Food Hydrocolloids 23 (2009) 1465–1472
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Food Hydrocolloids
journal homepage: www.elsevier.com/locate/foodhyd
Impacts of acid-methanol treatment and annealing on the enzymatic
resistance of corn starches
Jheng-Hua Lin a, Shu-Wen Wang b, Yung-Ho Chang b, *
a
b
Department of Hospitality Management, MingDao University, 369 Wen-Hwa Road, Peetow 52345, Taiwan
Department of Food and Nutrition, Providence University, 200 Chung-Chi Road, Shalu 43301, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 13 June 2008
Accepted 4 August 2008
Normal corn, Hylon V and Hylon VII starches were acid-methanol-treated at 25 C for 1–30 days in
methanol containing 0.36% HCl, and then annealing at 50 C for 72 h in excess water. The rapid digestible
starch (RDS), slow digestible starch (SDS) and resistant starch (RS) contents of starch before and after
treatments were determined. The molecular structure, thermal properties, double helix content and
relative crystallinity of starch were observed for elucidating the impacts of acid-methanol treatment and
annealing, as well as the molecular structure, on the enzymatic resistance of starch. Results showed that
the weight-average degree of polymerization of acid-methanol-treated corn starches ranged from
884 103 to 404, 778 103 to 299 and 337 103 to 250 anhydrous glucose units for normal corn, Hylon
V and Hylon VII starches, respectively. Annealing increased the RS content of starch, and the increment of
RS increased with decreasing molecular size of starch. Furthermore, the change in RS content after
treatments depended on the content and weight-average chain length of amylose fraction of starch. The
RS content of starch after treatments increased from 19.2 to 56.2%, 69.9 to 86.1%, and 73.1 to 89.1% for
normal corn, Hylon V and Hylon VII starches, respectively. The gelatinization peak temperature and
double helix content of starch increased after acid-methanol treatment or annealing. Results demonstrate that the degradation of starch, causing by acid-methanol treatment, enhances the mobility and
realignment of starch chains in molecules during treatments and further increases the enzymatic
resistance of starch granules.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Enzymatic resistance
Resistant starch
Molecular structure
Acid-methanol treatment
Annealing
1. Introduction
Resistant starch (RS) is defined as the sum of starch and
products of starch degradation not absorbed in the small intestine
of healthy individuals (Englyst, Kingman, & Cummings, 1992), and
has been classified into different types. They are type I, resulting
from physical inaccessibility in intact tissues or particulate
materials; type II, resulting from the physical structure of the raw
starch granules; type III, resulting from the physical structure of
associated starch molecules after cooking; and type IV, resulting
from chemical modification that interferes with the enzyme
digestion (Eerlingen & Delcour, 1995). RS is recognized having
a physiology effect similar to that of dietary fiber (Biliaderis, 1991;
Brown, 1996); therefore, the nutritional implication and the
impact of processing treatments on RS content in starchy foods
have gained the attention of food technologists (Brumovsky &
Thompson, 2001).
* Corresponding author. Tel.: þ886 4 2632 8001x15302; fax: þ886 4 2653 0027.
E-mail address: [email protected] (Y.-H. Chang).
0268-005X/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodhyd.2008.08.001
Annealing is a physical treatment on starch granules in presence
of heat, temperature between the glass transition temperature
and the gelatinization onset temperature (To), and excess water,
generally higher than 60% w/w (Tester, Debon, & Sommerville,
2000). During annealing, the increasing in molecular mobility of
starch within the amorphous regions leads to a molecular reorganization (Atichokudomchai, Varavinit, & Chinachoti, 2002; Hoover
& Vasanthan, 1994; Jacobs, Eerlingen, Rouseu, Colonna, & Delcour,
1998; Seow & Vasanti-Nair, 1994; Shi, Capitani, Trzasko, & Jeffcoat,
1998; Tester et al., 2000). The mobility of amylose chains also
increases during annealing and results in the formation of double
helices from interactions between amylose–amylose and/or
amylose–amylopectin (Hoover & Vasanthan, 1994; Jacobs et al.,
1998). The physically reorganization causes a more perfect crystalline of starch granules (Tester & Debon, 2000), consequently the
To of starch increases and the gelatinization temperature range (Tr)
decreases (Jacobs & Delcour, 1998; Tester & Debon, 2000; Tester,
Debon, & Karkalas, 1998). The changes in Tr of starch during
annealing could result from lengthening of the double helices,
which are not optimized during biosynthesis (Genkina, Wasserman,
& Yuryev, 2004; Qi, Tester, Snape, & Ansell, 2005; Tester et al., 1998,
2000).
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J.-H. Lin et al. / Food Hydrocolloids 23 (2009) 1465–1472
In an acid-alcohol treatment, starch granules are acid-treated in
alcohols. The glycosidic linkages of starch, especially those in
amorphous regions, are acid-hydrolyzed during treatment (Lin, Lee,
& Chang, 2003; Lin, Lii, & Chang, 2005). The molecular weight and
chain length of starch molecules decrease after treated (Lin &
Chang, 2006; Lin et al., 2003, 2005; Ma & Robyt, 1987), but the
recovery of starch granules after acid-treated in alcohols can be
higher than 90% (Chang, Lin, & Chang, 2006; Fox & Robyt, 1992; Lin
et al., 2003, 2005; Ma & Robyt, 1987). Acid-alcohol treatment shows
a high feasibility in controlling the molecular degradation of starch
(Fox & Robyt, 1992; Lin & Chang, 2006). Starch granules with widely
different molecular sizes, degree of polymerization ranging from
106 to 102 anhydrous glucose unit (AGU), can be prepared by using
various conditions of acid-alcohol treatment, such as reaction
time, temperature, acid concentration, alcohol type and
alcohol concentration (Chang et al., 2006; Chang, Lin, & Lii, 2004;
Fox & Robyt, 1992; Lin et al., 2003, 2005; Robyt, Choe, Hahn, &
Fuchs, 1996).
Results of previous studies (Chang & Lin, 2007; Lin, Wang, &
Chang, 2008) indicate that degradation within starch granules
during acid-methanol treatment enhances the retrogradation of
gelatinized starch stored at 4 C, and increases the gelatinization
onset and peak (Tp) temperatures of starch after annealing. This
implies that the degradation of starch granules could improve the
mobility of starch chains during treatments, such as annealing.
Brumovsky and Thompson (2001) proposed that annealing could
be used to enhance the resistant starch level of starch by perfecting
its granular or molecular structures. The alternation in granular or
molecular structure of starch could further affect the enzymatic
resistance of the starch. However, no study has concerned the effect
of acid-alcohol treatment on the enzymatic resistance of starch yet,
and investigation on the difference in the enzymatic resistance of
starch with various molecular sizes is very rare.
The purpose of this study is to deliberate the impacts of acidmethanol treatment and annealing on the enzymatic resistance of
starch. Normal corn, Hylon V and Hylon VII starches were treated in
methanol containing 0.36% HCl at 25 C for 1–30 days, and the
prepared starches with different molecular sizes were annealed at
50 C for 72 h. The molecular size and chain length distribution of
native and acid-methanol-treated starches were determined, and
the enzymatic resistance, thermal properties, double helix content
and crystallinity of starch granules before and after annealing were
examined. Then the effect of molecular structure on the enzymatic
resistance of starch after treated with acid-methanol treatment and
annealing was investigated.
2. Materials and methods
2.1. Materials
Normal corn starch was obtained from Roquette Company
(Lestrem, France); Hylon V and Hylon VII starches were products of
National Starch and Chemical Company (Bridgewater, NJ).
Amyloglucosidase (EC 3.2.1.3, Cat. No. A-9913) and pancreatin (Cat.
No. P-7545) were obtained from Sigma-Aldrich (St. Louis, MO).
Isoamylase (EC 3.2.1.68) of Pseudomonas amyloderamosa (59,000 IU/
mg) was purchased from Hayashibara Biochemical Laboratories,
Inc. (Tokyo, Japan). All reagents used were of analytical grade.
2.2. Methods
2.2.1. Preparation of acid-methanol-treated starch
Acid-methanol treatment on starch was performed following
procedures in Lin et al. (2003). Starch (25 g) was suspended in
100 mL methanol. The suspension was stirred at 25 C and the
reaction was started by adding 1 mL of concentrated HCl (36%, w/v),
then was allowed to proceed for 1–30 days. The reaction was
stopped by adding 14 mL of 1 M NaHCO3 solution and cooled in an
ice-bath for 5 min. The treated starch was centrifuged at 3500g
for 5 min, washed four times with 50% ethanol, and air-oven dried
at 40 C.
2.2.2. Starch annealing
Annealing of starch was performed using the method described
by Lin et al. (2008). Starch granules (2 g, dry weight) were annealed
in excess water (48 g) at 50 C for 72 h. After annealing, starch was
recovered by centrifugation (3500g, 5 min), washed with cold
water and acetone, and air equilibrated at room temperature.
2.2.3. Average molecular weight
The weight-average degree of polymerization (DPw) of starch
was determined by high-performance size-exclusion chromatography (HPSEC) (Lin et al., 2003). The solution of starch was prepared
by precipitating from an aliquot of starch–DMSO solution (2.1 mL,
5 mg starch per mL DMSO) with excess absolute ethyl alcohol and
centrifuged at 4000g for 10 min. The precipitated amorphous
starch pellet was dissolved in de-ionized water (15 mL, 95 C) and
stirred with magnetic stirrer in boiling water bath for 30 min. The
starch solution was filtered through a 5.0 mm syringe filter (Millipore, Billerica, MA), and then the filtrate (100 mL) was injected into
an HPSEC system. The system consisted of an isocratic pump
(G1310A series, Hewlett Packard, Wilmington, DE), a multi-angle
laser light scattering (MALLS, model Dawn DSP, Wyatt Tech. Co.,
Santa Barbara, CA) and a refractive index (RI) detectors (HP1047A).
The columns used were PWH (guard column), G5000PW and
G4000PW (TSK-Gel, Tosoh, Tokyo, Japan) columns connected in
series and kept at 70 C. The mobile phase was 100 mM NaNO3
containing 0.02% NaN3 at a flow rate of 0.5 mL/min.
2.2.4. Chain length distribution
The chain length distribution of isoamylase-debranched starch
was also measured by HPSEC (Lin et al., 2003). The solution of
starch after debranched by isoamylase was injected (100 mL) into
the HPSEC system used for the determination of molecular weight,
except the columns used were one G3000PWXL and two
G2500PWXL (TSK-Gel, Tosoh) connected in series. The mobile
phase was 100 mM phosphate buffer (pH 6.2) containing 0.02%
NaN3 at a flow rate of 0.5 mL/min.
2.2.5. Enzymatic resistance
Enzymatic resistance of starch was determined using the
method of Englyst et al. (1992). Starch sample was controllingly
enzymatic-hydrolyzed by using of pancreatin and amyloglucosidase at 37 C up to 120 min. For evaluating the enzymatic resistance
of starch granules, the rapid digestible starch (RDS), slow digestible
starch (SDS) and resistant starch (RS) contents were obtained by
measuring the released glucose units using a glucose oxidase–
peroxidase assay. RDS was measured after incubation with
enzymatic hydrolysis for 20 min and RS is the starch not being
hydrolyzed after 120 min incubation. SDS is the starch being
hydrolyzed within 20–120 min incubation, which was calculated
from total starch content minus RDS and RS values.
2.2.6. Gelatinization thermal properties
Gelatinization thermal properties of starch were determined by
using of a differential scanning calorimeter (DSC 2910, TA Instruments, Surrey, England). Starch (about 2.5 mg, dry basis) was
weighed in the sample pan, mixed with distilled water (about
7.5 mg), and sealed. The samples were heated from 25 to 120 C at
a heating rate of 10 C/min. The gelatinization onset (To), peak (Tp)
and conclusion (Tc) temperatures together with the enthalpy
change of gelatinization (DH) were quantified.
J.-H. Lin et al. / Food Hydrocolloids 23 (2009) 1465–1472
1467
2.2.7. 13C NMR spectra
The 13C NMR spectra of the native and the acid-methanol-treated
starches before and after annealing were examined following
procedures described by Lin et al. (2008). The water content of
starch was adjusted over BaCl2 saturated salt solution (aw ¼ 0.90) at
25 C for 2 weeks. The CP-MAS 13C NMR spectra were recorded on
a Bruker DSX-400 spectrometer (Karlsruhe, Germany) equipped
with CP/MAS (cross-polarization magic angle spinning) accessories.
Dipolar decoupling was systematically used during the acquisition
sequence. The samples were spun at a rate of 7.5 kHz at room
temperature in a 4 mm ZrO2 rotor.
The percentage of double helix content (ordered parts) in
the starches was calculated following the method proposed by
Bogracheva, Wang, and Hedley (2001). The percentage of amorphous parts in native starch was calculated as: (PPA for the native
starch)/(PPA for amorphous starch) 100%. While, the double helix
content (%) equates to (100% amorphous part %). The PPA was
defined as the proportion of C4 peak fitting area relative to the total
area of the spectrum. The percentage of relative crystallinity was
calculated as the proportion of the fitting peak areas of the triplet
relative to the total area of the C1 spectrum (Paris, Bizot, Emery,
Buzaré, & Buléon, 1999).
2.2.8. Statistical analysis
Statistical comparison of means and simple correlation coefficients were conducted using the Student’s t test in a general linear
model (GLM) procedure of an SAS system (SAS Institute, Cary, NC).
3. Results and discussion
3.1. Molecular size
Table 1 summarizes the weight-average degree of polymerization (DPw) of corn starches before and after treated at 25 C in
methanol containing 0.36% HCl for different periods of time. Native
normal corn starch had the highest DPw among the starches
studied. The DPw of starch obviously decreased with increasing
time of acid-methanol treatment. In this study, the longest time of
acid-methanol treatment is 30 days, and the recovery of starch
granules after 30 days of treatment were all above 90%. Results in
Table 1 indicate that acid-alcohol treatment can be used to prepare
starch with different molecular sizes, and the longer treated time
the lower molecular size of starch prepared (Lin & Chang, 2006; Lin
et al., 2003, 2005; Ma & Robyt, 1987). The DPw of corn starches used
in annealing treatment and enzymatic resistance observation
ranged from 884 103 to 404, 778 103 to 299 and 337 103 to
250 AGU for normal corn, Hylon V and Hylon VII starches,
respectively.
Fig. 1. Chain length distributions of native starch (–) and starches after treated in
methanol containing 0.36% HCl at 25 C for 1 (C), 3 (B), 7 (:), 11 (6), 15 (;) and
30 (7) days, respectively.
3.2. Chain length distribution
The HPSEC profiles (Fig. 1) of isoamylase-debranched starches
show trimodal distributions. Generally, the f1 fraction consists of
Table 1
Weight-average degree of polymerization (DPw) of native and acid-methanoltreated corn starches
Starch
Normal corn
Hylon V
Hylon VII
Native
1 daya
3 days
7 days
11 days
15 days
30 days
883,980 29,299
200,593 2512
27,384 163
5237 4
1262 4
887 1
404 3
777,723 4218
100,028 1757
15,241 107
2869 40
882 8
602 2
299 3
337,229 7378
37,625 306
9324 135
2386 77
598 2
474 1
250 4
a
Time of acid-methanol treatment.
amylose, f2 fraction consists of the B2 and longer chains of amylopectin, and f3 fraction consists of the A and B1 chains of amylopectin. After acid-methanol treatment, the f1 peak shifted toward
smaller molecular size region and increased the content of f2
fraction. The weight percentage (%) and weight-average chain
length (CLw) of each fraction of starches before and after acidmethanol treatment are shown in Table 2. The weight percentage
and CLw of f1 fraction gradually decreased with increasing time of
acid-methanol treatment. However, the weight percentage and CLw
of f2 fraction showed a reverse tendency. The weight percentage of
f3 fraction increased with increasing time of acid-methanol treatment, while the CLw of f3 fraction remained constant or slightly
increased with increasing treatment time. The result indicates that
the amylose chains (f1 fraction) of starch degrades to shorter chains
during acid-methanol treatment, and leads to the increase of the
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J.-H. Lin et al. / Food Hydrocolloids 23 (2009) 1465–1472
Table 2
Weight percentage and weight-average chain length (CLw) of HPSEC fractions of isoamylase-debranched corn starches
Starch
f1
f2
f3
%
CLw
%
CLw
%
CLw
Normal corn
Native
1 daya
3 days
7 days
11 days
15 days
30 days
28.0 0.4
28.1 0.1
27.2 0.4
25.7 0.1
24.2 0.2
22.5 0.4
18.3 0.1
2390 43
1480 6
930 24
596 2
458 19
401 14
302 10
15.4 0.5
15.5 0.2
16.0 0.3
16.5 0.4
18.4 0.5
19.4 0.2
22.3 0.0
68.2 0.5
68.6 0.1
69.9 0.2
71.2 0.1
73.5 0.1
74.8 0.2
77.5 0.0
56.6 0.7
56.5 0.0
56.8 0.4
57.8 0.4
57.5 0.4
58.1 0.2
59.4 0.1
20.6 0.1
20.7 0.0
20.7 0.1
20.8 0.1
20.9 0.0
20.9 0.0
21.1 0.0
Hylon V
Native
1 day
3 days
7 days
11 days
15 days
30 days
40.1 0.5
39.6 0.0
37.4 0.8
32.8 0.2
30.8 0.2
29.4 0.1
18.3 0.4
1939 30
1417 16
928 11
684 4
558 15
556 4
388 5
31.8 0.1
32.2 0.3
33.9 0.5
37.1 0.1
39.1 0.1
39.9 0.0
47.3 0.1
84.9 0.3
85.5 0.2
86.9 0.1
88.9 0.1
90.3 0.0
90.3 0.1
91.0 0.2
28.1 0.7
28.2 0.3
28.7 0.4
30.1 0.1
30.1 0.1
30.7 0.0
34.4 0.5
22.1 0.1
22.5 0.1
22.6 0.2
22.7 0.0
22.9 0.1
22.9 0.0
23.1 0.1
Hylon VII
Native
1 day
3 days
7 days
11 days
15 days
30 days
44.5 0.5
42.7 0.4
41.4 0.9
35.6 0.5
31.0 0.8
28.3 0.2
18.7 0.5
1669 3
1044 10
805 3
612 5
526 40
474 4
379 13
35.8 0.1
37.5 0.1
39.3 0.9
43.3 0.1
47.1 0.4
49.2 0.1
56.6 0.4
90.7 0.3
91.6 0.1
93.5 0.1
94.3 0.1
95.1 0.2
95.4 0.0
94.5 0.2
19.7 0.4
19.7 0.4
19.3 0.0
21.1 0.4
21.9 0.4
22.5 0.2
24.7 0.1
22.3 0.2
22.4 0.2
22.7 0.2
22.6 0.2
22.8 0.1
22.8 0.1
23.2 0.0
a
Time of acid-methanol treatment.
weight percentage and CLw of f2 fraction. The B2 and longer chains
(f2 fraction) from amylopectin of starch also degraded after treated,
but the CLw of chains degraded from f2 fraction is similar to that of
f3 fraction (Chang et al., 2004, 2006). Therefore, no obvious change
in CLw of f3 after acid-methanol treatment was found.
3.3. Enzymatic resistance
The RS content of starches studied increased with increasing
amylose content of starch, while the SDS content of starches
deceased (Table 3). The RDS content of normal corn starch was
obviously higher than that of Hylon V or Hylon VII starch. The
changes in enzymatic resistance of starch either after acid-methanol treatment (i.e. before annealing) or after annealing are shown
in Fig. 2. Starch after short period of acid-methanol treatment (3, 11
or 3 days for normal corn, Hylon V and Hylon VII starches,
respectively) had negative values of DRS, that is the RS content of
starch granules after short period of acid-methanol treatment was
lower than that of the counterpart native starch. However, the RS
content of starch granules gradually increased with increasing time
of acid-methanol treatment (in other words, increased with
decreasing molecular size of starch) when the treatment time was
longer than 3, 11 or 3 days for normal corn, Hylon V and Hylon VII
starches, respectively. The RDS content of normal corn starch after
acid-methanol-treated slightly increased (DRDS 4.2%), but
different treatment times did not show obvious effect on the RDS
content. This denotes that the DRDS of normal corn starch did not
depend on the time of acid-methanol treatment. For Hylon V and
Hylon VII starches, the DRDS decreased with increasing time of
acid-methanol treatment except for the Hylon VII starch with 3
days of acid-methanol treatment. The DSDS for the starches studied
showed a decreasing tendency with the increase of acid-methanol
treatment time.
After annealing, the DRS of the acid-methanol-treated starches
was higher than that of the counterpart starch without annealing
(Fig. 2). This indicates that annealing increases the RS content of
starch. Result in Fig. 2 shows that the DRS of acid-methanol-treated
starch after annealing gradually increased with increasing time of
acid-methanol treatment. On the other hand, the DRDS and DSDS of
annealed starches decreased with increasing time of acid-methanol
treatment. Furthermore, the RDS and SDS contents of annealed
starch were lower than that of either the counterpart native starch
or the counterpart acid-methanol-treated starch without annealing, except for the Hylon V starch with 1–7 days of acid-methanol
treatment. Before annealing, the DSDS of Hylon V starch after acidmethanol-treated for 1–7 days were 2.0–2.9% higher than that of
the native Hylon V starch.
Results show that short period of acid-methanol treatment
increased the RDS content and decreased the SDS and RS contents
of starch (Fig. 2), which implies that starch after short period of
acid-methanol-treated is more sensitive on enzymatic hydrolysis.
This could be due to the degradation of starch granules, causing by
acid-methanol treatment, and further results in more accessible
granule structure (Lin et al., 2003, 2005) for enzymatic hydrolysis.
However, starch after long period of acid-methanol treatment had
higher RS content than that of the counterpart native starch, which
means that long period of acid-methanol treatment may induce reordering of starch molecules. The RS content of starch after acidmethanol treatment and annealing increased from 19.2 to 56.2%,
69.9 to 86.1%, and 73.1 to 89.1% for normal corn, Hylon V and Hylon
VII starches, respectively. This indicates that RS content of starch
not only depends on annealing, but also on the time of acidmethanol treatment. In other words, the RS content of starch
increases with decreasing molecular size of starch, especially for
Table 3
Contents of RS,a RDSb and SDSc of native corn starches
Starch
RS (%)
RDS (%)
SDS (%)
Normal corn
Hylon V
Hylon VII
19.2 0.3
69.9 1.1
73.1 0.8
19.7 1.2
9.7 1.0
11.0 0.3
61.1 1.0
20.4 1.0
15.9 0.5
a
b
c
Resistant starch.
Rapid digestible starch.
Slow digestible starch.
J.-H. Lin et al. / Food Hydrocolloids 23 (2009) 1465–1472
1469
3.4. Thermal properties
Fig. 2. Changes in enzymatic resistance of starches. (A) DRS (change of resistant starch
content, %) ¼ RS (%) of treated starch RS (%) of the counterpart native starch;
(B) DRDS (change of rapid digestible starch content, %) ¼ RDS (%) of treated starch RDS
(%) of the counterpart native starch; and (C) DSDS (change of slow digestible starch
content, %) ¼ SDS (%) of treated starch SDS (%) of the counterpart native starch.
the acid-methanol-treated starch with annealing. While the SDS
content of acid-methanol-treated starch after annealing shows
a reverse tendency. Lin et al. (2008) indicated that the decrease in
molecular size of starch, causing by acid-methanol treatment,
increased the mobility of starch molecules and enhanced the
realignment of starch chains during annealing, which further
increased the thermal stability of starch granules. Results in this
study demonstrate that decreasing in molecular size of starch
benefits the realignment of molecules during annealing; consequently, the SDS content in starch granules decreases and the RS
content increases.
The DSC thermograms of starch (Fig. 3) illustrate that the gelatinization thermal peak of starch after acid-methanol treatment
(before annealing) shifted slightly to lower temperature comparing
to that of the counterpart native starch, while shifted obviously to
higher temperature for starch after annealing. The To of starch
slightly decreased or remained constant after acid-methanoltreated (Table 4). Whereas, the Tp of acid-methanol-treated starch
increased with increasing time of acid-methanol treatment, except
for the normal corn starch with acid-methanol-treating for less
than 7 days and the Hylon V starch with 1 day of acid-methanol
treatment. The Tr of acid-methanol-treated starch was higher than
the counterpart native starch, and significantly increased with
increasing time of acid-methanol treatment. The DH slightly
increased or remained constant for normal corn and Hylon V
starches after acid-methanol treatment, while obvious increase in
DH of Hylon VII starch after acid-methanol treatment was found.
These results are inline with the observation on acid-methanoltreated rice starch (Lin et al., 2008). The increase in DH of starch,
especially for Hylon VII starch, implies the re-ordering of starch
chains during acid-methanol treatment.
After annealing at 50 C for 72 h, the To and Tp of native starch
and acid-methanol-treated starch increased obviously, except for
the Tp of Hylon VII starch which showed a lower Tp for starch after
annealing than the counterpart starch before annealing. Furthermore, the To and Tp of annealed starch showed an increasing
tendency with increasing time of acid-methanol treatment. In
contrast to the increase of To and Tp, the Tr of starch evidently
decreased after annealing. Nevertheless, the DH of annealed starch
was higher than that of the counterpart starch before annealing.
The difference in DH before and after annealing of the acid-methanol-treated normal corn and Hylon V starches were more obvious
than that of the counterpart native starch, while the difference in
DH of acid-methanol-treated Hylon VII starch before and after
annealing was less obvious than that of the native Hylon VII starch.
As shown in Table 2, after acid-methanol-treated for the same
period, Hylon VII starch had higher f2% and longer f2CLw than those
of the counterpart normal corn and Hylon V starches. On the other
hand, Hylon VII starch had the lowest f3% among the starches
studied. Results imply that more ordered structure of is formed for
Hylon VII starch during acid-methanol-treating, which leads to less
ordering process could occur during the annealing of starch.
Although To or Tp slightly decreased after acid-methanol treatment, the To and Tp of acid-methanol-treated starches after
annealing were higher than those of the counterpart native
starches either before or after annealing. The DH of acid-treated
starch after annealing was also higher than that of the counterpart
native starch after annealing. This infers that the degradation of
starch, causing by the acid-methanol treatment, improves the
mobility of starch chains, and further results in the reorder of starch
chains during treatments. Results also indicate that reducing of
molecule size of starch enhances the efficiency of annealing on
starch.
3.5. Double helix content and relative crystallinity
Fig. 4 shows the 13C CP/MAS NMR spectra of native normal corn
and Hylon VII starch and their acid-methanol-treated starches
before and after annealing. The C1 resonance (signal around
100 ppm) of the native or acid-methanol-treated normal corn
starched was triplet, which is a typical characteristic of A-type
starch. While the spectra of Hylon VII starches showed a doublet
peak for C1 resonance, which is characteristic of B-type starch
(Paris et al., 1999). Hylon V starch had similar spectra to that of
Hylon VII starch (data not shown).
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J.-H. Lin et al. / Food Hydrocolloids 23 (2009) 1465–1472
Fig. 3. DSC thermograms of native and acid-methanol-treated (for 1, 7 and 30 days) corn starches before and after annealing.
Table 4
Thermal properties of starches before and after annealing
Starch
To ( C)
Tp ( C)
Tr ( C)
DH (J/g)
Before
After
Before
After
Before
After
Before
After
Normal corn
Native
1 dayb
3 days
7 days
11 days
15 days
30 days
65.3aa
63.7b
63.4a
63.7b
63.8bc
64.0d
63.9cd
69.0f
69.9g
70.6h
71.1i
71.5j
71.8k
71.2i
69.7c
68.4a
68.4a
69.2bc
70.0d
70.8e
72.8g
72.0f
72.9g
73.9h
74.9i
75.6j
76.3k
77.2l
14.9b
17.0c
18.3d
19.9e
22.0f
25.7g
33.9i
11.9a
14.0b
13.9b
15.0b
18.0cd
19.5e
27.6h
11.1a
11.3abc
11.1ab
11.6bcd
11.7cd
11.4cd
11.8cd
11.9d
12.6e
12.8e
13.3f
13.4f
13.5f
13.7f
Hylon V
Native
1 day
3 days
7 days
11 days
15 days
30 days
68.5d
66.3a
66.1a
66.3a
67.0bc
66.6ab
67.4c
70.5e
71.3f
72.1g
72.8h
73.3hi
73.8i
73.4i
75.9b
75.2a
76.8b
78.1cd
78.1cd
78.7de
80.0fg
76.8bc
77.0bc
77.6bcd
78.1cd
78.9def
79.5efg
80.7g
38.7fg
40.0h
39.6gh
39.8gh
40.6hi
41.8ij
42.9j
37.8ef
35.4c
33.9bc
34.2bc
33.5b
32.1a
36.9de
13.1a
13.1a
13.0a
13.4ab
13.3a
13.2a
13.2a
13.5ab
13.6ab
14.1bc
14.4cd
14.7d
14.8d
14.4cd
Hylon VII
Native
1 day
3 days
7 days
11 days
15 days
30 days
66.8de
65.4b
64.4a
65.7bc
65.6b
66.4cd
67.4e
69.1f
70.7g
71.0g
71.8h
72.3i
72.9i
73.6j
–c
82.8b
82.8b
86.0c
86.6c
86.3c
86.7c
–
80.9a
79.9a
79.6a
81.0a
82.6b
83.8b
41.7b
44.2c
46.2d
47.5de
49.0ef
50.5f
50.1f
41.2b
40.4ab
40.1ab
38.7a
40.1ab
41.5b
41.7b
10.7a
10.9ab
11.2bc
11.2bc
12.1de
12.6f
12.8fg
11.5c
12.1d
12.0d
12.6ef
12.6ef
12.9fg
13.1g
a
b
c
Means followed by different letters within a DSC parameter of a starch differ significantly (p < 0.05).
Time of acid-methanol treatment.
Undetectable.
J.-H. Lin et al. / Food Hydrocolloids 23 (2009) 1465–1472
1471
after acid-methanol treatment; however, treatment time did not
show obvious effect on the double helix content of starch. After
annealing, the double helix content of starch was similar to or
slightly higher than that of the counterpart starch before annealing.
A comparison between starch before and after annealing shows the
double helix content of the native starch studied slightly increased
after annealing, which is from 28 to 31%, 18 to 21% and 21 to 26% for
normal corn, Hylon V and Hylon VII starches, respectively.
Furthermore, the double helix content of acid-methanol-treated
starch after annealing was obviously higher than that of the
counterpart native starch.
The comparison between effect of treatments on the double
helix content and the relative crystallinity of starch shows that the
treatments had less impact to the relative crystallinity of starch
(Table 5). The relative crystallinity of starch after acid-methanol
treatment was similar to that of the counterpart native starch.
Moreover, no obvious change in the relative crystallinity of starch
was found for starch after annealing. Tester et al. (1998) indicated
that the number of double helices remained constant after
annealing and proposed that annealing improved the crystalline
register of double helices, thereby ‘‘perfecting’’ starch crystallites
rather than promoting the formation of additional double helices.
Results of this study demonstrate that the degradation of starch
molecules in granules enhances the mobility of molecules during
treatments and leads to the alignment of starch chains. The alignment of starch chains during treatments may develop new double
helix, but not being organized perfectly to form new crystallites. The
increase of RS content of starch after treatments indicates that the
realignment of starch chains, which producing new double helixes
or perfecting starch crystalline, increases the enzyme resistance of
starch. Moreover, the changes in RS and double helix contents of
starch after treatments were more obvious than the change in
relative crystalline (Fig. 2 and Table 5). This reveals that the double
helix structure of starch might increase the resistance of starch to
enzymatic hydrolysis and thereby increases the RS content.
Fig. 4. NMR spectra of native and acid-methanol-treated (for 30 days) normal corn and
Hylon VII starches before and after annealing.
The double helix content of native normal corn, Hylon V and
Hylon VII starches were 28, 18 and 21%, respectively (Table 5).
Among the starches studied, normal corn starch had the highest
double helix content. The double helix content of starch increased
Table 5
Double helix content and relative crystallinity of starches before and after annealing
determined by 13C CP/MAS NMR
Starch
Normal corn
Before
Double helix content (%)a
Native
28
33
1 dayb
3 days
32
7 days
35
11 days
35
15 days
33
30 days
33
Relative crystallinity (%)a
Native
24
1 day
23
3 days
24
7 days
24
11 days
24
15 days
24
30 days
24
Hylon V
Hylon VII
After
Before
After
Before
After
31
37
39
34
39
36
38
18
26
25
25
23
25
26
21
31
30
30
27
30
30
21
26
29
31
31
29
31
26
29
33
35
36
37
37
23
23
24
24
25
25
25
19
19
21
19
21
20
20
20
21
22
20
22
22
21
19
20
19
20
21
21
21
19
21
19
20
21
21
21
a
The peak fitting correlation coefficient is at least 0.995, and the maximum
standard deviation is less than 5%.
b
Time of acid-methanol treatment.
3.6. Correlation between changes in enzymatic resistance and
molecular structure
Table 6 summarizes the correlations between molecular structure and changes in enzymatic resistance of starch after acidmethanol treatment and annealing for the starches studied. Results
show that the molecular size (DPw) of starch had a negative
correlation with DRS and a positive correlation with DRDS. This
reveals that the degradation of starch increases the mobility and
realignment of starch chains within granules during treatments.
Results also show that the content (f1%) and weight-average chain
length (f1CLw) of amylose negatively correlated with DRS, but
positively correlated with DRDS and DSDS. Gidley and Bulpin
Table 6
Correlations between changes in enzymatic resistance and molecular structure of
starch
Parametera
DRSb
DRDS
DSDS
DPw
f1%
f1CLw
f2%
f2CLw
f3%
f3CLw
0.433*
0.767***
0.709**
–
–
0.486*
–
0.507*
–
0.731**
0.733**
0.691**
0.437*
0.660**
–
0.756***
0.483*
0.478*
0.535*
0.765***
0.576**
*p < 0.05; **p < 0.01; ***p < 0.001; –, p > 0.05.
a
DPw is the weight-average degree of polymerization of starch. f1, f2 and f3% are
the weight percentage of f1, f2 and f3 fractions in HPSEC profiles of isoamylasedebranched starch, respectively. f1CLw, f2CLw and f3CLw are the weight-average
chain length of f1, f2 and f3 fractions, respectively.
b
DRS, DRDS and DSDS are the changes in contents of resistant starch, rapid
digestible starch, and slow digestible starch.
1472
J.-H. Lin et al. / Food Hydrocolloids 23 (2009) 1465–1472
(1989) indicated that amylose with high degree of polymerization
did not easily form order structure during retrogradation. In this
study, the acid-methanol treatment decreases the chain length of
amylose and improves the re-ordering of amylose chains, which
results in a high content of RS and low contents of SDS and RDS of
starch after treatments.
DSDS of starch positively correlated with the content (f2% and
f3%) and weight-average chain length (f2CLw and f3CLw) of
amylopectin chains, and a reverse tendency was found for DRDS.
While DRS did not show significant correlation with molecular
structure of amylopectin of starch, except a positive correlation was
found between DRS and f3% (content of A and B1 chains of
amylopectin) (p < 0.05). This indicates that the change of SDS
content during treatments depends on the molecular structure of
amylopectin.
4. Conclusions
The RS content of normal corn, Hylon V and Hylon VII starch
granules were profoundly increased after acid-methanol treatment
and annealing. The RS content of starch increased from 19.2 to
56.2%, 69.9 to 86.1%, and 73.1 to 89.1% for normal corn, Hylon V and
Hylon VII starches, respectively. After treated by acid-methanol
treatment and annealing, the Tp, double helix content and RS
content of starch increased, which indicates the realignment of
starch chains during treatments. After annealing, the RS content of
acid-methanol-treated starch increased with decreasing molecular
size of starch. This reveals that the degradation of starch, causing by
acid-methanol treatment, enhances the mobility and realignment
of starch chains during annealing and further increases the enzymatic resistance of starch granules. In addition to the molecular size
of starch, the RS content of starch after treated also increased with
decreasing content and weight-average chain length of amylose
fraction of starch. Results of this study demonstrate that the
combination process of acid-methanol treatment and annealing
can be used to increases the RS content of starch granules.
Acknowledgment
We thank the National Science Council (Taiwan), for financial
support (NSC 95-2313-B-126-007-MY3).
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