Exploring green proteins from pumpkin leaf biomass: assessing their potential as a novel alternative protein source and functional alterations via pH-Shift treatment

As sustainability continues to be a major global focus, the use of agro-industrial waste as a renewable resource presents a promising solution. In this study, the potential of pumpkin leaf biomass, a type of agro-industrial waste, will be explored as an alternative protein source using thermal coagulation. A key challenge is the accumulation of green proteins, a protein-rich byproduct. This waste fraction, which has been insufficiently investigated due to its limited functional properties, is the main focus of this research, aimed at enabling complete biomass utilization. The novelty of this research lies in the application of combined pH-shift and controlled heat treatment to improve the solubility of green proteins isolated from pumpkin leaf biomass - an underexplored protein fraction derived from agro-industrial waste. The obtained crude green protein powder, with a yield of 47.95 g/kg of leaf dry biomass, exhibited a satisfactory composition, containing 53.58% protein, a high-quality amino acid profile, and notable antioxidant properties. On the other hand, it exhibited low solubility, below 25%, across the pH range of 2-10, with the isoelectric point at pH 4.4. Following the application of an optimized pH-shift and heat treatment, green proteins solubility increased significantly, reaching 89.74% at pH 8, nearly 4.5 times higher than before treatment, showing low solubility only at the isoelectric point and excellent stability maintained in salt concentrations up to 1 M NaCl. A notable decrease in particle size was observed, from 1883 nm to 192 nm, leading to a more uniform particle size distribution. SDS-PAGE and FTIR deconvolution analyses indicated structural changes, possibly involving disulfide bond formation. The promising findings of this research, not only ensure complete biomass utilization, but also highlight the need for further investigation regarding the potential of green modified proteins, as the observed improvements open up numerous biotechnological applications. One potential direction is green protein-stabilized emulsions, supported by a significant increase in alpha-helix content associated with enhanced functional properties, which offers new possibilities for future research on stable and functional emulsions.