Impact of storage on starch digestibility and texture of a high-amylose wheat bread

Staling is a complex process that determines the shelf-life of baked products like bread. Breads made using high-amylose flour may elicit a lower glycaemic response, with benefits for health, however the impact of storage on novel high-amylose wheat foods structure are not known. We investigated the staling behaviour of high-amylose bread made from a starch branching enzyme II (sbeII) wheat mutant compared to a wild-type (WT) control, by measuring starch digestibility (susceptibility to amylolysis) and bread texture over time in different storage conditions. Breads prepared from sbeII and WT control wheat flours were subjected to fresh, refrigerated and frozen storage, and starch digestibility and crumb texture were measured up to three days. Starch from sbeII flour was characterised by a larger proportion of long chains resulting in increased amylose content, typical of sbeII mutant wheat. Starch in sbeII bread was less susceptible to amylolysis when freshly baked (~17% difference) and after storage (26%-28% difference, depending on the storage condition), compared to the WT control. Texture of freshly baked sbeII bread was similar to the WT control; storage conditions affected the progression of crumb firming and resilience to touch for both breads, but changes in crumb texture were less pronounced in sbeII bread. Overall, sbeII bread was less prone to staling than conventional WT bread during the first three days of storage, particularly when stored in the fridge or at room temperature.


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Bread made from white wheat flour is a staple food in many countries, however, its high glycaemic 27 potency can potentially harm cardiometabolic health over time (Livesey, et al., 2019). 28 The glycaemic response to bread reflects the digestibility of starch, the main dietary carbohydrate, so 29 developing wheat starch with greater resistance to digestive enzymes (such as α-amylase) may lead to 30 a viable healthier alternative to conventional white wheat bread, if product quality and shelf-life can 31 be preserved. 32 One promising approach is the use of novel types of wheat with increased amylose content and lower 33 starch digestibility (Hallström E properties and the progression of staling, however, previous studies reported conflicting results on the 61 achieve a similar starch content (~75 g) based on the total starch content of the flour. The same 96 process as used to produce sbeII and WT control doughs, later portioned into rolls and baked. Using a 97 Kenwood mixer (KM300, Kenwood UK) with a hook attachment, flour (WT = 57%, sbeII = 55%), caster 98 sugar (WT and sbeII = 2%), Allison's dry yeast (WT and sbeII = 2%) and water (WT = 37%, sbeII = 39%) 99 were mixed at low speed for one minute, after which Willow vegetable shortening (WT and sbeII = 2%) 100 was added. After three minutes of mixing, salt (WT and sbeII = 1%) was added and the dough was 101 mixed at increasing speed for five more minutes. 102 The dough was then fermented for two hours at ambient conditions (21 ֯ C, 41% Relative Humidity 103 (RH)), then rolls were shaped and proofed for 15 minutes at 40 ֯ C, 100% RH using a steam oven (DG 104 6001 GourmetStar, Miele, UK). Rolls were sprayed with water and baked at ~185 ֯ C for 20 minutes in a 105 pre-heated convection oven (Hotpoint HAE60P, Whirlpool UK) with a tray of water to provide steam, 106 to reach a core temperature of ~95 ֯ C. Batches were prepared under identical conditions and rolls (3 107 per batch) were paired by their baking position in the oven to ensure even baking. After baking, rolls 108 were left at room temperature to cool for two hours. 109

Allocation to storage conditions and sample preparation 110
After 2h cooling at room temperature, bread rolls were either analysed immediately (fresh, 0h) or 111 after 24h, 48h, and 72h of storage at room temperature (RT, +19 to +21°C), freezer temperature (-18 112 to -20°C) and fridge temperature (+3 to +5°C), n = 3 per condition. Samples stored in the fridge and 113 freezer were allowed to return to RT before analysis. A roll of ~165 g required approximately 3h to 114 reach RT at the core after freezer storage, and approximately 2h after fridge storage. 115 Figure 1 shows sample preparation of each bread genotypes for the different analyses. 116 For starch digestibility analysis, the structure of the bread was not preserved as the starch 117 characteristics were analysed at microstructural level. 118 Three bread rolls per bread type (sbeII and WT control) were produced, with a sample from each roll 119 subjected to each storage condition. After 2h of cooling at RT, the crust was removed and the crumb 120 was ground using a Kenwood mini-chopper food processor and sieved using a 1 mm sieve. The fraction 121 below 1 mm was used to determine starch digestibility in vitro. Starch susceptibility to amylolysis was 122 measured on fresh bread and bread stored for 24h, 48h, 72h at RT, fridge and freezer conditions, as 123 described above. 124 All samples were weighed out from fresh bread and then stored in the allocated conditions, with three 125 independent samples (from three rolls) per condition, Figure 1A. For samples stored in cold temperature (fridge and freezer), tubes were allowed to return to RT before analysis. Because of the 127 small amount of sample required for this type of analysis (~63 mg) compared to the weight of a whole 128 roll (~165 g), thawing time was proportionally adjusted. Samples from the fridge reached RT in 20 min 129 and samples from the freezer reached RT in 45 minutes. 130 For texture and moisture analyses, the macrostructure of the rolls was preserved as the crust plays a 131 role in moisture loss and crumb firming. Therefore, bread rolls were produced over three subsequent 132 days and assigned to a storage condition (n = 3 rolls per condition). Each roll was stored in fridge, 133 freezer or at RT in individual sealed bags. 134 Each analysis was carried out on three independent rolls, from different batches of dough to capture 135 batch-to-batch variability and ensure fair comparison between sbeII and WT control breads. After 136 storage and ahead of analysis, each roll was weighed then the crust was discarded, the core of the roll 137 was cut into four 5x5x5 cm cubes and was analysed immediately (leading to four technical replicates 138 for each measure), Figure 1B. Moisture by air-oven method AACC (44-15A), one stage procedure 139 (AACC International, 1999) was used to measure moisture in samples from the same rolls used for 140 texture analysis, using the remaining bread crumb (n = 3 independent samples per condition). 141 For microscopy, three rolls were produced per bread type (sbeII and WT control), samples of crust and 142 crumb (from the core of the roll) were taken when fresh (2h after baking) after which the rolls were 143 stored in the freezer for 7 days. After 7 days, each roll was left at RT for approx. 3h to reach ambient 144 temperature (+19 °C). After thawing, crust and crumb samples were taken from the core of the roll 145 and mixed with water to disrupt the crumb matrix and imaged. 146

Moisture of bread crumb during storage 158
Moisture content of bread crumb samples was measured by the air-oven drying (AACC 44-15A), one 159 stage procedure (AACC International, 1999). Samples were weighed out in metal tins immediately 160 after slicing to prevent moisture loss. These were then placed in the oven for exactly 16 hours, then 161 removed from the oven and left to cool for one hour in a desiccator before weighing the tins again. 162 Moisture content was calculated by subtracting the weight of the sample after drying in the oven to 163 that of the fresh sample for three independent replicates (one from each roll). 164 Moisture content of bread rolls during storage was also determined by weighing the rolls before and 165 after storage. 166 Godalming, UK) equipped with a 5-kg load cell and 50 mm compression plate (P50). Uniaxial 170 compression with 100 mm min-1 crosshead speed was applied to a 5 x 5 x 5 cm sample with crumb 171 hardness corresponding to the force N required for a 40% compression. Parameters of interest were 172 obtained using Exponent software (6.0, Stable Micro System, Godalming, UK), (

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Dough formulation was similar for sbeII and WT control breads, however sbeII flour required 229 additional water (39% for sbeII and 37% for WT control) to produce a workable dough. After mixing, 230 the dough was assessed visually and found to be well developed however, the WT control dough 231 appeared to be slightly stickier than the sbeII dough, in all batches produced. Both roll types 232 developed and baked well, reaching a core temperature of 95°C. 233 Rolls were made to deliver an equal starch content; the sbeII flour was characterised by a lower total 234 starch content, therefore the sbeII rolls were ~10 g larger than the WT control rolls (161.26 g ± 0.16 g, 235 155.73 g ± 0.40 g, respectively, mean ± SEM, n=3). 236

Moisture 237
Moisture changes are shown in Figure 2, data is shown in Supplementary Table 1  between sbeII and WT control bread is maintained throughout storage. 255 Starch digestibility was 51.7% in sbeII freshly baked bread compared to 61.4%, 61.7% and 60.4% when 256 sbeII bread was stored for 72h in the freezer, fridge and RT respectively. In freshly baked WT control 257 bread, starch digestibility was 62.4% and increased to 78.6%, 78.5% and 82.1% after 72h freezer, 258 fridge and RT storage, respectively. For samples stored at RT, some evidence of interaction between 259 storage and bread genotype was observed (p = 0.03). Here, C90 was higher after 48h storage at RT 260 compared to fresh bread. 261 These changes are relative to the starch digested within 90 minutes of incubation however, the model 262 suggests that digestion of sbell bread is slower compared to the WT control, and that at the end of the 263 reaction the differences between the breads would be negligible (as evidenced by estimates of the C∞ 264 parameter), ( Figure 3E). 265 The digestion rate (k) of starch in sbeII bread was overall lower compared to starch in WT control 266 bread (p = <0.001) and no effect of interaction between genotype and storage was found (p = 0.1). 267 Compared to fresh bread, RT and fridge storage affected the rate of starch digestion in both bread (p = 268 <0.001, both conditions) but not in breads stored in the freezer (p = 0.2), Figure 3A-C. 269 Considering the AUC of digestibility curves, besides the significant effect of the genotype (p = <0.001), 270 there was a significant effect of the fridge and freezer storage on AUC, compared to fresh bread (p = 271 0.002 and p = <0.001, respectively). There was some evidence of an interaction effect between storage 272 and genotype on AUC however, the magnitude of the effect is relatively small as the AUC summarises 273 rate (k) and extent of digestibility (C90), (p = 0.0007). 274 The estimated marginal means with 95% CI for all storage conditions are reported in Supplementary 275 Table 2. 276   There was less effect on bread hardness with freezing, with no difference in the effect of freezing 297 between genotypes. 298 Following storage, the sbell bread appeared slightly chewier than the WT, with the most marked 299 difference after 72h in the fridge or at RT (sbeII fridge 72h = 634.5, WT fridge 72h = 1088, p = 0.0002, 300 sbeII RT 72h = 593.9, WT RT 72h = 888.9, p = 0.004). 301 Resilience of sbeII breads was marginally lower than the WT control after 24h of freezer storage (sbeII 302 freezer 24h = 0.52, WT freezer 24h = 0.57, p = 0.003) but slightly higher than the WT control after 303 fridge storage (sbeII fridge 24h = 0.51% and WT fridge 24h = 0.47, p = 0.004). 304

Chain length distribution 305
Analysis of starch chain-length distribution showed differences in the molecular structure of amylose 306 and amylopectin from sbeII mutant wheat compared to the WT control. 307 The proportion of long amylopectin chains (DP 37 to 100) was higher in sbeII starch (19% ± 0.3%) 308 compared to the WT control (10.9% ± 0.7%) and presented two distinct peaks (Figure 4, B), in contrast 309 to a slight shoulder peak in WT starch. The sbeII starch was also characterised by a larger proportion of 310 amylose chains (DP 100 -1600) compared to the WT control (26.3% ± 3.4, 15.8% ± 2.7%, respectively). 311 The percentage of short chains (DP<25) in mutants was lower in the sbeII mutant compared to the WT 312 control (Figure 4, C). 313 Chain length proportions per DP fraction are reported as Mean ± SD, n = 3. All fractions can be found 314 in Supplementary Table 4. 315

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with error bars = 95% CI, based on DP as described by Hanashiro et al. (Hanashiro, et al., 1996) and Vilaplana et al. (Vilaplana,  In freshly baked bread, only few starch granules in both WT and sbeII crumb were visibly birefringent 328 suggesting loss of starch semi-crystalline structure during baking ( Figure 5.B). 329 Breads were then stored for 7 days in the freezer and thawed once. A larger number of granules with a 330 birefringent pattern was observed in the crumb WT control bread, while almost no granules with such 331 pattern were visible in the crumb of frozen sbeII bread, Figure 5.C. 332 The crust of breads was also included in the analysis. Here, the birefringence was less evident in sbeII 333 starch granules from fresh crust compared to the WT control granules, possibly due to different levels