Protein Nanocomposite Based on Whey Protein Nanofibrils in

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Protein/Protein Nanocomposite Based on Whey Protein Nanofibrils in a Whey Protein Matrix Xinchen Ye, Kristina Junel, Mikael Gällstedt, Maud Langton, Xin-Feng Wei, Christofer Lendel, and Mikael S. Hedenqvist ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b00330 • Publication Date (Web): 15 Mar 2018 Downloaded from http://pubs.acs.org on March 16, 2018

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ACS Sustainable Chemistry & Engineering

Protein/Protein Nanocomposite Based on Whey Protein Nanofibrils in a Whey Protein Matrix

Xinchen Ye,a Kristina Junel,b Mikael Gällstedt,c Maud Langton,d Xin-Feng Wei,a Christofer Lendel,e* and Mikael S. Hedenqvista* a

Department Fibre and Polymer Technology, School of Engineering Sciences in Chemistry,

Biotechnology and Health, KTH Royal Institute of Technology, SE–100 44 Stockholm. Sweden b

c

RISE Bioeconomy Innventia AB, Drottning Kristinas väg 61, SE-114 86 Stockholm, Sweden

SIG Combibloc, Vasagatan 7, SE-111 20 Stockholm, Sweden

d

Department of Molecular Sciences, SLU Swedish Agricultural University, Box 7015, 750 07

Uppsala, Sweden e

Department of Chemistry, School of Engineering Sciences in Chemistry, Biotechnology and

Health, KTH Royal Institute of Technology, SE–100 44 Stockholm, Sweden *

Mikael S. Hedenqvist: [email protected], 468-790-7645; Christofer Lendel: [email protected], 468-

790-8554

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ACS Sustainable Chemistry & Engineering 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

Abstract This article describes nanocomposite films with separately grown protein nanofibrils (PNFs) in a non-fibrillar protein matrix from the same protein starting material (whey). Tensile tests on the glycerol-plasticised films indicate an increased elastic modulus and a decreased extensibility with increasing content of PNFs, although the films are still ductile at the maximum PNFs content (15 wt.%). Infrared spectroscopy confirms that the strongly hydrogen-bonded β-sheets in the PNFs are retained in the composites. The films appear with a PNFs-induced undulated upper surface. It is shown that micrometre-scale spatial variations in the glycerol distribution are not the cause of these undulations. Instead, the undulations seem to be a feature of the PNFs material itself. It was also shown that, apart from plasticising the protein film, the presence of glycerol seemed to favour to some extent exfoliation of stacked β-sheets in the proteins, as revealed by X-ray diffraction.

Keywords protein fibrils, whey, nanocomposite, protein structure, mechanical properties


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Introduction Improved competitiveness of bioplastics relative to petroleum-based plastics is important for the development of a more sustainable society. In this effort, nanotechnology affords the opportunity to improve the material properties of bioplastics. One such track is the development of nanofibrils-reinforced composite materials.1 This approach may be even more powerful if both the matrix and nanofibrils are of similar origin, providing compatibility between them. A hitherto unexplored route is to prepare a blend of protein nanofibrils with a matrix of the same protein. The potential for using natural plant and animal-based proteins for the manufacturing of bio-based materials is well established. For example, milk and maize/corn proteins were already being used in bioplastics in the mid-20th century, and soybean protein have been exploited in textiles2 and cars.3 Protein-rich industrial side streams offer cheap raw materials for bioplastic production.4,5 However, protein-based materials often suffer from inferior physical and mechanical properties. It has been shown that the addition of nanoparticles (e.g., nanoclays or carbon nanotubes) can improve the performance of these materials.6,7 Proteins have also gained attention in nanotechnology due to their ability to self-assemble into highly ordered protein nanofibrils (PNFs).8 These structures, also known as amyloid fibrils, were first highlighted as structures associated with human diseases.9 An increasing number of investigations demonstrate, however, that the same type of structures are utilized by nature to create high-performance materials.9 Amyloid fibrils are typically 5-10 nm wide, up to several micrometres long and have a high content of β-sheet structure, in which the chains segments are oriented perpendicular to the fibril axis.10 Investigations of the mechanical properties of individual fibrils show that they are high performance materials with a specific tensile strength of the same magnitude as steel.11,12 At the same time, the stiffness (elastic modulus) of the nanofibril -3ACS Paragon Plus Environment


can vary over two orders of magnitude depending on the structure of the fibril.13 Films cast from PNFs solutions were observed to have elastic moduli on the same order of magnitude as the fibrils.14 The potential of using PNFs in functional materials/composites has been shown in pioneering work in applications for water purification,15,16 biosensors,17,18 biomimetic bone,19 hybrid aerogels20-22 and catalytic membranes.23 The effects of using PNFs in different polymer matrices have been also reported. The addition of less than 1 wt.% bovine insulin nanofibrils to poly(vinyl alcohol) (PVOH) resulted in changes in the microstructure and mechanical properties, specifically increasing the stiffness of the film.24 Notably, the same amount of non-fibrillar bovine insulin did not result in the same changes in properties, illustrating the impact of nanofibril structure. Biodegradable composites of PLLA and hen egg white lysozyme fibrils have also been produced and evaluated.25 Surprisingly, both the Tg and the elongation at break increased in the presence of these fibrils.25 It has also been shown that for 10 wt.% hen egg white lysozyme fibrils in a silicone elastomer, there was an increase in the elastic modulus that was at least twice that obtained with the same amount of carbon nanotubes.26 Pectin has been used together with β-lactoglobulin fibrils to help the latter assemble into nanoring-structures.27,28 Rather than blending protein nanofibrils with different matrices, Claunch et al.29 showed that it is possible to obtain a fully self-assembled fibre-reinforced protein. The researchers grew wheat gluten fibres from a hydrolysed wheat gluten matrix and obtained a material with a large improvement in stiffness and strength compared to the matrix material. However, it should be noted though that their fibres were significantly larger than typical amyloid fibrils (0.5-1.5 mm long and 10 µm thick).


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However, there are no reports on the blending of PNFs with a protein matrix. To fill this gap in the published research, this study was initiated; we explored the possibility of improving the mechanical properties of a plasticized protein (in this case, whey protein) using PNFs of the same protein to form a homo-nanocomposite, i.e., a protein/protein nanocomposite. In addition to mechanical properties, we also characterized the morphology (using scanning electron microscopy) and the protein structure (using IR spectroscopy and X-ray diffraction) of the composite. The plasticizer content is usually presented as a single “overall” value for protein materials. At a finer scale, however, the distribution of the plasticizer inside the sample may not be uniform, and this may also be the case for the protein structure. Such local variations may be critical for the film’s properties. In this work, we present a detailed IR analysis of both the plasticizer distribution and protein structure variation within cast protein films, as well as the properties of this type of protein nanocomposite.

Materials and Methods Preparation of Protein Nanofibrils (PNFs). Whey protein isolate (WPI) (Lacprodan Di9224) was kindly provided by Arla Food Ingredients. WPI with a concentration of 100 g/L was dissolved in 0.1 M hydrochloric acid (HCl), and the solution was dialysed against 0.01 M HCl (pH 2), using a membrane with a 6-8 kDa molecular weight cut-off (Spectrum laboratories, Rancho Dominguez. CA), for 24 h at room temperature. The solution was diluted to 40 g/L and incubated at 90 °C for a period of 3 days. To remove non-fibrillated proteins, some PNFs solution was dialysed extensively using a 100-kDa molecular weight cut-off membrane (Spectrum laboratories. Rancho Dominguez. CA). “Dialysed PNFs”, in the rest of this report, refers to the



material resulting from the 100-kDa cutoff dialysis, and “non-dialysed PNFs” refers to PNFs without dialysis. Preparation of WPI and Nanocomposite Films. The “matrix” solution of non-fibrillar WPI was prepared by dissolving 50 g WPI powder into 250 mL MilliQ water (pH 7). The solution was degassed under vacuum for 1 h. The relative protein concentration in the fibrillar and non-fibrillar solutions was assessed by UV absorbance at 280 nm. Glycerol, corresponding to 50 wt.% of the protein content, was added to the PNFs-free and PNFs solutions. Thereafter, the pH of the PNFs solution was rapidly adjusted to 7 with the addition of 2 M NaOH, and the solution was degassed under vacuum. For each film, solution samples containing a total of 2.4 g protein and 1.2 g glycerol were prepared using different ratios of PNFs (dialysed and nondialysed) and non-fibrillar WPI. These samples were heated to 90 °C for 30 min (to allow denaturation of the matrix proteins) and cooled under ambient conditions to room temperature. The mixture was poured into plastic 90-mm-diameter petri dishes (Saarstedt), coated with a Bytac® Teflon/aluminium film, and dried into films under ambient conditions (temperature between 20-25 °C and relative humidity between 20 and 65 %). The average thickness of the dry cast films was ca. 340 µm. Films containing only PNFs and glycerol were also prepared as references for the characterization experiments. The materials are identified as follows: WPI-(D or N)-Y, where D and N refer to dialysed and non-dialysed samples; Y refers to the PNFs content in wt.%. The PNFs-free material is referred to WPI-0. Atomic Force Microscopy (AFM). The formation of PNFs was confirmed by AFM using a Dimension FastScan instrument (Bruker) operating in tapping mode. Samples were diluted 1:1000 in 10 mM HCl, and 25 µL of this solution was applied onto a freshly cleaved mica surface.


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The samples were then dried in air. FastScan A cantilevers (Bruker) were used for the experiments, and the images were investigated using Nanoscope 1.5 software. Mechanical Testing. Prior to thickness measurements and tensile tests, the films were conditioned for 2 days at 23 ± 0.5 °C and 50 ± 3 % relative humidity (RH). Tensile tests were performed in the same conditioned climate. The film thickness was determined using an STFIthickness tester (M201. Stockholm. Sweden) with a measuring accuracy of 0.001 mm. The thickness was determined by six point measurements of each film, and the mean values were used to calculate the stress and E-modulus. Tensile properties were determined according to ASTM D 882-02. Dumbbell-shaped specimens were punched out from the films with a length and width of the narrow section of 16 and 4 mm, respectively. The tensile tests were performed using an MTS FlexTest60 tensile tester with the MTS 793 software programme and a 500 N cell. The measurements were performed with a crosshead speed of 100 mm/min and an initial clampto-clamp distance of 30 mm to calculate the (nominal) strain. The number of replicates ranged between 3 and 12. The E-modulus was calculated as the stress-strain slope between the strains, corresponding to 10 % and 35 % of maximum stress in the tensile measurement. Scanning Electron Microscopy (SEM). The top and cross-sectional surfaces of the films were observed in a Hitachi S-4800 cold-field-emission SEM. The cross-sections of the surfaces were obtained by freeze-fracturing films that had been immersed in liquid nitrogen. Before the SEM analysis, the samples were sputtered with a platinum/palladium (60/40) alloy using a Cressington 208RH high-resolution sputter. IR Spectroscopy. IR spectroscopy was performed on a Perkin-Elmer Spotlight 400 FTIR imaging system equipped with a liquid-nitrogen-cooled mercury-cadmium-telluride detector. The spectra were recorded from 4000 cm-1 to 750 cm-1 with 16 scans at a resolution of 4 cm-1 in ATR mode. The IR spectra were modified to allow for deconvolution using Spectrum 10.5.1 software, -7ACS Paragon Plus Environment


with an enhancement factor (γ) of 2 and a smoothing filter of 70 %. The baseline-corrected spectra (1700 cm-1 to 1580 cm-1), were loaded into the Origin 9.1 software and deconvoluted into nine Gaussian peaks with fixed wavenumbers. Peak assignments were obtained from Cho et al.30 Prior to measurement the specimens were dried in a desiccator containing silica gel for at least three days. X-ray Diffraction (XRD). X-ray diffractograms of the WPI films were obtained using an ARL X’TRA X-ray diffractometer with Cu Κα radiation (wavelength = 1.54 Å) at a current of 44 mA and a voltage of 45 kV. The data were collected between 2 and 50˚, with an interval of 0.1˚.

Results and Discussion Preparation of Protein Nanofibrils and Nanocomposite Films. Whey PNFs were prepared by incubation of the solution at pH 2 and 90 °C for three days. Under these conditions, nanofibrils with a typical diameter of 4 nm and lengths up to several micrometres were formed (Figure 1a).31 We have studied the fibril formation in detail in a recent work32 and we could confirm that peptide hydrolysis occurs simultaneously with the fibrillation reaction,33 resulting in considerable amounts of short peptides that can affect the properties of the prepared composite materials. This phenomenon was also observed in this study (vide infra). The removal of nonfibrillar material was confirmed by a substantially reduced protein concentration of the dialysed PNFs solution, corresponding to ca. 25 % of that of the non-dialysed solution (including dilution effects). Hence, approximately three-fourths of the protein starting material was not incorporated into the fibrils. The upper film surface shifted from completely flat in the film without PNFs (WPI-0) to undulating in the presence of PNFs, leading to a surface that scattered light and a less optically -8ACS Paragon Plus Environment

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transparent film (Figure 1b, Figure 2). This undulation was observed also in films with 100 % dialysed or non-dialysed PNFs, with and without glycerol plasticizer. Hence, the undulation appears to primarily be a feature of the PNFs itself. Mechanical Properties. Tensile tests were performed to reveal the effect of the protein nanofibrils on the mechanical properties of the whey protein films. Typical force-extension curves are shown in Figure 3. The fibril-free sample showed a curve with extensive plastic deformation beyond the yield (maximum force) point, resulting in a force-extension slope that was nearly constant, until complete failure was reached at zero force (Figure 3a). As the PNFs content increased, the force-extension curve changed, primarily in the post-yield point region, showing a more distinct fracture point with a relatively sharp drop in force (Figure 3b). Considering the entire range between 0 and 15 wt.% PNFs, the stiffness (modulus) increased with increasing PNFs content in both non-dialysed and dialysed samples (Table 1, Figure S1 in the supporting information). The maximum modulus was 1.9-fold (WPI-N-10) and 2.1-fold (WPI-D-15) that of the fibril-free (WPI-0) sample. In an attempt to estimate the modulus of 100 % glycerol-plasticised fibril material (E1) the upper bound parallel relationship (Ec=v1 E1+v2 E2)34 was fitted to the dialysed protein composite moduli (Ec) in Table 1. v refer to the volume fraction. It is assumed here that the density of the PNFs and the WPI components are the same. The upper bound was used since the data was not possible to fit with the lower bound relationship. Using a modulus of 26 MPa for the WPI component (E2) yielded an upper bound modulus (E1) for the plasticised PNFs of 200 MPa. The modulus of pure β-lactoglobulin PNFs has been reported to be on the order of 3-5 GPa, depending on the method used to determine it and number of filaments in the fibril.35,36 The modulus of a protein film/plate (wheat gluten) can in some cases decrease by as much as two orders of magnitude when 30 wt.% glycerol is added



to it.37 Hence the estimated upper bound modulus of the plasticised PNFs films is not unreasonable. Again, considering the entire range of PNFs content, the strength (yield stress) values showed a small increase with increasing PNFs content, but the changes were essentially not significant. The overall strength (ca. 1 MPa) was similar to that presented earlier for whey/glycerol films with the same glycerol content.38 As observed in Table 1, ductility (strain at break) decreased with increasing PNFs content (over the entire PNFs range) for both dialysed and non-dialysed samples. However, the films were still ductile (~ 20 % strain at break for both dialysed and non-dialysed samples) at 15 wt.% PNFs content. Protein Secondary Structure. The protein secondary structure was investigated by deconvolution of the amide I band (1700 – 1580 cm-1).39,40 The total absorbance and nine resolved peaks in the amide I region are shown for the non-fibrillated (WPI-0) and fully fibrillated (WPI-D-100) samples in Figure 4a and b; there is a noticeable difference in the shape of the total absorbance. The relative contributions of the nine peaks for all samples are given in Table S1 (Supporting information) and are plotted in groups with the same structural origin as a function of PNFs content in Figure 5. Five IR-spectra were recorded randomly along the films on both the upper and lower surfaces (IR sampling depth is ca. 1-5 µm, depending on the wavenumber;41 the sampled surface area is 6.25 x 6.25 µm2). As observed in Figure 5, the content of strongly hydrogen-bonded β-sheets increased with increasing PNFs content, and the effect was greater for the dialysed samples. This was accompanied by a decrease in the amount of weakly hydrogen bonded β-sheets; effects were again greater for the dialysed samples. In fact, the content of the two types of β-sheets was relatively independent of the content of non-dialysed PNFs for the 0-15 wt.% PNFs range (Figure 5b). The difference in the protein secondary structure between the dialysed and non-dialysed samples illustrates the effects on the overall

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protein secondary structure of the removal of smaller peptide fragments during dialysis. The amount of unordered and α-helix-rich structures (1658-1644 cm-1) decreased with increasing PNFs content, and the size of the decrease was essentially independent of whether samples were dialysed (considering the entire range of PNFs). Notably, there was a near-linear increase in the average modulus with increasing PNFs content in the dialysed samples (Table 1 and Figure S1a) coinciding with a near linear increase (considering 0-15 wt.% PNFs) in the content of strongly hydrogen-bonded β-sheets (Figure 5a). The multiple measurements for the IR absorption were primarily taken to investigate whether the presence of the undulations in the film correlated with a more spatially heterogeneous protein secondary structure. The latter could be due to, for example, a proteinmolecular phase separation within the PNFs component or between the PNFs and the WPI matrix components, and/or spatial variation in protein aggregation. The variation in protein secondary structure (size of the error bars in Figure 5) was in most cases larger in the presence of PNFs compared to without PNFs (WPI-0). Hence it is possible that the observed undulations developed due to a spatially heterogeneous protein structure. However, there was no unique relationship between the size of the structure variation and the PNFs content. There was also no systematic difference in the size of the structure variation between the upper and lower surfaces (not shown specifically here). For WPI-0, the content of unordered/α-helix-rich components and strongly and weakly hydrogen bonded β-sheets varied with 24, 10 and 6 %, respectively. These percentages refer to the error bars in Figure 5, divided by the average values. The maximum variation of the unordered/α-helix-rich components in the films was 46 % among the non-dialyzed samples (WPI-N-100) and 54 % for the dialyzed samples (WPI-D-100). The variation observed for the



content of strongly hydrogen bonded β-sheets was: 50 % (WPI-N-5) and 30 % (WPI-D-100), and for the weakly hydrogen bonded β-sheets: 58 % (WPI-N-100) and 32 % (WPI-D-2.5). The PNFs solutions/dispersions were more viscous than the non-fibrillar whey solution, indicating fibrillar entanglement effects and possible PNFs aggregation. It is likely that these effects were enhanced during the drying process and generated the undulation in the final films. However, the cross-sections of the cast films under SEM, showed a dense, feature-less morphology, indicating a solid cast film without any apparent phase separation of non-fibril and PNFs components, at least on a micro-to-macro scale corresponding to the size of the undulations (Figure 2). Interestingly, Lara et al.42 have observed that the lyzosome amyloid fibril structure becomes undulated with a periodicity of ca. 250 nm, which is on a significantly smaller length scale than the undulations that we observe here. It is tempting to suggest that the small length scale undulations may lead to the larger scale undulations observed here. Whether this smallscale undulation causes on overall larger spatial variation in protein structure, compared with the fibril free material (as observed by IR), and a larger scale undulation, or if the latter is caused primarily by protein aggregation/internal PNFs phase separation is not possible to ascertain here. Glycerol Distribution. Many studies on cast protein films with plasticizers such as glycerol have been reported, but there are very few studies that consider the spatial distribution of the plasticizer within the film.43-45 To determine whether the undulations on the upper side of the film were associated with variations in glycerol concentration (i.e., due to phase separation of glycerol and protein), the IR spectra, recorded to evaluate the spatial variation in protein structure above, were analysed with a focus on the glycerol peaks. The normalized concentration of glycerol was obtained by determining the ratio of the glycerol peak (850 cm-1) and the whey protein amide I peak (the whole amide I absorbance region). The method was previously used by


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Mangavel et al.46 and Cho et al.30 As observed from the standard deviation values in Table 2, the variation in glycerol content along the film was not systematically larger in PNFs samples compared to the PNFs-free sample (WPI-0). In addition, the variation was not systematically different on the upper and lower surfaces. Hence, the undulated structure was not due to, or the cause of, a spatial variation in the glycerol content. It should be noted that even optically (and also observed with SEM) homogeneous “standard-prepared” plasticized protein-based films (WPI-0) show spatial variation in the protein structure and the plasticizer content at the IR resolution used. In fact, the largest variation in glycerol concentration was observed for the non-fibrillated sample (Table 2; WPI-0 upper surface: 36 % (size of the standard deviation error bar divided by the average value)). X-ray Diffraction. X-ray diffraction was used to examine whether the material remained fully amorphous during the casting process and whether any structural differences between the samples could be observed. The pristine WPI powder showed two broad peaks: a major peak (P1) at 2θ ≈ 20˚ and a smaller peak (P2) at ca. 9˚ (Figure 6).47,48 P1 refers to an “average” distance between neighbouring atom pairs (λ ≈ 4-4.5 Å)49,50 and P2 corresponds to the distance between adjacent stacked β-sheets (λ ≈ 10 Å), frequently observed in PNFs.51,52 The fact that this peak appeared in the pristine WPI powder suggests that similar stacks of β-sheets also occur in native whey proteins. In fact, the PNFs-free film without glycerol (WPI-0 no gly) showed an almost identical curve shape as the pristine WPI powder (Figure 6a), showing that the β-sheet structure before and after film preparation (without PNFs) is similar. The size of P2 (relative to P1) was larger in the glycerol-free non-dialysed 100 % PNFs sample than in the “WPI-0 no gly” sample, and the largest P2 was observed for the same sample with dialysis (WPI-D-100 no gly). The large P2 shows that PNFs consisted of numerous stacked β-sheets. Interestingly, in the presence of - 13 ACS Paragon Plus Environment


glycerol, the size of P2 decreased for all three samples (Figure 6a, WPI-D-100/WPI-N-100/WPI0). Since glycerol contributes to P1 but not P2, the relative size of P2 decreases (dilution effect) in the presence of glycerol.50, 53 However, P2 was small, or even absent, in all glycerol-containing samples—including films with mixed PNFs and non-fibrillar WPI (Figures 6b and c) and it did not increase with increasing PNFs content (within the 0-15 wt.% PNFs range). Hence, apart from the dilution effect, glycerol also seemed to favour a certain degree of exfoliation of the β-sheets (i.e., decrease in the amount of stacked sheets) in the final films, for all systems investigated. It is possible that this exfoliation can have an effect on the mechanical properties of the composite, although the plasticisation is the dominant feature when adding glycerol.

Conclusions The addition of whey PNFs into the whey matrix, prepared by the mixed in strategy, changed the mechanical (mainly stiffness and ductility) and optical properties, as well as the film microstructure and protein structure. Along with the improvement in stiffness with increasing PNFs content (0-15 wt.% PNFs), undulations in the upper film surface with a larger protein structure spatial variation also appeared. A hypothesis is that the undulations were caused by smaller-scale undulations of the fibril structure and/or a phase separation of the specific PNFs material. The latter was supported by that the undulations occurred also for pure PNFs films. However, the presence, and any spatial variation, of the glycerol plasticiser did not affect the film microstructure. In fact, our data show that even optically homogeneous and clear plasticized protein cast films show a local, micrometre-scale variation in glycerol content. Notably, glycerol seemed to not only provide ductility to the protein films; X-ray data indicated that its presence


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also favoured, to some extent, β-sheet exfoliation from β-sheet stacks, which may have altered the overall mechanical features of the films.

Acknowledgements Financial support from the China Scholarship Council and Formas (Grant 213-2014-1389) is gratefully acknowledged. Qiong Wu is thanked for assisting with the SEM work.

Associated Content Supporting Information Relative content of secondary structure in the amide I region; plots of modulus (a), stress (b) and extensibility (c) as a function of PNFs content (PDF)

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Figure 1. (a) AFM image of whey protein nanofibrils, (b) PNFs-free film (WPI-0, right) and a dialysed sample with 15 wt.% PNFs (WPI-D-15, left).


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Figure 2. SEM images of cross-sections of WPI-0 (a) and WPI-N-15 (b) films.



Figure 3. Typical force-extension curves of WPI-0 (a) and WPI-D-15 (b).


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Table 1. Mechanical properties of WPI films Samples WPI-0

Modulus (MPa) a 26 ± 8 a

WPI-N-2.5

19 ± 6

WPI-N-5

30 ± 7

WPI-N-10

49 ± 5

WPI-N-15

42 ± 8

WPI-0

26 ± 8

WPI-D-2.5

28 ± 5

WPI-D-5

33 ± 15

WPI-D-10

43 ± 1

WPI-D-15

54 ± 10

Stress (MPa) a 1.0 ± 0.4 0.9 ± 0.05

a

1.0 ± 0.3

b

1.6 ± 0.1

b

1.2 ± 0.1

a,b a

1.0 ± 0.4 1.0 ± 0.3

a

b,c c

1.1 ± 0.3 1.1 ± 0.2 1.2 ± 0.3

a

a b a,b a a a a a

Strain (%) a 43 ± 9 46 ± 4 32 ± 6 26 ± 5 24 ± 5 43 ± 9 28 ± 6

a b,c c c a b

32 ± 13 21 ± 8 21 ± 5

a,b

b b

± values are standard deviations; Values having the same letter are not signiﬁcantly different (Tukey HSD test at a signiﬁcance level of 0.05).



Figure 4. FTIR spectra of the amide I region of WPI-0 (a) and WPI-D-100 (b) samples, resolved into nine Gaussian peaks. The thick line is the experimental curve, and blue, red and green peaks refer to strongly hydrogen-bonded β-sheets, weakly hydrogen-bonded β-sheets and helix/unordered structures, respectively.


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Figure 5. Relative size of resolved IR peaks, grouped into those originating from strongly hydrogen-bonded β-sheets (strongly), weakly hydrogen-bonded β-sheets (weakly) and αhelix/unordered structures (α-helix/unordered). N and D refer to non-dialysed and dialysed films, respectively. Error bars correspond to standard deviation.



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Table 2. Glycerol normalized concentration of upper and lower film surface PNFs (%) 0 2.5 5 10 15

Non-dialysed Upper 0.067 ± 0.012 0.072 ± 0.004 0.068 ± 0.004 0.075 ± 0.005 0.068 ± 0.010

Lower 0.073 ± 0.005 0.078 ± 0.008 0.073 ± 0.010 0.065 ± 0.005 0.072 ± 0.004

Dialysed Upper 0.078 ± 0.008 0.070 ± 0.006 0.068 ± 0.008 0.068 ± 0.004

± values are standard deviations.


Lower 0.075 ± 0.005 0.073 ± 0.005 0.078 ± 0.008 0.085 ± 0.010

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Figure 6. X-ray diffraction normalized curves of (a) whey protein powder, non-fibrillated WPI and 100 % fibrillated films with and without glycerol, and (b) dialyzed and (c) non-dialyzed films (no gly = no glycerol).



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Synopsis: A new type of bioplastic nanocomposite based on nanofibrils and a matrix of the same protein, in this case the large byproduct from food production (whey).


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Protein Nanocomposite Based on Whey Protein Nanofibrils in

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