Defatting Method Shapes the Digestive Performance and Bioaccessibility of Brazil Nut Protein Concentrates

This study proposed to evaluate the impact of different defatting routes on the digestive performance of Brazil nut (Bertholletia excelsa) protein concentrate (~50% protein). The cold-pressed cake was subjected to three different extractions for the complete removal of residual oil: Soxhlet extraction with hexane (CPPH sample) or bioethanol (CPPB sample) at the boiling point of each solvent (15:1 v/m ratio for 8 h), and extraction using supercritical CO₂ (sc-CO₂, CPPS sample) at 40 °C and 300 bar for 4.5 h. After desolventization (50 °C/48 h), the concentrates were subjected to simulated dynamic gastrointestinal digestion using the INFOGEST method. The obtained fractions (jejunum, ileum, and undigested) were evaluated for yield, soluble proteins (Lowry), hydrolysis degree (TNBSA), total phenolic compounds (Folin-Ciocalteu), antioxidant activity (inhibition of ABTS and DPPH radicals), and antihypertensive activity (ACE inhibition). All analyses were performed at 37 °C, and the data were analyzed using analysis of variance (ANOVA) and Tukey's test (p < 0.05). The results revealed that the choice of solvent directly impacts the digestive behavior of the matrix. The volumetric yield was mainly concentrated in the intestinal phases (jejunum and ileum) for all extractions (~78 to 84%). However, sc-CO₂ altered the release profile: the CPPS sample exhibited a significant increase in protein solubility, reaching 39.0 ± 1.7% in the jejunum and 30.5 ± 2.5% in the ileum, surpassing the use of hexane (~33.3% and ~18.4%) and bioethanol (~26.5% and ~23.8%). The hydrolysis degree demonstrated high enzymatic digestibility for all samples (>75%). Statistically, the overall content of phenolic compounds was equal among the obtaining methods (~190 mg GAE/100 g) (p > 0.05). However, supercritical extraction increased the bioaccessibility of these compounds in the absorption phases (jejunum: ~64.8 mg GAE/100 g; ileum: ~53.9 mg GAE/100 g), whereas in conventional methods, the majority remained trapped and undigested in the residual fraction (retaining up to ~86.4 mg GAE/100 g in the bioethanol-treated matrix). Regarding bioactive properties, the ABTS assay indicated high antioxidant performance for all matrices, suggesting that the peptides and other antioxidant compounds released are mainly hydrophilic. The DPPH assay demonstrated that CPPS released peptides with high antioxidant activity in the intestinal fractions (up to ~681 µmol Trolox/g). Antihypertensive activity was also favored by the supercritical technology: CPPS achieved ~92.9% ACE inhibition in the jejunum and ~82.8% in the ileum, outperforming hexane (~85.3% and ~71.1%) and bioethanol (~77.7% and ~41.3%). Thus, the substitution of organic solvents with sc-CO₂ favorably modifies the protein matrix, facilitating solubilization and enhancing the bioaccessibility of phenolics and functional peptides during simulated digestion.

Acknowledgments: CAPES (Finance Code 001 and 88887.847010/2023-00), FAPESP (2022/10368-4, 2024/23581-3, and 2025/15916-8), and CNPq (306753/2025-1).