2.2.2. Solid state fermentation (SSF)
After removing impurities, the soybean samples (50 g) were
washed, soakedin de-ionizedwater (15)at4 ◦Cfor approximately
24 h until the weight increase to about twice of the original dry
weight. After being drained, the soaked beans were mixed with 5 g
NaCl salt and 1 g sucrose in a 250-ml flask, then placed in autoclave
at 121 ◦C for 15 min. 2ml bacterial inoculums prepared above or
2ml of natto bacteria liquid (prepared by suspending 0.1 g of natto
bacteria in 10–20 ml of sterilized water) were used to inoculate.
The mixture was stirred with a sterilized spatula so that the natto
bacteria will be evenly distributed, followed by static inoculation at
37 ◦C for 24 h. To extract the enzyme for assaying, 50 ml sterilized
de-ionizedwater (15)was used for extraction. The solid material
and bacteria were removed by filtering through cotton cloth, followed
by centrifugation at 5600×g, 4 ◦C for 20 min. After removing
bacteria, the liquid extract of crude enzyme was used to assay for
milk-clotting activity.