**1. Introduction**

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

Zhang, L., & Zeng, M. (2008). Proteins as sources of materials, In: *Monomers, Polymers and* 

Zhang, L. (2008). *Physicochemical, morphological, and adhesion properties of sodium bisulfite* 

 <http://krex.k-state.edu/dspace/bitstream/2097/1707/1/Lu%20Zhang2008.pdf> Zhang, S., Zhao, S., Tian, Y., Li, F.-X. & Yu, J.-Y. (2009). Preparation of cellulose/soy protein

Elsevier, ISBN 9780080453163, Oxford, Boston

University. April 20, 2011, Available from:

Vol. 59, No. 2, pp. 106-107. ISSN 1434-3584

*Composites from Renewable Resources*, M. N. Belgacem and A. Gandini, pp. 479-493,

*modified soy protein components – B. S. Thesis.* Kansas, Manhattan: Kansas State

isolate blend biofibers via direct dissolving approach, *Chemical Fibers International*,

Soybean curd residue is a residue of soy milk processing in which most soluble nutrients of soybean are extracted to liquid phase, and thus major carbon sources of the residue are insoluble fibers (O'tool, 1999) which amount to 40.2- 43.6 % on a dry matter basis (Van der Riet et al.,1989). Approximately 700,000 tons of the soybean curd residue were produced annually as a byproduct of *tofu* manufacturing in Japan and most of them is incinerated as an industrial waste. We re-utilized the soybean curd residue as a solid substrate of solidstate fermentation (SSF) using *Bacillus subtilis* (Mizumoto et al., 2006).

The insoluble fibers of soybean consist of cellulose, hemicellulose and lignin. Cellulose is the most abundant biological polymer on earth and is the major constituent of the plant cell wall. This lineal polymer is composed of D-glucose subunits linked by β-1,4 glycosidic bonds forming cellobiose molecules and the long chains are linked together by hydrogen bonds and van der Waals forces (Perez et al., 2002). Hemicellulose is a complex of polymeric carbohydrates which contains xylan, xyloglucan, (heteropolymer of D-xylose and Dglucose), glucomannan (heteropolymer of D-glucose and D-mannose), galactoglucomannan (heteropolymer of D-galactose, D-glucose and D-mannose) and arabinogalactan (heteropolymer of D-galactose, D-glucose and arabinose). Among them, xylan, a complex polysaccharide comprising a backbone of xylose residues linked by β-1,4-glycosidic bonds, is the major component. Xylan is the second most abundant polysaccharide in nature, accounting for approximately one-third of all renewable organic carbon on earth (Collins et al., 2005). Lignin is an amorphous non-water soluble and optically inactive heteropolymer. It consists of phenylpropane units joined together by different types of linkages (Perez et al., 2002) Although lignin is the most abundant polymer in wood fiber along with cellulose, its content in non-wood fiber such as straw, grass and seed hull is low (Sun & Cheng, 2002). The lignin content in the soybean seed coat is reported to be low (Krzyzanowski et al., 2001), and thus it is speculated that the soybean curd residue contains relatively small amount of lignin.

*B. subtilis* has ability to produce several antibiotics with a variety of structures, especially peptides that are either ribosomally or non-ribosomally synthesized (Leclere et al., 2005; Ongena et al., 2005; Stein, 2005). We previously isolated several strains of *B. subtilis* and the

Characterization of Enzymes Associated

**2.4.1 Acid detergent fiber** 

**2.4.2 Neutral detergent fiber** 

**2.4 Acid and neutral detergent fiber analysis** 

with Degradation of Insoluble Fiber of Soybean Curd Residue by *Bacillus subtilis* 525

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.

room temperature in a desiccator, the weight of the crucible was measured.

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

**2.4.3 Calculation of content of insoluble fibers** 

regarded as the content of hemicellulose.

flasks were incubated at 30°C at 120 spm.

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

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

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

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

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 enzymes were purified and characterized.
