**2.4.2 Neutral detergent fiber**

524 Recent Trends for Enhancing the Diversity and Quality of Soybean Products

wild strains and their derivatives suppressed 26 types of plant pathogen *in vitro* (Phae et al., 1990) and a fungal disease *in vivo* (Asaka & Shoda, 1996) by producing three lipopeptide antibiotics, iturin A, surfactin and plipastatin (Asaka & Shoda, 1996; Hiraoka et al., 1992; Tsuge et al., 1996, 1999). The suppressive effect of one of the isolates, *B. subtilis* RB14, was mainly associated with the cyclolipopeptide antibiotic iturin A, which contains seven amino acids and one -amino acid. *B. subtilis* RB14-CS, a derivative of the original strain RB14 and a sole producer of iturin A, produced iturin A in SSF using the soybean curd residue 3-fold higher than in submerged fermentation (SmF) (Mizumoto et al., 2006). This suggests that RB14-CS could degrade some kinds of insoluble fibers in soybean curd residue and utilize them as carbon sources during SSF. In this chapter, insoluble fibers in soybean curd residue that RB14-CS could degrade during SSF were clarified and the fiber-degrading

*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

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

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

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

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

and is a co-producer of the antibiotics iturin A and surfactin (Asaka & Shoda, 1996).

enzymes were purified and characterized.

**2. Materials and methods** 

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

flask was used as a sample for analysis.

**2.1 Strain** 

as a seed for SSF.

control.

The content of neutral detergent fiber which contained mainly cellulose, lignin, and hemicellulose was analyzed in the following manner (Van Soest, et al., 1991). In a 300 mLround bottom flask, 0.45 – 0.55 g of ground sample, 50 mL of neutral detergent solution (13.5 g of sodium dodecyl sulfate, 8.38 g of EDTA disodium salt, 3.07 g of NaB4O7・10H2O, 5.18 g of Na2HPO4・12H2O and 4.5 mL of tryethylene glycol per 450 mL) and 0.5 g of sodium sulfite were mixed. The flask was placed in an oil bath under the cold water condenser and boiled for 5 min. After 5 min of boiling, 2 mL of α-amylase solution, which consists of heat-stable α-amylase (Kleistase T10S; Daiwa Kasei, Shiga, Japan) and 50 mM sodium phosphate buffer (pH 6.0) (1:39 [vol/vol]), were mixed. Then, the sample was refluxed for 60 min. After approximately 30 min, the inside of flask was washed down with minimal amount of neutral detergent solution. After refluxed, the sample was filtrated under reduced pressure with a tared Gooch crucible. The crucible was filled with 2 mL of α-amylase solution and hot water, and incubated for at least 2 min. Then, the crucible was washed twice with hot water, and then twice with acetone. The crucible was dried at 105°C overnight. After cooled to room temperature in a desiccator, the weight of the crucible was measured.

#### **2.4.3 Calculation of content of insoluble fibers**

As the amount of acid detergent fiber was regarded as total amount of cellulose and lignin, the amount of the neutral detergent fiber minus the amount of acid detergent fiber was regarded as the content of hemicellulose.

### **2.5 Iturin A production in liquid culture using insoluble fibers**

In a 200-mL conical flask, 40 mL of liquid medium consisting of 10 g of fibrous carbon sources, 10 g of Polypepton S, 1 g of KH2PO4 and 0.5 g of MgSO4・7H2O (per liter) (pH 6.8) was prepared. As fibrous carbon sources, xylan (Tokyo Chemical Industry, Tokyo, Japan), avicel, carboxymethyl cellulose, and pectin were used. As a control carbon source, glucose was used. Four hundreds μL of a seeding culture was inoculated into the medium and the flasks were incubated at 30°C at 120 spm.

For measurement of iturin A concentration, 1 mL of culture broth was acidified to pH 2.0 with 12 N HCl. Iturin A was collected by centrifugation at 18,000 ×*g*, at 4°C for 10 min, and extracted with 1 mL of methanol. The extract was injected into a high-performance liquid chromatography (HPLC) with a column (Chromolith Performance RP-18eb 4.6 mm

Characterization of Enzymes Associated

mass cut-off 10 kDa; Advantec, Tokyo, Japan).

mL/min and a volume of one fraction was 2 mL.

**2.10 Molecular mass determination** 

**2.11 N-terminal sequence analysis** 

with Degradation of Insoluble Fiber of Soybean Curd Residue by *Bacillus subtilis* 527

supernatant to 30 % saturation, and the precipitate was removed by centrifugation. Then, ammonium sulfate was added to 70 % saturation. The precipitate was recovered by centrifugation, suspended in 50 mM MES buffer (pH 6.0) and dialyzed overnight against the same buffer. Then the sample was concentrated by ultrafiltration with YM10 (molecular

The concentrate was applied to a CM-Toyopearl column (1.3 cm Φ×8.3 cm; Tosoh, Tokyo, Japan) pre-equilibrated with buffer A (50 mM MES buffer, pH 6.0), and fractions were eluted with a continuous linear gradient of 0-0.5 M NaCl in buffer A (total volume 120 mL). The flow speed and the volume of one fraction were 4 mL/min and 8 mL, respectively. In this process, xylanase activity was detected in two fractions, one of which was trapped in the column (Fraction I) and the other was not trapped in the column but passed through

Fraction I was concentrated using Centriprep YM-10 (molecular mass cut-off 10 kDa; Millipore, Tokyo, Japan), diluted with buffer A and applied to a RESOURCES column (0.6 cm Φ×3.0 cm; Pharmacia Biotech, Uppsala, Sweden) pre-equilibrated with buffer A. Fractions were eluted with a continuous linear gradient of 0-0.15 M NaCl in buffer A (total volume 30 mL). The flow speed and the volume of one fraction were 1 mL/min and 1 mL, respectively. The xylanase active fractions were concentrated with Centriprep YM-10 and applied to a Superdex 75 column (1.6Φ×60 cm; Amersham Bioscience, Tokyo, Japan) preequilibrated with buffer A containing 0.2 M NaCl. The elution was carried at a flow rate of 1

The pH of the Fraction II was adjusted to 9.5 by adding NaOH and applied to a QAE-Toyopearl (1.6Φ×3.7 cm; Tosoh) pre-equilibrated with buffer B (25 mM piperazine buffer, pH 9.5), and fractions were eluted with a continuous linear gradient of 0-0.5 M NaCl in buffer B (total volume 120 mL). The flow speed and the volume of one fraction were 4 mL/min and 8 mL, respectively. The xylanase active fractions were concentrated with Centriprep YM-10, and fractions were diluted with buffer B and applied to a QAE column. Step elution was performed with 0.07 M NaCl (total elution volume 96 mL). The flow speed

The xylanase active fractions were supplied to the subsequent Butyl-Toyopearl chromatography. A column of Butyl-Toyopearl (1.6Φ×4.5 cm; Tosoh) pre-equilibrated with 25 mM piperazine buffer containing 1 M ammonium sulfate was used. Ammonium sulfate was added to the active fractions and its concentration was adjusted to 1 M. This solution was then applied to the column and the elution was carried out with a linear gradient of 1-0 M ammonium sulfate in 25 mM Piperazine buffer (total volume 180 mL). The flow speed

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed with a 12.5 % gel in accordance with the Laemmli method (Laemmli, 1970). M. W. Marker "Daiichi" II (Daiichi Pure Chemicals, Tokyo, Japan) was used as a molecular mass marker.

SDS-PAGE of xylanases was performed according to the above-described method and then the xylanases on the gel were electroblotted to a commercial membrane (Immobilon-P;

After electrophoresis, the gel was stained with Coomassie brilliant blue R-250 (CBB).

(Fraction II). These fractions were subjected to further purification processes.

and the volume of one fraction were 4 mL/min and 8 mL, respectively.

and the volume of one fraction were 4.5 mL/min and 9 mL, respectively.

diameter× 100 mm height, Merck, Germany) to determine iturin A concentrations. The HPCL system was operated at a flow rate of 2.0 mL/min with acetonitrile-10 mM ammonium acetate (65:35 [vol/vol]) at a column temperature of 40°C. The elution was monitored at 205 nm by a UV detector (880-UV, Intelligent UV/VIS Detector, Jasco, Tokyo, Japan).

Although iturin A has 8 homologues with different side-chain structures (Asaka & Shoda, 1996), the concentration of iturin A was defined as the total amount of five major homologues. The correlation between the peak heights and the concentration of pure iturin A (Sigma-Aldorich, Tokyo, Japan) was used for quantification. Iturin A concentration was expressed as μg/ g initial wet soybean curd residue.
