LWT - Food Science and Technology 145 (2021) 111518

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LWT
journal homepage: www.elsevier.com/locate/lwt

Comparison of different thermal treatments on the physicochemical

properties of Apios fortunei used for yellow wine fermentation
Shichao Bian a, b, Enbo Xu c, Xi Fu a, b, Zhengyu Jin a, b, d, Aiquan Jiao a, b, d, *
a
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, China
b
School of Food Science and Technology, Jiangnan University, Wuxi, 214122, China
c
College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou, 310058, China
d
Collaborative Innovation Center of Food Safety and Quality Control in Jiangsu Province, Jiangnan University, Wuxi, 214122, China

A R T I C L E I N F O A B S T R A C T

Keywords: A novel material (Apios fortunei), which has a high starch content and potential healthcare functions, was used for
Apios fortunei yellow wine fermentation. To determine how the tuber’s composition would be modified by thermal treatments
Extrusion and how those modifications would affect fermentation, the changes in the physicochemical properties of the
Starch crystallinity
A. fortunei tuber after steam cooking, traditional extrusion (extrusion without enzyme) and enzymatic extrusion
Amylase
Fermentation efficiency
(introduce the amylase into the extrusion) were investigated. The results showed a low degree of gelatinization
(70.56%) in the steam-cooked sample, and the fermentation broth of the traditionally extruded sample was
dense. However, the enzymatically extruded sample was almost completely gelatinized (99.09%) with an
amorphous starch structure measured via X-ray diffraction and Fourier-transform infrared spectroscopy. The
high water solubility index (61.66%), low water absorption index (1.68 g/g) and viscosity (<60 cP), indicated
that the broth of enzymatically extruded sample had a fine fluidity. The results of the final wines indicated that
enzymatic extrusion significantly increased the fermentation efficiency, from 62.22% (steam cooking) and
63.87% (traditional extrusion) to 73.47%. Meanwhile, enzymatic extrusion improved the quality characteristics
of the final wines, including total amino acid (4559.20 mg/L), soluble solid content (7.35 g/100 g) and total acid
(10.66 g/L).

1. Introduction functions, applying it to the process of fermenting Chinese yellow wine

Apios is a genus of perennial, twining, herbaceous plants, which have
a disjunct distribution between eastern Asia and North America. The
tubers of Apios species are regarded as useful sources of flavonoids, and,
therefore, they have great economic potential (Chu et al., 2019; Kim &
Jin, 2019; Li et al., 2014). A member of the Apios genus, Apios fortunei,
widely grows in China, and it is recorded in traditional pharmacopoeia
for its medicinal value (Zhao, 1963, p. 344). Additionally, the peeled
A. fortunei tuber contains approximately 75 g/100 g starch and 8 g/100
g soluble sugar, and its content of resistant starch after gelatinization is
only about 1.6 g/100 g, indicating that the tuber is an important starch
resource (Wang, Guo, Fan, Feng, & Wei, 2018). A. fortunei is mostly
cultivated in Heze city, with an annual output of 500 tons in 2020, to
date, there have been few valuable applications for A. fortunei tubers
outside of specialized diets. However, this research suggests that, as
A. fortunei is an excellent starch resource with potential healthcare
may be a suitable method of producing value-added products.
Yellow wine is one of the world’s three ancient wines, and it exhibits
various flavors due to its diverse ingredients and the Qū starter culture
(Jiao, Xu, & Jin, 2017). Qu is usually made of rice or wheat, after the
inoculation of the mold and a period of cultivation, Qu is rich in a wide
variety of microorganisms as well as various enzymes, for saccharifica­
tion and fermentation (Kuo, Shieh, Huang, David Wang, & Huang,
2019). Differing from the traditional types of yellow wine, which are
mostly fermented with rice, corn, or wheat, special yellow wines are
fermented with novel starch-based materials, while maintaining the
style of Chinese yellow wine, according to Chinese National Standard
GB/T 13662–2018 (SAC/TC 471, 2018). However, to date, no report has
considered the cooking and processing of A. fortunei for wine produc­
tion, and, if A. fortunei is to be a suitable source of starch for special
yellow wines, it is necessary to investigate what changes may occur in its
physicochemical properties after thermal treatments.

* Corresponding author. Collaborative Innovation Center of Food Safety and Quality Control in Jiangsu Province, Jiangnan University, Wuxi, 214122, China.
E-mail address: jiaoaq@jiangnan.edu.cn (A. Jiao).

https://doi.org/10.1016/j.lwt.2021.111518
Received 21 January 2021; Received in revised form 11 April 2021; Accepted 15 April 2021
Available online 16 April 2021
0023-6438/© 2021 Elsevier Ltd. All rights reserved.
S. Bian et al.
Additionally, the material soaking and steam cooking stages of
traditional winemaking material pretreatment consume huge amounts
of energy and water. In recent years, pretreatment via extrusion has
been widely applied to ethanol fermentation, and it has been proven that
extrusion, with or without enzymes, can effectively improve fermenta­
tion efficiency under proper conditions (Kuo et al., 2019; Xu et al.,
2014).
As an integrated process of mixing, heating, and forming, twin-screw
extrusion technology has been widely used in food areas due to its
unique high pressure, shear action, high efficiency, and low cost (Li,
Jiao, et al., 2019; Sun et al., 2019). The extrusion conditions can be
divided into two categories: (1) severe extrusion conditions (lower
moisture or higher temperature), in which the raw materials are usually
puffed and reach a high degree of gelatinization, and (2) mild extrusion
conditions (higher moisture or lower temperature), in which the raw
materials are squeezed, shaped, or pregelatinized (Rangira, Gu, Ek, &
Ganjyal, 2020; Sun et al., 2018). Introducing various enzymes with
different properties enriches the extrusion process, turning the extrusion
machine into a biological reaction container and causing it to play a
more effective role as the source of high mechanical action (Wu, Jiao,
Xu, Chen, & Jin, 2020; Xu, Campanella, et al., 2020).
In this research, three different thermal process methods—i.e., steam
cooking, traditional extrusion, and enzymatic extrusion—were used to
investigate the effect of thermal treatments on the physicochemical
properties of whole A. fortunei tubers (skin and flesh). The thermally
treated product was also fermented to produce special yellow wine. The
results aid the current understanding of how the A. fortunei tuber
changes during thermal processing and broaden the plant’s produc­
tiveness by successfully using it to make special yellow wine as a new
value-added product.
2. Materials and methods
2.1. Materials and chemicals
Powder from whole A. fortunei tubers (67.7 g/100 g starch with 25.3
g/100 g amylopectin, 14.9 g/100 g protein, 11.2 g/100 g water, 3.4 g/
100 g crude fiber, 3.3 g/100 g ash, and 2.3 g/100 g lipid) was obtained
from the Tianxiang Luohanshen Professional Cooperative (Heze, China).
Liquid thermostable α-amylase (Termamyl SC 120 L), extracted from
Bacillus licheniformis with an activity of 120 KUN/g at an optimum pH
(6–8) and optimum temperature (80–90 ◦ C) for enzymatic extrusion,
was purchased from Novozymes (China). Taka-amylase was purchased
from URchem (China). Wheat Qū (54.5 g/100 g starch with 22.5 g/100 g
amylose, 16.0 g/100 g water, 12.6 g/100 g protein, 3.9 g/100 g crude
fiber, 3.1 g/100 g ash, and 1.2 g/100 g lipid) was obtained from Jinfeng
Wine Co. Ltd. (Shanghai, China), and dry yeast was purchased from
Angel Yeast Co., Ltd. (Shanghai, China). The reagents used for high-
performance liquid chromatography (HPLC) were at chromatographic
levels, and the other reagents and chemicals were of reagent grade and
available on the market.
2.2. Treatment of raw materials
Three treatments—steam cooking, traditional extrusion, and enzy­
matic extrusion—were designed for observing the influence of heat,
shear action, and enzymatic hydrolysis on the raw material, respec­
tively. First, the raw material was humidified to 50 g/100 g moisture
content and then cooked with steam for 20 min to obtain the steam-
cooked sample (SCS). Prior to extrusion, the original A. fortunei pow­
der was pre-humidified to 25 g/100 g moisture content and stored at
4 ◦ C for 12 h to ensure that the water was evenly distributed. For
enzymatic extrusion, the liquid amylase (2‰ of the dry basis) was first
dissolved in water (enzyme: water = 1:104) and then premixed with the
A. fortunei powder as it was being pre-humidified to 25 g/100 g moisture
content. As with the traditional extrusion process, this mixture was
2
LWT 145 (2021) 111518
stored at 4 ◦ C for 12 h. A twin-screw extruder (FMHE36-24, Fumake Co.,
Hunan, China) was used to carry out the extrusion experiment. The
diameter of the extruder was 36 mm, and its length-to-diameter ratio
was 24:1. The extrusion parameters were set as follows: final extrusion
moisture content of 50 g/100 g; screw speed of 100 rpm; feeding rate of
4 kg/h; temperature profiles of 60, 70, 80, 90 and 110 ◦ C. The extrusion
operating conditions for the enzymatic extrusion were the same as those
for the traditional extrusion. Thus, the traditionally extruded sample
(TES) and the enzymatically extruded sample (EES) were obtained.
A part of the treated samples was immediately dried in a 40 ◦ C oven
until their moisture content reached about 10 g/100 g. After that, the
dried sample was broken into powder for subsequent physicochemical
testing. While another part of the undried samples was used for
fermentation before they cooled down. Additionally, the raw material
was used in physicochemical properties tests for comparation, showing
the modifications of the thermal processes. For fermentation tests, on
account of that the raw material is not suitable for fermentation (the
ungelatinized starch is quite difficult for saccharification), only the three
treated A. fortunei were conducted.
2.3. Water absorption index and water solubility index
The water absorption index (WAI) and water solubility index (WSI)
were calculated according to the procedure reported by Hagenimana,
Ding, and Fang (2006), with some modifications. 1 g of the sample was
weighed (m1) and dissolved with 12 mL of 0.2 mol/L sodium acetate
buffer (pH 3.5 to inactivate the residual enzymes) in a 50 mL centrifuge
tube (pre-weighed) and incubated for 30 min in a thermostatic shaking
water bath (30 ◦ C, 160 rpm). Then, after centrifugation (6500 g, 15
min), the supernatant was transferred to a pre-weighed glass dish and
dried in an oven at 105 ◦ C to a constant weight. The weight of the su­
pernatant (M1) and precipitate (M3) were used to calculate the WAI and
the WSI. 12 mL of sodium acetate buffer was also placed in the oven at
105 ◦ C and dried to a constant weight (M2). The WAI and the WSI were
calculated as:
M1 − M2
WSI% = × 100%
m1
/
M3
WAI g g=
m1
2.4. Differential scanning calorimetry
Differential scanning calorimetry (DSC) (SII; Nanotechnology Corp.,
Tokyo, Japan) was adopted to measure the gelatinization enthalpies
according to the method described by Li, Rashed, et al. (2019), with
some minor modifications. 3 mg of a sample were quantitatively
weighed and put into a stainless steel pan, and twice the weight of 0.2
mol/L sodium acetate buffer (pH = 3.5) was added. After the pan was
sealed, it was stored at 4 ◦ C for 12 h to ensure that the moisture was
evenly distributed. All the samples were heat from 25 ◦ C to 125 ◦ C at a
rate of 10 ◦ C/min. The resulting spectrogram was processed via the
Universal Analysis Program (TA Instruments Inc., NY) to determine the
enthalpy (ΔH), and the degree of gelatinization (DG) was calculated
with the following formula:
ΔH0 − ΔH
DG% = × 100%
ΔH0
where ΔH and ΔH0 were the enthalpy changes of the treated and raw
material, respectively.
2.5. Degree of gelatinization
As the raw material for this research was powder obtained from the
entire A. fortunei tuber, the powder’s composition, except for the starch,
S. Bian et al.
could have affected the enthalpy changes during the DSC instrument
heating process. Therefore, the enzymatic method, reported as Yang
et al. (2020), was conducted for comparison to measure the DG. 0.5 g of
a powder sample was quantitatively weighed and dispersed in a 50 mL
conical flask with 20 mL of deionized water. The starch from the sample
was completely gelatinized by boiling the sample in a water bath for 20
min (oscillating every 5 min). After the sample was cooled, 5 mL of 3
g/100 g Taka-amylase was added, and the sample was incubated for 2 h
in thermostatic shaking water bath (37 ◦ C, 100 rpm). Then, 1 mL of
hydrochloric acids (HCl; 1 mol/L) was added to inactivate the enzyme,
followed by 1 mL of sodium hydroxide (NaOH; 1 mol/L) to balance the
pH. Finally, the reducing sugar content was measured (C0) referring to
the method described by Miller (1959). In addition, 0.5 g of a sample
was taken quantitatively and measured with the same steps as above,
minus the process of boiling in the water bath. In this case, the reducing
sugar content was denoted as C1, and the final DG formula was defined
as follows:
C1
DG% = × 100%
C0
Due to the specificity of amylase, the influence of other components
on the final results could be minimized.
2.6. Rapid viscosity analyzer
The pasting properties were measured via a rapid viscosity analyzer
(RVA) (RVA-4500, Newport Scientific Pty. Ltd., Sidney, Australia),
following the method described by Yildiz et al. (2013). Briefly, 3 g of a
sample (14 g/100 g water content) were weighed and quickly mixed
with 25 g of a 0.2 mol/L sodium acetate buffer (pH = 3.5) in an
aluminum canister. For the first 10 s, the mixture was blended at a high
speed of 960 rpm, followed by a steady speed of 160 rpm till the end. The
initial temperature of the instrument was 50 ◦ C, and gradually raised to
95 ◦ C at a rate of 12 ◦ C/min. It was held at 95 ◦ C for 2 min and then
lowered to 50 ◦ C at the rate of 12 ◦ C/min, where it was held for another
2 min. Important parameters—including peak viscosity (PV), through
viscosity (TV), final viscosity (FV), breakdown viscosity (BDV), setback
viscosity (SBV), and peak time (PT) were recorded.
To detect the initial fermentation viscosity (IFV), the procedure re­
ported by Poonsrisawat et al. (2014) was followed and modified to
simulate the initial fermentdation environment. Briefly, 5 g of the
treated samples (db) with Qū (10 g/100 g, db) and yeast (0.1 g/100 g,
db) were mixed with deionized water at a ratio of 1:4.5. The instrument
procedure was as follows. The solution was premixed at 960 rpm for the
first 10 s and then maintained at 160 rpm till the end. The temperature
was also held at 30 ◦ C, and the total measuring time was 12 min. The
final viscosity was defined as the IFV.
2.7. X-ray diffraction and Fourier-transform infrared spectroscopy with
attenuated total reflection
The long-range order of the starch crystallization structure was
tested via X-ray diffraction (XRD) (D2 PHASER, Bruker AXS Inc., Ger­
many) according to the method reported by Wu et al. (2020), with some
modifications. CuKα radiation was used, and the diffractograms were
recorded from 4◦ to 40◦ (2θ) at a scan rate of 3.6◦ /min. The diffracto­
grams were analyzed and the relative crystallinity calculated by Jade 6.5
(Materials Data Inc., Livermore, CA).
Fourier-transform infrared spectroscopy with attenuated total
reflection (FTIR-ATR) (NEXUS, Thermo Nicolet Corporation, USA) was
used to observe the short-range order of the starch crystalline structure.
The scanning wave number ranged from 4000 cm− 1 to 650 cm− 1, and
the scan was run 32 times at a 5 cm− 1 resolution. Also, the air back­
ground was deducted. The obtained infrared spectrogram was decon­
volved with an enhancement factor of 1.9 and a peak width of 38 cm− 1
set according to the method described by Man et al. (2012).
3
LWT 145 (2021) 111518
2.8. Scanning electron microscopy
Scanning electron microscopy (SEM) (SU8100, Hitachi Science Sys­
tems Inc., Tokyo, Japan) was used to observe the samples’ microstruc­
ture. The dried samples were fixed uniformly and dispersedly on a
special sample table, which had been pre-coated with conductive tape
and coated platinum with a sputtering coater. Afterwards, the micro­
structures were observed at 2500 × magnification coefficients.
2.9. Fermentation of special yellow wine
60 g of a treated sample (db) was placed in a container selected for
fermentation (500 mL jar) with 10 g/100 g wheat Qū and 0.1 g/100 g
yeast. Deionized water was added at a ratio of 1:4.5 and mixed thor­
oughly. The jar was sealed with a filter membrane, which allows air to
pass through while isolating bacteria. The fermentation process was
conducted in a multistage, programmable, artificial climate incubator
for chief fermentation (3 d, 30 ◦ C) and post fermentation (11 d, 15 ◦ C).
After fermentation, four layers of sterile gauze were used for filtration,
followed by centrifugation at 6500 g for 20 min. The supernatant was
collected and heated to 85 ◦ C for 15 min to inactive the bacteria and
enzymes. The wine was then stored in a refrigerator at 4 ◦ C.
2.10. Ethanol yield and fermentation efficiency
During fermentation, yeast uses glucose for anaerobic respiration
and generates one molecule of CO2 while producing one molecule of
ethanol. The mass loss of CO2 can be used as the basis for continuously
reflecting the changes of ethanol production in the wine, which has been
proved to be a feasible method (Watanabe, Ota, Nitta, Akao, & Shimoi,
2011). The fermentation jars were weighed periodically to measure the
mass loss of CO2, and the weight of steam evaporation was taken into
account and deducted. The ethanol yield (EY) and fermentation effi­
ciency (FE) were calculated through the weightlessness of CO2 as
follows:
46 × total ​ lost ​ CO2 (g)/44
EY% = × 100%
mass of A. ​ fortunei and Qū (g)
46 × total ​ lost ​ CO2 (g)/44
FE% = × 100%
(starch mass of A. ​ fortunei ​ and Qū (g) × θ
The EY was defined as the percentage of the ethanol (calculated by
the weightlessness of CO2, where 44 and 46 were the relative molecular
weights of CO2 and ethanol respectively) based on the mass of the
original A. fortunei (db) and Qū (db). A similar calculation method was
used for FE (Xu et al., 2014). θ was the theoretical conversion value of
starch into ethanol: 0.5679 (g/g).
2.11. Total sugar, total acid, and soluble solid content
Total sugar was determined according to the method presented by
Miller (1959). Total acid (calculated as lactic acid, M = 90 g/mol) was
determined by titration using calibrated NaOH (C = 1.0761 mol/L), and
an Abbe refractometer (TYPE WAY-2 W, INESA Instrument Co.,
Shanghai) was used to measure soluble solid content.
2.12. Analysis of amino acid
The amino acid in the final wine was analyzed via HPLC (Agilent
Tech Inc., CA, USA) with an ODS HYPERSIL C18 column (250 mm × 4.0
mm, 5 μm, Agilent Tech Inc., CA, USA) according to the method
described by Yu, Zhao, Li, Tian, and Ma (2015). Mobile phase A (27.6
mM sodium acetate: triethylamine: tetrahydrofuran = 1000 mL: 0.22
mL: 5 mL) and mobile phase B (80.9 mM sodium acetate: acetonitrile:
methanol = 200 mL: 400 mL: 400 mL) were used for gradient elution at a
flow rate of 1 mL/min. A UV detector (VWD) was employed to detect
S. Bian et al.
and analyze the eluent. The detection wavelengths for the amino acid
and proline were 338 nm and 262 nm, respectively.
2.13. Statistical analysis
All the experiments, except the SEM, were repeated three times, with
the results expressed as mean ± standard deviation. Data were analyzed
by one-way analysis of variance (ANOVA) and p < 0.05 was considered
to be statistically significant, which was performed by SPSS 26.0 (IBM
Inc., Armonk, NY).
3. Results and discussion
3.1. Functional properties, degree of gelatinization, and pasting properties
The functional properties (the WAI and WSI) and DG of raw material
and the treated samples are shown in Table 1. The WAI is generally
considered to be closely related to the DG, and it reflects a material’s
water-holding capacity, while the WSI can be used to measure the
degradation degree of polymer materials during processing. Therefore,
the WAI and WSI values have opposite effects on the viscosity of
fermentation broth. Compared with the raw material, the WAI of SCS
and TES significantly increased due to the gelatinization of starch during
processing, which destroyed the molecular hydrogen bonds and allowed
more water to enter the interior structure. However, when the degree of
dextrinization and starch melting was greater than that of gelatinization,
the WAI decreased (Sarawong, Schoenlechner, Sekiguchi, Berghofer, &
Ng, 2014). Additionally, due to the degradation of the starch macro­
molecules, the WSI of the EES was much higher (11.4 times) than that of
the raw material, and this is similar to the enzymatically extruded rice
(Xu et al., 2014). After steam cooking and traditional extrusion, the WSI
of the SCS and TES increased to about twice that of the raw material,
indicating that the heat treatment slightly degraded the components of
A. fortunei and increased the soluble content of the small molecules.
Moreover, the WSI of the SCS was approximately 2% higher than that of
the TES. Though the starch polymers may undergo significant degra­
dation during extrusion, as described by Ye et al. (2018), the content of
the soluble degradant does not increase under mild extrusion conditions.
DG measurements are quite important for ethanol fermentation. As
the orderly structure of natural starch is resistant to the amylolytic
enzyme (Xu, Campanella, et al., 2020), a higher DG is desired as it
benefits the fermenting microbial community. During the starch gela­
tinization process, the destruction of the crystalline structure is
accompanied by enthalpy changes. A more orderly crystalline structure
must be broken by higher energy. Thus, the ΔH is generally used as a
quantitative basis for calculating the degree of gelatinization. The DG
values of the SCS and TES were 59.99% and 72.73%, respectively (by
DSC), and the DG of the EES was 97.45%. As starch is the substrate of
amylase, the introduction of α-amylase will, no doubt, increase the
gelatinization degree. Similar results have been reported by Xu et al.
(2017), which showed, under mild extrusion conditions in the presence
Table 1
Functional properties, degree of gelatinization and pasting propertiesb of the raw material and the samples with the treatments of steam cooking, traditional extrusion
and enzymatic extrusiona.
Samplec WAI (g/g)d WSI (%)d DG (%)d
DSCd Taka-amylase
RM 2.66 ± 0.03a 5.42 ± 0.76a – –
SCS 2.88 ± 0.11b 12.63 ± 1.04b 59.99 ± 2.33a 70.56 ± 0.78a
TES 3.44 ± 0.09c 10.70 ± 1.06c 72.73 ± 1.47b 84.45 ± 1.35b
EES 1.68 ± 0.03d 61.66 ± 0.71d 97.45 ± 0.57c 99.09 ± 0.33c
a
Values are the averages of three replicates, and different alphabets indicate statistical difference (p < 0.05).
b
PV, peak viscosity; TV, through viscosity; BDV, breakdown viscosity; FV, final viscosity; SBV, setback viscosity; PT, peak time; IFV, initial fermentation viscosity.
c
RM, raw material; SCS, steam-cooked sample; TES, traditionally extruded sample; EES, enzymatically extruded sample.
d
WAI, water absorption index; WSI, water solubility index; DG, degree of gelatinization; DSC, differential scanning calorimetry.
LWT 145 (2021) 111518
of α-amylase, that the DG increased from 80% to 99%—illustrating that
introducing trace amounts of α-amylase to the extrusion process can
effectively increase the DG.
The DG of the SCS and TES obtained via the enzymatic method were
greater than those measured through DSC by around 10%. This may be
attributed to the non-starch components of A. fortunei. The protein, in
particular, was quite endothermal during denaturation and greatly
influenced the enthalpy changes in the DSC, leading to a larger ΔH and,
thus, to a smaller DG measurement. Therefore, DSC must be used
modestly to measure the DG of starch-based materials. However, there is
little difference between the DG of the EES as measured by DSC and
enzymolysis (97.45% and 99.09%, respectively), which, once again,
proves the promotional effect enzymatic extrusion has on DG via the
introduction of α-amylase in appropriate amounts during extrusion.
The pasting profiles of the raw material and the treated samples are
shown in Fig. 1A. The viscosity of the original sample was the highest,
and, after processing, it decreased to varying degrees. The viscosity of
the EES was the lowest, and all the viscosity values dropped below 10 cP.
Similar viscosity profiles were reported in the enzymatically extruded
broken rice (Xu, Campanella, et al., 2020). Generally, the degree of
starch gelatinization and particle destruction play a decisive role in
viscosity (Bryant, Kadan, Champagne, Vinyard, & Boykin, 2001). During
the gelatinization process, ungelatinized starch granules rapidly swell
with water absorption, which increases the friction between the parti­
cles and results in quickly increasing viscosity. Enzymolysis and shear
action led to quite high DG and WSI values in the EES, which un­
doubtedly reduced its viscosity. Both the SCS and TES showed flat
pasting profiles without obvious peak viscosities, as evidenced in
Fig. 1A, and the viscosity values kept increasing until 11 min. This may
have been related to the newly formed crystalline peak observed via
XRD (detailed below), which prevented water absorption and delayed
gelatinization, resulting in a longer swelling time for the starch granules
and an increase in PT (Roman, Dura, Martinez, Rosell, & Gomez, 2016;
Sun, Han, Wang, & Xiong, 2014).
It is also worth noting that the DG of the TES was higher than that of
the SCS but that the TES had a higher viscosity. This may have occurred
because that (1) the TES has the higher WAI, the system was denser
while the same amount of water was added; (2) the DG is the key factor
influencing viscosity in RVA measurement; however, the protein, fiber,
and ash components in the A. fortunei tuber powder weakened the
dominant role of starch in the whole system. Commonly, insoluble
protein and ash loosen the overall structure, causing viscosity to
decrease; however, due to high pressure and high shear action, extrusion
can modify various components. For fiber, (1) partially insoluble fiber is
converted into soluble fiber, and (2) the water absorption and swelling
capacity of the insoluble fiber improves, resulting in a higher viscosity
across the system (Sandrin, Mejía, Caon, & de Francisco, 2019; Schmid,
Karbstein, & Emin, 2020). These factors make the final viscosity mea­
surement less predictable and controllable than that obtained in a pure
starch system.
Compared with PV, IFV can more directly reflect the influence
PV (cP) TV (cP) BDV (cP) FV (cP) SBV (cP) PT (min) IFV (cP)
898 ± 3a 522 ± 3a 376 ± 1a 721 ± 3a 199 ± 2a 4.8 ± 0.0a –
55 ± 5b 50 ± 6b 5 ± 1b 80 ± 4b 30 ± 1b 6.9 ± 0.1b 87 ± 3a
106 ± 3c 101 ± 4c 5 ± 1b 144 ± 4c 43 ± 0c 6.9 ± 0.0b 213 ± 9b
7 ± 0d 5 ± 1d 2 ± 1c 8 ± 1d 3 ± 1d 1.1 ± 0.0c 55 ± 6c
4
S. Bian et al.
Fig. 1. RVA (A), XRD (B), original (C) and deconvolved (D) FTIR profiles of raw material (RM), and A. fortunei pretreated by steam cooking (SC), traditional
extrusion (TE) and enzymatic extrusion (EE). Values are the averages of three replicates, and different alphabets indicate statistical difference (p < 0.05).
differently treated samples have on fermentation. In fermentation, a
broth with excessive viscosity leads to a dense substrate, causing
nonuniform fermentation. It is also not beneficial to the discharge of CO2
produced by yeast respiration; thus, yeast growth is inhibited. Among
the products obtained via the three different processing methods, the
EES had the lowest IFV (55 cP), followed by the SCS (87 cP) and the TES
(213 cP).
3.2. XRD and FTIR-ATR
The XRD patterns of different samples are shown in Fig. 1B. The
major peaks at 5.6◦ , 15◦ , 17◦ (2θ), the shoulder peak at 18◦ (2θ), and the
wide peak at 23◦ (2θ) were observed in the raw material, and the XRD
pattern is similar to that of A. fortunei starch sample (Wang et al., 2018).
Among these, the peak at 5.6◦ (2θ) was the characteristic peak of B-type
crystallization, while the strong peak at 15◦ (2θ), the adjacent peaks at
17◦ and 18◦ (2θ), and the single peak at 23◦ (2θ) were the characteristic
peaks of A-type crystallization—indicating that the A. fortunei tuber
undergoes a C-type starch crystallization. The peak at 5.6◦ (2θ) in the
raw material disappeared after the thermal treatments. The peaks at 17◦
and 23◦ (2θ), as well as the weak peak at 15◦ (2θ), could still be observed
in the SCS. Due to the higher degree of starch gelatinization, the crys­
talline structure of the TES was more severely destroyed; thus, the in­
tensity of the peaks at 17◦ and 23◦ (2θ) were weakened. The peak at 20◦
(2θ) was observed in both the SCS and the TES, which is the charac­
teristic peak of the V-type crystallization caused by amylose-lipid com­
plexes or the complex aggregation of gelatinized starch (Chen et al.,
2017). The only peak at 20◦ (2θ) was observed in the EES because, in
that sample, the starch was almost completely gelatinized, indicating the
sample’s amorphous structure (Kuo et al., 2019). The maximum relative
crystallinity (RC) of the raw material was 25.6%, and the RC of the SCS
was 15.8%, higher than that of the TES (13.0%). The lowest RC (7.9%)
was observed in the EES.
5
LWT 145 (2021) 111518
Fig. 1C shows the FTIR profiles of the raw material and the treated
samples. There was no significant difference among the spectrograms of
the treated samples. The FTIR spectrum, after deconvolution, of
1100–900 cm− 1 is shown in Fig. 1D. The characteristic absorbance peaks
at 1047 cm− 1 and 995 cm− 1 were positively correlated with the crys­
tallinity of starch, while the peak at 1022 cm− 1 was positively correlated
with the amorphous region. Therefore, the absorbance ratios of A1047/
1022 and A995/1022 could be used to determine the order of the
crystalline structure (Kuo et al., 2019; Wu et al., 2020). The raw material
had the highest ratios of A1047/A1022 (0.49) and A995/A1022 (1.32).
These decreased to 0.46 and 1.17, respectively, in the SCS and 0.44 and
1.14, respectively, in the TES. The lowest A1047/A1022 and
A995/A1022 ratios were obtained in the EES: 0.34 and 0.96, respec­
tively. The short-range order of the starch crystallization structure
measured via FTIR was in agreement with the long-range order
measured by XRD, indicating that the enzymatic extrusion had the
strongest modification effect, transforming the crystalline structure of
the starch into an amorphous form. This may be beneficial to the
enzymolysis and saccharification during fermentation.
3.3. SEM
To further understand the influence of different thermal processing
methods on the A. fortunei tuber, SEM was used to examine the micro­
structure of the A. fortunei surface. It also explored how the role of heat,
high shear action, and enzymatic hydrolysis affect the A. fortunei pro­
cessing. As shown in Fig. 2A, in the raw material, ellipsoidal starch
granules of different sizes were evident. In addition, the non-starch
components of A. fortunei mostly adhered to the starch granules in
irregular shapes, making their surfaces rough and uneven. After thermal
processing, the individual, small-sized starch particles were gelatinized
and agglomerated into larger blocks, as the continuous and rough sur­
face of the SCS sample is observed in Fig. 2B. The TES surface was
S. Bian et al. LWT 145 (2021) 111518

Fig. 2. SEM micrographs of raw material (A), steam-cooked (B), traditionally extruded (C) and enzymatically extruded (D) samples (2500 × ).

smoother than the SCS surface, as shown in Fig. 2C. In addition, due to
Table 2
the high shear action during extrusion, obvious cracks appeared on the
Fermentation efficiency, ethanol yield during the fermentation and the quality
TES surface. Similar cracks were observed in the extruded high-starchy
characteristics of the yellow wines with the pretreatments of steam cooking,
sample reported by Vieyra, Martínez, and Méndez (2011). As can be traditional extrusion and enzymatic extrusiona.
seen in Fig. 2D, the EES surface was similar to that of the TES (i.e.,
Sample Fermentation Ethanol Total Total Soluble
continuous and smooth with cracks). However, due to the introduction
efficiency (%) yield (%) acid sugar solid
of amylase, partial areas became uneven, exhibiting holes caused by (g/L) (g/L) content (g/
enzymatic hydrolysis. Similar porous surface of the enzymatically 100 g)
extruded wheat starch was reported by Li, Rashed, et al. (2019). This Steam 62.22 ± 2.77a 26.48 ± 11.82 2.19 ± 7.05 ±
porous structure may have been beneficial to the further enzymolysis cooking 1.18a ± 0.08a 0.05b
and degradation of starch during fermentation. 0.20a
Traditional 63.87 ± 1.31a 27.19 ± 11.93 2.03 ± 7.19 ±
extrusion 0.56a 0.21a 0.06 ab
3.4. Fermentation of special yellow wine
±
0.11a
Enzymatic 73.47 ± 1.40b 31.27 ± 10.66 2.69 ± 7.35 ±
The samples obtained via the three thermal treatment methods were extrusion 0.60b ± 0.10b 0.13a
used for to ferment special yellow wine. FE, EY, and other quality 0.14b
characteristics are shown in Table 2, and the total weight loss of CO2 a
Values are the averages of three replicates, and different alphabets indicate
during the fermentation process is depicted in Fig. 3. When dispersing statistical difference (p < 0.05).
the materials in water, different states were observed due to the physi­
cochemical properties of the treated A. fortunei samples. With its high conducive to enzyme saccharification by microorganisms; (2) Via
WSI and low viscosity, the EES was dispersed easily, while the dispersion extrusion with α-amylase, the starch was partially degraded, making it
for the SCS, with its low WSI, was more difficult. However, the TES was quickly enter the rapid fermentation phase and leading to higher
quite water-resistant. And after a glass rod was used to stir and disperse fermentation efficiency in the chief fermentation stage, as shown in
the sample into the water, the broth became dense due to its high WAI, Fig. 3. Therefore, the yeast in the fermentation broth could reproduce
which is unfavorable for fermentation. Therefore, though the DG of the rapidly at the initial stage and become the dominant strain, inhibiting
TES was higher than that of the SCS, the FE (63.87%, 62.22% of the TES the reproduction of other undesired acid producing bacteria. Thus, the
and SCS, respectively) and EY (27.19%, 26.48% of the TES and SCS, total acid content of the resulting wine was lower. The similar promo­
respectively) were similar. However, the FE and EY of the samples tion of FE and the decrease of total acid were observed in the fermen­
treated with enzymatic extrusion were significantly increased, reaching tation of the enzymatically extruded rice (Xu et al., 2014). The total acid
73.47% and 31.27%, respectively. Compared with the former two was mainly produced by yeast and lactic acid bacteria to control po­
sample types, the FE of the enzymatically extruded sample increased by tential microbial contamination during fermentation (Xu et al., 2014);
15–18%. The main reasons are as follows: (1) Enzymatic extrusion however, excessive total acid may be a disadvantage to the wine’s taste,
increased the DG and severely damaged the crystallization of the orig­ and it is better for the total acid to be less than 10 g/L, according to
inal starch, as gelatinized starch with an amorphous structure is more

6
S. Bian et al. LWT 145 (2021) 111518

Table 3 wines produced in this research are shown in Table 3. Among the free
Amino acid composition of the yellow wines with the pretreatments of steam amino acids, glu, asp, pro, and ala were the major players, accounting
cooking, traditional extrusion and enzymatic extrusion.a. for more than 10% of the total amino acids, and the thermal treatments
Amino acid (mg/ Steam cooking Traditional Enzymatic had little influence on the composition of the amino acids in the wines.
L) extrusion extrusion However, analysis of variance (ANOVA) and Duncan’s multiple range
ser 21.24 ± 1.63a 22.57 ± 1.08a 21.62 ± 1.24a test revealed that levels of amino acids—such as asp, glu, ala, and
gly 166.25 ± 4.43ab 172.81 ± 5.3b 159.06 ± 5.11a his—were significantly increased in the wine fermented with the TES
thr 131.18 ± 7.41a 138.95 ± 6.33a 127.89 ± 4.62a and EES compared to that fermented with the SCS. The total amino acid
ala 460.28 ± 13.85b 551.71 ± 26.25a 539.31 ± 18.19a
content of the wines showed that the protein modifications across the
cys-s 2.58 ± 0.08a 2.87 ± 0.15b 2.57 ± 0.07a
met 79.46 ± 0.54 ab 81.01 ± 4.13b 74.80 ± 1.63a three treatment methods could be ranked as follows: traditional extru­
pro 969.49 ± 90.36a 987.06 ± 59.47a 1110.85 ± 109.38a sion > enzymatic extrusion > steam cooking. This was because the high
his 138.11 ± 1.58c 177.80 ± 7.51b 156.99 ± 2.27a shear action in extrusion played a dominant role in protein degradation.
arg 4.55 ± 0.21b 4.87 ± 0.52b 6.69 ± 0.58a However, in enzymatic extrusion, the lubrication of rapid starch
val 227.26 ± 4.63a 237.19 ± 8.99a 224.52 ± 4.72a
phe 203.13 ± 4.67b 219.98 ± 7.09a 212.10 ± 4.72 ab
degradation and liquefaction greatly alleviated the shear effect on the
ile 135.57 ± 6.33a 138.71 ± 4.43a 130.89 ± 3.74a protein degradation.
leu 298.13 ± 5.92b 317.86 ± 8.61a 307.15 ± 6.62 ab
lys 210.98 ± 6.77a 234.23 ± 9.07b 213.44 ± 2.85a 4. Conclusion
asp 491.83 ± 18.59a 602.78 ± 58.75b 468.68 ± 12.79a
glu 688.43 ± 16.03a 746.83 ± 36.97b 673.97 ± 18.85a
tyr 129.23 ± 25.63a 114.44 ± 11.15a 128.66 ± 8.57a In this study, the influence of three different thermal process
Total amino 4357.69 ± 4751.71 ± 127.31a 4559.20 ± 173.59 methods on the A. fortunei tuber were investigated. The non-starch
acids 60.64b ab components of the A. fortunei powder greatly influenced the viscosity
a
Values are the averages of three replicates, and different alphabets indicate of the samples and the DG, as measured by RVA and DSC, respectively.
statistical difference (p < 0.05). The SEM micrographs directly reflected the different starch results
under the action of heat, heat-shear and heat-shear-enzymolysis syner­
gism. Partial areas with porous structures were observed in the EES,
indicating starch degradation. The DG of the SCS (70.56%) was rela­
tively low, which is unfavorable for the saccharification of starch and
subsequent fermentation. A higher DG (84.45%) was found in the TES,
but it was accompanied by a higher WAI (3.44 g/g), leading to an
excessively dense fermentation broth, which is a disadvantage for the
diffusion of microorganisms in fermentation. However, enzymatic
extrusion greatly improved the situation. Due to the introduction of
α-amylase, the starch in the EES was almost completely gelatinized
(99.09%), as evidenced by the amorphous structure measured via XRD
and FTIR, which was beneficial to simultaneous saccharification and
fermentation. For the EES, partial starch was degraded in the extruder,
leading to a relatively low WAI (1.68 g/g) and an extremely high WSI
(61.66%). Thus, the EES fermentation broth had a fine fluidity.
The results of the final yellow wines indicated that enzymatic
extrusion significantly increased the FE (73.47%) and EY (31.27%) and
improved the quality of the wine, including increasing the solid soluble
content (7.35 g/100 g) and the level of total amino acids (4559.20 mg/
L) while decreasing the excessive total acid (10.66 g/L). This proves the
feasibility and potential of applying enzymatic extrusion to the pre­
treatment of A. fortunei tubers in special yellow wine fermentation.
Fig. 3. Measurement of total lost CO2 during the fermentation of A. fortunei
pretreated by steam cooking (SC), traditional extrusion (TE) and enzymatic CRediT authorship contribution statement
extrusion (EE). Values are the averages of three replicates.
Shichao Bian: Methodology, Writing – original draft. Enbo Xu:
Chinese National Standard GB/T 13662–2018 (SAC/TC 471, 2018). It Writing – review & editing. Xi Fu: Investigation. Zhengyu Jin: Super­
can also be seen in Fig. 3 that chief fermentation is the main stage of vision, Funding acquisition. Aiquan Jiao: Conceptualization, Re­
ethanol production, while ethanol content increases slowly during post sources, Funding acquisition.
fermentation.
Chinese yellow wine contains various small molecules with nutri­ Declaration of competing interest
ents, such as sugars, peptides, phenols, organic acids, etc. (Xu et al.,
2014). Thus, soluble solid content is also an important evaluation index We declare that we have no financial and personal relationships with
for yellow wine. The soluble solid content of the wine fermented with other people or organizations that can inappropriately influence our
the SCS was the lowest, at 7.05 g/100 g. Due to the thermal pretreatment work, there is no professional or other personal interest of any nature or
involving shear action and enzymolysis, the soluble solid content of the kind in any product, service and/or company that could be construed as
wine fermented with the TES and EES slightly increased, reaching 7.19 influencing the position presented in, or the review of the manuscript
g/100 g and 7.35 g/100 g, respectively, which can be attributed to the entitled “Comparison of different thermal treatments on the physico­
degradation of the raw material’s starch and protein. chemical properties of Apios fortunei used for yellow wine
As amino acids are the precursors to the synthesis of aldehydes, fermentation”.
ketoacids, esters and higher alcohols, which have a great influence on
sensory properties, the composition of amino acids play an important
role in yellow wine (Yu et al., 2015). The amino acid analyses of the final

7
S. Bian et al.
Acknowledgements
This study was supported by the National “Thirteenth Five-Year”
Plan for Science & Technology Support of China
(No.2016YFD0400304), Jiangsu Agricultural Science and Technology
Innovation Fund (CX(17)2022), the Science & Technology Pillar Pro­
gram of Jiangsu Province (BE2018304) and Jiangsu Key Laboratory of
Advanced Food Manufacturing Equipment & Technology (FM-201904).
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