A Novel Non-Cyanide Dried Cassava Mash (DCM) as a Potential Functional Food Ingredient: Immunomodulatory Activity and Subacute Toxicity Assessment

Cassava is a staple food with significant nutritional value, but its cyanogenic glycosides pose health risks. This study assesses a novel Non-Cyanide Dried Cassava Mash (DCM) , produced according to the process disclosed in PCT/IB2025/062215 (patent pending), for its immunomodulatory activity and safety as a potential functional food ingredient. DCM was characterized for basic safety (cyanide, microbiological quality) and functional properties. Immunomodulatory effects were evaluated in male F1 hybrid (CBA × C57BL/6) mice following 8-day oral administration of freshly prepared DCM (5 g/100 mL drinking water). Outcomes included spleen and thymus mass, spleen cellularity, antibody-forming cells (AOC), and splenic cytokine levels (IL-2, IFN-γ, IL-4, IL-10) by ELISA. A 28-day subacute oral toxicity study was performed in rats (OECD 407). DCM had undetectable cyanogenic potential (< 1.0 mg HCN eq/kg) and no pathogenic microorganisms. Short-term oral DCM significantly increased spleen cellularity (210 ± 6.3 × 10⁶ vs. 183 ± 8.8 × 10⁶; p < 0.05) and enhanced Con A-stimulated splenic IL-2 (78.5 ± 8.4 vs. 45.2 ± 6.1 pg/mL; p = 0.008) and IFN-γ (315 ± 22 vs. 210 ± 18 pg/mL; p = 0.003), with no change in IL-4 or IL-10. A trend toward increased AOC was observed (302 ± 26.0 × 10³ vs. 265 ± 5.6 × 10³; p = 0.12). The 28-day toxicity study showed no adverse effects on body weight, hematological, hepatic, or renal markers. In conclusion, DCM exhibits Th1-biased immunostimulatory activity in a murine model and is non-toxic following subacute oral administration. These results support the safety and potential of DCM as a functional food ingredient with immunomodulatory benefits.