**2. Materials and methods**

#### **2.1 Strain**

*B. subtilis* RB14-CS which is a spontaneous mutant derived from RB14-C is a single iturin A producer. *B. subtilis* RB14-C is a streptomycin-resistant mutant from a parent strain RB14 and is a co-producer of the antibiotics iturin A and surfactin (Asaka & Shoda, 1996).

#### **2.2 Solid-state fermentation (SSF)**

The detail of SSF was described in the previous paper (Mizumoto et al., 2006). The L medium used for the growth of the bacterium contained 10 g of Polypepton (Nippon Pharmaceutical Co., Tokyo, Japan), 5 g of yeast extract and 5 g of NaCl (per liter). One ml of L medium culture broth after 24 h cultivation at 30°C was inoculated into 100 ml of number 3S (no. 3S) medium consisting of 30 g of Polypepton S (Nippon Pharmaceutical Co., Tokyo), 10 g of glucose, 1 g of KH2PO4, and 0.5 g of MgSO4・7H2O (per liter) (pH 6.8), and the culture was incubated at 120 strokes per minute (spm) at 30°C for 24 h in a shaking flask and used as a seed for SSF.

The soybean curd residue was supplied from a *tofu* company in Tokyo and stored at - 20°C. Each of fifteen grams of thawed soybean curd residue was placed in a 100-ml conical flask and autoclaved twice at 120°C for 20 min at an interval of 8-12 h to kill spore-forming microorganisms inhabiting the material. After cooling to room temperature, the following solutions were added as nutrient supplements for every 15 g of soybean curd residue and moisture content was adjusted to 79%: 833 L of 0.45 g glucose /ml, 75 L of 1 M KH2PO4, 150 L of 1 M MgSO4·7H2O and 367 L of deionized distilled water. Then, 3 mL of an RB14- CS culture grown in no. 3S medium was added to 15 g of soybean curd residue and mixed with a stainless steel spatula. All flasks were incubated statically in a water incubator at 25°C, and at a specified time, one flask was taken and the whole soybean curd residue in a flask was used as a sample for analysis.

#### **2.3 Preparation of samples for acid and neutral detergent fiber analysis**

After 5 days of SSF by *B. subtilis* RB14-CS, the whole solid culture was dried by microwave and ground by using a pestle and a mortar. Raw soybean curd residue was used as a control.

#### **2.4 Acid and neutral detergent fiber analysis 2.4.1 Acid detergent fiber**

The content of acid detergent fiber, which contains mainly cellulose and lignin, was analyzed in the following manner (Van Soest, 1963). In a 150 mL-flat bottom flask, 0.45 – 0.55 g of ground sample was weighed using micro-balance and 50 mL of acid detergent solution (20 g/L cetyl trimethylammonium bromide in 0.5 M sulfuric acid) was mixed. The flask was placed in an oil bath under the cold water condenser and boiled within 5-10 min. Sample was refluxed for 60 min from onset of boil. After approximately 30 min, the inside of flask was washed with minimal amount of acid detergent solution. After refluxed, sample was filtrated under reduced pressure with a tared Gooch crucible. The crucible was washed twice with hot water, then twice with acetone and was dried at 105°C overnight. After cooled to room temperature in a desiccator, the weight of the crucible was measured.
