INFLUENCE OF MIXING ON QUALITY OF GLUTEN-FREE BREAD

bs_bs_banner Journal of Food Quality ISSN 1745-4557 INFLUENCE OF MIXING ON QUALITY OF GLUTEN-FREE BREAD MANUEL GÓMEZ 1, MARÍA TALEGÓN and ESTHER DE LA HERA Food Technology Area, E.T.S. Ingenierías Agrarias, Universidad de Valladolid, 34004 Palencia, Spain 1 Corresponding author. TEL: +34 (9) 79-108495; FAX: +34 (9) 79-108302; EMAIL: pallares@iaf.uva.es Received for Publication May 30, 2012 Accepted for Publication November 15, 2012 10.1111/jfq.12014 ABSTRACT Interest in research into gluten-free bakery products has been increasing in recent years. In this paper, we have focused on the effect of mixing on two different gluten-free bread formulas (80 and 110% hydration), studying bread quality parameters. In less hydrated breads, no significant differences were found depending on the mixing arm (flat beater or dough hook), but mixing time influenced the specific volume of bread, being these higher while mixing time increased. Both mixer arm and mixing speed were found to have a significant effect on bread volume and texture in more hydrated dough, achieving higher specific volumes and softer breads with the wire whip compared with the flat beater, with lower mixing speeds and longer mixing time. In more hydrated breads, proofing time improved bread specific volume, but in less hydrated breads the effect was the opposite. This effect was remarked in longer mixing times. PRACTICAL APPLICATIONS This paper demonstrates the importance of mixing time and the type of mixer device in gluten-free bread making, something not studied so far. It also shows that this influence is different from which gluten-bread kneading has, where the mechanical work is essential for the development of the protein network. Based on the results of this study, the mixing process of the gluten-free elaborations can be optimized, allowing obtaining breads with higher volume and lower hardness, therefore increasing the final quality of the bread, one of the major problems of the gluten-free breads. INTRODUCTION Celiac disease is due to a permanent intolerance to prolamines present in certain cereals such as wheat, rye, barley and possibly oat. Ingestion of these substances causes intestinal mucosal damage and a reduction in the absorption of important nutrients. The only effective treatment for celiac disease is to maintain a strict gluten-free diet, which leads to recovery of the intestinal mucosa (Farrell and Kelly 2002; Green and Jabri 2003). Recent research has detected an incidence of celiac disease of one in 130 to one in 200 in the general population in industrialized countries (Sollid 2002; Fasano et al. 2003). Improvement in the quality of glutenfree products is therefore a challenge for modern society. Gluten proteins have an essential function in certain products, such as bread, as they are responsible for the formation of a cohesive, extensible and elastic dough that is able to retain the gas produced during fermentation (Gan et al. 1995; Singh and MacRitchie 2001). This fact makes it difficult to achieve high-quality gluten-free bread. Research into gluten-free bakery products has focused on adapting gluten-free flours and their blends (Sanchez et al. 2002; Sivaramakrishnan et al. 2004; Schober et al. 2005; Brites et al. 2008; Renzetti and Arendt 2009) and on the use of hydrocolloids as gluten substitutes (Ylimaki et al. 1991; Lazaridou et al. 2007; Mezaize et al. 2009). In addition, a number of enzymes have been found to improve the quality of gluten-free bread (Gujral et al. 2003; Gujral and Rosell 2004a,b; Moore et al. 2006; Renzetti and Arendt 2009). However, there has been little research into the influence of other process-related factors, particularly mixing and dough formation. The rheofermentometer is an instrument that provides information on gas production and retention and on the change in height of the dough during proofing. It is a useful device for studying differences in bread dough behavior 139

INFLUENCE OF MIXING ON GLUTEN-FREE BREAD QUALITY M. GÓMEZ, M. TALEGÓN AND E. DE LA HERA (Czuchajowska and Pomeranz 1993) and has been used to determine the effects of additives (Gujral and Singh 1999; Gomez et al. 2004), freezing (El-Hady et al. 1996) and mixing parameters (Potus et al. 1994; Huang et al. 2008) on doughs. However, those studies focused on conventional, wheat-flour-based doughs. The traditional rheofermentometer method is designed for wheat-flour doughs and includes a piston with a weight placed on top of it to reduce rising of the dough. Gluten-free bread dough has never been studied using the rheofermentometer. In the present study, we have investigated the effect of dough hydration, mixing time and type of mixer on proofing tolerance and final quality in gluten-free breads. The use of a modified rheofermentographic analysis to predict performance during proofing was also studied. MATERIALS AND METHODS Materials Short grain rice flour (11.5% moisture, 6.9% protein, 0.65% ash, less than 150 mm) was supplied by Harinera Castellana S.A., Medina del Campo, Valladolid, Spain. Commercial dry yeast (Saf-instant, Lesaffre, Lille, France) was used. Salt, sugar and sunflower oil were purchased from the local market. Hydroxypropyl methylcellulose (HPMC) (Methocel K4M, Dow Chemical, Midland, MI) was used as an additive. HPMC has 22.7% methyl groups and 11.2% hydroxypropyl groups, and the viscosity of a 2% solution in water is 4,664 mpa at 20C. Methods Gluten-Free Bread Production. A straight dough process was performed using a Kitchen-Aid Professional mixer (KPM5, KitchenAid, St. Joseph, MI) with a wire whip (K5AWWC), dough hook (K45DH) and flat beater (K45B). Two different formulas were tested. The following ingredients (as percentage on rice flour basis) were used in both formulas: sunflower oil (6%), sucrose (5%), salt (2%), instant yeast (3%) and HPMC (2%); however, in one formula, the proportion of water added was 80%, whereas in the other it was 110%. In both cases, the instant yeast was first rehydrated in 300 ml of water. The mechanical work on the dough with 80% water was done with a dough hook or flat beater for periods of 2, 4, 8 and 12 min at speeds 2 and 4. The process was performed twice for each type of dough. Dough mixing with the flat beater at speed 4 for 8 and 12 min could not be completed due to system overheating. This gave us 28 doughs (two accessories four mixing times two speeds two repetitions with four aborted doughs). The mechanical work on the doughs with 110% water was done with either the flat beater or the wire whip for periods of 2, 4 and 8 min and at speeds 2 and 4. Again, the process was performed twice. A total of 24 doughs were therefore elaborated (two accessories three mixing times two speeds two repetitions). The doughs were molded into aluminum tins of 232 108 43.5 mm: 400 g into each tin in the case of the dough with 80% water, but only 350 g in the case of the dough with 110% water due to a greater volume increase during proofing. Tins were place into a proofing chamber at 30C and 90% relative humidity for 50 or 90 min. After proofing, the breads were baked in an electric oven for 40 min at 190C. After baking, the breads were unmolded, cooled for 1 h at room temperature and then packed in sealed polyethylene bags to prevent staling. All measurements were made at 24 h. Rheofermentographic Analysis. The effect of the different mixing conditions on dough proofing was determined using a rheofermentometer (Chopin, Villeneuvela-Garenne, France), obtaining information on dough development and gas production during fermentation (Czuchajowska and Pomeranz 1993). In contrast to the traditional method, the weight of dough was reduced to 200 g and the weights were removed; the dough used in the analysis was the same as was used for baking. Proofing temperature was set at 30C, as in the bread making. The proofing conditions were therefore as similar as possible to those used in the doughs that were baked; the weights were removed from the piston due to the weakness of this kind of dough compared with wheat-flour doughs. Bread Quality Evaluation. Bread volume was determined using a laser sensor with the BVM-L 370 volume analyzer (TexVol Instruments, Viken, Sweden). The bread specific volume was calculated as the ratio between the volume of the bread and its weight. Measurements were made in duplicate. Crumb texture was determined using a TA-XT2 texture analyzer (Stable Microsystems, Surrey, UK) with the Texture Expert software. A 25-mm diameter cylindrical aluminum probe was used in a Texture Profile Analysis double compression test to penetrate to 50% depth, with a test speed of 2 mm/s and a 30-s delay between the first and second compressions. Firmness (N), cohesiveness and springiness were calculated. Measurements were made on two central slices (20 mm thickness) of two breads from each dough 24 h after baking. Statistical Analysis Differences between breads were studied by a one-way analysis of variance (ANOVA). A multifactorial analysis was also carried out to improve discussion. Fisher s least significant difference method was used to describe means with 95% confidence intervals. The statistical analysis was 140

M. GÓMEZ, M. TALEGÓN AND E. DE LA HERA INFLUENCE OF MIXING ON GLUTEN-FREE BREAD QUALITY TABLE 1. SPECIFIC VOLUME AND TEXTURE PARAMETERS OF BREADS MADE WITH A DOUGH HYDRATION OF 80% AT 50 AND 90 MIN OF PROOFING TIME, USING TWO MIXING CONDITIONS Kneading Specific volume (cm 3 /g) Firmness (N) Cohesiveness Springiness Arm Speed Time (min) 50 min 90 min 50 min 90 min 50 min 90 min 50 min 90 min Dough hook 2 2 2.14o 2.10o 11.10ghijk 11.70ijklm 0.45a 0.46ab 0.89a 0.90ab Dough hook 2 4 2.59efghij 2.50ghijk 7.82bcde 9.73efghij 0.48abcd 0.50bcdef 0.91abc 0.93bcd Dough hook 2 8 3.11b 2.59efghij 4.94ab 10.97fghij 0.49abcde 0.50bcdef 0.89a 0.94cd Dough hook 2 12 2.84cd 2.47ijkl 5.94abcd 12.62jklm 0.52defg 0.50bcdef 0.93bcd 0.95d Dough hook 4 2 2.43jklm 2.42jklm 9.29efghi 8.70cdefgh 0.51cdef 0.48abcd 0.94cd 0.91abc Dough hook 4 4 2.69defgh 2.26mno 6.90bcde 12.72klm 0.49abcde 0.48abcd 0.93bcd 0.91abc Dough hook 4 8 2.53fghijk 2.28lmno 11.55hijkl 14.5lm 0.47abc 0.46ab 0.93bcd 0.93bcd Dough hook 4 12 2.75cde 2.40jklm 7.44bcde 12.7klm 0.55g 0.54fg 0.91abc 0.92bcd Flat beater 2 2 2.16no 2.24mno 18.41n 14.52m 0.45a 0.48abcd 0.90ab 0.89a Flat beater 2 4 2.77cde 2.49hijk 6.80bcde 8.83defghi 0.48abcd 0.47abc 0.93bcd 0.91abc Flat beater 2 8 2.72def 2.36klmn 8.49cdefg 10.86fghij 0.53efg 0.52defg 0.91abc 0.91abc Flat beater 2 12 3.47a 2.65defghi 3.24a 8.67cdefgh 0.50bcdef 0.50bcdef 0.91abc 0.92bcd Flat beater 4 2 2.95bc 2.65defghi 5.81abc 8.07cdef 0.53efg 0.52defg 0.89a 0.90ab Flat beater 4 4 2.7defg 2.51ghijk 5.85abc 9.31efghi 0.49abcde 0.48abcd 0.91abc 0.90ab Standard deviation 0.07 1.02 0.01 0.02 Within each pair of columns (50 90 min), data with the same letter are not significantly different at the P < 0.05 level. performed with the Statgraphics Plus V5.1 software (Statpoint Technologies, Inc., Warrenton, VA). RESULTS AND DISCUSSION The specific volumes of the breads with 80 and 110% water are shown in Tables 1 and 2. In the breads with 80% water, the largest volumes were achieved with the breads proofed for 50 min. However, the proofing time did not affect volume in breads with insufficient mixing, such as those elaborated with the dough hook for 2 min (at speed 2 or 4) or with flat beater at speed 2 for 2 min; these breads had the lowest volumes after proofing for 50 min. Nor were differences in volume observed with the distinct proofing times in the breads mixed with flat beater at speed 4 for 4 min. It appears that these breads achieved optimal proofing but that their structure subsequently broke up leading to gas escape and a loss of volume during excess proofing. This finding may be observed in the rheofermentometer curves for doughs elaborated at speed 2 (Fig. 1). As can be seen, doughs mixed for longer show a greater rise early in proofing and achieve higher volumes, but they also reach the optimum point sooner and the dough starts to fall earlier than those mixed for shorter times. It may also be seen that doughs mixed for less time do not fall when they reach their maximum height, as was also observed with the TABLE 2. SPECIFIC VOLUME AND TEXTURE PARAMETERS OF BREADS MADE WITH A DOUGH HYDRATION OF 110% AT 50 AND 90 MIN OF PROOFING TIME, USING TWO MIXING CONDITIONS Kneading Specific volume (cm 3 /g) Firmness (N) Cohesiveness Springiness Arm Speed Time (min) 50 min 90 min 50 min 90 min 50 min 90 min 50 min 90 min Flat beater 2 2 2.71no 3.58g 4.00hi 2.18de 0.47a 0.53bc 0.89def 0.87bcde Flat beater 2 4 3.03k 4.07e 2.44ef 1.25bc 0.54bcd 0.58efg 0.89def 0.85abcd Flat beater 2 8 3.28j 4.42c 2.23de 0.84ab 0.56cdef 0.59fg 0.88cde 0.93f Flat beater 4 2 2.66o 3.47gh 4.29i 2.63ef 0.54bcd 0.55bcde 0.88cde 0.92ef Flat beater 4 4 2.88m 4.11de 3.18fg 1.00abc 0.56cdef 0.60fg 0.89def 0.91ef Flat beater 4 8 3.22j 4.24d 2.49ef 1.38bc 0.58efg 0.57def 0.84abcd 0.91ef Wire whip 2 2 2.72no 3.83f 3.63ghi 1.35bc 0.52b 0.57def 0.94f 0.91ef Wire whip 2 4 3.29ij 4.61b 1.63cd 0.93abc 0.57def 0.54bcd 0.87bcde 0.87bcde Wire whip 2 8 4.06e 5.23a 0.89abc 0.38a 0.60fg 0.52b 0.87bcde 0.81ab Wire whip 4 2 3.43hi 4.62b 1.39bc 0.69ab 0.58efg 0.54bcd 0.85abcd 0.80a Wire whip 4 4 2.75mno 4.16de 3.66ghi 0.84ab 0.60fg 0.59fg 0.84abcd 0.82abc Wire whip 4 8 2.82mn 3.77f 3.43gh 0.98abc 0.62g 0.60fg 0.82abc 0.87bcde Standard deviation 0.05 0.26 0.01 0.02 Within each pair of columns (50 90 min), data with the same letter are not significantly different at the P < 0.05 level. 141

INFLUENCE OF MIXING ON GLUTEN-FREE BREAD QUALITY M. GÓMEZ, M. TALEGÓN AND E. DE LA HERA FIG. 1. DOUGH DEVELOPMENT (A) AND GAS PRODUCTION (B) OF 80%-HYDRATED DOUGHS KNEADED WITH FLAT BEATER AT SPEED 2 Two-minute (straight gray line), four-minute (straight black line) and eight-minute (broken gray line) mixing. bread specific volume. It appears that optimal proofing depends on the volume that develops during proofing, and that the earlier the optimum point is reached, the faster the increase in volume. In fact, Fig. 1 also shows how doughs mixed for longer produce more gas in the early stages of proofing. This may have two causes. First, the oxygenation produced during dough mixing helps the yeast to adapt to its surrounding and to start reproduction under aerobic conditions, which is more effective than anaerobic reproduction in terms of yeast colony growth. Second, the increase in dough temperature during mixing favors proofing in the typical range for this kind of dough (20 40C). This influence of the mixing conditions on yeast activity has already been described by Gelinas (2006). In addition, doughs mixed for shorter times presented a brief arrest of fermentation in the first 15 min; this could be because fermentation initially uses the added sugars (sucrose), which are easily fermented, but when this source was exhausted, fermentation would continue by using the maltose generated by amylase enzymes. Short mixing times would not give the amylases sufficient time to produce enough maltose. Longer mixing times (8 min) could therefore reduce or even abolish this arrest of fermentation. With regard to the stand mixer used, no significant differences were observed between the flat beater and the dough hook when preparing the 80%-hydrated doughs, proofed for 50 min (optimum time). However, it should be pointed out that dough resistance when using the flat beater at speed 4 gave rise to problems of overheating if mixing was prolonged. A minimum necessary mixing time was found to exist, evidenced by the smaller volumes achieved by doughs kneaded for shorter times at speed 2. When mixing speed is increased, these time differences became less clear, indicating that the minimum necessary mixing was achieved in a little over 2 min. In contrast to what occurs with traditional bread doughs, a longer mixing time did not lead to falling doughs but rather there was an increase in volume or this was irregular (no clear tendency was observed). In wheat-based doughs, the negative effect from overmixing is believed to be due to a damage of the gluten network, which is responsible for gas retention during fermentation (Pyler and Gorton 2009). Gas retention in gluten-free doughs, however, is based on dough consistency, which does not decrease during mixing, and on air-bubble distribution within the dough. Even so, mixing can influence not only air incorporation into the dough but also yeast adaptation and reproduction in the initial stages and subsequent CO 2 production, as has already been mentioned. It therefore appears that there is a minimum mixing time necessary to get optimum air incorporation but that this is not then affected if excess mixing occurs. In contrast to what occurs with the 80%-hydrated doughs, the 110%-hydrated doughs reached their greatest volume after 90 min of proofing in all cases; this matches with the rheofermentometer results (Figs. 2 and 3) and the significant differences in multifactorial ANOVA. These figures show how the 110%-hydrated doughs reached their optimum state later than the 80%-hydrated doughs, but the fall in the 110%-hydrated doughs is more marked than in the 80%-hydrated doughs. This would suggest that the excess proofing of more hydrated doughs will have a more noticeable effect than in less hydrated doughs. It thus seems that the more fluid structure with more air incorporated can expand for a longer period, that is, it accepts greater expansion of the air incorporated during the mixing process, but the structure is very weak at the moment it breaks. In a pan loaf, volume loss will not be as evident as in the rheofermentometer as the dough is supported by the tin, whereas in the rheofermentometer its lack of consistency allows it to overflow through the gap between the rheofermentometer basket and the piston. It has to be taken into account that although the resistance weight (2 kg) used in the usual rheofermentometer analysis was removed, the piston alone weighs around 250 g. Because of this, the heights attained in the rheofermentometer are not comparable with the volume of the breads made using the same doughs, as this small weight has a greater effect on the less 142

M. GÓMEZ, M. TALEGÓN AND E. DE LA HERA INFLUENCE OF MIXING ON GLUTEN-FREE BREAD QUALITY FIG. 2. DOUGH DEVELOPMENT (A) AND GAS PRODUCTION (B) OF 110%-HYDRATED DOUGHS KNEADED WITH FLAT BEATER AT SPEED 2 Two-minute (straight gray line), four-minute (straight black line) and eight-minute (broken gray line) mixing. consistent 110% doughs than on the 80% doughs. In fact, the 110%-hydrated breads were found to achieve larger volumes than the 80% ones (though the differences are not visible on the rheofermentometer curves). Thus, with elaboration of the two doughs using the flat beater and with 50-min proofing (the optimum for the 80%-hydrated doughs), the 110%-hydrated breads achieved a 10% higher mean specific volume; however, if we compare the highest specific volume attained, the mean difference was of 49%. In the case of 110%-hydrated doughs, there was less variation in gas production during proofing, particularly with the flat beater-mixed doughs; this may be because the lower consistency of these doughs offers less resistance to the movement of the flat beater movement with the result that the dough temperature does not rise to the same degree as in the doughs with 80% water. The difference of temperature from 80% hydrated dough to 110% dough is 0.8C for 2 min, 1.2C for 4 min, 1.3C for 8 min and 1.6C for 12-min mixing. It is also possible that aeration could be greater and could reach a sufficient level with less mixing time. Nonetheless, as occurred with doughs with 80% water, a brief halt in gas production was observed during the first 15 min in doughs kneaded for shorter times. Thus, as occurs with dough temperature, the mixing time is important as it modifies the proofing process, though, in contrast to glutencontaining doughs, in which yeast activity could alter the gluten network, the effect of temperature in this case is due mainly to its influence on gas production and, to a lesser degree, on dough consistency. In doughs with 110% water, a minimum mixing time was found to be necessary, as the doughs elaborated at the minimum speed for the shortest time were those that achieved least volume. Multifactorial ANOVA showed significant differences between 8-min mixing and 2 and 4, but not between 2- and 4-min mixing. However, while bread volume increased with mixing when using the flat beater or wire whip at speed 2, use of the wire whip at speed 4 produced the opposite effect. Multifactorial ANOVA showed breads mixed at speed 2 significantly greater in volume than those mixed at speed 4. The doughs that achieved the highest-volume breads were those mixed with the wire whip at speed 2 for 4 8 min or at speed 4 for 2 min. These were the doughs that showed greatest differences compared with those mixed with the flat beater. It appears that the wire whip achieves greater air incorporation and that the bubbles are smaller and more uniformly distributed, producing a more stable structure that tolerates longer proofing times. In contrast to what occurs with the 80%-hydrated doughs, when gas production is faster in these doughs, optimal proofing does not occur earlier and this kind of dough FIG. 3. DOUGH DEVELOPMENT (A) AND GAS PRODUCTION (B) OF 110%-HYDRATED DOUGHS KNEADED WITH WIRE WHIP AT SPEED 2 Two-minute (straight gray line), four-minute (straight black line) and eight-minute (broken gray line) mixing. 143

INFLUENCE OF MIXING ON GLUTEN-FREE BREAD QUALITY M. GÓMEZ, M. TALEGÓN AND E. DE LA HERA shows greater tolerance to excess proofing times, which indicates that not only gas production is affected but also the internal dough structure. Furthermore, greater gas production is observed compared with doughs mixed with the flat beater, which may be due to the greater oxygenation of the doughs elaborated with the wire whip, particularly when mixing times are increased. As commented above, there is no gluten network, and bread volume develops due to gas incorporation in the mixing and mixing process and by its subsequent retention during proofing and baking; this does not depend on a protein network but on dough viscosity and bubble size. In breads with 110% water, the structure does not appear to break down during fermentation, but ideal mixing conditions for incorporation of a larger quantity of air and bubble shape do exist and need to be taken into account in each case, depending on the hydration and the stand mixer. Variations in mixing conditions can lead to differences of up to 50% in the volume. Specifically, the wire whip gives better results than the flat beater, a finding that cannot be ascribed exclusively to the previously mentioned effect on proofing, but is also related to internal dough structure and air incorporation into the batter; the wire whip creates a structure with more bubbles with a more uniform distribution. Many authors have stated that glutenfree bread doughs are similar to cake batters (Moore et al. 2004; Schober et al. 2005; Arendt et al. 2009), in which the mixing process affects batter structure and the final volume of the products (Conforti 2006). In fact, Tan et al. (2012), in studies of layer and sponge cakes, also found that an excessive mixing speed could have a negative effect on the specific volume of cakes and that longer mixing times improved the specific volume achieved; this coincides with our observations in 110%-hydrated doughs that, due to their greater fluidity, show a behavior more similar to cake batters than the 80%-hydrated doughs. As for bread texture, Tables 1 and 2 show the effect of mixing on the firmness, cohesiveness and springiness of 80%- and 110%-hydrated breads. Gumminess has not been included as it presents a significant correlation (99%) with firmness both in 80%-hydrated doughs (r = 0.98) and 110% ones (r = 0.99); the effects on gumminess are therefore the same as those on firmness. These high correlations have already been reported in previous studies, both with bread (Gomez et al. 2008) and with cakes (Gomez et al. 2009). Resilience has likewise been omitted as it shows a significant correlation (99.9%) with cohesiveness in both kinds of doughs (r = 0.94 for 80%-hydrated doughs and r = 0.86 for 110% ones). Furthermore, a significant correlation (99.9%) also exists between bread specific volume and firmness (r =-0.80 for 80%-hydrated breads and r =-0.88 for 110%- hydrated breads). The changes in bread firmness thus determine what occurs with specific volume. The 80%-hydrated breads show less firmness at 50-min proofing than at 90 min and greater firmness when mixing time does not exceed 2 min (speed 2, 50-min proofing); there was no difference between 4- and 8-min mixing times. The breads from the 110%-hydrated doughs showed less firmness at 90-min proofing and with mixing times over 2 min (except those achieved with the wire whip at speed 4). Generally, use of the wire whip produced less firmness than the flat beater, with much greater differences than were observed in the specific volume. This supports the idea that mixing with the wire whip has an important influence on internal dough structure. Moreover, multifactorial ANOVA shows significant greatest firmness in breads mixed with flat beater than those mixed with wire whip in 110% hydrated doughs. The differences in bread cohesiveness and springiness were smaller than the differences in firmness and there was no clear trend; in fact, no significant differences were observed even with the different proofing times. However, the springiness in breads elaborated with 110%-hydrated doughs mixed with the wire whip appeared to be less than the springiness of those from doughs kneaded with flat beater, which adds further support to the influence of the mixing arm on internal dough structure. CONCLUSIONS Based on our results, it may be stated that mixing has a major influence on the final characteristics of gluten-free breads through its effect either on fermentative activity or on dough structure. 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