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Bread fortified with anthocyanin-rich extract

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Article in Food Chemistry April 2016

DOI: 10.1016/j.foodchem.2015.09.113

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 Food Chemistry 196 (2016) 910916

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Bread fortified with anthocyanin-rich extract from black rice as

nutraceutical sources: Its quality attributes and in vitro digestibility
Xiaonan Sui a, Yan Zhang a, Weibiao Zhou a,b,
a
Food Science and Technology Programme, c/o Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543, Singapore
b
National University of Singapore (Suzhou) Research Institute, 377 Linquan Street, Suzhou Industrial Park, Jiangsu 215123, Peoples Republic of China

a r t i c l e i n f o a b s t r a c t

Article history: Anthocyanin-rich black rice extract powder (ABREP) as a nutraceutical source was fortified into bread.
Received 29 May 2015 The quality and digestibility behaviors of bread with ABREP were evaluated through instrumental and
Received in revised form 27 September in vitro digestion studies. The quality of bread with 2% of ABREP was not significantly (p > 0.05) different
2015
from the control bread; however, increasing the ABREP level to 4% caused less elasticity and higher den-
Accepted 28 September 2015
Available online 9 October 2015
sity of bread. A mathematical model was further developed to systemically describe the trajectory of
bread digestion. The digestion rates of bread with ABREP were found to be reduced by 12.8%, 14.1%,
and 20.5% for bread with 1%, 2%, and 4% of ABREP, respectively. Results of the study suggest that the
Keywords:
Anthocyanin-rich black rice extract powder
fortification of anthocyanins into bread could be an alternative way to produce functional bread with a
Anthocyanins lower digestion rate and extra health benefits.
Bread 2015 Elsevier Ltd. All rights reserved.
Quality attributes
Digestibility
Health benefits

1. Introduction and blue colors observed in nature (Manach, Scalbert, Morand,

Remesy, & Jimenez, 2004). Fruits and vegetables, such as blueber-
As a staple food for many people, bread is popular across the ries, grapes, blackberries, purple cabbage, black rice, and purple
world. Bread is a carbohydrate-rich product, which contains a high potatoes, are naturally rich in anthocyanins. The well-known
amount of rapidly digestible starch, and therefore many of them healthy property of anthocyanins is their antioxidant capacity due
have a high glycemic index (GI). Due to the rapid digestion of to their peculiar chemical structure that can react with reactive oxy-
bread, people likely consume excessive bread more than their body gen species (ROS), such as superoxide, singlet oxygen, peroxide,
requires to make up the hungry feel (Therdthai & Zhou, 2014). The hydrogen peroxide, and hydroxyl radical (Bueno et al., 2012). Apart
excessive consumption of bread could increase the risk of over- from this, recent studies reported that anthocyanins also had an
weight and obesity, and therefore their associated diseases, such inhibitory activity against digestive enzymes. McDougall et al.
as Type II diabetes (Bautista-Castao & Serra-Majem, 2012). (2005) tested the inhibitory activity of several extracts from soft
Among the diverse pharmacological strategies for the treatment fruits against a-glucosidase, and they observed that the
of Type II diabetes, the inhibition of digestive enzymes by plant- anthocyanins-rich fraction effectively inhibited a-glucosidase.
extracted enzyme inhibitors is one of the most promising methods. Matsui et al. (2001) found that anthocyanins from various plants
As such, the significance of plant-based enzyme inhibitors for the had a potential function to inhibit a-amylase, and to suppress the
modulation of diabetes has been studied for many years (McCue, increase in postprandial glucose level from starch.
Kwon, & Shetty, 2005). Among the different plant extracts, antho- However, knowledge of using anthocyanins as active ingredi-
cyanins are recently attracted a substantial amount of interests ents in real food systems is very limited. Hence, in this study, we
due to their health-promoting potency. Anthocyanins belonging to aimed to develop a real food product fortified with anthocyanins.
the group of flavonoids are naturally occurring pigments in fruits Bread was selected as the real food system and the carrier of antho-
and vegetables, which are responsible for the orange, red, violet, cyanins, because of its popularity around the world. Meanwhile,
the current approaches for developing health-promoting bread
are dominated by adding whole grains and fibers in bread, partly
Corresponding author at: Food Science and Technology Programme, c/o
Department of Chemistry, National University of Singapore, 3 Science Drive 3,
aiming to slow down its digestion among several health benefits.
Singapore 117543, Singapore. The feasibility of fortifying anthocyanins into bread to achieve a
E-mail address: chmzwb@nus.edu.sg (W. Zhou). slow digestion property as well as the quality attributes of such

http://dx.doi.org/10.1016/j.foodchem.2015.09.113
0308-8146/ 2015 Elsevier Ltd. All rights reserved.
 X. Sui et al. / Food Chemistry 196 (2016) 910916 911

bread were scientifically evaluated. The anthocyanins-fortified Varimixer, Globe, US). The dough was then divided and molded
bread may bring health benefits to consumers who seek for a to 50 g each using an electronic molder (DR Robot, Daub Bakery
healthier alternative to normal type of bread. Results of this study Machinery B.V., Holland), followed by 70 min of proofing at 40 C
might serve as a guideline for bread manufacturers with regards to and 85% relative humidity in a proofer (Climatic chamber-KBF,
the level of anthocyanins in bread to achieve a desired digestibility Binder, Germany) before being baked in an oven (Eurofours,
without compromising its quality. France). The baking temperature was set to 200 C and the baking
duration was 8 min. Despite of the severe baking condition (i.e.
2. Materials and methods 200 C for 8 min), a large amount of anthocyanins, circa 79% of
cyanidin-3-glucoside, was retained in bread crumb after the baking
2.1. Experimental materials according to our previous study (Sui et al., 2015).

Anthocyanin-rich black rice extract powder (ABREP) was pur- 2.5. Specific volume test
chased from Shaanxi Taiji Huaqing Technology Co., Ltd., China.
Bread flour containing 13% protein was purchased from Prima Bread volume is an important indicator for evaluating the qual-
Ltd., Singapore. Pure cane sugar (fine grain, NTUC Fairprice Cooper- ity attributes of bread (Ananingsih, Gao, & Zhou, 2013). The vol-
ative Ltd., Singapore), fine salt (NTUC Fairprice Cooperative Ltd., umes of bread dough and freshly baked bread fortified with 0%,
Singapore), vegetable shortening (Bake King, Gim Hin Lee Ltd., Sin- 1%, 2%, and 4% of ABREP were measured using a Volscan Profiler
gapore), and instant dry active yeast (Saccharomyces cerevisiae, S.I. (VSP 600; Stable Micro System Ltd., Surrey, UK). The specific vol-
Lesaffre, France) were purchased from a local supermarket. Pepsin ume was then derived from dividing bread volume (cm3) by its
(from porcine gastric mucosa, product number P6887), a-amylase weight (g).
(type VI-B, from porcine pancreas, product number A3176), 3,5-
dinitrosalicylic acid reagent (DNS) were purchased from Sigma 2.6. Texture attributes study
Aldrich (SigmaAldrich, MO, USA). Cyanidin-3-glucoside standard
was purchased from Polyphenols Laboratories (Sandnes, Norway). The texture attributes (hardness, springiness, cohesiveness,
chewiness, and resilience) of bread crumb with and without ABREP
were evaluated using a texture analyzer (TA.XTplus; Stable Micro
2.2. Farinograph test
System Ltd., Godalming, UK) according to the AIB standard proce-
dure AIB CAKE2/P1 (AIB Standard Procedure, 2011). Since gummi-
The farinograph test is commonly applied in estimating the
ness is not an appropriate measurement for baked foods, and also
amount of water required to make dough of good quality, and eval-
gumminess is mutually exclusive with chewiness, results of gum-
uating the effects of ingredients on flour mixing properties (Center,
miness were not included in this study.
2004). Bread flour fortified with different levels of ABREP was sep-
arately loaded on a Farinograph-E equipped with a S50 mixer and
2.7. In vitro digestibility study
sigma blades (Brabender, Duisburg, Germany). Farinograph test
was conducted according to the constant flour weight procedure
The in vitro digestibility study was conducted according to the
of the AACC Method 54-21 (AACC, 2000). The farinograph indices
study of Wolter, Hager, Zannini, and Arendt (2014) with some
of water absorption of flour with and without ABREP, dough devel-
modifications. The dosage of pepsin and a-amylase was adopted
opment time, and dough stability were determined.
according to the recommended amount from a recently published
review (Minekus et al., 2014). After baking, freshly baked bread
2.3. Extensograph test was placed on metal plates for cooling down to room temperature.
As bread crumb was the focus of this study, bread crust was man-
The extensograph test is often used in determining the resis- ually removed from the bread. The bread crumb fortified with 0%,
tance and extensibility of dough as well as the effect of additives 1%, 2%, and 4% of ABREP was respectively freeze-dried and then
on dough performance (Center, 2004). Freshly prepared bread ground into powder. To keep the same amount of starch in each
dough pieces containing 1.2% salt and different levels of ABREP test, 500 mg of control bread crumb powder, 504.6, 509.2, and
were tested using an Extensograph-E (Brabender, Duisburg, 518.3 mg of bread crumb powder fortified with 1%, 2%, and 4% of
Germany) according to the AACC Method 54-10 (AACC, 2000). ABREP, respectively, were placed in 5 mL of phosphate buffer con-
The following extensograph indices were determined: energy, taining 40 ppm of CaCl2, followed by pH adjustment (pH 1.5) using
resistance to extension (at 5 cm), extensibility, and ratio number. HCl (1 M). Pepsin (3.5 mg, 32004500 units/mg) was subsequently
added into the mixture to start hydrolysis. The mixture was incu-
2.4. The preparation of bread bated in a shaking water bath at 37 C for 1 h. At the end of 1 h, the
pH of the mixture was adjusted to 6.9 using K2CO3 (0.1 g/mL) to
Black rice was reported to contain a large amount of antho- stop the reaction and achieve the pH condition for the following
cyanins in its aleurone layer (Lee, 2010). Commercially purchased enzymatic reaction. The simulation of small intestine digestion
anthocyanin-rich black rice extract powder (ABREP) was used as was carried out by adding 55.5 mg of a-amylase (21.6 units/mg)
the source of anthocyanins. The type and content of anthocyanins into the mixture followed by transferring the mixture into a dialy-
in the ABREP have already been identified and quantified in our sis tubing (cut-off size 7000 kDa). The dialysis tubing was then
previous study (Sui & Zhou, 2014). ABREP was added to 100 g of placed into a blue cap bottle containing 100 mL of phosphate buf-
plain bread flour at the levels of 0%, 1%, 2%, and 4% (weight of fer with CaCl2 (40 ppm). The reaction was started by incubating the
ABREP/weight of plain bread flour). Bread dough was prepared blue cap bottle in the shaking water bath at 37 C. Although a
using the procedure reported in our previous study (Sui, Yap, & digestion time of 23 h was normally recommended in the litera-
Zhou, 2015). Briefly, bread flour with added ABREP was mixed with ture (Minekus et al., 2014), to thoroughly evaluate the trajectory
water (water amount was adjusted according to the farinograph of inhibitory activity of anthocyanins on a-amylase so that mathe-
results as shown in Table 1), 4 g sugar, 3 g shortening, 1.2 g salt, matical modeling could be conducted subsequently, the duration
and 1 g instant dry active yeast at a slow speed for 1 min followed of digestion in our study had been extended to 24 h. An aliquot
by an intensive mixing for 5 min in a mixer (WAG-RN20, of dialysate was withdrawn at 0, 5, 10, 15, 20, 30, 40, 50, 60, 90,
912 X. Sui et al. / Food Chemistry 196 (2016) 910916

Table 1
Farinograph and extensograph analysis of bread flour fortified with different levels of ABREP.

Sample Water absorption (%) Development time (min) Stability (min) Energy (cm2) Resistance (BU) Extensibility (mm) Ratio number**
* c b a a a a
Control (0%) 63 0.5 8.1 0.8 14.4 0.6 109.97 7.76 1,144.33 23.46 84.4 2.95 13.56 0.20a
1% 64.6 0.2b 8.2 0.2b 6.3 0.3c 117.97 1.35a 1,160.33 54.37a 76.20 2.93ab 15.25 1.12a
2% 65.4 0.7b 8.8 0.3b 5.6 0.5c 86.33 13.18b 946.33 94.71b 70.07 6.10bc 13.51 0.92a
4% 67.8 0.2a 13.2 0.4a 7.4 0.2b 56.10 0.40c 570.67 23.97c 63.40 3.03c 9.01 0.33c

Values in the same column followed by different superscript letters are significantly different from each other (p < 0.05).
*
Content of ABREP in bread flour.
**
Ratio number: resistance/extensibility.

120, 150, 180, 720, and 1440 min and replaced with the same the developed mathematical model was examined using RMSE
amount of fresh buffer. The concentration of released reducing sug- (Eq. (2)) as well as R2 (Eq. (3)).
ars (RRSs) in the withdrawn dialysate was measured according to s
the method described by Miller (1959). The withdrawn dialysate Pn Mod;i 2
i0 P t  PExp;i
at 180, 720, and 1440 min was further subjected to quantifying RMSE t
2
n
anthocyanins. In brief, equal volume of DNS reagent and with-
drawn dialysate sample were mixed in a capped Eppendorf tube where PMod
t is modeled value; P Exp
t is experimental value; n is the
and heated in boiling water for 15 min to develop the red-brown number of experimental value.
color. After cooling to room temperature, the absorbance of the
mixture was recorded at 575 nm using a spectrophotometer DF  RMSE2
R2 1  3
(UVMini 1240, Shimadzu, Kyoto, Japan). RRSs were expressed as SST
maltose equivalents using a maltose calibration curve of where DF represents the degree of freedom; and SST represents the
y 2:244x  0:196 with R2 = 0.995, where y represents the absor- total sum of squares.
bance and x represents the concentration of maltose in mg/mL.
2.10. Statistical analysis
2.8. Measurement of anthocyanins
All experiments were performed in triplicate, and results were
During the dialysis, anthocyanins were slowly released from expressed as mean standard deviation. One-way analysis of vari-
bread particles into dialysate. The released anthocyanins represent ance (ANOVA) with Tukey multiple comparisons test at p < 0.05
those that will be absorbed and transported into the human circu- was performed to identify the significant differences within groups
latory system (McGhie, Ainge, Barnett, Cooney, & Jensen, 2003). using SPSS software (version 22.0, SPSS Inc., IL, USA).
The quantification of released anthocyanins was conducted accord-
ing to the method in our previous study (Sui, Dong, & Zhou, 2014). 3. Results and discussion
An aliquot of 1 mL of dialysate sample was subjected to passing
through a Whatman 0.45 lm Nylon filter. The injection volume 3.1. Farinograph analysis
was 50 lL. The measurement was conducted using a reserved-
phase C18 Sunfire column (250  4.6 mm i.d., 5 lm; Waters, The farinograph results are presented in Table 1 showing the
Wexford, Ireland) connected to a Shimadzu high-performance liq- effect of ABREP on the quality attributes of bread dough. The water
uid chromatography (HPLC) system (Shimadzu, Kyoto, Japan) with absorption of bread flour fortified with ABREP was significantly
a diode array detector (DAD). The flow rate and oven temperature (p < 0.05) higher than the control and increased with increasing
were 1 mL/min and 25 C, respectively. Two mobile phases were the level of ABREP in bread flour. Especially, the water absorption
used for separation: mobile phase I consisted of 5% formic acid was increased by 4.8% (from 63% to 67.8%) when the level of ABREP
(v/v) in DI water; and mobile phase II consisted of 100% acetoni- increased from 0% to 4%. Similar tendency was also reported by
trile. A gradient elution program was designed to separate the Sudha, Baskaran, and Leelavathi (2007) in their study evaluating
samples: 100% mobile phase I for 5 min, 90% at 20th min, 87% at the influence of apple pomace on dough properties in cake making.
40th min, 80% at 44th min, 75% at 50th min and 0% at 55th min. They found that the water absorption was increased from 60.1% to
Data collection was performed at 520 nm, and the quantification 70.6% with increasing apple pomace content in flour from 0% to
of anthocyanins was accomplished by external calibration. 15%. The increased water absorption could be due to the fortified
ABREP which is able to interact with water to form hydrogen bonds
2.9. Mathematical modeling (Henry, 1996). The development time of bread flour fortified with
4% of ABREP was statistically higher than those of the control and
For the purpose of describing and predicting the digestion the flours fortified with 1% and 2% of ABREP. As expected, the con-
behavior of bread fortified with ABREP, the trajectory of bread trol flour had the highest stability of 14.4 min. Among the variety
digestion course was modeled using Eq. (1). of gluten proteins, cysteine accounts for 2%. Despite its low con-
tent, cysteine significantly influences the structure and functional-
Pt  Pf ity of gluten through reacting with another cysteine residue to
expki t 1
P0  Pf form a disulfide bond (SS) (Wieser, 2007). These disulphide bonds
are responsible to stabilize the structure of gluten which can be
where P0 , P t , and P f are the concentration of RRSs (maltose equiva- mobilized through SH/SS interchange reactions (Sivam,
lent mg/mL) in dialysate at time 0, t, and 1440 min, respectively; ki Sun-Waterhouse, Quek, & Perera, 2010). The anthocyanins acted
is the ABREP inhibition constant (min1); and t is time (min). The as antioxidants during dough development, which facilitated the
value of ki was obtained through linear-least-squares fit of Eq. (1) SH/SS interchange reactions to yield less amount of SS bonds,
to experimental data using Matlab software (version 8.1.0.604 thereby decreasing the number of cross-links between aggregated
R2013a, the MathWorks Inc., Massachusetts, USA). The quality of proteins and weakening the gluten matrix. Thus, flour fortified
 X. Sui et al. / Food Chemistry 196 (2016) 910916 913

with higher level of ABREP showed a longer development time and (2.39 cm3/g) than control bread (2.79 cm3/g). Bread making heavily
lower stability than the control flour. relies on oxidizing and reducing (redox) agents, such as ascorbic
acid and azodicarbonamide as oxidizing agents and sodium
3.2. Extensograph analysis metabisulfite and L-cysteine as reducing agents (Sahi, 2014). The
anthocyanins within ABREP acting as reducing agents would lead
The strength and weakness of bread dough are important to imbalanced viscoelastic and poor gas retention properties of
indicators of its baking performance, which can be evaluated by bread, resulting in low volume.
extensograph. Extensograph is widely used to determine the resis-
tance and extensibility of bread dough through measuring the 3.4. Texture analysis
force required to stretch the dough with a specially designed hook
until it breaks (Rahnama, Mohammadzadeh Milani, & Gohari The impact of different levels of ABREP on the quality attributes
Ardabili, 2015). The extensograph indices showing the effect of of bread crumb is shown in Table 2. The hardness and chewiness of
ABREP on bread dough quality are provided in Table 1. The energy bread crumb were positively related to the level of added ABREP,
values, which reveal the resistance ability of bread dough against and significant (p < 0.05) increases in hardness were found. As for
deformation forces, significantly (p < 0.05) decreased from 109.97 springiness, cohesiveness, and resilience, significant decreases
to 56.1 cm2 upon increasing the ABREP level from 0% to 4%, indicat- were observed with increasing level of added ABREP. The hardness
ing a weaker gluten network. This observation was further proved is defined as the peak force of the first compression of product
by the decreased value of resistance (from 1144 to 570 BU) and (Bourne, 2002). The significantly increased hardness value, from
extensibility (from 84.4 to 63.4 mm), as well as the ratio number 284 to 457 N for the control bread and the bread fortified with
(from 13.56 to 9.01) which is a measure of bread dough strength 4% ABREP, respectively, might be due to the poor gas retention
and a lower ratio number means that less force is required to ability of weak gluten networks, resulting a tight structure of bread
stretch the bread dough. crumb. Our results are in agreement with Wang et al. (2007) in
It was reported that certain phenolic compounds, such as ferulic their study evaluating changes in crumb appearance, texture attri-
acid, increased dough breakdown rate and simultaneously made butes, and taste profiles of bread fortified with green tea extracts.
dough more sticky (Holtekjlen & Knutsen, 2011, chap. 32). Ferulic They observed an increase in hardness and stickiness with increas-
acid has been well studied for its effect on gluten, which is through ing level of green tea extracts.
crosslinking pentosans or directly linking to the gluten, resulting in As for chewiness, bread with and without ABREP didnt show
a decrease in gluten strength and therefore dough extensibility significant differences. Springiness is used to indicate how well a
(Wang, van Vliet, & Hamer, 2004). Despite that the mechanism of food product physically springs back after it has been deformed
anthocyanins interactions with gluten network has not been during the first compression; cohesiveness indicates how well
clearly elucidated, we propose that the decreases in resistance & the product withstands a second deformation relative to how it
extensibility might be due to the less number of SS bonds being behaves under the first deformation, and resilience demonstrates
formed during bread dough development through anthocyanins how well a product fights to regain its original shape which can
interference with SH/SS interchange reactions. be regarded as instant springiness (Bourne, 2002). The significantly
decreased springiness, cohesiveness, and resilience of ABREP
3.3. Specific volume analysis fortified bread might be due to the weaker and less elastic gluten
structure of the bread caused by the anthocyanins within ABREP.
Bread volume is an important indicator for the quality of bread A recently published study investigating the quality attributes
since it provides quantitative measurement which strongly corre- of bread fortified with quinoa leaves showed that an increase in
lates to other bread quality attributes (Hathorn, Biswas, Gichuhi, the percentage of quinoa leaves (up to 5%) in wheat dough resulted
& Bovell-Benjamin, 2008; Wang, Zhou, & Isabelle, 2007). Results in an increase in bread crumb hardness and a decrease in bread
of the specific volume of bread dough and freshly baked bread with volume (Swieca, Seczyk, Gawlik-Dziki, & Dziki, 2014). Besides,
different levels of ABREP are shown in Table 2. No significant dif- Gawlik-Dziki, Dziki, Baraniak, and Lin (2009) reported that an addi-
ference (p > 0.05) was found among the freshly prepared bread tion of 2.5% Tartary buckwheat plant extract slightly decreased
dough with and without ABREP. However, after baking, the control bread quality; however, an addition of 5% caused a large decrease
bread had the highest specific volume of 3.5 cm3/g. Increasing the in bread quality. Our study showed that, in terms of the bread
level of ABREP in bread flour, the specific volume of baked bread quality attributes, bread with 2% of ABREP was not significantly
was successively decreased to 2.89 cm3/g for 4% ABREP which (p > 0.05) different from the control; however, the addition level
was significantly (p < 0.05) lower than the control bread. of 4% ABREP affected the quality attributes of bread in a negative
These results are consistent with those of the farinograph and way: bread crumb exhibited less elasticity and had a higher den-
extensograph analyses of bread dough quality attributes. Our sity. This was due to the reducing ability of anthocyanins that
observations are in agreement with Han and Koh (2011) that they yielded less amount of SS bonds, thereby weakening the bread
found the addition of phenolic acids reduced bread volumes and matrix structure. Besides, the side chain amino groups of peptides
bread fortified with caffeic acid had a significantly lower volume are known to be able to react with polyphenols under oxidizing

Table 2
Specific volume of dough and texture analysis of bread crumb fortified with different levels of ABREP.

Sample Specific volume (cm3/g) Hardness (N) Springiness Cohesiveness Chewiness (N) Resilience
Before proofing After baking
Control (0%)* 1.03 0.03a 3.50 0.21a 284.31 9.93c 0.96 0.02a 0.81 0.02a 220.03 11.70a 0.43 0.03a
1% 1.03 0.01a 3.42 0.17a 360.70 37.05b 0.93 0.03a 0.78 0.00ab 261.18 18.87a 0.42 0.01a
2% 1.03 0.01a 3.24 0.24ab 403.19 10.91ab 0.90 0.04ab 0.70 0.04b 254.88 32.52a 0.32 0.04b
4% 1.03 0.02a 2.89 0.07b 457.05 23.49a 0.85 0.03b 0.72 0.03b 280.43 26.31a 0.33 0.00b

Values in the same column followed by different superscript letters are significantly different from each other (p < 0.05).
*
Content of ABREP in bread flour.
914 X. Sui et al. / Food Chemistry 196 (2016) 910916

conditions, which leads to formation of cross-links in proteins. The 3.6. Mathematical modeling
cross-linked protein gel has greater mechanical strength and
reduced swelling properties (Strauss & Gibson, 2004). As shown in Fig. 1, the values of RRSs were very close to each
other, albeit the ANOVA test showed significant differences among
the data at some sampling points, indicating the complicated task
3.5. In vitro digestibility analysis of discriminating digestion curves. Mathematical modeling of
digestion profiles allows a more effective description, and enables
Epidemiological studies and associated meta-analyses suggest efficient comparison among digestion rates.
that diets rich in plant polyphenols offer some protections against The regressed rate coefficients (ki ) from the nonlinear regres-
the development of cancers, cardiovascular diseases, diabetes, sion of Eq. (1) are shown in Table 3. The ki values of bread fortified
osteoporosis, and neurodegenerative diseases to the people who with 1 (0.0068 min1), 2 (0.0067 min1), and 4% (0.0062 min1) of
consume them for a long term (Arts & Hollman, 2005). As shown ABREP were lower than the control bread (0.0078 min1), indicat-
in Fig. 1, the concentration of RRSs increased over time in the early ing slower digestion rates. This study demonstrates, for the first
stage of dialysis and then leveled off until reaching its highest time, that the digestion rate of bread could be reduced by as much
value of 2.1 mg/mL at 1440 min. During the first 120 min of dialy- as 20.5% through fortifying the bread with 4% of ABREP, whereas
sis, the concentration of RRSs for the control bread and the bread the reduction rate was 12.8% and 14.1% for the bread with 1%
fortified with 1%, 2%, and 4% of ABREP lied very close to each other. and 2% of ABREP, respectively. The mathematical models (dash
However, significant (p < 0.05) differences in the concentration of lines) developed based on each regressed rate coefficients and
RRSs were observed at 150 and 180 min of dialysis. Nevertheless, Eq. (1), were plotted together with the experimental data for the
when the dialysis time was prolonged to 720 and 1440 min, no sig- control bread and the bread fortified with 1%, 2%, and 4% of ABREP
nificant differences in the concentration of RRSs was observed. (Fig. 2). The low values of RMSE and high values of R2 indicate that
Specifically speaking, at the dialysis time of 150 min, the concen- the developed models could satisfactorily describe the digestion
tration of RRSs was reduced, in comparison to the control bread profiles of bread fortified with ABREP. To further validate the mod-
(1.56 mg/mL), by 8.8%, 14.2%, and 24.5% for bread fortified with els, the modeled and the experimental data sets were plotted in a
1%, 2%, and 4% of ABREP, respectively. At the dialysis time of scatter chart against a 45-degree line (Fig. 2, inserted graphs) that
180 min, the concentration of RRSs for breads fortified with 1%, showed a good agreement between the modeled and the experi-
2%, and 4% ABREP was 6.31%, 10.14%, and 17.45%, respectively, mental data, indicating the good quality of the developed models.
lower than the control bread (1.71 mg/mL). These observations
indicated that the inhibition of a-amylase during the in vitro diges- 3.7. Released anthocyanins in dialysates
tion process was increased with increasing amount of ABREP in
bread. Cyanidin-3-glucoside was the most dominant anthocyanin in
Our results are in agreement with Hargrove, Greenspan, Hartle, the ABREP, which accounted for up to 89.6% of total anthocyanins
and Dowd (2011), who found that either tannin or black sorghum (Sui, Bary, & Zhou, 2016). Therefore, the ratio of released cyanidin-
phenolic extracts (without tannins) inhibited a-amylase, and the 3-glucoside was taken as an indicator for the changes in total
inhibition activity increased as the concentration of phenolic anthocyanins as the amount of other anthocyanins in the dialy-
extracts increased. The fortification of bread using phenolics from sates was too small to be detected. The ratio of released
plant extracts has been proved to be an effective way to slow down cyanidin-3-glucoside was expressed as a percentage of the
the digestion rate of bread. Phenolics derived from green tea were detected amount of cyanidin-3-glucoside in dialysate relative to
reported to interact with amylose via hydrogen bonding, resulting the retained amount of cyanidin-3-glucoside in bread crumb after
in a decreased digestibility of starch (Chai, Wang, & Zhang, 2013). baking, and was calculated using Eq. (4).
Similarly, Swieca et al. (2014) reported that the starch digestibility
of bread fortified with 5% of quinoa leaves was lowered by 11.8% Ratio of released cyanidin-3-glusocide%
with respect to control bread. Dcy3glu
 100 4
M crumb  Rcy3glu

where Dcy3glu is the amount of cyanidin-3-glucoside in dialysate

(mg); Mcrumb is the weight (mg) of bread crumb (which equals to
500 mg in this study); and Rcy3glu is the content (%) of retained
cyanidin-3-glucoside in bread crumb after baking.
As shown in Fig. 3, the ratio of released of cyanidin-3-glucoside
for the bread fortified with 1%, 2%, and 4% of ABREP, respectively,
were all increased with an increase of the dialysis time from 3 to
24 h. Bread fortified with 2% and 4% of ABREP had much higher
ratio of released cyanidin-3-glucoside than that of bread fortified
with 1% of ABREP after both 3 and 24 h of dialysis. At the end of

Table 3
Mathematical modeling parameters and regressed rate coefficients (ki , min1).

Control (0%)* 1% 2% 4%
P0 0 0 0 0
Pf 2.1301 2.1129 2.0675 2.0383
ki 0.0078 0.0068 0.0067 0.0062
R2 0.9891 0.9860 0.9903 0.9936
RMSE 0.0722 0.0794 0.0637 0.0498
*
Fig. 1. The digestion trajectories of bread fortified with and without ABREP. Content of ABREP in bread flour.
 X. Sui et al. / Food Chemistry 196 (2016) 910916 915

Fig. 2. Developed mathematical models and validation plots for modeled data against experimental data.

1%, 2%, and 4% of ABREP, respectively). The lower ratio of released

cyanidin-3-glucoside in the first 23 h of dialysis in our study
might be caused by the bread making procedure which could have
enhanced the possible interactions between anthocyanins and food
compounds, such as protein and starch. As discussed earlier, these
interactions contributed partially to the slow digestibility of bread.
Besides, differences in the experimental procedures and setup such
as dialysis tubing, would have resulted in different mass transfer
rates during the dialysis, and thereby different amounts of released
anthocyanins at a particular sampling time. Caution is therefore
needed to compare such data in the literature. It is also worthy
to point out that this is the first time reporting the ratio of released
cyanidin-3-glucoside from digesting a whole food (i.e. bread)
fortified with anthocyanins.

4. Conclusions

In conclusion, the quality attributes of bread as well as the

digestibility of bread were evidently influenced by the addition
of ABREP. Through developing a mathematical model, the trajec-
Fig. 3. Ratio of released cyanidin-3-glucoside from bread particles into dialysate tory of bread digestion course was successfully modeled, which
during 24 h digestion.
showed that fortification of ABREP reduced the digestibility rate
of bread. These results indicated that anthocyanins fortification
dialysis (24 h), the ratio of released cyanidin-3-glucoside for the may provide new opportunities to produce functional bread by
bread with 4% of ABREP was approximately 6.5%, whereas the ratio slowing down its digestion rate, thereby providing extra health
for the bread fortified with 1% and 2% of ABREP were lower, at 2.7% benefits to consumers.
and 5.9%, respectively. McDougall, Dobson, Smith, Blake, and
Stewart (2005) reported that about 5% of total anthocyanins was Acknowledgements
detected in dialysate at the end of 2 h digestion of diluted raspber-
ries syrup. This amount of released anthocyanins in dialysate Supports from ASTAR through research grant SERC 112 177
stayed either unaffected or increased when they co-digested rasp- 0033 and the National University of Singapore (Suzhou) Research
berries with foodstuffs (white bread, breakfast cereal, minced beef Institute under the grant number NUSRI2011-007 and Jiangsu
steak, and ice cream). In our study, at the end of 3 h dialysis, only a Province under the Scientific Research Platform scheme are grate-
small amount of cyanidin-3-glucoside were detected in the fully acknowledged. The first author also likes to thank China
dialysates (e.g. the ratio of released cyanidin-3-glucoside after Scholarship Council (CSC) and the National University of Singapore
3 h dialysis were 0.31%, 1.27%, and 2.84% for bread fortified with (NUS) for financial support.
916 X. Sui et al. / Food Chemistry 196 (2016) 910916

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