Ultrasound pretreatment and fermentation temperature improve phytochemical, antioxidant capacity, and mineral bioaccessibility in tempeh under simulated digestion

• Fermentation of tempeh at the temperature of 36 °C significantly enhances phenolic and flavonoid content, antioxidant capacity, and calcium concentration. • Ultrasound pretreatment prior to fermentation at 36 °C improves flavonoid bioaccessibility and calcium release under simulated gastrointestinal conditions. • Flavonoid levels exhibit a strong positive correlation with calcium release following in vitro digestion. This study investigated how ultrasound pretreatment and controlled fermentation temperature influence phytonutrient release, antioxidant capacity, and mineral bioaccessibility in tempeh under simulated gastrointestinal conditions. Soybeans underwent ultrasound pretreatment soaking and cooking, followed by fermentation with Rhizopus oligosporus for 48 h at 30 °C or 36 °C. Total phenolic content (TPC), total flavonoid content (TFC), antioxidant activity (FRAP and CUPRAC), and mineral bioaccessibility (calcium, magnesium, and iron) were measured in both undigested and in vitro digested samples. Correlation analyses were conducted to explore functional interactions between phytonutrients, antioxidant potential, and mineral release. Compared with unfermented soybeans, tempeh fermented at 36 °C showed significantly higher TPC, TFC, FRAP, CUPRAC, and calcium concentrations, while magnesium and iron levels remained unchanged. Ultrasound pretreatment soaking and cooking with fermentation at 36 °C promoted TPC and TFC accumulation before digestion and yielded the highest TFC, FRAP, and calcium levels after simulated digestion. Bioaccessibility analysis revealed that TFC, calcium, and iron were significantly higher in ultrasound pretreatment soaking and cooking fermented at 36 °C. A strong positive correlation was observed between flavonoid levels and calcium release ( r = 0.929, p < 0.001). The combination of ultrasound pretreatment and elevated fermentation temperature enhance the release and bioaccessibility of flavonoids, calcium, and iron in tempeh. The strong correlation between flavonoid content and calcium release underscores their interrelated roles. These findings highlight ultrasound pretreatment combined with controlled fermentation temperature as a practical food processing strategy to improve the nutritional profile, bioaccessibility, and functional quality of plant‑based fermented foods under simulated gastrointestinal conditions.