formation from caprine whey protein concentrate: physicochemical and emulsifying properties

formation from caprine whey protein concentrate: physicochemical and emulsifying properties | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 3 October 2025 V1 Latest version Share on formation from caprine whey protein concentrate: physicochemical and emulsifying properties Authors : Carolina Ayunta 0000-0003-3588-1613 [email protected] , Axel Hollmann , María Puppo , and Laura Iturriaga 0000-0002-0942-7751 Authors Info & Affiliations https://doi.org/10.22541/au.175952145.51247685/v1 171 views 100 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract This study aimed to produce fibrils from goat whey protein concentrate (FWPCC), characterize their properties, and compare them with bovine whey protein fibrils (FWPCB). Goat whey protein concentrate (WPC) was obtained from sweet goat whey by ultrafiltration and diafiltration, and subsequently converted into fibrils at pH 2. The physicochemical and emulsifying properties of both native WPC and its fibrils (FWPC) were systematically evaluated. Transmission electron microscopy (TEM) revealed distinct morphological differences: FWPCB formed long, linear fibrils without visible aggregates, whereas FWPCC fibrils were thinner and showed stronger inter-fibrillar interactions, assembling into rope-like or dense network structures. Zeta potential measurements indicated that fibrillation increased the positive charge of proteins, enhancing electrostatic repulsion and preventing visible aggregation. Fibril suspensions exhibited higher turbidity than native WPC, which decreased in the presence of SDS, highlighting the role of hydrophobic interactions in fibril stability. Electrophoretic analysis confirmed protein denaturation and fibril formation after heat treatment, while surface hydrophobicity remained unchanged. No significant differences were observed in the emulsifying activity index (EAI), although FWPCB showed a lower creaming index. Overall, despite morphological differences, goat whey proteins fibrils exhibited physicochemical and functional properties comparable to their bovine control, supporting their potential use as an alternative protein nanostructure. Fibril formation from caprine whey protein concentrate: physicochemical and emulsifying properties Carolina Anabel Ayuntaa,b*, Axel Hollmannb, María Cecilia Puppoc, Laura Beatriz Iturriagaa,b a Facultad de Agronomía y Agroindustrias, Universidad Nacional de Santiago del Estero, Av. Belgrano sur 1912 (CP 4200), Santiago del Estero, Argentina. b CIBAAL (UNSE‑CONICET), Centro de Investigación en Biofísica Aplicada y Alimentos, Univ. Nacional de Santiago del Estero, Ruta Nacional 9 km 1.125 Villa El Zanjón (CP 4206), Santiago del Estero, Argentina. c CIDCA (UNLP‑CIC‑CONICET), Facultad de Ciencias Exactas, Univ. Nacional de La Plata, Calle 47 y 116, La Plata, Buenos Aires, Argentina. Corresponding author Carolina Anabel Ayunta. Centro de Investigación en Biofísica Aplicada y Alimentos, Instituto de Ciencias y Tecnología de Alimentos, Univ. Nacional de Santiago del Estero, RN 9 km 1.125 Villa El Zanjón (4206) Santiago del Estero, Argentina. Tel: 54 385 5171511. E-mail: [email protected] Short Title Caprine whey protein fibrils: physicochemical and emulsifying properties Abstract This study aimed to produce fibrils from goat whey protein concentrate (FWPCC), characterize their properties, and compare them with bovine whey protein fibrils (FWPCB). Goat whey protein concentrate (WPC) was obtained from sweet goat whey by ultrafiltration and diafiltration, and subsequently converted into fibrils at pH 2. The physicochemical and emulsifying properties of both native WPC and its fibrils (FWPC) were systematically evaluated. Transmission electron microscopy (TEM) revealed distinct morphological differences: FWPCB formed long, linear fibrils without visible aggregates, whereas FWPCC fibrils were thinner and showed stronger inter-fibrillar interactions, assembling into rope-like or dense network structures. Zeta potential measurements indicated that fibrillation increased the positive charge of proteins, enhancing electrostatic repulsion and preventing visible aggregation. Fibril suspensions exhibited higher turbidity than native WPC, which decreased in the presence of SDS, highlighting the role of hydrophobic interactions in fibril stability. Electrophoretic analysis confirmed protein denaturation and fibril formation after heat treatment, while surface hydrophobicity remained unchanged. No significant differences were observed in the emulsifying activity index (EAI), although FWPCB showed a lower creaming index. Overall, despite morphological differences, goat whey proteins fibrils exhibited physicochemical and functional properties comparable to their bovine control, supporting their potential use as an alternative protein nanostructure. Keywords Caprine whey proteins concentrate, fibril, physicochemical properties, emulsifying properties. Introduction Whey is a dairy by-product that constitutes a valuable source of high-quality proteins. For this reason, the recovery of these proteins in the form of whey protein concentrates (WPC) or isolates (WPI) has considerable technological and nutritional importance. Both WPC and WPI are widely used as food ingredients or additives due to their excellent functional properties. In recent years, increasing attention has been directed toward the self-assembly of food proteins into fibrillar structures as a means of enhancing their technological functionality (Jansens et al., 2019; Mohammadian & Madadlou, 2018). Numerous proteins of animal and plant origin have been shown to form amyloid-like fibrils under controlled temperature and pH conditions. These include egg proteins such as ovalbumin and lysozyme, milk proteins like whey protein isolate (WPI), and plant-derived proteins from soy and peas (Akkermans et al., 2008; Bolder et al., 2007; Munialo et al., 2014; Veerman et al., 2003). Protein fibrils are elongated, anisotropic aggregates typically measuring 1–10 μm in length and less than 100 nm in diameter, and are therefore often referred to as nanofibers (Josefsson et al., 2019; Wei & Huang, 2019). Their high aspect ratio and rigid, rod-like morphology confer a large excluded volume, which enhances their potential as functional ingredients in food applications (Peng et al., 2016). For whey proteins, fibril formation is commonly achieved by prolonged heating (10–24 h) at temperatures above 75–80 °C under strongly acidic conditions (pH ≤ 2). These conditions promote partial hydrolysis followed by the self-assembly of peptides into fibrillar structures (Arnaudov et al., 2003; Durand et al., 2002; Gosal et al., 2002; Loveday et al., 2012a). Compared with their native counterparts, food-grade protein fibrils are biocompatible, non-toxic, and exhibit improved functionalities such as foaming, emulsifying, gelling, and water-binding capacities, as well as antioxidant activity (Fameau & Salonen, 2014; Mantovani et al., 2018; Mohammadian & Madadlou, 2016; Moayedzadeh et al., 2015). Their production is relatively cost-effective, which further increases their attractiveness for food industry applications (Meng et al., 2022). Specifically, fibrillation of whey proteins converts the native globular conformation into fibrillar structures, thereby increasing viscosity and water retention compared with the untreated protein (Rathod et al., 2021). Among milk proteins, bovine β-lactoglobulin has been extensively used as a model for studying fibril formation. Under acidic and high-temperature conditions, the protein undergoes partial unfolding, exposing β-sheet regions that promote aggregation stabilized by hydrogen bonds. The fibrillation process is strongly influenced by factors such as pH, ionic strength, and incubation time, which determine both the kinetics of assembly and the final morphology of the fibrils (Dave et al., 2013; Kroes-Nijboer et al., 2011). At pH ≤ 3 and temperatures ≥ 75 °C, β-lactoglobulin rapidly assembles into amyloid-like nanofibrils (Dave et al., 2013). Because the protein retains considerable tertiary structure, partial unfolding is necessary to enable fibril formation, allowing β-sheet domains to align and stack through hydrogen bonding. Low ionic strength and acidic conditions provide sufficient electrostatic repulsion between positively charged monomers and β-lg peptides, reducing random aggregation. The resulting fibrils have diameters below 10 nm and lengths that can exceed 10 μm. Their rigidity and morphology are highly dependent on ionic strength: heating at low ionic strength yields long, semi-flexible fibrils, whereas the presence of salts promotes the formation of much shorter, flexible, worm-like fibrils. Acidic conditions at elevated temperatures also induce partial hydrolysis, which facilitates fibril assembly (Mishra et al., 2007). However, excessive hydrolysis may reduce fibril yield by cleaving non-core regions essential for assembly. For example, Akkermans et al. (2008) demonstrated that hydrolysis of β-lg by AspN proteinase at pH 8 and 37 °C, followed by incubation of the hydrolysate at pH 2 and room temperature, produced fibrils several hundred nanometers in length. While fibrillation of bovine whey protein isolates (WPI) has been studied, information on fibrils derived from whey protein concentrate (WPC) and caprine whey proteins remains scarce. Since goat milk proteins differ in composition from those of bovine milk, investigating their fibrillation capacity and resulting functionalities is essential to assess their potential as alternative ingredients in food systems. In this context, the objective of the present study was to evaluate the production, physicochemical characteristics, and emulsifying properties of caprine whey protein fibrils (FWPCC) obtained from caprine whey protein concentrate (WPCC), and to compare them with bovine whey protein fibrils (FWPCB). The fibrils were characterized using transmission electron microscopy, zeta potential, surface hydrophobicity, turbidity analysis, and SDS-PAGE electrophoresis, and their emulsifying properties were assessed in comparison with a commercial bovine whey protein concentrate (WPCB). Materials and methods 2.1. Materials Sweet caprine whey (pH > 5.6) was supplied by a local goat cheese-making factory (La Carola, Santiago del Estero, Argentina). The bovine whey protein concentrate (WPCB) (Arla Foods, Videbaek, Denmark) was utilized as control sample (composition chemical on a dry basis was: proteins 63%, lipids 9%, lactose 22.8%, and ash 3.9%). Sunflower oil (Cocinero® Molinos Rio de la Plata S.A., Argentina) was purchased from a local supermarket and used without further purification. The chemical reactants used were of analytical grade. 2.2. Preparation of caprine whey protein concentrates WPCC was prepared using a laboratory-scale ultrafiltration (UF) process (Fig. 1 A). The whey was centrifuged at 1300×g for 10 min at 4 °C mainly to reduce the lipid content. After centrifugation, the skimmed whey was ultrafiltered using a 10 kDa MW cut-off membrane (Vivaflow 200, Sartorius, Göttingen, Germany). The UF process was defined with a volumetric concentration factor (VCF) equal to 7 and the concentrate was diafiltrated in five discontinuous stages. The concentrate was freeze-dried using a Lyph-LockTM (Labconco Corporation, Kansas City, USA) freeze-dryer. The composition of WPCC on a dry basis was as follows: proteins 64.60%, lipids 14.79%, lactose 18.83%, and ash 1.77% (Ayunta et al., 2019). 2.3. Fibril formation Protein dispersions (1% w/v) were prepared by dispersing WPC power in distilled water, using magnetic stirring at room temperature for 1 h and stored at 4ºC for 24 h to complete their hydration. Then the pH of the solutions was adjusted to pH 2 with 6 N HCl, centrifuged at 15000 xg for 20 minutes at 4°C, for removing non-soluble components (Mantovani et al., 2018; Tang et al., 2018). The WPC dispersions were then subjected to heat treatment in a water bath for 20 h at 90°C, followed by cooled to room temperature (20ºC) using an ice bath. All WPC fibril dispersions (FWPC) were kept refrigerated at 4°C until the time of determinations (Fig. 1 B). 2.4. Characterization of FWPC 2.4.1. Transmission electron microscopy (TEM) TEM micrographs were obtained by placing a droplet of the FWPC dispersions onto a carbon support film on a copper grid. The excess of water was removed using a filter paper. Subsequently, the negative staining technique was performed with 2% (w/v) uranyl acetate (Mantovani et al., 2018). Electron micrographs were taken using Zeiss EM 109T Transmission Electron Microscope (Carl Zeiss AG, Oberkochen, Germany) equipped with Gatan ES1000W digital camera (Gatan, Inc., Pleasanton, CA, USA). 2.4.2. Surface hydrophobicity Surface hydrophobicity (So) was determined using the fluorescence spectroscopy methods described by Liu et al. (2014) and Feng et al. (2019), employing 8-anilino-1-naphthalenesulfonic acid (ANS) as a probe. A 50 μL aliquot of WPC or FWPC suspension (1% w/v) was mixed with 600 μL of 10 mM phosphate buffer (pH 7.4) containing ANS at a final concentration of 100 μM. The mixture was incubated in the dark for 3 minutes. Fluorescence spectra were recorded using a Cary Eclipse fluorescence spectrometer (Agilent Technologies Inc., Santa Clara, CA, USA). The excitation wavelength was set at 370 nm, and emission spectra were collected from 400 to 600 nm. The excitation and emission slit widths were both set at 5 nm. All measurements were performed in triplicate. 2.4.3. ζ-potential The measurements of ζ-potential of WPC and FWPC dispersions were determined using Horiba SZ-100 nanoparticle analyzer (Horiba Ltd., Kyoto, Japan). The samples were diluted by taking one millilitre of sample (1% w/v) and adding ultrapure water until reaching a final volume of 15 ml as suggested Ayunta et al. (2019).The results of each measurement are the average of the zeta potential of 30 measurements. The complete experiment was carried out in triplicate for each sample. 2.4.4. Turbidity measurements Turbidity measurements were performed according to the method proposed by Groleau et al. (2003), WPC and FWPC dispersions at pH 2 were analyzed at 25°C, in the absence and the presence of SDS (5% w/v) (Motovani et al., 2018). The mixture was allowed to react for 90 minutes at room temperature and then the absorbance was measured at 500 nm in a Jasco V630 UV–visible spectrophotometer (Jasco International Co., Tokyo, Japan). The determinations were performed in triplicate. 2.4.5. Electrophoresis SDS-PAGE WPC dispersions and FWPC dispersions were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in absence of β-mercaptoethanol as reducing agent according to the method described by Laemmli (1970). Continuous and stacking gels of 15 and 4% of acrylamide, respectively, were prepared. An aliquot of each sample containing 25 μg of protein was taken and diluted in 10 μl of phosphate buffer at pH 7 and 10 μl of 0.5 M Tris-HCl buffer (pH 6.8) containing SDS (0.4% w/v), glycerol and bromophenol blue (0.012% w/v). Then, aliquots (14 μl) were loaded in each lane. The gels were run in a Tris-Glycine running buffer (pH 8.3) containing 0.1% SDS at 15 mA for 1.5 h approximately. Proteins were stained by Coomassie Brilliant Blue R250 (0.2%) in a solvent (methyl alcohol: distilled water: acetic acid, 42:42:16% v/v) and discoloured with a solvent mixture (ethanol: acetic acid: water, 25:10:65 v/v/v). The molecular weight of samples was determined using the protein standard PageRuler Plus Prestained Protein Ladder™ (Thermo Fisher Scientific Inc., Vilnius, Lithuania) containing molecular weight markers from 10 to 250 kDa. 2.5. Emulsifying properties 2.5.1. Emulsion preparation Oil-in-water emulsions were prepared using sunflower oil and protein solutions (WPC and FWPC) at a volume fraction (Ф) of 0.7. Emulsification was carried out at 20.000 rpm for 5 min using an Ultra-Turrax T25 homogenizer (IKA Labortechnik, Staufen, Germany) at 25 °C. 2.5.2. Emulsion activity index (EAI) The EAI were determined by the turbidimetric technique described by Pearce and Kinsella (1978). The freshly prepared emulsion (0.1 g) was mixed with 19.9 ml of 0.1% (w/v) sodium dodecyl sulfate (SDS) solution. The absorbance of the diluted emulsions was measured at λ = 500 nm using a Jasco V630 UV-visible spectrophotometer (Jasco International Co., Tokyo, Japan). The EAI (m2/g) of the emulsions prepared with WPC and FWPC dispersions was calculated using the following equation: \[EAI\ (m^{2}/g)=\frac{\left(4,406\ x\ A\ x\ D\right)}{\left(L\ x\ Ф\ x\ C\right)}\] Where A is the absorbance at 500 nm, D is the dilution factor, L is the path length of the cuvette (1 cm), C is the weight of protein per unit volume of aqueous phase before the emulsion was formed (g/m3), and Ф is the volume fraction of dispersed phase (v/v). 2.5.3. Creaming index The stability of the emulsions against creaming was determined immediately after preparation by placing 10 ml of emulsion in a graduated test tube (internal diameter 8 mm, height 130 mm) with lids to avoid evaporation; sodium azide (0.1% w/v) was added to prevent microbial growth. The samples were stored in an oven at 25 °C for 14 days. The emulsions separated into an upper layer of cream and a lower layer of serum (Hs). The height of the cream phase (Hc) and the total height of the emulsion (Ht) were measured until 14 days. The cremation index (CI) was calculated according to Keowmaneechai & McClements (2002) with the following equation: \[\text{IC}\left(\%\right)=\frac{\text{Hc}}{\text{Ht}}x100\] 2.6. Statistical Analysis Data were evaluated using the one-way analysis of variance (ANOVA). Tukey’s test of multiple comparisons was employed to compare mean values when the significant variance was found by ANOVA. The significance degree was established at a level of p<0.05. Results were statistically analysed using the InfoStat-version 2008 software (Grupo InfoStat, FCA-UNCo, Argentina). Results and Discussion 3.1. Transmission Electron Microscopy (TEM) Transmission electron micrographs (TEM) of FWPCs are shown in Fig. 2. The fibrils formed differed in both morphology and thickness depending on whether the protein was of bovine or caprine origin. In the micrographs of FWPCB (Fig. 2A), long linear fibrils were clearly visible, with no evidence of aggregate formation. Similar observations have been previously reported for whey protein fibrils (Loveday et al., 2012a; Mantovani et al., 2018) and for β-lactoglobulin (Loveday et al., 2012b). In contrast, fibrils obtained from caprine whey protein concentrate (FWPCC) (Fig. 2B) also appeared long but exhibited smaller diameters and greater interactions with neighboring fibrils, assembling into rope-like or network-like structures that were denser than those observed in Fig. 2A. 3.2. Zeta Potential Figure 3 shows the zeta potential (ZP) of WPC and FWPC suspensions at pH 2. Both systems exhibited a markedly positive surface charge (> +20 mV), attributed to the protonation of amino groups under acidic conditions, as the pH is well below the isoelectric point of serum proteins (4.7 for WPCC and 4.25 for WPCB; Ayunta et al., (2019). Following heat treatment, a significant increase in ZP was observed, reaching 44.35 mV in FWPCC and 40.85 mV in FWPCB, which can be explained by protein unfolding and the consequent exposure of charged groups (Mantovani et al., 2018). Fan et al. (2021) and Mantovani et al. (2018) reported similar results for whey protein isolate nanofibrils. During fibril elongation, the assembly of monomers into β-sheet structures may further expose or bury charged residues, thereby modifying the surface charge density (Koo et al., 2018; Zhao et al., 2021). At pH 2, the fibril suspensions produced more stable dispersions, since particles with zeta potentials greater than +20 mV or lower than −20 mV are generally considered stable in colloidal systems (Feng et al., 2019). These high ZP values support the absence of aggregates in the TEM micrographs (Fig. 2), as they promote electrostatic repulsion among fibrils. 3.3. Surface hydrophobicity Figure 4 shows the ANS fluorescence intensity of WPC suspensions (before heating) and FWPC suspensions (after heating). Similar values were observed across all samples, regardless of protein source or heat treatment, which could be explained by the acidic conditions (pH 2). At this pH, globular proteins are partially unfolded, exposing hydrophobic groups through electrostatic repulsion (Moreno et al., 2005). Accordingly, Ayunta et al. (2019) reported higher S o values at pH 2, with greater values for WPCB compared to WPCC. However, Mantovani et al. (2018) and Fan et al. (2021) observed that heating WPI dispersions increased fluorescence intensity, suggesting that fibrillar systems are more hydrophobic than the native proteins. Thus, an increase in So would generally be expected following heat treatment. However, under prolonged heating (90 °C, 20 h) at acidic pH, the formation of fibrillar aggregates (Fig. 2) would be detected, where many hydrophobic sites become buried within aggregates, reducing their accessibility to ANS. This behavior agree with Lee et al. (2018); these authors demonstrated that soluble α-synuclein oligomers exhibited more exposed hydrophobic surfaces than their fibrillar forms, implying that compact aggregates reduce the accessibility of hydrophobic patches. Furthermore, the high turbidity of FWPC samples (Fig. 5) likely reduced the apparent fluorescence intensity due to light scattering. 3.4. Turbidity measurements Figure 5 shows the turbidity of the WPC suspensions (control) and after the fibrillation process (FWPC) bovine and caprine, both with and without SDS. As anticipated, WPC suspensions exhibited lower turbidity than FWPC, given that turbidity at 500 nm is an indirect indicator of aggregates or large supramolecular structures, such as fibrils. The addition of SDS resulted in a decreased turbidity across all systems. When present above its critical micelle concentration (CMC), SDS is known to disrupt protein aggregates by weakening hydrophobic interactions and introducing additional negative charges to the protein molecules (Mantovani et al., 2018). For WPC, this decrease may be attributed to disaggregation into smaller, soluble structures. In the FWPC suspensions, the decrease in turbidity indicates that the fibrillar aggregates were dissolved by SDS. This occurs because SDS binds to hydrophobic regions, disrupting the non-covalent interactions that maintain the fibril structure, thereby promoting solubilization and reducing large light-scattering structures. This observation strongly suggests that hydrophobic interactions play a significant role in the formation of fibrillar aggregates. Furthermore, the turbidity of fibril dispersions mixed with SDS can be used to estimate the contribution of hydrophobic interactions to fibril aggregation (Groleau et al., 2003). 3.5. Electrophoresis (SDS-PAGE) The electrophoretic profiles of the WPC and FWPC suspensions are shown in Fig. 6. The profiles observed in lanes 1 and 3 (WPCB and WPCC) were similar, displaying prominent bands at approximately 14 kDa and 18 kDa, corresponding to α-lactalbumin and β-lactoglobulin, respectively, the major whey proteins. A band of approximately 66 kDa corresponding to serum albumin, as well as a band above 250 kDa more intense in WPCC were also detected, the latter likely representing a soluble aggregate. In addition, a faint band of approximately 75 kDa was observed in the WPCC suspension (lane 3), corresponding to Lactoferrin (Lf). These results are consistent with those reported by Ayunta et al. (2019). As for the fibril suspensions (lane 2 and 4) after heat treatment the individual protein bands disappeared and exhibited diffuse bands with lower molecular weight (approximately 18 kDa –10 kDa), indicating degradation into small peptides during heating under acidic conditions (Norouzi et al., 2025). Similarly, Rathod and Amamcharla (2020) reported consistent findings, attributing the diffuse band to protein denaturation and the subsequent transformation of individual proteins into fibrils . 3.6. Emulsion Properties Figure 7 presents the EAI and CI of WPC and FWPC suspensions. The EAI reflects the degree to which protein molecules occupy the surface of oil droplets in a dilute emulsion system. As shown in Figure 7A, no significant differences were detected in EAI values, either between samples or between protein types (fibrillated vs. non-fibrillated), indicating that emulsion formation is not influenced by the protein type or its conformation. Fang et al. (2021) reported comparable findings, with only a slight increase in EAI for emulsions stabilized with WPI nanofibers compared to those with WPI of bovine origin. Although protein fibrils are generally expected to enhance EAI due to their unfolded conformation and exposure of hydrophobic groups, which can facilitate interactions with the oil phase and stabilize emulsions, all systems exhibited strongly positive ZP values. Furthermore, no significant differences in surface hydrophobicity were observed between WPC and FWPC samples, regardless of their bovine or caprine origin. On the other hand, Feng et al. (2019) observed an increase in the emulsifying activity index of emulsions prepared with FWPI compared to those with WPI due to emulsions were ultrasonicated, process that may have contributed to the formation of smaller droplets, favoring the emulsion formation. Regarding the CI (%) during storage (Fig. 7B), the emulsions stabilized with WPCB, WPCC, and FWPCC showed similar values (28-29%). In contrast, the emulsions made with FWPCB presented a considerably lower CI (≈10.5%), reflecting greater physical stability. This behavior could be explained by the presence of WPCB fibrils in isolated or individual form, which facilitates their unfolding, diffusion, and reorientation at the water-oil interfaces, thus favoring emulsion stabilization. Mantovani et al. (2018) reported that the CI values were similar and above 70% in emulsions containing WPI and FWPI, in emulsions with an oil content of 20% (w/v). The results obtained in this study confirm the formation of fibrils from caprine and bovine whey protein concentrates by heat treatment under acidic conditions. Transmission electron microscopy observations revealed long, well-defined fibrils in both systems, although the goat whey-derived fibrils displayed smaller diameters and a tendency to organize into denser networks. These findings were complemented by zeta potential values, which showed high positive charges (>20 mV), which increased after the fibrillation process, favoring electrostatic repulsion and preventing the formation of visible aggregates. Turbidity measurements showed that the fibril suspensions exhibited greater light scattering compared to the controls, and that the addition of SDS significantly reduced turbidity, suggesting that hydrophobic interactions play a key role in stabilizing fibril aggregates. Furthermore, electrophoretic profiles confirmed protein denaturation and the conversion of individual proteins into fibrillar structures following heat treatment. Regarding surface hydrophobicity, no differences were observed between the samples. 0.1 Finally, emulsification assays revealed that fibrils obtained from bovine whey protein concentrate significantly improved emulsion stability, as evidenced by a lower creaming index. 0.2 These results highlight the potential of caprine whey protein fibrils, as an alternative to bovine whey proteins as emulsion stabilizing agents, opening up prospects for their application in the development of functional foods and more stable colloidal systems. 0.3 Author Contributions Carolina Anabel Ayunta: conceptualization, investigation, writing – original draft preparation, funding acquisition. Axel Hollmann: investigation, conceptualization. Maria Cecilia Puppo: writing – review and editing, supervision. Laura Beatriz Iturriaga: writing – review and editing, supervision and funding acquisition. All authors have read and approved the final draft of the manuscript. 0.4 Ethics Statement 0.1 Finally, emulsification assays revealed that fibrils obtained from bovine whey protein concentrate significantly improved emulsion stability, as evidenced by a lower creaming index. The authors have nothing to report. Conflicts of Interest The authors declare no conflicts of interest. Acknowledgements The authors acknowledge Universidad Nacional de Santiago del Estero ( 23/A303-A-2024, 23/A310- Bint-2024) . Consejo Nacional de Investigaciones Científicas y Técnicas ( PIO-CONICET 14520140100005CO ), and Centro de Investigación en Biofísica Aplicada y Alimentos (CIBAAL) for the financial support. References Akkermans C, Van der Goot AJ, Venema P, Van der Linden E, Boom RM. Properties of protein fibrils in whey protein isolate solutions: microstructure, flow behaviour and gelation. Int Dairy J. 2008;18(10–11):1034–42. doi:10.1016/j.idairyj.2008.05.006. Arnaudov LN, De Vries R, Ippel H, Van Mierlo CPM. Multiple steps during the formation of β-lactoglobulin fibrils. Biomacromolecules. 2003;4(6):1614–22. doi: 10.1021/bm034096b. Ayunta CA, Quinzio CM, Puppo MC, Iturriaga LB. Physicochemical properties of caprine and commercial bovine whey protein concentrate. J Food Meas Charact. 2019;13:1783–93. doi:10.1007/s11694-019-00194-5. Bolder SG, Sagis LMC, Venema P, Van der Linden E. Effect of stirring and seeding on whey protein fibril formation. J Agric Food Chem. 2007;55(14):5661–9. doi:10.1021/jf063351r. Dave AC, Loveday SM, Anema SG, Loo TS, Norris GE, Jameson GB, et al. β-Lactoglobulin self-assembly: structural changes in early stages and disulfide bonding in fibrils. J Agric Food Chem. 2013;61(33):7817–28. doi: 10.1021/jf401084f. Durand D, Gimel JC, Nicolai T. Aggregation, gelation and phase separation of heat denatured globular proteins. Physica A Statistical Mechanics and its Applications 2002;304(1–2):253–65. doi:10.1016/S0378-4371(01)00514-3. Fameau AL, Salonen A. Effect of particles and aggregated structures on the foam stability and aging. C R Phys. 2014;15(8–9):748–60. doi:10.1016/j.crhy.2014.09.009. Fan Y, Peng G, Pang X, Wen Z, Yi J. Physicochemical, emulsifying, and interfacial properties of different whey protein aggregates obtained by thermal treatment. LWT. 2021;149:111904. doi:10.1016/j.lwt.2021.111904. Feng Z, Li L, Zhang Y, Li X, Liu C, Jiang B, et al. Formation of whey protein isolate nanofibrils by endoproteinase GluC and their emulsifying properties. Food Hydrocolloids. 2019;94:71–9. doi:10.1016/j.foodhyd.2019.03.004. Gosal WS, Clark AH, Pudney PDA, Ross-Murphy SB. Novel amyloid fibrillar networks derived from a globular protein: β-lactoglobulin. Langmuir. 2002;18(19):7174–81. doi: 10.1021/la025531a. Groleau PE, Morin P, Gauthier SF, Pouliot Y. Effect of physicochemical conditions on peptide–peptide interactions in a tryptic hydrolysate of β-lactoglobulin and identification of aggregating peptides. J Agric Food Chem. 2003;51(15):4370–5. doi: 10.1021/jf0259720. Jansens KJA, Rombouts I, Grootaert C, Brijs K, Van Camp J, Van der Meeren P, et al. Rational design of amyloid-like fibrillary structures for tailoring food protein techno-functionality and their potential health implications. Compr Rev Food Sci Food Saf. 2019;18(1):84–105. doi: 10.1111/1541-4337.12404. Josefsson L, Cronhamn M, Ekman M, Widehammar H, Emmer Å, Lendel C. Structural basis for the formation of soy protein nanofibrils. RSC Adv. 2019;9(11):6310–9. doi:10.1039/c8ra10610j. Keowmaneechai E, McClements DJ. Influence of EDTA and citrate on physicochemical properties of whey protein-stabilized oil-in-water emulsions containing CaCl2. J Agric Food Chem. 2002;50(23):7145–53. doi: 10.1021/jf020489a. Klemaszewski JL, Kinsella JE. Sulfitolysis of whey proteins: effects on emulsion properties. J Agric Food Chem. 1991;39(6):1033–6. doi: 10.1021/jf00006a005. Koo CK, Chung C, Picard R, Ogren T, Mutilangi W, McClements DJ. Modulation of physical properties of microfluidized whey protein fibrils with chitosan. Food Res Int. 2018;113:149–55. doi:10.1016/j.foodres.2018.07.012. Kroes-Nijboer A, Venema P, Bouman J, Van der Linden E. Influence of protein hydrolysis on the growth kinetics of β-lg fibrils. Langmuir. 2011; 27(10):5753–61. doi:10.1021/la104797u. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227(5259):680–5. doi:10.1038/227680a0. Lee JE et al. Mapping Surface Hydrophobicity of α-Synuclein Oligomers at the Nanoscale. Nano Letters . 2018; 18 (12), 7494-750. doi: 10.1021/acs.nanolett.8b02916. Liu C, Liu W, Feng Z, Li D. Aggregation of Whey Protein Hydrolysate Using Alcalase 2.4 L. PLoS ONE. 2014; 9(10): e109439. doi: 10.1371/journal.pone.0109439. Loveday SM, Su J, Rao MA, Anema SG, Singh H. Whey protein nanofibrils: the environment–morphology–functionality relationship in lyophilization, rehydration, and seeding. J Agric Food Chem. 2012a;60(21):5229–36. doi:10.1021/jf300367k. Loveday SM, Wang XL, Rao MA, Anema SG, Singh H. β-Lactoglobulin nanofibrils: effect of temperature on fibril formation kinetics, fibril morphology and the rheological properties of fibril dispersions. Food Hydrocolloids. 2012b;27(1):242–9. doi:10.1016/j.foodhyd.2011.07.001. Mantovani RA, Furtado GF, Netto FM, Lopes Cunha R. Assessing the potential of whey protein fibril as emulsifier. J Food Eng. 2018; 223:99–108. doi:10.1016/j.jfoodeng.2017.12.006. Meng Y, Wei Z, Xue C. Protein fibrils from different food sources: a review of fibrillation conditions, properties, applications and research trends. Trends Food Sci Technol. 2022;121:59–75. doi:10.1016/j.tifs.2022.01.031. Moayedzadeh S, Madadlou A. Formation mechanisms, handling and digestibility of food protein nanofibrils. Trends Food Sci Technol. 2015;45(1):50–9. doi:10.1016/j.tifs.2015.05.006. Mohammadian M, Madadlou A. Cold-set hydrogels made of whey protein nanofibrils with different divalent cations. Int J Biol Macromol. 2016;89:499–506. doi:10.1016/j.ijbiomac.2016.05.014. Mohammadian M, Madadlou A. Technological functionality and biological properties of food protein nanofibrils formed by heating at acidic condition. Trends Food Sci Technol. 2018;75:115–28. doi:10.1016/j.tifs.2018.03.013. Moreno FJ, Mackie AR, Mills ENC. Phospholipid Interactions Protect the Milk Allergen α-Lactalbumin from Proteolysis during in Vitro Digestion. Journal of Agricultural and Food Chemistry. 2005; 53 (25), 9810-9816.doi: 10.1021/jf0515227. Munialo CD, Martin AH, Van der Linden E, De Jongh HHJ. Fibril formation from pea protein and subsequent gel formation. J Agric Food Chem. 2014;62(11):2418–27. doi: 10.1021/jf4055215. Norouzi M, Hesarinejad MA, Kadkhodaee R, Nishinari K, Gao Z. Catalytic nucleation effect of the insoluble fraction of Persian gum on amyloid fibrillation of whey protein isolate. Food Hydrocolloids. 2025;163:111148. doi:10.1016/j.foodhyd.2025.111148. Pearce KN, Kinsella JE. Emulsifying properties of food proteins: evaluation of a turbidimetric technique. J Agric Food Chem. 1978;26(3):716–23. doi:10.1021/jf60217a041. Peng J, Simon JR, Venema P, Van der Linden E. Protein fibrils induce emulsion stabilization. Langmuir. 2016;32(9):2164–74. doi: 10.1021/acs.langmuir.5b04341. Rathod R, Amamcharla J. Process development for a novel milk protein concentrate with whey proteins as fibrils. J Dairy Sci. 2021;104(4):4094–107. doi: 10.3168/jds.2020-19409. Veerman C, De Schiffart G, Sagis LMC, Van der Linden E. Irreversible self-assembly of ovalbumin into fibrils and the resulting network rheology. Int J Biol Macromol. 2003;33(1–3):121–7. doi: 10.1016/s0141-8130(03)00076-x. Wei Z, Huang Q. Assembly of iron-bound ovotransferrin amyloid fibrils. Food Hydrocolloids. 2019;89:579–89. doi:10.1016/j.foodhyd.2018.11.028. Zhao Y, Wang C, Lu W, Sun C, Zhu X, Fang Y. Evolution of physicochemical and antioxidant properties of whey protein isolate during fibrillization process. Food Chem. 2021;357:129751. doi:10.1016/j.foodchem.2021.129751. Figure Legends Fig. 1 A. Process diagram for obtaining caprine whey protein concentrate (WPCC) B. Process diagram for obtaining fibrils whey protein concentrate (FWPC) Fig. 2 Transmission Electron Microscopy (TEM) micrographs of carpine and bovine WPC fibrils A and B. Scale bar represents 100 nm. C and D. Scale bar represents 200 nm. Fig. 3 Zeta potential of WPC and WPC fibril (FWPC) suspensions of bovine and caprine at pH 2 Fig. 4 ANS fluorescence intensity at 480 nm of WPC suspension and FWPC suspension (after fibrillation process ). ( A ) WPCB and FWPCB; ( B ) WPCC and FWPCC Fig. 5 Turbidity (A500nm) measured in WPC dispersions (WPCB, WPCC) and WPC fibril dispersions (FWPCB, FWPCC), with (5%) and without (0%) SDS Fig. 6 SDS-PAGE profiles of emulsions of bovine and caprine WPC. (M) Commercial molecular weight marker, (1) WPCB (2) FWPCB (3) WPCC (4) FWPCC Fig. 7 (A) Emulsion activity index (EAI), (B) Creaming index (CI) during storage at 25 °C for 14 days of emulsion formulated with WPC or FWPC bovine and caprine . Same letters indicate not statistical differences (p<0,05) Supplementary Material File (figures fibrils of wpc.pdf) Download 411.55 KB Information & Authors Information Version history V1 Version 1 03 October 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords dairy proteins functional properties Authors Affiliations Carolina Ayunta 0000-0003-3588-1613 [email protected] Centro de Investigacion en Biofisica Aplicada y Alimentos View all articles by this author Axel Hollmann Centro de Investigacion en Biofisica Aplicada y Alimentos View all articles by this author María Puppo Centro de Investigacion y Desarrollo en Criotecnologia de Alimentos View all articles by this author Laura Iturriaga 0000-0002-0942-7751 Centro de Investigacion en Biofisica Aplicada y Alimentos View all articles by this author Metrics & Citations Metrics Article Usage 171 views 100 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Carolina Ayunta, Axel Hollmann, María Puppo, et al. formation from caprine whey protein concentrate: physicochemical and emulsifying properties. Authorea . 03 October 2025. 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