Whey protein concentrates (WPC) with remarkable functional properties are obtained by removing non-protein ingredients from whey, which is a by-product of cheese making. (Henriques et al., Reference Henriques, Gomes, Pereira, Nedović, Raspor, Lević, Tumbas Šaponjac and Barbosa-Cánovas2016). WPC contains 35–80% protein (including β-lactoglobulin and α-lactalbumin as major components).WPC can be used as food additives, foaming agents, emulsifiers, thickening agents, gelatinizers and nutraceuticals due to their functional properties and biological activity (Brandenberg et al., Reference Brandenberg, Morr and Weller1992; Banerjee and Chen, Reference Banerjee and Chen1995; Perez et al., Reference Perez, Carrara, Sánchez, Santiago and Patino2010; de Castro et al., Reference de Castro, Domingues, Ohara, Okuro, dos Santos, Brexó and Sato2017). Whey protein-based films are extensively researched as they generally demonstrate better physical properties and barrier properties to oxygen and aroma transmission than other protein-based films (such as caseinates, soy protein isolate and wheat) or polysaccharide-based films (such as chitosan, starch and cellulose) (Kumari et al., Reference Kumari, Mahajan, Joshi and Gupta2017; Sukyai et al., Reference Sukyai, Anongjanya, Bunyahwuthakul, Kongsin, Harnkarnsujarit, Sukatta, Sothornvit and Chollakup2018).The properties of the films are influenced by protein concentration, heat treatment, pH, salt concentration (Pérez-Gago et al., Reference Pérez-Gago, Nadaud and Krochta1999; Ayadi et al., Reference Ayadi, Leuliet, Chopard, Berthou and Lebouché2004; Mchugh et al., Reference Mchugh, Aujard and Krochta2010). However, whey protein films have some limitations in respect of their unfavorable mechanical properties and high permeability of water-vapor (Henriques et al., Reference Henriques, Gomes, Pereira, Nedović, Raspor, Lević, Tumbas Šaponjac and Barbosa-Cánovas2016).
The polymeric films such as whey protein-based films are formed by cohesion and adhesion of composite which relates to the structural and chemical properties of aggregation. Heating causes unfolding of whey proteins and exposure of hydrophobic residues, whilst the cross-linked network of protein increases the cohesion and rigidity of film (Pérez-Gago et al., Reference Pérez-Gago, Nadaud and Krochta1999). Two types of distinctly different morphologies are formed during heating: amorphous and ordered aggregation according to pH (Nicolai and Durand, Reference Nicolai and Durand2013). Based on previous studies, WPC (Gao et al., Reference Gao, Xu, Ju and Zhao2013; Xu et al., Reference Xu, Wang, Dong, Cheng, Kong and Tan2016), whey protein isolate (Bolder et al., Reference Bolder, Vasbinder, Sagis and van der Linden2007b; Mantovani et al., Reference Mantovani, Pinheiro, Vicente and Cunha2017), β-lactoglobulin (Dave et al., Reference Dave, Loveday, Anema, Jameson and Singh2015; Nicolai et al., Reference Nicolai, Britten and Schmitt2011) and several other proteins have the ability to form fibrils at a pH value (such as pH2.0) far away from the isoelectric point and at low ionic strengths, by incubation above their denaturation temperature (commonly at 90°C) for about 10 h. These fibrils which are normally composed of 2−6 protofilaments twisted together with nanometric diameter and several micron lengths, are rich in β-sheets that run parallel to the axis of fiber (Nelson et al., Reference Nelson, Sawaya, Balbirnie, Madsen, Riekel, Grothe and Eisenberg2005). The fibrillar structures show prominent mechanical properties, such as high elasticity, stiffness, and resistance (Adamcik and Mezzenga, Reference Adamcik and Mezzenga2011). Stable and rigid structures have been regarded as a powerful tool to fabricate high-performance nanostructured materials (Knowles et al., Reference Knowles, Fitzpatrick, Meehan, Mott, Vendruscolo, Dobson and Welland2007). The rigidity derives from the intermolecular organized hydrogen-bond network oriented by side-chain interactions (Liu et al., Reference Liu, Busuttil, Zhang, Yang, Wang, Besenbacher and Dong2011).
Knowles and his colleagues (Knowles et al., Reference Knowles, Oppenheim, Buell, Chirgadze and Welland2010; Knowles and Buehler, Reference Knowles and Buehler2011) manufactured free-standing films from self-assembled β-lactoglobulin and hen egg-white lysozyme fibrils. These highly rigid films showed Young's modulus of up to 5–7 GPa and well-ordered structure that aligned other unstructured constituents (such as fluorophores) within the nanostructured films. Films formed from β-LG nanofibrils presented enhanced transparency and decreased moisture content and were able to expand the shelf life of fresh-cut apple by inhibiting the loss of total phenolic content, browning, and water consumption (Feng et al., Reference Feng, Wu, Liu, Li, Jiang and Zhang2018). Lysozyme nanofilm (Wang et al., Reference Wang, Ha, Gu, Li, Zhang and Yang2016) and Aβ16−22 nanofibrils films (Pan et al., Reference Pan, Liu, Zhang, Yu and Dong2012) had high optical transparency, steady adhesion force and were said to be environmental-friendly. In recent years, there has been an increasing amount of literature on hybrid nanocomposite films with modified catalytic efficiency, mechanical and electronic properties as well as on biological devices fabricated by combining fibrils with metal nanoparticles (Bolisetty et al., Reference Bolisetty, Arcari, Adamcik and Mezzenga2015), or with polyvinyl alcohol (PVOH) (Pilkington et al., Reference Pilkington, Roberts, Meade and Gerrard2010; Rao et al., Reference Rao, Meade, Healy, Sutton, Larsen, Staiger and Gerrard2012) or graphene (Li et al., Reference Li, Adamcik and Mezzenga2012).
Electrical conductivity is an important characteristic of the functional polymer film, which can be potentially applied in conductive biosensors (Gao et al., Reference Gao, Wen, Wang, Yan, Zhao, Lei and Miao2012; Abdel-Karim et al., Reference Abdel-Karim, Salama and Hassan2018). However, there is little published information about the electrical conductivity of self-assembly fibril film. The present work compared the two types of films (WPC fibril film with pH 2.0 and conventional WPC film with pH 6.5) regarding electrical conductivity, film protein solubility, mechanical properties and oil resistance.
Materials and methods
Materials
Wpc-80 with 76.93% protein content was purchased from Hilmar Cheese Company (Hilmar, California, United States). Analytical grade reagents were used in all cases and obtained from local suppliers.
WPC fibrils formation
Wpc fibrils were prepared according to the procedure used by Xu et al. (Reference Xu, Wang, Dong, Cheng, Kong and Tan2016). WPC (5% w/v) was stirred into deionized water containing different concentrations of CaCl2, then the dispersion was adjusted to pH 2.0 by the addition of 6 m HCl. To remove non-fibrillated proteins, WPC solution was centrifuged at 19 000 × g for 20 min at 4°C. The resulting supernatant was collected and the protein content determined by Kjeldahl analysis (N × 6.38).The solution was diluted to the protein concentration of 3.0% (w/v) with deionized water (pH 2.0)and heated at 90°C for 10 h to form mature fibrils. In order to study the effect of CaCl2 concentration, WPC dispersions with ionic strength of 20, 40, 60, 80, 100, 150, 200, 250 and 300 mm were prepared by adding CaCl2 to protein solutions with protein concentration of 3.0% (w/v).
Film formations
Conventional WPC film (pH 6.5): 3% w/v WPC solution was prepared. Then gelatin and glycerol were added as plasticizers with the relative weight of 60 and 50% (w/w protein) (Le et al., Reference Le, Letendre, Ispasszabo, Mateescu, Delmaspatterson, Yu and Lacroix2000; Schmid, Reference Schmid2013). The well-stirred solution was heated in a water bath (90°C) for 30 min and cooled to room temperature after heating. The solution was poured over the glass plate and left to dry for 24 h at room temperature. The film was detached from the surface and conditioned at 50% relative humidity (RH) and room temperature for 48 h prior to testing.
WPC fibril film (pH 2.0): 3% w/v WPC fibril solution was prepared. Then gelatin and glycerol were added as plasticizers with the relative weight of 60 and 50% (w/w protein). After heating at 90°C for 30 min, the cooled film-making solution was cast and conditioned in the same way as conventional WPC film.
Conventional WPC film (pH 2.0): In order to compare with the WPC fibril film, conventional WPC film with pH 2.0 was fabricated. 3.0% (w/v) WPC solution (pH 6.5) with gelatin (60% w/w protein) and glycerol (50% w/w protein) was incubated at 90°C for 30 min. After cooling, the pH value of the solution was adjusted to 2.0 by the use of 6 m HCl and cast as conventional WPC film.
Electrical conductivity measurement
The electrical conductivities of the film-forming solution and film were measured at room temperature using a silver probe method with a high resistivity meter (HIOKI LCR HiTESTER 3532-50, Japan). The frequency and voltage were 1000 Hz and 0.5 V. The values of electrical conductivity σ(S/m) were calculated using the following equation:
$${\rm \sigma } = \displaystyle{L \over {RA}}\;$$where A is the cross-section area of the measured surface(m2), L is the length of the sample in the direction of measurement(m), R is the resistivity(Ω).
Film protein solubility
2.0 g WPC fibril film was dissolved in deionized water (pH 2.0) for 5, 10, 15, 20, 25, 30 min. The solution of the film was centrifuged at 4000 r/min for 20 min. The gelatin film with the same gelatin concentration as the WPC fibril film was fabricated to avoid the influence of protein solubility in gelatin. The solubility was calculated using the following formula:
$$M = \displaystyle{{M_2-m} \over {M_1-m}} \times 100\% $$where m is the protein weight of gelatin in the supernatant(g); M 1 is the initial protein weight of film; M 2 is the protein weight of supernatant after dissolved(g).
Mechanical properties
The tensile strength (TS) and percent elongation at break (E/B) with a size of 30 × 100 mm were measured by XLW (M) Auto Tensile Tester (Labthink International, Inc., China) at 25°C and 50% RH. The test speed was 50 mm/min.
Oil permeability
A tube containing 5 ml oil was sealed with the sample film and inverted on the filter paper for a week. The oil permeability coefficient P 0 (g mm/m2/d) was calculated according to the formula:
$$P_0 = \displaystyle{{\Delta m \times T} \over {S \times R}}$$where △m is the change in weight of the filter paper(g); T is the film thickness(mm); S is the film area (m2); R is the test time (d) (Iwata et al., Reference Iwata, Ishizaki, Handa and Tanaka2000).
Thioflavin T (Th T) fluorescence assay
A stock solution was made by adding 8 mg Th T into 10 ml of phosphate buffer (10 mm phosphate and 150 mm NaCl, pH 7.0). These dispersions were filtered through a 0.22 μm syringe filter and stored at 4°C in the dark. Working solution was diluted 50-times with the same buffer before utilized. Fibril sample (50 μl) and Th T working solution (5 ml) were mixed for at least 1 min and measured using a Hitachi F4500 fluorescence spectrometer (Tokyo, Japan) at the excitation wavelength of 446 nm and the emission wavelength of 490 nm.
Surface hydrophobicity
Ans (8-anilinonaphthalene-1-sulfonic acid ammonium salt) as a fluorescent probe was used to measure the surface hydrophobicity of samples. Protein solutions were gradually diluted to a concentration range from 0.005 to 0.1% with 10 mm phosphate buffer of pH 6.7. Aliquots of the solution (6 ml) were added to 20 μl ANS solution (8 mm ANS and 10 mm phosphate buffer at pH 7.0) and stood in the dark for 15 min before the analysis. Hitachi F4500 fluorescence spectrometer was used to measure absolute fluorescence intensity with excitation wavelength of 39 nm and emission wavelength of 47 nm. Surface hydrophobicity was determined with the initial slope of the fluorescence intensity against protein concentration.
ζ-Potential
ζ-Potential of the solution was measured using a Malvern Zetasizer Nano ZS90 (Malvern Instruments, Worcestershire, UK) at 25°C. The samples were diluted to a concentration of 0.1% (w/v) with deionized water (the same pH as the sample) before measurements. Refractive index values were set at 1.450, the viscosity of fibril samples was set at 0.8872 cP.
Transmission electron microscopy (TEM)
Sample solutions were prepared for TEM by diluted to 0.03% w/w protein with deionized water. A drop of the solution was transferred to a 200-mesh carbon-coated copper grid and removed the excess sample with filter paper after 20 min. TEM micrographs were operated at 100 kV using a H-7650 transmission electron microscope(Hitachi, Tokyo, Japan).
Statistical analysis
All experiments were performed in triplicate and the data are expressed as means ± standard deviation (sd) of three replicates. All data were analyzed by one-way analysis of variance (ANOVA) using SPSS (16.0) software (IBM software, NY, USA). Significant differences of P < 0.05 was used and determined by Duncan's multiple range test.
Results and discussion
The electrical conductivity of WPC fibril and WPC fibril film
The electrical conductivity was different among the three types of WPC films with different aggregation structures (Fig. 1a). The electrical conductivity of WPC fibril film increased by 123% as compared with that of conventional WPC film (pH 6.5), and 73% as compared with that of conventional WPC film (pH 2.0). The electrical conductivity of WPC fibril film-forming solution (pH 2.0) which was higher than that of conventional WPC film-forming solution (pH 6.5), significantly increased (P < 0.05) from 0.280 to 0.373 S/m after 20 h of heating (Fig. 1b). These results indicate that the improved electrical conductivity is possibly attributable to the forming of fibrillar structures at pH 2.0, instead of adding HCl.
(a) Electrical conductivity of WPC fibril film (pH 2.0), conventional WPC film (pH 6.5) and conventional WPC film (pH 2.0). (b) Electrical conductivity of the film-forming solution (prepared by heating 3% (w/v) WPC at 90°C with pH 2.0 or 6.5) at different incubation time. Different letters above the bars indicate a statistically significant differences (P < 0.05).
Factors for the conductivity of fibril solution
Fibril morphology and quantity are known to be affected by protein concentration (Schokker et al., Reference Schokker, Singh, Pinder and Creamer2000), pH, ionic strength and valency (Loveday et al., Reference Loveday, Wang, Rao, Anema, Creamer and Singh2010) as well as by stirring and seeding (Bolder et al., Reference Bolder, Sagis, Venema and van der Linden2007a). The influence of protein concentration and CaCl2 concentration on the electrical conductivity of our WPC fibril solution (pH 2.0) and conventional WPC solution (pH 6.5) are shown in Fig. 2. The electrical conductivity of both solutions improved significantly (P < 0.05) as protein concentration increased from 3% to 6%. The reduction in electrical conductivity at 7% could be attributed to gelation at high protein concentration. The conductivity of WPC fiber is better than that of conventional WPC (pH 6.5), moreover, the variation of conductivity between the two solutions increased from 0.174 to 0.273 S/m with increasing protein concentration (Fig. 2a).
Electrical conductivity of WPC fibril solution (pH 2.0) and conventional WPC solution (pH 6.5) as a function of (a) protein concentration, (b) CaCl2 concentration. Different letters above the bars indicate significant differences (P < 0.05).
Calcium ions can potentially lead to the change of electrical conductivity according to shield electrostatic interactions, and in addition can change charge interactions and induce different morphologies (Ramos et al., Reference Ramos, Pereira, Martins, Rodrigues, Fucinos, Teixeira, Pastrana, Malcata and Vicente2017). The electrical conductivity of the fibril and conventional solutions improved significantly (P < 0.05) with the increase of CaCl2 concentration (Fig. 2b). Since, in previous work, fibril solutions have appeared to undergo phase separation and films have become disrupted with excessive CaCl2 (Bolisetty et al., Reference Bolisetty, Harnau, Jung and Mezzenga2012), 100 mm CaCl2 was chosen in follow-up research with consideration of both electrical conductivity and integrity of the film.
Protein solubility for WPC fibril film
Wpc fibril film (pH 2.0) was more soluble due to the relatively weak noncovalent interactions among proteins (Fig. 3a) (Pérez-Gago et al., Reference Pérez-Gago, Nadaud and Krochta1999; Oboroceanu et al., Reference Oboroceanu, Wang and Brodkorb2010). However, water resistance and insolubility are required for film-forming and product integrity (Rhim et al., Reference Rhim, Gennadios, Handa, Weller and Hanna2000). A decrease of film protein solubility with increasing protein concentration was due to high cohesion from the unfolding of protein molecules and exposing hydrophobic groups formerly buried inside the molecules (Pérez-Gago et al., Reference Pérez-Gago, Nadaud and Krochta1999). Mature fibril solution was adjusted to different pH before it was cast. The solubility of film manufactured by diverse pH of film-forming solutions is presented in Fig. 3b. The film protein solubility of WPC fibril solutions at pH 2.0 was reduced from 51.62 to 30.94% by adjusting pH of film-forming solutions to 5 (pI range of WPC), and the maximum was recorded at pH 7. Increasing the CaCl2 concentration from 0 to 100 mm improved aggregation by electrostatic screening, increased the yield and viscosity of fibrils (Loveday et al., Reference Loveday, Su, Rao, Anema and Singh2011), resulting in a decreased solubility from 51.62 to 33.60% (Fig. 3c).
Protein solubility from WPC fibril film (prepared by 3%(w/v) WPC heating at 90°C for 10 h, pH2.0) as a function of (a) protein concentration, (b) pH 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0 and (c) CaCl2 concentration.
Characteristic parameters of films
Considering the effects of protein concentration, pH and CaCl2 concentration on electrical conductivity and film protein solubility, WPC fibril film was prepared from 6%(w/v) protein concentration with 100 mm CaCl2 at pH 2.0, and conventional WPC film was prepared from the equal concentration of protein and CaCl2 at pH 6.5. The results of characteristic parameters (electrical conductivity, film protein solubility, tensile strength, elongation at break and oil permeability) for two types of films are presented in Table 1. It can be observed that the electrical conductivity of the WPC fibril film (pH 2.0) was increased by almost 72% as compared with that of conventional WPC film (pH 6.5). WPC fibril film showed superior mechanical properties (tensile strength, elongation at break) and oil permeability compared with conventional WPC film, probably due to its highly ordered hydrogen-bonding network (Paparcone et al., Reference Paparcone, Keten and Buehler2010; Xu and Buehler, Reference Xu and Buehler2010). However, the solubility was found to be higher for the WPC fibril film. The variation of two films was associated with the structure, surface hydrophobicity and net charge, which we will discuss below.
Electrical conductivity, film protein solubility, tensile strength, elongation at break and oil permeability of two types of films
WPC, whey protein concentrate; TS, tensile strength; E/B, elongation at break.
a–bDifferent letters in the same column indicate significant differences (P < 0.05).
Th T fluorescence and morphology of protein solutions
The morphology of different aggregates from two protein solutions was visualized using TEM (Fig. 4b, c). WPC self-assembled into long and straight fibrils by reason of electrostatic repulsive forces, while it formed amorphous aggregate when heated at pH 6.5 (van der Linden and Venema, Reference van der Linden and Venema2007). The β-sheets-rich structures of WPC fibrils can be measured by Th T fluorescence assay, as Th T which binds to the grooves on the β-sheets causes an increase in fluorescence intensity. Fig. 4a shows the typical sigmoidal growth kinetics in Th T fluorescence intensity upon fibrillation of WPC: the lag phase (about 0–2 h), growth phase or elongation phase (2–6 h) and plateau phase (6–10 h). However, conventional WPC (pH 6.5) did not significantly increase in fluorescence upon heating. These highly ordered and stable β-sheets structures with parallel or antiparallel orientation, probably contribute to the excellent mechanical properties (Knowles et al., Reference Knowles, Oppenheim, Buell, Chirgadze and Welland2010) and conductivity of fibril films.
(a) Th T fluorescence intensity of WPC fibril (prepared by 3%(w/v) WPC heating at 90°C, pH 2.0)and conventional WPC (prepared by 3%(w/v) WPC heating at 90°C, pH 6.5)as a function of time; (b) TEM of 3% conventional WPC forming at pH 6.5, heated for 0.5 h at 90°C; (c) TEM of 3%(w/v) WPC fibrils forming at pH 2.0, heated for 10 h at 90°C. Scale bar corresponds to 1 μm.
Surface hydrophobicity of protein solutions
The differences in electrical conductivity of films fabricated by the two types of aggregate structures may be related to the unfolding of protein and exposure of hydrophobic regions. ANS, which can bind to hydrophobic groups of protein, is considered to be representative of surface hydrophobicity (Mantovani et al., Reference Mantovani, de Figueiredo Furtado, Netto and Cunha2018). The surface hydrophobicity showed 2.5-fold and 1.85-fold increase during WPC fibrillation process and conventional aggregation process (Fig. 5). The surface hydrophobicity results indicated that fibril-like structure showed more exposed hydrophobic groups which resulted in the increase of electrical conductivity.
Surface hydrophobicity of WPC fibril (prepared by 3%(w/v) WPC heating at 90°C, pH 2.0) and conventional WPC (prepared by 3%(w/v) WPC heating at 90°C, pH 6.5) as a function of time. Different letters above the bars indicate significant differences (P < 0.05).
ζ- potential of protein solutions
Heat-induced aggregation of WPC brought about significant changes not only in the surface hydrophobicity but also in net charge. Jordens et al. (Reference Jordens, Ruhs, Sieber, Isa, Fischer and Mezzenga2014) reported that the fibrils forming at pH 2 with high charge, aligned into nematic domains due to their adsorption at interfaces, created an elastic interface. Positive surface charge (+19.47 mV) of WPC was increased to +32.43 mV during WPC fibrillation, resulting from protein unfolding and the exposure of charged groups. On the contrary, the maximum increase of the conventional aggregation was up to 45%. Therefore, comparing with the conventional structure of aggregation, the fibril structure was the reason for high electrical conductivity (Fig. 6).
ζ- potential of WPC fibril (prepared by 3%(w/v) WPC heating at 90°C, pH 2.0) and conventional WPC (prepared by 3%(w/v) WPC heating at 90°C, pH 6.5) as a function of time.
In conclusion, compared with conventional WPC film (pH 6.5), WPC fibril film (pH 2.0) achieved a 1.31-fold increase in electrical conductivity, higher tensile strength and elongation at break as well as superior oil resistance. It was demonstrated that the increased electrical conductivity of WPC fibril film (pH 2.0) probably resulted from the well-ordered β-sheets-rich structures, the exposure of hydrophobic amino acid and a higher surface charge.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (Grant No. 31471682) and the 13th National Five-year Topic of China (Grant No. 2017YFD0401200).
