Investigating surface properties of a blend of phycocyanin and chia mucilage for its possible applications in dispersed systems

Investigating surface properties of a blend of phycocyanin and chia mucilage for its possible applications in dispersed systems María Vela-Albarr´ an a , Luis A. Trujillo-Cayado a , Francisco Carrillo a , Jenifer Santos b , Nuria Calero a,* a Departamento de Ingeniería Química, Escuela Polit´ ecnica Superior, Universidad de Sevilla, c/Virgen de ´ Africa, 7, 41011, Sevilla, Spain b Departamento de Ciencias de la Salud y Biom´ edicas, Facultad de Ciencias de la Salud, Universidad Loyola Andalucía, Avda. de las Universidades s/n, Dos Hermanas, 41704, Sevilla, Spain ARTICLE INFO Keywords: Foam Surface properties Surface rheology Phycocyanin Chia mucilage ABSTRACT The study of the effect of protein-polysaccharide interactions on the properties of air-water and oil-water interfaces has special relevance in the development of processed dispersions, such as foams or emulsions. Specifically, the analysis of the interfacial behaviour of mixtures that exhibit associative interactions. A surface characterization of a system consisted of a 1:1 mixture of phycocyanin and chia mucilage was studied by comparison with both unitary aqueous systems. Surface activity was assessed through surface tension measurements, revealing that the mixture achieved a surface tension of approximately 52 mN/m at 0.1 wt%, like pure phycocyanin, despite its lower protein content. Interfacial rheology showed a significant enhancement in viscoelastic properties for the mixture, with elastic and viscous moduli (G ′ and G ″ ) approximately two orders of magnitude higher than those of phycocyanin alone, indicating a reinforced gel-like behaviour. In contrast, chia mucilage alone formed a fluid-like film with G ″ >G ′ . Zeta potential measurements confirmed that the mixture retained a high negative surface charge (like phycocyanin), ensuring colloidal stability. The foaming capacity increased with concentration for phycocyanin, which produced the greatest volume expansion. In the 1:1 phycocyanin–chia mucilage blend, overrun was intermediate but protein-efficient up to a concentration of 0.25 wt%, beyond which the increasing bulk viscosity curtailed further aeration. Chia mucilage alone consistently displayed the lowest foaming capacity. These findings demonstrate a cooperative interaction between phycocyanin and chia mucilage, resulting in improved surface structuring and mechanical strength, making the blend a promising candidate for sustainable, bio-based stabilizers in dispersed systems. 1. Introduction The formulation of dispersed systems, such as emulsions, requires the presence of two fundamental components to extend the lifespan of thermodynamically unstable systems: emulsifiers and stabilizers [1]. Emulsifiers, amphiphilic substances like surfactants or proteins with surface activity, are essential for reducing interfacial tension and stabilizing the mixture of immiscible phases, such as oil and water. On the other hand, stabilizers, commonly polysaccharides, can form three-dimensional networks that increase the viscosity of the continuous phase, contributing to the physical stability of the emulsion [2]. Surface characterization of food emulsifiers has emerged as a critical research area in the food industry, which directly impact the texture, appearance, mouthfeel, and overall stability of emulsified foods [3,4]. This knowledge is essential to optimize the performance of emulsifiers in food formulations and ensure product stability during processing, storage, and consumption. Additionally, surface characterization techniques allow for valuable insights into the molecular structure, composition, morphology, and interfacial behavior of emulsifiers, which aids in designing dispersed systems, such as emulsions with desired properties and functionality. At the same time, the push towards more sustainable food production has led to an increased use of natural and renewable biological resources. The growing global demand for healthier, environmentally friendly food products has highlighted the importance of these resources in meeting consumer needs while preserving ecosystems and biodiversity for future generations [5]. Biological materials derived from plants, animals, and microorganisms offer significant advantages as sustainable * Corresponding author. E-mail address: [email protected] (N. Calero). Contents lists available at ScienceDirect Journal of Agriculture and Food Research journal homepage: www.sciencedirect.com/journal/journal-of-agriculture-and-food-research https://doi.org/10.1016/j.jafr.2025.102260 Received 26 April 2025; Received in revised form 31 July 2025; Accepted 10 August 2025 Journal of Agriculture and Food Research 23 (2025) 102260 Available online 11 August 2025 2666-1543/© 2025 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/bync-nd/4.0/ ). alternatives to synthetic ingredients. In particular, natural resources, such as those derived from algae and plants, provide essential functional properties for food production, including thickening, gelling, stabilizing, and emulsifying. These properties are key to developing foods with desirable textures, stability, and sensory qualities [6,7]. However, the complexity of these natural substances, often mixtures of compounds such as proteins and polysaccharides, makes studying their properties more challenging. This is the case for phycocyanin and chia mucilage, natural substances analysed in this study, which represent both a challenge and an opportunity in developing sustainable disperse systems with excellent functional properties. Phycocyanin, a natural blue pigment found in certain species of cyanobacteria (commonly referred to as blue-green algae), has gained significant attention in the food industry for its multifunctional properties, particularly as an emulsifier [8]. This compound not only imparts a visually appealing colour to food products but also exhibits emulsifying capacity due to its high protein content (40–60 wt%) and substantial polysaccharide fraction (40–60 wt%) [9]. These components contribute to its amphiphilic nature, enabling it to reduce interfacial tension and stabilize emulsions. However, the coexistence of proteins and polysaccharides also introduces complexity to its surface behavior. Protein–polysaccharide interactions can enhance surface stability by promoting the formation of stronger interfacial films [10,11], although in some cases they may lead to destabilization depending on the nature of the interactions [12]. As a natural alternative to synthetic emulsifiers such as polysorbates (E432–E436), sorbitan esters (E491–E495) or sucrose esters (E473), phycocyanin supports the formation and stabilization of dispersed systems, improving texture, mouthfeel, and product quality while aligning with clean-label trends [13]. Its biocompatibility and safety profile make it suitable for a wide range of applications, including dairy products, beverages, dressings, sauces, and baked goods. In addition to its functional role, phycocyanin contributes to the nutritional profile of food products through its antioxidant and anti-inflammatory properties [14,15], as well as its content of vitamins and essential amino acids [16,17], which support overall health and well-being. Its antioxidant activity may also help reduce oxidative stress and inflammation, potentially lowering the risk of chronic diseases [18]. The emulsifying potential of phycocyanin has been demonstrated in various systems. For instance, Zhong et al. [19] showed that phycocyanin–chitosan complexes reduce interfacial tension, improving emulsion stability, digestibility, and resistance to environmental stress. Similarly, Tello et al. demonstrated that phycocyanin effectively stabilizes emulsions by protecting the interface and preventing coalescence, while its combination with diutan gum has been shown to reduce creaming and enhance stability [9]. These findings highlight the relevance of phycocyanin–polysaccharide complexes in improving emulsifying performance in food systems. Chia seeds, derived from Salvia hispanica L. and native to southern Mexico and Guatemala, are highly valued for their rich nutritional profile, which includes proteins, fatty acids, carbohydrates, dietary fibre, minerals, and antioxidant compounds [20], earning them the designation of a “superfood” due to their numerous health benefits [21]. When hydrated, chia seeds release a gel-like substance known as mucilage, which is primarily composed of polysaccharides and exhibits excellent thickening properties [22]. This mucilage has been traditionally used as a natural stabilizer in various food products such as soups, sauces, baked goods, and beverages, often replacing conventional thickeners like corn-starch or gelatine. In addition to its polysaccharide content, chia mucilage contains a moderate amount of protein (~11.5 wt%), which imparts surface-active properties and contributes to its emulsifying capacity. This makes it particularly useful in stabilizing formulations like dressings, dips, and spreads, where maintaining consistency and preventing phase separation is essential. Furthermore, chia mucilage is rich in soluble fibre, which supports digestive health, helps regulate cholesterol levels, and acts as a prebiotic [22], making it attractive for health-conscious consumers. Chia mucilage was selected over other commonly used polysaccharides, such as chitosan, gum arabic, xanthan gum, and guar gum, due to its distinctive combination of functional and nutritional attributes. It exhibits both emulsifying and thickening capabilities, is plantderived, and is readily soluble at neutral pH, making it compatible with a wide range of formulation conditions. Moreover, it contributes dietary fiber and a moderate amount of protein, which are not typically present in significant quantities in other hydrocolloids. In contrast, chitosan is of animal origin and requires acidic conditions for solubilization, limiting its applicability in neutral or mildly alkaline systems. Gum arabic, xanthan gum, and guar gum are widely used as stabilizers or thickeners, but their nutritional contributions are largely restricted to soluble fiber with limited physiological benefits. Chia mucilage, therefore, offers a multifunctional alternative that aligns with clean-label and vegan product development, while also enhancing the nutritional profile of the final formulation. In recent years, numerous studies have analysed the role of chia mucilage as a stabilizer and emulsifier, as well as its application in dispersed systems with potential use in food products [23–27]. However, despite these investigations, the mechanism by which chia mucilage functions as a stabilizing and emulsifying ingredient remains not well defined, and much less when combined with other components. Given their unique compositions, both phycocyanin and chia mucilage offer promising opportunities for the development of new emulsifiers and stabilizers for complex dispersed systems. These natural hydrocolloids fulfil the essential function of stabilizing food emulsions while also providing a host of health benefits, making them highly valuable for health-conscious consumers. However, the combination of phycocyanin and chia mucilage has not yet been explored. The complexity of their molecular compositions, combining proteins, polysaccharides, and other bioactive compounds, presents unique challenges for surface characterization of these systems. This study provides novel insights into the surface properties of phycocyanin and chia mucilage, exploring their behaviour when combined. Unlike previous research that has primarily examined isolated proteins or polysaccharides, this work highlights the cooperative interactions between these natural emulsifiers, demonstrating their role in enhancing stability and functionality. By characterizing their molecular interactions, this study broadens the understanding of their potential applications in stabilizing complex food dispersions. The findings not only emphasize their capacity to improve food product quality and stability but also contribute to the growing body of knowledge on sustainable and health-promoting food ingredients. Moreover, this study can serve as a starting point for future research that delves deeper into the role of chia and phycocyanin as emulsifiers and stabilizers, as well as their application in specific dispersed systems. 2. Materials and methods 2.1. Materials Phycocyanin (PC), extracted from the cyanobacterium Arthrospira platensis, was supplied by EcoSpirulina S.L., based in Valencia, Spain. According to the certificate of analysis provided by the supplier, the powder exhibited a purity of ≥95 % phycocyanin and contained the following: protein 50.1 wt%, polysaccharides 43.0 wt%, moisture 4.5 wt % and ash 2.4 wt%. Whole organic chia seeds (Salvia hispanica L.) were purchased from a local retailer in Seville, Spain. The seeds were used without undergoing any additional thermal or chemical pretreatment prior to the mucilage extraction protocol described in Section 2.2. All aqueous solutions were prepared with Milli-Q water. 2.2. Mucilage extraction, characterization and sample preparation In order to achieve the highest possible yield in the extraction of chia mucilage, a procedure based on the combination of recent experiences M. Vela-Albarr´ an et al. Journal of Agriculture and Food Research 23 (2025) 102260 2 with relatively high yields (6–12 %) has been developed [26,28,29]. First, 150 g of chia seeds and water were weighed in a 1:20 ratio. Next, the pH of the water was adjusted to 8 with the help of a NaOH solution. Subsequently, the water was heated to a temperature of 85 ◦C in a stirred and isothermal reactor at atmospheric pressure. Once the temperature was reached, the chia seeds were added and left to heat with continuous stirring for 2 h. This process resulted in the swelling of the mucilage, which emerged from the interior of the seed and remained adhered to its surface. Once this period of time has elapsed, the seeds were allowed to cool to room temperature and were then placed in small plastic jars to freeze at a temperature of −40 ◦C for 24 h. Once the requisite temperature was reached, the seeds were freeze-dried in an ultrafreezer model TSX400 (Thermo Fisher Scientific Inc., Waltham, MA, EE. UU.) for 48 or 72 h (condensation temperature −80 ◦C and vacuum 0.114 mBar). After this period, and ensuring that all the water had been removed, a solid mucilage was produced that was attached to the chia seeds and must be separated. The seeds were then separated from the mucilage using a sieve with a mesh size of 0.3 mm, with slight friction applied to the mixture as it passed through the sieve. This process allowed the mucilage to pass through the sieve in powder form, while the seeds remained on the sieve. The extraction yield obtained was 9.81 %. Moisture content was determined gravimetrically by oven-drying at 105 ◦C until constant weight (AACC Method 44–15.02). Total ash content was determined by incineration at 550 ◦C for 4 h (AACC Method 08–01.01). Mineral profile was analysed using ICP-OES. Samples (0.5 g) were digested at 105 ◦C for 90 min in nitric and hydrochloric acid, then analysed for Na, K, Ca, Mg, P, S, Fe, Cu, Zn, Mn, B, Mo, Cr, Ni, Cd, and Pb using a Thermo Scientific iCAP 7000 Series. Protein content was determined by DUMAS, where nitrogen content was multiplied by 6.25. Fat content was determined by the Soxhlet method (AACC Method 30–25.01). Total dietary fiber (TDF) was measured using the enzymaticgravimetric method (AACC Method 32–05.0). Carbohydrates were calculated by difference, and total energy content was determined according to EU Council Directive 1169/2011. Dissolutions of varying concentrations of phycocyanin, chia mucilage, and their 1:1 mixture were prepared. The initial concentrations (0.4 wt% for phycocyanin and the mixture, and 1 wt% for chia mucilage) were chosen as starting points to create systematic dilutions by halving the concentration at each step. Surface tension measurements were conducted at different concentrations to determine the saturation concentration of the three studied systems. Each solution was divided into two 20 g portions: one reserved for further analysis, and the other diluted with 20 g of water to obtain the subsequent concentrations. The pH of the solutions under study was in the range of 6.5–7.0, as they were prepared using Milli-Q water without further pH adjustment. 2.3. Surface tension Surface tension was determined at different concentrations to obtain the adsorption curve, which allows determining the saturation concentration of the surface. For this purpose, the surface tension was determined using the dynamic Wilhelmy platinum plate method, which involves inserting the plate into the sample and slowly raising it until it is completely separated from the liquid. Before this occurs, the interface is bent on both sides of the plate. The stress exerted by the interface at this point is quantified and balanced with the lifting force of the plate. Measurements were conducted using a Sigma 701 tensiometer (KSV, Castlemead, UK) with the SGSErver software. The surface tension was monitored during at least 30 min until a constant value was attained. A constant value was reached when no change greater than 0.1 mN/m was observed over a period of five consecutive minutes. 2.4. Interfacial rheology The rheological properties were evaluated to ascertain the structural integrity of the interfaces. The rheological properties were quantified at the solution interface across a range of concentrations. This rheological study was conducted through dynamic oscillatory tests using a DHR3 rheometer (TA Instruments, New Castle, DE, USA) and a ring-shaped accessory. The results were obtained by means of frequency sweep tests at room temperature (20 ±2 ◦C). Prior to this, strain sweep tests were carried out at 0.1 Hz (0.628 rad/s) in order to determine the critical strain (the last strain in the linear viscoelastic range). For the systems with only phycocyanin and chia, two concentrations were analysed: the saturation concentration (0.1 wt% for phycocyanin and 0.4 wt% for chia mucilage) and a concentration above saturation (0.2 wt% for phycocyanin and 0.6 wt% for chia mucilage). In contrast, for the mixture, only the saturation concentration (0.1 wt% for the mixture) was analysed since it is sufficient to determine the interactions between both components without complicating the system. The measurements were performed the day after sample preparation. Once the sample was placed in the measurement cuvette, measurements were taken starting from that time and monitored for 1 h to control the formation of the surface film. 2.5. Z-potential and conductivity The Z-potential of the proteins in solution was determined by surface charge detection at pH =7 by a Zetasizer Nano ZS zeta potential analyzer (Malvern Instruments, Malvern, UK). To this end, different samples were prepared by dispersing phycocyanin, chia mucilage or the mixture of both. This measurement provides insight into the stability of a particle and indicates the potential required to penetrate the surrounding ion layer of the particle to destabilize it. The electrical conductivity measured at 25 ±1 ◦C was 366 ±10 μ S cm −1 for the phycocyanin dispersion, 360 ±8 μ S cm −1 for the chia-mucilage dispersion, and 451 ±12 μ S cm −1 for the 1:1 phycocyanin-chia mixture, corresponding to ionic strengths below ~10 mM. 2.6. Foaming capacity and stability Foaming capacity was evaluated following a protocol adapted from Coffman and García (1977) [30]. Dispersions of phycocyanin, chia-seed mucilage, and their 1:1 mixture were prepared at 0.1–1 wt% in 100 mL of Milli-Q water. Each dispersion was subjected to high-shear homogenization for 60 s at 13500 rpm using an Ultra-Turrax T25 digital homogeniser (IKA-Werke GmbH & Co. KG, Staufen, Germany) to generate foam. The initial liquid volume of each dispersion (V 1 =100 mL) and the total volume of liquid and foam post-homogenization (V 2 ), were recorded. The percentage increase in volume was subsequently expressed as a result of the volume change. Volume change (%) = (V2−V1)⋅100 This procedure was repeated for all concentrations to examine how biopolymer content influences foam formation. 2.7. Statistical analysis All experiments were conducted in triplicate to ensure the reliability and reproducibility of the results. Standard deviations are represented either as error bars in the figures (when visible) or as percentage values in the figure captions. 3. Results and discussion The adsorption and rearrangement of the surface-active agents contained in phycocyanin, chia mucilage and the mixture of both (1:1 ratio) on the air/water surface were studied. Surface tension over time was measured with a plate tensiometer until a constant value was reached, which marked the achievement of pseudo-equilibrium. These measurements were carried out at different concentrations for each of the samples studied in order to obtain the adsorption curve. For this M. Vela-Albarr´ an et al. Journal of Agriculture and Food Research 23 (2025) 102260 3 purpose, the surface tension at equilibrium is plotted against different concentrations of phycocyanin, chia mucilage and the mixture of both (1:1 ratio) (Fig. 1). It can be seen that, for all the systems, an increase in concentration caused a decrease in surface tension until an approximately constant value was reached, which corresponds to the saturation of the air-water surface. At very low concentrations of all these systems, a drastic drop in surface tension was observed, with a more gradual decrease at higher concentrations until the asymptotic value was reached. This evolution has previously been associated with an increase in the concentration of the surface-active agent at the air-water interface, resulting in the aforementioned decrease in surface tension [31]. However, once the surface was saturated, higher concentrations of surface agents did not result in a significant further decrease in surface tension [32]. At concentrations below the saturation concentration the surface was not completely covered by the surface-active agent, and the decrease in tension with increasing concentration may be associated with the existence of interactions in the already adsorbed molecules after diffusion. However, above the saturation concentration, both the surface-active molecules from phycocyanin and chia mucilage must migrate throughout the solution and oriented in an arrangement that allows each of the components to interact with its most favourable environment. The molecules can form aggregates in which their hydrophobic portions are oriented towards the interior of the aggregate and the hydrophilic portion towards the aqueous solution. The concentration at which aggregates form in such biopolymeric surfactants is called the critical aggregate concentration (CAC) [33]. On the other hand, the saturation concentration is also known as the adsorption efficiency (AE) and the minimum surface tension in the plateau zone as the surface activity (SA). In this case, as shown in Fig. 1, the AE of phycocyanin was approximately 0.1 wt% while in the case of chia mucilage it was approximately equal to 0.4 wt%. The decrease in surface tension of chia may be related to the presence of a certain amount of protein (11.54 wt%) and it is widely recognised that the amphiphilic nature of proteins allows them to reduce surface tension at the air/water interface [34]. The difference in AE between chia and phycocyanin can be explained by considering the higher protein concentration in the seaweed extract compared to chia mucilage, 50.1 wt% and 11.54 wt% respectively (Table 1). Thus, the AE of chia is 4 times higher than that of phycocyanin (0.4 wt%), for which the protein content in chia. In addition, chia polysaccharides possess a notably elevated molar mass, thereby imparting substantial bulk viscosity. This, in turn, retards the diffusive transport of macromolecules towards the interface. Furthermore, following adsorption, the extended, highly hydrated chains have the capacity to reorganise into polymer "brush" layers. These layers are able to hinder further adsorption events and delay the build-up of viscoelastic structure. Regarding SA, chia mucilage had a lower value (SA ≈43 mN/m) than phycocyanin (SA ≈50 mN/m). This may seem contradictory given the higher protein content of phycocyanin. However, it is well known that the presence of surface or dissolved impurities affects the surface tension of the liquid and that the equilibrium surface tension decreases as the impurity concentration increases. The chia used is a mucilage extracted from the seed in our laboratories, which was not further purified. This would explain the presence of various types of impurities, such as lipids, which would cause a greater decrease in tension than would be expected. It has been demonstrated that, at pseudo-equilibrium, the phycocyanin–chia mucilage blend attains an approximate AE of 0.1 wt% and an approximate SA of 52 mN/m (as for phycocyanin). However, across the concentration range, the blend’s equilibrium surface tension closely follows that of chia mucilage (practically coinciding with it), except at 0.4 % wt.%, where a slight deviation is evident. This fact indicates that, although the protein content of the mixture was lower than that of phycocyanin, the behaviour in terms of adsorption was similar, suggesting a positive cooperative interaction between the surface-active molecules of both substances at the interface [11]. While the study of surface tension provides information on the adsorption of molecules on the surface, surface rheology depends on both adsorption and intermolecular interactions between the molecules that form the surface film [35]. To study the nature of the surface film, the evolution of the viscoelastic properties of the air-water interface for samples of phycocyanin, chia gum and mixtures of the two was studied using linear viscoelastic surface shear measurements. Firstly, the elastic modulus (G ′ ) and viscous modulus (G ″ ) of the surface films were measured as a function of time to investigate the adsorption, ageing and structural reorganisation of molecules of phycocyanin, chia mucilage and mixtures of both adsorbed on the air/water surface. Fig. 2A and B shows the values of the viscoelastic properties, elastic modulus (G ′ ) and viscous modulus (G ″ ), obtained from low amplitude oscillatory shear (s-SAOS) tests at a frequency of 0.1 Hz for phycocyanin adsorbed at the air-water interface as a function of adsorption time at the saturated concentration, 0.1 wt% (Fig. 2A) and above (supersaturated concentration), 0.2 wt% (Fig. 2B). In Fig. 2A, corresponding to the saturation concentration, it can be observed that the values of G ′ and G ″ were constant as a function of time, indicating that the pseudo-equilibrium was reached immediately, there being no structural changes on the surface since the film was formed from the beginning [36]. However, for a concentration higher than saturation, the adsorption of phycocyanin molecules on the air/water surface implied a rapid increase in the surface shear elastic and viscous moduli (G’ s and G ″ s ) with a tendency to reach a plateau value, indicating the pseudo-equilibrium [37]. Furthermore, both moduli showed similar values from the beginning up to about 1500 s. However, at longer times, the elastic modulus became progressively higher than the viscous modulus. This result is related to the adsorption of the phycocyanin on the surface, which induces the formation of a film based on the proteins contained in the phycocyanin. As a consequence, the tan δ s (=G’’/G’) values decreased (data not shown), indicating the increase of the elastic character as a consequence of the adsorption, leading either to the appearance of strong interactions on the surface or to the formation of a densely compressed surface. Similar behaviour has previously been reported for other protein-based systems [38]. The difference in behaviour for saturated (Fig. 2A) and supersaturated (Fig. 2B) systems can be understood by considering that, as expected, at higher concentrations there was a greater steric hindrance, which would hinder the mobility of molecules to reach the surface, thus Fig. 1. Evolution of surface tension of aqueous solutions of phycocyanin, chia mucilage and the mixture of both (1:1) at different concentration levels (0–1 wt%). M. Vela-Albarr´ an et al. Journal of Agriculture and Food Research 23 (2025) 102260 4 requiring a certain time to reach pseudo-equilibrium of the surface [39]. The linear viscoelastic behaviour of a 2D surface can be described by analogy with the behaviour of a material in 3D space. Consequently, the linear viscoelastic behaviour of the surface can be obtained when the total applied interfacial shear stress or strain, for a given strain history, is sufficiently small so that the surface structure remains unchanged [37]. Stress sweeps were performed to investigate the strength, possible fracture mechanisms of the surface films and to determine the linear viscoelastic range (LVR) (data not shown). Surface shear rheology can directly provide relevant information on the specific linear viscoelastic moduli of the surface layer, allowing the evolution of the adsorption of the surface agents to be followed and the mechanical spectra at pseudo-equilibrium to be obtained. Thus, all the mechanical spectra were obtained at a strain value below the critical strain and therefore at the LVR. Fig. 3A and B shows the evolution of the mechanical spectra of the surface obtained during the adsorption of the phycocyanin protein adsorbed on the surface until the pseudo-equilibrium was reached. For this purpose, the evolution of the surface structure was analysed by frequency sweeps (0.01–0.1 Hz) at different times (t =0, 15, 15, 30, 45, 60 min) and with a constant deformation value of 0.12 % (within the LVR) for concentrations of 0.1 wt% (saturation) and 0.2 wt% (supersaturation) of phycocyanin. The response found for the saturation concentration of phycocyanin (Fig. 3A) suggests an elastic-dominant gel-like behaviour of the superficial layer, with the elastic component G’ s exceeding the viscous component G ″ s over the whole frequency range studied. This behaviour corresponds to the plateau zone of the mechanical [37]. Moreover, no differences were observed between the values of G ′ and G ″ with time at the different frequencies studied, indicating that the surface film was formed instantaneously. This result is consistent with those obtained in the time sweeps previously analysed. Attention should be paid to these measurements as the surface films had relatively low moduli due to the low torque limit of the rheometer, which contributes noise to the measurements [40–42]. In the study at a concentration higher than the saturation concentration (Fig. 3B), an increase in the value of the components G ′ and G ″ Table 1 Nutritional composition of chia mucilage. Humidity (%) Ashes (%) Proteins (%) Fats (%) Carbohydrates (%) Fibers (%) Energy (kcal) 8.04 ±0.01 9.50 ±0.21 11.54 ±0.24 1.40 ±0.21 69.53 a 15.05 ±0.73 336.83 a Calculated by difference. Fig. 2. Surface shear viscoelastic parameters with adsorption time of phycocyanin aqueous solutions for (A) saturated (0.1 wt%) and (B) supersaturated (0.2 wt%) systems. The standard deviation of the measured parameters was lower than 7 %. Fig. 3. Mechanical spectra with adsorption time of phycocyanin aqueous solutions for (A) saturated (0.1 wt%) and (B) supersaturated (0.2 wt%) systems. The standard deviation of the measured parameters was lower than 5 %. M. Vela-Albarr´ an et al. Journal of Agriculture and Food Research 23 (2025) 102260 5 with time was observed. At t =0, G ″ was higher than G ′ , indicating that the viscous component is more important than the elastic component. This can be explained by the fact that the surface film was still forming and was not very structured. As the time increased, an increase in both components was observed until G ′ values were higher than G ″ , indicating that the surface film was forming. At time t =60 min, an asymptotic value of the viscoelastic moduli of the surface appeared to have been reached (Fig. 2B) and almost equal values of the viscoelastic moduli with respect to t =45 min (Fig. 3B). This indicates that a pseudo-equilibrium had been reached and that the film formed on the surface by the phycocyanin molecules had a gel-like behaviour, with G ′ above G ″ , with a tendency to cross over at low frequencies (end of the relaxation zone and plateau zone). As for the phycocyanin concentration, it is noteworthy that the viscoelastic properties increased significantly as the concentration increases from saturation to supersaturation. Both G ′ and G ″ increased by 1–2 orders of magnitude. This could be related to the formation of multilayers on the surface [43]. Fig. 4A and B shows the values of the viscoelastic properties obtained from small amplitude oscillatory shear (s-SAOS) tests at a frequency of 0.1 Hz for chia mucilage adsorbed on the A/W surface as a function of adsorption time, at the saturation concentration, 0.4 wt% (Fig. 4A) and above, 0.6 wt% (Fig. 4B). Fig. 4A shows a predominance of G ″ over G ′ over the whole-time range studied, contrary to that observed for phycocyanin in Fig. 2A. The delta tangent values were always greater than 1 and decreased significantly with time, indicating that the adsorbed film formed by the chia molecules was predominantly viscous (G’ <G ″ ) and that its elasticity apparently improved during adsorption (data not shown). This could be related to the limited number of hydrophobic fragments (proteins). Thus, it would be relatively difficult for the molecules to anchor to the surface and the chia molecular chains could only form a fluid-like film with weak intermolecular interactions [44]. In addition, and also in contrast to phycocyanin, an adsorption equilibrium was not reached because both modules were still evolving at the end of the tests. As a consequence, a continuous increase of G ′ and G ″ was observed over time, without reaching a plateau value. This may be related to the fact that chia mucilage has a very low protein content and therefore required a long time for the proteins to diffuse to the surface. In relation to Fig. 4B, corresponding to the supersaturated concentration, it is again observed that the viscous component predominates over the elastic one. However, in this case pseudo-equilibrium was actually reached in the time range studied, possibly because the amount of protein present was somewhat higher. Fig. 5A and B shows the evolution of the mechanical surface oscillatory shear spectra obtained during the adsorption of the chia mucilage protein adsorbed on the surface. In this case, a constant strain value of 0.12 % was also used. According to this study, for the saturation concentration of chia mucilage (0.4 wt%), there were significant differences Fig. 4. Surface shear viscoelastic parameters with adsorption time of chia mucilage aqueous solutions for (A) saturated (0.4 wt%) and (B) supersaturated (0.6 wt%) systems. The standard deviation of the measured parameters was lower than 7 %. Fig. 5. Mechanical spectra with adsorption time of chia mucilage aqueous solutions for (A) saturated (0.4 wt%) and (B) supersaturated (0.6 wt%) systems. The standard deviation of the measured parameters was lower than 5 %. M. Vela-Albarr´ an et al. Journal of Agriculture and Food Research 23 (2025) 102260 6 between the values of G ′ and G ″ with time at the different frequencies studied, as expected from the results obtained in the time sweep previously analysed. Moreover, the G ″ component is higher than G ′ , indicating that the viscous component is more important than the elastic component, a behaviour different from that observed for the phycocyanin solutions at the saturation concentration level. This behaviour may be related to the low concentration of proteins present in chia, which, similarly to native corn fibre gum (CFG), limits the ability of the molecules to anchor effectively at the interface. As a result, the adsorbed molecular chains tend to form a fluid-like interfacial layer with weak intermolecular interactions [44]. The general mechanism of adsorption and formation of these surface films can be deduced from the above measurements and their analysis. The chia molecules diffuse and migrate to the air/water surface to form the surface film, resulting in an increased surface viscoelastic response. Hydrophobic groups extend into the air and hydrophilic fragments extend into the aqueous phase, resulting in steric hindrance. The superficial viscoelastic response can be influenced by diffusion, migration, rearrangement and conformational change of the polymer chains together with intermolecular interactions [45]. In this case, it is confirmed that low content of proteins present in chia are responsible for the surface behaviour of chia and must be somehow affected by the presence of the polymer chains that constitute the main component of chia. This explains the significant differences found in the behaviour of phycocyanin, whose main component is protein. Finally, the surface viscoelastic properties of the system formed by the phycocyanin-chia mixture were studied by means of both time and frequency sweeps, respectively (Fig. 6A and B). Fig. 6A shows the evolution with time of the surface viscoelastic parameters of an aqueous solution of a 1:1 phycocyanin-chia mucilage mixture at a concentration of 0.1 wt%, which corresponded to the saturation concentration of phycocyanin when formulated alone (0.1 wt%). The first thing to note is that the values obtained for both G ′ and G ″ were much higher than those obtained for the system with phycocyanin alone, by about two orders of magnitude. Moreover, in this case a certain time was required to reach constant values of G ′ and G ″ , indicating a pseudo-equilibrium at the surface [37]. This is comparable to the results obtained for the system with only phycocyanin at a concentration above the saturation concentration. The cause can be attributed to steric reasons due to the presence of polymer chains interacting with the proteins in the interface. This would also explain the abrupt increase in the viscoelastic modulus values of the mixed system compared to the phycocyanin system. This type of protein-polysaccharide interaction, which produces a positive synergy at the air/water interface, has been extensively studied [10,11]. Thus, the results from both surface tension and surface rheology measurements indicate complementary and cooperative behaviour between the two components. While surface tension reflects the adsorption capacity at the interface, surface rheology offers additional insights into the nature and strength of intermolecular interactions within the interfacial film [46]. Together, these analyses suggest cooperative behaviour between the molecules, despite the different perspectives they provide on the interfacial dynamics. Additionally, a recently published study on the interfacial behaviour of the same systems at the oil-water interface further supports these findings, observing similar cooperation between the components at the interface. This cooperative interaction translates into emulsions. When phycocyanin and chia mucilage were combined at a 50:50 ratio, the resulting mixed system displayed good long-term stability, which was attributed to the protein–polysaccharide interactions at the interface and in the continuous phase [47]. However, it should be remembered that the surface tension variation is related to the adsorption of molecules on the surface and that surface rheology is not only related to this but also it provides information about the intermolecular interactions between the molecules that form the surface film [46]. Finally, low-amplitude oscillatory shear (s-SAOS) tests were carried out at a frequency of 0.1 Hz and with a constant strain value within LVR of 0.12 %. Fig. 6B shows the values of the viscoelastic properties for the phycocyanin-chia mucilage mixture. It is observed that at 0.1 wt% of the phycocyanin-chia mucilage mixture, a slight increase in the values of G ′ and G ″ with adsorption time was observed, indicative of the evolution in the creation of the film on the surface, as explained in the previous figure. Moreover, it was observed that, in the whole frequency range, the G ′ component is above G ″ , which indicates that the elastic behaviour prevails over the viscous one and presents a pattern of behaviour similar to the supersaturated phycocyanin system. This behaviour is associated with gels and corresponds to the plateau zone of the mechanical spectrum. Comparing these results with those shown in Fig. 2A (corresponding to the system with phycocyanin at surface saturation concentration), it is worth noting that the values of the viscoelastic moduli of the mixture are significantly higher. This again reveals the existence of interactions between phycocyanin and chia, which causes positive cooperative interactions in the viscoelastic properties, improving them significantly [11]. The zeta potential measurements shown in Fig. 7 provide valuable insights into the electrostatic stability and interfacial properties of phycocyanin, chia mucilage and their 1:1 mixture in aqueous systems. The observed differences in zeta potential between these three systems highlight the influence of protein-polysaccharide interactions on surface charge, which is a key factor in understanding their potential applications in dispersed systems such as emulsions and foams. Phycocyanin exhibited a zeta potential value indicating a relatively high surface Fig. 6. (A) Surface shear viscoelastic parameters with adsorption time and (B) Mechanical spectra with adsorption time of aqueous solutions of a 1:1 phycocyanin/chia mucilage mixture (0.1 wt%). The standard deviation of the measured parameters was lower than 5 %. M. Vela-Albarr´ an et al. Journal of Agriculture and Food Research 23 (2025) 102260 7 charge, consistent with its amphiphilic nature due to its protein and polysaccharide content. This charge contributes to its ability to stabilize dispersed systems by electrostatic repulsion, preventing aggregation and phase separation. The high negative charge suggests that phycocyanin molecules remain well dispersed in solution, promoting enhanced surface activity, as evidenced by the surface tension and interfacial rheology results discussed in the manuscript. Conversely, chia mucilage exhibited a lower absolute zeta potential value. This can be attributed to the predominantly polysaccharide composition of chia mucilage which, although containing some protein fractions, does not have the same amphiphilic character as phycocyanin. While a negative Z potential in protein-polysaccharide mixtures can indicate high electrostatic stability due to greater repulsion between the negatively charged molecules, it could also lead to the destabilization of dispersed systems due to desorption of the protein from the interface. This observation is consistent with the viscoelastic properties of chia mucilage at the airwater interface, suggesting the formation of a fluid-like film with weak intermolecular interactions. Interestingly, the phycocyanin-chia mucilage mixture showed an intermediate zeta potential value, closer to that of phycocyanin. This finding suggests that the presence of phycocyanin dominates the surface charge properties of the mixture, maintaining sufficient electrostatic repulsion to prevent aggregation. The comparable zeta potential to pure phycocyanin is particularly significant as it indicates that even with dilution of the protein fraction in the presence of chia mucilage, the overall charge remains stable. This observation is consistent with the surface tension results, which showed that the mixture had similar adsorption efficiency (AE) and surface activity (SA) to phycocyanin alone. In addition, the enhanced viscoelastic properties observed in the interfacial rheology further support the existence of cooperative interactions between phycocyanin and chia mucilage, strengthening the mechanical integrity of the adsorbed film. From a functional point of view, these results suggest that the phycocyanin-chia mucilage blend could serve as an effective stabilizing agent in dispersed systems. The retention of a high zeta potential value implies that the blend could maintain colloidal stability in formulations, reducing the risk of flocculation or coalescence. Furthermore, the rheological improvement observed in the blend underlines the potential of protein-polysaccharide interactions in strengthening interfacial films, a desirable property in food, pharmaceutical and cosmetic emulsions. Fig. 8 clearly shows that the foaming capacity of all three systems (phycocyanin, chia mucilage and the 1:1 mixture) increases with concentration, although the rate and extent -of this increase differ significantly between them. Within the 0.1–1 % by weight range, phycocyanin produces the greatest volume expansion. This reflects the rapid diffusion and adsorption of its abundant amphiphilic proteins. These proteins reduce interfacial tension and generate a cohesive, viscoelastic film. This film is capable of trapping air efficiently. By contrast, chia mucilage exhibits the lowest foaming capacity within this entire range. Although chia mucilage reaches a lower equilibrium surface tension than phycocyanin, its high molar mass polysaccharides slow adsorption and form a predominantly viscous interfacial layer (G ″ >G ′ ), while strongly increasing bulk viscosity. Under the 60 s rotor–stator whipping used here, these kinetic and rheological factors suppress air incorporation, explaining the lower overrun of pure chia despite lower surface tension. In the blend, cooperative protein–polysaccharide interactions stiffen the interfacial film, but the progressive rise of bulk viscosity above 0.25 wt% limits further overrun. While the mixture does not surpass phycocyanin in terms of absolute foam volume, its performance far exceeds that of chia mucilage. Above this concentration, and probably due to the high viscosity of the medium, the foaming capacity decreases. This is consistent with the interfacial rheology data, which show that cooperative interactions between proteins and polysaccharides give the mixed film a significantly higher elastic modulus than phycocyanin alone (G ′ and G ″ are two orders of magnitude higher than phycocyanin at 0.1 wt%). This reinforcement facilitates bubble retention without the need for additional proteins. Overall, the foam results suggest that chia mucilage primarily acts as a supporting polysaccharide matrix that moderates the viscosity of the solution and reinforces the interfacial network. Meanwhile, the actual foaming capacity is determined by the protein fraction contributed by phycocyanin. All foams generated from phycocyanin, chia mucilage, and their 1:1 blend disintegrated within 1 h, with the foam column completely collapsing before 60 min. This transient stability is consistent with the interfacial rheology: phycocyanin forms a thin, gel-like film whose elastic and viscous moduli plateau after around 60 min, indicating modest structural reinforcement. In contrast, chia mucilage produces a predominantly viscous, weakly connected layer (G ″ >G ′ ). Finally, the nutritional composition of chia mucilage (Table 1) provides important insights into its potential functional contributions to surface properties. The mucilage exhibited a relatively high carbohydrate content (69.53 wt%), consisting mainly of polysaccharides, which are known for their ability to influence interfacial behavior by modifying viscosity and forming weak surface films. In addition, the presence of protein (11.54 wt%) suggests a degree of surface activity that could contribute to the interfacial phenomena observed. The moisture content (8.04 wt%) and ash content (9.50 wt%) indicate that the mucilage Fig. 7. Zeta potential values of aqueous solutions of phycocyanin, chia mucilage and the mixture of both (1:1). Fig. 8. Effect of concentration on foam capacity of aqueous solutions of phycocyanin, chia mucilage and the mixture of both (1:1). M. Vela-Albarr´ an et al. Journal of Agriculture and Food Research 23 (2025) 102260 8 retains a considerable amount of bound water and minerals, which may affect its solubility and interaction with other biopolymers in aqueous systems. The lipid content (1.40 wt%) is relatively low, suggesting that any surfactant behavior is primarily driven by the polysaccharideprotein matrix rather than the lipid fractions. These compositional features are directly related to the surface properties observed in the rheological and interfacial analyses. The predominance of polysaccharides in chia mucilage contributes to the formation of a fluid-like film at the air-water interface, as shown by the viscoelastic measurements (Fig. 4A and B), where the viscous modulus (G ″ ) consistently exceeds the elastic modulus (G ′ ). This behavior is characteristic of weakly structured interfacial films, which contrasts with the stronger, gel-like surface layers observed for phycocyanin. In addition, the moderate protein content, although lower than that of phycocyanin, is likely to play a role in the adsorption dynamics. Although it does not lead to a highly elastic interfacial network, it still contributes to a reduction in surface tension as seen in the adsorption curves (Fig. 1). The results support the notion that chia mucilage alone is not sufficient to form a robust interfacial film, but may act as a co-stabilizing agent when combined with proteins such as phycocyanin. These findings highlight the potential applications of the phycocyanin-chia mucilage mixture in the stabilization of complex dispersed systems, such as emulsions and foams in the food, pharmaceutical, and cosmetic industries. The cooperative interaction between phycocyanin and chia mucilage results in improved surface structuring and mechanical strength, making the blend a promising candidate for sustainable, bio-based stabilizers in dispersed systems. Additionally, the natural origin and functional properties of both components make this system an attractive alternative to synthetic stabilizers, aligning with the growing demand for clean-label and sustainable ingredients. Further studies on its behaviour under different processing conditions could pave the way for its practical implementation in industrial applications. 4. Conclusions The interfacial behavior of a 1:1 blend of phycocyanin and chia mucilage reveals that, despite its lower protein content, the mixture achieves the same surface tension reduction as pure phycocyanin (approximately 52 mN/m at 0.1 wt %). Of particular significance is the observation that cooperative protein–polysaccharide interactions at the air–water interface result in a substantial enhancement of the mechanical strength of the interfacial film. The elastic and viscous moduli of the mixed system exceed those of phycocyanin alone by approximately two orders of magnitude, thereby yielding an elastic-dominant, gel-like network. Zeta potential measurements have been used to demonstrate that phycocyanin governs the surface charge of the blend, thereby maintaining colloidal stability while reinforcing surface structuring. Although all foams collapsed within 60 min, the blend’s ability to sustain high overrun with a reduced protein load highlights its promise as a clean-label foaming aid for products that require rapid aeration yet only transient stability, such as instant beverages or short-life desserts. The findings demonstrate that blending phycocyanin with chia mucilage offers a simple, sustainable strategy to produce bio-based emulsifiers that combine efficient adsorption with superior interfacial rheology. The findings of this study demonstrate cooperative interfacial strengthening in phycocyanin–chia mixtures (elastic-dominant films with markedly higher moduli at equal total concentration), thus supporting their promise as clean-label stabilizers for emulsions and short-lived foams. However, the generation and persistence of foams are processdependent, and are constrained by bulk viscosity at higher mucilage contents. It is evident that additional optimization (e.g. concentration, whipping protocol, or ionic conditions) would be required for the creation of long-lasting foams. This will allow for a comprehensive assessment of their applicability in industrial processes. Nonetheless, the present findings were obtained exclusively at the air–water interface under neutral pH and ambient temperature. Interfacial and rheological responses may diverge in oil–water geometries, acidic environments or at elevated temperatures. These variables must be quantified before the biomaterial can be confidently translated into industrial processes. Ongoing work is therefore directed towards extending the present framework to such technologically relevant conditions. CRediT authorship contribution statement María Vela-Albarr´ an: Visualization, Software, Investigation. Luis A. Trujillo-Cayado: Writing – review & editing, Writing – original draft, Visualization, Resources, Project administration, Methodology, Funding acquisition. Francisco Carrillo: Writing – original draft, Validation, Software, Investigation, Formal analysis, Data curation. Jenifer Santos: Writing – review & editing, Resources, Investigation, Funding acquisition. Nuria Calero: Writing – review & editing, Writing – original draft, Visualization, Resources, Project administration, Methodology, Investigation, Funding acquisition. Data availability statement The data that support the findings of this study are available from the corresponding author upon reasonable request. Declaration of generative AI and AI-assisted technologies in the writing process We acknowledge the use of generative AI and AI-assisted technologies in the writing process, specifically Copilot, licensed by the University of Seville, to enhance language clarity. All content has been reviewed and validated by the authors to ensure accuracy and integrity. Funding This research was funded by Ministerio de Innovaci´ on y Ciencia (Gobierno de Espa˜ na, grant number TED2021-131246B), FEDER Programme (European Commision) and by Programa Ram´ on y Cajal (Ministerio de Innovaci´ on y Ciencia, Gobierno de Espa˜ na). Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Data availability Data will be made available on request. References [1] E. Dickinson, An Introduction to Food Colloids, Oxford university press, Oxford New York Tokyo, 1992. [2] E. Dickinson, Hydrocolloids as emulsifiers and emulsion stabilizers, Food Hydrocoll. 23 (2009) 1473–1482, https://doi.org/10.1016/j. foodhyd.2008.08.005. [3] T. Karbowiak, F. Debeaufort, A. 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