مقایسه اثر بر همکنش کنژوگه ایزوله پروتئین سویا با گالیک اسید و تانیک اسید بر ویژگی های ساختاری و فعالیت آنتی اکسیدانی آن

Comparison of the Conjugation interaction of Isolated Soy Protein with Gallic Acid and Tannic Acid on Its Structural Properties and Antioxidant Activity
Extended abstract
Background: In the food industry, proteins play a crucial role in food systems. However, protein instability in these systems poses a significant challenge to the food industry and drives researchers toward improving protein functional properties. Soy protein isolate (SPI), as a plant-based protein with high nutritional value and a potential substitute for animal proteins, faces challenges due to its instability against heat, pH changes, and other food processing conditions (Sui et al., 2021). Recently, studies have shown that protein modification through conjugation with polyphenols can significantly enhance their functional properties and health benefits. Polyphenols, widely studied for their antioxidant, health-promoting, and therapeutic effects, are secondary metabolites derived from plants and are abundant in fruits, vegetables, and cereal grains. Among phenolic compounds, phenolic acids are important members. Tannic acid (TA) and gallic acid (GA) are two common phenolic acids structurally related. TA, an ester of gallic acid and D-glucose, has multiple phenolic groups and a high molecular weight, whereas GA consists of a single phenolic ring and has a significantly lower molecular weight (Hanger et al., 2014). Both GA and TA contain pyrogallol and carboxyl groups, imparting various biological activities such as antioxidant, antimicrobial, and anticancer properties (Heleno et al., 2015). The hydroxyl groups in GA and TA easily convert to quinones, exhibiting their antioxidant activity (Liu et al., 2019). Research indicates that GA and TA can interact with macromolecules like proteins and polysaccharides, improving their stability and functional properties for broader applications. Protein-polyphenol interactions include covalent and non-covalent bonds. Compared to non-covalent interactions, covalent bonds are usually irreversible and more resistant to environmental changes (Chubinski et al., 2017). Common methods to form covalent bonds (conjugation) between proteins and polyphenols include enzymatic and non-enzymatic approaches. Enzymatic methods are environmentally friendly but complex and costly. Among non-enzymatic methods, alkaline reaction is more common, where polyphenols first convert to quinones and then react with proteins to form protein-polyphenol conjugates (Liu et al., 2019). Considering the importance of improving the functional properties of soy protein isolate for broader industrial applications and the effective role of polyphenols in enhancing these properties, a detailed study of the conjugate interactions between these compounds is essential. Among various polyphenols, gallic acid and tannic acid, due to their different structures and notable biological activities, are suitable candidates for investigation. Despite previous studies, comprehensive knowledge regarding the effects of different concentrations of these two polyphenols on the functional, antioxidant, and structural properties of soy protein isolate remains limited. Therefore, this study aims to examine and compare the effects of gallic acid and tannic acid at various concentrations on the properties of soy protein isolate conjugates, seeking to determine optimal conditions to facilitate broader industrial applications of these conjugates in the food industry.
Objective: Given the importance of enhancing the functional properties of soy protein isolate for expanding its industrial applications and the significant role of polyphenols in improving these properties, a precise investigation of conjugate interactions between these compounds is indispensable. Gallic acid and tannic acid, due to their distinct structures and prominent biological activities, are appropriate candidates for study. Despite previous research, there is still no comprehensive understanding of how different concentrations of these two polyphenols affect the functional, antioxidant, and structural properties of soy protein isolate. Thus, this research is designed to investigate and compare the effects of gallic acid and tannic acid at various levels on soy protein isolate conjugate properties and aims to identify optimal conditions for wider use of these conjugates in the food industry.
Method: Preparation of dual conjugates SPI-TA and SPI-GA was carried out as follows: First, a 2% (w/v) soy protein isolate solution was stirred at room temperature for 4 hours, and its pH was adjusted to 9 using 0.1 M NaOH. Separate solutions of tannic acid and gallic acid were prepared at 5%, 10%, and 15% (w/w protein). Next, polyphenol solutions at various concentrations were mixed with the protein solution at a 1:1 ratio. The pH of the resulting solutions was again adjusted to 9 with 0.1 M NaOH and stirred at room temperature for 24 hours using a magnetic stirrer. To remove free phenolic acids, the solutions were dialyzed against distilled water for 48 hours. The dialyzed solutions were then frozen and lyophilized at -80°C under vacuum for 24 hours (Yi et al., 2021). The extent of protein-phenol conjugation was determined by the Folin–Ciocalteu assay following the method of Qiu et al. (2021). Covalent bonding formation was confirmed via SDS-PAGE analysis, which revealed molecular weight shifts indicative of phenolic compound attachment. FTIR spectroscopy was employed to assess structural alterations in protein secondary structures by monitoring amide bond vibrations. Antioxidant capacity was quantified using the DPPH radical scavenging assay. Emulsifying properties were evaluated by measuring emulsion activity index (EAI) and emulsion stability index (ESI), while emulsion stability was further characterized by zeta potential and droplet size analysis using dynamic light scattering (DLS). Colorimetric analysis was performed to quantify changes in color parameters (L*, a*, b*). All experiments were conducted in triplicate, and data were statistically analyzed using factorial ANOVA and Duncan’s multiple range test at a 5% significance level
Results:In this study, the conjugation percentage, serving as an indicator of the extent of covalent bond formation between the free amino groups of proteins and the phenolic groups of acids, was quantified using the Folin–Ciocalteu assay. The results demonstrated a significant increase in the conjugation percentage with rising phenolic acid concentration up to 10%, after which the values plateaued, indicating saturation of binding sites. This increase reflects enhanced covalent crosslinking between protein molecules and phenolic compounds, leading to more stable conjugates.Notably, samples containing tannic acid (TA) exhibited significantly higher conjugation percentages compared to those with gallic acid (GA), attributable primarily to TA’s greater molecular weight and higher number of phenolic rings. This structural complexity of TA facilitates more extensive covalent bonding with protein molecules, potentially resulting in enhanced protein stability and more pronounced conformational changes.Fourier-transform infrared spectroscopy (FTIR) analysis revealed significant alterations in the protein amide bands (Amide A, I, and II), which correspond to carbonyl, NH groups, and secondary protein structures. These spectral changes confirm modifications in protein conformation and the formation of new covalent bonds upon reaction with phenolic acids.SDS-PAGE electrophoresis further corroborated covalent conjugate formation, as major protein bands in conjugated samples shifted toward higher molecular weights relative to the pure protein controls, indicating successful attachment of phenolic moieties to the protein backbone.Functional properties essential for food industry applications, including emulsifying activity index (EAI) and emulsion stability index (ESI), showed marked improvement in conjugate samples. Specifically, at a 10% phenolic acid concentration, both EAI and ESI increased significantly. This enhancement is likely due to surface modifications and elevated surface charge on the proteins, supported by increased zeta potential measurements, which reduce particle aggregation and thereby improve emulsion stability. Antioxidant capacity, evaluated via the DPPH radical scavenging assay, revealed that all conjugate samples exhibited substantially higher free radical inhibition compared to native protein. Increasing phenolic acid concentration correlated positively with antioxidant activity, indicating efficient transfer of antioxidant functionalities from polyphenols to proteins through conjugation. Overall, SPI-TA samples demonstrated superior antioxidant properties relative to SPI-GA, consistent with the higher phenolic content and structural characteristics of tannic acid. Colorimetric analysis showed reductions in L* (lightness), a* (red-green axis), and b* (yellow-blue axis) values post-conjugation, indicative of sample darkening and shifts toward greenish and yellowish hues. These changes are typically attributed to polyphenol oxidation and the formation of new pigments within the conjugates, a factor that should be carefully considered for industrial applications. This color shift primarily results from reactions between polyphenol-derived quinones and amino acid residues. Statistical analysis identified SPI-GA 10% and SPI-TA 10% treatments as optimal conditions for maximizing improvements in the structural, functional, and antioxidant properties of soy protein isolate. These samples exhibited the highest conjugation percentages, strongest antioxidant activity, and most favorable emulsifying characteristics, highlighting their potential for industrial-scale applications.
Conclusion: Based on the findings, covalent conjugation between soy protein isolate and phenolic acids, particularly tannic acid, is an effective and economical method to enhance protein functional properties and stability. These improvements can facilitate broader applications of soy protein isolate in food and pharmaceutical industries. Furthermore, this research provides a better understanding of protein-polyphenol interaction mechanisms and the role of polyphenol concentration in controlling final properties of conjugate products. Due to their high antioxidant activity, these conjugates can be used as natural and healthy additives in food formulations to improve product stability and quality.