Purification and characterisation of the native black soldier fly larva protein

Abstract

The larvae of the black soldier fly (BSF) are one of the promising edible insect species for industrial food production, showing great potential as a sustainable alternative to conventional protein sources. To fully utilize the potential of insect protein as food, it is important to refine and enhance its functional characteristics. This study aimed to investigate the effects of various purification and characterization techniques, such as salting in/out, ultrafiltration, and ultrasound treatments, on native BSF larva proteins. The goal was to enhance their functional and nutritional properties for potential food-related applications. Protein isolates were extracted using alkaline extraction and acid precipitation (AAF), salting-in (AAFSI), salting-out (AAFSO), and salting-in-out (AAFSISO) coupled with ultrafiltration. Additionally, BSF larval proteins were subjected to ultrasound treatment at varying power levels (150 W, 200 W, 250 W, and 300 W) and treatment durations (10, 20, 30, and 40 min). The physicochemical properties, such as colour change, amino acids, zeta potential and sulfhydryl group levels, were measured. Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), x-ray diffraction and circular dichroism (CD) were used to analyze protein structures, while functional properties like solubility, surface hydrophobicity, water binding capacity, oil binding capacity, foaming capacity, and foam stability were evaluated. The extraction method had a significant impact (p < 0.05) on the crude protein content of the isolates. For example, the AAF extraction yielded 71.25% protein, which increased to 85.02% with the addition of salting-out-assisted treatment. Furthermore, the total essential amino acid content was 320.48 mg/g for AAF, 300.40 mg/g for AAFSI, 286.15 mg/g for AAFSO, and 296.88 mg/g for AAFSISO. These values all exceed the World Health Organization's recommended minimum of 277 mg/g for adult daily requirements. The zeta potentials of all samples were found to be significantly negative (p < 0.05), with values ranging from -9.81 to -26.86 mV, with no significant differences (p > 0.05) observed in the levels of exposed sulfhydryl groups between AAF and AAFSI proteins. FTIR of protein extracts derived from AAFSO and AAFSISO revealed significant N–H free stretching vibrations, indicating structural transformations confirmed by CD analysis. Furthermore, this study thoroughly examined the influence of alkaline extraction and isoelectric precipitation methods assisted by salting-in (1% NaCl) and salting-out (80% (NH4)2SO4) techniques on the functional properties of BSF larvae proteins. The addition of (NH4)2SO4 significantly (p < 0.05) improved protein solubility, particularly at pH 2, suggesting its potential use in acidic food systems. Correlation analysis demonstrated a significant positive relationship (p < 0.05) between various functional properties, including foaming capacity, foam stability, emulsification properties, solubility, and surface hydrophobicity, emphasizing their interconnected nature and potential applications in food formulations. Foaming capacity and foam stability showed a positive relationship with protein solubility at pH 4 (r = 0.88, p < 0.05; r = 0.74, p < 0.01, respectively). Additionally, the impact of ultrasound treatment on enhancing the functional and nutritional attributes of BSF larval proteins was investigated. Ultrasound, a promising technique for modifying protein characteristics, was employed at different power levels (150 W, 200 W, 250 W, and 300 W) and treatment durations (10, 20, 30, and 40 min), inducing structural modifications and enhancing functional properties. Ultrasound treatment at varying power levels and durations significantly increased the lightness (L*), solubility, surface hydrophobicity, water-binding capacity, oil-binding capacity, foaming capacity, and foam stability of BSF larval proteins. These improvements were most pronounced at higher ultrasound power and longer treatment times. P200T10 exhibited higher lightness (L* = 74.99, p > 0.05) and reduced redness (a* = 2.55, p < 0.05) compared to the other isolates. Optimal conditions, such as 200 W for 40 min (foaming) and 250 W for 10-20 min (emulsification), maximized these properties. Excessive treatment, however, could lead to protein aggregation and reduce functionality. FS showed a positive correlation with solubility (r = 0.70, p < 0.001), supporting the idea that ultrasound disrupts disulfide bonds and exposes sulfhydryl groups. However, FS and exposed SH exhibited weak or negative correlations with WBC (r = -0.29, p < 0.05) and OBC (r = 0.35, p < 0.05), suggesting that while these structural changes improve solubility, they may decrease binding capacities The integration of salting-out extractions, ultrafiltration, and ultrasound treatments effectively enhanced the functional and nutritional properties of BSF larval proteins. These techniques produced insect proteins with excellent nutritional quality and functional properties, making them suitable as sustainable functional ingredients for various food-related applications. Future research should focus on optimizing these extraction and treatment techniques to ensure scalable and commercially viable production of BSF larval proteins. This includes fine-tuning ultrasound parameters and exploring additional treatment combinations to maximize the potential of BSF larvae as a sustainable and functional protein source in the global protein market.