Textural Comparisons of Gluten-Free and Wheat ... - Cereal Chemistry

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Michelle M. Moore,1,2 Tilman J. Schober,1,2 Peter Dockery,3 and Elke K. Arendt1,4. ABSTRACT. Cereal Chem. 81(5):567–575. Studies were conducted with ...
Textural Comparisons of Gluten-Free and Wheat-Based Doughs, Batters, and Breads Michelle M. Moore,1,2 Tilman J. Schober,1,2 Peter Dockery,3 and Elke K. Arendt1,4 ABSTRACT

Cereal Chem. 81(5):567–575

Studies were conducted with two newly developed gluten-free bread recipes. One was based on corn starch (relative amount 54), brown rice (25), soya (12.5), and buckwheat flour (8.5), while the other contained brown rice flour (50), skim milk powder (37.5), whole egg (30), potato (25), and corn starch (12.5), and soya flour (12.5). The hydrocolloids used were xanthan gum (1.25) and xanthan (0.9) plus konjac gum (1.5), respectively. Wheat bread and gluten-free bread made from commercial flour mix were included for comparison. Baking tests showed that wheat and the bread made from the commercial flour mix yielded significantly higher loaf volumes (P < 0.01). All the gluten-free breads were brittle

after two days of storage, detectable by the occurrence of fracture, and the decrease in springiness (P < 0.01), cohesiveness (P < 0.01), and resilience (P < 0.01) derived from texture profile analysis. However, these changes were generally less pronounced for the dairy-based gluten-free bread, indicating a better keeping quality. Confocal laser-scanning microscopy showed that the dairy-based gluten-free bread crumb contained networklike structures resembling the gluten network in wheat bread crumb. It was concluded that the formation of a continuous protein phase is critical for an improved keeping quality of gluten-free bread.

In recent years there has been a slow and steady increase in consumer interest in wheat-free foods, driven in part by an increasing awareness of the relatively unfamiliar condition known as celiac disease (CD) (Lovis 2003). CD, also known as gluten-sensitive enteropathy, is characterized by inflammation of the small-intestinal mucosa that results from a genetically based immunologic intolerance to ingested gluten (Murray 1999). In Europe, the prevalence of CD has been estimated to be 1 in 300 to 1 to 500 persons, but recent population-based screening studies suggest that the prevalence may be as high as 1 in 100 (Mustalahti et al 2002). Recent studies show that CD is as frequent in the United States as in Europe (Fasano and Catassi 2001). The symptoms of CD can develop at any age. These symptoms include, severe symptoms of malabsorption such as steatorrhoea, abdominal discomfort, weight loss or gain, tiredness, anemia, and severe diarrhea (Feighery 1999; Murray 1999; Fasano and Catassi 2001). These authors suggest that the only way that CD can be treated is the total lifelong avoidance of gluten ingestion. The deleterious proteins gliadins (wheat), secalins (rye), hordeins (barley), and possibly avenins (oats) are the predominant grains containing toxic peptides (Murray 1999; Fasano and Catassi 2001; Kasarda 2001) and should be avoided. A review presented by Kasarda (2001) suggests that species that are less closely related to wheat such as sorghum, millet, teff, ragi, and Job’s tears are relatively safe grasses. Persons with CD are unable to consume some of the most common products on the market today including breads, baked goods, and other food products made with wheat flour (Lovis 2003). To satisfy the demand for high-quality bread, the gluten-free breads must have quality characteristics similar to those of wheat flour bread (Ylimaki et al 1991). The development of such breads is difficult in view of the fact that gluten is the main structure-forming protein in wheat flour, responsible for the elastic and extensible properties needed to produce good quality bread (Ylimaki et al 1991) The main objective of this study was to produce gluten-free breads that are comparable to wheat bread and also to compare their structure with a gluten-free bread from a commercial flour mix. Another aspect of this study was to develop healthier breads that contain higher protein and dietary fiber levels and not only

starch. From this, two new recipes were developed: a dairy-based gluten-free bread and a nondairy-based gluten-free bread. These recipes were selected from a series of 200 trials for their sensory properties as well as their nutritional improvements and food structure. The purpose of developing a nondairy-based gluten-free bread was because ≈50% of the CD sufferers are intolerant to lactose (Murray 1999). Because the villi in the small intestine of CD sufferers are damaged, they cannot produce the enzyme lactase required to break down the lactose sugar molecule (Murray 1999). Therefore they are unable to digest lactose. The dairy-based gluten-free bread, containing more complex cereals, was developed with a view to improving the nutritional and keeping qualities of bread for nonlactose intolerant CD sufferers. Breads were studied over a five-day storage period using texture profile analysis (TPA) to determine the staling rate associated with the breads. Microscopic images of the dough, batters, and bread crumbs were generated using confocal laser-scanning microscopy (CLSM) to develop a fundamental understanding of the structure of gluten-free batter and gluten-free bread as a good basis for further improvements. The advantage of CLSM is its ability to produce optical sections of a three-dimensional specimen without damaging the structure (Dürrenberger et al 2001). To supplement the insight gained by CLSM, the rheological properties of the batters and dough were compared using extrusion and penetration tests.

1

Department of Food and Nutritional Sciences, University College Cork, Ireland. National Food Biotechnology Centre, University College Cork, Ireland. Department of Anatomy, University College Cork, Ireland. 4 Corresponding author. Phone: +353-21-4902064. Fax: +353-21-4270213. E-mail: [email protected] 2

MATERIALS AND METHODS Baker’s flour and trytamyl gluten-free flour mix (Odlum Group, Dublin, Ireland) were used in conjunction with instant dried yeast (Mauripan, Burns Philip Food Ltd., UK). Salt (Salt Union, Weston Point, Cheshire, UK), sugar (Suicra, Ireland), and ascorbic acid (BDH, Poole, UK) were also incorporated into the dough and the batters. The nondairy gluten-free bread and dairy gluten-free bread were prepared using brown rice flour and buckwheat flour (Doves Farm Foods Ltd, Berkshire, UK), corn starch (National Starch and Chemical), soya flour, potato flour (Wholefood Wholesalers, Dublin, Ireland), xanthan gum (Quest International, Holland), skim milk powder (Dairygold, Mitchelstown, Ireland), sugar syrup (Tate & Lyle Sugars, Cheshire, England), konjac powder (The Mill, Cork, Ireland), baking powder (Supercook, Leeds, UK), eggs (locally supplied), and tap water.

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Publication no. C-2004-0712-03R. © 2004 American Association of Cereal Chemists, Inc.

Baking Tests The ingredients used for wheat bread (W), commercial glutenfree flour mix bread (C), nondairy gluten-free bread (ND), and dairy gluten-free bread (D) are listed in Table I. The appropriate Vol. 81, No. 5, 2004

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crude protein (% db) and moisture levels are included. Moisture and crude protein contents were determined by Standard 110/1 (ICC 1976) and Approved Method 46-30 (AACC 2000), respectively. For all the recipes, the yeast was prefermented by suspending it in a water and sugar solution for 20 min for revitalization. Dough W, based on 3,000 g of flour, was mixed in a 30-qt capacity planetary mixer (Hunt 30, Lancashire, UK) with a dough hook for 1 min at a disk speed of 44 rpm (shaft 88 rpm) and 7 min at a disk speed of 135 rpm (shaft 270 rpm). Water temperature was ≈18–20°C. Dough W was rested in bulk for 30 min in a proofer/retarder (Koma Popular, Koma, Roermond, The Netherlands) at 30°C and 85% rh, scaled into 400-g portions, rounded manually, and rested further in the proofer for 10 min at 30°C and 85% rh. Dough W was then molded in a small-scale molder (Machinefabriek Holtkamp B.V., Almelo, Holland), placed into tins 180 mm × 120 mm × 60 mm (Sasa UK Ltd., Middlesex, UK), and proofed at 30°C and 85% rh for 50 min. The gluten-free breads and batters were prepared with the same 30-qt capacity planetary mixer but equipped with a batter attachment and mixed for 30 sec at a disk speed of 44 rpm (shaft 88 rpm) and 60 sec at a disk speed of 135 rpm (shaft 270 rpm). Batters were based on 3,000 g of gluten-free flour mix in bread C and 2,000 g of flour in breads ND and D. The water temperature was 18–20°C. The batter was then scaled to 500 g into nine baking tins (height 7 cm, top and bottom length and breadth 16.5 cm x 10.5 cm and 13.5 cm x 7.5 cm, respectively) and proofed at 30°C and 85% rh for a period of 40 min for breads ND and C and 30 min for bread D. Baking was done at 230°C top heat and 230°C bottom heat for 30 min for bread W, 190°C top heat and 190°C bottom heat for 45 min for breads C and ND, and 180°C top heat and 180°C bottom heat for 45 min for bread D in a deck oven (MIWE, Arnstein, Germany). The oven was preinjected with steam (0.3 L of water) and after loading was again steamed with 0.7 L of water. The loaves were depanned and allowed to cool for 120 min on cooling racks at room temperature. The loaves for analyses at day 2 and 5 were

then packaged in containers (polystyrol, ethylene vinyl alcohol, polyethylene) under modified atmosphere (60% N2 and 40% CO2) and stored at 21°C. Rheology of Batters and Doughs The batters and dough were prepared as described for the baking experiments but without the addition of yeast and prefermentation step. Dough and batter hardness characteristics were evaluated with a texture analyzer (TA-XT2i, Stable Micro Systems, Surrey, UK) equipped with the forward extrusion cell HPP/FE. For batter extrusion, 200 g of sample was filled into the extrusion vessel and the air pockets were removed with a spoon. For further removal of randomly distributed air, the sample was precompressed using the controls on the texture analyzer, while partly closing the 10-mm diameter nozzle. Afterward, the extrusion force was measured at a test speed of 1.0 mm/sec over a distance of 20 mm, using the 5-kg load cell of the texture analyzer. The average force measured after reaching a plateau (8–18 mm) was used as an indicator of batter firmness. Three repetitions made with the same batter batch were averaged into one replicate value. For extrusion of the wheat dough, 30 min of rest in bulk at room temperature was allowed, the extrusion cell was filled with 200 g of sample and measured after 5, 10, and 60 min. For the dough, the 25-kg load cell was used. For batter penetration, 200 g of batter was penetrated by a 20-mm cylinder probe at a test speed of 1.0 mm/sec over a distance of 10 mm and the maximum force was recorded. CLSM Safranin O dye (Sigma Chemicals Co., St. Louis, MO) was added to the respective recipes at a rate of 0.002% flour weight basis (fwb) to stain the protein and the starch. The dye was solubilized in the water before mixing to ensure homogenous distribution. For microscopy of the batters, the batters were prepared as described for the baking test without the yeast. Each sample was placed onto a welled slide using a needle before the

TABLE I Bread Recipes,a Moisture, and Protein Contents Ingredients Wheat flour (W) Commercial gluten-free flour mix (C)c Brown rice flour Potato starch Corn starch Buckwheat flour Soya flour Skim milk powder Baking powder Salt Yeast Sugar Sugar syrup Xanthan gum Konjac powder Ascorbic acid Water Egg Sum Total moisture in recipe Total solid matter in recipe Ratio of moisture to solid matter Total nitrogen in recipe (% wb)e a

W 100.0 ... ... ... ... ... ... ... ... 2.0 1.5 1.5 ... ... ... 0.0 61.0 ... 166.0 73.0 93.0 0.78 1.20

C ... 100.0 ... ... ... ... ... ... ... ... 2.0 2.0 ... ... ... ... 63.0 ... 167.0 74.9 92.1 0.81 0.32

ND ... ... 25.0 ... 54.0 8.5 12.5 ... ... 1.8 2.0 1.0 2.5 1.3 ... ... 105.0 ... 213.5 117.7 95.8 1.23 0.62

D ... ... 50.0 25.0 12.5 ... 12.5 37.5 2.0 1.8 3.5 1.0 ... 0.9 1.5 ... 105.0 30.0 283.2 142.9 140.3 1.00 1.50

Moisture (%) 12.0 11.9 12.4 18.7 12.7 13.5 9.1 5.9 . . .d ... ... ... 19.5 ... ... ... 100.0 73.5 ... ... ... ... ...

Nitrogen (% wb)b 1.99 0.53 1.34 0.03 0.02 1.86 6.48 5.69 . . .d ... ... ... ... ... ... ... ... 2.05 ... ... ... ... ...

Relative amounts (% fwb), total of brown rice flour, corn starch, buckwheat flour, and soya flour (ND) and brown rice flour, potato starch, corn starch, and soya flour (D) calculated as flour. b Two replicates, standard deviation ≤ 0.01 except for corn starch (0.02). c Composition (list of ingredients): wheat starch (Codex Alimentarius Quality), milk solids, modified corn starch, soya flour, glucose, salt, stabilizer, methylhydroxyproply cellulose, iron, thiamine, riboflavin, niacin. d Moisture and protein content of minor ingredients were ignored. e Based on sum of ingredients. 568

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Analysis of Variance Baking tests and rheological experiments were conducted with the different bread and batter types (W, C, ND, D) according to a randomized block design with three blocks to achieve three replicates. Rheological measurements with dough W yielded overload, therefore only batters C, ND, and D were compared in rheology. For the measurement of bread texture, each batch of nine bread loaves (one replicate of each bread type) was subdivided three times into three pieces and these were randomly assigned to the three different storage times. A split-plot design resulted with three blocks (replicates) and four main plots (treatments for breads W, C, ND, D) that were subdivided into three split plots (days 0, 2, 5). Analysis of variance for the randomized block and split-plot design was made as described in Mead and Curnow (1983), calculating the respective mean squares and F-ratios. Multiple comparisons were made with least significant differences. LSD values were calculated with the respective t-values (two-tailed, P = 0.01) and standard errors were multiplied. Standard errors were

application of a glass coverslip. For microscopy of the bread crumb, the breads were baked with the dye incorporated into them as for the baking experiments. After the bread was cooled for 2 hr, a small sample (≈1 mm side length) of each was taken from the center of the crumb, immersed in oil in a welled slice, and covered with a glass coverslip. A confocal laser-scanning system (MRC1024, Biorad, UK) mounted on an upright microscope (Axioskop, Zeiss, Germany) with ×10 and ×40 water immersion objectives was used for the batter images. For bread crumb images, ×10 and ×63 oil immersion objectives were used. Fluorescence images (λexc = 488 nm, λem = 540 nm) of a z-series of optical sections were acquired by scanning the sample along the optical axis in 2-µm steps for the ×10 objective and 1-µm steps for the ×40 and ×63 objectives. Bread Analysis A series of bread analyses was performed with three loaves at day 0, after 2 hr of cooling, before packaging. Loaf weight and volume (rapeseed displacement method) were determined. Bake loss and loaf specific volume (mL/g) were calculated. Crust color was determined with a chromameter (CR-300, Minolta, Osaka, Japan). For crumb texture analysis, bread was sliced transversely using a slice regulator and a bread knife to obtain uniform slices of 25-mm thickness. Two bread slices taken from the center of each loaf were used to evaluate the crumb texture. Images of the bread were captured using a flatbed scanner (ScanJet4c, Hewlett Packard) and supporting software (Desk Scan II, Hewlett Packard). The brightness levels were adjusted to 150 units and contrast to 170 units using software controls (Crowley 2000). Texture profile analysis (TPA) was performed using a universal testing machine (TA-XT2i, Stable Micro Systems, Surrey, UK) equipped with a 25-kg load cell and a 35-mm aluminum cylindrical probe. The settings used were a test speed of 2.0 mm/sec with a trigger force of 20 g to compress the middle of the bread crumb to 60% of its original height. Water activity was determined with material taken from the center of the crumb (Aqua Lab CX-2, Decagon Devices, Pullman, WA). All measurements obtained with the three loaves from one batch were averaged into one replicate value. TPA was repeated with three loaves each at day 2 (50 hr) and day 5 (122 hr) after baking.

2s 2 / r

(1)

for comparisons of bread/batter types within the randomized block design,    2 2 2 (b − 1)s b + s a  /(rb )    

(2)

comparisons between bread types at the same storage time within the split plot design, and 2

2sb / r

(3)

comparisons between the same bread type at different storage times within the split plot design where s2 is error mean square; r is number of blocks and replicates; b is the number of split plot levels; sb2 is the split-plot error mean square; sa2 is the main plot error mean square. The respective degrees of freedom for the tvalues were 6 (batter rheology 4), 6, and 16, respectively. Unless otherwise mentioned, all significance statements are based on P < 0.01.

TABLE II Batter Rheology and Baking Characteristics of Breadsa Wb Extrusion (N) Loaf specific volume (mL/g) Bake loss (%) Water activity of crumb

>295 3.18 ± 0.06a 9.87 ± 0.12a 0.963 ± 0.001a

Cc 5.49 ± 0.16a 2.83 ± 0.09a 12.07 ± 0.09b 0.964 ± 0.001a

De

NDd 6.18 ± 0.62a 1.87 ± 0.08b 11.03 ± 0.18c 0.977 ± 0.003b

11.48 ± 0.44b 2.08 ± 0.01b 9.20 ± 0.06d 0.969 ± 0.002a

a

Mean value ± standard error of three replicates. Mean values followed by a common letter within the same row are not significantly different (P < 0.01). Wheat bread. c Commercial gluten-free bread flour mix. d Nondairy gluten-free bread. e Dairy gluten-free bread. b

TABLE III Mean Squares for Textural Characteristics of Four Bread Types During Storagea

Main plot Block Bread type Main plot error Split plot Storage time Bread type × storage time Split plot error a

Mean Square Values

Degrees of Freedom

Hardness

Resilience

2 3 6

2.268 252.693*** 5.685

0.002028* 0.018444*** 0.000195

2 6 16

506.416*** 8.127 3.276

0.238987*** 0.035180*** 0.000355

Cohesiveness

Springiness

Chewiness

0.000629 0.046439*** 0.000584

0.002787 0.034573*** 0.000683

0.160 58.534*** 0.318

0.208472*** 0.046392*** 0.000215

0.147193*** 0.019284*** 0.000651

0.389 8.162*** 0.294

*, **, *** = F-ratio significant at P < 0.05, 0.01, or 0.001, respectively. Vol. 81, No. 5, 2004

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RESULTS Two gluten-free bread recipes were developed from a series of 200 preliminary experiments, one containing no dairy ingredients (nondairy gluten-free, ND) and one containing a variety of ingredients with cereals, especially skim milk powder and egg

Fig. 1. Crumb hardness values of loaves baked from wheat (W), commercial gluten-free flour mix (C), nondairy gluten-free (ND), and dairy gluten-free (D) formulations during a five-day storage period. Mean values ± standard error of three replicates. Mean values labeled with a common lower case letter within the same bread type are not significantly different (P < 0.01). Mean values for a given storage time labeled with a common upper case letter are not significantly different (P < 0.01).

(dairy gluten-free, D). These breads were compared with a wheat bread (W) and gluten-free bread made from a commercial glutenfree flour mix (C). Table I lists the recipes, the moisture and protein contents of the individual ingredients, and the calculated moisture and protein levels in the total recipes. The commercial gluten-free flour mix (C), according to supplier information, is based on wheat starch, milk solids, modified corn starch, soya flour, and methyl-hydroxypropyl-cellulose. The ratio of moisture-to-solid matter in batter C is most similar to dough W; however, the nitrogen content is only ≈25% of dough W. Both newly developed recipes are characterized by higher nitrogen contents. Batter ND has higher nitrogen content than batter C but distinctly lower than batter W, whereas batter D has higher nitrogen content than batter W. Complex ingredients with high protein content in the newly developed recipes account for higher total nitrogen contents in ND brown rice flour (25%), buckwheat flour (8.5%), and soya flour (12.5%), in D brown rice flour (50%) and soya flour (12.5%) (Table I). In addition, D contains skim milk powder and egg as protein sources. Hydrocolloids used were xanthan gum (ND) and xanthan gum plus konjac gum (D). D and especially ND contain comparatively high water levels, as reflected by the moisture-to-solid matter ratio (Table I). Batter Consistency The consistencies of three batters (C, ND, D) were compared with each other and also with wheat (W) dough. Two methods were tested: 1) extrusion through a nozzle and 2) penetration with a cylinder probe. Regarding extrusion, a significantly greater force

Fig. 2A. Crumb fracturability values of loaves baked from wheat (W), commercial gluten-free flour mix (C), nondairy gluten-free (ND), and dairy gluten-free (D) formulations during a five-day storage period. B and C show TPA raw data curves for individual slices of ND bread. 570

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was required for batter D than for C and ND, whereas no significant difference was found between the latter two (Table II). Extrusion of dough W resulted in forces that exceeded the maximum load of the instrument (295 N ≈ 30 kg). This was also the case after prolonged relaxation times of up to 105 min. Penetration yielded no significant differences between the batters (data not shown). Baking Results and Texture In baking tests, the baking parameters for loaf specific volume, bake loss, water activity, and crust color were measured 2 hr after baking. Textural parameters from TPA (including hardness, fracturability, resilience, cohesiveness, springiness, and chewiness) were monitored over a five-day storage period, applying a split-plot design (main plot treatments were bread types; split-plot treatments were storage for 0, 2, 5 days). Table II summarizes the baking parameters obtained for the four bread types. Table III (split-plot) and Figs. 1–6 show the results of texture measurements. Loaf specific volume was significantly higher for breads W and C than for ND and D. Bake loss was significantly different between all breads; it was highest for C and lowest for D. Water activity was significantly higher only for ND, whereas the other breads showed no significant differences. Crust color showed no significant differences globally (data not shown). Digital images of bread crumb and whole breads are shown Figs. 7 and 8, respectively.

Fig. 3. Crumb cohesiveness values of loaves baked from wheat (W), commercial gluten-free flour mix (C), nondairy gluten-free (ND), and dairy gluten-free (D) formulations during a five-day storage period. Mean values ± standard error of three replicates. Mean values labeled with a common lower case letter within the same bread type are not significantly different (P < 0.01). Mean values for a given storage time labeled with a common upper case letter are not significantly different (P < 0.01).

Fig. 4. Crumb springiness values of loaves baked from wheat dough (W), commercial gluten-free flour mix (C), nondairy gluten-free (ND), and dairy gluten-free (D) formulations during a five-day storage period. Mean values ± standard error of three replicates. Mean values labeled with a common lower case letter within the same bread type are not significantly different (P < 0.01). Mean values for a given storage time labeled with a common upper case letter are not significantly different (P < 0.01).

With the TPA of the crumb, analysis of variance revealed for most textural parameters that bread type (W, C, ND, D) and storage time (0, 2, 5 days) had highly significant (P < 0.001) effects and that there were also highly significant interactions between them. However, hardness showed no significant interactions, whereas chewiness showed no significant time effects. Fracture occurred only for gluten-free breads (C, ND, D) at day 2 and 5. High random errors could be observed when measuring the force at which fracture took place (Fig. 2A). With some individual bread slices, no fracture occurred, whereas with the remainder of the bread slices, fracture could be either detected before the peak force (Fig. 2B), or the force at the fracture point was equivalent to the peak force of the first compression cycle (Fig. 2C). In the latter case, fracturability equaled hardness and the peak force occurred before the probe reached its deepest point. At day 0 for all bread types, and additionally at day 2 and 5 for bread W, no fracture occurred at all (force and variance were 0 at these points). Because equal variances for all bread types at all storage days could not be assumed, fracturability could not be included in the analysis of variance. Hardness of bread D was significantly higher at all days of storage in comparison with the other breads, except that at day 5, the difference from ND was no longer significant (Fig. 1). Furthermore, the hardness of all breads clearly increased over storage time, although the increase from day 2 to day 5 was no longer significant for breads C and D. Despite the fact that analysis of variance showed no interactions,

Fig. 5. Crumb resilience values of loaves baked from wheat (W), commercial gluten-free flour mix (C), nondairy gluten-free (ND), and dairy gluten-free (D) formulations during a five-day storage period. Mean values ± standard error of three replicates. Mean values labeled with a common lower case letter within the same bread type are not significantly different (P < 0.01). Mean values for a given storage time labeled with a common upper case letter are not significantly different (P < 0.01).

Fig. 6. Crumb chewiness values of loaves baked from wheat (W), commercial gluten-free flour mix (C), nondairy gluten-free (ND), and dairy gluten-free (D) formulations during a five-day storage period. Mean values ± standard error of three replicates. Mean values for a given storage time labeled with a common upper case letter are not significantly different (P < 0.01). Vol. 81, No. 5, 2004

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a certain trend can be seen in Fig. 1 that the increase in firmness over storage time is less pronounced for bread D than for the other bread types. Cohesiveness, springiness, and resilience showed comparable trends (Figs. 3–5). These parameters showed little dependence on storage time for bread W, whereas for the gluten-free breads C and ND, and less so for D, they decreased over storage time. This decrease was significant between all days of storage for cohesiveness and springiness, and significant between day 0 and day 2 only for resilience. The fact that the changes over storage time depended on bread type for all three parameters (W behaved differently from C, ND, and D, and D showed weaker effects than C and ND) is in agreement with the interactions found by analysis of variance (Table III). With regard to differences between bread types, it is remarkable that at day 0, W did not show the highest values for all three parameters, whereas at day 2 and day 5, it was significantly higher than all other treat-

Fig. 7. Digital images of wheat bread crumb (W), commercial gluten-free bread crumb (C), nondairy gluten-free bread crumb (ND), and dairy gluten-free bread crumb (D).

ments. Furthermore, at day 2 and day 5, D was significantly higher than C and ND in springiness and cohesiveness. Chewiness (Fig. 6) was characterized by the clearest interactions; it increased over storage time for W, decreased for D, and varied little for C and ND. Averaged over the different bread types, however, storage time showed no significant effect on chewiness, the F-test was not significant (Table III). Chewiness was generally high for W and D and low for C and ND, although this also depended on the storage day (Fig. 6). CLSM The microstructures of batters, dough W, and breads were studied by CLSM (Figs. 9 and 10). To identify the individual components in the batters (potato starch, corn starch, brown rice flour, buckwheat flour, soya flour, and skim milk powder), suspensions of these components were prepared and observed under the microscope (data not shown). The microstructure of dough W at ×10 was characterized by an oriented gluten network. At ×10, individual strands were visible, between which, more amorphous gluten areas were located. Gluten appeared very bright, the fluorescent dye Safranin O was strongly adsorbed. At ×40, globular starch granules (dark) with ≈10–20 µm diameters were visible embedded in gluten. These adsorbed the dye obviously to a lesser degree than protein (gluten) and thus appear dark. Batter C at ×10 showed mainly starch granules (wheat starch, according to information from the supplier, see Table I) visible as light grey globules with diameters of ≈20 µm. No oriented network was visible at ×10 but isolated white areas were found (protein in wheat starch and soya flour). At ×40, besides the greyish wheat starch granules (diameter ≈10–20 µm as in dough W), these white protein areas were found again along with some fine structures. Batter ND was similar to the batter C, however the white protein areas were more frequent (×10), reflecting the higher (soya) protein content of the recipe. Corn starch (54% of the flour basis, Table I) appeared as slightly smaller granules compared with the wheat starch (×40). Batter D appeared most heterogeneous at ×10, with numerous white areas (soya protein) and large (≤50 µm) dark starch granules (potato starch). At ×40, a continuous white area (milk and egg protein) was visible, in which starch granules of different sizes were embedded.

Fig. 8. Digital images of wheat bread (W), commercial gluten-free bread (C), nondairy gluten-free bread (ND), and dairy gluten-free bread (D). 572

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The bread crumbs were studied at ×10 and ×63, however, at ×10, no characteristic differences were found between the bread types. Pore walls ≈0.1–1 mm thick that surrounded the voids were visible (data not shown). At ×63, pore walls in bread crumb W were characterized by a sponge-like structure of continuous associated strands (denatured gluten). Globular dark bodies embedded in these white strands might either be voids or deformed (gelatinized) starch granules. In crumb C, only diffuse, greyish areas could be found (probably gelatinized starch), overall lacking any network-like structure. Crumb ND appeared similar, however, in the grey areas, globular structures were still visible, and also white, well-stained areas. Overall, crumb ND resembled batter ND. It appears plausible that due to gelatinization, the corn starch granules increase in size and partly loose integrity, but are still visible in crumb ND. No continuous white protein network was visible. Crumb D showed greatest similarity to crumb W. Well-stained protein structures were visible with film-like areas and strands. Dark bodies (diameter ≈20 µm) embedded in W were either voids or gelatinized starch. DISCUSSION The present study included three different recipes, all of which resulted in acceptable gluten-free breads. However, the bread structure was achieved in different ways. The commercial (C) flour mix contained mainly starch with little protein originating from the soya and dairy ingredients present. The newly developed nondairy bread (ND) was rich in whole meal cereals and soya flour and thus higher in protein, although still much lower than a typical wheat (W) formulation. The dairy bread (D), however, had the highest protein levels due to the presence of soya flour, skim milk powder, egg, and whole meal cereals (brown rice flour).

Fig. 9. Confocal laser-scanning micrographs of wheat dough (W), commercial gluten-free batter (C), nondairy gluten-free batter (ND), and dairy gluten-free batter (D) stained with Safranin O dye. Magnification bar ×10 (100 µm, left column); ×40 (50 µm, right column).

Most of the differences between the breads can be explained by the characteristic properties of starch, protein, or fiber. Gelatinized starch tends to regain a microcrystalline form at room temperature (retrogradation). Many researchers have found that the firming of wheat bread crumb is influenced by many factors, including protein content, moisture, temperature, loaf specific volume, and flour content (Ponte et al 1962; Axford et al 1968; Maleki et al 1980). Changes in crumb properties associated with staling include an increase in crumbliness of the crumb, starch crystallinity, opacity, firmness, loss of flavor, and a decrease in soluble starch and hydration capacity of the crumb (Herz 1965; D’Appolonia and Morad 1981). Retrogradation in wheat bread solely depended on temperature and moisture. Zeleznak and Hoseney (1986), Davidou et al (1996), and Rogers et al (1998) reported that bread moisture content influenced the firming rate and starch retrogradation during storage of bread. Rogers et al (1998) reported that the firming rate of wheat bread was retarded in bread that contained a higher moisture content than that of wheat bread of lower moisture. The high water activity of the ND bread reported here did not, however, affect a retarded rate of firming. Bran particles from brown rice flour or buckwheat contain high concentrations of fiber and swell extensively. Bran supplementation usually weakens the structure and baking quality of wheat dough and decreases bread volume and elasticity of the crumb (Pomeranz et al 1977; Gan et al 1992). For wheat, Gomez et al (2003) concluded that water absorption was increased with the addition of dietary fiber, which in turn may explain the resulting low volume. Therefore, in analogy, a higher water level was necessary in ND batter to obtain a consistency comparable to that of batter C due to the swelling of such particles. The significantly higher firmness of batter D in comparison with that of batter C, despite a higher water content reflects its high egg, soya, and milk protein content, the high percentage of brown rice flour, and the addition of konjac flour plus xanthan gum. Dairy ingredients are used in bread for nutritional benefits including calcium content and protein efficiency ratio and functional benefits including flavor and texture enhancements and storage improvement (Kenny et al 2001). The significantly higher firmness of batter D in comparison with that of C, despite a higher water content, reflects its high protein content. Konjac gum is derived from the tuber of Amorphophallus konjac (Nishinari and Takahashi 2003). Konjac gum contains a high molecular weight glucomannon consisting of mannose and glucose in a molar ratio of ≈3:2 with beta-1,4-linkages (Tye 1991). Xanthan gum is a high molecular

Fig. 10. Confocal laser-scanning micrographs of wheat bread crumb (W), commercial gluten-free bread crumb (C), nondairy gluten-free bread crumb (ND), and dairy gluten-free bread crumb (D) stained with Safranin O dye. Magnification bar ×10 (100 µm, left column); ×63 (50 µm, right column). Vol. 81, No. 5, 2004

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weight natural exopolysaccharide produced by the bacterium Xanthomonas campestris (Su et al 2003). Xanthan gum forms a single- or double-stranded helix (Sato 1984; Rodd 2000) and is classified as a cold-set gel. Hydrocolloids according to Toufeile et al (1994), are added as gluten substitutes in the formulation of gluten-free breads because these gums could act as polymeric substances that mimic the viscoelastic properties of gluten in wheat bread dough. Research reported by Tye (1991) showed that konjac flour and xanthan gum interact to form a gel with unique viscoelastic properties. This author also reported that konjac flour interacts with most starches, thus increasing the viscosity of the systems. Nevertheless, all batters, including batter D, were softer than dough W, which could not be extruded under the conditions applied. Wheat gluten is unique in its ability to form a large, aggregated, viscoelastic network (Paolo et al 2001). However, the lack of gluten does not preclude the achievement of a high volume, as demonstrated by bread C, which was mainly dominated by starch. The main drawbacks of this approach are of nutritional nature because important components such as fiber and naturally occurring vitamins are lacking. Furthermore, such starch-based breads are characterized by a relatively flat “starchy” aroma. The presence of whole meal cereals adds aroma and dietary fiber, however at the expense of volume (Krishnan et al 1987; Chen et al 1988; Pomeranz et al 1997; Sievert et al 1990). In bread ND, CLSM illustrated that the structure was visibly dominated by starch, which will result in the formation of a weak gel structure. As explained above, bran particles or fiber reduce the volume even in whole meal wheat breads, where the structure is stabilized by gluten. When no gluten is present, this negative effect on volume can be assumed to be even worse. In agreement with these findings, breads D and ND were lower in volume in comparison with C, although D was slightly higher than ND. The texture and staling showed several common traits characteristic of all gluten-free breads. All were brittle at day 2 and also at day 5, unlike bread W. This was reflected by different texture parameters. Fracture occurred during the first bite; that is, the bread structure was no longer cohesive as it cracked or crumbled under the probe. Consequently, the measured cohesiveness derived from the two-bite curve (ratio of the positive force area during second compression to that during first compression) (Bourne 1978) was also much lower at day 2 and day 5. Springiness was also lower at day 2 and day 5; that is, the bread recovered less in height during the first and second bite (Bourne 1978) as it lost elasticity during storage. The reduction in resilience also characterizes loss of elasticity. Whereas in the determination of the springiness, the bread crumb can recover for 5 sec between the two compression cycles, in the determination of resilience, the ratio of the area under curve of the second half of the first cycle (upward stroke) to the first half (downward stroke) is measured. For high resilience, a relatively quick, instantaneous recovery is required, whereas delayed recovery also may contribute to springiness. In agreement with this consideration, resilience was more quickly lost over storage time; it strongly dropped from day 0 to day 2 and only slightly and nonsignificantly from day 2 to day 5. In contrast, springiness, dropped more gradually over storage time; that is, a delayed recovery could be maintained even longer in stale bread. These various changes characterizing brittleness are in agreement with what was reported by Toufeili et al (1994) for gluten-free flat bread. These authors found that after overnight storage, all gluten-free formulations cracked extensively on application of stress, had a markedly reduced ability to resist shearing forces, and were notably less cohesive when compared with regular bread aged for a similar time period. In the present study, bread W showed none of these changes, except a slight drop in resilience after five days of storage in accordance with what was found by other authors (Nussinovitch et al 1992). Only the increase in firmness was the same as for the 574

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gluten-free breads. Although bread W was not most elastic and cohesive at day 0, it did preserve these properties to day 5 and thus exceeded the gluten-free breads at day 2 and day 5. In wheat bread, it may be considered that a continuous elastic structure of denatured gluten strands surrounding the starch may mask some of the changes originating from starch retrogradation. It has also been reported that gluten may serve as a reservoir to buffer any changes in the hydration capacity of starch and retard crumb firmness development (Gray and BeMiller 2003) A similar situation may be anticipated for bread D. It was the only gluten-free bread where CLSM showed continuous film-like protein structures similar to gluten. Because milk and soy proteins in the other recipes did not show such films, it is most likely that egg protein is responsible for this. Regarding egg proteins, interfacial film formation is essential for foam and emulsion development. In foaming systems, the formation of strong cohesive viscoelastic films are essential for stable foaming (Kato et al 1990). Despite the common staling patterns of all gluten-free breads described so far, several effects were weaker for bread D relative to breads C and ND; hardness was generally higher (reflecting the stronger structure caused by egg protein as seen in CLSM), but the increase over time was less (reflecting the lower starch content). Accordingly, resilience, cohesiveness, and springiness were better maintained. Hence, bread D remained elastic for a longer period of time and overall became less brittle during storage. Even though a firm structure might be regarded as a disadvantage of bread (bread D), this property coincides with the elastic and cohesiveness characteristics recorded for wheat bread. The chewiness was the most indicative characteristic of bread D. It is calculated by multiplying hardness, cohesiveness, and springiness (Bourne 1978), and reflects the time required to masticate a solid food to ready for swallowing (Cauvain 1987). Chewiness was much higher for bread D than for C and ND. The low values of C and ND at all days reflected the fact that a strong increase in firmness was compensated by a strong drop in cohesiveness and also in springiness; that is, less chewing is required because the bread breaks in the mouth like a biscuit. In contrast, bread D had a higher chewiness value that was more comparable to bread W after day 2 and day 5. The overall better keeping quality of the high protein bread D was its outstanding advantage. CONCLUSIONS Starch-based gluten-free breads may achieve a high volume, but at the expense of quick staling. Increased water levels in combination with whole meal cereals could not notably delay the staling. However, the addition of the right protein sources in sufficient amounts improved the keeping quality. The formation of a continuous protein phase and film-like structures appears to be critical because they can partially mask changes caused by starch retrogradation. Due to its high resolution and its ability to scan through thick samples, confocal laser-scanning microscopy proved very useful in detecting these desirable protein structure elements in bread crumb. ACKNOWLEDGMENTS This project was funded by the Irish government under the National Development Plan, 2000-2006. We would like to thank Charmaine Clarke and Tom Hannon for valuable contributions made to this article. LITERATURE CITED American Association of Cereal Chemists. 2000. Approved Methods of the AACC, 10th Ed. Method 46-30. The Association: St. Paul, MN. Arendt, E. K., O’Brien, C. M., Schober, T. J., Gallagher, E., and Gormley, T. R. 2002. Development of gluten-free cereal products. Farm Food 2127. Axford, D. W. E., Colwell, K. H., Confor, S. J., and Elton, G. A. H. 1968.

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[Received November 14, 2003. Accepted March 23, 2004.]

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