Improving gluten-free bread quality through the application of enzymes

Eimear Gallagher

EIMEAR GALLAGHER Ashtown Food Research Centre Teagasc, Ashtown Dublin 15, Ireland

ABSTRACT: In response to an increasing demand for high quality gluten-free bread, research in the area is growing. To date, much work has focussed on replacing wheat flour with alternative gluten-free flours and starches such as rice, potato, corn and maize, and combining these with gums and hydrocolloids. The use of enzymes in the bakery industry is not a new concept; alpha amylase, transglutaminase, glucose oxidase, lipase and laccase have been used for many years as natural processing aids and bread improvers. However, their use in gluten-free formulations has not been explored in depth. This paper reviews the most recent advances made in the application of enzyme preparations in gluten-free bread formulations. WHEAT DouGH, WHEAT BrEAD AnD THE IMPorTAnCE oF GluTEn Bread has been, for several thousand years, one of the major constituents of the human diet, and baking yeast-leavened bread is one of the oldest biotechnological processes (1). Traditionally, bread is made from wheat flour, but can also be produced from different types of cereal flours such as rye, barley or oats. Hard wheat flour is normally used for bread making, and is composed of ~ 82 percent starch, 12.5 percent protein, 3.5 percent fibre, 1.5 percent lipid, and 0.5 percent ash (minerals) (2). The most important proteins in wheat flour are gliadins and glutenins based on their role in bread making. As the gluten proteins, they represent between 80-85 percent of total wheat protein. The gliadins are monomeric and the glutenins are polymeric. Gluten proteins are largely insoluble in water (1). During dough mixing, wheat flour is hydrated, and due to the input of mechanical energy, discrete masses of gluten protein are disrupted. They are transformed into a cohesive visco-elastic gluten protein network (3). Gliadins and glutenins fulfil different roles during the formation of bread dough. Due to their large size, glutenin polymers form a continuous, network that provides strength (resistance to deformation) and elasticity to the dough. The gliadins are believed to act as plasticizers for the glutenin polymeric system. In this way, they provide plasticity/viscosity to wheat flour doughs (4, 5). The viscoelastic protein network in the fermenting dough plays a major role in retaining the carbon dioxide produced during proofing and during the initial stages of baking. These gas retention properties in turn determine loaf volume and crumb structure of the resulting bread. During the baking phase, changes which occur in the gluten proteins are most probably changes in protein surface hydrophobicity, sulphydryl/disulphide inter-changes and formation of new disulphide cross-links (6, 7). As a result of these heat-induced changes as well as those of the starch, the typical foam structure of baked bread is formed. disease only occurs in 1 in 1500 of the population. However, an average worldwide prevalence of 1:266 has now been estimated, based on accurate serologic screening studies (8). When gluten is ingested by a celiac sufferer, inflammation of the small intestine leads to the malabsorption of several important nutrients including iron, folic acid, calcium and fatsoluble vitamins (9, 10). The presence of gluten activates the immune system to attack the delicate lining of the small bowel (this is responsible for absorbing nutrients and vitamins). The only effective treatment for celiac disease is a strict life-long adherence to a gluten-free diet. The formulation of gluten-free bakery products presents a complex challenge to both the researcher and the baker and studies addressing this challenge have been reported (11). other studies investigated a range of starch, protein and fibre sources and also hydrocolloids which might be used to replace gluten (12). It was previously believed that the protein component of wheat could be completely removed from the starch component. However, it is now thought that this is almost impossible, and wheat starch will still contain trace amounts of gluten. Today, the most popular bread formulations are predominantly based on ingredients such as rice, corn and potato flours. Making bread with these ingredients however necessitates the use of gums or hydrocolloids that will, in some respects, mimic the properties of gluten.

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GluTEn InTolErAnCE AnD THE MAnuFACTurE oF GluTEnFrEE BrEAD Many people suffer from celiac disease (CD). It is also known as celiac sprue or gluten-sensitive enteropathy. CD is a lifelong intolerance to the gliadin fraction of wheat and the prolamins of rye (secalins), barley (hordeins) and possibly oats (avidins). Until recently, it was thought that celiac

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Table 1. Enzyme Function in Baked Goods (obel, 2001).

APPlICATIon oF EnzyMES In WHEAT BrEAD Enzymes are specialized proteins that function as biochemical catalysts. They are essential in the human body for tasks such as transmitting genetic information and synthesizing molecules (13). However, enzymes have also been used as exogenous ingredients in the baking industry for many years, acting as processing aids during bread making and enhancing the quality of the final products. The application of a range of enzymes in wheat bread is given in Table 1. Benefits such as improved dough properties, softer bread crumb, extended shelf life, increased loaf volume and uniform end-product quality have been reported in wheat breads following the application of enzymes (14-17). Many enzymes occur naturally in grains and flour, but when used as an added ingredient, most are obtained from a microbial source. Also, enzymes are considered to be safe alternatives to chemical compounds, having a GRAS (generally recognized as safe) status and are inactive after baking as their protein structure is denatured during bread making. Those frequently used in bread making are -amylases from different origins (cereal, fungal and microbial) for improving the baking properties and flavour of breads (18). As well as having a beneficial effect on dough

process ability and bread characteristics, enzymes also have a secondary effect on flavour formation (19). Rosell (20) employed transglutaminase (TG) and glucose oxidase (Go) to modify the storage proteins in wheat and to improve dough rheological properties and gluten strength. The authors found that while treatment with Go did change the gluten quality and dough properties, the protein profile was relatively unaffected. TG had a more pronounced effect on the protein properties. The effectiveness of -amylases has been attributed to the inhibition or blocking of starch-protein complexes by dextrins, thus affecting the rate of starch retrogradation (21). Hug-Iten (22) studied the effects of a selection amylases on the degradation of starch and how they reduced the rate of bread firming/ staling. Xylanases (also referred to as endoxylanases, endob-1, 4-xylanases, pentosanases or hemicelluloses), are assigned to distinct families based upon their hydrolytic activities, physicochemical properties and structures. These improved dough flexibility and mach inability, and enhanced loaf volume and crumb grain. In particular, the potential of family 8 and 11 xylanases has been proven (23). It should also be noted that inadequate or inappropriate use of enzyme preparations in dough formulations can adversely affect dough and bread quality (24). Excess enzyme activity affects the colour and flavour of the product, usually perceived as an overly dark, hard crust with the presence of "off notes" in the flavour profile (25). Over-dosage with amylase, particularly bacterial -amylase, can lead to an excessive degree of dextrinization and to sticky, gummy breads (26). The effect of too much enzyme activity in dough is perceived as a slack, sometimes sticky dough, having less elasticity. During proofing, the dough may rise faster due to additional fermentable sugars.

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APPlICATIon oF EnzyMES In GluTEn-FrEE BrEAD Despite the success of enzyme applications in wheat products, only a small number of reports have been published on their use in gluten-free systems. As already mentioned, the demand for high quality gluten-free baked goods has paralleled the increasing incidence of those suffering from celiac disease. However, the ingredients used in the formulation of gluten-free breads are highly refined, and bread formulations which are based predominantly on starch result in viscous (rather than viscoelastic) batters prebaking, and dry, crumbly loaves post-baking. Also, the rate of staling is significantly more rapid than in their wheatcontaining counterparts. It is only recently that the potential of enzymes (to aid with structure formation, and synergise with bread quality improvers) has been explored.

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Transglutaminase (TGase) is a transferase that can modify protein functionality and promote cross linking of proteins from different origins. The action of TGase leads indirectly to a conversion of soluble proteins to insoluble high molecular weight protein polymers through the formation of disulphide covalent cross links (27). A recent study showed how the introduction of TGase in gluten-free bread formulations modified the viscoelastic properties of the batters and improved the quality of the resulting baked loaves (28). In particular, confocal microscopy showed the formation of a distinct protein network between skim milk and whole egg powder in the presence of 1 and 10 U TGase/g of protein. In another study, the capacity of TGase in a rice fl our bread formulation (containing HPMC) resulted in polymerization of the rice proteins through the action of the enzyme. The cross linking proteins resulted in an elastic dough that was able to retain carbon dioxide gas during fermentation, and baked breads with enhanced volume and crumb texture (29). The protein source, and hence the availability of glutamine and lysine residues has been highlighted, as the amino acid residues act as the substrate for protein cross linking. Whilst studying the action of TGase with a range of gluten-free flours, Renzetti (30) found that buckwheat and brown rice flours proved optimal substrates for TGase action. In these cases, dough rheological properties were improved, and resulting breads also had enhanced structure and texture. The addition of TGase only slightly effected formulations comprised of oat, sorghum and teff flours, and the enzyme had negative effects on the pseudo plastic behaviour of doughs from corn flour. However the resulting breads were of high quality. Marco (31) also studied the effect of protein isolates (from pea, soybean, egg albumen, whey) and TGase on the protein functionality of rice flour. The presence of TGase had a significant effect on the elastic modulus (G) of the doughs. Also, when soybean or whey proteins were blended with rice flour, a reduction in the amount of amino groups present confirmed protein cross linking catalysed by TGase. In a very recent study, the same authors used texture profile analysis and microscopy to show how protein isolates in the presence of TGase can strengthen a protein network (32). An in-depth proteomic study showed how large protein polymers, linked by isopeptide and disulfide bonds were formed from rice flour and pea protein isolates which were treated with TGase. The gluten-free dough which was formed had a higher nutritional value (due to the protein isolate) and an improved amino acid composition (33). The effectiveness of -amylase in retarding the staling of bread crumb has been discussed many times. The most popular explanation attributed the softening effects of the enzyme to a reduction of the starch level (as it has been degraded to dextrins) available to retrograde (34), and also to the interference by the dextrins to the protein-protein and starch-protein entanglements (35), thus retarding

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crumb staling. The incorporation of -amylase in gluten-free chapattis resulted in a non significant increase in dough extensibility properties and a marked improvement in the texture of the product. Shelf-life was also increased as the enzyme contributed to a reduction in the rate of starch retrogradation (36). Lee (37) determined the presence of alpha amylase in a gluten-free bread formulation served to significantly enhance loaf volume and reduce the rate of crumb staling. Similar beneficial results were found by the present author, when a maltogenic alpha amylase was applied to a gluten-free bread formulation based on rice flour and potato starch (38). A study by the Novozymes company revealed that a dosage of -amylase of 600 MAN/U/kg gluten-free premix resulted in no differences in the dough characteristics (therefore the mixing process need not be altered), yet a significant improvement in crumb softness, elasticity and shelf life of a gluten-free bread was observed (39). Cyclodextrin glycosyl transferase or cyclodextrinase (CGTase) functions by cleaving -1,4 glycosidic linkages in starch molecules, simultaneously linking the reducing and non-reducing ends, producing cyclic molecules (40). These cyclodextrins have a hydrophilic surface and hydrophobic interior, and can form complexes with fatty acids and emulsifiers, thus affecting rheological properties of the starch. For glutenfree bread formulations, particularly those containing rice

flour, the presence of CGTase could reduce the hydrophilic nature of the rice proteins by hydrolyzing and cycling the starch present (41). one particular study has shown that an optimal gluten-free bread (with good loaf volume and soft crumb texture) could be formulated with rice flour in the presence of CGTase (42). The authors concluded that the action of CGTase with the rice proteins decreased the hydrophobic environment, and complexes were formed with lipids and proteins during the bread making process. CGTase has also been shown to reduce the rate of staling of gluten-free Table 2. Recent studies involving enzymes in gluten-free formulations. breads (43). The application of glucose oxidase (Go) enhances 4. M. Cornet, Y. Popineau et al., Journal of Cereal Science, 19, pp. both wheat bread and gluten-free bread properties. Go 131-139 (1994). is a natural, less controversial alternative to chemical 5. B.S. Khatkar, A.E. Bell et al., Journal of Cereal Science, 22, pp. 29-44 oxidising agents. In a rice bread-based formulation (also (1995). containing HPMC), the presence of the enzyme lowered 6. J.D. Schofield, R.C. Bottomley et al., Journal of Cereal Science, 1, the concentration of thiol and amino groups, and cross pp. 241-253 (1983). linking with the rice proteins was observed. When used at 7. P.L. Weegels, A.M.G. de Groot et al., Journal of Cereal Science, 0.01, 0.02 and 0.03 percent of flour weight, the resulting 19, pp. 39-47 (1994). Farinograph dough consistency values increased to an 8. A. Fasano, C. Catassi, Gastroenterology, 120, pp. 636-651 (2001). optimal point (0.02 percent). Above this point, it was 9. C.F. Feighery, British Medical Journal, 319, pp. 236-239 (1999). 10. C.P. Kelly, C. Feighery et al., Advances in Internal Medicine, 35, pp. proposed that excess H 2 o 2 generated by the Go may 341-364 (1999). have decreased the viscosity of the water soluble fraction. 11. E. Gallagher, T.R. Gormley et al., Journal of Food Engineering, 56, Fundamental rheology showed that the enzyme increased pp. 153-161 (2003). the firmness and elasticity of the doughs, and breads. 12. E. Gallagher, T.R. Gormley et al., Trends in Food Science and Improved volume and soft crumb were produced at levels Technology, 15, pp. 18-23 (2004). of 2 percent HPMC and 0.01 percent Go (44). Laccase is 13. L. obel, Cereal Foods World, 46(9), pp. 396-398 (2001). a copper-containing polyphenol oxidase (45), and has 14. J.M. Sanz Panella, C. Collar et al., Journal of Cereal Science, 48, been utilised in the bakery industry to enhance the pp. 715-721 (2008). properties of wheat bread, functioning by generating an 15. P.A. Caballero, M. Gmez et al., Journal of Food Engineering, 81, oxidising effect on constituents of the dough, thus pp. 42-52 (2007). 16. C. Collar, C. Bollan et al., Journal of Food Engineering, 70, pp. 479improving the strength of the dough structure and the 488 (2005). resulting baked product. The network-forming properties 17. A.H. Barrett, G. Marando et al., Cereal Chemistry, 82(2), pp. 152of laccase could also prove beneficial in gluten-free 157 (2005). systems. A summary of enzymes used in recent gluten-free 18. C.M. Rosell, M. Haros et al., European Food Research Technology, studies is given in Table 2.
19. 212, pp. 364-368 (2001). M.A. Martnez-Anaya, Journal of Agricultural and Food Chemistry, 44(9), pp. 2469-2480 (1996). C.M. Rosell, J. Wang et al., Cereal Chemistry, 80(1), pp. 52-55 (2003). H.W. Van Dam, J.D. Hille, Cereal Foods World, 37, pp. 245-251 (1992). S. Hug-Iten, F. Escher et al., Cereal Chemistry, 80(6), pp. 654-661 (2003). T. Collins, A. Hoyoux et al., Journal of Cereal Science, 43, pp. 79-84 (2006). Qi Si, J. Cereal Foods World, 42(10), pp. 802-807 (1997). P.R. Matthewson, Cereal Foods World, 45(3), pp. 98-101 (2000). L.K. Bowles, Amylolytic enzymes. Baked Goods Freshness: Technology, Evaluation and inhibition of staling, pp. 105-130 (1996). C. Larre, P.S. Denery et al., J. Cereal Chemistry, 77, pp. 32-38 (2000). M.M. Moore, M. Heinbockel et al., Cereal Chemistry, 83(1), pp. 28-36 (2006). H.S. Gujral, C.M. Rosell, Journal of Cereal Science, 39, pp. 225-230 (2004). S. Renzetti, F. Dal Bello et al., Journal of Cereal Science, 48(1), pp. 33-45 (2008).

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ConCluSIon The use of enzymes is not a new concept in the bakery industry. They have been used for many years as natural, effective ingredients to improve the processability and baked characteristics of wheat breads. In gluten-free bread manufacture, their use is less widespread. However their potential has been documented in recent studies. As the incidence of celiac disease continues to increase, a simultaneous increasing demand for good quality glutenfree products is taking place. Future research will explore more extensively the application of enzymes in this area, as studies to date have generated encouraging results.

20. 21. 22. 23. 24. 25. 26.

27. 28.

rEFErEnCES AnD noTES

1. 2. 3. H. Goesaert, K. Brijs et al., Trends in Food Science & Technology, 16, pp. 12-30 (2005). P.R. Mathewson, Cereal Foods World, 45, pp. 98-101 (2000). H. Singh, F. MacRitchie, Journal of Cereal Science, 33, pp. 231-243 (2001).

29. 30.

Readers interested in a complete list of references are kindly invited to write to the author at: Eimear.Gallagher@teagasc.ie

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