2.2. Red Bean Fermentation

Red beans were washed and soaked for twelve hrs. After drying, water of twice the weight of red beans and 1% lactic acid were added, and followed by cooking at 100 °C for 30 min, and the cooked beans were then dried and cooled to 37 °C. A total of 100 g of cooked red beans was placed on an aluminum plate (10 × 20 cm) with an aluminum cover and thoroughly mixed with 5 mL of R. microsporus var. oligosporus IFO 8631 (ATCC 22959) containing 1 × 10⁶/mL spores. The mixture was incubated for two days in an aerobic environment at 37 °C. The fermented tempeh product was then freeze-dried for two days and ground to a powder for analysis. The above experiment was repeated at 30 °C. Samples were prepared according to a previously-described method with some modifications [25]. Briefly, one gram of fermented red beans was extracted using 15 mL of 70% alcohol and shaken for 1 min and put into 4 °C cold room for one hour and then centrifuged at 6000× g for 20 min. The whole extracting procedure was repeated three times before the supernatant was extracted and concentrated using a rotary evaporator (IKA RV10, Staufen im Breisgau, Germany) to 15 mL and stored in refrigerator.