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Project

Surface modification of plant protein based nanoparticles: a tool to improve their functional properties

Interest in replacing animal by plant proteins in food systems is increasing. For various reasons, such replacement is unfortunately not straightforward. A main hurdle in this regard is the often limited solubility of plant proteins in aqueous media, which notably reduces their applicability in many foods. A possible solution is to produce protein nanoparticles (NPs) that are colloidally stable in aqueous systems. The solubility profile of cereal prolamins (such as wheat gliadin and maize zein) enables the fabrication of such NPs (GNPs and ZNPs, respectively) via liquid anti-solvent precipitation (LAS). In such procedures, proteins are aggregated in a controlled fashion by a decrease in solvent quality to form homogenous aqueous NP dispersions.

GNPs and ZNPs are potential novel food ingredients in i.a., encapsulation applications or the stabilization of food emulsions and foams. Thus far, the ability of GNPs and ZNPs to stabilize foams has been investigated to a limited extent. However, it is known that foams sustained by GNP (constituents) have good stability in a narrow pH range. ZNPs, in contrast, have very limited foaming properties irrespective of the pH. Mechanisms underpinning the foaming properties of such NPs are still poorly understood. Finally, such NPs have only limited colloidal stability under food system-relevant conditions. This notably limits their potential application of GNPs/ZNPs in food systems.

Against this background, this doctoral dissertation had a two-fold aim. A first aim was to optimize the production of highly functional cereal prolamin NPs (based on gliadin, zein, or their blends) with good foaming properties and high colloidal stability under food system-relevant conditions. A second aim was to unravel the mechanisms underpinning the foaming properties of such NPs and thus, their structure-functionality relationship.

First, NPs based on gliadin, commercial zein, and zein extracted on a laboratory scale from maize flour (GNPs, CS-ZNPs, and LS-ZNPs, respectively) were produced via an optimized LAS procedure, and their foaming properties were investigated over a wide pH range (4.0 to 10.0). More specifically, the ability of NPs to form foam (defined as foaming capacity, FC) and to stabilize the produced foam over time (defined as foam stability, FS) was investigated.

The FC provided by CS-ZNPs was very low regardless of the pH (4.0 – 10.0), while a FC of LS-ZNPs was pH-dependent. Indeed, relatively low FC was observed for LS-ZNPs at pH values between 4.0 and 8.0, but a considerable amount of foam could be formed at pH 10.0. GNPs, in contrast, possessed a good FC irrespective of the pH. FC of proteins or NPs based thereon is often linked to the rate at which they adsorb at the air-water (A-W) interface, which in turn can be associated with their size, surface charge, and surface hydrophobicity. However, there were no clear correlations between the rate of adsorption of the different NPs and the FCs they provided. An additional mechanism i.e., a bridging de-wetting effect, was hypothesized to negatively affect the FC of CS-/LS-ZNPs. This may induce coalescence of adjacent air bubbles. As this was not noted for GNPs, which had a solid particle-like structure, it is hypothesized that the coacervate-like morphology of CS-/LS ZNPs may facilitate this antifoaming effect. This, however, needs further investigation.

As almost no foam could be produced from CS-ZNP suspensions, the ability of CS-ZNP to provide FS was not further investigated. FS brought about by LS-ZNPs was pH-dependent and increased with pH. At pH 4.0, LS-ZNPs formed coherent films at the A-W interface (as indicated by surface dilatational rheology measurements) but only limited FS was observed. At pH 8.0 and 10.0, LS-ZNPs stabilized foams more effectively even if at these pH values the formed interfacial films displayed only limited viscoelasticity. This suggested that the formation of interfacial films was not the main mechanism underpinning the FS of LS-ZNPs. It was hypothesized that the good FS provided by LS-ZNPs at higher pH related to electrostatic and steric effects between interfaces of adjacent air bubbles. Polar lipids present in the zein powder used may have also contributed to FS. Bridging de-wetting upon liquid film thinning during foam drainage, may also have resulted in lower FS. This phenomenon was more likely at play at pH 4.0 as supported by foam fractionation experiments, in which it was shown that more NP material was present in the foam phase and thus in its thin liquid films at pH 4.0 than at higher pH. The extent to which GNPs effectively stabilized foams was also pH-dependent, but substantially different trends were observed than for LS-ZNPs. The best FS was observed at pH 6.0 and 8.0, i.e., close to the gliadin isoelectric point (pI). This was explained as resulting from extensive gliadin (NP) mutual interactions upon their adsorption at the A-W interface and thus, the formation of coherent films thereat. At pH values further from the gliadin pI, repulsive electrostatic effects between adsorbed proteins (NP constituents) limited the extent of interfacial film formation resulting in low FS.

Next, it was hypothesized that co-precipitation of proteins displaying different mechanisms of foam stabilization may result in NPs with improved foaming properties. When the foaming properties of hybrid NPs based on gliadin and LS-zein (GZNPs) were investigated, they all provided FCs comparable to those of GNPs and irrespective of the gliadin-to-zein ratio used during their production. Thus, gliadin to a large degree determined the FC linked to GZNPs. In contrast, FS brought about by GZNPs seemed to originate from both protein types as intermediate values to those associated with GNPs and LS-ZNPs were noted. Good FS provided by GZNPs (which was comparable to that of GNPs) was noted at pH 8.0 and 10.0, despite the limited coherence of the formed interfacial films at these pH values. Thus, the good FS brought about by GZNPs was attributed to (limited) interfacial film formation, electrostatic effects and the contribution of endogenous lipids present in the system. At pH 4.0, the FS provided by GZNPs increased when less gliadin was used during their production which was linked to a higher coherence of the formed interfacial film.

It is worthwhile to mention that the FS noted with all the above-mentioned NP types was still relatively limited at pH 4.0 and needed further improvement. A modification strategy of GNPs based on their incubation with the protein crosslinking enzyme transglutaminase (TG) was therefore investigated. While TG treatment had no notable impact on the FC of GNPs, it positively affected their ability to provide FS at pH 4.5. This effect was linked to a strengthening of the interfacial protein films by TG-catalyzed formation of (iso)peptide bonds between GNP constituents but also to incorporation of lysine residues into these newly formed bonds that otherwise would carry a positive charge. The latter effect facilitated GNP (constituent) interactions at the interface. At pH 9.0, i.e., under conditions where unmodified GNPs provided good FS, no notable impact of TG on the functionality of GNPs was noted.

To better understand the contributions of electrostatic effects to the interfacial and foaming properties of NPs based on gliadin, it was co-precipitated in a modified LAS procedure with chitosan, a polysaccharide comprised of glucosamine units, resulting in gliadin – chitosan NP formation (GCNPs). However, at pH 4.5, i.e., where the free amino groups of gliadin and chitosan were protonated, FS provided by such GCNPs was very low. It was hence concluded that chitosan did not contribute positively to FS by increasing the electrostatic repulsion and steric hindrance between adjacent gas cells. Moreover, the presence of chitosan charged groups at the interface weakened the film due to the limited protein interaction thereat and thus worsened interface/FS.

Interestingly, although GCNPs provided limited foaming properties, they did supply excellent colloidal stability. At pH 4.0, regular GNPs were stable for up to 10 days but precipitated rapidly at pH 6.0. GCNPs, however, remained stable over the course of 17 days of storage at both these pH values. Moreover, while regular GNPs precipitated very rapidly at NaCl concentrations as low as 0.02 M, GCNPs remained stable at up to 1.5 M NaCl. Such excellent resistance against precipitation in the presence of salt has, to the best of the author’s knowledge, not been reported earlier for similar (un)modified NPs based on gliadins.

In conclusion, the work in this doctoral dissertation led to a better understanding of the mechanisms underpinning the foaming properties of cereal prolamin NPs. Moreover, a modification strategy of GNPs involving the use of TG was developed. Use of the enzyme notably improved their foaming properties at pH 4.5. In follow-up work, the foaming properties of such (TG-modified) GNPs in more complex matrices reflecting real food systems should be investigated. GCNPs, despite their poor foaming properties, hold great potential in applications for which high colloidal stability is necessary e.g., in the encapsulation of bioactive compounds. However, further investigation in this context would be, of course, required.

Date:16 Oct 2018 →  18 Dec 2023
Keywords:protein nanoparticles
Disciplines:Microbiology, Systems biology, Laboratory medicine, Biomaterials engineering, Biological system engineering, Biomechanical engineering, Other (bio)medical engineering, Environmental engineering and biotechnology, Industrial biotechnology, Other biotechnology, bio-engineering and biosystem engineering, Other chemical sciences, Nutrition and dietetics, Agricultural animal production, Food sciences and (bio)technology
Project type:PhD project