Nonlinear Surface Dilatational Rheology and Foaming Behavior of

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Non-linear surface dilatational rheology and foaming behavior of protein and protein fibrillar aggregates in the presence of natural surfactant Zhili Wan, Xiao-Quan Yang, and Leonard Martin Sagis Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b00446 • Publication Date (Web): 04 Apr 2016 Downloaded from http://pubs.acs.org on April 6, 2016

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Non-linear surface dilatational rheology and foaming behavior of protein and

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protein fibrillar aggregates in the presence of natural surfactant

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Zhili Wan,1,2 Xiaoquan Yang,1 and Leonard M. C. Sagis*,2,3 1

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Research and Development Center of Food Proteins, Department of Food Science and Technology, South China University of Technology, Guangzhou 510640, People’s Republic of China

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Laboratory of Physics and Physical Chemistry of Foods, Wageningen University, Bornse Weilanden

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9, 6708WG Wageningen, The Netherlands 3

ETH Zürich, Department of Materials, Polymer Physics, Leopold-Ruzicka-Weg 4,

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8093 Zurich, Switzerland

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AUTHOR INFORMATION

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Zhili Wan: [email protected]

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Xiaoquan Yang: [email protected]

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Leonard M. C. Sagis (*corresponding author): [email protected]; Fax: +31 317 483669; Tel: +31

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317 485023

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ABSTRACT

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In this study, the surface and foaming properties of native soy glycinin (11S) and its heat-induced

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fibrillar aggregates, in the presence of natural surfactant steviol glycoside (STE), were investigated

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and compared at pH 7.0, to determine the impact of protein structure modification on

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protein-surfactant interfacial interactions. The adsorption at, and non-linear dilatational rheological

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behavior of the air-water interface were studied by combining drop shape analysis tensiometry,

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ellipsometry and large amplitude oscillatory dilatational rheology. Lissajous plots of surface pressure

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versus deformation were used to analyze the surface rheological response in terms of interfacial

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microstructure. The heat treatment generates a mixture of long fibrils and unconverted peptides. The

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presence of small peptides in 11S fibril samples resulted in a faster adsorption kinetics than that of

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native 11S. The addition of STE, affected the adsorption of 11S significantly, whereas no apparent

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effect on the adsorption of the 11S fibril-peptide system was observed. The rheological response of

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interfaces stabilized by 11S-STE mixtures also differed significantly from the response for 11S

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fibril-peptide-STE mixtures. For 11S, the STE reduces the degree of strain hardening in extension

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and increases strain hardening in compression, suggesting the interfacial structure may change from

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a surface gel to a mixed phase of protein patches and STE domains. The foams generated from the

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mixtures displayed comparable foam stability to that of pure 11S. For 11S fibril–peptide mixtures

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STE only significantly affects the response in extension, where the degree of strain softening is

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decreased compared to the pure fibril–peptide system. The foam stability of the fibril–peptide system

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was significantly reduced by STE. These findings indicate that fibrillization of globular proteins

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could be a potential strategy to modify the complex surface and foaming behaviors of

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protein-surfactant mixtures.

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INTRODUCTION Protein-surfactant mixtures are widely used in many practical applications, such as food, 2

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cosmetics, and pharmaceutical products.1-3 For example, most foamed or emulsified food products

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contain mixtures of proteins and low-molecular weight surfactants. Both synergistic and antagonistic

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effects can occur in protein-surfactant mixtures, due to the interactions between these molecules and

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their different interface stabilization mechanisms.1, 4-7 Therefore, it is essential to understand the

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complex surface behavior of mixed protein-surfactant systems, which is closely related to their

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functional properties, such as the foamability and foam stability. In recent years, the adsorption of

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protein-surfactant mixtures have been extensively studied,8, 9 and it was found that the structure and

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composition of mixed adsorption layers generally depend on the interactions between proteins and

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surfactants, which can result in the formation of surface complexes with different properties.8, 10

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Among these studies, emphasis is mainly paid to the use of different types of protein (e.g. globular or

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random coil) and surfactant (e.g. ionic or non-ionic), or changing the bulk concentration of surfactant

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(below or above its CMC).8-13 These variations have been demonstrated to alter the protein-surfactant

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interactions and thus provide the interface with a different microstructure and mechanical properties.

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It is well known that a modification of protein molecules by chemical or physical treatment,

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leading to more exposed hydrophobicity,14 can strongly influence the adsorption behavior at the

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air-water interface. Considering the fact that the protein-surfactant interactions are mainly driven by

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hydrophobic and/or electrostatic forces,8, 9 such structural modifications of proteins would inevitably

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affect their interactions with surfactants. Therefore, for a specific mixture of protein and surfactant, it

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could be feasible to control their mutual interactions at the interface through the modification of

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protein structure itself, which may be another potential strategy to modify the foaming and foam

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stabilization of mixtures. In food processing, the heat treatment of many food proteins, like

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β-lactoglobulin and soy glycinin, at different pHs and ionic strengths can induce the formation of

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supramolecular aggregates with various structures, which include fibrillar structures (protein

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fibrils).15-17 Such fibrils, like their source proteins, have been shown to be efficient stabilizers of

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air-water and oil-water interfaces.18-20 However, the fibrils and native proteins exhibit very different 3

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behaviors at both interfaces due to their different structural characteristics. For example, fibrils can

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form interfaces with a higher dilatational elastic modulus than the native protein, while native

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proteins seem to be more efficient in lowering the surface tension.18, 19 Therefore, it is of interest to

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understand the surface and the functional behaviors (e.g. foam stabilization) of protein fibrils in the

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presence of surfactants, which may be different from those of native protein mixed system. However,

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the effects of interactions between protein fibrils and surfactant at the interface on functional

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behavior so far has not received much attention in the research literature.

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Recently, naturally occurring surface-active substances have attracted increased interest due to

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their safety and intrinsic biodegradability. These natural surfactants, such as saponins, exhibit

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remarkable surface activity due to their amphiphilic structures, and in most cases are also essential

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bioactive ingredients for the human body.21 Thus, the unique combination of surface properties and

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biological activity makes these bi-functional natural surfactants particularly attractive for application

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in the food, cosmetics and pharmaceutical fields. Most recently, researchers have demonstrated that

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triterpenoid saponins (Quillaja) can form highly elastic layer at the air-water interface, but the

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adsorption layers of steroid saponins (Yucca) are purely viscous, due to different molecular

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structures.22, 23 The adsorption kinetics of mixtures of Quillaja saponins with a globular protein

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(β-lactoglobulin) at the air−water and oil−water interfaces has also been described in detail.24

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Another group of terpenoids, diterpenoid steviol glycosides (STE), a noncaloric natural sweetener in

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food products, also show notable surface activity and can be developed into a new type of natural

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surfactants, due to their similar amphiphilic molecular structures to triterpenoid saponins.25 In a

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recent study, we characterized the adsorption and dilatational rheological properties of STE, which

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are similar to those observed for many common low molecular weight surfactants.26, 27 We also found

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that, in the presence of globular protein (soy protein), the protein-STE mixtures exhibit synergistic

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effects in reducing interfacial tension of both air-water and oil-water interfaces at low STE

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concentrations, probably due to protein/STE interfacial complex formation.26, 27 The formed mixed 4

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interface shows an increased response to deformations, accompanied with a plateau in surface

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elasticity during time sweeps. The foams or emulsions stabilized by the protein-STE mixtures exhibit

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superior properties compared to the individual components, such as remarkable foamability and

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long-term emulsion stability.26, 27 Despite the promising applications of mixed protein-STE systems

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in industrial formulations, the surface properties of such systems are still not sufficiently well

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characterized, and as a consequence, very little information is available about the detailed

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microstructure and mechanical properties of mixed protein−STE adsorbed layers. Such knowledge is

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essential in understanding foam and emulsion formation and stabilization for these mixtures.28, 29

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Currently, most studies on surface rheology (especially surface dilatational rheology) focus on the

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small deformation regime with a linear stress response to applied deformations, aiming to investigate

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the structural properties of interfaces close to equilibrium. From a practical point of view, these

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measurements do not reflect the real conditions of formation and evolution of foams and emulsions,

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since most of these systems are exposed to far from equilibrium conditions (large and fast

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deformations) during their production and processing, where responses of interfaces are often highly

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non-linear.28 Therefore, a rheological characterization of the non-linear response of interfaces is far

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more relevant to understand the surface behavior of multiphase systems. In a recent paper, we have

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adopted non-linear surface dilatational rheology as a tool to understand the rheological response of

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air-water interfaces stabilized by oligofructose fatty acid esters, and found that the interfaces

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stabilized by these esters display a highly non-linear response to high dilatational amplitudes.30 We

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observed that, at the highest applied amplitudes (up to 30%), the interface is strain softening during

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extension and strain hardening during compression, by analyzing the Lissajous plots of surface

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pressure versus strain. This rheological response of these interfaces was attributed to the formation of

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a 2D soft glass phase by the esters.30 This particular example demonstrates that the use of Lissajous

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plots in surface dilatational rheology can increase the understanding of the link between surface

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rheological response and interfacial microstructure. 5

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As mentioned above, detailed information about the microstructure and mechanical properties of

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mixed protein−STE adsorbed layers is limited, and in particular their non-linear surface dilatational

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behavior has so far not been examined. Therefore, the major aims of the current work are (1) to

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characterize the non-linear dilatational rheological properties of native protein-STE mixtures at the

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air-water interface; (2) to compare the surface properties of native protein and the protein

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fibril-peptide system, respectively, in the presence of STE, with the aim to assess the impact of

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protein structure modification on the protein-surfactant interactions at the interface; (3) to investigate

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and compare the links between surface behavior and the functional properties (i.e. foamability and

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foam stability) of these two mixed systems. To achieve our aims, we first prepared a protein

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fibril-peptide system by heat-induced fibrillar aggregation of soy glycinin (11S), the major and pure

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globulin of soybeans. This protein was chosen because its structural and functional properties have

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been extensively studied. Previous studies have also shown that most properties of soy glycinin

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fibrils are similar to β-lactoglobulin and whey protein isolate fibrils.17 We performed surface

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rheological measurements at high dilatational amplitudes from 10% to 30%, to study the non-linear

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rheological response of adsorption layers of 11S protein, the 11S fibril-peptide system, and their

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mixtures with STE. We used Lissajous plots to extract quantitative information on the nonlinearity in

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the response of interfaces. In addition, ellipsometry was used to measure the adsorbed amounts of

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proteins and STE at the air-water interface. Finally, the foaming properties of these two mixed

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systems were measured and compared.

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EXPERIMENTAL SECTION

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Materials. Defatted soy flour was provided by Shandong Yuwang Industrial and Commercial Co.,

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Ltd., China. The protein content of soy flour was 55.10% (determined by micro-Kjeldahl method, N

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× 6.25, dry basis). Soy glycinin (11S) was prepared according to a previously method by Yuan et

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al.31 The protein content of 11S was 97.39%, determined by using Dumas analysis (N × 6.25) using a

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Flash EA 1112 NC analyzer (Thermo Fisher Scientific Inc., Waltham, MA, U.S.A.). Sodium dodecyl 6

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sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that the purity of isolated

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11S was above 94.5% (data not shown). STE (95.82% total diterpenic glycosides, containing 60%

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stevioside and 30% rebaudioside A, 3.07% moisture, and 0.13% ash) was purchased from Jining

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Aoxing Stevia Products Co., Ltd., China. All other chemicals used were of analytical grade.

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Solution Preparation and Fibril Formation. The native 11S and STE solutions were prepared in

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10 mM phosphate buffer solution (pH 7.0) using Milli-Q water at room temperature (20 °C). The

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native 11S solution was obtained by dissolving the protein in phosphate buffer (10 mM, pH 7.0) for 2

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h under mild magnetic stirring and then left overnight at 4 °C to allow complete hydration. The

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obtained solution was then centrifuged at 10000g for 30 min at 4 °C to remove any insoluble

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materials (around 3.5% of the protein sample). The protein concentration of solutions was

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determined using Dumas analysis (N × 6.25). From now on, we refer in this manuscript to this native

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protein system as “11S”.

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Preparation of 11S fibril-peptide system was carried out according to a previously described

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protocol.17 Briefly, native 11S solutions were made by dissolving the protein in Milli-Q water. The

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pH of the protein solution was adjusted to pH 2.0 with 6 M HCl solution, followed by centrifugation

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(10000g, 30 min, 4 °C) to remove any undissolved materials. The obtained 11S solution (2 wt%) was

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placed in small glass vials (20 mL) and then incubated at 85 °C for 20 h under continuous stirring at

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300 rpm by using a metal heating and stirring plate. After heat treatment, samples were immediately

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cooled in an ice bath and then stored at 4 °C for further use, typically within 1 week. The formed

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protein fibrils are long semiflexible strands with a thickness of a few nanometers, a persistence

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length of about 2.3 µm, and a contour length of about 1 µm.17 From now on, we refer in this

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manuscript to this fibril system as “11S fibril-peptide system”. This system is a mixture of

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approximately 20% pure fibrils and 80% unconverted peptides, determined according to the method

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described in previous papers.17, 32 7

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To prepare the mixtures of 11S/11S fibril-peptide system and STE, separate stock solutions of

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11S/11S fibril-peptide system and STE with doubled concentrations were first prepared. The final

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mixed systems were obtained by mixing these stock solutions (1:1 by weight) up to the required

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concentration and stirring for a further 30 min before any tests. All measurements were performed at

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constant protein concentration (0.1 wt%) and varying STE concentration. The pH of all systems was

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fixed at 7.0. Based on our previous studies, at high STE concentrations (above its CMC of 0.5 wt%),

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the surface and foaming properties of mixed systems would be dominated by STE molecules.27

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Therefore, in the present work, the STE concentrations used were below its CMC (0.1 and 0.25%).

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Surface Tension and Surface Dilatational Rheology. The surface tension and surface dilatational

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modulus of the studied samples in 10 mM phosphate buffer (pH 7.0, ionic strength I = 25 mM) at the

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air-water interface were determined using a Profile Analysis Tensiometer (PAT-1M, Sinterface

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Technologies, Germany). A drop of sample solution was formed in a rectangular glass cuvette and

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monitored with a video camera. The surface tension (γ) was calculated from the shape analysis of a

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pendent drop according to the Gauss-Laplace equation. The dynamic surface tension of all the

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solutions was monitored for 180 min at a constant area of 25 mm2 of the drop. After this period,

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quasi-equilibrium conditions were obtained and the dilatational rheology measurements were then

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performed.

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To investigate the rheological response at high deformations, dilatational amplitude sweeps from

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10% to 30% deformation were performed at a constant frequency of 0.1 Hz. In addition to the

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amplitude sweeps, a frequency sweep was performed after 180 min of equilibrium time. The

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amplitude was constant at 10% and the frequency was increased stepwise from 0.005 Hz to 0.1 Hz.

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The slope of a double logarithmic plot of complex surface dilatational modulus versus frequency was

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determined using a linear regression fit. The complex modulus was calculated via a Fourier

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transform from the surface tension variation as a response to the generated harmonic area oscillations 8

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(extension and compression) of the drop surface. All experiments were performed at 20 °C. Reported

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values represent the average of 3-7 measurements.

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Lissajous Plots. The analysis of dilatational amplitude sweeps was performed according to the

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method developed by Van Kempen et al.30 and Rühs et al.33 The results of amplitude sweeps were

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presented in the form of Lissajous plots, of the surface pressure (Π = γ − γ0) versus deformation

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(δA/A0). Here δA = A - A0, γ and A are the surface tension and area of the deformed interface, and γ0

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and A0 are the surface tension and area of the non-deformed interface. For the analysis of these

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curves, we defined the following four factors: EL,E, defined as the large strain modulus in extension,

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EM,E, defined as the minimum strain modulus in extension, EL,C, defined as the large strain modulus

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in compression, EM,C, defined as the minimum strain modulus in compression. The method for

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determining these factors has been discussed in detail in a previous study.30 Based on these moduli,

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two nonlinearity parameters can be defined, one for the extension part of the cycle, Sext = (EL,E -

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EM,E)/EL,E, and one for the compression part, Scom = (EL,C - EM,C)/EL,C. For both factors, S = 0 may be

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interpreted as a linear elastic response, S > 0 indicates strain hardening behavior of the interface, and

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S < 0 corresponds to intracycle strain softening. With these factors as a function of applied strain, the

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degree of nonlinearity in the response of interfaces during extension and compression can be

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quantitatively analyzed.

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Ellipsometry. The adsorbed amount and the adsorption layer thickness were measured by null

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ellipsometry using a Multiskop instrument (Optrel GBR, Sinzing, Germany). The details of this

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apparatus and the procedure to calculate adsorbed amount and layer thickness have been given

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elsewhere.34 The light source was a He-Ne laser with a wavelength of 632.8 nm, and the incidence

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angle of the light was set at 50º. The changes of ellipsometric angles (∆ and Ψ) due to the adsorption

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of materials at the air-water interface were measured and further fitted by using a three-layer model. 9

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This model assumes a single homogeneous adsorption layer characterized by an average refractive

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index (nad) and a thickness (δad) between two homogenous phases (water and air). The adsorbed

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amount (Г, mg/m2) can be calculated according to the following equation:34

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Г =

     



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where nbuffer (nbuffer = nwater) is the refractive index of buffer solution and dn/dc is the refractive index

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increment of the system under investigation. The measurements were performed in triplicate.

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Foaming Properties. The foaming properties of the studied systems were measured using a

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Foamscan (Teclis IT-Concept, Longessaigne, France). An initial volume of 60 mL of solutions was

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foamed by sparging nitrogen at a constant gas flow rate of 400 mL/min through a metal frit (60 mm

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diameter, pore size 27 ± 2 µm, 100 µm distance between centres of pores, square lattice). Bubbling

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was stopped after a volume of 400 cm3 of foam was obtained. The foam formation, the drainage of

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liquid from the foam, and foam stability were followed by conductivity and optical measurements of

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the foam column. Series of images of the foam were taken every 20 s by a CCD camera to observe

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the changes of bubbles. The bubble size was measured at the middle of the foam column using a

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CCD camera and a prism and lighting arrangement at the surface of the foam tube. The time for the

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foam to drain 50% of its initial liquid content is defined as the foam liquid stability (s). The time

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required for the foam volume to reduce to half of its initial volume (half-life, t1/2) was used as an

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indicator for the foam stability. Reported values are averages of at least three individual

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measurements. All experiments were performed at 20 °C.

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Statistical Analysis. Unless specified otherwise, three independent trials were performed, each with

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a new batch of sample preparation. Analysis of variance (ANOVA) of the data was performed using

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the SPSS 19.0 statistical analysis system. A Duncan Test was used for comparison of mean values

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among three treatments using a level of significance of 5%. 10

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RESULTS AND DISCUSSION

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Adsorption Behavior. To investigate the impact of protein structure modification and STE

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concentration on the surface and subsequent foaming properties of protein-STE mixtures, the

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adsorption of the studied systems at the air-water interface was first determined using pendant drop

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tensiometer and ellipsometry.

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Surface Tension. Figure 1 shows the time evolution of surface tension of 11S/11S fibril-peptide

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system-STE mixtures at the air-water interface. As seen from Figure 1, for all systems, the surface

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tension gradually decreased with adsorption time and after 3 h the surface tension values became

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relatively constant as the surface was already saturated. However, these two mixed systems exhibited

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different adsorption kinetics. For native 11S-STE mixtures, pure 11S showed a high initial surface

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tension value (1 s, ~70 mN/m), indicating its slow adsorption at the interface. This should be mainly

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due to the large molecular size and rigid structure of 11S, thus hindering the fast diffusion to the

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interface. Upon the addition of STE (0.1 and 0.25%), the initial surface tension values of 11S-STE

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mixtures significantly decreased, suggesting that the presence of STE greatly accelerates the

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adsorption of 11S. Previous studies have demonstrated that the STE can bind to 11S by nonspecific

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hydrophobic interactions, which can promote the partial dissociation of 11S and loosen its rigid

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structure, and thus facilitate the migration of 11S to the interface.26, 27 After 3 h of adsorption, the

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mixtures still had lower equilibrated surface tension values than that of pure 11S, however, the

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observed difference between these values (48.3-50.3 mN/m) was not very obvious, which points to

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the interfaces with similar surface density and composition.

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For 11S fibril-peptide-STE mixtures, the initial (1 s) and equilibrated (3 h) surface tension values

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of 11S fibril-peptide system were 53.5 mN/m and 48.1 mN/m, respectively, which are much lower

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than those of native 11S, indicating a faster adsorption kinetics for the 11S fibril system. The 11S 11

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fibril-peptide system is a transparent solution at pH 2.0, consisting of long fibrils and small peptides.

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After adjustment to pH 7.0, this system became a little turbid (data not shown), which is mainly

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attributed to the presence of some small peptide aggregates due to the reduced electrostatic repulsive

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forces (data not shown). Therefore, at pH 7.0, the 11S fibril-peptide system should consist of pure

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fibrils, small peptides and some small peptide aggregates.32 We attribute the faster adsorption

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kinetics of the 11S fibril system (Figure 1) to the presence of small peptides in this system.

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Compared to large fibrils (and also native 11S), these small peptides have a faster diffusion rate

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toward the interface, decreasing the surface tension more rapidly and thus dominating the initial

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adsorption process of the 11S fibril system. Similar behaviors were also observed in the adsorption

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kinetics of β-lactoglobulin long semiflexible and short rod-like fibril systems at the air-water

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interface.18 The presence of STE in the 11S fibril-peptide system did not affect the adsorption

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kinetics significantly, apart from a slight decrease in the equilibrated (3 h) surface tension values

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(46.5-48.3 mN/m). This is very different from what we observed in native 11S-STE mixtures. Either

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the STE added to the 11S fibril-peptide system did not interact with the peptides or fibrils, or the

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interactions did not affect the structure of the interface in a significant manner. Note that the ability

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of pure STE (0.25%) to reduce surface tension is lower than that of 11S fibril-peptide system (0.1%)

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(data not shown).

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Ellipsometry. Figure 2 shows the dynamic adsorbed amount (Г, Figure 2A) and thickness (δad, Figure

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2B) of adsorption layers formed from the mixtures of 11S/11S fibril-peptide system with STE. As

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can be seen in Figure 2A, for all investigated systems, Г gradually increased with time, indicating the

298

materials accumulation at the interface and the subsequent formation of the adsorption layer. These

299

two mixtures exhibited rather different kinetics for the adsorbed amount. For 11S-STE mixtures, in

300

all cases, Г initially increased rapidly, followed by a slow gradual increase. After 104 s the adsorbed

301

amount is still increasing and has not reached a steady state value. For the 11S fibril-peptide-STE 12

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302

systems, Г appears to reach a steady state much faster than for the 11S-STE mixtures. This is

303

consistent with the surface tension results (Figure 1), in which the 11S fibril-peptide-STE mixtures

304

showed a much larger initial drop than 11S-STE mixtures. The values for Г for the 11S

305

fibril-peptide-STE mixtures (2.7-2.9 mg/m2) were lower than those of 11S-STE mixtures (4.2-4.6

306

mg/m2). However, the 11S fibril-peptide-STE mixtures showed a higher ability to reduce the surface

307

tension than 11S-STE mixtures (Figure 1). The faster adsorption, the lower adsorbed amount, and the

308

larger decrease in surface tension all suggest that for the 11S fibril-peptide-STE mixtures a

309

substantial amount of small peptides is adsorbed at the interface.

310

For pure 11S, the measured Г value after 3 h of adsorption was 4.2 mg/m2, which is higher than

311

the average Г reported for a monolayer of most proteins (2.0-3.0 mg/m2),1 suggesting the possibility

312

of multilayer formation at the bulk concentration used here (0.1%). This hypothesis is supported by

313

the δad value of 6.9 nm for 11S after 3 h (Figure 2B), which is also higher than the average δad for

314

monolayers of most proteins (about 4.0 nm). With increasing STE concentration, compared to pure

315

11S, the initial Г of mixtures decreased (Figure 2A). This points to a slower initial adsorption, and

316

could be the result of complex formation between the 11S and STE. The final Г was not affected

317

significantly by STE. Compared to pure 11S, the δad values for 11S-STE mixtures increased

318

(especially at 0.25% STE), as shown in Figure 2B. This suggests conformational changes of the

319

protein molecules in the surface layer due to their interactions with STE.26, 27 This hypothesis is

320

supported by the decreased refractive index of the adsorption layer for mixtures (especially at 0.25%

321

STE) (data not shown). This increase of the δad with increasing surfactant concentration was also

322

observed in other protein/surfactant mixtures.11, 35

323

For 11S fibril-peptide-STE mixtures, the impact of STE on Г and δad was much smaller than in

324

11S-STE mixtures. Addition of STE to the fibril-peptide system, leads to a slight decrease in Г

325

values (Figure 2A), and no obvious changes in the δad were found (Figure 2B). Note that the δad value

326

(7.9 nm) for the 11S fibril-peptide system was very high, suggesting the possibility of a multilayer 13

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327

formation. Multilayer formation was also observed for β-lactoglobulin fibril-peptide systems.36, 37 In

328

view of the complexity of the11S fibril-peptide system, it is a challenging task to obtain clear insight

329

into the contribution of each individual compound (peptide materials, pure fibrils and STE) during

330

the adsorption. However, when combining the results of the surface tension measurements and

331

ellipsometry the adsorption layer formed from 11S fibril-peptide-STE mixtures appears to be more

332

complex and heterogeneous than that of 11S-STE mixtures.

333 334

Dilatational Rheological Behavior.

335

Frequency Sweeps. To gain more insight about the microstructure and mechanical properties of

336

interfaces stabilized by the 11S-STE and 11S fibril-peptide-STE mixtures, dilatational surface

337

rheology measurements (frequency and amplitude sweeps) were performed. Figure 3A shows the

338

results of the frequency sweeps applied to interfaces stabilized by these two mixtures. For all

339

investigated cases, the dilatational storage modulus (E′) was much higher than the loss modulus (E″)

340

(data not shown) and the loss tangent (E″/E′) values were also low (around 0.03-0.06), suggesting

341

that all the interfaces exhibit a highly elastic response at such frequencies. It can be seen from Figure

342

3A that for all systems the complex dilatational modulus increased with increasing frequency,

343

showing a similar frequency dependence of the modulus. This behavior is caused by relaxation

344

mechanisms at the interface, which include the exchange of molecules between the bulk solution and

345

the surface, and in-plane structural rearrangements of the surface layer.38

346

The slope of a double logarithmic plot of complex surface dilatational modulus as a function of

347

frequency was determined, as shown in Figure 3B. A slope of 0.5 indicates the dilatational elasticity

348

is predominantly determined by the rate of diffusional exchange of surfactant between the interface

349

and the bulk phase, and as shown by the Lucassen van den Tempel model,39 is a typical value for the

350

low frequency response of interfaces stabilized by low molecular weight surfactants which adsorb

351

reversibly at an interface. A slope that approaches 0 implies a completely elastic response, and is 14

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352

often an indication that the surface active materials have self-assembled into a highly elastic film,

353

with a dilatational modulus determined by in-plane interactions. However, low molecular weight

354

surfactants which do not self-assemble into elastic films may also have a low slope, when the

355

frequencies are sufficiently high, such that the rate of deformation of the interface is much faster than

356

the rate of diffusional exchange. These two types of interfaces can be distinguished by amplitude

357

variations, since for the former interfaces the dilatational modulus tends to be strongly strain

358

amplitude dependent, whereas the latter is relatively insensitive to strain amplitude variations.

359

As can be seen from Figure 3B, the difference in the slope values for pure 11S and 11S

360

fibril-peptide system was not significant, and the slope values (about 0.10) were much lower than the

361

value of 0.5 predicted by the Lucassen van den Tempel model,39 indicating that both the interfaces

362

stabilized by pure 11S and the 11S fibril-peptide system exhibit similar mostly elastic solid-like

363

behavior. It should be noted that the dilatational modulus of the 11S fibril-peptide system was higher

364

than that of native 11S (Figure 3A), implying that a stronger surface layer is formed by the 11S

365

fibril-peptide system. Previous studies have reported that the values of dilatational and shear

366

complex surface modulus are higher for β-lactoglobulin fibrils, as compared to the native proteins.18,

367

33

368

network, thus increasing the surface elasticity. The interfacial layer was demonstrated to be stronger

369

for β-lactoglobulin fibril-peptide systems (mixture of peptides, monomers and fibrils) as compared to

370

pure fibrils, due to the contribution of the smaller molecules to the interfacial modulus.18 Also, the

371

polydispersity of the long fibrils contributes to enhancing their packing at the surface, thus

372

strengthening the surface layer.18-20 For the 11S fibril-peptide system, the interactions between

373

peptides and fibrils adsorbed at the surface are also likely to be a factor in the results shown in Figure

374

3A. This will be further discussed in the next paragraphs.

This is caused by the large size of long fibrils, which contributes to the formation of an interfacial

375

From Figure 3B, it can be clearly seen that the impact of STE on the slope values of 11S-STE and

376

11S fibril-peptide-STE mixtures is different. For 11S-STE mixtures, with increasing STE 15

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377

concentration, especially at 0.25% STE, the slope values increased significantly. The slope for the

378

11S-0.25%STE mixture was about 0.20, which points neither to a diffusion-controlled elasticity nor

379

to the presence of a highly elastic layer. This surface layer is clearly less elastic compared to pure

380

11S, with much lower values for the dilatational modulus (Figure 3A), in agreement with previous

381

studies.27 This is most likely due to the presence of STE at the interface, which could break down the

382

in-plane protein intermolecular interactions and partly disrupt the interfacial network, thus reducing

383

the viscoelastic properties.27 This is consistent with the results of the adsorption layer thickness for

384

the 11S-0.25%STE mixture (Figure 2B).

385

In contrast, for 11S fibril-peptide-STE mixtures, the impact of STE on the slope values was not

386

significant, in agreement with the results of surface tension (Figure 1) and ellipsometry (Figure 2),

387

suggesting that the interfaces still exhibited mostly elastic behavior. However, it is noted that the

388

dilatational modulus slightly decreased with increasing STE content (especially at 0.25% STE), but

389

to a much lesser extent than for the 11S-STE mixtures (Figure 3B). Thus, on the basis of these results,

390

we can speculate that, for the 11S fibril-peptide-STE mixtures, the presence of STE domains could

391

also weaken the surface layer, leading to the decrease in modulus; however, they cannot disrupt the

392

interfacial network as much as in the 11S stabilized interface, due to the stronger surface layer

393

formed by 11S fibril-peptide system (Figure 3A), and thus the interfaces still retain a predominantly

394

elastic behavior. We need to point out here that at a strain amplitude of 10% the data for the 11S

395

fibril-peptide-STE mixtures are already showing nonlinearities (as we will also see in the discussion

396

of the amplitude sweeps). Hence the first harmonic based moduli presented here may be somewhat

397

inaccurate. Since reducing the amplitude to lower values results in a very noisy signal for the surface

398

pressure, we have opted to compare all frequency sweeps at 10% amplitude.

399 400

Amplitude Sweeps. While frequency sweeps are often performed in dilatational surface rheology,

401

amplitude sweeps are much less commonly used. However, when the dilatational modulus is 16

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402

expected to be the result of any in-plane structure formation at the interface, the interfacial

403

microstructure may be affected when the deformation amplitude is high. Therefore, to investigate the

404

dependence of the rheological properties on amplitude, amplitude sweeps were performed. The

405

amplitude was increased from 10% to 30%. Figure 4 shows the results of the complex surface

406

dilatational modulus versus amplitude for the interfaces formed from 11S-STE and 11S

407

fibril-peptide-STE mixtures. As can be seen, the surface dilatational modulus continuously decreased

408

with increasing amplitude for all the systems, except the 11S-0.25% STE mixture. This nonlinear

409

rheological response suggests that the interfacial microstructure is affected by the high degree of

410

deformation. For the 11S-0.25% STE mixture, based on the low dilatational modulus during

411

amplitude sweeps, and the fact that the modulus is relatively strain independent, combined with the

412

observation in Figure 3B, where a significantly higher frequency dependence of the modulus was

413

observed, we can conclude that the response of this interface is significantly affected by diffusive

414

exchange of the STE, and to a lesser extent by in-plane interactions between the 11S proteins.

415

Considering the significant nonlinearity in the dilatational rheological response at high strain

416

amplitude, the values for the dilatational modulus determined by the tensiometer, based on the

417

intensity and phase of the first harmonic of the Fourier transform of the surface pressure signal, are

418

bound to be inaccurate. As pointed out by Ewoldt et al.40, any nonlinearity present in an unprocessed

419

stress signal is disregarded in a first harmonic based analysis. Moreover, although the amplitude

420

sweep plot unveils the amplitude dependence of the modulus, any further information on the

421

rheological response of surface during extension and compression, which is closely related to the

422

interfacial structure, cannot be obtained. Therefore, in the following section, we use Lissajous plots

423

of surface pressure versus deformation to help interpret surface dilatational behavior during

424

amplitude sweeps. Previous studies also demonstrated that Lissajous plots are very useful in

425

analyzing the nonlinearities in large amplitude surface shear and dilatational experiments.30, 33, 37, 41

426 17

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427

Lissajous Plots. Figure 5 shows the Lissajous plots of surface pressure against deformation during

428

amplitude sweeps for 11S-STE and 11S fibril-peptide-STE mixtures. The shape of the Lissajous plots

429

strongly depends on the deformation amplitude and the concentration of STE. For 11S, at an

430

amplitude of 10%, the Lissajous plot shows only minor asymmetries, and the S factors for extension

431

and compression in Figure 6 are roughly equal to zero (within the margin of error of the

432

measurement), indicating a close to linear viscoelastic response (predominantly elastic), which is

433

consistent with our previous studies,27 where the amplitude of 10% was still within the linear

434

viscoelastic regime. With increasing amplitude, the plots became increasingly asymmetric, and the

435

interfaces display strain hardening both in extension and compression, where the effect is stronger in

436

compression. At an amplitude of 30%, it can be seen that the maximum surface pressure observed in

437

compression was much higher than that observed in extension. For the 11S-STE mixtures, the

438

addition of STE, particularly at the highest concentration, results at low amplitude (10%) in a mild

439

strain softening behavior in extension, and at high amplitudes, in a decrease in strain hardening,

440

compared to pure 11S. In the compression part of the cycle we observe an increase in the degree of

441

strain hardening, for all amplitudes. These results are in agreement with the findings in Figures 3 and

442

4, and confirm that the STE is disrupting the elastic film formed by pure 11S.

443

Compared to 11S, the 11S fibril-peptide system displayed a different shape of the Lissajous plots.

444

As can be seen, even at an amplitude of 10%, the Lissajous plot showed pronounced asymmetries,

445

showing a non-linear viscoelastic behavior. In compression the maximum surface pressure was

446

higher than that observed in extension and the plot also became narrower, which implies that the

447

surface is strain hardening upon compression. At the lower amplitudes, a widening of the Lissajous

448

plot was observed in extension and this suggests that the surface had a more viscous response in this

449

part of the cycle (strain softening). However, at higher amplitudes (25% and 30%), the extension

450

phase of the plot became increasingly narrower, which points to a strain hardening upon extension.

451

This is confirmed by the S values for extension and compression, which are all positive in 18

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452

compression (Figure 6B), and in extension they are negative for small amplitudes and become

453

positive for amplitudes of 25% and above (Figure 6A). This type of behavior suggests that these

454

interfaces, which are composed of fibrils and small peptides, have a segregated structure composed

455

of patches of fibrils and patches of small peptides. Upon compression, the smaller peptides are likely

456

to be pushed out of the interface, leading to an increase in the surface fraction of fibrils, and

457

consequently to an increase in elasticity. In extension the surface fraction of small peptides increase,

458

leading to a softening of the structure. The fact that there is still a mild degree of hardening in

459

extension for the largest amplitudes indicates that this segregated interfacial network still has a high

460

degree of connectivity at these high amplitudes, and is not completely disrupted. Similar behavior is

461

also observed in the oil-water interface stabilized by ovalbumin fibrils.42 Previous work by Jordens et

462

al.36 has shown that protein fibrils can form a complex interfacial mesostructure, consisting of

463

nematic domains and isotropic domains.

464

For 11S fibril-peptide-STE mixtures, with increasing STE concentrations, the degree of strain

465

softening in the extension phase of the plots was somewhat reduced, especially in the amplitude

466

range from 10% to 20%. In compression the STE had only a minor effect. We saw that at 0.25% the

467

STE does decrease the modulus of 11S fibril-peptide system-stabilized interfaces, but apparently it

468

does not markedly change the nature of the microstructure of the interface, since even for the

469

fibril-peptide system itself the interface is most likely already in a segregated state. This analysis

470

needs to be further confirmed by structural characterization methods, such as Brewster angle

471

microscopy (BAM) and particle tracking observed by microscopy.43

472 473

Foaming Properties. We now attempt to relate the surface behavior of 11S/11S fibril-peptide-STE

474

mixtures to the formation and stability of macroscopic foams formed from these systems with the

475

same compositions as used in the study of the surface properties. Here, we address foamability in

476

terms of initial bubble size (at 0 s). As can be seen from Figure 7A, the bubbles formed from pure 19

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477

11S showed a large initial bubble size, and the bubble shape appeared to be polyhedral, which

478

suggests that the foamability of 11S is very low, and most of the liquid may already drain during

479

foam creation. This is in good agreement with our previous study,27 showing that the foamability of

480

11S is also low in a whipping test. In general, high 11S concentration (2% or more) is required to

481

make good foams. For 11S-STE mixtures, even in the presence of 0.1% STE, a uniform bubble size

482

distribution with small spherical bubbles was observed. Also, the liquid films appear to be thicker

483

than those of pure 11S. This implies that the foamability of 11S is significantly increased by STE.

484

Compared to 11S, the 11S fibril-peptide system exhibited better foamability since a more uniform

485

bubble size distribution with smaller bubbles was found in the initial bubble image (Figure 7B). In

486

addition, the foam stability of the 11S fibril-peptide system appeared to be better than that of 11S by

487

comparing the images of the bubbles with time, which will be further discussed in the following

488

paragraphs. Similar to 11S-STE mixtures, the 11S fibril-peptide-STE mixtures also showed better

489

foamability with smaller bubble size and thicker liquid channels than those of the pure 11S

490

fibril-peptide system.

491

To evaluate the foam stability, the foam volume and liquid volume in the foams were measured as

492

a function of time. Figure 8A shows the variation of liquid volume in the foams generated from

493

11S/11S fibril-peptide-STE mixtures. The maximum of the liquid volume in the foam formed from

494

11S is very low (about 10 mL) compared to foam prepared from the 11S fibril-peptide system. The

495

presence of STE clearly increased the maximum in the liquid volume in the foams of mixtures. These

496

results are consistent with the observations of the liquid films in initial bubble images (Figure 7). In

497

general, a high liquid volume in the foams favors their stabilization, and hence the rate of drainage is

498

important to assess foam stability. Therefore, the foam liquid stability (the time in which 50% of the

499

initial liquid volume has drained) was calculated and plotted in Figure 8B. It can be clearly seen that,

500

foams prepared with the 11S fibril-peptide system have higher foam liquid stability than those of 11S.

501

It is interesting to note that the effect of STE on the foam liquid stability of 11S and the 11S 20

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502

fibril-peptide system is different. With increasing STE concentration, the foam liquid stability of

503

11S-STE mixtures increased, whereas a reverse trend occurred in the 11 fibril-peptide-STE mixtures.

504

This suggests that the presence of STE can decrease the liquid drainage rate of 11S, but increase the

505

rate of drainage in 11S fibril-peptide system stabilized foam. Significantly different effects of STE on

506

foams prepared with 11S and the 11S fibril-peptide system are also observed in the foam decay

507

curves (Figure 8C) and the half-life time (t1/2) of foams (Figure 8D). For 11S-STE mixtures, with

508

increasing STE concentration, no apparent difference was observed in foam decay rates and t1/2

509

values. In contrast, for 11S fibril-peptide-STE mixtures, a substantial increase in decay rates and

510

decrease in t1/2 values were observed with increasing STE concentration. This is in line with the

511

images of the bubbles with time (Figure 7) and the data on liquid drainage (Figures 8A and B). On

512

the basis of these results, it can be concluded that, in the presence of STE (0.1-0.25%), the 11S-STE

513

mixtures produce more stable foams compared to 11S, in agreement with the previous whipping test

514

results,27 whereas the stability of foams prepared from the 11S fibril-peptide system is decreased by

515

the addition of STE. It is noteworthy to mention that, even at the low protein concentration used here

516

(0.1%), the 11S fibril-peptide system still exhibited impressive foam stability, with a t1/2 value of ~13

517

h, which is much higher than that of native 11S at the same concentration. This implies that the 11S

518

fibril-peptide system at pH 7.0 is a promising protein material to make very stable foams when

519

higher concentration of 11S fibril-peptide system (1% or more) are used.

520 521

General Discussion. Combining the results of the dynamic surface tension measurements and

522

ellipsometry (Figures 1 and 2A), with the results for the foamability we see a strong correlation

523

between the adsorption behavior (especially the initial surface tension) of 11S-STE mixtures and 11S

524

fibril-peptide-STE mixtures (Figure 7), and their foamability. Compared to the 11S system, the 11S

525

fibril-peptide system has a much lower initial surface tension, and also a much smaller bubble size,

526

just after foam preparation. For both systems the addition of STE lowers the initial surface tension, 21

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527

and the resulting foams also have a smaller initial bubble size. The effect of STE is much more

528

significant for the 11S system, than for 11S fibril-peptide system (Figures 1 and 2), which may be

529

due to the different structural characteristics and complexity of the protein systems, which affect

530

their interactions with STE. The adsorption properties (surface tension decay, adsorbed amount, and

531

layer thickness) of native 11S is most likely affected by STE (Figures 1 and 2) due to hydrophobic

532

binding of 11S with STE,27 which affects the migration of 11S toward the interface, leading to a

533

more pronounced and faster drop in surface tension, and thus improving its foamability (Figure 7). In

534

contrast, there is only a small effect of STE on the surface tension (Figure 1) and adsorption layer

535

properties (Figure 2) of the 11S fibril-peptide system. Considering that the source protein for the

536

fibril-peptide system has already been considerably denatured by the heat treatment involved in their

537

production, the STE might be not able to interact with peptide materials or long fibrils as strongly as

538

with the monomeric 11S. As a result, the adsorption behavior of the 11S fibril-peptide system was

539

not markedly affected by STE (Figures 1 and 2).

540

Surface viscoelasticity is generally believed to play an important role in foam stability.1, 9, 44, 45 For

541

pure 11S, the interfaces displayed relatively high moduli, a low frequency dependence of the

542

modulus, and fairly elastic Lissajous plots with strain hardening both in extension and compression

543

(Figures 3-6). Based on these findings, we can conclude that a highly elastic gel structure at the

544

surface is formed by 11S, which could protect the bubbles against coalescence. Thus, despite poor

545

foamability of pure 11S (Figure 7), the formed foam remained relatively stable, with a foam half-life

546

of about 1 hour (Figure 8). In the presence of STE, interestingly, although there was a dramatic

547

decrease in surface modulus (especially at 0.25% STE) due to the disruption of the protein network

548

by STE (Figures 3 and 4), no apparent effect on the foam stability of the mixtures was observed as

549

compared to pure 11S (Figure 8). This finding implies that the magnitude of the surface dilatational

550

modulus is not a major factor affecting the foam stability of the 11S-STE mixtures. It is often stated

551

and observed that protein-surfactant mixtures can give more stable foams than proteins alone.1, 44 The 22

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552

addition of surfactants will make the rather rigid protein layer more flexible and mobile, and the

553

formed mixed surface layer might then be much better able to respond to applied deformations,

554

which would have a positive effect on the foam stability due to a quicker recovery of the interface.1,

555

44

556

the Lissajous plots of 11S-STE mixtures reveal that the STE reduces strain hardening in extension

557

and increases strain hardening in compression, suggesting the interfacial structure may change from

558

a surface gel to a mixed phase of protein patches and STE domains. Upon extension, the strain

559

hardening decreases due to the fast exchange of STE domains from the surface to the bulk, whereas

560

the protein patches may form a soft glass/gel phase during compression, thus increasing strain

561

hardening (Figures 5 and 6). These results suggest the mixed surface layer stabilized by 11S and STE

562

domains could not only provide a relatively high elastic response but would also be less susceptible

563

to rupture when high amplitude deformations are applied. These changes in surface properties are

564

believed to contribute to the increased foam stability of protein-surfactant mixtures,1, 9, 44 and may

565

also be a factor in the stability of the 11S-STE foams (Figure 8).

This view is supported by the analysis of the Lissajous plots (Figures 5 and 6). As discussed above,

566

Compared to native 11S, the 11S fibril-peptide system displayed rather different rheological

567

properties with a higher dilatational modulus and a more complex non-linear response to applied

568

deformations (Figures 3-6). The fibril-peptide system still displays strain hardening in compression,

569

but with increasing amplitude, the response in extension changes from initially strain softening to

570

strain hardening, suggesting the occurrence of rearrangement in the interfacial structure. This result,

571

together with the low frequency dependence and high dilatational modulus (Figures 3 and 4),

572

indicates that when a higher degree of extension is applied, the interactions among components

573

(peptides and long fibrils) at the interface may become stronger due to more available network points,

574

thus enhancing the elastic response. Previous studies have shown that long fibrils can form a surface

575

structure in which highly ordered (nematic) domains coexist with domains with randomly oriented

576

fibrils, and at high deformations a substantial fraction of these nematic domains will have converted 23

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577

to random domains.36, 42 These rheological behaviors suggest that the 11S fibril-peptide system could

578

provide very stable foams. This is convincingly confirmed by the observation that the 11S

579

fibril-peptide system displayed much higher foam stability (t1/2 at about 13 h) than that of native 11S

580

(Figure 8). The effect of STE on its rheological response is different from that observed in 11S-STE

581

mixtures. The decrease in dilatational modulus at the highest STE concentration is less pronounced

582

as for 11S. The Lissajous plot results showed that the behavior in compression is not significantly

583

affected, and the STE mainly affects the response behavior of 11S fibril-peptide system in extension,

584

where the strain softening observed at lower amplitudes is decreased by STE (Figures 5 and 6). A

585

possible explanation could be that the STE causes a small shift in the relative surface fractions of

586

nematic and random domains. In view of all of these results, it appears that the disruption of the

587

interfacial structure by STE for the 11S fibril-peptide system is not as extensive as for native 11S, but

588

surprisingly, the presence of STE leads to a very significant reduction in foam stability of the 11S

589

fibril-peptide system, particularly at the highest STE concentration (Figure 8). Hence a clear relation

590

between surface dilatational rheological properties and foam stability for 11S fibril-peptide-STE

591

mixtures, cannot be established from our results. In Figure 8 we can observe that for the 11S

592

fibril-peptide system the drainage is markedly slower than for 11S, and the foams have a much

593

higher foam liquid stability. Possibly the fibrils are not just providing stability to the foam by

594

imparting elasticity to the air-water interfaces, but also by affecting the properties of the bulk of the

595

thin liquid films. At pH 7 aggregates of peptides and fibrils may also be present in the aqueous phase,

596

and these could become trapped in the thin films and plateau borders of the foam, significantly

597

slowing down the drainage from the foam. To confirm this hypothesis, thin film stability

598

measurements could be performed, for example in a Scheludko cell.

599 600 601

CONCLUSIONS We have studied and compared the adsorption and dilatational rheological behaviors of native 11S 24

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and its heat-induced fibrillar aggregates at pH 7.0, in the presence of natural surfactant STE.

603

Compared to native 11S, the 11S fibril-peptide system showed faster adsorption kinetics, which

604

could be due to the presence of small peptide materials in the fibril-peptide system. The adsorption

605

properties of 11S were significantly affected by STE, most likely due to hydrophobic binding of 11S

606

with STE. No apparent effect of STE on the adsorption of the 11S fibril-peptide system was observed,

607

implying that the interactions of STE with the peptide materials or fibrils are not significantly

608

affecting the structure of the interface. The adsorption behavior of these two mixtures appears to

609

affect their foamability strongly.

610

The foams formed from the mixtures of 11S and STE showed comparable foam stability to that of

611

pure 11S. For the 11S fibril-peptide system the foam stability of the fibril-peptide system was

612

significantly reduced by STE. A clear link between surface properties and foam stability could not be

613

established. Foams produced from the 11S fibril-peptide system were considerably more stable than

614

those prepared from native 11S, at the same bulk concentration. Our results indicate that fibril

615

formation of globular protein could be a potential strategy to modify the complex surface and

616

foaming behaviors of proteins and protein-surfactant mixtures.

617 618

ACKNOWLEDGMENTS

619

This work is supported by grants from the Chinese National Natural Science Foundation (Serial

620

numbers: 31371744, 31301432, and 31130042). We also thank the China Scholarship Council (CSC)

621

research program for providing funding for Zhili Wan.

622 623

REFERENCES

624

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625 626

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Equilibrium of adsorption of mixed milk protein/surfactant solutions at the water/air interface.

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(12) Petkova, R.; Tcholakova, S.; Denkov, N. D. Foaming and foam stability for mixed

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(14) Wierenga, P. A.; Meinders, M. B.; Egmond, M. R.; Voragen, F. A.; de Jongh, H. H. Protein

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exposed hydrophobicity reduces the kinetic barrier for adsorption of ovalbumin to the air-water

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(15) Arnaudov, L. N.; de Vries, R.; Ippel, H.; van Mierlo, C. P. Multiple steps during the formation of β-lactoglobulin fibrils. Biomacromolecules 2003, 4, 1614-1622. (16) Nicolai, T.; Britten, M.; Schmitt, C. β-Lactoglobulin and WPI aggregates: formation, structure and applications. Food Hydrocolloids 2011, 25, 1945-1962.

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(17) Akkermans, C.; Van der Goot, A.; Venema, P.; Gruppen, H.; Vereijken, J.; Van der Linden, E.;

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Boom, R. Micrometer-sized fibrillar protein aggregates from soy glycinin and soy protein

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isolate. J. Agric. Food Chem. 2007, 55, 9877-9882.

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(18) Jung, J.-M.; Gunes, D. Z.; Mezzenga, R. Interfacial activity and interfacial shear rheology of

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native β-lactoglobulin monomers and their heat-induced fibers. Langmuir 2010, 26,

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(19) Humblet-Hua, N.-P. K.; van der Linden, E.; Sagis, L. M. Surface rheological properties of

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liquid–liquid interfaces stabilized by protein fibrillar aggregates and protein–polysaccharide

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complexes. Soft Matter 2013, 9, 2154-2165.

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(20) Isa, L.; Jung, J.-M.; Mezzenga, R. Unravelling adsorption and alignment of amyloid fibrils at interfaces by probe particle tracking. Soft Matter 2011, 7, 8127-8134. (21) Güçlü-Üstündağ, Ö.; Mazza, G. Saponins: properties, applications and processing. Crit. Rev. Food Sci. Nutr. 2007, 47, 231-258. 27

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(22) Stanimirova, R.; Marinova, K.; Tcholakova, S.; Denkov, N.; Stoyanov, S.; Pelan, E. Surface rheology of saponin adsorption layers. Langmuir 2011, 27, 12486-12498. (23) Golemanov, K.; Tcholakova, S.; Denkov, N.; Pelan, E.; Stoyanov, S. D. Surface shear rheology of saponin adsorption layers. Langmuir 2012, 28, 12071-12084.

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(24) Piotrowski, M.; Lewandowska, J.; Wojciechowski, K. Biosurfactant–protein mixtures: Quillaja

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Bark Saponin at water/air and water/oil interfaces in presence of β-lactoglobulin. J. Phys. Chem.

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B 2012, 116, (16), 4843-4850.

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(25) Wan, Z. L.; Wang, J. M.; Wang, L. Y.; Yang, X. Q.; Yuan, Y. Enhanced physical and oxidative

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stabilities

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protein-based

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incorporation

of

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stevioside–resveratrol complex. J. Agric. Food Chem. 2013, 61, 4433-4440.

a

water-soluble

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(26) Wan, Z. L.; Wang, L. Y.; Wang, J. M.; Zhou, Q.; Yuan, Y.; Yang, X. Q., Synergistic interfacial

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properties of soy protein–stevioside mixtures: relationship to emulsion stability. Food

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Hydrocolloids 2014, 39, 127-135.

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(27) Wan, Z. L.; Wang, L. Y.; Wang, J. M.; Yuan, Y.; Yang, X. Q., Synergistic foaming and surface

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properties of a weakly interacting mixture of soy glycinin and biosurfactant stevioside. J. Agric.

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Food Chem. 2014, 62, 6834-6843.

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(28) Sagis, L. M. Dynamic properties of interfaces in soft matter: experiments and theory. Rev. Mod. Phys. 2011, 83, 1367. (29) Sagis, L. M.; Scholten, E. Complex interfaces in food: structure and mechanical properties. Trends Food Sci. Technol. 2014, 37, 59-71.

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(30) van Kempen, S. E.; Schols, H. A.; van der Linden, E.; Sagis, L. M. Non-linear surface

698

dilatational rheology as a tool for understanding microstructures of air/water interfaces

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stabilized by oligofructose fatty acid esters. Soft Matter 2013, 9, 9579-9592.

700

(31) Yuan, Y.; Wan, Z. L.; Yin, S. W.; Teng, Z.; Yang, X. Q.; Qi, J. R.; Wang, X. Y. Formation and

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dynamic interfacial adsorption of glycinin/chitosan soluble complex at acidic pH: relationship to 28

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mixed emulsion stability. Food Hydrocolloids 2013, 31, 85-93.

703

(32) Kroes-Nijboer, A.; Sawalha, H.; Venema, P.; Bot, A.; Flöter, E.; den Adel, R.; Bouwman, W. G.;

704

van der Linden, E. Stability of aqueous food grade fibrillar systems against pH change. Faraday

705

Discuss. 2012, 158, 125-138.

706

(33) Rühs, P. A.; Affolter, C.; Windhab, E. J.; Fischer, P. Shear and dilatational linear and nonlinear

707

subphase controlled interfacial rheology of β-lactoglobulin fibrils and their derivatives. J. Rheol.

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2013, 57, 1003-1022.

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(34) De Feijter, J.; Benjamins, d. J.; Veer, F. Ellipsometry as a tool to study the adsorption behavior

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of synthetic and biopolymers at the air–water interface. Biopolymers 1978, 17, 1759-1772.

711

(35) Mackie, A. R.; Gunning, A. P.; Wilde, P. J.; Morris, V. J. Competitive displacement of

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β-lactoglobulin from the air/water interface by sodium dodecyl sulfate. Langmuir 2000, 16,

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8176-8181.

714

(36) Jordens, S.; Isa, L.; Usov, I.; Mezzenga, R. Non-equilibrium nature of two-dimensional

715

isotropic and nematic coexistence in amyloid fibrils at liquid interfaces. Nat. Commun. 2013, 4,

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(37) Rühs, P.; Scheuble, N.; Windhab, E.; Fischer, P. Protein adsorption and interfacial rheology interfering in dilatational experiment. Eur. Phys. J. Sp. Top. 2013, 222, 47-60.

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(38) Freer, E. M.; Yim, K. S.; Fuller, G. G.; Radke, C. J. Shear and dilatational relaxation

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mechanisms of globular and flexible proteins at the hexadecane/water interface. Langmuir 2004,

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20, 10159-10167.

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(39) Lucassen, J.; Van Den Tempel, M. Dynamic measurements of dilational properties of a liquid interface. Chem. Eng. Sci. 1972, 27, 1283-1291. (40) Ewoldt, R. H.; Hosoi, A.; McKinley, G. H. New measures for characterizing nonlinear viscoelasticity in large amplitude oscillatory shear. J. Rheol. 2008, 52, 1427-1458. (41) Erni, P.; Parker, A. Nonlinear viscoelasticity and shear localization at complex fluid interfaces. 29

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Langmuir 2012, 28, 7757-7767.

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(42) Sagis, L.; Humblet-Hua, K.; van Kempen, S. Nonlinear stress deformation behavior of

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interfaces stabilized by food-based ingredients. J. Phys.: Condens. Matter 2014, 26, 464105.

730

(43) van der Linden, E.; Sagis, L.; Venema, P. Rheo-optics and food systems. Curr. Opin. Colloid

731 732 733 734 735

Interface Sci. 2003, 8, 349-358. (44) Langevin, D. Aqueous foams: a field of investigation at the frontier between chemistry and physics. ChemPhysChem 2008, 9, 510-522. (45) Georgieva, D.; Cagna, A.; Langevin, D. Link between surface elasticity and foam stability. Soft Matter 2009, 5, 2063-2071.

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752

Figure 1. Surface tension as a function of time for mixed solutions (pH 7.0, ionic strength I = 25

753

mM) of 0.1% 11S (native or fibril-peptide system) with different STE concentrations (0-0.25%) at

754

the air-water interface. The experiments were performed at 20 °C.

755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 31

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771

Figure 2. (A) Dynamic adsorbed amount (Г, mg/m2) and (B) thickness (δad, nm) of adsorption

772

layers formed from mixed solutions (pH 7.0, I = 25 mM) of 0.1% 11S (native or fibril-peptide

773

system) with different STE concentrations (0-0.25%) at 20 °C.

774

775 776 777 778 779 780 781 782 783 32

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784

Figure 3. (A) Complex surface dilatational modulus as a function of frequency for air-water

785

interfaces stabilized by mixtures (pH 7.0, I = 25 mM) of 0.1% 11S (native or fibril-peptide system)

786

with different STE concentrations (0-0.25%) at 20 °C. (B) Slope of a double logarithmic plot of the

787

modulus versus frequency. Frequency: 0.005-0.1 Hz. Amplitude of deformation: 10%.

788

789 790 791 792 793 794 795 796 33

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797

Figure 4. Complex surface dilatational modulus as a function of amplitude for air-water interfaces

798

stabilized by mixtures (pH 7.0, I = 25 mM) of 0.1% 11S (native or fibril-peptide system) with

799

different STE concentrations (0-0.25%) at 20 °C. Frequency: 0.1 Hz. Amplitude of deformation:

800

10-30%.

801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 34

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816

Figure 5. Lissajous plots obtained during amplitude sweeps (10-30%) of air-water interfaces

817

stabilized by mixtures (pH 7.0, I = 25 mM) of native 11S (above) or 11S fibril-peptide system

818

(below) with different STE concentrations (0-0.25%) at 20 °C. The protein concentration is fixed at

819

0.1%.

820

821 822 823 824 825 826 35

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827

Figure 6. S factor during extension (A) and during compression (B), determined for amplitude

828

sweeps (10-30%) of air-water interfaces stabilized by mixtures (pH 7.0, I = 25 mM) of 0.1% 11S

829

(native or fibril-peptide system) with different STE concentrations (0-0.25%) at 20 °C.

830

831 832 833 834 835 836 837 838 839 36

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840

Figure 7. The time evolution of air bubbles within foams formed by mixtures (pH 7.0, I = 25 mM)

841

of 0.1% 11S (native or fibril-peptide system) with different STE concentrations (0-0.25%) at 20 ºC.

842

The magnification is same for all images (1 cm × 1 cm).

843

844 845 846 847 848 849 850 851 852 37

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853

Figure 8. (A) Liquid volume as a function of time in foams generated from mixtures (pH 7.0, I =

854

25 mM) of 0.1% 11S (native or fibril-peptide system) with different STE concentrations (0-0.25%) at

855

20 ºC. (B) Liquid stability (s), (C) foam decay curves, and (D) half-life time (t1/2, h) of these foams.

856

857 858 859

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