Food Research International 37 (2004) 7581 www.elsevier.

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Improvement of the breadmaking quality of rice our by glucose oxidase

Hardeep Singh Gujral a, Cristina M. Rosell
a

b,*

Department of Food Science and Technology. Guru Nanak Dev University, Amritsar, 143005, India b Food Science Department, Instituto de Agroqumica y Tecnologa de Alimentos (IATA-CSIC), PO Box 73, 46100 Burjassot, Valencia, Spain Received 8 August 2003; accepted 11 August 2003

Abstract Bread from rice our is very dicult to bake as it lacks gluten like proteins but modication of the rice our proteins with enzymes like glucose oxidase (GO) to improve its breadmaking properties is an interesting option. In this study the eect of GO on rice our dough rheology, protein modication and bread quality has been reported. GO modied the rice our proteins by lowering the thiol and amino group concentration. Protein modication was also conrmed by the changes observed in the free zone capillary electrophoregrams of the rice glutelins. The addition of GO promoted an increase in the elastic and viscous modulus. Rice bread with better specic volume and texture was obtained with GO addition allowing the decrease of the hydroxypropylmethylcellulose (HPMC) levels in the rice bread recipe. 2003 Elsevier Ltd. All rights reserved.
Keywords: Rice proteins; Glucose oxidase; Protein crosslinking; FZCE; Dynamic rheology; Bread

1. Introduction Rice our has been found to be one of the most suitable cereal grain ours for preparing foods for celiac patients. The suitability of rice our is attributed to its low levels of prolamins, since the peptides released from the breakdown of the prolamins act as toxins for individuals suering from celiac disease. As a result cereals containing prolamins (wheat, rye, barley and oats) cannot be consumed and the only preventive measure is to keep the diet of the celiac patient as gluten free as possible. Since rice possesses unique nutritional, hypoallergenic, colourless and bland taste properties, its use in baby foods, puddings and especially in development of foods for gluten intolerant patients has been increasing. However, the use of rice our in breadmaking is still limited because rice proteins are unable to retain the gas produced during the fermentation process. The storage proteins of wheat are unique because they are also the functional proteins. The wheat prolamins
Corresponding author. Tel.: +34-96-390-0022; fax: 34-96-3636301. E-mail address: crosell@iata.csic.es (C.M. Rosell). 0963-9969/$ - see front matter 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2003.08.001
*

(prolamins 4050%) are extremely sticky and responsible for the viscosity and extensibility in a dough system whereas the glutelins provide elasticity. The prolamins and glutelins combine through covalent and non covalent bonds to form the gluten complex resulting in viscoelastic dough that has the ability to retain gas and produce a light baked product (Lindsay & Skerritt, 1999). Rice on the other hand has very little prolamins (2.53.5%), as a result a viscoelastic dough is not formed when rice our is kneaded with water. Therefore the gases produced during proong and baking cannot be retained and the resulting product has a low specic volume, which does not resemble wheat bread. The incorporation of hydroxypropylmethylcellulose (HPMC) in rice our has made it possible to produce bread from rice our (Gujral, Guardiola, Carbonell, & Rosell, 2003; Gujral, Haros, & Rosell, in press; Haque & Morris, 1994; Nishita, Roberts, & Bean, 1976) with a specic volume comparable to that of wheat bread. The HPMC is able to provide the rice our dough with the lm forming and CO2 entrapping properties resulting in a product with a high specic volume. Other hydrocolloids like CMC and xanthan gum have also been tried (Kang, Choi, & Choi, 1997; Kulp,

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Hepburn, & Lehmann, 1974), although they were not able to replace HPMC by providing similar gas retaining and lm forming properties. HPMC has been used at levels of 3.55.3% fb (Gujral et al., 2003; Ylimaki, Hawrysh, Hardin, & Thomson, 1988) in rice bread formulas; therefore any reduction in its use could signicantly aect the economics of rice bread production. Protein crosslinking or the formation of covalent bonds between polypeptide chains is a means of modifying the protein functionality and simultaneously broadening its applications in dierent processes (Gerrard, 2002). A type of covalent crosslink commonly formed during mixing of wheat our and water are the disulde bonds formed by oxidative coupling of two cysteine residues (Lindsay et al., 1999). The contribution of dityrosine crosslinks to the protein network in gluten has been reported (Tilley, Benjamin, Bagorogoza, Moses, Prakash, & Kwen, 2001). Chemical oxidants are frequently added to our to improve bread-making performance, although lately their relationship with cancer disease incidence is decreasing their use. Currently, the enzymes are replacing the chemical oxidants in numerous food applications. Glucose oxidase (GO) catalyses the oxidation of glucose to gluconic acid and hydrogen peroxide, which in wheat our either, causes the formation of disulphide bonds between proteins (Haaralsita & Pullinen, 1992) or the tyrosine crosslinks. The use of GO in combination with other enzymes and surfactants for the production of wheat bread has been reported (Haarasilta, Pullinen, Vaeisaenen, & Tammersalo, 1989; Haarassilta & Vaeisaenen, 1989; Nakai, Takami, Tanaka, & Takasaki, 1995). Oxidative enzymes like GO, peroxidase and laccase are presently being used in breadmaking (Hilhorst, Dunnewind, Orsel, Stegeman, van Vliet, Gruppen, & Schols, 1999; Minussi, Pastore, & Duran, 2002; van Oort, van Straaten, & Laane, 1995; van Oort, 1996). Improvements in the wheat bread loaf volume and crumb grain has been obtained by adding glucose oxidase (Vemulapalli, Miller, & Hoseney, 1998). Despite GO modied wheat our proteins (Aja, Wang, & Rosell, 2003; Rosell, Wang, Aja, Bean, & Lookhart, 2003), the mechanism by which GO improves bread quality is still not completely understood. The objectives of the present investigation were to determine the extent of modication produced by GO on rice our proteins. There may be a possibility of crosslinking the proteins and this could improve the functionality of rice proteins and in consequence the volume and crumb texture of bread made from rice our.

with 12.8% moisture content, 0.57% ash, and 8.83% protein content was used. HPMC (Methocel K4M) was obtained from Dow Chemical Company (Michigan, USA). Vegetable seed oil, compressed yeast (Lessafre, Spain), commercial sugar and salt were obtained from the local market. The glucose oxidase (10000 BG) was generously gifted from Novo Nordisk (Madrid, Spain). All reagents were of analytical grade. 2.1. Quantication of thiol groups Changes in the thiol (SH) groups were determined as describe by Prasada Rao, Vatlasa, and Haridas Rao (2002). Trisglycine (TrisGly) buer was prepared by dissolving 10.4g Tris, 6.9 g glycine and 1.2g ethylene diamine tetraacetic (EDTA) in 1.0 litre of water and pH was adjusted to 8.0. GuHCl/TrisGly solution contained 5 M guanidine hydrochloride. Ellmans reagent contained 4 mg of 5,50 dithiobis-2-nitrobenzoic acid in 1.0 ml TrisGly buer pH 8.0 and was freshly prepared each day. Rice our dough (200 mg) in the presence and absence of GO (control) was suspended in 1.0 ml of GuHCl/Tris-Gly solution, vortexed for 10 min and centrifuged at 16,000 g for 5 min. The clear supernatant (100 ll) was added to 150 ll of GuHCl/Tris-Gly solution and 50 ll of Ellmans reagent and absorbance read at 412 nm. Results were calculated against cysteine standard curve. Values obtained were the means of four replicates. 2.2. Quantication of amino groups Changes in free amino groups were determined by spectrophotometric assay using the o-phthaldiadehyde (OPA) method reported by Nielsen, Petersen, and Dambmann (2001). OPA (40 mg) previously dissolved in 1.0 ml of ethanol. Di-Na-tetraborate decahydrate (1.905 g, TB) and sodium dodecyl sulfate (50 mg, SDS) were dissolved in 40 ml of distilled water. For the OPA reagent preparation the two solutions were mixed together and volume made up to 50 ml with distilled water. This OPA reagent was stored in a dark bottle at 4 C. One part of 2-mercapto-ethanol (ME) (5%) was mixed with 21.27 parts of the OPA reagent just before the quantication. All reagents were AR grade and were purchased from Sigma-Aldrich (USA). Rice our dough, obtained by mixing 100 mg rice our with 90 ll of distilled water, was suspended in 1.0 ml KCl solution (0.1 M and pH 1.0), vortexed (10 min) and centrifuged at 16,000 g for 5 min. Then 50 ll of the clear supernatant were added to 250 ll of OPA reagent containing ME and the absorbance read at 340 nm in a microplate reader. The results were calculated against a serine standard curve. When GO eect was studied the enzyme (0.01, 0.02 and 0.03%, our basis)

2. Materials and methods Commercial rice our obtained from Huici Leidan S.A (Navarra, Spain) was used in this study. Rice our

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was added to the rice our. Four replicates were made for each determination. 2.3. Free zone capillary electrophoresis analysis of rice glutelins Glutelins were extracted from control and treated (0.01, 0.02 and 0.03% GO, our basis) rice our dough (200 mg). The albumins, globulins and prolamins were removed as reported earlier (Bean, Bietz, & Lookhart, 1998; Rosell et al., 2003). The precipitate was extracted for 30 min with 600 ll of dithiothreitol (DTT) propanol solution (65 mM DTT in 50%, v/v, 1-propanol) and then centrifuged at 16,000 g for 5 min. The supernatant containing the glutelins were analysed by free zone capillary electrophoresis (FZCE) in a Beckham MDQ instrument (Beckham, Fullerton, CA). Uncoated fused silica capillaries (Composite Metal Services Ltd., Worcester, UK) of 50 lm i.d.27 cm (20 cm LD ) were used for all separations. FZCE conditions were 50 mM iminodiacetic acid (IDA) containing 26 mM lauryl sulfobetaine (SB 312), 20% (v/v) acetonitrile and 0.05% (w/v) hydroxypropylmethylcellulose at 45 C and 30 kV, the optimum separation conditions described by Bean and Lookhart (2000). 2.4. Determination of rice dough rheology Dynamic rheological measurements were performed on a controlled stress rheometer (Rheostress 1, Thermo Haake, Germany). The rice dough was prepared by mixing rice our (50 g) along with the enzyme (when added) and 45 ml water in the farinograph (Brabender, Germany). The mixing was carried out for 15 min after addition of water. The rice dough was placed between parallel plates (60 mm diameter) and the gap was adjusted to 1 mm. Vaseline oil was used to coat the outer edges to prevent drying of sample. The dough was allowed to rest for 5 min so that residual stresses could relax. A frequency sweep from 0.01 to 10 Hz was performed at a constant stress of 2 Pa at 30 C (Gujral et al., 2003). Preliminary trials indicated that the stress in this range was not injurious to the dough structure. The dough structure was evaluated by comparing log log plots of G0 and G00 with frequency. 2.5. Breadmaking and bread characteristics Rice our (500 g), HPMC (2%, our basis, when added), sugar (7.5%, our basis), salt (2%, our basis), yeast (3%, our basis) and 450 ml water were blended in the bowl of the Hobart mixer (N50, Hobart, Canada). Oil (6%, our basis) was then added and mixed with all the ingredients. GO when added was incorporated at levels of 0.01, 0.02 and 0.03% (our weight basis) to the our before the mixing. A portion of dough (100 g) was

placed in the 50 g bowl of the farinograph (Brabender, Germany) to determine dough consistency. Then, dough pieces of 100 g were put in well-greased pans (measuring 70 40 mm), proofed for 60 min at 30 C and 80% RH and then baked at 175 C for 40 min. Bread was removed from the pans and cooled at room temperature. Bread quality evaluation was carried out by determining weight, volume (rapeseed displacement), specic volume and crumb hardness. Crumb hardness was measured in a Texture Analyzer TA-XT2i (Stable Micro Systems, Surrey, UK) after 24 h of baking. A bread slice of 20 mm thickness was compressed to 50% of its original height at a crosshead speed of 1 mm/s with a cylindrical stainless steel probe having a diameter of 25 mm. The peak force of compression was reported as hardness. 2.6. Statistical analysis Multiple sample comparison was statistically analysed with the Statgraphics Plus 5.0. Fishers least signicant dierences (LSD) test was used to describe means at the 5% signicance level.

3. Results and discussion 3.1. Rice dough consistency and dynamic rheology Rice dough consistency was determined in the presence and absence of HPMC and adding dierent levels of glucose oxidase, in order to assess the extent to which the protein crosslinks could replace the use of the hydrocolloid. In Table 1 it can be observed that the dough consistency decreased with lowering HPMC concentration. The HPMC has the ability to bind large quantities of water and thus increase the resistance during mixing. Reduction in HPMC levels resulted in a

Table 1 Consistency of rice our treated with dierent concentration of GO in the absence and presence of HPMC HPMC (% fb)a 0 GO concentration (% fb)a 0 0.01 0.02 0.03 0 0.01 0.02 0.03 0 Farinograph consistency (BU)b 70 80 80 95 220 160 155 150 330

4
a b

fb: our basis. BU: Brabender units.

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dough, which oered less resistance to mixing and thus lower consistency. The addition of GO in absence of HPMC produced a progressive increase in dough consistency with the enzyme concentration. However, in the presence of 2% of HPMC the dough consistency decreased with increasing level of GO. The drop in dough consistency was most signicant when 0.01% of GO was incorporated along with 2% HPMC. GO up to a certain level increases the viscosity of water soluble fraction extracted from wheat dough but further addition of GO caused a signicant drop in relative viscosity (Vemulapalli & Hoseney, 1998). Painter and Neukom (1968) reported that excess of oxidant results in either no gel formation or a gel that forms and quickly dissolves. Excessive H2 O2 generated by GO may have decreased the viscosity of the water soluble fraction. The mechanism by which this occurs is not clearly understood. In the absence of HPMC the increasing levels of GO brought about a slight increase in the dough consistency. HPMC might be part of the water soluble fraction being aected in some way by the GO. The H2 O2 generated by GO in the presence of the native peroxidase in the rice our (Navasero, Baun, & Juliano, 1975) may have been responsible for the increase in the viscosity. The dynamic rheological studies of the rice our dough showed that the elastic modulus (G0 ) was higher than the viscous modulus (G00 ), which suggests a solid elastic like behavior of the rice our dough. Both moduli were frequency dependent and augmented with increasing frequency and that eect being more pronounced at higher frequencies. Both the viscous and elastic moduli increased with increasing levels of GO indicating that more force was needed to deform doughs containing glucose oxidase (Fig. 1). The changes in the dough rheology may be attributed to the ability of hydrogen peroxide to bring about the gelation of water soluble pentosans in the rice our as has been suggested in wheat our dough (Crowe & Rasper, 1988; Hoseney & Faubion, 1981). An increase in the elastic and viscous moduli of wheat our dough was observed with the

addition of glucose oxidase (Dunnewind, van Vliet, & Orsel, 2002; Vemulapalli et al., 1998). Appreciable amounts of pentosans are present in milled rice our (Mod, Conkerton, Ory, & Normand, 1978; Shibuya & Iwasaki, 1978). Gel formation limits water mobility and gives drier dough and changes dough rheology. 3.2. Protein modication of rice our with glucose oxidase The free zone capillary electrophoresis (FZCE) of the rice our proteins also revealed that the rice proteins were being modied. Glutelins are the predominant proteins in rice and the electrophoregrams (Fig. 2) revealed that the height and area under the peaks decreased with increasing GO concentration, indicating that the interactions among the proteins might result in higher polymers with reduced solubility, in consequence the amount of glutelins extracted would decrease as was conrmed by the FZCE electophoregrams. Rosell et al. (2003) reported the modication of wheat storage proteins (glutelins and prolamins) with GO. The levels of prolamins in rice are too low and therefore the electrophoregrams obtained were not good enough to be studied. The rice glutelins appear to be a good substrate for the glucose oxidase. In order to conrm the interaction between groups due to the glucose oxidase activity, the free amino and thiol groups were measured (Fig. 3). The thiol group concentration showed a decrease with increasing level of addition of GO to the rice our dough. The thiol group concentration decreased by almost 41.3% when GO was added at 0.01% level. Addition at higher levels of 0.02 and 0.03% further lowered the thiol group concentration however the decrease was not signicant. The thiol content of the SDS soluble protein fraction was reported to decrease by the addition of GO to wheat our dough (Vemulapalli et al., 1998). They reported

Fig. 1. Eect of GO on the elastic and viscous modulus of rice our dough. Numbers are referred to the enzyme concentration used in the treatment (%, w/w, our basis).

Fig. 2. Eect of dierent concentrations of glucose oxidase on the FZCE of rice glutelins. Numbers are referred to the enzyme concentration used in the treatment (%, w/w, our basis). Separations were in an uncoated capillary 50 lm 27 cm long (20 cm LD ) at 45 C and 30kV. Samples were pressure injected at 1.5 psi for 4 s.

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Fig. 3. Changes in free amino (d) and thiol (n) groups of rice proteins treated with dierent GO concentrations. Error bars indicate the standard deviation of four replicates.

that the thiol content of the water soluble fraction extracted from the wheat our decreased gradually but signicantly with increasing levels of GO. This enzyme causes the oxidation of the free sulfhydryl units from gluten protein giving disulde linkages, in consequence stronger dough is obtained with greater resistance to mechanical shock, better oven spring and larger loaf volume. H2 O2 produced by GO is involved in the oxidation of the SH groups with the SH content showing a decrease in the presence of the enzyme (Vemulapalli et al., 1998). Pentosans are able to absorb high amounts of water through interchain associations involving oxidative coupling and chain entanglements. The decrease in SH groups could also be attributed to its reaction with the activated double bond of the ferulic acid and thus resulting in a linkage between arabinoxylans and adjacent protein molecules (Hoseney & Faubion, 1981). Proteins also participate in the gelation since the gel formation contains about 25% protein (Neukom & Markwalder, 1978). The amino group concentration showed a progressive decrease by the addition of GO (Fig. 3). The reduction might be attributed to the formation of tyrosine cross links due to the increased proximity of the amino acids as a result of the disulde links promoted by the GO. A 13.2% decrease in the amino group concentration was brought about by the addition of GO at 0.01% level whereas the incorporation of higher enzyme levels further lowered the amino group concentration though not signicantly. 3.3. Bread quality Acceptable bread can be obtained from rice our by incorporating HPMC at levels of 4% (Gujral et al., 2003; Gujral et al., in press). The HPMC can eectively retain the CO2 produced during the fermentation and is able to provide the structure so as to result in a light

baked product. In the presence of 4% HPMC it was possible to obtain bread loaves with 2.5 cm3 /g specic volume. Lowering the HPMC levels in the rice bread recipe lead to deterioration of the rice bread specic volume, the decrease being 26.0 and 41.6% when HPMC was lowered from 4 to 2 and 0% respectively (Fig. 4). The addition of GO improved the specic volume of the bread, obtaining increasing specic volume by raising the enzyme concentration. However the specic volume reached at the highest enzyme concentration tested was around 2.0 cm3 /g, which is lower that the one obtained with 4% of HPMC. In the presence of 2% HPMC and 0.01% GO the bread specic volume was higher than that of bread made with only 4% HPMC. At higher levels of addition (0.02 and 0.03%) the GO lead to a non signicant increase in the bread specic volume. The decrease in specic volume at 2% HPMC and increasing GO concentration correlated with the decrease in farinograph dough consistency. The rheological and protein modication produced in the rice our dough by the addition of GO lead to an improvement in the bread specic volume. It has been reported that the addition of GO to wheat our resulted in higher volume and improved crumb grain characteristics (Vemulapalli et al., 1998). The protein crosslinks promoted by GO, evident from the changes in the thiol and amino group concentration and changed rice glutelin electrophoregrams, increase elastic and viscous modulus of the dough and likely constitute an articial network of proteins similar to the gluten formed by wheat proteins. Regarding rice bread crumb in the absence of HPMC, the rmness was lowered with increase in the GO concentration (Fig. 5). This correlated with the increase in specic volume obtained in the presence of increasing GO concentrations. However, a further improvement in the crumb rmness was obtained when 0.01% GO was

Fig. 4. Eect of glucose oxidase on the specic volume of rice bread made with two dierent levels of HPMC. Error bars indicate the standard deviation of four replicates.

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H.S. Gujral, C.M. Rosell / Food Research International 37 (2004) 7581 Crowe, N. L., & Rasper, V. F. (1988). The ability of choline and choline related oxidants to induce oxidative gelation in wheat our pentosans. Journal of Cereal Science, 7, 283294. Dunnewind, B., van Vliet, T., & Orsel, R. (2002). Eect of oxidative enzymes on bulk rheological properties of wheat our doughs. Journal of Cereal Science, 36, 357366. Gerrard, J. A. (2002). Protein-protein crosslinking in food methods, consequences, applications. Trends in Food Science and Technology, 13, 389397. Gujral, H. S., Guardiola, I., Carbonell, J. V., & Rosell, C. M. (2003). Eect of cyclodextrin glycoxyl transferase on dough rheology and bread quality from rice our. Journal of Agricultural and Food Chemistry, 51, 38143818. Gujral, H.S., Haros, M., & Rosell, C.M. Starch hydrolysing enzymes for retarding the staling of rice bread. Cereal Chemistry (in press). Haarasilta, S., Pullinen, T., Vaeisaenen, S., & Tammersalo, K.I. (1989). A method of improving the properties of dough and the quality of bread. European patent 0338452A1. Haarasilta, S., & Vaeisaenen, S. (1989). Method for improving our dough. European patent 0321811A1. Haarasilta, S., & Pullinen, T. (1992). Novel enzyme combination. A new tool to improve baking results. Agro Food industry Hi Tech, (Italy), 3, 1213. Haque, A., & Morris, E. R. (1994). Combined use of ispaghula and HPMC to replace or augment gluten in bread making. Food Research International, 27, 379393. Hilhorst, R., Dunnewind, B., Orsel, R., Stegeman, P., van Vliet, T., Gruppen, H., & Schols, H. A. (1999). Baking performance, rheology, and chemical composition of wheat dough and gluten aected by xylanase and oxidative enzymes. Food Chemical Toxicology, 64, 808813. Hoseney, R. C., & Faubion, J. M. (1981). A mechanism of oxidative gelation of wheat our water soluble pentosans. Cereal Chemistry, 58, 421424. Kang, M. Y., Choi, Y. H., & Choi, H. C. (1997). Eects of gums, fats and glutens adding on processing and quality of milled rice bread. Korean Journal of Food Science and Technology, 29, 700704. Kulp, K., Hepburn, F. N., & Lehmann, T. A. (1974). Preparation of bread without gluten. Bakers Digest, 48, 3437, 58. Lindsay, M. P., & Skerritt, J. H. (1999). The glutenin macropolymer of wheat our doughs structure-function perspectives. Trends in Food Science and Technology, 10, 247253. Minussi, R. C., Pastore, G. M., & Durn, N. (2002). Potential a applications of laccase in the food industry. Trends in Food Science and Technology, 13, 205216. Mod, R. R., Conkerton, E. J., Ory, R. L., & Normand, F. L. (1978). Hemicellulose composition of dietary bre of milled rice and rice bran. Journal of Agricultural and Food Chemistry, 26, 10311035. Nakai, K., Takami, K., Tanaka, N., & Takasaki, Y. (1995). Bread quality improving composition and bread producing process using the same. European patent 0686348A1. Navasero, E. P., Baun, L. C., & Juliano, B. O. (1975). Grain dormancy peroxidase activity and oxygen uptake in orryza sativa. Phytochemistry, 14, 18991902. Neukom, H., & Markwalder, H. U. (1978). Oxidative gelation of wheat our pentosans a new way of crosslinking polymers. Cereals Foods World, 23, 374376. Nielsen, P. M., Petersen, D., & Dambmann, C. (2001). Improved method for determining food protein degree of hydrolysis. Journal Food Science, 66, 642646. Nishita, K. D., Roberts, R. L., & Bean, M. M. (1976). Development of a yeast leavened rice bread formula. Cereal Chemistry, 53, 626635. Painter, T. J., & Neukom, H. (1968). The mechanism of oxidative gelation of a glycoprotein from wheat our. Biochem Biophys Acta, 158, 363381. Prasada Rao, U. J. S., Vatlasa, C. N., & Haridas, Rao (2002). Changes in protein characteristics during the processing of wheat into akes. European Food Research and Technology, 215, 322326.

Fig. 5. Bread crumb hardness of rice bread added with dierent concentrations of glucose oxidase and two levels of HPMC. Error bars indicate the standard deviation of four replicates.

incorporated to the bread containing 2% HPMC, the crumb rmness lead to a 60.4% decrease, indicating that the improvement in the specic volume lowered the crumb rmness. The addition of higher amounts of GO yielded softer crumbs. It can be concluded that glucose oxidase an oxidizing enzyme can be incorporated into the rice bread formula to improve bread quality. The GO brings about the crosslinking of rice protein, and in consequence, modication of the elastic and viscous behavior of the rice dough. In the presence of GO the levels of HPMC required to produce acceptable rice bread can be lowered, economizing the process.

Acknowledgements This work was nancially supported by Comisin o Interministerial de Ciencia y Tecnologa Project (MCYT, AGL2002-04093-C03-02) and Consejo Superior de Investigaciones Cientcas (CSIC), Spain. H.S. Gujral would also like to thank Ministerio de Educacin, Cultura y Deporte (Spain) for his grant. o

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