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Whole-wheat flour particle size influences dough

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Cite this: DOI: 10.1039/c9fo02587a

properties, bread structure and in vitro starch
digestibility†
Suyun Lin, a,b Jing Gao, a,b Xiaoxuan Jin,a,b Yong Wang,c,d Zhizhong Dong,c
Jian Yingc and Weibiao Zhou *a,b

Received 4th November 2019,
Accepted 18th March 2020
DOI: 10.1039/c9fo02587a
rsc.li/food-function
Whole-wheat flour (WWF) is increasingly popular because of the health benefits of whole grains. This
study investigated the effect of WWF particle size on dough properties, bread quality and in vitro starch
digestibility. WWF was made from intact whole grain directly. Three WWF particle sizes were examined,
including coarse, medium and fine with a mean size of 1315, 450 and 199 µm, respectively. The dough
made from WWF of a larger particle size exhibited lower extensibility and stability, and subsequently the
bread had a more compact structure (i.e. lower open porosity and thicker cell thickness), smaller specific
volume and harder texture, which were regarded as poor quality attributes. On the other hand, the bread
made from the fine WWF exhibited a higher amount of released glucose than those made from the
coarse and medium WWFs. Moreover, the particle size of bread bolus showed no significant effect on
in vitro starch digestion. The whole study demonstrated that the particle size of WWF plays a critical role
in determining both bread quality and digestibility.

1. Introduction devised to enhance the quality of whole grain products will

A whole grain consists of intact, ground, cracked, or flaked car-
yopses, whose principal anatomical components – starchy
endosperm, germ, and bran – are present in the same relative
proportions as they exist in the intact caryopsis.1 Hence, it has
been regarded as a primary source of dietary fibre and other
various nutritional and functional compounds (which exist
mainly in bran and germ) including vitamins, minerals and
bioactive phytochemicals.2 Furthermore, it has been proved
that whole grain intake is positively related to the prevention
of many chronic diseases such as certain types of cancer, type
2 diabetes, and cardiovascular diseases.2,3 However, the fact
that foods containing whole grains or made from whole grain
flour typically show an inferior structure and sensory quality
has led to limited consumer acceptance. As a result, strategies
a
Department of Food Science and Technology, National University of Singapore,
Science Drive 2, Singapore 117542, Singapore. E-mail: weibiao@nus.edu.sg
b
National University of Singapore (Suzhou) Research Institute, 377 Linquan Street,
Suzhou Industrial Park, Jiangsu 215123, China
c
COFCO Nutrition & Health Research Institute, No.4 Road, Future Science and
Technology Park South, Beijing 102209, China
d
Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing
Technology & Business University, Road 11st Fucheng, Beijing 100048, China
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c9fo02587a
definitely arouse the interest of food manufacturers.
In general, the particle size of whole-wheat flour (WWF)
was recognized as a significant factor impacting flour func-
tionality and product quality.4 Wang et al.5 reported that the
reduction of WWF particle size markedly increased the
maximum resistance to extension and extensibility of cracker
dough. Liu et al.6 noticed that when the median particle sizes
of WWF reduced from 175 μm to 130 μm, the breaking force
and extensibility of tortilla significantly increased, suggesting
an improvement of the WWF tortilla quality. Moreover, the
grain particle size was reported to influence starch digestion.
Blasel et al.7 observed that with each 100 μm increase in the
corn grain particle size, the degree of starch accessed by
α-amylase declined by 26.8 g kg−1. Concerning bread, it was
identified that the proportions of slowly digestible starch and
resistant starch in gluten-free bread made from coarse rice
flour were larger than that of bread made from fine rice flour.8
Bread, as one of the most widely consumed grain-based
foods, is an important carrier for the beneficial intake of
whole grains. Its physical structure was considered as one of
the factors impacting the glycemic index, implying that the
more compact the structure, the lower the glycemic response.9
Therefore, it is urgent to investigate the effect of WWF particle
size on the quality of bread, especially the structure.
Nonetheless, there is insufficient knowledge on either how
such a structure can become favourable when producing

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whole-wheat bread, or whether by manipulating the particle
size of WWF it is possible to impact the baked bread structure
and regulate its starch hydrolysis. Furthermore, WWF directly
produced from grinding intact whole grains can be composi-
tionally different from those made by reincorporating bran
into refined flour which was used in most studies in the
literature.5,6,10
Therefore, this research attempts to investigate the effect of
WWF particle size (coarse, medium and fine) on dough pro-
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perties, one of the most pivotal factors during bread making,
as well as on the physical quality and starch digestibility of
whole-wheat bread. All WWFs were obtained from directly
grinding intact whole grain. These results will provide manu-
facturers with a scientific guideline to produce higher quality
whole-wheat bread by using WWF, and help consumers to
better understand the digestion profile of whole-wheat bread.
2. Materials and methods
2.1. Materials
Coarse whole grain wheat flour (Prima, Singapore; 57.1%
carbohydrate, 15.5% dietary fibre, 15.3% protein, 10% moist-
ure, and 1.9% fat) was purchased from a local supermarket.
Table salt, sugar, shortening (Radman, Singapore), and dry
yeast (Algict Bruggeman N.V., Belgium) were bought from a
local market (Singapore). The digestive enzymes of α-amylase
(A3176, from porcine pancreas), pepsin (P7000, from porcine
gastric mucosa), pancreatin (P7545, from porcine pancreas),
and amyloglucosidase (A7095, from Aspergillus niger) were all
bought from Sigma Aldrich (St Louis, MO, USA), along with
porcine bile extract (B8631) and dialysis tubing membrane
(D9777, cut-off 1400 Da, avg. flat width 25 mm).
2.2. Physicochemical characterisation
The coarse whole grain wheat flour defined as coarse WWF
was then ground into medium and fine WWFs using a pulver-
izing machine (Rong Tsong, RT-N12HK, Taiwan) operated at
4000 rpm for 30 s and 8000 rpm for 30 s, respectively. Refined
wheat flour (Prima, Singapore) was taken as the control (11.7%
protein). Particle size distribution of the four types of flour was
measured by using a laser diffraction particle size analyzer
(LA-950, HORIBA, Japan). Water retention capacity (WRC) of
flour was measured according to AACC Method 56-11 (2000).
The measurements of contents of wet gluten and damaged
starch were conducted as stated in AACC Method 38-10 and
AACC Method 76-30, respectively.
2.3. Characterization of dough properties
2.3.1. Farinographic test. Farinograph characteristics of
the flours were investigated according to AACC method 54-21
by using the Farinograph-E with a 50 g bowl (Brabender,
Duisburg, Germany). Triplicate tests were conducted to obtain
the following parameters: water absorption (WA, percentage of
water required to reach the desired dough consistency), dough
development time (DDT, time to achieve peak consistency),
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stability (time that dough consistency remains at the desired
consistency), mixing tolerance index (MTI, consistency differ-
ence between the height at peak and that 5 min later) and
time to breakdown (time to leave the desired consistency).
2.3.2. Extensograph test. The extensibility of dough was
determined by utilizing an Extensograph-E (Brabender,
Duisburg, Germany) based on the AACC Method 54-10.
Herein, the parameters of the maximum resistance to exten-
sion (Rm, the maximum height of the curve), extensibility (E,
the length of the curve) and ratio of Rm to E (Rm/E) were
measured in triplicate.
2.3.3. Rheofermentometer analysis. The characteristics of
dough development and gas production during fermentation
were investigated by applying a Rheofermentometer F3
(Chopin Technologies Ltd, Paris, France). Briefly, the dough
piece (315 g) was put into a movable steel basket with a piston
(2000 g) laying on the top and measured at 40 °C for 3 h. Then
the final dough height (h), the maximum dough height (Hm),
dough weakening coefficient (WC, (Hm − h)/Hm), the time
when gas starts to escape from the dough (Tx), the total gas
production volume (Vt), and coefficient of gas retention (CR)
were determined in triplicate.
2.3.4. Amylograph test. The gelatinisation property of flour
was determined by utilizing an Amylograph-E (Brabender,
Germany) according to AACC Method 22-10. Briefly, a flour
slurry was prepared by mixing 90 g WWF with 450 mL distilled
water and put into a stirring bowl. The amylograph then con-
tinuously recorded the changes of viscosity of the flour slurry
during heating (from 30 to 95 °C) with a rate of 1.5 °C min−1.
The following parameters were obtained from the amylogram:
beginning-of-gelatinisation temperature (Tb) means the temp-
erature at which starch starts gelatinising, gelatinisation
maximum (Gm) means the peak viscosity in amylograph unit
(AU), and gelatinisation temperature (Tg) means the tempera-
ture at which the peak viscosity is obtained. The test was con-
ducted in triplicate.
2.4. Bread preparation
Baked bread was prepared by slightly modifying the previous
method,11 and the formula included the following: flour,
1000 g; sugar, 30 g; yeast, 10 g; salt, 20 g; shortening, 30 g;
water (adjusted by WA obtained from the farinograph test). All
ingredients were mixed at 45 rpm for 1 min by using a spiral
mixer (WAG-RN20, Varimixer Globe, USA), and subsequently
mixed at 100 rpm for another 4 min to produce dough
samples. After resting for 15 min at ambient temperature, seg-
ments of dough were produced by using an automatic dough
divider (DR Robot, Daub Bakery Machinery B.V., Holland).
Dough pieces (60 g) were then rounded and proofed in a fer-
mentation cabinet (Climatic chamber-KBF, Binder, Germany)
for 40 min (40 °C, 85% relative humanity). Finally, the proofed
samples were put into an oven (MS01T04-2, Eurofours, France)
for baking (200 °C, 10 min). After 1 h of cooling at ambient
temperature, the bread was stored in sealed plastic bags for
further analyses.
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Food & Function
2.5. Quality evaluation of bread
2.5.1. Specific volume and the maximum height. Specific
volume (SV) of bread is the ratio of the volume to the weight.
Bread volume and its maximum height were detected by using
a Volscan Profiler (VSP 600, Stable Micro System Ltd, Surrey, U.
K.). The moisture content of bread was measured based on the
AACC method 44-01 (2000).
2.5.2 Crumb structure. Structural characteristics of the
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bread crumb were evaluated by using an X-ray microtomogra-
phy (μCT) device (Skyscan 1076, Bruker, Kontich, Belgium).
The sample was made by cutting a 1.5 × 1.5 × 1.5 cm cube
from the center of the bread crumb, which was then sealed in
a plastic bag to prevent water loss. The X-ray beam emitter was
tested at a voltage of 65 kV per 153 μA. During the scanning
process, the sample was rotated by a total of 360° (rotation
step set at 0.2°) with a pixel size of 9 μm to get radiographs
with ideal resolution. The radiographs were then reconstructed
by using NRecon reconstruction software (v. 1.6.1) to obtain
cross-sectional images. Cell walls of the solid matrix appeared
grey, and air cells appeared black. The cross-sectional images
were then analyzed by using CTAn software (v. 1.4.1, Bruker
microCT, Belgium), and finally elements including mean cell
size, cell wall thickness, open porosity, and total porosity were
obtained.
2.5.3. Textural profile analysis. Textural characteristics of
bread were determined utilizing a Texture Analyzer (TA-XT2,
Stable Micro Systems, Surry, U.K.) assembled with a 20 mm
diameter cylindrical probe. 2 cm thick bread slices were cut
vertically from each bread. The main test conditions of the
double compression analysis were as follows: test speed, 2 mm
s−1; trigger force, 5 g; 5 s gap between compressions; and com-
pression height, 40% of the original height. The hardness (N,
peak force in the first compression), resilience (withdrawal of
the first compression), cohesiveness (ratio of area work of two
compressions) and chewiness (calculated from hardness ×
cohesiveness × ratio of time to reach the peak force in two
compressions) of the bread were quantified. For each type of
bread, twenty-four samples from three independent batches
were analyzed.
2.6. In vitro starch digestion
A three-phase oral-gastro-intestinal in vitro digestion analysis
was carried out to simulate human digestion. All stimulated
digestive fluids used in this study were prepared according to
an international consensus.12 Bread samples were applied to
in vitro digestion analysis 24 h after baking. The in vitro diges-
tion procedure was modified from the method of Sui et al.13
Briefly, for the oral phase, in order to find out whether the
particle size of bread bolus would influence its in vitro digesti-
bility, bread crumb (without crust) was blended into fragments
of three different ranges of sizes: F1 (0.5–3 mm), F2
(5–10 mm), and F3 (15–30 mm). To mimic the chewing
process, the crumb fragments (5 g) were mixed with 5 mL oral
phase fluid (final concentration: 75 U mL−1 α-amylase and
0.75 mM CaCl2) in a beaker, and after vortexing for 20 s, the
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beaker was put in a water bath (37 °C) for 100 s. Subsequently,
8 mL gastric phase fluid (final concentration: 2000 U mL−1
pepsin and 0.075 mM CaCl2) was added into the mixture and
further incubated for 2 h under constant stirring at 450 rpm
(37 °C water bath). Then the simulation of small intestine
digestion was started through mixing with 16 mL intestinal
phase fluid (final concentration: 100 U mL−1 trypsin, 21 U
mL−1 amyloglucosidase, 0.3 mM CaCl2, 10 mM bile). The
mixture was subsequently transferred to a 30 cm dialysis tube
to mimic sugar release in the intestine. The reaction was con-
stantly performed in a water bath (37 °C) with continuous stir-
ring at 450 rpm for 6 h. During this phase, 0.5 mL dialysate
was taken at 0th, 5th, 10th, 15th, 20th, 30th, 40th, 50th, 60th, 90th,
120th, 150th, 180th, 240th, and 360th min and the same amount
of phosphate buffer solution was added immediately. The
amount of released reducing sugars in the dialysate was quan-
tified by reacting with the reagent of 3,5-dinitrosalicylic acid
(DNS). Briefly, DNS regent and dialysate sample (v/v 1 : 1) were
mixed thoroughly in a tube which then was put into boiling
water and incubated for 15 min. After cooling (in the dark) for
10 min, its absorbance was measured (λ = 575 nm) using a
spectrophotometer (UV Mini 1240, Shimadzu, Kyoto, Japan).
In this study, the amount of released sugar was stated as
glucose equivalents calculated from a glucose calibration curve
(y = 0.7929x − 0.0096, R2 = 0.9967) constructed earlier.
Two kinetic models were applied to analyze the in vitro
experimental data in this study. The first model (Goñi model)
is a first-order model and eqn (1) was proposed by Goñi
et al.:14
C ¼ C1 ð1  e kt Þ ð1Þ
Here, C represents the concentration of glucose at time t, k
denotes a first-order kinetic constant, i.e., the digestion rate
coefficient of starch, and C∞ means the estimated final con-
centration of glucose.
For the second model (LOS model), by formatting eqn (1) in
logarithmic form shown as eqn (2), a Logarithm of Slope (LOS)
plot can be obtained:15
 
dC
ln ¼ lnðC1 kÞ  kt: ð2Þ
dt
From this linear plot, the values of two digestibility para-
meters C∞ and k can be obtained. Although this model is
mathematically equivalent to the first one, it provides a novel
approach for conveniently estimating the parameters, which
help study the mechanisms of starch amylolysis and identify
whether the digestion is a single-phase process or two-phase
system. Briefly, if the starch fractions are digested at different
rates, the LOS plot displays two or more linear phases with
different slopes and distinct k.16
2.7. Statistical analysis
Measurements were conducted at least three times and the
results were expressed as mean values and standard deviations
(SD). One-way analysis of variance (ANOVA) and Duncan’s post
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hoc test were performed using SPSS 16.0 (IBM, Chicago, USA) perties and bread qualities was determined by using Pearson’s
to examine the significant difference (P < 0.05) between correlation analysis.
samples. The correlation between flour properties, dough pro-

3. Results and discussion

3.1. Physicochemical properties of WWFs
The particle size distribution and mean particle size of the
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flour samples are presented in Fig. 1 (with an inserted table).

Fig. 1 Particle size distribution of control flour, coarse, medium and
fine WWFs. The values are the means of percentage and the bar line is
SD (n = 6). The inset table shows the values of mean size; significant
difference (P < 0.05) is indicated by different letters.
Coarse WWF has a higher proportion (71%) of particles over
1000 μm than the medium (12%) and fine (0%) WWF. Besides,
the proportion of particles smaller than 100 μm was the
highest in the control flour (84%), followed by the fine (61%)
and the medium WWFs (25%) and the lowest in coarse flour
(4%). The mean particle size of the coarse, the medium and
the fine WWFs was 1315.6 μm, 450.5 μm and 199.1 μm,
respectively, which were significantly larger than that of the
control flour (61.2 μm) (inserted table in Fig. 1). Hence, it
could be concluded that the three types of WWFs and the
control showed considerable differences in the distribution of
particle size.
It can be seen from Table 1 that all WWFs had larger WRC
than the control flour. This might be due to the higher fibre
content in WWFs which can bind more water as compared to
the control flour.17 In addition, the WRC values declined with
the diminishing particle size of WWF (P < 0.05). Specifically,

Table 1 Physicochemical properties of control, coarse, medium and fine flour, and properties (obtained from farinograph, extensograph, rheofer-
mentometer and amylograph) of the corresponding dough samples. The values are mean with SD (n ≥ 3)

Control dough Coarse dough Medium dough Fine dough

Physicochemical properties
WRC (%) 61.05 ± 3.80d 104.63 ± 4.20a 86.21 ± 4.10b 75.67 ± 4.10c
Damaged starch (%) 6.80 ± 0.10b 4.68 ± 0.12d 5.69 ± 0.20c 7.22 ± 0.30a
Wet gluten (%) 34.10 ± 0.46a 20.87 ± 0.40d 25.17 ± 0.15c 30.70 ± 0.53b
Farinographic properties
WA (%) 62.4 ± 0.2d 73.2 ± 0.4a 71.8 ± 0.5b 70.5 ± 0.5c
DDT (min) 9.7 ± 0.9c 7.5 ± 0.3c 15.1 ± 2.1b 25.7 ± 1.5a
Stability (min) 17.0 ± 1.1b 10.2 ± 0.6c 16.4 ± 1.8b 20.8 ± 2.3a
MTI (FU) 17.9 ± 4.3a 18.8 ± 1.6a 18.0 ± 2.6a 11.3 ± 1.5b
Time to breakdown (min) 18.1 ± 0.2c 15.7 ± 0.6c 25.7 ± 1.7b 36.5 ± 5.7a
Extensibility properties
Rm (BU) 580 ± 15a 205 ± 13d 266 ± 25c 523 ± 21b
E (mm) 148 ± 15a 79 ± 15b 131 ± 12a 156 ± 15a
Rm/E 3.95 ± 0.10a 2.59 ± 0.12c 2.03 ± 0.20d 3.35 ± 0.30b
Rheofermentation properties
Hm (mm) 75.6 ± 0.6a 16.1 ± 1.2d 25.3 ± 1.3c 37.9 ± 1.0b
h (mm) 59.2 ± 0.7a 4.4 ± 1.1d 10.5 ± 1.6c 24.2 ± 2.3b
WC (%) 21.1 ± 0.7d 72.2 ± 8.0a 65.3 ± 0.5b 43.5 ± 2.4c
Tx (min) 32.7 ± 2.9a 19.5 ± 0.0b 19.5 ± 0.5b 23.0 ± 1.7b
Vt (mL) 2814 ± 59a 2498 ± 69c 2506 ± 92c 2668 ± 50b
CR (%) 59.0 ± 0.7b 55.5 ± 0.9c 59.3 ± 0.7b 62.7 ± 0.6a
Gelatinisation properties
Tb (°C) 60.9 ± 0.4c 85.1 ± 1.7a 78.1 ± 0.8b 62.6 ± 0.4c
Tg (°C) 90.0 ± 0.2b 91.8 ± 0.3a 92.1 ± 0.3a 89.9 ± 0.1b
Gm (AU) 1621.3 ± 24.2b 918.3 ± 30.5c 1407.7 ± 7.5b 2024.7 ± 24.2a
a–d
Mean values within the same row denoted by different superscript letters differ significantly (P < 0.05). WRC = water retention capacity; WA =
water absorption; DDT = dough development time; MTI = mixing tolerance index; Rm = maximum resistance to extension; E = extensibility; Rm/E
= Rm to E ratio; Hm = maximum dough height; h = final dough height; WC = dough weakening coefficient; Tx = time when gas starts to escape
from the dough; Vt = total gas production volume; CR = coefficient of gas retention; Tb = beginning-of-gelatinisation temperature; Tg = gelatinisa-
tion temperature; Gm = gelatinisation maximum.

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the coarse WWF had the highest WRC value (104.6%) com-
pared to the medium (86.2%) and the fine WWFs (75.7%).
This might be attributed to the depressive impact of grinding
on the capacity of fibre in WWF to bind water. That is, grind-
ing could reduce the room available for free water through
destroying the structure of the fibre matrix, and thus the
ground fibre would be unable to imbibe water as much as the
coarse fibre.18
The percentages of damaged starch and wet gluten of the
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flours are shown in Table 1. The control flour had a higher
percentage of damaged starch and wet gluten than the WWFs.
Among the WWFs, the amount of damaged starch increased
from 4.68% to 5.69% and to 7.22% with reducing the flour
particle size from coarse to medium and to fine, respectively.
An increasing trend in the damaged starch content was also
reported by Angelidis et al.19 when they milled commercial
soft wheat flour (D50 = 127.45 μm) to smaller sizes (D50 = 62.30,
22.94 and 11.4 μm). In fact, the content of damaged starch can
significantly impact the dough properties and end-product
qualities. For instance, Liu et al.20 reported that an excess
content of damaged starch may induce higher water absorp-
tion, forming a softer and stickier dough, which can’t main-
tain dough expansion during proofing, finally resulting in a
smaller bread volume.
The wet gluten content indicates the aggregating ability of
gluten proteins.21 The fine WWF contained the highest
amount of wet gluten (30.7%), followed by the medium
(25.2%) and the coarse WWF (20.9%), indicating that the wet
gluten content increased with the decreasing particle size of
the WWFs. This was due to that the larger the particle size, the
harder the gluten protein aggregation. Barak et al.22 also
reported a slight increase in wet gluten content with reducing
the flour particle sizes from >150 μm to <100 μm.
3.2. Effects of particle size on dough properties
3.2.1. Influence on farinograph properties. The influence
of WWF particle size on the mixing behaviour of dough can be
determined by using a farinograph and the main results are
presented in Table 1.
WA plays a key role in bread making. The WWFs showed
relatively higher values of WA than the control flour. It was
likely because of the greater number of hydroxyl groups in the
fibre structure which would interact with more water by hydro-
gen bonding.17 Within the WWFs, the WA value decreased
from 73.2% to 70.5% with reducing the particle size of WWFs
from coarse to fine, resulting from the decreasing water-
binding capacity of the fibre.23
DDT and stability time can indicate the strength of dough,
with their greater values implying a stronger dough.24 The
values of stability time, DDT and time to breakdown all
increased significantly with the reducing particle size of WWF
(Table 1), suggesting increasing dough strength. This result
could be explained by the less destructive effect of fine par-
ticles on the gluten network as compared to coarser
particles.25
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Moreover, flours which have good tolerance to mixing
exhibit lower a MTI, indicating a stronger dough.26 The fine
WWF showed the lowest MTI value (11.3 FU) among the three
WWFs (Table 1), implying that the dough made from the fine
WWF exhibited the highest resistance to mechanical damage.
3.2.2. Influence on dough extensibility. In terms of bread
proofing and baking, desirable dough extensibility and ade-
quate elasticity are expected to support an ample expansion of
gas bubbles, and necessary for preventing the break of
bubbles and developing a bread with an appealing volume and
a smooth and elastic internal structure.27 As summarized in
Table 1, compared to the dough made from WWFs, the control
dough showed higher values of Rm, E and Rm/E. This is consist-
ent with a previous report which found that the presence of
fibre unfavorably impacted on forming a gluten network
because of its diluting effect on gluten protein and the fibre–
gluten interaction.23,28,29
Additionally, the dough made from the fine WWF exhibited
larger values of both Rm (523.0 BU) and E (156.0 mm) than the
medium (266.0 BU, 131.0 mm) and the coarse WWFs (205.0
BU, 79.0 mm). It was reported that the presence of bran par-
ticles would hinder the aggregation of flour proteins, leading
to a poorly developed gluten network.30 The larger the bran
particles were, the more obvious the inhibition presented.
Furthermore, previous studies also showed that dough extensi-
bility and resistance had a positive correlation with bread
specific volume.31
3.2.3. Influence on rheofermentation characteristic.
Fermentation is a vital phase during bread making, in which
the dough development, gas production and retention
together impact the aerated structure of bread.32
Dough development depends on both yeast gassing power
and gluten network strength. It is characterized by the para-
meters of Hm, h and WC which shows the proportion of decrease
in volume during fermentation. Dough with higher values of Hm
and h is regarded to maintain more gas, and a lower WC value
suggests a higher dough stability during proofing.33
As presented in Table 1, the dough samples made from the
WWFs showed significantly lower Hm and h values and a
higher WC value than the control dough. Moreover, with the
decrease of WWF particle size (from coarse, medium to fine),
the whole-wheat dough development was promoted as indi-
cated by increases in the values of Hm (from 16.1, 25.3 to
37.9 mm) and h (from 4.4, 10.5 to 24.2 mm) and a decrease in
the value of WC (from 72.2, 65.3 to 43.5 mm).
These results indicated that the dough samples made from
the WWFs, compared with the control dough, had worse gas
retaining ability and dough stability. It might be explained by
the interactions between gluten and fibre in the WWFs which
impeded the free expansion of dough during proofing.24 On
the other hand, finer WWFs had stronger dough strength,
leading to a lower degree of dough collapse during proofing,
which might be due to that the finer fibre size showed less
influence on the formation of a gluten network. These obser-
vations were consistent with the results of the farinograph test
(section 3.2.1).
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Gassing power of yeast is characterised by the values of
total gas production volume (Vt), the coefficient of gas reten-
tion (CR) and the time when gas starts to escape (Tx). A larger
gassing power relates to a higher yeast activity, implying a
higher Vt.33 A higher Tx means a higher dough permeability to
carbon dioxide.
As also shown in Table 1, the control dough had a higher Tx
value (32.7 min) than the dough samples made from the
WWFs (19.5–23.0 min), indicating that the dough made from
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pectin more easily, causing an increment in the viscosity of
starch paste.35
Significant decreases were also noticed in the case of the Tb
value (from 85.1, 78.1 to 62.6 °C) upon reduction of WWF par-
ticle size from coarse, medium to fine. The higher Tb value of
the coarse WWF might result from its lower content of
damaged starch granules which required more energy for gela-
tinisation to occur.36
Statistically, the Tg values of the coarse and the medium

the WWFs had a higher permeability to carbon dioxide than
the control. This might be explained by the speculation that
fibres in the whole-wheat dough physically disrupted the
gluten protein matrix through acting as points of weakness or
stress focus within the expanding dough cell walls.24 No sig-
nificant difference was found in the Tx value among the whole-
wheat dough samples, suggesting that the WWF particle size
did not affect the dough permeability. Nevertheless, the Vt and
CR values of whole-wheat doughs increased obviously with the
reduction of WWF particle size from coarse to fine. This
increased gassing power might be related to their increasing
content of damaged starch, which was more favorable for
forming dextrins as fermentable sugars for yeast growth.33 On
the other hand, the increased gas retention ability might
resulted from the improving dough extensibility as shown in
section 3.2.2, which helped prevent air cells from collapsing
during dough expansion, resulting in greater gas retention.
3.2.4. Influence on gelatinisation properties. Starch gelati-
nisation is an important process, because gelatinised starch in
the dough, along with the gluten, forms the membranes of gas
cells in bread crumb.34 Amylograph results of the starch
pasting properties including Tb, Tg, and Gm are presented in
Table 1.
The coarse WWF showed the lowest Gm (918.3 AU) followed
by the medium (1407.7 AU) and then the fine (2024.7 AU)
WWFs. The increasing Gm value could be explained by the
increasing content of damaged starch granules which
absorbed water, swelled and leached out amylose and amylo-
WWFs (91.8 and 92.1 °C) showed no significant difference but
were slightly larger than those of the fine WWF and the
control flour (89.9 and 90.0 °C). As the Tg of the wheat flour
slurry was thought to depend on hydrogen bonds through
which starch molecules are associated with each other,36 it
could be concluded that the influence of WWF particle size on
hydrogen bonds between starch molecules was very limited.
3.3. Effect of particle size on bread quality attributes
3.3.1 Specific volume (SV) and maximum height of bread.
SV is a very important property of bread quality. As presented
in Table 2, the whole-wheat bread had a smaller SV and the
maximum height compared to the control bread. This might
be due to the fibre particles in the WWFs which disrupted the
gluten network, reduced the dough extensibility and restricted
gas retention and gas bubble expansion, decreasing the SV of
bread.37 These were consistent with the results in sections
3.2.2 and 3.2.3. For the whole-wheat bread, both SV and
maximum height increased with the reduction of WWF par-
ticle size. This was supported by the result that reducing WWF
particle size enhanced the Rm and the ability of gas retention
(Table 1) of dough, which would prevent air cells from collap-
sing and lead to a larger SV. The increasing trend of the
maximum height of bread agreed with the trend of Hm during
dough fermentation (Table 1). Furthermore, as shown in
Table 1S,† a positive correlation was found between the SV of
bread and the gas retention of dough and its wet gluten
content. This indicates that the SV of bread could be well pre-

Table 2 Quality characterization (specific volume, maximum height, and morphological and textural parameters) of bread samples made from
control, coarse, medium and fine flour. Values are mean with SD (n ≥ 3)

Control bread Coarse bread Medium bread Fine bread

−1 a d c
Specific volume (mL g ) 4.23 ± 0.17 2.41 ± 0.25 3.02 ± 0.09 3.76 ± 0.15b
Maximum height (mm) 81.46 ± 0.81a 73.58 ± 0.52c 79.40 ± 1.12b 79.76 ± 0.76b
Morphological parametersa
Total porosity (%) 72.55 ± 1.90a 57.56 ± 0.98c 63.48 ± 0.72b 63.77 ± 3.53b
Closed porosity (%) 0.16 ± 0.01c 0.64 ± 0.05a 0.27 ± 0.06b 0.16 ± 0.04c
Open porosity (%) 72.43 ± 1.91a 57.06 ± 1.07c 63.37 ± 0.74b 63.67 ± 3.45b
Cell wall thickness (mm) 0.09 ± 0.03c 0.23 ± 0.05a 0.17 ± 0.01b 0.11 ± 0.03c
Cell size (mm) 1.05 ± 0.14a 0.27 ± 0.15c 0.87 ± 0.08b 0.86 ± 0.05b
Textural parametersb
Hardness (N) 2.52 ± 0.34d 16.23 ± 2.75a 8.09 ± 1.22b 5.20 ± 0.99c
Chewiness 224.1 ± 52.6d 976.1 ± 100.7a 568.0 ± 89.3b 413.9 ± 78.8c
Cohesiveness 0.83 ± 0.01a 0.69 ± 0.0.03d 0.74 ± 0.0.03c 0.79 ± 0.03b
Resilience 0.44 ± 0.01a 0.32 ± 0.02d 0.37 ± 0.01c 0.43 ± 0.02b
a–c
Mean values of the parameters of breads within the same row signified by different superscript letters differ significantly (P < 0.05).
a
Morphological parameters were obtained from image analysis of X-ray µCT measurements. b Textural parameters were obtained from texture
profile analysis (TPA).

Food Funct. This journal is © The Royal Society of Chemistry 2020


Food & Function
dicted by the gluten content of flour and the extensibility of
dough.38
3.3.2 Structure of bread. Typical images of the bread
crumbs are shown in Fig. 2. Morphological parameters includ-
ing cell wall thickness, mean cell diameter, and porosity (total,
open and closed) are listed in Table 2. For the whole-wheat
bread, bread made from the coarse WWF had lower values of
total porosity, open porosity, and cell size, but larger values of
closed porosity and cell wall thickness than the bread made
Published on 23 March 2020. Downloaded by University of Birmingham on 4/16/2020 12:33:26 PM.
from the other two WFFs. Porosity is a valuable parameter for
describing the crumb structure, and larger porosity suggests a
more open texture.39 As a consequence, with the thinnest
(0.12 mm) and the largest cells (0.86 mm), bread made from
the fine WWF appeared to be the most porous bread.
Conversely, bread made from the coarse WWF had a denser
crumb structure with the thickest walls (0.23 mm) and the
smallest air cells (0.27 mm). Therefore, it could be concluded
that the reduction of WWF particle size induced a more
porous crumb with larger and thinner cells. This might be
attributed to the increasing ability of gas generation and pore
enlargement as supported by the results of rheofermentation
(section 3.2.3).
Additionally, the mouthfeel of bread was reported to be
related to cell properties.40 Finer, thinner-walled uniform cells
Fig. 2 Slice photos (first column), crumb images (second column) and typical cross-sectional X-ray µCT binary images (third column) of bread
samples (control, coarse, medium, and fine).
This journal is © The Royal Society of Chemistry 2020
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Paper
produce a softer and more elastic texture than coarser, thicker-
walled cell structures.40 Thus, the decreasing WWF particle
size was expected to improve the bread mouthfeel.
3.3.3 Texture of bread. Regarding bread, hardness is
usually regarded as an index for the total textural attributes,
and soft texture is generally an appealing quality. The hardness
values of whole-wheat bread significantly decreased from 16.2
to 8.1 to 5.2 N with the diminishing WWF particle size from
coarse to medium to fine (Table 2). Wang et al.28 also reported
that a smaller reconstituted WWF particle size resulted in a
relatively lower hardness in Chinese southern-type steamed
bread. Moreover, the size reduction was inversely correlated
with hardness (r = −0.964, Table 1S†), but positively correlated
with the SV (r = 0.998, Table 1S†) of bread, suggesting that
bread made from coarser WWF had higher hardness and
smaller SV. The changes in bread chewiness exhibited the
same pattern as hardness, which is in agreement with the find-
ings of previous studies.41 Hence, WWF of a smaller particle
size was more favourable for producing soft whole-wheat bread.
Bread cohesiveness is an indicator of the internal resistance
or cohesion of the bread structure, while resilience is com-
monly related to the bread crumb elasticity.42 As shown in
Table 2, both cohesiveness and resilience increased with a
reduction in WWF particle size. This phenomenon can be
Food Funct.
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Paper Food & Function

explained by the fact that the dough made from the fine WWF
had higher extensibility and resistance than that made from
the coarse WWF (Table 1). In contract, Wang et al.28 reported
that steam bread (made from reconstituted WWF) comprising
fine bran had a lower cohesiveness value than that comprising
coarse bran. This might be due to two reasons; the steam
bread has a different making process from baked bread in this
study, and secondly, the impact of reconstituted WWF on end-
products is different from WWF made directly from whole
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ticle size on the properties of starch digestibility in bread was
assessed. The LOS model was used to assess whether the
starch hydrolysis followed a single or two-phase process.
Table 3 summarizes the estimated values of starch digestion
rate coefficient (k) and final concentration of glucose (C∞)
obtained from these two models. Firstly, the LOS analysis
showed that the digestion was a continuing single-phase
process with a single k value; that is, the reaction rate constant
remained unchanged throughout the whole small intestinal

wheat grain. Furthermore, bread with high cohesiveness is
expected as it can readily form a bolus during chewing rather
than disintegrates in the chewing process, while low cohesive-
ness implies enhanced vulnerability of bread being fractured
or crumbled.43
3.4 Effects of particle size on in vitro starch digestibility
By fitting the experimental data (Fig. 3A) to two models (i.e.
LOS model and Goñi model), the potential impact of flour par-
digestion process. Secondly, the values of k and C∞ calculated
from these two models showed a similar trend.
Considering the values of R2 (>0.9), better fitting was
obtained by applying the Goñi model. Specifically, the k values
of whole-wheat bread (from 0.0032 to 0.0052 min−1) were
smaller or slightly smaller than that of the control bread
(0.0060 min−1). Additionally, bread made from the coarse and
the medium WWFs had higher k values than the bread made
from the fine WWF. The dietary fibre in WWF might act as a

Fig. 3 (A) Release of starch (experimental data) of four bread samples (control, coarse, medium and fine) in an in vitro digestion system represented
as white, blue, black and gray respectively. (B) Scanning images of bolus fragments with different particle sizes: F1 (0.5–3 mm), F2 (5–10 mm), and
F3 (15–30 mm). Values of C∞ (C) and k (D) of three bolus fragments (F1, F2, and F3) within each bread (control, coarse, medium, and fine), C∞: the
estimated final concentration of glucose; k: the first order kinetic constant. The bar line is SD (n = 3). The same letters above the bars denote no sig-
nificant difference among the three fragments within each type of bread (P > 0.05).

Food Funct. This journal is © The Royal Society of Chemistry 2020


Food & Function
Table 3 The first order kinetic constant (k) and the estimated final con-
centrations of glucose (C∞) of bread (control, coarse, medium, and fine)
in an in vitro digestion system. Values are mean with SD (n = 3)
Kinetic parameters
Model Bread sample k (min−1) C∞ (mg mL−1) R2
Goñi Control 0.0060 ± 0.0002a 22.16 ± 1.11b 0.9902
Coarse 0.0052 ± 0.0007b 19.12 ± 0.83c 0.9940
Medium 0.0052 ± 0.0016ab 16.53 ± 3.49c 0.9669
Published on 23 March 2020. Downloaded by University of Birmingham on 4/16/2020 12:33:26 PM.
Fine 0.0032 ± 0.0002c 35.29 ± 1.65a 0.9994
LOS Control 0.0068 ± 0.0007A 20.41 ± 1.31B 0.6213
Coarse 0.0057 ± 0.0006AB 18.53 ± 0.71C 0.6441
Medium 0.0041 ± 0.0016BC 16.46 ± 7.94BC 0.5768
Fine 0.0033 ± 0.0006C 34.19 ± 4.54A 0.7528
a–c
Mean values of the parameters (Goñi model) of bread within the
same column denoted by different superscript letters differ signifi-
cantly (P < 0.05). A−CMean values of the parameters (LOS model) of
breads within the same column denoted by different superscript
letters differ significantly (P < 0.05).
sponge and have the capacity to absorb water, which was likely
to form an entangled network, therefore impeding the
diffusion of amylase into a starch granule and decreasing the
rate of digestion.44 On the other hand, the relatively integrated
fibre in the coarse and the medium WWFs could absorb and
retain more water than that in the fine WWF as described in
sections 3.1 and 3.2.1, which might be favorable for the
migration of enzyme molecules from the solution into the
bread matrix and attacking starch molecules, resulting a
higher k value.
It was also noted that bread made from the coarse and the
medium WWFs had lower C∞ values than the one made from
the fine WWF. One explanation would be that the entangled
network of fibre in bread made from the coarse and the
medium WWFs affected the starch granule swelling and conse-
quently enzyme action, which eventually reduced the total
starch hydrolysis. In addition, the more compact structure in
the former two kinds of bread (as explained in section 3.3.2)
could be another important factor, in which the particle state
was maintained longer during the digestive process and the
area accessible to α-amylase was thus reduced, finally leading
to a lower glycemic response.9 Interestingly, bread made from
the fine WWF showed a higher C∞ value than the control
bread, considering their similar structure, which led us to
suggest that insoluble fibre particles in the fine WWF could
impact the continuity of the gluten network, which increased
the vulnerability of starch against digestive enzymes.9
On the other hand, with the aim to study the impact of
bolus size on the starch digestibility in bread, three size ranges
of bolus fragments (F1, F2 and F3) were prepared from each
type of bread sample (Fig. 3B). Their particle size ranges were
0.5–3 mm, 5–10 mm, and 15–30 mm, respectively. The values
of k and corresponding C∞ (Goñi model) relating to the three
different bolus fragments (F1, F2, and F3) are presented in
Fig. 3C and D. Both C∞ and k values showed no significant
difference among F1, F2, and F3 within each type of bread. It
This journal is © The Royal Society of Chemistry 2020
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Paper
could be concluded that the influence of bread bolus particle
size (with ranges of 0.5–3 mm, 5–10 mm, and 15–30 mm) on
its in vitro starch digestibility was not significant. Nevertheless,
Zou et al.45 found a rapid increase in digestion rate constant
when pasta was ground into a powder (0.1–1 mm) before being
cooked. As they summarized, the sequential digestion (from
the outer region towards the inner region) through the pasta
structure reduced the reaction area of starch hydrolysis, but
once the pasta structure was destroyed after grinding, many of
the starch granules were no longer fully encapsulated within
gluten, thus accelerating their digestibility. Moreover,
Ranawana et al.46 reported that the significant inter-individual
difference of particle size distribution of chewed rice might
have an effect on the in vitro and in vivo glycemic potency, as
they found that the in vitro starch digestion of rice reduced
with its increasing particle size (from <0.5 mm to >2 mm).
Considering the bread digestion in this study, while the
compact micro-structures in pasta and rice may impede the
accessibility of starch granules to enzymes by decreasing the
reaction area, the situation was different in bread, as its more
porous structure (without grinding) had provided enough wide
reaction areas for the hydrolysis of starch. Thus, the influence
of bolus particle size on starch digestion was less profound for
bread than for pasta and rice.
4. Conclusions
In conclusion, the dough properties, bread quality attributes
and in vitro starch digestion were all significantly influenced
by the particle size of WWFs. With the decrease of WWF par-
ticle size, DDT and stability increased, indicating a stronger
gluten network of dough, which increased its gas retention
capacity during proofing. Consequently, bread made from
coarser WWF had a smaller SV and a more compact crumb
structure, resulting in a less appealing texture which was
harder and had lower cohesiveness.
However, because of the entangled network of fibre in the
food matrix and the more compact crumb microstructure
which reduced the accessibility of starch granules to enzymes,
bread made from the fine WWF showed a larger C∞, which
was not favorable for those who want to control their blood
glucose. Moreover, the particle size of bread bolus within the
range of 0.3–30 mm had no significant impact on in vitro
starch digestion. Thus, the particle size of WWFs is a signifi-
cant factor that needs to be considered in making whole-wheat
bread, and this indicates that too coarse or too fine WWFs are
both undesirable. Further investigations should be conducted
to find the optimal range of WWF particle size for producing
whole-wheat bread with a targeted level of both texture and
low starch digestibility.
Conflicts of interest
There are no conflicts to declare.
Food Funct.

Paper
Acknowledgements
The research funding from the National Key Research and
Development Program of China (International S&T
Cooperation Program, ISTCP 2016YFE0101200), COFCO
Corporation through project 101011201600614 at the National
University of Singapore (Suzhou) Research Institute, Singapore
Ministry of Education through Academic Research Fund Tier 1
R-143-000-677-114, and the Agency for Science, Technology
Published on 23 March 2020. Downloaded by University of Birmingham on 4/16/2020 12:33:26 PM.
and Research (A*STAR), Singapore, through grant H18/01/a0/
G11 is gratefully acknowledged. The first and the third authors
would also like to thank the National University of Singapore
for its financial support.
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