- Email: [email protected]
Functionality of rice flour modified with a microbial transglutaminase
Journal of Cereal Science 39 (2004) 225–230 www.elsevier.com/locate/jnlabr/yjcrs
Functionality of rice flour modified with a microbial transglutaminase Hardeep Singh Gujrala, Cristina M. Rosellb,* a Department of Food Science and Technology, Guru Nanak Dev University, Amritsar 143005, India Laboratorio de Cereales, Department of Food Science, Instituto de Agroquı´mica y Tecnologı´a de Alimentos (IATA-CSIC), P.O. Box 73, 46100 Burjassot, Valencia, Spain
b
Received 17 June 2003; revised 13 October 2003; accepted 13 October 2003
Abstract Rice flour is one of the most valuable cereal flours from a nutritional viewpoint. However, its use is limited to unfermented baked products since rice proteins are unable to hold gas produced during fermentation. Protein functionality can be modified by cross-linking. Rice flour was treated with different concentrations of a microbial transglutaminase (TG). The extent of the modification was evaluated by quantifying amino and thiol groups. The addition of TG improved the dynamic rheological properties of rice flour doughs, resulting in a progressive increase of the viscous ðG00 Þ and elastic ðG0 Þ moduli with increase in TG concentration. The improvement in rice protein functionality became evident in breadmaking, since it was possible to obtain rice bread with an increased specific volume and softer crumb at 1% TG level in the presence of 2% hydroxypropylmethylcellulose. Analyses showed that rice proteins are polymerised through the TG reaction, providing a protein network necessary for holding the gas produced in fermentation. q 2003 Elsevier Ltd. All rights reserved. Keywords: Rice proteins; Transglutaminase; Protein cross-linking; Dynamic rheology; Bread
1. Introduction Flour from rice (Oryza sativa) possesses unique nutritional properties, it is hypoallergenic, colourless and has a bland taste. Compared with other cereals (Bechtel and Juliano, 1980) rice has a higher lysine content and its glutelin has a more evenly balanced amino acid profile than wheat prolamin, which is deficient in lysine and tryptophan. However, rice proteins lack the ability to form the necessary network for holding the gas produced during the fermentation during baking. The storage proteins of wheat (Triticum aestivum) are unique because they are also the functional proteins in breadmaking. The prolamins of wheat, the gliadins, that comprise 40– 50% of the proteins, are extremely sticky and responsible for the cohesiveness of doughs, whereas the glutelins, named glutenins, provide resistance to extension. Abbreviations: DDT, dithiothreitol; FZCE, free zone capillary electrophoresis; HPMC, hydroxypropylmethylcellulose; HMW-GS, high molecular weight glutenin subunits; LMW-GS, low molecular weight glutenin subunits; ME, 2-mercaptoethanol; OPA, o-phthaldialdehyde; TG, transglutaminase. * Corresponding author. Tel.: þ 34-96-390-0022; fax: þ34-96-363-6301. E-mail address: [email protected] (C.M. Rosell). 0733-5210/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.jcs.2003.10.004
The prolamins and glutelins combine during mixing to form the gluten complex resulting in a viscoelastic dough, which has the ability to retain gas and produce a light baked product. Rice on the other hand is low in prolamins (2.5 –3.5%) and consequently a viscoelastic dough is not formed when rice flour is kneaded with water. Consequently, the gases produced during proofing and baking are not retained and the resulting product has a low specific volume, and does not resemble wheat bread. The incorporation of various hydrocolloids into dough formulations has made it possible to produce bread from rice flour (Kang et al., 1997; Kulp et al., 1974; Nishita et al., 1976). Hydroxypropylmethylcellulose (HPMC) has been found to be the most suitable hydrocolloid and yields rice bread with a specific volume comparable to that of wheat bread (Gujral et al., 2003a,b; Nishita et al., 1976). However, applications of rice flour could be broadened if rice proteins could be covalently cross-linked, either intramolecularly or intermolecularly, to produce a stable network. Such crosslinking would modify the functional properties of the proteins without damaging their nutritional quality (Seguro et al., 1998), and may improve their traditional properties (Gerrard et al., 1998). Protein cross-linking may be promoted by chemical or enzymatic reactions (Gerrard,
226
H.S. Gujral, C.M. Rosell / Journal of Cereal Science 39 (2004) 225–230
2002). The latter are more favoured due to increasing consumer awareness about the use of chemicals in foods. Transglutaminase (TG) (EC 2.3.2.13), a g-glutamyltransferase, catalyses the reaction between an 1-amino group on protein-bound lysine residues and a g-carboxyamide groups on protein-bound glutamine residues leading to covalent cross-linking of the proteins. TG has been used to modify flour proteins for improving their functionality. The action of TG also leads indirectly to a conversion of soluble proteins to insoluble high molecular weight protein polymers through formation of disulphide covalent crosslinks (Larre et al., 2000). TG has found applications in a diverse range of foods and related food products (Kuraishi et al., 2001). TG catalyses the formation of homologous and heterologous polymers between milk, meat, soybean and wheat gluten proteins (Babiker, 2000; Basman et al., 2002b; Motoki and Seguro, 1998). Low levels of TG have improving effects on wheat bread crumb and crust characteristics (Basman et al., 2002a). TG action also transforms weak gluten into a strong gluten through its effects on rheological behaviour (Larre et al., 2000). TG improves dough elasticity (Losche, 1995) and its beneficial effects in breadmaking are similar to those produced by oxidising improvers (Gerrard et al., 1998). We have also recently reported the modification of wheat flour proteins by the addition of TG (Rosell et al., 2003). Taken together these studies suggest the possibility of generating crosslinks between proteins in rice flour by the incorporation of TG that may improve dough characteristics resulting in bread with improved volume and crumb. Since the enzyme is inactivated during the baking process it is a better alternative to chemical additives. The objectives of the present investigation were to explore the extent of protein modification brought about by incorporation of TG into rice flour. In addition the functionality of the resulting protein network was tested in a bread making process.
ethanol. In a separate solution 1.905 g of di-sodiumtetraborate decahydrate and 50 mg of sodium dodecylsulfate were dissolved in 40 ml of distilled water. The two solutions were mixed and volume brought to 50 ml with distilled water. This OPA reagent was stored in a dark bottle in a refrigerator. One part of 2-mercaptoethanol (ME) was mixed with 21.27 parts of the OPA reagent just before the use in the assay. All reagents were AR grade and were from Sigma-Aldrich (USA). Rice flour dough, obtained by mixing 100 mg rice flour with 90 ml of distilled water, was suspended in 1.0 ml KCl solution (0.1 M and pH 1.0), mixed on a vortex mixer (10 min) and then centrifuged at 16,000g for 5 min. To 50 ml of the clear supernatant 250 ml of OPA reagent containing ME was added in a microtitre plate and absorbance read at 340 nm. The results were calculated against a serine standard curve. In studies on the effect of TG the enzyme was added to the rice flour at 0.5, 1.0, 1.5% (flour basis). Four replicates were made for each determination. 2.2. Quantification of thiol groups Changes in the thiol groups were determined using Ellman’s reagent (Prasada Rao et al., 2002). Tris –glycine (Tris– Gly) buffer was prepared by dissolving 10.4 g Tris, 6.9 g glycine and 1.2 g EDTA in 1 l of water and pH was adjusted to 8.0. GuHCl/Tris –Gly solution contains 5 M guanidine hydrochloride. Ellman’s reagent contained 4 mg of 5,50 dithiobis-2-nitrobenzoic acid in 1 ml Tris– Gly buffer pH 8.0 and was freshly prepared each day. Rice flour dough (200 mg), in the presence and absence of TG (control) was suspended in 1 ml of GuHCl/Tris– Gly solution, mixed on a vortex mixer for 10 min and centrifuged at 16,000g for 5 min. GuHCl/Tris –Gly solution (150 ml) and Ellman’s reagent (50 ml) were added to 100 ml of the clear supernatant and the absorbance read at 412 nm. Results were calculated against a cysteine standard curve. Values recorded were the mean of four replicates.
2. Materials and methods 2.3. Rheological measurements Commercial rice flour was from Huici Leidan S.A (Navarra, Spain). The rice flour had moisture, ash, and protein of 12.80, 0.57, 8.83%, respectively, and the amylose content was 21.90%. HPMC, Methocel K4M, was from Dow Chemical Company (Michigan, USA). Vegetable seed oil, compressed yeast (Lessafre, Spain), commercial sugar and salt were from the local market. The TG was from Ajinomoto (Japan) and had an activity of 100 units/g. All reagents were of analytical grade. 2.1. Quantification of amino groups Changes in free amino groups were determined spectrophotometrically using the o-phthaldialdehyde (OPA) (Nielsen et al., 2001). OPA (40 mg) was dissolved in 1 ml of
Dynamic rheological measurements were performed on a controlled stress rheometer (Rheostress 1, Thermo Haake, Germany). The rice dough was prepared by mixing rice flour (50 g) with enzyme (when added) and 45 ml water in a 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 was used to coat the outer edges to prevent drying of sample. The dough was allowed to rest for 5 min to allow relaxation of residual stresses. A frequency sweep from 0.01 to 10 Hz was performed at a constant stress of 2 Pa at 30 8C (Gujral et al., 2003a). Preliminary trials indicated that the stress in this range was not injurious to the dough structure. The dough structure was
H.S. Gujral, C.M. Rosell / Journal of Cereal Science 39 (2004) 225–230
227
evaluated by comparing log –log plots of G0 and G00 with frequency. The means of two replicates are reported. 2.4. Breadmaking Rice flour 500 g and HPMC (2%, flour basis) were blended and added to the bowl of a Hobart mixer (N50, Hobart, Canada) containing sugar (7.5%, flour basis), sodium chloride (2%, flour basis) and yeast (3%, flour basis) dissolved individually in 450 ml of water. Oil (6%, flour basis) was added and the ingredients mixed for 1 min at speed 2 with the paddle attachment, followed by 30 s mixing at speed 3. TG was incorporated at levels of 0.5, 1.0 and 1.5% (flour weight basis). Dough consistency was measured on dough (100 g) in a 50 g Farinograph (Brabender, Germany) bowl. For baking tests a constant weight of dough (100 g) was weighed into well-greased pans (measuring 70 by 40 mm), proofed for 60 min at 30 8C and 80%RH and then baked at 175 8C for 40 min. Bread was removed from the pans and cooled at room temperature. 2.5. Bread quality evaluation The following bread characteristics were assessed: weight, volume (rapeseed displacement), specific volume (volume/weight). Crumb hardness was determined in a Texture Analyzer TA-XT2i (Stable Micro Systems, Surrey, UK) after 24 h after baking. A bread slice of 20 mm thickness was compressed to 50% of its original height at a cross-head speed of 1 mm/s with a cylindrical stainless steel probe (diameter of 25 mm). The peak force of compression was reported as hardness. Results are the mean of three replicates from two different sets of breads. 2.6. Statistical analysis Multiple sample comparison was statistically analysed with the Statgraphics Plus 5.0. Fisher’s least significant differences (LSD) test was used to describe means at the 5% significance level.
3. Results and discussion 3.1. Effect of TG treatment on rice flour proteins Transglutaminase (TG) catalyses the cross-linking between glutamine and lysine residues in proteins, resulting in their polymerisation and a parallel reduction of protein solubility. Rosell et al. (2003) reported that TG induced several changes in free zone capillary electrophoresis (FZCE) of wheat glutenin with a progressive decrease in the peaks with increasing TG concentration. In the case of wheat flour all the gluten proteins were substrates for the TG but the reactivity of the high molecular weight glutenin subunits (HMW-GS) was highest since they contain higher
Fig. 1. Changes in free amino groups of rice proteins with and without transglutaminase addition. Error bars indicate the standard deviation of four replicates. Different letters above the histogram bars indicate significant differences ðp , 0:05Þ:
amounts of lysine/mol of protein than the gliadins or the low molecular weight glutenin subunits (LMW-GS) (Larre et al., 1993, 2000). Barley proteins, mainly glutelins, are also cross-linked by TG to yield polymers of high molecular weight (Basman et al., 2002b). The protein modification produced by the action of the TG leading to the reduction of free amino group content was measured by the changes in the amount of free amino groups before and after TG treatment (Fig. 1). A progressive decrease in the amount of free amino groups was observed on addition of TG up to 1.0%. Beyond that concentration no significant differences in the amount of free amino groups were detected. This could be due to the disappearance of the lysine groups exposed to the enzyme reaction. The free amino group quantification is in agreement with previous results of glutelin electrophoresis and confirms the action of TG on the rice proteins. The cross-linking reaction may bring some amino acids closer to each other as the polymeric protein molecules become more compact. Thus, the sulphur containing amino acids may come close to each other leading to the formation of S –S bonds by oxidation. In fact there was a significant decrease in thiol group concentration from 4.844 to 3.873 mM/g flour when TG was added at 5% (Fig. 2). These values are similar to those reported for wheat flour (Beveridge et al., 1974). The decrease in thiol groups was more significant at a TG concentration of 0.5% compared with 1 and 1.5%. The decrease in the amount of amino groups clearly indicates the cross-linking catalysed by TG and the reduction in thiol group concentration suggests the formation of disulphide bonds is most likely favoured by the proximity of the cross-linked polypeptide chains. 3.2. Effect of TG on the dynamic rheology of rice dough Dynamic rheology measurements were made on the rice dough containing flour, enzyme and water. These studies
228
H.S. Gujral, C.M. Rosell / Journal of Cereal Science 39 (2004) 225–230
the G0 and G00 values of gluten increase with increasing level of TG whereas the tan d values decrease (Larre et al., 2000). They also reported that protein cross-linking by TG was efficient in gluten despite its low lysine content. The complex modulus ðGp Þ was used to describe the effect of TG on sound and bug damaged wheat flour dough (Koksel et al., 2001). The complex modulus combines both the elastic and viscous components ððGp Þ2 ¼ ðG0 Þ2 þ ðG00 Þ2 Þ: In rice flour dough the Gp increased with increasing TG concentration (data not shown) and also increased with increasing frequency. These oscillatory measurements clearly indicate that TG brings about changes in the rice flour dough structure by forming cross-links between proteins. The increase in the elastic and viscous behaviour of the dough could improve its bread making properties. Fig. 2. Changes in free thiol groups of rice proteins treated with different transglutaminase concentrations. Error bars indicate the standard deviation of four replicates. Different letters above the histogram bars indicate significant differences ðp , 0:05Þ:
showed that the elastic modulus ðG0 Þ was higher than the viscous modulus ðG00 Þ; which suggests a solid, elastic-like behaviour of the rice flour dough (Fig. 3). The viscous modulus was frequency-dependent and increased with increasing frequency. Addition of TG increased both the elastic and viscous moduli. The magnitude of the moduli increased with increasing TG concentration indicating that the dough became stronger as TG increased (Fig. 3). The elastic modulus remained higher than the viscous modulus at all TG concentrations studied. The decrease in the tan d ðG00 =G0 Þ with increasing levels of TG indicated that the dough became stiffer and more rigid because of the crosslinking (data not shown). Viscoelastic properties of dough are greatly dependent on the continuous protein phase; therefore, an increase in the average molecular weight of the protein due to TG activity would lead to an increase in the elastic and viscous modulus. It has been reported that
3.3. Bread dough consistency The addition of TG modified the dough consistency as measured by the Farinograph (Table 1). Higher consistencies were found with increasing TG concentrations. Increasing levels of HPMC also increased the consistency; which is attributed to its ability to bind water. The increase in dough consistency with increasing TG indicates that more water is being bound by the modified proteins in the dough structure. The increase in wheat bread dough consistency by TG was reported earlier by Gerrard et al. (1998) and was attributed to the hydrolysis of glutamine residues to glutamic acid residues, resulting in a decrease of the hydrophobic environment, thus increasing the capacity of the protein matrix to hold water. The dough development time and dough stability of wheat flour dough treated with TG increased (Basman et al., 2002a) with increasing levels of TG (up to 0.5%) indicating that the strength of the dough and dough extensibility increased, but at higher levels TG had the reverse effect. In spite of this the consistency increase produced in rice flour dough was relatively small; suggesting that the TG was promoting the changes in dough consistency measured by the Farinograph. Table 1 Rice flour dough consistency and bread quality as affected by TG concentration
Fig. 3. Effect of transglutaminase on the elastic and viscous moduli of rice flour dough. Numbers are referred to the enzyme concentration used in the treatment (%, w/w, flour basis).
HPMC (%)
TG concentration (%fb)
0
0 0.5 1.0 1.5
70 80 90 90
2
0 0.5 1.0 1.5
200 200 210 220
fb, Flour basis; BU, Brabender units.
Farinograph consistency (BU)
H.S. Gujral, C.M. Rosell / Journal of Cereal Science 39 (2004) 225–230
229
3.4. Rice bread quality An acceptable bread can be made by incorporating HPMC into rice bread recipes at levels of 4% (Gujral et al., 2003a,b), where this hydrocolloid provides the gas-retaining network. When HPMC was lowered to levels of 2%, or omitted altogether, the specific volume decreased by 26.0 and 41.6%, respectively (Fig. 4). HPMC is responsible for the gas-retaining- and film-forming properties in the absence of gluten, and a deterioration in the specific volume at lower HPMC levels. Protein cross-linking in rice flour doughs produced by TG could result in the formation of a protein network. This would be indicated by the bread specific volume if the network was sufficient to retain the carbon dioxide produced during the fermentation process. The specific volume of bread made with 2% HPMC increased by 46.5% with the incorporation of TG at 1% level in the dough formula. This specific volume was even higher than that obtained in bread with 4% HPMC (2.5 cm3/ g). This indicated that TG at 1% level and HPMC at 2% levels had synergistic effects and improved specific volume. At higher levels (i.e. 1.5%) TG did not promote any further improvement but on the contrary had a deteriorating effect. In the absence of HPMC, TG was able to improve the specific volume up to 1% level, but the volumes were low and therefore some HPMC was required in the formula to produce acceptable bread. Improvement in wheat bread volume by TG with 0.5% being the optimal concentration has been reported (Basman et al., 2002a,b) but negative effects were observed at higher concentrations. They attributed this behaviour to the ability of the TG to transform weak gluten into a strong one and suggest that at high TG concentrations excessive cross-linking produced an over-strong dough. The same explanation might be applied to describe the effect of TG on rice doughs because
Fig. 5. Bread crumb hardness of rice bread added with different concentrations of transglutaminase. Error bars indicate the standard deviation of four replicates.
an optimum TG concentration was found at 1.0% and a negative effect was observed at higher enzyme levels. The addition of TG up to 1% level lowered crumb firmness sand this effect was observed at the two levels of HPMC tested (Fig. 5). Bread crumb firmness without HPMC was highest and was lowered by increasing TG levels. This effect can be explained partially by the inverse relationship between crumb firmness and specific volume. Crumb firmness at 2% HPMC was 975.3 g but was lowered by 53.51% by incorporating TG at 1% level. Low levels of TG have been reported to have improving effects on wheat bread crumb characteristics while higher levels (1 –1.5%) had detrimental effects (Basman et al., 2002a). Rice flour bread with 1% TG and 2% HPMC had a higher specific volume but also a higher crumb firmness as compared to bread with 4% HPMC, without TG (314 g). This could be attributed to the TG induced cross-links in the protein matrix and hence increased strength of the crumb. Addition of TG to wheat bread produces a marked increase in crumb strength (Gerrard et al., 1998).
4. Conclusions
Fig. 4. Effect of transglutaminase on the specific volume of rice bread. Error bars indicate the standard deviation of four replicates.
TG cross-links rice proteins as suggested by the decrease in the free amino groups. This cross-linking results in a dough with an improved elastic and viscous behaviour. The improvement in the viscoelastic properties of the rice dough was associated with an improvement in the ability of rice flour to retain the carbon dioxide produced during proofing, resulting in rice bread with higher specific volume and crumb strength. TG can thus partially replace the HPMC in the baking of rice bread. Thus by adding TG to rice flour doughs fermented baked products could be produced.
230
H.S. Gujral, C.M. Rosell / Journal of Cereal Science 39 (2004) 225–230
Acknowledgements This work was financially supported by Comisio´n Interministerial de Ciencia y Tecnologı´a Project (MCYT, AGL2002-04093-C03-02 ALI) and Consejo Superior de Investigaciones Cientı´ficas (CSIC), Spain. H.S. Gujral would also like to thank Ministerio de Educacio´n, Cultura y Deporte (Spain) for his grant.
References Babiker, E.E., 2000. Effect of transglutaminase treatment on the functional properties of native and chymotrypsin-digested soy protein. Food Chemistry 70, 139 –145. Basman, A., Koksel, H., Ng, K.W., 2002a. Effects of increasing levels of transglutaminase on the rheological properties and bread quality characteristics of two wheat flours. European Food Research and Technology 215, 419 –424. Basman, A., Koksel, H., Ng, K.W., 2002b. Effects of transglutaminase on SDS-PAGE patterns of wheat, soy, and barley proteins and their blends. Journal of Food Science 67, 2654– 2658. Bechtel, D.B., Juliano, B.O., 1980. Formation of protein bodies in the starchy endosperm of rice (Oryza sativa): a reinvestigation. Annals of Botany London 45, 503 –509. Beveridge, T., Toma, S.J., Nakai, S., 1974. Determination of Sulfhydryland disulfide-groups in some food proteins using Ellmans reagents. Journal of Food Science 39, 49–51. Gerrard, J.A., 2002. Protein – protein crosslinking in food: methods, consequences, applications. Trends in Food Science and Technology 13, 389 –397. Gerrard, J.A., Fayle, S.E., Wilson, A.J., Newberry, M.P., Ross, M., Kavale, S., 1998. Dough properties and crumb strength of white pan bread as affected by microbial transglutaminase. Journal of Food Science 63, 472– 475. Gujral, H.S., Guardiola, I., Carbonell, J.V., Rosell, C.M., 2003a. Effect of cyclodextrin glycoxyl transferase on dough rheology and bread quality
from rice flour. Journal of Agriculture and Food Chemistry 51, 3814–3818. Gujral, H.S., Haros, M., Rosell, C.M., 2003b. Starch hydrolysing enzymes for retarding the staling of rice bread. Cereal Chemistry 80, 750 –754. Kang, M.Y., Choi, Y.H., Choi, H.C., 1997. Effects of gums, fats and glutens adding on processing and quality of milled rice bread. Korean Journal of Food Science and Technology 29, 700–704. Koksel, H., Sivri, D., Ng, P.K.W., Steffe, J.F., 2001. Effects of transglutaminase enzyme on fundamental rheological properties of sound and bug-damaged wheat flour doughs. Cereal Chemistry 78, 26 –30. Kulp, K., Hepburn, F.N., Lehmann, T.A., 1974. Preparation of bread without gluten. Bakers’ Digest 48, 34–37. see also p. 58. Kuraishi, C., Yamazaki, K., Susa, Y., 2001. Transglutaminase: its utilization in the food industry. Food Reviews International 17, 221 –246. Larre, C., Chiarello, M., Dudek, S., Chenu, M., Guegen, J., 1993. Action of transglutaminase on constitutive polypeptides of pea legumin. Journal of Agriculture and Food Chemistry 41, 1816– 1820. Larre, C., Denery, P.S., Popineau, Y., Deshayes, G., Desserme, C., Lefebvre, J., 2000. Biochemical analysis and rheological properties of gluten modified by transglutaminase. Cereal Chemistry 77, 32 –38. Losche, I.K., 1995. Enzymes in baking. The World of Ingredients May– June, 22–25. Motoki, M., Seguro, K., 1998. Transglutaminase and its use for food processing. Trends in Food Science and Technology 9, 204–210. Nielsen, P.M., Petersen, D., Dambmann, C., 2001. Improved method for determining food protein degree of hydrolysis. Journal of Food Science 66, 642–646. Nishita, K.D., Roberts, R.L., Bean, M.M., 1976. Development of a yeast leavened rice bread formula. Cereal Chemistry 53, 626–635. Prasada Rao, U.J.S., Vatlasa, C.N., Haridas Rao, P., 2002. Changes in protein characteristics during the processing of wheat into flakes. European Food Research and Technology 215, 322 –326. Rosell, C.M., Wang, J., Aja, S., Bean, S., Lookhart, G., 2003. Wheat flour proteins as affected by transglutaminase and glucose oxidase. Cereal Chemistry 80, 52 –55. Seguro, K., Kumazawa, Y., Kuraishi, C., Sakamoto, H., Motoki, M., 1996. The 1-(g-glutamyl) lysine moiety in crosslinked casein is an available source for lysine for rats. Journal of Nutrition 126, 2557–2562.
Functionality of rice flour modified with a microbial transglutaminase Hardeep Singh Gujrala, Cristina M. Rosellb,* a Department of Food Science and Technology, Guru Nanak Dev University, Amritsar 143005, India Laboratorio de Cereales, Department of Food Science, Instituto de Agroquı´mica y Tecnologı´a de Alimentos (IATA-CSIC), P.O. Box 73, 46100 Burjassot, Valencia, Spain
b
Received 17 June 2003; revised 13 October 2003; accepted 13 October 2003
Abstract Rice flour is one of the most valuable cereal flours from a nutritional viewpoint. However, its use is limited to unfermented baked products since rice proteins are unable to hold gas produced during fermentation. Protein functionality can be modified by cross-linking. Rice flour was treated with different concentrations of a microbial transglutaminase (TG). The extent of the modification was evaluated by quantifying amino and thiol groups. The addition of TG improved the dynamic rheological properties of rice flour doughs, resulting in a progressive increase of the viscous ðG00 Þ and elastic ðG0 Þ moduli with increase in TG concentration. The improvement in rice protein functionality became evident in breadmaking, since it was possible to obtain rice bread with an increased specific volume and softer crumb at 1% TG level in the presence of 2% hydroxypropylmethylcellulose. Analyses showed that rice proteins are polymerised through the TG reaction, providing a protein network necessary for holding the gas produced in fermentation. q 2003 Elsevier Ltd. All rights reserved. Keywords: Rice proteins; Transglutaminase; Protein cross-linking; Dynamic rheology; Bread
1. Introduction Flour from rice (Oryza sativa) possesses unique nutritional properties, it is hypoallergenic, colourless and has a bland taste. Compared with other cereals (Bechtel and Juliano, 1980) rice has a higher lysine content and its glutelin has a more evenly balanced amino acid profile than wheat prolamin, which is deficient in lysine and tryptophan. However, rice proteins lack the ability to form the necessary network for holding the gas produced during the fermentation during baking. The storage proteins of wheat (Triticum aestivum) are unique because they are also the functional proteins in breadmaking. The prolamins of wheat, the gliadins, that comprise 40– 50% of the proteins, are extremely sticky and responsible for the cohesiveness of doughs, whereas the glutelins, named glutenins, provide resistance to extension. Abbreviations: DDT, dithiothreitol; FZCE, free zone capillary electrophoresis; HPMC, hydroxypropylmethylcellulose; HMW-GS, high molecular weight glutenin subunits; LMW-GS, low molecular weight glutenin subunits; ME, 2-mercaptoethanol; OPA, o-phthaldialdehyde; TG, transglutaminase. * Corresponding author. Tel.: þ 34-96-390-0022; fax: þ34-96-363-6301. E-mail address: [email protected] (C.M. Rosell). 0733-5210/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.jcs.2003.10.004
The prolamins and glutelins combine during mixing to form the gluten complex resulting in a viscoelastic dough, which has the ability to retain gas and produce a light baked product. Rice on the other hand is low in prolamins (2.5 –3.5%) and consequently a viscoelastic dough is not formed when rice flour is kneaded with water. Consequently, the gases produced during proofing and baking are not retained and the resulting product has a low specific volume, and does not resemble wheat bread. The incorporation of various hydrocolloids into dough formulations has made it possible to produce bread from rice flour (Kang et al., 1997; Kulp et al., 1974; Nishita et al., 1976). Hydroxypropylmethylcellulose (HPMC) has been found to be the most suitable hydrocolloid and yields rice bread with a specific volume comparable to that of wheat bread (Gujral et al., 2003a,b; Nishita et al., 1976). However, applications of rice flour could be broadened if rice proteins could be covalently cross-linked, either intramolecularly or intermolecularly, to produce a stable network. Such crosslinking would modify the functional properties of the proteins without damaging their nutritional quality (Seguro et al., 1998), and may improve their traditional properties (Gerrard et al., 1998). Protein cross-linking may be promoted by chemical or enzymatic reactions (Gerrard,
226
H.S. Gujral, C.M. Rosell / Journal of Cereal Science 39 (2004) 225–230
2002). The latter are more favoured due to increasing consumer awareness about the use of chemicals in foods. Transglutaminase (TG) (EC 2.3.2.13), a g-glutamyltransferase, catalyses the reaction between an 1-amino group on protein-bound lysine residues and a g-carboxyamide groups on protein-bound glutamine residues leading to covalent cross-linking of the proteins. TG has been used to modify flour proteins for improving their functionality. The action of TG also leads indirectly to a conversion of soluble proteins to insoluble high molecular weight protein polymers through formation of disulphide covalent crosslinks (Larre et al., 2000). TG has found applications in a diverse range of foods and related food products (Kuraishi et al., 2001). TG catalyses the formation of homologous and heterologous polymers between milk, meat, soybean and wheat gluten proteins (Babiker, 2000; Basman et al., 2002b; Motoki and Seguro, 1998). Low levels of TG have improving effects on wheat bread crumb and crust characteristics (Basman et al., 2002a). TG action also transforms weak gluten into a strong gluten through its effects on rheological behaviour (Larre et al., 2000). TG improves dough elasticity (Losche, 1995) and its beneficial effects in breadmaking are similar to those produced by oxidising improvers (Gerrard et al., 1998). We have also recently reported the modification of wheat flour proteins by the addition of TG (Rosell et al., 2003). Taken together these studies suggest the possibility of generating crosslinks between proteins in rice flour by the incorporation of TG that may improve dough characteristics resulting in bread with improved volume and crumb. Since the enzyme is inactivated during the baking process it is a better alternative to chemical additives. The objectives of the present investigation were to explore the extent of protein modification brought about by incorporation of TG into rice flour. In addition the functionality of the resulting protein network was tested in a bread making process.
ethanol. In a separate solution 1.905 g of di-sodiumtetraborate decahydrate and 50 mg of sodium dodecylsulfate were dissolved in 40 ml of distilled water. The two solutions were mixed and volume brought to 50 ml with distilled water. This OPA reagent was stored in a dark bottle in a refrigerator. One part of 2-mercaptoethanol (ME) was mixed with 21.27 parts of the OPA reagent just before the use in the assay. All reagents were AR grade and were from Sigma-Aldrich (USA). Rice flour dough, obtained by mixing 100 mg rice flour with 90 ml of distilled water, was suspended in 1.0 ml KCl solution (0.1 M and pH 1.0), mixed on a vortex mixer (10 min) and then centrifuged at 16,000g for 5 min. To 50 ml of the clear supernatant 250 ml of OPA reagent containing ME was added in a microtitre plate and absorbance read at 340 nm. The results were calculated against a serine standard curve. In studies on the effect of TG the enzyme was added to the rice flour at 0.5, 1.0, 1.5% (flour basis). Four replicates were made for each determination. 2.2. Quantification of thiol groups Changes in the thiol groups were determined using Ellman’s reagent (Prasada Rao et al., 2002). Tris –glycine (Tris– Gly) buffer was prepared by dissolving 10.4 g Tris, 6.9 g glycine and 1.2 g EDTA in 1 l of water and pH was adjusted to 8.0. GuHCl/Tris –Gly solution contains 5 M guanidine hydrochloride. Ellman’s reagent contained 4 mg of 5,50 dithiobis-2-nitrobenzoic acid in 1 ml Tris– Gly buffer pH 8.0 and was freshly prepared each day. Rice flour dough (200 mg), in the presence and absence of TG (control) was suspended in 1 ml of GuHCl/Tris– Gly solution, mixed on a vortex mixer for 10 min and centrifuged at 16,000g for 5 min. GuHCl/Tris –Gly solution (150 ml) and Ellman’s reagent (50 ml) were added to 100 ml of the clear supernatant and the absorbance read at 412 nm. Results were calculated against a cysteine standard curve. Values recorded were the mean of four replicates.
2. Materials and methods 2.3. Rheological measurements Commercial rice flour was from Huici Leidan S.A (Navarra, Spain). The rice flour had moisture, ash, and protein of 12.80, 0.57, 8.83%, respectively, and the amylose content was 21.90%. HPMC, Methocel K4M, was from Dow Chemical Company (Michigan, USA). Vegetable seed oil, compressed yeast (Lessafre, Spain), commercial sugar and salt were from the local market. The TG was from Ajinomoto (Japan) and had an activity of 100 units/g. All reagents were of analytical grade. 2.1. Quantification of amino groups Changes in free amino groups were determined spectrophotometrically using the o-phthaldialdehyde (OPA) (Nielsen et al., 2001). OPA (40 mg) was dissolved in 1 ml of
Dynamic rheological measurements were performed on a controlled stress rheometer (Rheostress 1, Thermo Haake, Germany). The rice dough was prepared by mixing rice flour (50 g) with enzyme (when added) and 45 ml water in a 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 was used to coat the outer edges to prevent drying of sample. The dough was allowed to rest for 5 min to allow relaxation of residual stresses. A frequency sweep from 0.01 to 10 Hz was performed at a constant stress of 2 Pa at 30 8C (Gujral et al., 2003a). Preliminary trials indicated that the stress in this range was not injurious to the dough structure. The dough structure was
H.S. Gujral, C.M. Rosell / Journal of Cereal Science 39 (2004) 225–230
227
evaluated by comparing log –log plots of G0 and G00 with frequency. The means of two replicates are reported. 2.4. Breadmaking Rice flour 500 g and HPMC (2%, flour basis) were blended and added to the bowl of a Hobart mixer (N50, Hobart, Canada) containing sugar (7.5%, flour basis), sodium chloride (2%, flour basis) and yeast (3%, flour basis) dissolved individually in 450 ml of water. Oil (6%, flour basis) was added and the ingredients mixed for 1 min at speed 2 with the paddle attachment, followed by 30 s mixing at speed 3. TG was incorporated at levels of 0.5, 1.0 and 1.5% (flour weight basis). Dough consistency was measured on dough (100 g) in a 50 g Farinograph (Brabender, Germany) bowl. For baking tests a constant weight of dough (100 g) was weighed into well-greased pans (measuring 70 by 40 mm), proofed for 60 min at 30 8C and 80%RH and then baked at 175 8C for 40 min. Bread was removed from the pans and cooled at room temperature. 2.5. Bread quality evaluation The following bread characteristics were assessed: weight, volume (rapeseed displacement), specific volume (volume/weight). Crumb hardness was determined in a Texture Analyzer TA-XT2i (Stable Micro Systems, Surrey, UK) after 24 h after baking. A bread slice of 20 mm thickness was compressed to 50% of its original height at a cross-head speed of 1 mm/s with a cylindrical stainless steel probe (diameter of 25 mm). The peak force of compression was reported as hardness. Results are the mean of three replicates from two different sets of breads. 2.6. Statistical analysis Multiple sample comparison was statistically analysed with the Statgraphics Plus 5.0. Fisher’s least significant differences (LSD) test was used to describe means at the 5% significance level.
3. Results and discussion 3.1. Effect of TG treatment on rice flour proteins Transglutaminase (TG) catalyses the cross-linking between glutamine and lysine residues in proteins, resulting in their polymerisation and a parallel reduction of protein solubility. Rosell et al. (2003) reported that TG induced several changes in free zone capillary electrophoresis (FZCE) of wheat glutenin with a progressive decrease in the peaks with increasing TG concentration. In the case of wheat flour all the gluten proteins were substrates for the TG but the reactivity of the high molecular weight glutenin subunits (HMW-GS) was highest since they contain higher
Fig. 1. Changes in free amino groups of rice proteins with and without transglutaminase addition. Error bars indicate the standard deviation of four replicates. Different letters above the histogram bars indicate significant differences ðp , 0:05Þ:
amounts of lysine/mol of protein than the gliadins or the low molecular weight glutenin subunits (LMW-GS) (Larre et al., 1993, 2000). Barley proteins, mainly glutelins, are also cross-linked by TG to yield polymers of high molecular weight (Basman et al., 2002b). The protein modification produced by the action of the TG leading to the reduction of free amino group content was measured by the changes in the amount of free amino groups before and after TG treatment (Fig. 1). A progressive decrease in the amount of free amino groups was observed on addition of TG up to 1.0%. Beyond that concentration no significant differences in the amount of free amino groups were detected. This could be due to the disappearance of the lysine groups exposed to the enzyme reaction. The free amino group quantification is in agreement with previous results of glutelin electrophoresis and confirms the action of TG on the rice proteins. The cross-linking reaction may bring some amino acids closer to each other as the polymeric protein molecules become more compact. Thus, the sulphur containing amino acids may come close to each other leading to the formation of S –S bonds by oxidation. In fact there was a significant decrease in thiol group concentration from 4.844 to 3.873 mM/g flour when TG was added at 5% (Fig. 2). These values are similar to those reported for wheat flour (Beveridge et al., 1974). The decrease in thiol groups was more significant at a TG concentration of 0.5% compared with 1 and 1.5%. The decrease in the amount of amino groups clearly indicates the cross-linking catalysed by TG and the reduction in thiol group concentration suggests the formation of disulphide bonds is most likely favoured by the proximity of the cross-linked polypeptide chains. 3.2. Effect of TG on the dynamic rheology of rice dough Dynamic rheology measurements were made on the rice dough containing flour, enzyme and water. These studies
228
H.S. Gujral, C.M. Rosell / Journal of Cereal Science 39 (2004) 225–230
the G0 and G00 values of gluten increase with increasing level of TG whereas the tan d values decrease (Larre et al., 2000). They also reported that protein cross-linking by TG was efficient in gluten despite its low lysine content. The complex modulus ðGp Þ was used to describe the effect of TG on sound and bug damaged wheat flour dough (Koksel et al., 2001). The complex modulus combines both the elastic and viscous components ððGp Þ2 ¼ ðG0 Þ2 þ ðG00 Þ2 Þ: In rice flour dough the Gp increased with increasing TG concentration (data not shown) and also increased with increasing frequency. These oscillatory measurements clearly indicate that TG brings about changes in the rice flour dough structure by forming cross-links between proteins. The increase in the elastic and viscous behaviour of the dough could improve its bread making properties. Fig. 2. Changes in free thiol groups of rice proteins treated with different transglutaminase concentrations. Error bars indicate the standard deviation of four replicates. Different letters above the histogram bars indicate significant differences ðp , 0:05Þ:
showed that the elastic modulus ðG0 Þ was higher than the viscous modulus ðG00 Þ; which suggests a solid, elastic-like behaviour of the rice flour dough (Fig. 3). The viscous modulus was frequency-dependent and increased with increasing frequency. Addition of TG increased both the elastic and viscous moduli. The magnitude of the moduli increased with increasing TG concentration indicating that the dough became stronger as TG increased (Fig. 3). The elastic modulus remained higher than the viscous modulus at all TG concentrations studied. The decrease in the tan d ðG00 =G0 Þ with increasing levels of TG indicated that the dough became stiffer and more rigid because of the crosslinking (data not shown). Viscoelastic properties of dough are greatly dependent on the continuous protein phase; therefore, an increase in the average molecular weight of the protein due to TG activity would lead to an increase in the elastic and viscous modulus. It has been reported that
3.3. Bread dough consistency The addition of TG modified the dough consistency as measured by the Farinograph (Table 1). Higher consistencies were found with increasing TG concentrations. Increasing levels of HPMC also increased the consistency; which is attributed to its ability to bind water. The increase in dough consistency with increasing TG indicates that more water is being bound by the modified proteins in the dough structure. The increase in wheat bread dough consistency by TG was reported earlier by Gerrard et al. (1998) and was attributed to the hydrolysis of glutamine residues to glutamic acid residues, resulting in a decrease of the hydrophobic environment, thus increasing the capacity of the protein matrix to hold water. The dough development time and dough stability of wheat flour dough treated with TG increased (Basman et al., 2002a) with increasing levels of TG (up to 0.5%) indicating that the strength of the dough and dough extensibility increased, but at higher levels TG had the reverse effect. In spite of this the consistency increase produced in rice flour dough was relatively small; suggesting that the TG was promoting the changes in dough consistency measured by the Farinograph. Table 1 Rice flour dough consistency and bread quality as affected by TG concentration
Fig. 3. Effect of transglutaminase on the elastic and viscous moduli of rice flour dough. Numbers are referred to the enzyme concentration used in the treatment (%, w/w, flour basis).
HPMC (%)
TG concentration (%fb)
0
0 0.5 1.0 1.5
70 80 90 90
2
0 0.5 1.0 1.5
200 200 210 220
fb, Flour basis; BU, Brabender units.
Farinograph consistency (BU)
H.S. Gujral, C.M. Rosell / Journal of Cereal Science 39 (2004) 225–230
229
3.4. Rice bread quality An acceptable bread can be made by incorporating HPMC into rice bread recipes at levels of 4% (Gujral et al., 2003a,b), where this hydrocolloid provides the gas-retaining network. When HPMC was lowered to levels of 2%, or omitted altogether, the specific volume decreased by 26.0 and 41.6%, respectively (Fig. 4). HPMC is responsible for the gas-retaining- and film-forming properties in the absence of gluten, and a deterioration in the specific volume at lower HPMC levels. Protein cross-linking in rice flour doughs produced by TG could result in the formation of a protein network. This would be indicated by the bread specific volume if the network was sufficient to retain the carbon dioxide produced during the fermentation process. The specific volume of bread made with 2% HPMC increased by 46.5% with the incorporation of TG at 1% level in the dough formula. This specific volume was even higher than that obtained in bread with 4% HPMC (2.5 cm3/ g). This indicated that TG at 1% level and HPMC at 2% levels had synergistic effects and improved specific volume. At higher levels (i.e. 1.5%) TG did not promote any further improvement but on the contrary had a deteriorating effect. In the absence of HPMC, TG was able to improve the specific volume up to 1% level, but the volumes were low and therefore some HPMC was required in the formula to produce acceptable bread. Improvement in wheat bread volume by TG with 0.5% being the optimal concentration has been reported (Basman et al., 2002a,b) but negative effects were observed at higher concentrations. They attributed this behaviour to the ability of the TG to transform weak gluten into a strong one and suggest that at high TG concentrations excessive cross-linking produced an over-strong dough. The same explanation might be applied to describe the effect of TG on rice doughs because
Fig. 5. Bread crumb hardness of rice bread added with different concentrations of transglutaminase. Error bars indicate the standard deviation of four replicates.
an optimum TG concentration was found at 1.0% and a negative effect was observed at higher enzyme levels. The addition of TG up to 1% level lowered crumb firmness sand this effect was observed at the two levels of HPMC tested (Fig. 5). Bread crumb firmness without HPMC was highest and was lowered by increasing TG levels. This effect can be explained partially by the inverse relationship between crumb firmness and specific volume. Crumb firmness at 2% HPMC was 975.3 g but was lowered by 53.51% by incorporating TG at 1% level. Low levels of TG have been reported to have improving effects on wheat bread crumb characteristics while higher levels (1 –1.5%) had detrimental effects (Basman et al., 2002a). Rice flour bread with 1% TG and 2% HPMC had a higher specific volume but also a higher crumb firmness as compared to bread with 4% HPMC, without TG (314 g). This could be attributed to the TG induced cross-links in the protein matrix and hence increased strength of the crumb. Addition of TG to wheat bread produces a marked increase in crumb strength (Gerrard et al., 1998).
4. Conclusions
Fig. 4. Effect of transglutaminase on the specific volume of rice bread. Error bars indicate the standard deviation of four replicates.
TG cross-links rice proteins as suggested by the decrease in the free amino groups. This cross-linking results in a dough with an improved elastic and viscous behaviour. The improvement in the viscoelastic properties of the rice dough was associated with an improvement in the ability of rice flour to retain the carbon dioxide produced during proofing, resulting in rice bread with higher specific volume and crumb strength. TG can thus partially replace the HPMC in the baking of rice bread. Thus by adding TG to rice flour doughs fermented baked products could be produced.
230
H.S. Gujral, C.M. Rosell / Journal of Cereal Science 39 (2004) 225–230
Acknowledgements This work was financially supported by Comisio´n Interministerial de Ciencia y Tecnologı´a Project (MCYT, AGL2002-04093-C03-02 ALI) and Consejo Superior de Investigaciones Cientı´ficas (CSIC), Spain. H.S. Gujral would also like to thank Ministerio de Educacio´n, Cultura y Deporte (Spain) for his grant.
References Babiker, E.E., 2000. Effect of transglutaminase treatment on the functional properties of native and chymotrypsin-digested soy protein. Food Chemistry 70, 139 –145. Basman, A., Koksel, H., Ng, K.W., 2002a. Effects of increasing levels of transglutaminase on the rheological properties and bread quality characteristics of two wheat flours. European Food Research and Technology 215, 419 –424. Basman, A., Koksel, H., Ng, K.W., 2002b. Effects of transglutaminase on SDS-PAGE patterns of wheat, soy, and barley proteins and their blends. Journal of Food Science 67, 2654– 2658. Bechtel, D.B., Juliano, B.O., 1980. Formation of protein bodies in the starchy endosperm of rice (Oryza sativa): a reinvestigation. Annals of Botany London 45, 503 –509. Beveridge, T., Toma, S.J., Nakai, S., 1974. Determination of Sulfhydryland disulfide-groups in some food proteins using Ellmans reagents. Journal of Food Science 39, 49–51. Gerrard, J.A., 2002. Protein – protein crosslinking in food: methods, consequences, applications. Trends in Food Science and Technology 13, 389 –397. Gerrard, J.A., Fayle, S.E., Wilson, A.J., Newberry, M.P., Ross, M., Kavale, S., 1998. Dough properties and crumb strength of white pan bread as affected by microbial transglutaminase. Journal of Food Science 63, 472– 475. Gujral, H.S., Guardiola, I., Carbonell, J.V., Rosell, C.M., 2003a. Effect of cyclodextrin glycoxyl transferase on dough rheology and bread quality
from rice flour. Journal of Agriculture and Food Chemistry 51, 3814–3818. Gujral, H.S., Haros, M., Rosell, C.M., 2003b. Starch hydrolysing enzymes for retarding the staling of rice bread. Cereal Chemistry 80, 750 –754. Kang, M.Y., Choi, Y.H., Choi, H.C., 1997. Effects of gums, fats and glutens adding on processing and quality of milled rice bread. Korean Journal of Food Science and Technology 29, 700–704. Koksel, H., Sivri, D., Ng, P.K.W., Steffe, J.F., 2001. Effects of transglutaminase enzyme on fundamental rheological properties of sound and bug-damaged wheat flour doughs. Cereal Chemistry 78, 26 –30. Kulp, K., Hepburn, F.N., Lehmann, T.A., 1974. Preparation of bread without gluten. Bakers’ Digest 48, 34–37. see also p. 58. Kuraishi, C., Yamazaki, K., Susa, Y., 2001. Transglutaminase: its utilization in the food industry. Food Reviews International 17, 221 –246. Larre, C., Chiarello, M., Dudek, S., Chenu, M., Guegen, J., 1993. Action of transglutaminase on constitutive polypeptides of pea legumin. Journal of Agriculture and Food Chemistry 41, 1816– 1820. Larre, C., Denery, P.S., Popineau, Y., Deshayes, G., Desserme, C., Lefebvre, J., 2000. Biochemical analysis and rheological properties of gluten modified by transglutaminase. Cereal Chemistry 77, 32 –38. Losche, I.K., 1995. Enzymes in baking. The World of Ingredients May– June, 22–25. Motoki, M., Seguro, K., 1998. Transglutaminase and its use for food processing. Trends in Food Science and Technology 9, 204–210. Nielsen, P.M., Petersen, D., Dambmann, C., 2001. Improved method for determining food protein degree of hydrolysis. Journal of Food Science 66, 642–646. Nishita, K.D., Roberts, R.L., Bean, M.M., 1976. Development of a yeast leavened rice bread formula. Cereal Chemistry 53, 626–635. Prasada Rao, U.J.S., Vatlasa, C.N., Haridas Rao, P., 2002. Changes in protein characteristics during the processing of wheat into flakes. European Food Research and Technology 215, 322 –326. Rosell, C.M., Wang, J., Aja, S., Bean, S., Lookhart, G., 2003. Wheat flour proteins as affected by transglutaminase and glucose oxidase. Cereal Chemistry 80, 52 –55. Seguro, K., Kumazawa, Y., Kuraishi, C., Sakamoto, H., Motoki, M., 1996. The 1-(g-glutamyl) lysine moiety in crosslinked casein is an available source for lysine for rats. Journal of Nutrition 126, 2557–2562.