transition from α-helix to β-sheet was impeditive for nanoparticle formation. Compared with native SPI, the SPNPs showed more rapid adsorptions toward the oil-water interface, suggesting the potential good emulsifying property. Meanwhile, enhanced surface hydrophobicity and hydrolysis of α′α subunits into polypeptides endowed SPNPs with superior antioxidative capacity. These findings are expected to expand the potential of partial enzymatic hydrolysis as an efficient strategy to design and construct multifunctional soy protein nanoparticles for foods, cosmetics, and pharmaceutical applications.

Introduction

Hydrolysis is widely used to modify protein structures with the aim to improve their functional applications in food industry, such as solubility, viscosity, gelation, fat emulsification, and foaming (Panyam & Kilara, 1996; Wouters, Rombouts, Fierens, Brijs, & Delcour, 2016). In more recent decades, the exposure of amphiphilic structures upon hydrolysis has attracted increasing attention due to the formation of various shape-specific self-assembled nanostructures, such as fibers, micelles, vesicles, nanotubes, nanospheres, etc. (Du et al., 2019a, Du et al., 2019b; Ipsen & Otte, 2007; Jansens et al., 2019; Jiang et al., 2018). These nanostructures have well broaden the utilization of specific food proteins in the fields of functional foods, pharmaceuticals and nutraceuticals, as encapsulation and delivery systems, or as building blocks to further modulate the physicochemical and sensory properties of the food matrices (Jones & McClements, 2010; Sozer & Kokini, 2009).

It is known that chemical hydrolysis, specific pressure-temperature-induced hydrolysis and enzyme catalyzed hydrolysis, alone or in combination, have been proved to generate amphiphilic hydrolysates/peptides (Du et al., 2019a, Du et al., 2019b; Lara, Adamcik, Jordens, & Mezzenga, 2011; Yin, Rastogi, Terry, & Popescu, 2007), which are known to spontaneously self-assemble into a range of well-defined, hierarchical nanostructures mainly through non-covalent interactions including hydrogen bonding, van der Waals, electrostatic, hydrophobic, and π-π stacking interactions (Qin et al., 2011; Zhang et al., 2018). Chemical hydrolysis by heating (80–90 °C) protein suspension (i.e., β-lactoglobulin, α-lactalbumin, ovalbumin and lysozyme, etc.) for several hours (2–30 h) at acid pH with/without cationic templates has been widely reported to fabricate oriented nanostructures like nanospheres, nanofibrils and nanotubes (Esmaeilzadeh, Fakhroueian, & Miran Beigi, 2012; Ipsen & Otte, 2007; Lara et al., 2011). Meanwhile, superheated water under specific pressure-temperature conditions can also hydrolysis protein through scission of the protein chain to yield oligopeptides. Yin et al. reported that the dissolution of keratins could self-assemble into a hierarchical architecture, which can further be used as important building blocks for the synthesis of a range of novel polymers (Yin et al., 2007). Apart from the chemical hydrolysis with relative harsh conditions or the superheated water with high energy input, proteolysis triggered by enzyme is uniquely chemo-, regio-, and enantioselective and is conducted under mild conditions, which appears to be an advantageous alternative for nanostructure fabrication (Toledano, Williams, Jayawarna, & Ulijn, 2006). Akkermans et al. found that AspN endoproteinase could cleave the peptide bonds in β-Lactoglobulin with N-terminal assigned to aspartic acid residues and release peptides similar to that obtained by heat-induced acid hydrolysis, facilitating molecular assembling (Akkermans, Venema, van der Goot, Boom, & van der Linden, 2008). Ipsen and co-workers have done a series of researches on α-lactalbumin and found that it could self-assemble into varying nanostructures like nanotubes after partial hydrolysis by Glu- and Asp-specific proteases from Bacillus licheniformis (BLP) in the presence of calcium (Geng, Kirkensgaard, Arleth, Otte, & Ipsen, 2019; Ipsen & Otte, 2007). Li and co-workers later found that enzymatically partially hydrolyzed α-lactalbumin peptides could self-assemble into micelles for bioactive encapsulation (Y. Du et al., 2019a, Du et al., 2019b; Jiang et al., 2018). Du et al. found that amphiphilic egg yolk peptide formed upon trypsin treatment could self-assemble into spherical micellar nanoparticles, promisingly showing a potential use as natural edible nano-Pickering emulsifier (Du et al., 2019a, Du et al., 2019b). Nevertheless, taken the relative complex structures of food proteins (especially plant proteins) into consideration, knowledge on how enzymatic hydrolysis influences the assembly of hydrolysates remains quite limited.

So far as concerned, the protease used, the substrate proteins applied, the degree of hydrolysis (DH) obtained and even the way to inactivate enzymes all have profound effects on the assembling of hydrolysates. Gao et al. found that the ability for whey protein to form nanostructures varied after treating with four different proteases (trypsin, protease A, pepsin, and protease M) (Gao, Xu, Ju, & Zhao, 2013). Nicklas et al. reported that collagen nanoparticles in different sizes were obtained upon Alcalase treatment at different time intervals from 18 to 168 h (Nicklas, Schatton, Heinemann, Hanke, & Kreuter, 2009). When compared with animal proteins, the preparation of “environmentally economic” plant proteins-based nano structures through enzymatic hydrolysis was less investigated, possibly due to their more compact structures and more complex compositions. Athamneh and Barone found that tryptic treatment on wheat gluten to different DHs led to different assembling behaviors, and partial glutamine-rich peptides/subunits from gliadin could form building blocks at nano scale. Interestingly, thermal inactivation after tryptic hydrolysis provided a driving force to initiated the self-assembly process (Athamneh & Barone, 2009). Recently, we successfully fabricated multifunctional soy peptide nanoparticles via Alcalase-induced peptide aggregation (Zhang et al., 2018), well suggesting that partial enzymatic hydrolysis could be a useful tool to fabricate soy protein-based nanoparticles.

In the present study, we continuously explored the potential of enzymatic hydrolysis as an efficient approach for the fabrication of soy protein nanoparticles (SPNPs) from the assembly of partially hydrolyzed proteins. Three types of widely used enzymes including Flavorzyme, Alcalase and Protamex, which can cleave different peptide bonds, were selected to hydrolyze soy proteins to different degrees (DH, 3%, 7% and 11%). DLS, FE-SEM, and TEM were used to characterize the particle size distributions and morphologies of the obtained SPNPs. SDS-PAGE patterns, secondary structures as revealed by circular dichroism (CD), and interactive forces maintaining particle structure were then analyzed. The interfacial and antioxidant properties of these SPNPS were also evaluated. We expect that these findings could expand the potential of enzymatic hydrolysis as an efficient strategy to design and construct multifunctional soy protein nanoparticles for foods, cosmetics, and pharmaceutical applications.