Contribution of Long Fibrils and Peptides to Surface and Foaming

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Contribution of long fibrils and peptides to surface and foaming behavior of soy protein fibril system Zhili Wan, Xiao-Quan Yang, and Leonard Martin C. Sagis Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b01511 • Publication Date (Web): 25 Jul 2016 Downloaded from http://pubs.acs.org on August 4, 2016

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Contribution of long fibrils and peptides to surface and foaming behavior of soy

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protein fibril system

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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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Leonard M. C. Sagis* (corresponding author)

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E-mail: [email protected]; Fax: +31 317 483669; Tel: +31 317 485023

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ACS Paragon Plus Environment

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ABSTRACT: When soy glycinin (11S) is heated for prolonged time at pH 2 (20 h at 85 °C), a

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mixture is formed consisting of long semiflexible 11S fibrils and small peptides. The surface and

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foaming properties of this mixture were investigated at different pHs, and compared to the behavior

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of pure fibrils and pure peptides, to determine the individual contributions of these two factions to

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the behavior of the mixture. The adsorption of these three systems at air-water interfaces, and the

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resulting surface rheological properties were studied by combining drop shape analysis tensiometry,

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ellipsometry and surface large amplitude oscillatory dilatational (LAOD) rheology. Lissajous plots of

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

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of interfacial microstructure. Our results show that the adsorption kinetics, dilatational rheological

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properties, and the foaming behavior of the mixture were mainly dominated by the small peptides in

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the fibril system. Compared to pH 2, the fibril mixture at pH 5 and 7 provides much better foam

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stability, and appears to be a very promising protein material to make stable foams, even at low

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protein concentration (0.1 wt%). The presence of fibril clusters and peptide aggregates at pH 5 and 7

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contributed to foam stability of the mixture. In contrast, pure fibril formed an interface with a highly

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pH-responsive adsorption and rheological behavior, and the foamability and foam stability of the

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pure fibrils were very poor.

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INTRODUCTION

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Protein adsorption at the interface plays a crucial role in many industrial processes, for example, in

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the formation and stabilization of multiphase food systems.1-3 In systems such as foams, upon

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adsorption, globular food proteins are known to form highly elastic networks around air bubbles,

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which appear to be able to stabilize foams against drainage, bubble coalescence and coarsening.2-6

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Studies on the adsorption of globular proteins at the air-liquid interface have been carried out

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extensively, and suggest that the changes in molecular structure of proteins could strongly affect their

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adsorption behavior and thereby the foaming properties.7-14 For example, several papers have

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reported that even a slight modification of protein molecules by a physical or chemical treatment can

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enhance their adsorption kinetics.9-13 These treatments can lead to more exposed hydrophobicity or

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lower net charge of proteins, which can decrease the energy barrier to adsorption at the interface, and

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thus lead to faster adsorption kinetics.9, 10, 15, 16 The faster adsorption process generally contributes

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positively to the foaming performance of globular food proteins.

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In food processing, the heat treatment of many food proteins, like whey proteins and soy proteins,

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at different pHs and ionic strengths can induce structural changes and lead to the formation of

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supramolecular aggregates with various structures, such as fibrillar structures.17-19 The long,

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semiflexible protein fibrillar aggregates obtained by heat treatment (80-90 °C) at pH 2 and low ionic

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strength have attracted increasing interest as functional ingredients in food products, due to their high

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aspect ratio, which could make them ideal as thickening or gelling agents.20 Such fibrils, like their

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source proteins, have also been shown to be efficient stabilizers for foams or emulsions.21,

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Oboroceanu et al. reported that fibrillized whey protein isolate (WPI) dispersion had better foaming

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capacity and foam stability than the native protein, and the fibril length (long and short) did not

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significantly affect the foaming properties.23 They also found that WPI fibril dispersions at pH 7 had

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better foaming properties than at pH 2. It is well-known that the formation of fibrils, through the heat

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denaturation of proteins at pH 2, consists of a two-step process.17, 24 First, the proteins are hydrolyzed

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into small peptides, and then part of the peptides is assembled into fibrils. Thus, the obtained fibril


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dispersions actually contain a mixture of pure fibrils and unconverted peptides. The small peptides

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are expected to contribute to the foaming properties of fibril systems due to their amphiphilic

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structure. So far, no studies have appeared quantifying the contributions of these peptides to the

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foaming behavior of fibril systems.

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Recently, the interfacial properties of air-water and oil-water interfaces stabilized by fibrillar

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aggregates from various proteins have been studied.21, 22, 25-28 Jung et al. showed that the adsorption

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kinetics of β-lactoglobulin fibril systems at both air-water and oil-water interfaces were faster than

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those of native protein, and the adsorption process of pure fibrils seemed to be independent of their

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length or flexibility.21 Based on surface shear and dilatational rheology measurements, these fibrillar

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aggregates were found to be able to form a highly elastic interface with solid-like behavior, and long

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semiflexible fibrils exhibited the highest modulus. Similar findings have been obtained in the case of

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lysozyme fibrils.22 Through the use of subphase exchange interfacial rheology, Rühs et al. studied the

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influence of pH on the interfacial rheological properties of long β-lactoglobulin fibrils, and found the

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highest interfacial moduli were observed at pH values close to the isoelectric point (pH 5-6).25 In

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addition, the influence of unconverted molecules (peptides and protein monomers) on the adsorption

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kinetics and interfacial viscoelasticity of the fibril system were also investigated.21 From the results,

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it was shown that these unconverted molecules could increase the rate of adsorption and interfacial

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modulus of the fibril system. These studies suggest that the microstructure and rheological properties

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of interfaces stabilized by protein fibrillar aggregates are modified not only by the length and

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flexibility, but also by their polydispersity.21, 28

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As a result of the high specific surface area, it is known that the macroscopic dynamic behavior of

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foams can be affected strongly by surface properties, such as surface tension and surface rheological

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parameters.29 However, studies on the link between surface behavior and foam properties of protein

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fibrillar aggregates have not been carried out to date. From an industrial point of view, the whole

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protein fibril mixture is a more relevant material to be used as a foaming agent, since separation of

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peptides and fibrils can be costly. However, it is important to have knowledge about the surface and


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foaming properties of pure long fibrils and unconverted peptides, to understand their individual

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contributions to the complex fibril system. Therefore, in this work, we first characterized the surface

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properties of pure fibrils, unconverted peptides, and the whole fibril system in terms of adsorption

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kinetics and dilatational rheology at the air-water interface, and then we further investigated the

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foaming properties of all three systems, to assess the links between surface behavior and foam

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properties. We have chosen to use soy proteins (soy glycinin, 11S) to produce long semiflexible

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fibrillar aggregates at pH 2, since they are widely used in different research fields, relatively low-cost,

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and biocompatible. Previous studies have shown that most properties of soy glycinin fibrils are

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similar to β-lactoglobulin and whey protein isolate fibrils.19 Since most food products have a pH

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between 4 and 7, we investigated the influence of pH changes on the surface and foaming properties

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of the fibril systems. The adsorption kinetics at the air-water interface were determined by drop

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shape analysis tensiometry and ellipsometry. Subsequently, we performed a detailed study of the

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frequency and amplitude dependence of the surface dilatational modulus, to obtain information about

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the microstructure of interfaces formed by pure fibrils, peptides, and the whole fibril system,

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respectively. We used the Lissajous plots to quantify the degree of nonlinearity in the rheological

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response of interfaces. Finally, we evaluated foam formation and stability of these three systems and

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correlated foam properties with the results of adsorption and surface dilatational rheology.

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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.30 The protein content of 11S was 97.98%, 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

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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). All other chemicals used were of analytical grade.


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Preparation of 11S fibril system. Preparation of the 11S fibril system was carried out according to a

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previously described protocol.19 Briefly, protein stock solutions of 11S were made by dissolving the

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protein in Millipore water. The pH of the solution was adjusted to pH 2.0 with 6 M HCl solution,

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followed by centrifugation (10000 g, 30 min, 4 °C) to remove undissolved materials (around 3.5% of

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the protein sample). The protein concentration of solutions was determined using Dumas analysis (N

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× 6.25). The obtained 11S solution (2 wt%) was placed in small glass vials (20 mL) and then

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incubated at 85 °C for 20 h under continuous stirring at 300 rpm by using a metal heating and stirring

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plate. After heat treatment, samples were immediately cooled in an ice bath and then stored at 4 °C

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for further use, typically within 1 week. The formed 11S fibrils are long and semiflexible with a

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thickness of a few nanometers, a persistence length of about 2.3 µm, and a contour length of about 1

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µm.19

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Separation of fibrils and unconverted peptides. Fibrils and unconverted peptides were separated

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according to a previously described method.19, 31 The fibril solution was first diluted to a protein

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concentration of 0.2 wt% with HCl solution of pH 2. The solution was divided into centrifugal filters

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(Amicon Ultra 100 K–15 Centrifugal Filters) and centrifuged at 3000 g and 15 °C for 30 min

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(Allegra X-22R Centrifuge, Beckman Coulter). The retentate (containing the fibrils) was washed

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twice with fresh pH 2 HCl solution after each centrifugation to remove unconverted protein left in

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the retentate. The filtrate was recovered after each washing step, and after the last washing step, the

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retentate was recovered. The protein concentration in the three filtrates was measured using Dumas

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analysis, and in the third filtrate no protein was detected anymore. Further analysis was also done on

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the recovered retentate solution. The difference between the total amount of protein and the amount

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of protein in the retentate was used to calculate the amount of protein converted into fibrils. Based on

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this method, we found that the prepared 11S fibril system consists of approximately 20% fibrils and

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80% unconverted peptides.

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Surface tension and surface dilatational rheology. The surface tension and surface dilatational

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


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

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

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

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all the solutions was monitored for 180 min at a constant area of 25 mm2 of the drop. The surface

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tension data was converted into surface pressure by using the relation Π (t) = γ (water) – γ (t), where

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γ (water) = 72 mN m-1 is the surface tension value of pure water. All measurements were performed

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at a bulk protein concentration of 0.1 wt%.

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After 180 min of adsorption, quasi-equilibrium conditions of interface were obtained and then the

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following dilatational rheology measurements were performed: (1) A frequency sweep was

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performed with frequencies between 0.005 and 1 Hz, at a constant amplitude of 10%. The slope of a

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double logarithmic plot of complex surface dilatational modulus versus frequency was determined

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using linear regression. For higher frequencies, the fit used to establish the first-harmonic Fourier

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moduli was not always of sufficient quality, which reduced the accuracy of values for the moduli.

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Therefore, the regression fit was based on the frequency range from 0.005 Hz to 0.1 Hz. (2) To

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investigate the rheological response as a function of dilatational amplitude, amplitude sweeps were

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performed with deformations ranging from 1.5% to 30%, at a frequency of 0.1 Hz. The complex

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modulus was calculated from the intensity and phase of the first harmonic of a Fourier transform of

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the oscillatory surface pressure signal. All experiments were performed at 20 °C and at a bulk protein

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concentration of 0.1 wt%. Reported values represent the average of 3-7 measurements.

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Lissajous plots. The large amplitude dilatational measurements were also analyzed with a method

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developed by Van Kempen et al.32 Lissajous plots were constructed of the surface pressure (Π = γ −

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γ0) versus deformation (δA/A0), where δA = A - A0, γ and A are the surface tension and area of the

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deformed interface, and γ0 and A0 are the surface tension and area of the non-deformed interface. For

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the analysis of the curves, we defined the following four factors: EL,E, defined as the large strain

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

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large strain modulus in compression, and EM,C, defined as the minimum strain modulus in


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compression. The method for determining these factors has been discussed in detail in a previous

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study.32 Based on these moduli, we further defined two nonlinearity parameters, one for the extension

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part of the cycle, Sexp = (EL,E - EM,E)/EL,E, and one for the compression part, Scom = (EL,C - EM,C)/EL,C.

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For both factors, S = 0 may be interpreted as a linear elastic response, S > 0 indicates strain

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hardening behavior in the interface, and S < 0 corresponds to intracycle strain softening. With these

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factors, we could quantitatively analyze the degree of nonlinearity in the response of interfaces

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during extension and compression.

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Ellipsometry. The adsorbed amount and the adsorption layer thickness after 3 h were measured by

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null 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.33 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 protein at the air-water interface were measured, and fitted using a three-layer model. This model

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assumes a single homogeneous adsorption layer characterized by the average refractive index (nad)

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and the thickness (δad) between two homogenous phases (water and air). Subsequently, the adsorbed

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amount (Г, mg/m2) can be calculated according to the following equation:33

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Г =

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where nwater is the refractive index of water (1.333) and dn/dc is the refractive index increment of the

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protein in water (0.185 mL g-1). The measurements were performed at a bulk protein concentration of

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0.1 wt%, and performed in triplicate.

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Foamability and foam stability. The foaming properties of protein fibrillar aggregates were

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measured using a Foamscan device (Teclis IT-Concept, Longessaigne, France). An initial volume of

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60 mL of solutions was foamed by sparging nitrogen at a constant gas flow rate of 400 mL/min

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through a metal frit (60 mm diameter, pore size 27 ± 2 µm, 100 µm distance between centres of pores,

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square lattice). Bubbling was stopped after a volume of 400 cm3 of foam was obtained. The foam

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formation, the drainage of liquid from the foam, and foam stability were monitored by a combination ACS Paragon Plus Environment

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of optical (CCD camera) and conductivity measurements. Series of images of the foam were taken

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every 20 s by a CCD camera to observe the changes of bubbles. The bubble size was measured at the

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middle of the foam column using a CCD camera and a prism and lighting arrangement at the surface

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of foam tube. The time for the foam to drain 50% of its initial liquid content is defined as the foam

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liquid stability (s). The time required for the foam volume to reduce to half of its initial volume

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(half-life, t1/2) was used as an indication of foam stability. Reported values are averages of at least

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three individual measurements. All experiments were performed at 20 °C and at a bulk protein

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concentration of 0.1 wt%.

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RESULTS AND DISCUSSION

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In this work, the adsorption and dilatational rheological properties of pure fibrils, unconverted

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peptides and the whole mixed fibril system at the air-water interface, as well as the foaming

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properties of these three systems, were investigated, to determine the individual contributions of

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fibrils and unconverted peptides to surface and foaming properties of the complex fibril system. To

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study the influence of pH changes on these properties, the pHs of all three systems were adjusted to

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pH 2, 5 and 7, respectively. All systems had the same protein concentration of 0.1 wt%. As shown in

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Figure 1S (see Supporting Information, SI), these three systems were stable and transparent at pH 2

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due to the high electrostatic repulsive force (Table 1). The fibril system and pure peptide became

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turbid at pH 5 and 7, especially at pH 5, since their net charge is close to zero at pH 5 (Table 1).

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However, the pure fibrils stayed relatively transparent at pH 5 and 7. Previous studies have shown

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that the turbidity of β-lactoglobulin fibril systems around pH 5 could be decreased drastically by

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removing the unconverted peptides from the system, and thus confirmed that the unconverted

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peptides are the main cause for the turbidity.31 Despite the relatively transparent appearance, pure

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fibrils actually formed dilute clusters around pH 5 since the surface charge of the fibrils was also

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close to zero at this pH (Table 1).31 We conclude that, at pH 5, the 11S fibril system predominantly

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consists of dilute fibril clusters and peptide aggregates, and at pH 7 free peptides, peptide aggregates,


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dilute fibril clusters, and individual fibrils coexist. These pronounced changes on the structure and

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net charge of all the systems as a function of pH are expected to have a significant influence on their

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adsorption kinetics and thus foaming properties, and the results are described in the following

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sections.

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Adsorption behavior of 11S fibril system, pure fibrils, and unconverted peptides.

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Adsorption kinetics. Figure 1 shows the time evolution of surface pressure (Π) of the fibril system,

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pure fibrils, and pure peptide at the air-water interface. As can be seen, at all investigated pHs, the Π

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curves for the fibril system and pure peptide almost completely overlapped, and they differed

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significantly from those for pure fibrils (especially at pH 5 and 7). As mentioned above, the prepared

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fibril system consists of approximately 20% pure fibrils and 80% unconverted peptides, therefore,

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the adsorption process of the 11S fibril system at air-water interface was seen to be mainly

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dominated by the high proportion of unconverted peptides. This is not surprising, since, compared to

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large fibrils (long fibrils at pH 2, and fibril clusters at pH 5 and 7), these small peptide materials (free

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peptides and/or peptide aggregates) should have faster mass transport rate toward the interface,

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decreasing the surface tension more rapidly, thus dominating the adsorption kinetics (especially the

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initial adsorption) of the fibril system. However, it should be noted that, at pH 2, although pure fibril

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had a lower Π during the initial phase of adsorption, pure fibril and the fibril system showed similar

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final Π, which was slightly higher than that of pure peptide, implying that pure fibril may contribute

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to the equilibrium surface properties of the fibril system. A similar observation was reported for

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β-lactoglobulin fibrils at pH 2, a system which also consists of a mix of fibrils and small peptides,

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and for which an interfacial structure was proposed which consists either of a multilayer with the

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peptides directly at the interface and the fibrils attached below this primary layer, or a mixed

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interface where both species are adsorbed at the interface together.28, 34

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As expected, the adsorption of all three systems at the air-water interface was affected by pH

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changes. The effect of pH on the fibril system and pure peptide was different from that observed in


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pure fibril (Figure 1). At both pH 5 and 7, the fibril system and pure peptide exhibited similar

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adsorption kinetics, and reached nearly identical values for the surface pressure after 3 h (about 25

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mN/m). At pH 2 these systems had much slower adsorption and reached only a surface pressure of

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18 mN/m. Pure fibrils showed a markedly different effect of pH on adsorption kinetics, especially in

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the initial adsorption stage. The initial adsorption rate for pure fibrils was faster at pH 7 than at both

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pH 2 and 5. At pH 5, there was an obvious lag-time of around 10 s for pure fibrils (Figure 1B).

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However, the subsequent increase in Π was faster than the increase observed at pH 2 and 7. It is

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known that the initial adsorption kinetics of proteins is generally limited by their diffusion from the

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bulk to the interface. The diffusion of proteins to air-water interfaces can be affected by a subtle

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balance between molecular size, shape, surface charge and surface hydrophobicity.9,

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previously mentioned, the fibril system and pure peptides are highly positively charged at pH 2

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(Table 1), and therefore the electrostatic repulsion is strong, which would lead to a higher

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electrostatic adsorption barrier. This is probably the main reason that the fibril system and pure

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peptides showed a faster initial adsorption and higher surface pressure values at both pH 5 and 7 than

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at pH 2. This analysis is supported by previous studies, which showed that around the iso-electric

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point (pI) proteins appear to be more surface active and have faster adsorption kinetics.16, 36, 37

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However, for pure fibrils, the observed Π curve at pH 5 seems to contradict these earlier observations.

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As can be seen from Figure 1B, almost no adsorption (increase in Π) was observed in the initial 10 s

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(lag-time), although the surface charge of the fibrils was close to zero at pH 5 (Table 1). Since the

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fibrils formed dilute clusters at pH 5, we attributed the presence of the so-called lag phase to the

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large size of these fibril clusters, which hindered their migration to the interface. Once they have

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diffused to the interface, a very fast increase in Π was observed since the electrostatic repulsion

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energy barrier for adsorption is low (Figure 1B). The fact that the fibrils exhibited faster initial

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adsorption at pH 7 than at pH 2, can be attributed to the lower energy barrier to adsorption at the

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former pH, since at pH 2 the fibrils have a higher charge. It is described extensively that the

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adsorption kinetics, especially the initial adsorption, is well correlated to the foam formation of


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As

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proteins.2, 16 Therefore, we expect these observed differences in adsorption kinetics would affect the

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foaming properties.

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Ellipsometry. Figure 2 shows the adsorbed amount (Г) and thickness (δad) of adsorbed layers formed

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from all systems after 3 h of adsorption. As can be seen, the fibril system and pure peptides showed

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similar trends in changes of Г and δad at all three pHs. This can be explained by their similar

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adsorption processes at the air-water interface (Figure 1). The fibril system and pure peptides

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exhibited similar values for Г (around 3.0 mg/m2) and δad (around 8 nm) at both pH 5 and 7, which

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differed from the values at pH 2 (Г~2.0 mg/m2, δad~10-12 nm). This indicates that a higher packing

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density of adsorbed proteins at the interface was obtained at pH 5 and 7. As previously discussed,

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there was a lower electrostatic adsorption barrier and thus a faster adsorption for proteins at pH 5 and

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7 (Figure 1). Also, there was less electrostatic repulsion between adsorbed proteins, which can

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increase their packing density at the interface.10, 16 A higher density of the adsorbed layer could

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provide stronger steric stabilization, thus contributing to the foam stability. It may also enhance the

301

surface shear and dilatational moduli. For pure fibrils, the changes of Г and δad as a function of pH

302

differed from those observed for pure peptide. As can be seen from Figure 2A, for pure fibrils the

303

adsorbed amount and layer thickness are less sensitive to changes in pH: these showed a small

304

decrease in Г upon increasing the pH from 2 to pH 5 and pH 7, with no significant change in layer

305

thickness (within the margin of error of the measurement), suggesting a slightly higher packing

306

density of adsorbed fibrils at pH 2. This result is consistent with the surface pressure measurements,

307

since pure fibril at all three pHs showed similar final surface pressures (Figure 1).

308 309

Nonlinear dilatational rheological properties.

310

To gain more information about the microstructure and mechanical properties of interfaces stabilized

311

by these systems, surface dilatational rheology measurements including frequency and amplitude

312

sweeps were performed. A detailed discussion of these sweeps is given in the Supplementary


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313

Information (SI). In the strain sweeps we observed that even at small deformation amplitudes the

314

response of the interfaces stabilized by all systems was in the nonlinear regime (Figure 3S, SI). The

315

moduli shown in these sweeps were determined from a Fourier Transform of the oscillatory surface

316

pressure signal, in particular from the intensity and phase of the first harmonic of the Fourier

317

spectrum. As pointed out by Ewoldt et al., any nonlinearity that could be present in the raw signal

318

will be disregarded when using this first-harmonic based Fourier analysis.39 Therefore, the values of

319

the first harmonic moduli presented in Figure 3S (SI) are bound to be inaccurate. To more accurately

320

characterize the dilatational behavior, we instead used Lissajous plots of dynamic surface pressure

321

versus strain, to help interpret the dilatational behavior during amplitude sweeps. Previous studies

322

demonstrated that Lissajous plots are very useful in analyzing nonlinear dilatational behavior.26, 32, 40

323

The Lissajous plots of the amplitude sweeps for all systems at pH 2 and 7 are given in Figures 3A

324

and 3B, respectively (additional plots at intermediate amplitude values between 1.5 and 30% are

325

given in the Supplementary Information). While the amplitude sweep plots unveiled comparable

326

dilatational moduli for all systems at pH 2 (Figure 3S-A, SI), the Lissajous plots showed significant

327

differences for the fibril system, pure fibrils, and pure peptides, especially at higher amplitudes

328

(7.5-30%) (Figures 3 and 4S, SI). For all systems at pH 2, at amplitudes up to 5% the Lissajous plots

329

were characterized by a mostly elastic response, without pronounced asymmetries. At higher

330

amplitudes of 15% and 30%, the plots for the fibril system and pure peptides became increasingly

331

asymmetric, indicating different responses in extension and compression. It can be seen that the

332

maximum surface pressure observed in compression was much higher than that observed in

333

extension, and the plot also became narrower towards maximum compression, which indicates that

334

the surface is strain hardening upon compression. A widening of the plots was observed in extension

335

(compared to the compression phase), implying that the surface has a more viscous response (strain

336

softening) in extension. Compared to the fibril system and pure peptides, for pure fibrils the shape of

337

the Lissajous plots were less asymmetric, and the response was still mostly elastic when the

338

amplitude was increased. Relatively pronounced non-linearities only appeared at higher amplitudes


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

339

(15-30%), where the extension phase of the plots became narrower, implying a relatively small strain

340

hardening upon extension.

341

In addition, it can be seen that the shapes of the Lissajous plots for the fibril system and pure

342

peptides at pH 7 were similar to those obtained at pH 2 (Figures 3, 4S, and 5S, SI), but at pH 7 the

343

non-linearities appeared even at the smallest amplitude (1.5%) and the non-linear response seemed to

344

be more pronounced when the amplitude was increased from 7.5% to 30%. Additionally, it is worth

345

noting that compared to pure fibrils at pH 2, pure fibrils at pH 7 showed an obvious widening of the

346

Lissajous plots at small amplitudes (1.5-7.5%), indicating a relative increase in the viscous

347

components during deformation. At higher amplitudes (15-30%), the asymmetries became more

348

pronounced, with strain hardening both in extension and compression. This is consistent with the

349

results from the amplitude sweeps (Figure 3S, SI), which show that, at smaller amplitudes (1.5-7.5%),

350

the surface layer stabilized by pure fibrils had much higher dilatational modulus at pH 2 than at pH 7

351

(Figure 3S, SI). The pH may have affected the in-plane interactions between the fibrils, which could

352

have resulted in a decrease in in-plane cohesion of the interfacial microstructure, thus leading to

353

changes in strain softening or hardening behavior of the interface.

354 355

Quantitative analysis of Lissajous plots. To further quantify the degree of non-linearity, the

356

nonlinearity parameters Sext and Scom for both phases of the cycle were determined, as shown in

357

Figure 4. It can be seen that, at pH 2, the absence of non-linearity at amplitudes up to 5% for all the

358

systems was confirmed (Figures 4A and D). At 7.5 and 10% amplitude, the fibril system and pure

359

peptides showed pronounced non-linear responses, with strain hardening during compression and

360

strain softening during extension. When the amplitude was further increased from 15% to 30%, the

361

strain hardening in compression became increasingly larger, but the degree of strain softening in

362

extension was reduced, and finally, at 30% amplitude, the response in extension became strain

363

hardening. Combined with the low frequency dependence and high dilatational modulus during

364

frequency and amplitude sweeps (Figures 2S and 3S, SI), the most likely structure of the air-water


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365

interfaces formed by the peptides at pH 2 is a heterogeneous structure, in which areas of densely

366

packed peptides coexist with areas with a more dilute composition. Upon compression a shift occurs

367

in the fractions of dense and dilute domains, towards a higher fraction of dense domains, which is

368

responsible for the observed strain hardening in compression. Upon expansion the relative fraction of

369

dilute domains increases leading to strain softening. The decrease of the softening effect at higher

370

amplitudes (with even a mild strain hardening at 30% deformation, Figure 4A) suggests that the

371

dense domains are weakly connected. In view of the similarity between the large-amplitude response

372

of pure peptides stabilized interfaces and those stabilized by the complete fibril system, the structure

373

of the latter is also dominated by the peptides, which is supported by the similarities in adsorption

374

kinetics (Figure 1), the similarities in adsorbed amounts of protein (Figure 2), and the nearly identical

375

frequency response of both types of interfaces (Figure 2S, SI). This analysis needs to be further

376

confirmed by structural characterization methods, such as Brewster angle microscopy (BAM) and

377

particle tracking observed by microscopy.41

378

At pH 5 and 7, for interfaces stabilized by peptides or the fibril system, the degree of non-linearity

379

during extension and compression showed a similar tend to the one observed for pH 2, but with

380

significantly higher values for the S factors over the whole range of amplitudes (Figures 4B and E,

381

Figures 4C and F), indicating the interface had a stronger response to deformation. This may be

382

attributed to the enhanced interactions among components at the interface at both pH 5 and 7 due to

383

the reduced electrostatic repulsion.

384

For pure fibrils, at pH 2, at high amplitudes (15-30%), the interfaces showed a mild strain

385

hardening during extension and a near linear elastic response during compression (Figures 4A and D).

386

A possible explanation for the different responses of the pure fibrils may be due to a structural

387

transition in the interface induced by the deformation. Previous studies by Humblet-Hua et al.22 and

388

others27,

389

semi-flexible protein fibrils form a structure after adsorption to the interface in which nematic

390

domains and disordered domains coexist at the interface. Upon compression, the surface fraction of

28

have shown that, at relatively high bulk protein concentration (0.1 wt%), the long,


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391

nematic domains grows at the expense of the surface fraction of disordered domains, but if at the

392

maximum degree of compression we are still in the coexistence regime and do not cross into a fully

393

nematic structure, we would expect a near linear response with only a minor degree of hardening.

394

Upon extension the interfacial structure changes gradually into a more and more disordered state,

395

which in view of the relatively high electrostatic repulsion has only limited connectivity between the

396

fibrils, which results into a moderate degree of strain hardening during extension. At pH 5 and 7, the

397

strain hardening in compression and extension is clearly more pronounced, especially at high

398

amplitudes (20 and 30%) (Figures 4B and E, Figures 4C and F). The strain hardening both in

399

extension and compression indicates that a more disordered gel-like interfacial structure was formed

400

by pure fibril at these two pHs, which could be explained by the fact that the fibrils could more easily

401

aggregate and build a stronger network at the interface due to the reduced electrostatic repulsive

402

forces.25, 42

403 404

Foaming properties. In this section, we examine the relation between the obtained surface

405

properties of all protein systems to their macroscopic foaming behavior. Here, we use the initial

406

bubble size to assess the foamability of all systems as a function of pH. As can be seen from Figure 5,

407

at all investigated pHs, the bubbles formed from the fibril system and pure peptides showed

408

comparable initial bubble size (at 0 s), which was much smaller than that observed in pure fibrils.

409

This indicates that the fibril system and pure peptides had a better foamability. Additionally, the

410

initial bubbles of the fibril system and pure peptides were spherical, but the bubbles from pure fibrils

411

appeared to be polyhedral, implying the liquid drainage in foams formed from pure fibrils is much

412

faster than in the other foams. For the fibril system and (especially) pure peptides, at pH 5, the initial

413

bubbles had a smaller bubble size with a more uniform size distribution (Figure 5B), as compared to

414

the two systems at pH 2 and 7 (Figures 5A and C). For pure fibrils at pH 5, it should be noted that it

415

is difficult to produce foams with enough volume for measurements at the present fibril

416

concentration (0.1 wt%). For all systems, it can be seen that, with increasing time, the bubble size


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417

gradually increased and the bubble shapes started to transform from spherical to polyhedral due to

418

the destabilization processes, such as liquid drainage, coarsening, and coalescence.

419

The foam stability of all systems was evaluated by measuring the half-life time (t1/2) and liquid

420

stability of foams, as shown in Figures 6A and 6B, respectively. It can be clearly seen from Figure

421

6A that, at pH 2, the foam stability of pure peptides was significantly higher than the fibril system

422

and pure fibrils. Furthermore, at pH 5 and 7, both the fibril system and pure peptides could form

423

more stable foams with comparable foam stability. The t1/2 values for foams were around 10 h, which

424

were much higher than that of pure fibril, i.e. roughly five times higher (Figure 6A). Similar

425

observations were also reported by Oboroceanu et al., who found that a whey protein fibril system at

426

pH 7 had better foaming properties than at pH 2.23 For pure fibrils, the foam stability at pH 7 was

427

also shown to be much higher than that at pH 2, but pure fibrils appeared to be unable to make foams

428

effectively at pH 5. Delayed liquid drainage generally can contribute to foam stability. Therefore, the

429

foam liquid stability was calculated to assess the liquid drainage rate (Figure 6B). A high foam liquid

430

stability indicates a low liquid drainage rate. As can be seen, the pure fibrils had a much higher liquid

431

drainage rate than foams prepared from pure peptides and the full fibril system. Although the formed

432

foams were not very stable (Figure 6A), pure fibrils at pH 2 and 7 did show a capacity to delay liquid

433

drainage (Figure 6B). On the basis of these results, it can be concluded that, the foaming properties

434

of the fibril system appeared to be mainly determined by the high proportion of unconverted peptides

435

in the system, especially at pH 5 and 7 (Figure 6). This observation is in good agreement with the

436

results from adsorption and dilatational rheology measurements (Figures 1, 2, 2S, and 3S, SI).

437 438

CONCLUSIONS

439

In this work, we have studied the surface and foaming properties of an 11S soy protein fibril system,

440

consisting of a mix of long semi-flexible protein fibrils (20%), and unconverted peptides (80%). We

441

also study the purified 11S fibrils, and purified peptides of this system, with the aim to understand

442

the individual contributions of these two factions to the complex fibril system. We examined the


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

443

influence of pH changes on these properties of all three systems. From the surface pressure and

444

ellipsometry measurements at the air-water interface, we were able to shed light on two major

445

observations. First, the adsorption kinetics of the fibril system appeared to be mainly dominated by

446

its high proportion of unconverted peptides, especially in the initial adsorption, which could be

447

explained by the faster adsorption of smaller peptide materials at the interface. Second, the fibril

448

system and pure peptides showed faster adsorption kinetics and higher packing density at the

449

interface at pH 5 and 7 than at initial pH 2, due to the reduced electrostatic adsorption barrier. In

450

addition, compared to pure peptides, pure fibrils showed a much slower adsorption kinetics due to

451

the large size of fibril or fibril clusters. The results from surface dilatational rheology showed that,

452

again, the unconverted peptides mostly dominated the dilatational rheological properties of surface

453

layers stabilized by the fibril system. At all three pHs, the fibril system and pure peptide formed

454

interfaces with similarly high dilatational moduli, an identical low frequency dependence of the

455

modulus, and produced Lissajous plots with strain hardening during compression and strain softening

456

during extension. We argue that the rheological properties of interface are most likely the result of

457

the formation of a heterogeneous structure by the peptides, in which dense and dilute regimes coexist.

458

The presence of fibrils in this structure could neither be confirmed nor excluded, solely based on the

459

surface rheological results. The interface stabilized by pure fibrils had a highly pH-responsive

460

response behavior, and showed different rheological properties and interfacial microstructures as a

461

function of pH.

462

Not only the surface properties of the fibril system are dominated by peptides, it appears that the

463

foaming properties of the fibril system were also dominated by the unconverted peptides, and not by

464

the protein fibrils as previously thought. Compared to pH 2, the fibril system at pH 5 and 7 seem to

465

be a more promising protein material to make stable foams, even at low protein concentration (0.1

466

wt%), which is mainly due to the faster adsorption kinetics and the formation of highly elastic

467

surface layer by the peptide materials. Additionally, the presence of fibril clusters and peptide

468

aggregates at pH 5 and 7 may also contribute to the foam stability of fibril system. For pure fibrils,


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469

the foaming properties appeared to be mostly dependent on their adsorption kinetics, and both the

470

foamability and foam stability of pure fibrils were very poor.

471 472

ASSOCIATED CONTENT

473

Supporting Information

474

Visual observations of the fibril system, pure fibril, and pure peptide at pH 2, 5 and 7; complex

475

surface dilatational modulus as a function of frequency for interfaces stabilized by the fibril system,

476

pure fibril, and pure peptide at pH 2, 5 and 7; slope of a double logarithmic plot of the modulus

477

versus frequency; complex surface dilatational modulus as a function of amplitude for air-water

478

interfaces stabilized by the fibril system, pure fibril, and pure peptide at pH 2, 5 and 7; Lissajous

479

plots obtained during amplitude sweeps (1.5-30%) of air-water interfaces stabilized by the fibril

480

system, pure fibril, and pure peptide at pH 2; Lissajous plots obtained during amplitude sweeps

481

(1.5-30%) of air-water interfaces stabilized by the fibril system, pure fibril, and pure peptide at pH 7.

482

This material is available free of charge via the Internet at http://pubs.acs.org.

483 484

ACKNOWLEDGMENTS

485

The authors thank M. A. H. de Beus, M. Chen, and H. Baptist (Food Physics Group, Wageningen

486

University) for their help with the pendant drop tensiometer measurements and protein fibril

487

characterization, respectively, and R. J. B. M. Delahaije and F. J. Lech (Food Chemistry Group,

488

Wageningen University) for their help with the ellipsometry and foamscan experiments, respectively.

489

We also thank the China Scholarship Council (CSC) research program for providing funding for

490

Zhili Wan.

491 492

REFERENCES

493

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494

Food hydrocolloids 2003, 17, 25-39.


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

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

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

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Discuss. 2012, 158, 125-138.

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interfering in dilatational experiment. Eur. Phys. J. Sp. Top. 2013, 222, 47-60.

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Ivanov, I. B. Physico-chemical factors controlling the foamability and foam stability of milk proteins:

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Sodium caseinate and whey protein concentrates. Food Hydrocolloids 2009, 23, 1864-1876.

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(38) Lucassen, J.; Van Den Tempel, M. Dynamic measurements of dilational properties of a liquid

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interface. Chem. Eng. Sci. 1972, 27, 1283-1291.

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(39) Ewoldt, R. H.; Hosoi, A.; McKinley, G. H. New measures for characterizing nonlinear

584

viscoelasticity in large amplitude oscillatory shear. J. Rheol. 2008, 52, 1427-1458.

585

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

586

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

587

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

588

Interface Sci. 2003, 8, 349-358.

589

(42) Jordens, S.; Rühs, P. A.; Sieber, C.; Isa, L.; Fischer, P.; Mezzenga, R. Bridging the gap between

590

the nanostructural organization and macroscopic interfacial rheology of amyloid fibrils at liquid

591

interfaces. Langmuir 2014, 30, 10090-10097.

592

(43) Georgieva, D.; Cagna, A.; Langevin, D. Link between surface elasticity and foam stability. Soft

593

Matter 2009, 5, 2063-2071.

594

(44) Wilde, P. Interfaces: their role in foam and emulsion behaviour. Curr. Opin. Colloid Interface

595

Sci. 2000, 5, 176-181.

596

(45) Saint-Jalmes, A.; Peugeot, M.-L.; Ferraz, H.; Langevin, D. Differences between protein and

597

surfactant foams: microscopic properties, stability and coarsening. Colloids Surf., A 2005, 263,

598

219-225.


Langmuir

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599

(46) Rullier, B.; Novales, B.; Axelos, M. A. Effect of protein aggregates on foaming properties of

600

β-lactoglobulin. Colloids Surf., A 2008, 330, 96-102.

601


Page 24 of 31

Page 25 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

602 603

Langmuir

Table 1. Zeta potential (mV) of the fibril system, pure fibril, and pure peptide at pH 2, 5 and 7. Protein concentrations are 0.1 wt%. Zeta potential (mV) Sample pH 2

pH 5

pH 7

36.2 ± 2.4

4.3 ± 1.0

-14.4 ± 1.6

Pure fibril

36.4 ± 2.2

-2.6 ± 0.1

-24.2 ± 2.9

Pure peptide

25.1 ± 0.1

4.6 ± 1.1

-9.3 ± 0.8

Fibril system

604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

621 622

Figure 1. Surface pressure (Π) as a function of time for the fibril system, pure fibril, and pure peptide at pH 2 (A), 5 (B) and 7 (C). All systems have the same protein concentration of 0.1 wt%.

623

624

625 626 627 628 629


Page 26 of 31

Page 27 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

630

Figure 2. (A) Adsorbed amount (Г, mg/m2) and (B) thickness (δad, nm) of adsorption layers after 3

631

h formed from the fibril system, pure fibril, and pure peptide at pH 2, 5 and 7. All systems have the

632

same protein concentration of 0.1 wt%.

633

634 635 636 637 638 639 640


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

641

Figure 3. Lissajous plots obtained during amplitude sweeps (1.5, 15, and 30%) of air-water

642

interfaces stabilized by the fibril system, pure fibril, and pure peptide at pH 2 (A) and 7 (B). Protein

643

concentrations are 0.1 wt%.

644

645 646 647 648 649 650


Page 28 of 31

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Langmuir

651

Figure 4. S factor during extension (above the dotted line) and during compression (below the

652

dotted line), determined during amplitude sweeps (1.5-30%) of air-water interfaces stabilized by the

653

fibril system, pure fibril, and pure peptide at pH 2 (A, D), 5 (B, E) and 7 (C, F). All systems have the

654

same protein concentration of 0.1 wt%.

655 656 657 658 659 660 661 662 663 664 665 666 667

Figure 5. Time evolution of air bubbles within foams formed by the fibril system, pure fibril, and 29


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

668

pure peptide at pH 2 (A), 5 (B) and 7 (C). Protein concentrations are 0.1 wt%. The magnification is

669

same for all images (1 cm × 1 cm).

670

671

672 673 30


Page 30 of 31

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Langmuir

674

Figure 6. (A) Half-life time (t1/2, h) and (B) liquid stability (s) of foams generated from the fibril

675

system, pure fibril, and pure peptide at pH 2, 5 and 7. All systems have the same protein

676

concentrations of 0.1 wt%.

677

678 679

31


Contribution of Long Fibrils and Peptides to Surface and Foaming

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