Impact of Bambara groundnut protein modification on complexation behaviour with gum arabic for improved stability and functionality in food applications

Abstract

Protein-polysaccharides complexation has attracted considerable research interest over the past several decades, particularly regarding its role in the encapsulation of bioactive compounds, emulsion stabilization, meat mimetics, and fat replacers. However, complexes usually form within a narrow pH range characterized by weak interactions owing to a tightly packed structure and limited molecular flexibility of the native proteins. Consequently, this restricts the number of oppositely charged groups on globular proteins that can interact with polysaccharides and limits the applications of complexes in certain foods, particularly in acidic beverages. Enhancing the interactions between globular proteins and polysaccharides to achieve stable complex formation requires modifications to the protein structure, composition, and functional properties. Hence, the purpose of this study was to evaluate the influence of various protein modification methods on their physicochemical and structural properties, as well as how these structural changes affect their ability to form complexes with polysaccharides. To achieve this aim, pH-induced fractionation and ionic concentration were utilized as a protocol for extracting and structurally modifying Bambara protein prior to complexation with gum arabic. Additionally, limited trypsin hydrolysis and atmospheric cold plasma were done to modify the protein, and their impact on the stability and properties of complexes was investigated. Finally, encapsulation of riboflavin in a model beverage using Bambara protein hydrolysates (DH 5)- gum arabic soluble complex as a delivery system was determined. Response surface methodology was employed to optimise the pH-induced fractionation of Bambara protein. Thirteen experimental trials were conducted under varying extraction conditions of pH (2.0 to 9.0) and NaCl concentrations (0.0 to 0.6 M), which were considered as independent variables within a central composite design. Complexation behaviour was evaluated through turbidimetric analyses, while structural and conformational changes in fractionated proteins were examined to elucidate their interaction with gum-arabic. Bambara protein fraction obtained at pH 2.95, 0.28 M NaCl produced the most pronounced shift in pHopt (3.4) with spherical microparticles complex and 70% coacervate yield. The modification of protein-gum arabic complexation profile was corroborated by increased content of β-sheet, enriched legumin subunits, and basic amino acids of the protein fraction. Trypsin hydrolysis was done at a controlled degree of 2, 5, and 7.5 prior to complexation with gum arabic focusing on the critical pH structures formations. The hydrolysates were characterized using SEC-HPLC, zeta potential, surface hydrophobicity, and intrinsic fluorescence. Electrostatic interactions between the biopolymers were monitored through turbidimetry analysis. The stability property of the complexes was investigated using differential scanning calorimetry and rheometry. Among the hydrolysates, DH 5 produced the most stable soluble complexes over a broader pH range (7.0 - 4.3), compared to DH 2, DH 7.5, and the unhydrolyzed isolate showed enhanced binding strength and structural stability. This was evidenced by its rheological fingerprints, with DH 5 showing a more pronounced plateau value of Gʹ, indicating a stronger molecular interaction and binding strength between the hydrolysate and gum arabic. This suggests a higher degree of internal structure and makes the complex more stable. Additionally, DH 5 showed a shift in thermal transition temperature, increased negative charge, and surface hydrophobicity. Bambara protein was subjected to atmospheric cold plasma treatment (2, 5 and 10 min) prior to complexation with gum arabic. The impact of the exposure time on composition, structural changes of protein and complex behaviour was analysed. The optimum complexation, pHopt of cold plasma treated Bambara protein-gum arabic complexes significantly shifted to acidic pH following the treatment in an exposure time-dependent manner. The structural changes coincide with the observed shift from α-helix to β-sheet structure, indicating opening of the structure of protein. Crosslinking effects of cold plasma corroborated the breaking of the cross linking disulfide bonds and oxidative modification of proteins, respectively by the chemically reactive species. The variation in the protein composition was associated with a significant rise in positively charged amino acids throughout the cold plasma treatment compared to the untreated protein. The slight red shift in fluorescence intensity data of the plasma protein suggests partial unfolding of the protein structure, correlating with increased hydrophobicity and negative surface charge. The difference between the Gʹ and Gʺ values of the insoluble complex for the untreated proteins increased from < 1 log to ~ 1 log after 10 min exposure time indicating the formation of a stable network structure. The complexes formed at optimal pH (pHopt) of 3.2 and 3.0 could be useful in acidic beverages and may serve as fat replacers in low fat food products. Hence, cold plasma treatment for 5 min could be recommended for a more stable structure. The complexes formed at optimal pH (pHopt) of 3.2 and 3.0 could be useful in acidic beverages and may serve as fat replacers in low-fat food products. Hence, cold plasma treatment for 5 min could be recommended for a more stable structure. Three methods of protein modifications were employed in this study, pH fractionation in the presence of salt, limited enzymatic hydrolysis and emerging atmospheric cold plasma technology. Due to the observed structural changes following each method of modification, a choice of modification strategy depends on the intended objective. The pH fractionation with salt may be adequate for protein sources where high yield is required to produce mild acidic food products. Limited enzymatic hydrolysis and polysaccharides could be appropriate for food products with more stable and better network structure in a wider pH range while atmospheric cold plasma being a non-thermal treatment may be appropriate for heat sensitive food products and where crosslinking of the protein is required. The results of this study are uniquely differentiated by the key variables associated with each modification method, namely: exposure time in cold plasma treatment, enzyme selection in limited hydrolysis, and the range of pH and salt concentrations applied during the extraction fractionation process. This variability highlights the complexity of the research topic and suggests that further investigation is needed to fully understand the relationships between these variables. Additionally, there is a need to determine different results that could be produced when the variables are extended and their applications in other food products.