Process-induced structural changes in proteins of quinoa and the impact thereof on their functionalities

The pseudo-cereal quinoa (Chenopodium quinoa Willd.) has been cultivated for thousands of years in the Andean region. Over the past decade, it has gained interest in Western countries as nutritious gluten-free alternative for wheat in cereal-based food products. Furthermore, its protein has a well-balanced amino acid composition and is an excellent plant-based alternative for animal proteins rich in albumins and globulins.

The incorporation of quinoa (protein) into existing food product recipes and the development of novel quinoa-based food products requires insights on the structural and functional properties of its native protein as well as the impact of processing thereupon. Unfortunately, most studies available on this topic have been limited to quinoa protein isolates obtained by extraction at alkaline pH (8.0-12.0) and subsequent precipitation at isoelectric pH (4.0-5.5). In spite of the high protein yield and purity obtained with such extraction procedure, the harsh extraction conditions also induce protein denaturation and thus result in altered functional properties.

Against this background, the aim of this doctoral dissertation was to unravel the heat-induced denaturation, aggregation and functional properties of native quinoa protein. Given the potential of quinoa whole meal as plant-based alternative protein source in cereal-based food products and the overall competence profile of the research group where it was executed, this work was approached from a cereal-research perspective.

First, the extractability of protein from quinoa whole meal was investigated. It was demonstrated that the maximum protein yield that could be obtained from quinoa whole meal was 82% when using the denaturing agent sodium dodecyl sulfate (SDS) either or not containing the reducing agent dithiothreitol (DTT) and, optionally, also first applying an enzymatic or physical pretreatment. It was reasonably concluded that inert physical barriers prevent about 20% of the protein in quinoa whole meal from being extracted.

A mild extraction procedure with high protein yield and little protein denaturation was developed and optimized to obtain native protein from quinoa whole meal. The highest protein yield (64% of the total protein) with the least protein denaturation was obtained by twofold extraction (10 min) of quinoa whole meal with water followed by twofold extraction (10 min) with 0.4 mol/l sodium chloride. Albumins, globulins, prolamins and glutelins accounted for 41-50, 7-11, 4-6, and 14-16%, respectively, of the total protein in whole meal of four quinoa cultivars. Furthermore, aqueous quinoa extracts contained more than 50% of the total protein. These were both albumins and globulins as a result of quinoa whole meal containing a relatively high mineral content. Chloroplast and cytoplasm enzymes as well as legumin-like 11S and vicilin-like 7S globulins were identified. Therefore, it was concluded that aqueous quinoa extracts can serve as a reference for the total native protein in quinoa whole meal.

Quinoa seeds are usually consumed after boiling in excess water. Food products containing whole meal or protein concentrates/isolates often undergo one or more processing steps such as heating to obtain the final products. In a second part of this doctoral dissertation, the heat-induced denaturation and aggregation of native protein in aqueous quinoa extracts was investigated. Heat-induced changes in the level of total and covalent protein aggregation were evaluated by determining protein solubility losses in water and an SDS containing medium, respectively, using size separation chromatography. It was demonstrated that heating time only has an impact on the intensity of protein aggregation, while heating temperature and pH also determine the type of aggregates formed. Protein in aqueous quinoa extracts only aggregates when heated at pH 5.0 to 7.0. Non-covalent aggregation occurs when heating at temperatures not exceeding 70 °C and provided that the pH is close to the globulin isoelectric point (pH 5.0). Hydrogen bonds, hydrophobic and electrostatic interactions jointly contribute to the strength of these non-covalent aggregates. Covalent protein aggregation is maximal when heating for 15 min at 100 °C and pH 7.0. Under such conditions, oligomeric protein structures (ca. 100-500 kDa) are already formed after 1 min of heating and further polymerize to result in larger ones (> 500 kDa) after 5 min of heating. It was observed that mainly 7S and 11S globulins covalently aggregate and predominantly by disulfide (SS) cross-links. In contrast, 2S albumins are not involved in covalent protein aggregation.

Heat-induced denaturation and aggregation of protein in quinoa seeds and whole meal thereof were evaluated in the third part of this doctoral dissertation. The limited protein mobility in quinoa seeds and its presence in protein bodies makes that a significant portion (up to 37%) of the protein covalently aggregates when seeds are boiled for 15 min. 11S Globulin monomers first dissociate into their acidic and basic subunits which then further assemble into large-sized (> 500 kDa) covalent aggregates predominantly by SS cross-links. 2S Albumins are not involved in covalent aggregation and part of these proteins leach into the boiling water. In contrast, when seeds are ground to obtain whole meal, the presence of starch, dietary fiber and lipids and/or their structural organization in whole meal delays the heat-induced denaturation and hinders the aggregation of its protein. Globulins still dissociate into their subunits, but they next aggregate less and mainly small covalent aggregates (ca. 100-500 kDa) are formed during boiling of quinoa whole meal.

Protein structural and functional properties are closely related. In a fourth part, the air-water (A‑W) interfacial and foaming properties of native protein in aqueous quinoa extracts were evaluated. These are important functional properties in for example bread and cake making. Foams from aqueous quinoa extracts contain both albumins and 11S globulins. At pH 7.0, where both albumins and globulins are soluble, they cooperatively adsorb at the A‑W interface. Closer to the globulin isoelectric point (pH 5.0), only albumins adsorb at the A‑W interface, while globulins are dispersed in the liquid film between gas cells. It was concluded that the structural organization of 11S globulin acidic and basic subunits into their monomeric, trimeric or hexameric forms mainly determines the foaming capacity of aqueous quinoa extracts.

Heat-induced aggregation of globulins and hence enrichment of albumins causes the surface tension of the A‑W interface to increase. Furthermore, heating decreases the viscoelastic properties of the A‑W interface. Although processes of interfacial protein unfolding and subsequent protein-protein interaction differ from those of heat-induced denaturation and aggregation, it is suggested that quinoa albumins are not likely to interact at the A‑W interface but rather behave like colloidal particles upon adsorption. Furthermore, protein aggregates do not adsorb at the A‑W interface but are dispersed in the liquid film between gas cells and thus impact the steric stability of the foams. It is suggested that ordered covalent aggregates (formed at pH 7.0) provide steric stability, while random non-covalent aggregates (formed at pH 5.0) provide steric instability.

It was also demonstrated throughout this doctoral dissertation that the actual protein composition of a given quinoa cultivar affects the heat-induced denaturation, aggregation and foaming properties of their aqueous extracts. There are cultivar dependent variations in the stability of the SS bond connecting the acidic and basic subunits of the 11S globulin seed storage protein. Intact 11S globulin monomers more efficiently form covalent aggregates during hydrothermal treatment than their acidic and basic subunits. In contrast, lower molecular weight acidic and basic subunits are better foaming agents than intact 11S globulin monomers.

In the final part, as a proof-of-concept, it was demonstrated that wheat flour and 50% (w/w) of the egg white protein in cream cake can successfully be substituted by quinoa whole meal. The presence of preformed SS-linked protein aggregates in quinoa-based cream cake batters does not affect the level of SS cross-links present in the final cakes. Still, batters containing preformed SS-linked protein aggregates result in cream cakes with a low crumb cohesiveness and resilience and a high crumb springiness.

In conclusion, this doctoral dissertation provides a knowledge basis on the heat-induced denaturation, aggregation and foaming properties of native quinoa protein. These insights pave the way for the development of novel quinoa-based food products. Furthermore, it also serves as a basis for studying the structural and functional properties of native protein from phylogenetic related species such as amaranth (Amaranthus hypochondriacus), soybean (Glycine max L.) and yellow pea (Pisum sativum L.).