Optimising Enzymatic Cross-Linking: Impact on Physicochemical and Functional Properties of Lupin Flour and Soy Protein Isolate

Abstract

The growing demand for plant-based protein alternatives has driven interest in protein modifications to enhance their functional properties in food applications. Enzymatic cross-linking using laccases derived from Rhus vernicifera (LR) and transglutaminase (TG) offers a promising strategy to enhance protein solubility, emulsifying properties, and foaming properties of food proteins. This study varied the enzymatic reaction conditions, including enzyme concentration, pH, temperature, incubation time, and ferulic acid addition, for the most effective cross-linking between proteins in lupin flour (LF) and soy protein isolate (SPI), resulting in changes in physicochemical and functional properties of the cross-linked proteins. LR-induced cross-linking in lupin and soy proteins was most favourable at 142.5 U/100 mg protein, pH 6, and 20 °C, where ferulic acid enhanced cross-linking efficiency with prolonged incubation (20 h). TG-induced cross-linking in lupin and soy proteins was most favourable at 1.25 U/100 mg protein, pH 6 and 30 °C, where high-molecular-weight aggregates were observed. Cross-linking modified protein surface characteristics, increasing ζ-potential and particle size due to protein aggregation, while ferulic acid further enhanced polymerisation. Morphological analysis revealed a porous powder structure across all samples with increased porosity in cross-linked samples as evidenced by the predominance of small fragments within the particles. Prolonged incubation led to partial disaggregation in LR-treated samples unless they were stabilised by ferulic acid. Under mild conditions (1 h, pH 6, 20 °C), LR and ferulic acid-added samples showed minor and significant improvements in protein solubility and foaming stability, respectively. Additionally, a significant increase in foaming ability was observed in ferulic acid-added LR samples after prolonged incubation (20 h), compared to the corresponding control. In contrast, prolonged incubation (20 h) or TG treatment had a lower foaming stability compared to the mild LR treatment. Emulsifying ability and emulsion stability showed limited variation across treatments. These findings suggest that cross-linking conditions influence specific functional properties, highlighting the need for further optimisation to achieve desired protein functionality in food applications.

Keywords: enzymatic cross-linking; functional properties; laccase; lupin flour; physicochemical properties; plant proteins; soy protein isolate; transglutaminase.

Figure 1

Flowchart of experimental design for optimising cross-linking conditions in lupin and soy (LS) mixtures. LF—lupin flour; SPI—soy protein isolate; LR—laccase; TG—transglutaminase.

Figure 2

SDS-PAGE profile of soy protein isolate (SPI) and lupin flour (LF), showing the major protein bands: β-conglycinin and glycinin in SPI, and α, β, γ, and δ-conglutin in LF. Lane 1: Protein marker; Lane 2: SPI; Lane 3: LF.

Figure 3

SDS-PAGE profile showing the cross-linking patterns of lupin and soy (LS) protein mixtures treated with laccase (LR) or transglutaminase (TG) at pH 7 and 30 °C under varying incubation times (1, 5, 10, and 20 h—H). Lane 1: Protein marker; Lane 2: LS-CT-1H Lanes 3–6: LS-LR-1H/5H/10H/20H; Lanes 7–10: LS-TG-1H/5H/10H/20H. CT—Control (non-enzyme-treated). A: Faint bands above 100 kDa in LR-treated samples, indicating limited cross-linking. B–D: New bands in TG-treated samples at 20 h, suggesting enhanced protein modifications. Summary: TG treatment at 30 °C led to more visible cross-linking over time, with smearing and high molecular weight bands, unlike LR, which showed limited time-dependent enhancement.

Figure 4

SDS-PAGE profile showing the cross-linking patterns of lupin and soy (LS) protein mixtures treated with laccase (LR) or transglutaminase (TG) for 1 h at 30 °C at varying pH (4, 5, 6, 7, and 8). Lane 1: Protein marker; Lanes 2–6: LS-CT pH 4–8 (controls); Lanes 7–11: LS-LR pH 4–8 (LR-treated); Lanes 12–16: LS-TG pH 4–8 (TG-treated). CT—Control (non-enzyme-treated). Summary: TG-treated samples showed optimal cross-linking at pH 6 with dark smears and aggregates; LR treatments showed minimal changes across pH.

Figure 5

SDS-PAGE profile showing the effects of varying laccase (LR) (under the addition of ferulic acid—FeA), transglutaminase (TG) concentrations, and incubation temperatures on lupin and soy (LS) protein mixtures. Lane 1: Protein marker; Lane 2: LS-LR-285U-1H-30C; Lanes 3–5: LS-LR-71.25U/142.5U/285U-FeA-1H-30C; Lane 6: LS-LR-285U-1H-20C; Lanes 7–9: LS-LR-71.25U/142.5U/285U-FeA-1H-20C; Lane 10: LS-LR-285U-20H-20C; Lanes 11–13: LS-LR-71.25U/142.5U/285U-FeA-20H-20C; Lane 14: LS-CT-1H-30C; Lanes 15–18: LS-TG-1.25U/2.5U/5U/10U-1H-30C. CT—Control (non-enzyme-treated); U—Enzyme units (U/100 mg protein); H—Treatment time (h); C—Incubation temperature (°C). Summary: LR cross-linking was most effective at 20 °C with 142.5 U/100 mg protein and ferulic acid; TG achieved extensive cross-linking even at low concentrations (1.25 U/100 mg protein) at 30 °C.

Figure 6

ζ-potential of lupin flour (LF), soy protein isolate (SPI), and mixtures of LF and SPI: control (LS-CT), laccase-treated (LS-LR), and transglutaminase-treated (LS-TG). FeA—Ferulic acid-added; H—Treatment time (h); C—Incubation temperature (°C). Different letters (a–c) indicate significant differences among samples (p < 0.05).

Figure 7

Particle size distribution of lupin flour (LF), soy protein isolate (SPI), and mixtures of LF and SPI: control (LS-CT), laccase-treated (LS-LR), and transglutaminase-treated (LS-TG). FeA—Ferulic acid-added; H—Treatment time (h); C—Incubation temperature (°C).

Figure 8

Morphology of lupin flour (LF), soy protein isolate (SPI), non-enzyme-treated LF and SPI mixture (LS-CT), and enzyme-treated mixtures with laccase (LS-LR) and transglutaminase (LS-TG) at 20 °C and 30 °C, respectively. FeA—Ferulic acid-added; H—Treatment time (h); C—Incubation temperature (°C).

Figure 9

Protein solubility of lupin flour (LF), soy protein isolate (SPI), non-enzyme-treated LF and SPI mixture (LS-CT), laccase-treated mixture (LS-LR), and transglutaminase-treated mixture (LS-TG). FeA—Ferulic acid-added; H—Treatment time (h); C—Treatment temperature (°C). Different letters (a–e) indicate significant differences among samples (p < 0.05).

Figure 10

Emulsifying ability (a) and emulsion stability (b) of lupin flour (LF), soy protein isolate (SPI), non-enzyme-treated LF and SPI mixture (LS-CT), laccase-treated mixture (LS-LR), and transglutaminase-treated mixture (LS-TG). FeA—Ferulic acid-added; H—Treatment time (h); C—Treatment temperature (°C). Different letters (a–c) indicate significant differences among samples (p < 0.05).

Figure 11

Foaming ability (a) and foaming stability (b) of lupin flour (LF), soy protein isolate (SPI), and mixtures of LF and SPI: control (LS-CT), laccase-treated (LS-LR), and transglutaminase-treated (LS-TG). FeA—Ferulic acid-added; H—Treatment time (h); C—Treatment temperature (°C). Different letters (a–e) indicate significant differences among samples (p < 0.05).