Green Supercritical CO2 Technology Improves Digestive Performance and Bioactivities of Brazil Nut Protein Concentrates

This study proposed the use of supercritical CO₂ (sc-CO₂) as a green strategy to improve the digestive performance of Brazil nut (Bertholletia excelsa) protein concentrate (Control, ~50% protein (m/m)), obtained by cold-pressing followed by residual oil extraction using Soxhlet with bioethanol. In previous studies evaluating different operating conditions of temperature (40 to 60 °C) and pressure (100 to 300 bar), the treatment at 60 °C and 300 bar for 1 hour stood out for optimizing the techno-functional properties of the matrix. Based on this, the present study evaluated the behavior of this optimized condition (HTHP sample) compared to the control sample (without sc-CO₂) after simulated dynamic gastrointestinal digestion using the INFOGEST method. The resulting fractions, jejunum, ileum, and undigested, were characterized regarding yield, soluble proteins by the Lowry method, hydrolysis degree by TNBSA, total phenolic compounds by the Folin method, antioxidant activity by ABTS and DPPH radical inhibition, and antihypertensive activity by ACE inhibition. The analyses were performed at 37 °C and the data were analyzed using ANOVA and Tukey's test (p < 0.05). Regardless of the applied treatment, the yield of the collected fractions was higher in the jejunum and ileum, accounting for 40 to 45% each (p > 0.05). The undigested fraction represented only 10 to 15% of the total content. However, the sc-CO₂ treatment altered the release profile during digestion. The HTHP sample exhibited a significant increase in soluble proteins in the intestinal fractions, reaching 35.5 ± 1.7% in the jejunum (vs. 26.1 ± 2.1% in the Control) and 30.6 ± 1.2% in the ileum (vs. 23.7 ± 3.4% in the Control). The hydrolysis degree indicated efficient protein breakdown (>75% in the jejunum and ileum) in both samples. Despite the similar total phenolic content (~190 mg GAE/100 g), sc-CO₂ increased the bioaccessibility of these compounds in the jejunum (68.1 ± 1.4) and ileum (59.7 ± 5.0) (p < 0.05), whereas in the control, the majority remained undigested (89.4 ± 3.0). Regarding antioxidant activity, using the ABTS method, strong activity was observed for all samples (~10.4 mmol Trolox/g), also indicating a greater presence and action of hydrophilic antioxidant compounds. In turn, the DPPH assay revealed a release of peptides with higher antioxidant potential in the intestinal fractions, reaching averages of up to 700 µmol Trolox/g, regardless of the treatment. Antihypertensive activity was higher in the jejunum (~70–80%). However, the HTHP treatment promoted an increase in the activity of the ileum fraction (~64% vs. ~42% for the Control). Therefore, in addition to improving technological parameters, sc-CO₂ modifies the protein matrix, facilitating solubilization and enhancing the bioaccessibility of antioxidant and antihypertensive compounds during digestion.

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).