OPTIMIZATION OF ENZYMATIC HYDROLYSIS OF HERMETIA ILLUCENS LARVAL PROTEIN CONCENTRATE USING RESPONSE SURFACE METHODOLOGY

The utilization of alternative and highly sustainable protein matrix configurations, such as edible insects, has emerged as a premier pathway for the industrial biorefinery production of therapeutic functional ingredients. This comprehensive investigation establishes an optimized upstream chemical extraction and catalytic processing framework designed to maximize the generation of low-molecular-weight bioactive antioxidant peptide fractions derived from black soldier fly (Hermetia illucens) larvae (BSFL) protein concentrate. In the primary extraction phase, the raw centesimal composition of the baseline whole-larvae flour was thoroughly characterized, containing 61.81% moisture, 44.37% lipid, 6.50% ash, 8.03% structural chitin, and 28.76% crude protein on a dry matter basis. Through a sequential downstream strategy utilizing mechanical pressing, multi-stage hexane defatting, and alkaline pH-shifting solubilization, a highly purified concentrated protein isolate was successfully prepared, registering an absolute protein level of 67.64% while achieving a total protein extraction efficiency of 41.9%. Following substrate preparation, controlled enzymatic hydrolysis was executed using a commercial protease derived from Aspergillus oryzae to break down the dense raw matrices. This proteolysis system was systematically modeled and validated using a three-factor, five-level Central Composite Design (CCD) matrix combined with Response Surface Methodology (RSM). The independent operational parameters—comprising processing incubation temperature (38.18–71.81 °C), matrix pH (2.64–9.36), and reaction time (0.32–3.68 h) were quantitatively evaluated and balanced against three dependent variables: the degree of hydrolysis (DH, %), DPPH free-radical scavenging capacity, and Ferric Reducing Antioxidant Power (FRAP) metrics. The resulting quadratic linear regressions revealed excellent fit coefficients (R² ≥ 0.9964), confirming that the biological uncoiling and subsequent peptide generation are strictly governed by the linear vectors of environmental pH along with profound negative quadratic curvature constraints for both temperature and processing acidity. Multi-objective numerical optimization identified the ultimate operational vector to be a stable incubation temperature of 55.0 °C, an optimized aqueous pH of 7.76, and a duration of 2.0 hours. This precise factor combination shifts the extraction medium well away from the native isoelectric point of BSFL proteins, ensuring maximized substrate solubility and accelerating enzyme accessibility. At this optimal intersection, empirical validation campaigns yielded excellent response metrics, achieving a maximum degree of hydrolysis of 36.94 ± 0.15%, a DPPH radical scavenging capacity of 32.08 ± 0.12%, and a FRAP capacity of 31.35 ± 0.22 %.These results confirm a tight 99.7% compliance with mathematical model predictions. This complete framework demonstrates an efficient, scalable, and highly reproducible pathway to transform sustainable insect biomass into high-value antioxidant peptide formulations.

Funding: National Council for Scientific and Technological Development (CNPq); Coordination for the Improvement of Higher Education Personnel (CAPES, Finance Code 001); Carlos Chagas Filho Research Support Foundation of Rio de Janeiro State (FAPERJ); Funding Authority for Studies and Projects (Finep); and LetsFly.