Food Hydrocolloids 48 (2015) 189e196

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Food Hydrocolloids
journal homepage: www.elsevier.com/locate/foodhyd

The effect of starch concentration on the gelatinization and

liquefaction of corn starch
Zhaofeng Li a, b, c, Wenjing Liu b, Zhengbiao Gu a, b, c, *, Caiming Li b, Yan Hong a, b, c,
Li Cheng a, b
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
Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, Jiangsu 214122, China

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

Article history: Starch liquefaction is a key step in the industrial production of syrups, including those used in microbial
Received 12 July 2014 fermentation processes. As part of an effort to improve the efficiency of this process, the effects of starch
Received in revised form concentration on the gelatinization and liquefaction of corn starch were investigated. The results
2 February 2015
demonstrated that high starch concentrations in the slurry make it difficult to gelatinize the starch.
Accepted 23 February 2015
Available online 3 March 2015
Elevated starch concentrations inhibited swelling and disruption of starch granules and resulted in the
retention of starch crystallinity after heat treatment. It became very difficult to destroy the granular and
crystalline structure of the starch by heat treatment when the starch concentration exceeded 45%.
Keywords:
Corn starch
Furthermore, elevated starch concentration had negative effects on the liquefaction of corn starch
Starch concentration catalyzed by a thermostable a-amylase. The dextrose equivalent (DE) values of starch hydrolysates
Gelatinization decreased as the starch concentration increased. Nevertheless, increasing the starch concentration from
Liquefaction 30 to 45% caused only a slight reduction in the DE value of the hydrolysate, indicating that starch
DE value liquefaction at 45% starch could be comparable with that at 30%. Starch concentrations exceeding 45%
severely retarded starch liquefaction. The relatively low degree of starch liquefaction seen at high starch
concentrations, especially above 45%, was related to incomplete gelatinization of the starch, and low
mobility of the water molecules and polymeric chains in the reaction system. These results provide
additional insight into the feasibility of high-temperature liquefaction at high corn starch concentrations.
© 2015 Elsevier Ltd. All rights reserved.

1. Introduction generally involves gelatinization of the starch either before or

The enzymatic hydrolysis of starch is one of the most important
enzymatic reactions (Presecki, Blazevic, & Vasic-Racki, 2013).
Liquefaction is a key step in the starch hydrolysis process used to
produce syrups, including those used in the industrial production
of microbial fermentation products such as alcohol, organic acids,
amino acids, and antibiotics. Liquefaction is commonly achieved
through the dispersion of insoluble starch granules in an aqueous
solution, followed by partial hydrolysis at a relatively high tem-
perature using thermostable a-amylases, which are endogluca-
nases that catalyze the hydrolysis of internal a-1,4-glycosidic
linkages in starch (Presecki et al., 2013). Starch liquefaction
* Corresponding author. School of Food Science and Technology, Jiangnan Uni-
versity, Wuxi, Jiangsu 214122, China. Tel./fax: þ86 510 85329237.
E-mail address: zhengbiaogu@jiangnan.edu.cn (Z. Gu).
during the addition of the a-amylase, while gelatinization is
accompanied by swelling and disruption of the starch granules and
melting of their crystalline structure (Bogracheva, Morris, Ring, &
Hedley, 1998; Mandala & Bayas, 2004). Since native starch is hy-
drolyzed more slowly than gelatinized starch whose crystallinity
has been largely destroyed (Blazek & Gilbert, 2010), the degree of
starch liquefaction is closely related to starch gelatinization. The
other factors affecting starch liquefaction include the source and
concentration of the starch; the source and activity of the a-
amylase; the concentration of calcium ions, which are related to the
activation and stability of a-amylase; and other reaction conditions,
such as temperature and pH (Ariff, Asbi, Azudin, & Kennedy, 1997;
Baruque, Baruque, & Sant'Anna, 2000; Khedher, Bressollier, Urdaci,
Limam, & Marzouki, 2008; Presecki et al., 2013). Even though
substantial efforts have been devoted to the optimization of starch
liquefaction, such as use of high performance a-amylase, the
addition of calcium, optimization of process parameters, and starch

http://dx.doi.org/10.1016/j.foodhyd.2015.02.030
0268-005X/© 2015 Elsevier Ltd. All rights reserved.
190 Z. Li et al. / Food Hydrocolloids 48 (2015) 189e196
pretreatment (Nikoli
c, Mojovi
c, Rakin, Pejin, & Savi
c, 2008;
Presecki et al., 2013; Richardson et al., 2002), there is still a need
to improve this step, since the processes of syrup production and
fermentation can be made more efficient.
Native starch slurries derived from conventional corn wet
milling contain approximately 40% dry solids. They are generally
diluted to 25e35% dry solids before heating above the liquefaction
temperature (Bhargava, Frisner, & Johal, 2008; Lee, Shetty, &
Strohm, 2013). However, increased amounts of energy are
required to heat starch slurries of relatively low solid content and to
evaporate the excess water when concentrating the mash after
subsequent saccharification. In addition, efficient production of
fermentation products may require the culture medium to be
supplemented with an appropriate carbon source, which is often a
syrup containing a high concentration of fermentable saccharides
(above 40%). Furthermore, increasing the initial concentration of
starch slurry during liquefaction can yield higher productivity, and
higher enzyme stability (Baks, Kappen, Janssen, & Boom, 2008; de
Cordt, Hendrickx, Maesmans, & Tobback, 1994). Thus, it is desirable
to use more concentrated starch slurries.
Although the effects of the initial concentration on starch
liquefaction have been mentioned in previous reports (Baks et al.,
2008; Konsula & Liakopoulou-Kyriakides, 2004; Yankov, Dobreva,
Beschkov, & Emanuilova, 1986), the degree of liquefaction has not
been measured over a wide range of starch concentrations. In the
present study, the effects of starch concentration on the gelatini-
zation of corn starch were examined. Then, the high temperature
liquefaction of corn starch slurries was investigated over a wide
concentration range (10e60%, w/w, dry basis), with the expectation
that increasing the initial concentration of the starch slurry would
enhance hydrolysate productivity. The relationship between starch
gelatinization and liquefaction was also analyzed. The results pro-
vide additional insight into the feasibility for enzymatic liquefac-
tion of corn starch at high concentrations.
2. Materials and methods
2.1. Materials
Corn starch was obtained from Zhucheng Xingmao Corn
Developing Co., Ltd (Shandong, China). The thermostable a-
amylase from Bacillus subtilis was obtained from Genencor Inter-
national (18,100 U/mL; Palo Alto, CA, USA). Blue dextran, which was
prepared from dextran with an average molecular weight of
approximate 2.0  106 g/mol, was purchased from Sigma (St. Louis,
MO, USA). Soluble starch, dinitrosalicylic acid (DNS), hydrochloric
acid (HCl), sodium hydroxide, acetic acid, sodium acetate, iodine
(I2), and potassium iodide (KI) were purchased from Sinopharm
Chemical Reagent Co. (Shanghai, China).
2.2. Determination of the swelling factor
The swelling factor of the starch was measured using the blue
dextran dye exclusion method of Tester and Morrison (Tester &
Morrison, 1990), with some modifications. Slurries containing
10e60% (w/w, dry basis) starch were incubated at 90  C for 1 h.
After cooling, the samples originally containing 100 mg of dry
starch were weighed into 10-ml screw-cap tubes, distilled water
was added to a total of 5 mL, 0.5 mL of blue dextran solution (5 mg/
mL) was added, and then the contents were mixed by gently
inverting the closed tubes several times. After centrifugation at
5000  g for 5 min, the absorbance of the supernatant was
measured at 620 nm. The absorbance of reference tubes that con-
tained no starch was also measured. The swelling factor, reported
as a ratio of the volume of swollen granules to the volume of dry
starch, was calculated according to the method shown by Tester
and Morrison (Tester & Morrison, 1990).
2.3. Scanning electron microscopy analysis
Slurries containing 10e60% (w/w, dry basis) starch were pre-
pared for scanning electron microscopy (SEM) by incubation at
90  C for 1 h, cooling, and then freeze-drying. The freeze-dried
samples were coated with goldepalladium by using a sputter
coater (Denton Vacuum, LLC, Moorestown, NJ), and then viewed at
2000  resolution with a scanning electron microscope (S-3800N,
Hitachi Science Systems, Ltd., Japan) operating at an accelerating
voltage of 20 kV.
2.4. Light microscopy analysis
Slurries containing 10e60% (w/w, dry basis) starch were incu-
bated at 90  C for 1 h. After cooling, the diluted samples were
promptly viewed using an optical microscope (Model BX51,
Olympus Co., Japan) fitted with a polarized light filter and a digital
camera. Observations were conducted under cross-polarized light
using a 40 objective.
2.5. X-ray diffraction analysis
Slurries containing 10e60% (w/w, dry basis) starch were incu-
bated at 90  C for 1 h. After cooling, the samples were freeze-dried
and pulverized to a powder. X-ray diffractograms of these starch
powders were obtained with a copper anode X-ray tube using a
Rigaku D-Max- 2200 X-ray diffractometer (Rigaku Denki Co. Tokyo,
Japan). X-ray diffraction patterns were acquired at room tempera-
ture over the 2q range of 4e36 with a step size of 0.02 . Crystal-
linity was quantified by integrating the area under the fitted
crystalline peaks using MDI Jade 5.0 software.
2.6. Differential scanning calorimetry (DSC) analysis
The thermal properties of slurries containing 10e60% (w/w, dry
basis) starch were determined from DSC curves obtained using a
Pyris 1-DSC (PerkinElmer Corp., Norwalk, CT, USA). These samples
(approximately 6 mg) were accurately weighed and sealed in
aluminum DSC pans, and then equilibrated at 4  C for 24 h. This
equilibration resulted in an even distribution of water without
microbial fermentation, making the starch slurries more uniform.
Scans were performed from 30  C to 90  C at a constant rate of
10  C/min. Enthalpy changes (DH) were evaluated based on the area
of the main endothermic peak and expressed in terms of J/g of dry
starch. A sealed, empty crucible was used as a reference. All mea-
surements were performed in triplicate.
2.7. Determination of water molecule mobility
The measurement of spinespin relaxation time (T2) was per-
formed using an MQC-23 nuclear magnetic resonance (NMR)
spectrometer (Oxford Instruments LTD., England) operating at
25  C and a resonance frequency of 20.9 MHz. Slurries containing
10e60% (w/w, dry basis) starch were incubated at 90  C for 1 h.
After cooling to 25  C, the samples (3 g) were poured into a 10 mm
diameter NMR tube that was then sealed and immediately trans-
ferred to the NMR probe. The T2 values of water protons were
measured using the Carr-Purcell-Meiboom-Gill (CPMG) pulse
sequence. The pulse gap between the 90 and 180 pulses was
200 ms. Eight scans, each containing 1024 echoes, were averaged to
obtain the final signal. All measurements were performed in trip-
licate. Relaxation curves were obtained by fitting the data to the
 Z. Li et al. / Food Hydrocolloids 48 (2015) 189e196
exponential model described in Eq. (1), where T2 is spinespin
relaxation time and A0 is the relative measure of the amount of
water fractions corresponding to T2.


t
T2
A ¼ A0 e
Fitting was accomplished using 1st Opt software, version 1.5
(7D-Soft High Technology Inc., Beijing, China).
2.8. Liquefaction process of starch
Starch liquefaction was performed in the sample cup of a Bra-
bender Viscoamylograph (C.W. Brabender Instruments, Inc., South
Hackensack, NJ). Four hundred grams of slurries containing 10e60%
(w/w, dry basis) starch were prepared in distilled water, and
adjusted to pH 6.0. The temperature of the starch slurry was
increased to 60  C at a rate of 3.0  C/min, and then a thermostable
a-amylase (12 U/g of dry starch) was added to the reaction mixture.
Subsequently, the temperature was increased to 90  C at a rate of
1.0  C/min. Liquefaction was continued for 2 h at a constant
agitation speed of 200 rpm. At different time intervals, 5 mL of
starch hydrolysate was withdrawn for analysis. Liquefaction was
immediately stopped by chilling to 50  C followed by acidification
at pH 3.5 with 0.1 N HCl.
2.9. a-Amylase activity assay
Soluble starch (0.4 g) was dissolved in 25 mL of 50 mM sodium
acetate buffer (pH 6.0). The temperature of starch solution was
adjusted to 70  C in a water bath, 1 mL of appropriately diluted
enzyme solution was added, and the mixture was incubated at
70  C for 5 min. Then, 1 mL of reaction mixture was added to a
mixture containing 5 mL of iodine reagent (0.01% I2 in 4% KI) and
0.5 mL of 0.1 M HCl, which stopped the reaction. A complete assay
mixture without enzyme was used as a control. The degradation of
starch by the enzyme was determined by comparing the absor-
bance of the sample or the control at 660 nm with the absorbance
of a blank prepared by treating 1 mL of water with the same iodine/
HCl mixture. One unit of a-amylase activity was defined as the
amount of enzyme that caused a 10% reduction in the
starcheiodine color under the assay conditions.
2.10. Dextrose equivalent (DE) of the starch hydrolysate
The reducing sugar content of the starch hydrolysate was
determined using the DNS method (Bruner, 1964). The degree of
liquefaction is expressed as dextrose equivalent (DE), which is
defined as the total reducing sugars expressed as dextrose and
calculated as a percentage of the dry substance.
2.11. Changes in viscosity during starch liquefaction
During starch liquefaction, a Brabender Viscoamylograph (C.W.
Brabender Instruments, Inc., South Hackensack, NJ) was used to
determine the change in viscosity as a function of time.
2.12. Statistical analysis
Experimental results about swelling factor, T2 value, DE value,
and enthalpy are reported as the average of triplicate measure-
ments, along with their associated standard deviations. Differences
between treatments were analyzed for statistical significance using
the StudenteNewmaneKeuls (SNK) procedure, as implemented in
the statistical software package from SPSS Inc. (Chicago, IL, USA).
191
Differences resulting in values of p < 0.05 were considered statis-
tically significant.
3. Results and discussion
(1) 3.1. Effects of starch concentration on the swelling and disruption of
starch granules
3.1.1. Swelling factor analysis
Starch granules are generally insoluble in cold water. When
starch is heated in water, the starch granules absorb water and
swell (Ratnayake & Jackson, 2006). The extent of granule swelling
can be determined by measuring the swelling factor (Contreras

pez, Role
Lo
e, & Le Meste, 2004). After slurries containing
different concentrations of corn starch (no enzyme added) were
incubated at 90  C for 1 h, the swelling factors of the starch were
analyzed. As shown in Fig. 1, the swelling factors decreased grad-
ually with increasing starch concentration, suggesting that the
starch concentration had an inhibitory effect on granule swelling.
When the slurry contained 10% starch, the swelling factor reached
approximately 15, which was about 12% lower than that for the
slurry containing 2% (v/w, dry basis) starch (Li, Cai, Gu, & Shi, 2014).
Increasing the starch concentration from 10% to 45% resulted in a
decrease of approximately 70% in the swelling factor. When the
slurries reached starch concentrations of 50e60%, the swelling
factors were very low, indicating very little swelling of starch
granule during heat treatment.
3.1.2. SEM analysis
When heated in water, starch undergoes a transition process
during which the granules break down (Ratnayake & Jackson,
2009). Thus, heat treatment of a starch slurry is associated with
the disruption of granular structure, causing starch molecules to
dissolve in water. After slurries containing different concentrations
of starch were incubated at 90  C for 1 h, freeze-dried starch
samples were observed by SEM. As shown in Fig. 2, when the starch
concentration was 10e20%, heat treatment led to complete granule
disruption. Although increasing the starch concentration to
30e45% resulted in a viscous starch paste, almost no intact starch
granules appeared in the SEM images, indicating that most of the
starch granules were damaged by heat treatment to a certain
extent. A few intact starch granules could be observed by SEM
when the starch concentration reached 50e55%, indicating
incomplete granule disruption by heat treatment. When the slurry
Fig. 1. The effect of starch concentration on the swelling factor. Each value represents
the mean of three independent measurements. Bars with different letters on the top
are significantly different (p < 0.05).
192 Z. Li et al. / Food Hydrocolloids 48 (2015) 189e196

Fig. 2. SEM images of samples containing different concentrations of corn starch, after heat treatment at 90  C for 1 h, and then freeze drying. Magnification, 500.

containing 60% starch was analyzed, most of the starch granules
remained intact.
Continued heating generally causes starch granules to swell and
be disrupted to a greater extent. However, the starch concentration
had a substantial effect on the swelling and disruption of starch
granules, indicating varying degrees of starch gelatinization. The
increase in starch concentration, particularly to concentrations
above 45%, resulted in reduced extents of granule swelling and
disruption. It has been shown that starch granules are quite resis-
tant to enzymatic hydrolysis and are predominantly hydrolyzed by
surface abrasion (Asare et al., 2011; Siswoyo & Morita, 2003;
Srichuwong, Isono, Mishima, & Hisamatsu, 2005). Granule
swelling and disruption could greatly increase the susceptibility of
starch molecules within the granule to cleavage by amylase,
resulting in the generation of amorphous material within the starch
granule and an associated increase in granule porosity (Wang &
Copeland, 2013). Thus, the inhibited swelling and disruption of
starch granules, as characteristics of poor starch gelatinization,
would have negative effect on starch liquefaction.
3.2. Effects of starch concentration on the crystalline structure of
starch granules after heat treatment
3.2.1. Light microscopy analysis
Corn starch molecules are densely packed in starch granules,
resulting in a semi-crystalline state that gives rise to birefringence
 Z. Li et al. / Food Hydrocolloids 48 (2015) 189e196 193

under cross-polarized light (Chen, Huang, Tang, Chen, & Zhang,
2011). When starch is heated in water, the absorption of water
into the granules can destabilize their crystalline structure,
resulting in the loss of birefringence (Parker & Ring, 2001;
Ratnayake & Jackson, 2006). Therefore, a loss of birefringence is a
good indicator of the destruction of the crystalline structure
of starch granules. Polarized light micrographs of diluted
starch samples, obtained after slurries containing different starch
concentrations were incubated at 90  C for 1 h, are shown in Fig. 3.
The results demonstrated that the loss of birefringence was
controlled by the availability of water. Heat treatment of slurries
containing 10e20% starch led to the complete loss of starch granule
birefringence. When slurries containing 30e45% starch were heated,
almost no birefringence was observed in polarizing microscope
images, indicating that the crystalline structure of most starch
granules were destroyed, to some extent, by the heat treatment. As
slurries containing even higher concentrations of starch were heat-
ed, many starch granules retained their birefringence, indicating that
heat treatment could not completely destroy the crystalline struc-
ture of the starch granules present at high starch concentrations.
3.2.2. X-ray diffraction analysis
The crystalline properties of starch granules can be studied
using X-ray diffraction (Eliasson, 2006; Ratnayake & Jackson,

Fig. 3. Photomicrographs of samples containing different concentrations of corn starch, after heat treatment at 90  C for 1 h. Images were obtained at 400 using a polarizing
microscope.
194 Z. Li et al. / Food Hydrocolloids 48 (2015) 189e196
2008). The crystalline structures of starches can be classified into
three forms (A, B and C) on the basis of their X-ray diffraction
spectra (Cheetham & Tao, 1998). The X-ray diffraction patterns of
freeze-dried starch samples obtained after slurries containing
different concentrations of starch were incubated at 90  C for 1 h
are shown in Fig. 4. Native corn starch showed the strongest
diffraction peaks, at 2q values of 15.3 , 17.1, 18.2 and 23.5
(Fig. 4a), indicating that this starch had a characteristic A-type
crystalline form, which is consistent with previous reports (Koo,
Lee, & Lee, 2010; Sun, Zhao, Zeng, Li, & Li, 2010; Uthumporn,
Zaidul, & Karim, 2010). Compared with those of native starch, the
characteristic diffraction peaks of heat-treated starch decreased in
intensity, or disappeared (Fig. 4bei). During heating in excess
water, individual crystalline regions within starch molecules can
melt within a specific narrow temperature range (Jackson, 2003).
Heat treatment of a slurry containing 10% starch led to a complete
loss of characteristic diffraction peaks (Fig. 4i), indicating that the
heated and freeze-dried starch sample had almost no crystallinity.
When the slurries contained 20e45% starch, some of the charac-
teristic diffraction peaks were combined (Fig. 4eeh), indicating
that the heated and freeze-dried starch samples had relatively low
crystallinity. As slurries containing higher starch concentrations
were heated, the starch samples displayed A-type X-ray diffraction
patterns with different amounts of decrease in the intensity of the
characteristic diffraction peaks. These starch samples had a rela-
tively high degree of crystallinity. When the slurries contained 55%
or 60% starch, the relative crystallinities of starch samples reached
16.5% and 18.3%, respectively, which were comparable to that of
native corn starch (21.2%) Thus, the results obtained using X-ray
diffraction are largely consistent with those obtained using light
microscopy.
3.2.3. DSC analysis
The thermal properties of slurries containing different concen-
trations of starch were analyzed using DSC. The DSC enthalpy
represents the net sum of all endothermic processes that take place
during heating (Ratnayake, Otani, & Jackson, 2009). As shown in
Table 1, the enthalpy change upon starch gelatinization decreased
as the starch concentration increased. This resulted in an increase
in residual enthalpy, which is defined as the difference between the
enthalpic value for the restricted water sample and that for the high
moisture condition when gelatinization is complete. For slurries
containing high concentrations of starch, the residual enthalpy
existed because the starches contained too little moisture to permit
Fig. 4. X-ray diffraction patterns of samples containing different concentrations of
corn starch, after heat treatment at 90  C for 1 h a: native starch, b: 60%, c: 55%, d: 50%,
e: 45%, f: 40%, g: 30%, h: 20%, i: 10%.
gelatinization (Tester & Sommerville, 2001). Thus, the retention of
starch crystallinity can be evaluated by the residual enthalpy after
heat treatment (Tester & Sommerville, 2001). It is apparent that the
slurries containing higher concentrations of starch retained greater
crystallinity after heat-treatment. When the slurry contained >45%
starch, the retention of crystallinity reached >60%, suggesting that
more than half of the crystalline starch could not be destroyed by
heat treatment.
Starch gelatinization can be reflected in the melting of starch
crystallites (Zobel, Young, & Rocca, 1988). Increasing the starch con-
centration, especially to concentrations above 45%, severely reduced
the loss of crystallinity, suggesting incomplete gelatinization of the
starch. Previous reports have also shown that the crystalline lamellae
in starch granules were resistant to enzymatic erosion, and that
crystalline starch was much less susceptible to enzymatic hydrolysis
than amorphous material (Oates, 1997; Oates & Powell, 1996). Thus,
the retention of crystallinity retards starch liquefaction. In addition,
heat not only melts crystallite structures during gelatinization, but
also facilitates new molecular rearrangements or formation of new
bonds among molecules before gelatinization (Ratnayake & Jackson,
2007). As a result, incomplete gelatinization of starch at high con-
centrations might be accompanied by molecular rearrangements,
which are also unfavorable for starch liquefaction.
3.3. Effect of starch concentration on the mobility of water
molecules
Water molecules play an important role in the gelatinization of
starch. NMR spectroscopic techniques can be used to probe the
mobility of water molecules in aqueous solution. Constant spine-
spin relaxation times (T2s) are closely related to the mobility of
water molecules (Baranowska, Sikora, Kowalski, & Tomasik, 2008;
Ritota, Gianferri, Bucci, & Brosio, 2008; Wang, Gu, Li, Hong, &
Cheng, 2013). In general, aqueous solutions with higher T2 values
contain higher fractions of mobile water molecules.
To investigate the effect of starch concentration on the mobility
of water molecules, the slurries containing different concentrations
of corn starch were incubated at 90  C for 1 h, and then the T2
values of these starch pastes were determined. As shown in Fig. 5,
with the increase in starch concentration, the T2 values decreased
gradually, suggesting that the pastes with a high starch concen-
tration had lower water molecule mobilities. An increase in starch
concentration from 10% to 45% resulted in a >90% decrease in the T2
value of the starch paste. Although further increases in starch
concentration did not cause a noticeable decrease, the T2 values of
starch pastes at 50e60% were very low (<30 ms), indicating very
poor mobility of water molecules in the pastes.
Table 1
Thermal properties of slurries containing different concentrations of corn starch.
Starch concentration Enthalpy (J/g)c Residual enthalpyb
(%, w/w, dry basis)
(J/g) (%)
10 a e e
20 12.2 ± 0.5g 0 0
30 10.6 ± 0.4f 1.6 13.1
40 8.6 ± 0.3e 3.6 29.5
45 6.9 ± 0.3d 5.3 43.4
50 4.5 ± 0.2c 7.7 63.1
55 2.3 ± 0.1b 9.9 81.1
60 1.2 ± 0.1a 11.0 90.2
a
Not determined, primarily because of excessive moisture content.
b
Calculated using the difference in enthalpy between the endotherm at 20% and
other starch concentrations.
c
Values are means ± SD (n ¼ 3). Means with different online letters within the
same column are significantly different (p < 0.05).
 Z. Li et al. / Food Hydrocolloids 48 (2015) 189e196
Fig. 5. Dependence of the spinespin T2 value on the concentration of corn starch. Each
value represents the mean of three independent measurements. Bars with different
letters on the top are significantly different (p < 0.05).
Previous reports have shown that the rates of enzymatic re-
actions are closely related to diffusion in high viscosity aqueous
solutions (Cicerone & Soles, 2004; Neri, Pittia, Bertolo, Torreggiani,
& Sacchetti, 2010). However, diffusion would be impeded by rela-
tive low mobility of water molecules in the reaction system. Thus,
an increase in starch concentration would reduce the effective
diffusion of enzyme and substrate, which could negatively affect
the liquefaction of starch catalyzed by a thermostable a-amylase.
3.4. Effect of starch concentration on the DE values of starch
hydrolysate
During liquefaction, starch is converted to shorter chains and
less viscous dextrins (Henderson & Doyal, 2011). In this study, corn
starch was liquefied by the thermostable a-amylase at 12 U/g of dry
starch. As shown in Fig. 6, the DE values of starch hydrolysates
increased gradually with liquefaction time. When viewed at the
same liquefaction time, slurries with higher initial starch concen-
trations had lower DE values. As the starch concentration increased
from 10% to 30%, the degree of starch liquefaction decreased
noticeably. After 2 h of liquefaction, slurries with starch concen-
trations of 20% or 30% yielded starch hydrolysates with DE values 10
or 20% lower, respectively, than that seen in a hydrolysate produced
Fig. 6. The effect of corn starch concentration on the DE values of starch hydrolysate
during liquefaction. Each value represents the mean of three independent measure-
ments. Deviations from the mean were below 5% for all values displayed.
195
from a slurry with starch concentration of 10%. By comparison,
increasing the starch concentration from 30 to 45% caused an
approximately 7% reduction in the DE values of starch hydrolysate
after 2 h of liquefaction, indicating that starch liquefaction at an
initial concentration of 45% was comparable with that at 30%.
However, further increases in the initial concentration resulted in
much lower degrees of starch liquefaction. The DE values of starch
hydrolysates obtained by 2 h liquefaction of slurries containing 50
or 55% starch were approximately 25% or 45% lower, respectively,
than that of the hydrolysate produced from a slurry with starch
concentration of 45%. When the starch concentration reached 60%,
the starch paste could not be easily stirred during gelatinization,
resulting in a very low DE value of the starch hydrolysate after 2 h of
liquefaction (data not shown). Taken as a whole, these results
indicate that starch slurries that are less well liquefied correspond
to those that were less well gelatinizated by heat treatment and had
lower water molecule mobilities.
3.5. Effect of starch concentration on the viscosity profiles of starch
hydrolysate
During starch liquefaction, the viscosity of the starch paste
initially increased rapidly, primarily due to starch gelatinization
and shear-thickening, which results from the break-up of highly
concentrated gel-like starch clusters and resultant increase in the
effective starch concentration (Kim, Willett, Carriere, & Felker,
2002). Despite the relatively low degree of gelatinization, slurries
containing 40e50% starch might exhibit obvious shear-thickening
behaviors at a rapid agitation speed (200 rpm). The viscosity then
decreased with the liquefaction time, primarily due to the degra-
dation of starch molecules by the thermostable a-amylase. As
shown in Fig. 7, different initial starch concentrations in the slurry
resulted in distinct viscosity profiles. The higher the initial starch
concentration, the higher the viscosity of the starch pastes. If the
starch concentration increased from 10% to 40%, the peak viscosity
of the starch paste during liquefaction increased significantly.
When the starch concentration was controlled at 45% or 50%, the
Brabender viscoamylograph did not produce smooth viscosity
curves during liquefaction, probably because very high viscosity led
to difficulty in stirring the starch paste. A further increase in starch
concentration, to above 50%, resulted in a completely irregular
viscosity curve during gelatinization and liquefaction (data not
shown). When the starch concentration is 60%, a slurry with a very
low degree of gelatinization might exhibit a solid-like behavior
(Crawford et al., 2013).
Fig. 7. Changes in the viscosity profile during liquefaction of slurries containing
different starch concentrations. a: 10%; b: 20%; c, 30%; d, 40%; e, 45%; f, 50%.
196 Z. Li et al. / Food Hydrocolloids 48 (2015) 189e196
4. Conclusions
The starch concentration in the slurry had a substantial effect on
starch gelatinization. High starch concentrations inhibited swelling
and disruption of starch granules and caused retention of starch
crystallinity after heat treatment, to varying degrees. When the
starch concentration was above 45%, it was very difficult to destroy
the granule and crystalline structure of starch using heat treatment.
The starch concentration also substantially influenced the high-
temperature liquefaction of corn starch catalyzed by a thermo-
stable a-amylase. The relatively low degree of liquefaction seen at
high starch concentrations was intimately related to the incom-
plete gelatinization of the starch and low mobility of water mole-
cules in the reaction system. To improve the liquefaction of slurries
containing high concentrations of starch, it will be necessary to
increase the mobility of water molecules, to swell or damage starch
granules, and to destroy starch crystallinity by pretreatment.
Acknowledgments
This work was financially supported by the Twelfth Five-Year
National Key Technology Research and Development Program of
the Ministry of Science and Technology of China (No.
2012BAD34B07), and the National Natural Science Foundation of
China (No. 31371787).
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