28 CEREAL CHEMISTRY Network Formation in Gluten-Free Bread with Application of Transglutaminase Michelle M. Moore, 1,2 Meike Heinbockel, 2 Peter Dockery, 4 H. M. Ulmer, 1,2 and Elke K. Arendt 1,5 ABSTRACT Cereal Chem. 83(1):28–36 One of the main problems associated with gluten-free bread is obtaining a good structure. Transglutaminase (TGase), an enzyme that catalyzes acyl-transfer reactions through which proteins can be cross-linked could be a way to improve the structure of gluten-free breads. The objective of this study was to evaluate the impact of TGase at different levels (0, 0.1, 1, and 10 U of TGase/g of protein) on the quality of gluten-free bread. The recipe consisted of white rice flour (relative amount: 35), potato starch (30), corn flour (22.5), xanthan gum (1), and various protein sources (skim milk powder [SMP] [12.5], soya flour, and egg powder). The influence of the various proteins in combination with the different addition levels of TGase on bread quality (% bake loss, specific volume, color, texture, image characteristics, and total moisture) was determined. Confocal laser-scanning microscopy (CLSM) was used to evaluate the influence of TGase on the microstructure of the bread. Baking tests showed that TGase had an effect on the specific volume of the bread. For instance, the SMP bread with 10 U of enzyme contained the most com- pact structure, which was reflected in the crumb texture profile analysis results (highest values) (P < 0.05), digital image analysis (highest level of cells/cm 2 ) (P < 0.05), and CLSM micrographs (network formation). Finally, it can be concluded that it is possible to form a protein network in gluten-free bread with the addition of TGase. However the efficiency of the enzyme is dependent on both the protein source and the level of enzyme concentration. Celiac disease (CD) is a chronic enteropathy caused by the intake of gluten proteins from widely prevalent food sources such as wheat, rye, barley, and possibly oats. The ingestion of gluten induces an inflammatory response resulting in the destruction of the villous structure of the small intestine (Shan et al 2002). Currently, the only effective treatment for CD is the strict lifelong renunciation of gluten-containing foods (Feighery 1999). Because cereal products, especially breads, are the basic com- ponents of the diet in many countries, there is a high demand for gluten-free breads from persons with CD. In view of the fact that gluten is the major structure-forming protein present in wheat bread and is responsible for the viscoelastic properties, it is a challenge to produce high-quality gluten-free bread. Therefore, ingredients that have the ability to mimic the properties of gluten are generally used. According to Toufeili et al (1994) and Guarda et al (2003), the utilization of polymeric substances such as xanthan gum or hydroxypropylmethylcellulose (HPMC) is required for the production of gluten-free breads. Several studies have been conducted (O’Brien et al 2002a,b; Gallagher et al 2003, 2004; Moore et al 2004; Schober et al 2004) whereby novel ingredients such as dairy powders, sorghum, rice, starches, pseudocereals, etc., in combination with hydrocolloids replaced gluten. Another approach is the addition of different enzymes. Enzymes such as amylases, proteases, and xylanases were reported (Kirshnarau and Hoseney 1994; Harada et al 2000; Guarda et al 2003) to create positive effects on the quality of wheat bread. Transglutaminase (TGase) is a relatively new tool used in the manufacture of baked goods (Diez Poza 2002). TGase is an enzyme that catalyzes an acyl-transfer reaction between the γ-carboxy- amide group of peptide-bound glutamine residues and a variety of primary amines (Motoki and Seguro 1998). When protein-bound lysyl residues act as acyl receptors, intra- and intermolecular isopep- tide bonds are formed within the enzyme reaction. According to Motoki and Kumanzawa (2000), in the absence of primary amines in the reaction system, water is used as an acyl acceptor leading to a deamination of glutamine residues. Thus, the TGase can modify proteins by amine incorporation, cross-linking, and deamination. Generally, TGase is more associated with utilization in the pharm- aceutical, meat, and dairy industry. There are several different forms of TGase. However, for use in the baking industry, TGase is obtained using biotechnological techniques (Diez Poza 2002). Studies by Larré et al (1998, 2000) and Bauer et al (2003) have been conducted on wheat gluten where the influence of TGase was examined. Also, Gerrard et al (2000) reported that TGase has a positive effect on the specific volume of wheat-based croissants. Very little is reported on the effects of TGase on the structure of gluten-free breads. Therefore the main objective of this study was to study the effect of TGase on the quality and structure of gluten- free bread. Because TGase reacts differently with various protein sources and at different addition levels, this study examined both the effect of TGase on three different protein sources (soya flour, skim milk, and egg powder) as well as four different enzyme addi- tion levels (0, 0.1, 1, and 10 U of TGase/g of protein). Firstly, gluten-free bread was developed from a series of trials. Standard baking tests (specific volume, % bake loss, moisture, digital image analysis) were conducted on the breads in conjunction with a five- day staling trial using texture profile analysis (TPA). The micro- structure of the bread was examined by confocal laser-scanning microscopy (CLSM). This method produces optical sections through a three-dimensional specimen without damaging the structure (Lichtman 1994). MATERIALS AND METHODS Materials White rice flour (Doves Farm Foods, Berkshire, UK), potato starch and soya flour (Wholefood Wholesalers, Dublin, Ireland), and corn flour (National Starch and Chemical, Bridgewater, NJ) were used in conjunction with instant dried yeast (Mauripan, Burns Philip Food, Sydney, Australia). Salt (Salt Union, Weston Point, Cheshire, UK), sugar (Suicra, Ireland), xanthan gum (Quest International, Holland), skim milk powder (Dairygold, Mitchels- town, Ireland), whole egg powder (Healy Group, Dublin, Ireland), and tap water were also incorporated into the batters. TGase was obtained from Activia Ajinomoto Co. (Hamburg, Germany). Transglutaminase (TGase) Preparation The addition of TGase was calculated on the basis of the amount of crude protein present in each recipe. The recommended level of use for food is generally 1 U of TGase/g of protein. A 1 Department of Food and Nutritional Sciences, University College Cork, Ireland. 2 Bio-transfer Unit, University College Cork, Ireland. 4 Department of Anatomy, University College Cork, Ireland. 5 Corresponding author. Phone: +353-21-4902064. Fax: +353-21-4270213. E- mail: e.arendt@ucc.ie DOI: 10.1094 / CC-83-0028 © 2006 AACC International, Inc.