Food Chemistry 284 (2019) 118–124

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

Effect of purple yam flour substitution for wheat flour on in vitro starch T
digestibility of wheat bread
⁎ ⁎
Xia Liua,b,c, Kui Lua,b,c, Jinglin Yua, Les Copelandd, Shujun Wanga,b,c, , Shuo Wange,
a
State Key Laboratory of Food Nutrition and Safety, Tianjin University of Science & Technology, Tianjin 300457, China
b
Key Laboratory of Food Nutrition and Safety, Ministry of Education, Tianjin University of Science & Technology, Tianjin 300457, China
c
School of Food Engineering and Biotechnology, Tianjin University of Science & Technology, 300457, China
d
The University of Sydney, Sydney Institute of Agriculture, School of Life and Environmental Sciences, NSW 2006, Australia
e
Tianjin Key Laboratory of Food Science and Health, School of Medicine, Nankai University, Tianjin 300071, China

A R T I C LE I N FO A B S T R A C T

Keywords: This study aimed to investigate the effect of purple yam (Dioscorea alata L.) flour substitution for wheat flour on
Wheat bread in vitro starch digestibility of wheat bread. The enzyme-resistant starch content increased from 34.9% for wheat
Purple yam flour bread to 41.3% for bread made with 70% wheat flour and 30% purple yam flour. Meanwhile, the content of
Digestibility rapidly digestible starch and slowly digestible starch decreased with the addition of purple yam flour in the
Starch granules
bread. The results from differential scanning calorimetry, X-ray diffraction, polarized light and scanning electron
Resistant starch
microscopy showed that purple yam starch granules were not completely disrupted during bread baking. The
fluorescence intensity of α-amylase and amyloglucosidase was reduced in bread made with purple yam flour.
The decreased digestibility of starch in bread made with purple yam flour was due to the relatively intact starch
granules from purple yam flour and possibly an inhibitory effect on digestive enzymes by purple yam flour.

1. Introduction the food industry.

Starch digestion can be measured by in vitro and in vivo methods. In
There has been a significant increase in the consumption of high- vitro methods generally measure the rate and extent of starch digestion
carbohydrate foods as a result of the changing lifestyles and dietary during 2 h of incubation. Based on the extent of starch digestion, starch
patterns. Bread is a staple food consumed in different forms in many is classified into rapidly digested starch (RDS, digested within 20 min),
parts of the world (Lau, Soong, Zhou, & Henry, 2015). The consumption slowly digested starch (SDS, digested between 20 and 120 min), and
of bread has been increasing in the world due to its high nutritional resistant starch (RS, undigested after 120 min) (Englyst, Kingman, &
value, convenience to carry and eat, and low price. The presence of Cummings, 1992). There have been many studies on the effects of ad-
water and the high baking temperature for yeast-leavened breads re- ditives on the in vitro and in vivo digestion of wheat bread. The additives
sults in the gelatinisation of starch in the bread. Gelatinized starch is used include green tea catechins, sorghum flour, white bean extract,
digested quickly in the small intestine, causing a rapid increase in the barley β-glucan-enriched flour, crude malva nut gum, dietary fibre and
concentration of postprandial blood glucose and insulin (Borczak, wild grown fruits. The breads made with sorghum flour, dietary fibre
Sikora, Sikora, Kapusta-Duch, & Rosell, 2015). Hence, bread is in and wild grown fruits had a significantly lower content of rapidly di-
general a high glycemic index (GI) food although some wholegrain gestible starch (RDS) compared with those made only with wheat flour
breads exhibit low glycemic potential (Ranawana & Henry, 2013). The (Borczak, 2016; Borczak et al., 2015; Yousif, Nhepera, & Johnson,
long-term ingestion of high GI foods is considered to be a risk factor for 2012). Green tea catechins, white bean extract, barley β-glucan-en-
a range of chronic diseases, such as diabetes, cardiovascular disease, riched flour and crude malva nut gum appear to be potentially effective
hyperlipidemia and hypertension. In this context, the growing demand additives for reducing the GI value of breads (Gianinetti, 2012; Goh,
for novel, tasty and healthy foods has given rise to a new bread market 2015; Phimolsiripol, 2017; Udani, Singh, Barrett, & Preuss, 2009)
(Rinaldi, Paciulli, Dall'Asta, Cirlini, & Chiavaro, 2015). Finding alter- Purple yam, belonging to the species of Dioscorea alata L, has been
native functional ingredients to reduce the digestibility of starch in used as a food or traditional Chinese herbal medicine for many years.
bread without altering the original flavor of bread is of great interest to Apart from starch, which is the main component, yam tubers contain

⁎
Corresponding authors at: No 29 13th Avenue, Tianjin Economic and Developmental Area (TEDA), Tianjin 300457, China.
E-mail addresses: swang@tust.edu.cn (S. Wang), sjwang@tust.edu.cn (S. Wang).

https://doi.org/10.1016/j.foodchem.2019.01.025
Received 18 July 2018; Received in revised form 27 October 2018; Accepted 3 January 2019
Available online 16 January 2019
0308-8146/ © 2019 Elsevier Ltd. All rights reserved.
X. Liu et al. Food Chemistry 284 (2019) 118–124

many bioactive ingredients, such as mucilage, dioscin, allantoin, cho- mixture.

line, amino acids, and phenolic compounds (Bhandari, Kasai, &
Kawabata, 2003; Fang et al., 2011; Huang, Lai, Chen, Liu, & Wang, 2.3. Texture, color and specific bulk volume of breads
2010; Ma et al., 2017). While the documented physiological functions
of purple yam are widely accepted, there are few studies on the prop- The following analyses were performed on the freshly baked breads
erties of purple yam being used as functional ingredients in foods. Fang after cooling at 25 °C for 1 h. The texture profile analysis (TPA) of the
et al. (2011) investigated phenolic compounds in Chinese purple yam bread was carried out using a Texture Profile Analyzer (TA-XT.PLUS,
and their changes during vacuum frying. Moriya et al. (2015) studied Stable Micro System, Godalming, UK) according to a method described
the antioxidant activity of new acylated anthocyanins from purple yam. previously (Demirkesen, Mert, Sumnu, & Sahin, 2010). The bread
Recently, the effect of purple yam flours on the textural and sensory samples were compressed to 30% of its thickness at a speed of 1 mm/s
properties of salted noodles was investigated (Li, Huang, Yang, & Wang, with a cylindrical probe (diameter 100 mm). The crust color of the
2012). With the exception of this study, no further information is bread samples was measured using a CM-5 colorimeter (Konica Min-
available on the utilization of purple yam in food preparation. Pre- olta, Japan). A white standard color plate was used to calibrate the
liminary results in our group showed that preheated yam starch is di- instrument. A colorimetric definition using a CIELAB scale was used to
gested more slowly than preheated wheat starch, especially at low evaluate changes in crust color of bread samples. Lightness (L*) value
water content (Wang, Wang, Guo, Liu, & Wang, 2017). Thus, it may be runs from 0 (black) to 100 (white). a* represents red/green and b* re-
promising to use purple yam as a substitute in the preparation of starch- presents yellow/blue. The color values L*, a* and b* were expressed by
based foods to decrease the digestibility of starch and lower the GI of averaging 6 readings performed on the exposed surface. The volumes of
food products. the baked bread were measured using a TexVol bread volume measurer
In this paper, we aimed to investigate the effect of adding purple (BVM6630, Perten, Sweden). Specific volumes were then calculated by
yam flour to wheat bread on the in vitro enzymatic digestibility of dividing the measured volumes by bread mass.
starch. To the best of our knowledge, this is the first study to evaluate
the potential of purple yam as possible ingredient to reduce starch di- 2.4. In vitro starch digestion
gestibility in breads. The structural properties of starch in breads made
from mixtures of wheat flour and purple yam flour were examined In vitro starch digestibility was determined according to the pro-
using differential scanning calorimetry (DSC), X-ray diffraction (XRD), cedure of Wang, Li, Zhang, Wang, and Copeland (2017) with some
scanning electron microscopy (SEM) and polarized light microscope modifications. Starch content in bread samples was determined by total
(PLM) to gain a better understanding of the lower digestibility of starch starch assay kit (Megazyme, K-TSTA). The samples (containing 100 mg
in breads. The quenching of fluorescence of porcine pancreatic α- starch) were weighed into a 50 mL centrifuge tube, and incubated in
amylase and amyloglucosidase caused by purple yam flour was also 5 mL of a solution containing pepsin (1 mg/mL) in 0.01 M HCl (pH 2) at
examined. 37 °C with magnetic stirring (260 rpm) for 30 min. The solution was
neutralized with 5 mL of 0.01 M NaOH and mixed with 25 mL of sodium
2. Materials and methods acetate buffer (pH 6, 0.2 M) to stop enzyme reactions. Subsequently,
5 mL of simulated intestinal fluid containing porcine pancreatic 1600 U
2.1. Materials ɑ-amylase and 40 U amyloglucosidase was added to the above solution,
and incubated at 37 °C with continuous stirring (260 rpm) for 2 h. Ali-
All ingredients used in bread-making, including wheat flour (high quots (0.2 mL) were taken after 30 min of pepsin digestion, and after
gluten flour), purple yam, dried yeast, butter, salt and sugar, were the simulated intestinal digestion at 20, 40, 60, 80, 100 and 120 min
obtained from a local supermarket. The purple yams were washed, and mixed with 0.8 mL of anhydrous ethanol to inactivate the enzymes.
peeled, sliced thinly, freeze-dried, and ground into a powder which was The amount of glucose released was determined using the Megazyme
passed through a 150 μm sieve. The moisture content of the purple yam GOPOD kit. The percentage of hydrolyzed starch was calculated by
flour was 6.2%. Porcine pancreatic alpha-amylase (PPA, A3176, EC multiplying the glucose content with a factor of 0.9. The digestograms
3.2.1.1, type VI-B, 13 U/mg), pepsin (P7012, from porcine gastric of starch hydrolysis were fitted to the first-order rate equation (Dhital,
mucosa, ≥2500 U/mg), were purchased from Sigma Chemical Co. (St. Warren, Butterworth, Ellis, & Gidley, 2017; Goñi, Garcia-Alonso, &
Louis, Mo., U.S.A.). Amyloglucosidase (AMG, 3260 U/mL) and D- Saura-Calixto, 1997)
Glucose Assay Kit (glucose oxidase/peroxide, GOPOD format) were
purchased from Megazyme International Ireland Ltd. (Bray County, Ct = C∞ (1 − e−kt )
Wicklow, Ireland). Other chemical reagents were all of analytical grade.
where Ct is the amount of starch digested at time t (min), C∞ is the
estimated amount of starch digested at the reaction end point, and k
2.2. Bread preparation
(min−1) is the first-order rate coefficient. For ease of interpretation, C∞
was expressed as the final percentage of starch hydrolyzed. The value of
Purple yam flour was added to wheat flour at the following weight
k was calculated from the slope of a linear-least squares fit of a plot of ln
percentages of 0, 10, 20, 30, 40, and 50% (wheat flour basis, w/w). The
(1 − Ct/C∞) against t.
bread formula used was as follows: flour (10 g), dried yeast (0.1 g), salt
The amount of starch digested enzymically after 20 min (referred to
(0.15 g), sugar (0.6 g), water (6.3 mL), butter (0.3 g). Yeast was firstly
as rapidly digested starch, RDS), between 20 min and 120 min (referred
suspended in deionized water at 37 °C for 5 min before adding to the
to as slowly digested starch, SDS), and starch remaining after 120 min
flour. The dough formed after mixing the above ingredients by hand
(referred to as resistant starch, RS) was calculated using the following
was placed in a baking pan and proofed for 80 min at 29 °C and 75%
formula:
relative humidity (RH). Dough was kneaded after the first fermentation
and proofed for another 20 min at 29 °C and 75% RH. Dough was RDS (%) = (G20−FG) × 0.9 × 100
molded before the final proofing for another 40 min at 38 °C and 85%
RH. The proofed dough was baked for 4 min at 140 °C and then 6 min at SDS (%) = (G120 − G20) × 0.9 × 100
160 °C. The bread was cooled, freeze-dried, ground and passed through
RS (%) = [TG − FG] × 0.9 × 100 − (RDS + SDS)
a 150 μm sieve. The bread samples were subsequently named as (m%)
WF + (n%) PY, which means bread (B) made of m% wheat flour (WF) where G20 and G120 are the amount of glucose released after 20 min
and n% purple yam flour (PY). Three loaves were made from each and 120 min, respectively; FG is the amount of free glucose in the

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X. Liu et al. Food Chemistry 284 (2019) 118–124

Table 1
Color and structure parameters of breads.
100%WF + 0%PY 90%WF + 10%PY 80%WF + 20%PY 70%WF + 30%PY 60%WF + 40%PY 50%WF + 50%PY

Color analysis (crust)

L* 77.7 ± 1.1d 68.0 ± 1.0c 66.2 ± 2.3b 63.6 ± 0.8a 62.2 ± 1.1a 64.0 ± 0.8a
a* 3.8 ± 0.9a 10.9 ± 0.5b 12.8 ± 0.7c 14.6 ± 0.6d 16.3 ± 0.6e 16.9 ± 0.4e
b* 26.8 ± 1.3d 19.9 ± 2.3c 18.1 ± 1.1c 8.9 ± 0.5b 7.2 ± 2.3b 4.7 ± 1.1a

Volume and textural characteristics

SV (mL/g) 1.9 ± 0.0d 1.8 ± 0.0d 1.7 ± 0.0c 1.4 ± 0.1b 1.5 ± 0.1b 1.3 ± 0.1a
Hardness (N) 15.7 ± 1.9a 18.3 ± 0.9a 18.7 ± 0.7a 24.3 ± 1.9b 28.5 ± 0.7c 32.5 ± 0.7d
Gumminess 10.2 ± 0.3a 12.0 ± 0.5b 11.8 ± 0.9b 17.7 ± 0.5c 18.7 ± 0.0c 18.8 ± 0.3c
Chewiness 9.0 ± 0.2a 10.5 ± 0.4a 10.2 ± 0.7a 15.3 ± 0.3b 16.6 ± 0.5b 16.8 ± 1.6b

Values are means ± SD. Values with the same letters in the same column are not significantly different (p < 0.05). WF and PY represent wheat flour and purple yam
flour, respectively. m% WF + n% PY represents breads made of m% wheat flour (WF) and n% of purple yam flour (PY) . L*: lightness. a*: redness. b*: yellowness, SV:
specific volume.

undigested samples and TG the amount of total glucose released at the 2.9. Fluorescence quenching
end point of hydrolysis.
Fluorescence quenching of porcine pancreatic α-amylase and amy-
loglucosidase was analyzed using a fluorospectro photometer (Lumina,
2.5. Differential scanning calorimetry Thermo Fisher, U.S.A) according to Liu et al. (2017) with minor mod-
ifications. Bread samples (containing 100 mg starch) were weighed into
Thermal properties of the bread samples were examined using a centrifuge tubes, and 25 mL of 0.2 M sodium acetate buffer (pH 6) and
differential scanning calorimeter (200 F3, Netzsch, Germany) equipped 5 mL of simulated intestinal fluid containing porcine pancreatic alpha-
with a thermal analysis data station. Samples (approximately 3 mg, amylase (1600 U) and amyloglucosidase (40 U) were added and in-
8.7% moisture) were weighed accurately into a 40 μL aluminum pan. cubated at 37 °C with continuous stirring (260 rpm) for 30 min. The
Distilled water was added to obtain a flour/water ratio of 1:3 (w/v) in samples were centrifuged at 8000g for 5 min and the supernatant was
the DSC pans. The pans were sealed and allowed to stand overnight at used for testing. The sample (1 mL) was excited at 288 nm, with 2.5 nm
room temperature before DSC measurement. The samples were heated excitation and 5 nm emission slits, and fluorescence spectra were re-
from 20 to 120 °C heating rate of 10 °C/min. An empty aluminum pan corded between 310 and 400 nm.
was used as the reference. The onset (To), peak (Tp), conclusion (Tc)
temperatures, and gelatinization enthalpy change (△H) were obtained
2.10. Statistical analysis
through data recording software (Wang, Zhang, Wang, & Copeland,
2016). All measurements were performed in triplicate.
All analyses were performed at least in triplicate and the results are
reported as the mean values and standard deviations. In the case of
XRD, only one measurement was performed. One way analysis of var-
2.6. X-ray diffraction analysis (XRD)
iance (ANOVA) followed by post-hoc Duncan’s multiple range tests
(p < 0.05) was conducted to determine the significant differences
X-ray diffraction analysis was performed using a D/max-2500vk/pc
between mean values using the SPSS 17.0 Statistical Software Program
X-ray diffractometer (D8 Advance, Bruker, Germany) operating at
(SPSS Inc. Chicago, IL, USA).
40 kV and 40 mA. Samples were equilibrated over a saturated NaCl
solution at room temperature for one week before analysis. The X-ray
diffraction pattern was obtained from 4° to 40° (2θ) at a scanning speed 3. Results
of 2°/min and a step size of 0.02°. The relative crystallinity was cal-
culated using the software of TOPAS 5.0 (Wang, Wang, Liu, Wang, & 3.1. Color, specific volume and texture properties of breads
Copeland, 2017).
Color and texture parameters of breads are shown in Table 1. The L*
(lightness) values of breads decreased from 77.7 to 63.9 and the a*
2.7. Polarized light microscope (PLM) (redness) values increased from 3.8 to 16.9 with increasing amounts of
purple yam flour in breads, indicating that the color of the breads be-
A light microscope (DM-400M-LED, Leica, Germany) was used to came increasingly reddish-black. The b* values of breads decreased
observe the changes in starch granule birefringence before and after from 26.8 to 4.7 with increasing amounts of purple yam flour in breads,
baking. Approximately 12.5 mg of starch samples was weighed into the indicative of the increasing blueness of bread. The specific volume of
plastic tubes, and 1 mL of 50% glycerol in water was added and mixed breads decreased from 1.93 to 1.27 with increasing amount of purple
homogeneously. The suspension (20 μL) was applied onto a microscope yam flour in breads. The hardness and chewiness of breads increased
slide, covered with a coverslip and imaged under polarized light (Wang from 15.65 to 32.54 and from 8.99 to 16.79 with increasing amount of
et al., 2017). purple yam flour in breads, indicating that breads made with mixtures
of purple yam and wheat flours were harder to chew than wheat breads.
The increased hardness and chewiness with addition of purple yam
2.8. Scanning electron microscopy (SEM) flour in bread could be attributed to the inhibition of gluten network
formation and the reduction of the gas retention capacity of the dough
The wheat flour, purple yam flour and breads were mounted on a by purple yam flour (Ribotta, Arnulphi, Leon, & Anon, 2005). Sub-
stub using double-sided adhesive tape, sputter coated with gold in a stituting up to 20% of wheat flour with purple yam flour did not seem
sputter coater (JEC-3000FC, Tokyo, Japan). The samples were imaged to cause major changes to the color, specific volume and textural
(×800) using a scanning electron microscope (JSM-IT300LV, JEOL, properties of the breads. The effects of purple yam flour on these
Japan) at an accelerating voltage of 5 kV (Wang et al., 2017). properties were more noticeable at substitutions of 30% or greater.

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X. Liu et al. Food Chemistry 284 (2019) 118–124

increasing amounts of purple yam flour substituted for wheat flour in

the breads (Table 2). With 30–50% purple yam flour in the bread, RS
values were 41–42% (Table 2). These results indicated that the addition
of purple yam flour in breads decreased in vitro enzymatic digestibility
of starch. It has been reported that there is a positive correlation be-
tween RDS and GI and a negative correlation between RS and GI of
foods, respectively (Ek, Wang, Copeland, & Brand-Miller, 2014; Englyst,
Englyst, Hudson, Cole, & Cummings, 1999; Whitney & Simsek, 2017).
While RDS and SDS decreased and RS increased with the addition of
purple yam flour in breads, the first order digestion rate constant was
little affected by the addition of purple yam flour (Table 2), indicating
that starch was digested at a similar rate over the 2 h of incubation.

3.3. Thermal properties of flours and breads

The onset (To), peak (Tp), conclusion (Tc) temperatures and en-
thalpy change of gelatinization (ΔH) of wheat flour, purple yam flour
and bread samples are presented in Table 3. Wheat flour and purple
yam flour displayed a typical gelatinization endothermic transition in
the temperature ranges of 58.2–70.0 °C and 78.6–88.9 °C, respectively.
Purple yam flour presented a higher enthalpy change of 13.8 J/g than
did wheat flour (6.7 J/g). No endothermic transitions were observed for
100% wheat bread over a temperature range of 20–120 °C, indicative of
complete gelatinization of starch in wheat bread. With the addition of
purple yam flour, all bread samples presented an endotherm transition,
with To, Tp, and Tc in the ranges of 80.3–83.2 °C, 94.1–95.0 °C, and
99.7–102.2 °C, respectively. The gelatinization temperatures of breads
with purple yam flour were higher than those of the wheat flour and
purple yam flour. The higher thermal transition temperatures was at-
tributed to the melting of more stable starch crystallites that remained
after baking (Collar, Jiménez, Conte, & Piga, 2015; Wang et al., 2016).
The enthalpy change (ΔH) increased from 2.3 J/g for bread with 10%
purple yam flour to 4.5 J/g for bread with 50% purple yam flour. The
increases were not statistically significant for bread samples with
10∼40% purple yam flour. The above results showed that starch
granules in the purple yam flour were more stable to thermal processing
than those in wheat flour.

Fig. 1. Digestograms of breads fitted to first-order kinetics (A), and kinetic 3.4. X-ray diffraction analysis
constants of breads (B). m% WFs + n% PY represents breads made of m%
wheat flour (WF) and n% of purple yam flour (PY). (For interpretation of the The XRD patterns of wheat flour, purple yam flour and bread
references to color in this figure legend, the reader is referred to the web ver-
samples are illustrated in Fig. 2. Wheat flour displayed the typical A-
sion of this article.)
type diffraction pattern with two singlet peaks at about 15 and 23° (2θ),
and a doublet peak at around 17° and 18° (2θ). The weak peak at 20.0°
3.2. In vitro starch digestion (2θ) was attributable to amylose–lipid complex. Purple yam flour
showed a typical C-type diffraction pattern with strong reflections at
The in vitro enzymatic digestograms of bread samples are shown in about 15.3°, 17.3°, and 23.5° (2θ). The results are consistent with those
Fig. 1. All digestograms displayed a more rapid initial stage followed by for native yam starch, which showed a typical C-type diffraction pattern
a slower rate of hydrolysis (Fig. 1A). The digestograms fitted well to the with strong reflections at about 5.6°, 15.3°, 17.3°, and 23.5° (2θ) (Wang
first-order kinetic equation with correlation coefficients all above 0.98 et al., 2016). The relative crystallinity (RC) of wheat flour and purple
and a linear relationship between (1 − Ct/C∞) and digestion time yam flour were 23.2 and 28.7%, respectively.
(Fig. 1B). The RDS, SDS and RS contents of wheat bread, as defined The typical A-type diffraction pattern of 100% wheat bread almost
according to the terminology of Englyst et al. (1992), were 23.1, 42.0 disappeared, indicating that almost complete gelatinization of starches
and 34.9%, respectively. RDS and SDS decreased and RS increased with in 100% wheat bread, consistent with the DSC result. The peak at 20.0°

Table 2
Starch hydrolysis fractions and hydrolysis kinetic parameters of breads.
Samples RDS (%) SDS (%) RS (%) k (min−1)

100%WF + 0%PY 23.1 ± 0.6b 42.0 ± 1.6c 34.9 ± 1.1a 0.016 ± 0.001ab
90%WF + 10%PY 22.9 ± 1.4b 41.7 ± 0.9c 35.4 ± 0.9a 0.015 ± 0.001ab
80%WF + 20%PY 21.6 ± 0.6a 39.8 ± 0.7b 38.6 ± 0.9b 0.015 ± 0.001a
70%WF + 30%PY 21.4 ± 0.3a 37.4 ± 0.6a 41.3 ± 0.7c 0.017 ± 0.001b
60%WF + 40%PY 20.6 ± 0.4a 36.5 ± 1.0a 42.9 ± 0.9c 0.016 ± 0.001ab
50%WF + 50%PY 20.6 ± 0.1a 38.1 ± 1.2ab 41.3 ± 1.3c 0.015 ± 0.001a

Values are means ± SD. Values with the same letters in the same column are not significantly different (p < 0.05).

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X. Liu et al. Food Chemistry 284 (2019) 118–124

Table 3
Thermal transition parameters of native wheat flour, yam flour and breads.
Sample To (oC) Tp (oC) Tc (oC) ΔH (J/g)

WF 58.2 ± 0.3a 63.8 ± 0.2a 70.0 ± 0.2a 6.7 ± 0.2c

PY 78.6 ± 0.1b 83.6 ± 0.1b 88.9 ± 0.5b 13.8 ± 1.5d
100%WF + 0%PY ND ND ND ND
90%WF + 10%PY 82.0 ± 3.3bc 94.8 ± 0.3d 101.2 ± 0.4de 2.3 ± 0.9a
80%WF + 20%PY 80.3 ± 0.0bc 94.6 ± 0.4 cd 102.2 ± 0.2e 2.6 ± 0.3a
70%WF + 30%PY 83.2 ± 2.1c 95.0 ± 0.7d 101.6 ± 1.4e 3.0 ± 0.1a
60%WF + 40%PY 81.1 ± 1.6bc 94.4 ± 0.1 cd 100.2 ± 0.5 cd 2.9 ± 0.2a
50%WF + 50%PY 81.1 ± 0.5bc 94.1 ± 0.4c 99.7 ± 0.3c 4.5 ± 0.5b

Values are means ± SD. Values with the same letters in the same column are not significantly different (p < 0.05).
ND: not determined.

SEM images showed that starch granules in purple yam flour

(Fig. 3B′) were larger than wheat starch granules (Fig. 3A′). Starch
granules were observed to be disrupted completely in wheat bread
samples (Fig. 3C′). However, relatively intact starch granules, pre-
dominantly from purple yam flour, were observed in bread samples
with purple yam flour, and the numbers of starch granules were ob-
served to increase with increasing amount of purple yam flour in bread
samples (Fig. 3D′–3H′). Taking SEM and PLM results into account, we
can conclude that wheat starch granules were completely disrupted
while purple yam starch was only partly disrupted during baking.

4. Discussion

In the present study, we investigated the effect of substitution of

purple yam flour for wheat flour on in vitro digestibility of starch in
bread. The structural properties of starch in the breads were examined.
The results of DSC and XRD showed the long-range crystallinity of
Fig. 2. XRD patterns of wheat flour, purple yam flour and breads made from
starch in breads made with mixtures of two flours was greater than that
mixtures of the flours. WF and PY represent wheat flour and purple yam flour,
respectively. m% WF + n% PY represents breads made of m% wheat flour (WF) in 100% wheat bread. The breads with 50% purple yam flour showed a
and n% of purple yam flour (PY). RC: relative crystallinity. (For interpretation greater long-range crystallinity than other breads with 10∼40% purple
of the references to color in this figure legend, the reader is referred to the web yam flour. The bread made with wheat flour had no intact starch
version of this article.) granules under PLM and SEM, whereas the intact starch granules were
present in breads made with mixtures of the two flours. These structural
(2θ) for wheat bread sample became more prominent than that ob- analyses showed that purple yam starch was not completely disrupted
served for wheat flour, indicating the formation of amylose–lipid during baking, indicating their greater resistance to thermal processing
complexes during heating. In our previous studies, the heating of wheat than wheat starch, presumably due to the more compact structure of
flour–water mixtures led to the formation of amylose–lipid complexes purple yam starch than wheat starch. Previous studies have shown that
(Guo, Yu, Wang, Wang, & Copeland, 2018). There were small differ- C-type yam starch is more stable than A-type cereal starches when
ences in the intensity of diffraction peaks at the 15°, 17°, and 23° (2θ) subjected to non-thermal or thermal processing treatments (Wang,
and relative crystallinity for bread samples containing 10∼40% purple Wang, Wang, & Wang, 2017).
yam flour. However, a significant increase in relative crystallinity was With the addition of purple yam flour, the bread samples presented
observed as the amount of purple yam flour was 50% (Fig. 2), con- a lower amount of RDS and SDS and a higher amount of RS compared
sistent with DSC results. with 100% wheat bread. This indicated that breads with purple yam
flour had lower digestibility than 100% wheat bread. The RS content
was highest as the percentage of purple yam flour was 30∼50%. While
3.5. Granular morphology observed by PLM and SEM the digestion extent of starch was reduced, the digestion rate was little
affected by the addition of purple yam flour in breads. This result was
PLM and SEM images of wheat flour, purple yam flour and bread consistent with our previous finding that the digestion rate of cooked
samples are shown in Fig. 3. Native starch granules from wheat and starch was little affected by the degree of gelatinization or structural
purple yam flours showed the typical birefringent “Maltese crosses”, order in gelatinized starch (Wang et al., 2017).
characteristic of the radial arrangement of amylopectin crystallites To gain a further mechanistic insight of the effect of purple yam
within the granule (Fig. 3A and 3B). Starch granules in purple yam flour flour on starch digestibility, the fluorescence quenching of porcine
(Fig. 3B) were much larger and the Maltese crosses were clearer than pancreatic α-amylase and amyloglucosidase by the bread samples was
those of wheat starch granules (Fig. 3A). No “Maltese crosses” were analysed (Fig. S1). The fluorescence peak at 344 nm is a property of the
observed in 100% wheat bread (Fig. 3C), indicating that starch crys- tryptophan residues in ɑ-amylase and amyloglucosidase (Burstein,
tallites in bread samples were completely disrupted after baking. It is Vedenkina, & Ivkova, 1973). The fluorescence intensity of alpha-amy-
interesting to note that bread with purple yam flours showed clear lase and amyloglucosidase decreased with increasing amount of purple
“Maltese crosses”, and the number of the granules increased with in- yam flour in bread samples, indicating that these enzymes interacted
creasing amount of purple yam flour in breads (Fig. 3D-3H). These with constituents in the purple yam flour, and the effect increased with
observations further confirmed that starch granules in purple yam flour increasing amount of purple yam flour in the breads. Similar results
were not completely disrupted, consistent with DSC and XRD results. were also found that the green tea extract decreased the fluorescence

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X. Liu et al. Food Chemistry 284 (2019) 118–124

Fig. 3. PLM and SEM images of wheat flour (A) (A′), purple yam flour (B) (B′) and breads made from the flours: C (C′): 100%WF + 0%PY, D (D′): 90%WF + 10%PY,
E (E′): 80%WF + 20%PY, F (F′): 70%WF + 30%PY, G (G′): 60%WF + 40%PY, H: 50%WF + 50%PY. m% WF + n% PY represents breads made of m% wheat flour
(WF) and n% of purple yam flour (PY). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

intensity of a-amylase (Miao, Jiang, Jiang, Zhang, & Li, 2015). Taking Acknowledgements
the structure and fluorescence quenching results together, we can
conclude that the residual starch granules in bread samples with purple The authors gratefully acknowledge the financial support from the
yam flour and the inhibitory effect of breads containing purple yam National Key Research and Development Program of China
flour on enzyme activity were the factors that potentially reduced (2017YFD0400200), National Natural Science Foundation of China
starch digestibility of bread. (31871796, 31522043) and Natural Science Foundation of Tianjin City
Supplementary data associated with this article can be found, in the (17JCJQJC45600, 18ZYPTJC00020).
online version, at https://doi.org/10.1016/j.foodchem.2019.01.025.
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