مقایسه تأثیر اینولین و مالتودکسترین بر ویژگی‌های امولسیون‌های پیکرینگ پایدارشده با میکروژل پروتئین آب‌پنیر از طریق واکنش میلارد

Introduction: Pickering emulsions, an innovative emulsion system, have recently attracted significant attention. These emulsions achieve stability through the use of solid particles rather than synthetic surfactants, offering high resistance to coalescence. Unlike surfactants, these particles are insoluble in water and oil, and they stabilize the emulsion by forming a physical barrier at the oil-water interface, which prevents droplet merging. Such features make Pickering emulsions a sustainable, environmentally friendly, and safe choice for applications in the food, pharmaceutical, and cosmetic industries. With the rising demand among consumers for natural and safer products, there is increasing interest in developing biocompatible, particle-stabilized Pickering emulsions. In recent years, biopolymers—often referred to as “green” alternatives—have been explored to replace synthetic emulsifiers in food products. Biopolymers, including proteins and carbohydrates, offer nutritional value, natural origins, and biocompatibility, making them appealing as replacements for industrial emulsifiers. Proteins are useful for emulsification due to their amphiphilic nature but can be unstable under external processing conditions such as temperature, pH, and salts. Carbohydrates, on the other hand, enhance emulsion stability by increasing viscosity but are not surface-active enough to function as emulsifiers on their own. However, combining and modifying proteins and saccharides can enhance their emulsifying properties. Key modifications include forming protein microgel structures or conjugating proteins with carbohydrates via the Maillard reaction, which improves their functional stability, thermal resistance, and solubility. Whey protein, a cheese industry byproduct rich in essential amino acids, is known for its rapid absorption and high bioavailability. Due to its favorable functional properties, including three-dimensional network formation, strong particle bonding, and biocompatibility, whey protein microgels are considered a promising stabilizer for Pickering emulsions. Conjugating whey protein microgels with saccharides such as inulin and maltodextrin via the Maillard reaction can further enhance their emulsifying capabilities, affecting properties like surface tension, and improving stability against temperature and pH fluctuations. Inulin and maltodextrin are commonly used saccharides in food science. Maltodextrin, a tasteless, water-soluble oligosaccharide, is widely used due to its low cost and ease of production from corn starch. In contrast, inulin, a fructan polysaccharide with prebiotic benefits, is non-digestible and reaches the large intestine intact. It is valued for its ability to reduce calorie intake, lower blood lipids, and increase satiety, making it popular in low-calorie foods as a thickening and stabilizing agent. This study aims to investigate the effectiveness of whey protein microgel conjugates with inulin and maltodextrin in stabilizing Pickering emulsions. The primary focus is to assess the influence of saccharide type and the Maillard reaction on emulsion stability, structure, and physicochemical properties in systems with a high internal phase. The findings contribute to understanding polysaccharide roles in protein conjugates, supporting the development of more stable and safer Pickering emulsions for various industries.
Materials and Methods: Whey protein isolate was dissolved in Milli-Q water to a concentration of 40 mg/mL. The protein dispersion was stirred at 25 °C for 2 hours, then incubated at 4 °C for 12 hours to ensure full hydration. The pH was adjusted to 5.80 ± 0.05 using 1.0 M HCl with a pH-meter. Next, the dispersion was heated in a water bath at 85 °C for 15 minutes, without stirring, to form whey protein microgels (WPMs), and rapidly cooled to 4 °C in an ice-water bath. Freshly prepared WPM was then combined with inulin (In) and maltodextrin (Md) at a 2:2 protein-to-saccharide ratio, and these were labeled as In:WPM and Md:WPM, respectively. Additionally, a mixture containing both saccharides in a 2:1:1 protein:inulin:maltodextrin ratio was prepared and labeled In:Md:WPM. All mixtures were prepared at a total concentration of 4% w/w. The pH was adjusted to 8.0 using NaOH. The dispersions were then heated in a 90 °C water bath for 1 hour, followed by rapid cooling in an ice-water bath. Samples were dialyzed against Milli-Q water (pH-matched) using a 14,000 Da molecular weight cutoff membrane, and stored at 4 °C for further analysis. WPM heated without carbohydrate was labeled WPM. To ensure repeatability, three separate samples were prepared per run, with each experiment conducted in triplicate. After the Maillard reaction, degree of grafting (DG), particle size, zeta potential, and surface tension of the nanoparticles were measured. For HIPPE preparation, soybean oil (φoil = 0.80) was added to both conjugated and unconjugated WPM dispersions (pH = 7.0). Emulsification was achieved via homogenization at 24,000 rpm for 4 minutes using an Ultra-Turrax disperser, and the emulsions were analyzed on the same day. FTIR spectroscopy, droplet size, polydispersity index (PDI), emulsification activity index (EAI), emulsification stability index (ESI), creaming index, and viscosity of HIPPEs were also determined.
Results and Discussion: Regarding modification/conjugation degree, the Maillard reaction enabled covalent bonding between whey protein microgel and saccharides, with inulin (polysaccharide) showing a higher degree of conjugation than maltodextrin (oligosaccharide) due to its longer chain length and greater availability of carbonyl groups. Without carbohydrates, modification in WPM was minimal, likely from trace lactose interactions. The particle size increased with saccharide addition, with inulin contributing more significantly due to its larger chain, which expanded the WPM particles’ volume. This larger size supports stability in high internal-phase Pickering emulsions. After the Maillard reaction, increased zeta potential improved electrostatic repulsion and stability, as all samples carried a negative charge at pH 7 (above WPM’s isoelectric point). All conjugated WPM particles reduced surface tension, a critical factor for emulsion stability. To assess the emulsification performance and interfacial properties of emulsifiers, surface tension is an important parameter, as it is closely related to the stabilization of emulsions against coalescence. The adsorption mechanism of emulsifiers includes two steps: first, the emulsifiers diffuse and attach to newly formed oil droplets, resulting in a steep drop in surface tension. In the second stage, the emulsifiers rearrange at the interface, causing a slight decrease in surface tension until it levels off. Inulin showed a greater reduction in surface tension than maltodextrin due to its long, unbranched chain, which better covers the particle surfaces. In terms of HIPPEs, FTIR analysis indicated characteristic protein-related amide bands and triglyceride peaks from the oil phase, with changes in amide peaks supporting protein modification during glycation. Moreover, saccharide addition led to smaller oil droplets, which are more stable, as larger WPM particles occupied more space, restricting oil mobility and droplet size. WPM conjugated with carbohydrates, especially inulin, showed higher emulsion activity index than unconjugated WPM. Inulin's large and linear structure enabled better surface protection around oil droplets, enhancing stability. Similarly, conjugated WPM-stabilized emulsions had greater stability than unconjugated ones. Inulin's structure formed a stronger protective layer compared to maltodextrin, reducing droplet agglomeration and enhancing stability. Also, conjugation with saccharides, particularly inulin, increased viscosity and emulsion uniformity. The rheological properties of Pickering emulsions are influenced by two key factors: the viscosity of the continuous phase and the structural characteristics of the particles, such as their size, shape, and rigidity. Higher viscosity supports stability by increasing the interfacial area and promoting shear-thinning behavior. Due to the formation of products with higher molecular mass, hydrophilicity, surface charge, and steric hindrance , HIPPEs created using inulin-conjugated WPMs exhibited a higher consistency coefficient (K) and greater resistance to applied shear stress. Finally, the creaming index decreased with carbohydrate addition, particularly with a higher inulin-to-maltodextrin ratio, due to increased oil droplet density and viscosity, reflecting the positive impact of the Maillard reaction on stability and creaminess.
Conclusion: The findings of this study indicate that conjugating whey protein microgels (WPM) with inulin and maltodextrin via the Maillard reaction is an effective method for producing stable Pickering emulsions with a high internal phase. This covalent binding with polyols significantly enhances the surface activity of WPM, allowing it to be adsorbed at the oil-water interface and play a crucial role in stabilizing the emulsions. Among the compounds tested, WPM conjugates with inulin exhibited a greater impact on improving emulsion properties and stability. This enhanced functionality is likely due to inulin’s longer molecular chains, which provide stronger steric repulsion, lower surface tension, and form more stable interfacial layers compared to maltodextrin. Overall, this research highlights the substantial potential of the Maillard reaction for enhancing emulsion performance and boosting the emulsifying capacity of natural proteins by conjugating them with saccharides. Conjugated whey protein nanoparticles with inulin and maltodextrin show promise as an effective approach for creating stable Pickering emulsions with favorable physicochemical characteristics for applications in the food, pharmaceutical, and cosmetic industries. Additionally, these Pickering emulsions can serve as precursors or integral components in innovative products, such as oleogels, paving the way for further advancements in these fields.