RESEARCH ARTICLEEffect of debranching and storage condition on crystallinity and functional properties of cassava and potato starches Pham Van Hung1, Nguyen Thi Lan Phi2and Tran Thi Vy V
Trang 1RESEARCH ARTICLE
Effect of debranching and storage condition on
crystallinity and functional properties of
cassava and potato starches
Pham Van Hung1, Nguyen Thi Lan Phi2and Tran Thi Vy Vy1
HoChiMinh City, Vietnam
2
Faculty of Chemical Engineering, HoChiMinh City University of Technology, HoChiMinh City, Vietnam
Debranching starch by pullulanase is considered to improve the RS content of starch which is
widely used to produce the starch-based foods with high-health benefit impacts In this study,
the cassava and potato starches were debranched by pullulanase, followed by an autoclave
properties After debranching, the potato starch contained significantly higher CL (35.4
glucose units) than did the cassava starch (32.4 glucose units) The debranched cassava
and potato starches after retrogradation at the storage temperatures had a typical B-type
crystalline structure although the native cassava and potato starches exhibited the different
crystalline forms (A- and B-type, respectively) The RS contents of the debranched cassava
and potato starches significantly improved with higher RS content of the debranched potato
starch than that of the debranched cassava starch at the same storage condition The storage
temperature significantly affected the RS formation of the debranched starches with the
cassava and potato starches, respectively) The debranched starches had significantly lower
viscosities and paste clarities but higher solubilities than did the native starches As a result,
the debranched cassava and potato starches can be considered for use not only in functional
foods with enhanced health benefits but also in pharmaceutical and cosmetic industries
Received: February 29, 2012 Revised: May 15, 2012 Accepted: May 31, 2012
Keywords:
Cassava starch / Crystallinity / Debranched starch / Functional properties / Potato starch
1 Introduction
AM and AP play an important role in starch characteristics
and are considered to have great impact on the formation
of texture and quality of starch-based food products [1] AM
has been known as an essentially linear molecule joined by
rec-ognized that a fraction of the AM molecules is slightly branched by a-(1,6)-linkages [2, 3] AM has a MW of
degree of polymerization (DPn) of 324–4920 [7–11] In contrast, AP is a very large, highly branched chain
attached to a-(1,4)-bonds [4–6, 12], in which the MW of
[12] These chains can be classified as either the unbranched outermost chains (A) or the branched inner
Colour online: See the article online to view Figs 2–4 in colour.
Correspondence: Dr Pham Van Hung, School of Biotechnology,
International University, Vietnam National University in HoChiMinh
City, Quarter 6, Linh Trung Ward, Thu Duc District, HoChiMinh City,
Vietnam
E-mail: pvhung@hcmiu.edu.vn
Trang 2chains (B) In addition, there is a single chain (C) per
molecule which contains a sole reducing residue [13]
The B chains can be further divided in B1, B2, B3,
and B4 chains [8] Branch points occur at approximately
mol-ecules is typically within the range 1278–15 900 in which
AP unit chains are relatively short compared to AM
molecules with typically ranging in 17–35 units long on
average
The AP molecules are debranched by debranching
enzymes such as isoamylase or pullulanase which cleaves
[14, 15] Shi et al [16] reported that debranching of normal
starches releases a mixture of short and long linear chains
because both AM and AP in starch granules were
debranched, whereas debranching of wx starch releases
short linear side chains from AP During retrogradation, AM
or the linear chains formed from debranched AP can form
double helices by hydrogen bonds between molecules
resulting in rigid gels called crystallinity In nature, native
starch granules exhibited three main types of crystallinity,
the A-, B-, and C-type crystals, which can be monitored by
XRD analysis [17] After debranching and crystallization,
the short-chain AM from debranched wx maize starch
formed a typical A-type crystalline structure when
debranched at high concentration (25% solids) [18],
whereas a dilute starting concentration (5% solids) led
to the formation of B-type crystalline structure [19] The
debranching combined with physical treatment such as
ANN or heating and cooling cycles have been applied to
produce re-crystallized AM products, which had various
degrees of crystallinity and differing functional properties
The debranched and recrystallined starch products were
reported to have high amounts of slowly digestible starch
(SDS) [20, 21] or RSs [22–24] The solubility and
water-binding values of autoclaved and debranched samples of
the high-AM corn starches were higher than those of their
respective native starches, whereas autoclaving–storing
cycles after debranching caused decreases in peak,
breakdown, and final viscosity values [23] Debranching
caused decreases in DSC peak temperature (Tp) and
increases in enthalpy of the high-AM corn starches [23]
However, Cai and Shi [19] reported that all three crystalline
short chain AMs (CSCA) from wx wheat, wx maize, and wx
potato starches had larger melting temperature range (80–
1408C) as compared to did the native starches (59–958C),
in which the CSCA from debranched wx potato starch
displayed a higher peak melting temperature (116.28C)
than those from debranched wx wheat (99.78C) and wx
maize (99.98C) starches Although the relationship
between debranching and heat treatments and the
for-mation of RSs and their functional properties from
high-AM starches [23], banana starch [24], or wx starches
[18, 19] have been recently reported, the relationship between the debranching and autoclave treatment with
debranched starches as well as their functional properties
of cassava and potato starches has not been examined Therefore, the objective of this study is to investigate crystallinity forms, functional properties, and RS contents
of debranched and recrystallined starches by debranching cassava and potato starches using pullulanase, autoclave treatment, and storage at different temperatures
2 Materials and methods
2.1 Materials Cassava (Manihot esculenta) and potato (Solanum tuber-osum) grown in the southern part of Vietnam were used in this study Cassava and potato starches were isolated from raw cassava roots and potato tubes at the Laboratory of Applied Chemistry, International University, Vietnam The raw cassava roots and potato tubes were ground using an engine-driven drum grater Then the slurry was kept in a
(0.232 and 0.105 mm in aperture size) Resultant starches were washed thoroughly in clean water to remove the contaminant substances Finally, the starch sediment was recovered by centrifugation and dried in an oven at 408C to 10–11% moisture The isolated starches were deffated by hexane in a sohxlet system for 6 h before using for debranching
Pullulanase from Bacillus acidopullulyticus (400 U/mL), a-amylase from Aspergillus oryzae (30 U/mg), and amyloglucosidase from Aspergillus niger (300 U/mL) used in this study were purchased from Sigma–Aldrich
Co (St Louis, MO, USA) Other chemicals were pur-chased from Merck Co (Darmstadt, Germany)
2.2 Debranching and storage of starches Cassava and potato starches were debranched by pullu-lanase according to the method of Gonzalez-Soto et al [23] with slight modification as follows The deffated starch (10 g, db) was well mixed with 100 mL of acetate buffer 0.1 M (pH 5.2) and cooked in a boiling water bath (with stirring) for 10 min with continuously stirring The hot paste was then autoclaved at 1218C for 30 min using an auto-clave (model HV-85, Hirayama Manufacturer Corp., Japan) After autoclaving, the paste was cooled to 508C and mixed with pullulanase (20 U/g starch) The mixture was incubated with constant stirring for 24 h at 508C After
24 h, the debranched starch gel was autoclaved at 1218C
another 24 h The debranched starches were then dried at
Trang 3508C in an oven overnight and stored in closed glass
containers until further use
2.3 Scanning electron microscopy (SEM) of
starches
Appearances of the native starch granules and crystals
of the debranched starches were observed by a SEM
(JEOL-JSM-6480LV, Tokyo, Japan) The preparation
and operation procedures were carried out as previously
described [25] The samples were coated with Pt/Pd
and photographed at an accelerating potential of 10 kV
2.4 Measurements of iodine absorption of
starches
Blue value of the native and debranched cassava and
potato starches were measured as previously described
[25] Each starch was suspended in 1 M aqueous NaOH,
followed by heating in a boiling water bath with shaking
An aliquot of the solution was mixed with iodine solution
starch complex was measured at 680 nm AM content of
the starches was calculated based on a calibration curve of
a mixture of AM and AP according to the equation:
in which BV is the blue value of starches measured at
620 nm
2.5 Determination of degree of polymerization
of debranched starches
calculated as the difference between reducing residues
and total glucose concentration of the native starches,
whereas CL was calculated as the difference between
reducing residues and total glucose concentration of the
debranched starches The average number of chains per
2.6 X-ray diffraction pattern of starches
The crystalline structure of the native and debranched
cassava and potato starches was observed using an
X-ray diffractometer (Rigaku Co., Ltd., Rint-2000 type,
Tokyo, Japan) according to the operation described by
Hung and Morita [25] The XRD system was operated at
40 kV and 80 mA and diffractograms of the starches were
recorded from 28 2u to 358 2u with a scanning speed of
88/min and scanning step of 0.028
2.7 Resistant starch determination Resistant starch contents (%RS) of the native and debranched cassava and potato starches were measured based on the method of Englyst et al [27] with moderate modification as follows Starch (1 g, db) was mixed with
25 mL of acetate buffer (pH 6.0) and then boiled for 30 min
in a water bath After cooling to 378C, amylase solution (7000 U/g starch) was added and the slurry was incubated
at 378C for 2 h The suspension was then cooled to 258C and adjusted to pH 4.5 before adding amyloglucosidase (50 U/g starch) The mixture was then incubated at 608C for
the sediment The sediment was washed with distilled water for three times and then dried at 508C for 48 h The %RS3 was calculated as weight of remained residue (db) com-pared to that of the initial sample A blank with no starch was used to minus the contamination of the enzymes 2.8 Determination of pasting properties of starches
Viscosity of the debranched starches was measured using
a Brookfield Viscometer LVDV-E according to the official method described by the International Starch Institute (ISI17, Science Park Aarhus, Denmark) with slight modi-fication [28] A starch slurry (2% starch) was cooked in a boiling water bath for 15 min with continuously stirring and additional 15 min without stirring The paste was cooled down to 508C and measured the viscosity in centipoises (cP) at 508C at 50 rpm with spindle SC4-18
Paste clarity of starches was determined according to the method of Craig et al [29] Starch (0.05 g, db) was suspended in 5 mL of distilled water in a glass-stoppered tube Then the slurry was heated at 958C for 30 min with shaking every 5 min and cooled The clarity of paste was measured for transmittance (%T) using a spectro-photometer (UVD-2960, Labomed, USA) at 650 nm against a water blank
Solubility of starch (%) was determined as the method previously described by Singh and Singh with slight modification [30] The starch (0.5 g, db) was suspended
in 50 mL of distilled water and shaked thoroughly for
30 min on a rotary shaker The starch suspension was
the supernatant was taken in a preweighed beaker and dried in an air oven at 1108C for 4 h The cold water solubility
2.9 Statistical analysis
three separate determinations Comparison of means
Trang 4was performed by one-way analysis of variance (ANOVA)
followed by Duncan’s multiple comparison tests ( p<0.05)
using SPSS version 16 (SPSS Inc., Chicago, IL) system
3 Results and discussion
3.1 SEM of native and debranched starches
Figure 1 shows the granular appearances of the native
cassava and potato starches and their crystalline forms
after debranching Native cassava and potato starches
had naturally granular structures with smooth surfaces
Potato starch had oval-shaped granules, whereas cassava
starch had both spherical- and polygonal-shaped
gran-ules, which are smaller than those of potato starch
After debranching, both debranched cassava and potato
starches formed crystalline particles The crystalline
particles of the dried debranched starches were the same
for both cassava and potato starches and their sizes were
dependant on the sizes of sieves Thus, the treatment of
the native starches by debranching and autoclaving
caused the granular structure of starches broken down
to form short-chain linear AMs which were aggregated and
developed crystalline particles Gonzalez-Soto et al [23] reported that the samples stored at 48C for 24 h observed
a more compact structure than samples stored at higher temperatures The different structures of the debranched starches stored at different temperatures were due to the crystalline character present in those samples which might
be associated to a higher level of cavities or channels in the matrix of starches stored at the high temperature in con-trast with the topological structure of starches stored at lower temperatures In this study, the debranched starches stored at 258C for 24 h did not show any cavities or chan-nels in the matrix of crystalline particles
3.2 Amylose content and degree of polymerization of debranched starches Amylose content of native potato starch was significantly higher than that of native cassava starch resulting in the higher blue value of iodine–AM complex of potato starch than that of cassava starch (Table 1) The CLs of the debranched cassava and potato starches were 32.4 and 35.4 glucose units, respectively, whereas the average degree of polymerization were 1400 and 1520 glucose units for native cassava and potato starches, respectively
Figure 1 SEM of native and debranched cassava and potato starches NCS, native cassava starch; DCS, debranched cassava starch; NPS, native potato starch; DPS, debranched potato starch
Trang 5Thus, AP molecules of the native starches were mostly
debranched by pullulanase into the short-chain molecules
Morrison and Karkalas [30] reported that the CLs of AMs
fractionated from cassava and potato starches were 340
and 670 glucose units, respectively, which were
signifi-cantly higher than those of APs of cassava and potato
starches (21.2 and 22.0–23.9 glucose units, respectively)
These results indicate that the CLs of both AM and AP of the
cassava starch were lower than those of the potato starch,
which is consistent with the findings in this study Cai and Shi
[19] reported that the CLs of the debranched wx wheat, wx
maize, and wx potato starches were 28.1, 29.2, and 35.5
glucose units, respectively, after debranching with 1% of
cas-sava and potato starches had significantly higher CLs than
did the wx wheat and wx maize starches Although the
minimum chain length required to form starch double
heli-ces is 10 [31], the difference in the CL led to the differenheli-ces
in yield of crystallized product, peak melting temperature,
and RS content of the debranched starches [19]
3.3 X-ray diffraction patterns of starches
debranched cassava and potato starches are shown in
Fig 2 The native cassava starch had the typical A-type crystalline structure with the main peaks numbered 2a, 2b, 3b, 4a, 4b, 5a, 6a, and 7, whereas the native potato starch had the typical B-type crystalline structure with the main peaks numbered 1, 3a, 3b, 4a, 5a, 6a, 6b, and 7 as described by Zobel et al [17] After debranching, all debranched starches showed the B-type crystalline struc-tures with stronger peaks than did the native starches However, it is not clear difference in degree of crystallinity
of the debranched starches stored at different temperature both for cassava and potato starches These results are consistent with the previous studies [19, 23], who reported that the typical B-type structure was observed for all debranched starches even though the corresponding native starch had the A-type structure [19] or the C-type structure [23] However, a dual-stage crystallization phenomenon, B-type crystallization appeared during the first 8 h of incubation, followed by A-type crystallization between 16 and 24 h, was observed when debranched
at 25% solids and crystallized at 508C [18] These results indicate that debranching of starch at low tration (5 or 10% solids) or short time at higher concen-tration of starch produced a B-type crystalline form, otherwise the formation of A-type crystallites was observed
Starch
Structural indexes
b) Values with the same letter in the same column are not significantly different
Figure 2 XRD patterns of native and debranched cassava (A) and potato (B) starches at different storage conditions
þ48C, and 188C, respectively; NPS, native potato starch; DPS þ 25, DPS þ 4 and DPS 18, debranched potato starches
Trang 63.4 Resistant starch contents of debranched
starches
Resistant starch (RS3) content (%, db) of native and
debranched cassava and potato starches at different
stor-age conditions are shown in Fig 3 According to Englyst
et al [27], RS of the native starch was classified as RS2,
whereas the RS of the cooked and retrograded starch was
RS3 Therefore, the RS of the debranched starches in this
study was determined as RS3 After debranching and
of the starches was significantly increased as compared to
those of the native starches This result agreed with the
previous studies on formation of RS by debranching starch
using pullulanase or isoamylase [18, 19, 23, 24] Thus,
the aggregation and arrangement of double helices of the
short chain AMs in the debranched starches formed the
new crystalline structure (B-type) resulting in the increased
RS content The storage of sample at different
tempera-tures after autoclaved cycle showed significantly influence
retro-gradated than those stored at 4 and 258C This result
indicates that the speed of retrogradation significantly
affects the crystalline structure of double helices and
RS content of the short chain AMs In addition, the effect
of storage temperature on RS formation was also reported
by Gonzalez-Soto et al [23], who stated that when starchy
material was stored at high temperatures (i.e., 608C)
material was in a rubbery state resulting in a slow
retro-gradation process and low-RS content
In the native forms, the potato starch with the typical
the starches having the A-type crystallinity [18, 25] The result in this study also shows that the native potato starch after cooking still contained higher RS content than did
debranched potato starch was significantly higher than that of the debranched cassava starch at the same storage condition The differences might be due to the chain length distribution and degree of crystalline structure of the short chain AMs obtained by debranching of starches The debranched potato starch with significantly higher
CL (35.4 glucose units) formed double helices with more dense crystalline structure resulting in more resistance to enzyme digestion as compared to the debranched cas-sava starch having the CL about 32.4 glucose units This result is consistent with other studies on wx starches [19]
35 and 48% for the debranched cassava and potato
the debranching and autoclaving process followed by
high-RS content
3.5 Viscosity of debranched starches Viscosities (cP) of the native and debranched cassava and potato starches at different storage conditions are given in Table 2 In the native forms, the starch pastes made from the cooked cassava and potato starches were too viscous which were hardly measured by the Brookfield at high concentration (>2%) according to the method described above At 2% of starch concentration, the viscosities of native cassava and potato starches were 45.7 and 45.9 cP, respectively After debranching, the viscosities of pastes from the cooked debranched cassava and potato starches significantly reduced because of low-MWs of the linear chains presented in those starches The viscosities of the debranched starches at different storage condition
0
10
20
30
40
50
60
Storage temperature (oC)
Cassava Potato
Figure 3 RS content (%, db) of native and debranched
cassava and potato starches at different storage
Table 2 Viscosities (cP) of native and debranched
cassava and potato starches at different storage
a) Values with the same letter in the same column are not significantly different
188C
Trang 7were not significantly different However, the viscosities of
the debranched cassava starches were significantly lower
than those of the debranched potato starches at the same
storage conditions This result might be due to the lower
CL of the debranched cassava starch which reduced the
viscosity as compared to that of the debranched potato
starch
3.6 Paste clarity of native and debranched
starches
Figure 4 shows the results of paste clarity (%T) of native
and debranched cassava and potato starches at different
storage conditions Transparences of the pastes from the
debranched starches were significantly lower than those of
the native starches The remarkable changes from the
starch polymer solution at the beginning of debranching
to the cloudy slurry after 24 h of crystallization at 508C
were also observed by Cai et al [18] These results are due
to the small and fine crystallites formed by the double
helices of the short chain length of AMs of the debranched
starches as compared to the high molecules of AM and AP
of the native starches In this study, the lowest
transpar-ences of the pastes of the debranched starches stored at
48C were observed among the tested samples because
the smallest and finest crystallites were formed at this
temperature [29]
3.7 Solubility (%) of native and debranched
starches
Solubility of native and debranched cassava and potato
starches were determined by stirring the starches in
excess water at ambient temperature and the results
are shown in Table 3 The native starch hardly dissolved
in the water, whereas the debranched starches containing the short-chain molecules had higher solubilities The sol-ubilities of the debranched cassava starch (12.1–12.6%) were significantly higher than those of the debranched potato starch (9.8–10.2%) because the debranched cas-sava starch had lower short-chain molecules as compared
to the debranched potato starch The solubilities of the debranched starches stored at different temperatures were not significantly different indicating that the solubil-ities of the debranched starches were dependant on the chain length distribution rather than the form of crystallites
As a result, the debranched starches with high-RS contents and low viscosities and solubilities are considered
to be not only used in functional foods with enhanced health benefits but also used in pharmaceutical and cos-metic industries
4 Conclusions
The crystallinity and functional properties of cassava and potato starches after debranching by pullulanase and auto-clave treatment with different storage temperatures are successfully investigated in this study The debranched potato starch had higher CL and RS content than did the debranched cassava starch Both debranched cas-sava and potato starches exhibited the typical B-type structure with reduced viscosity and paste clarity and increased solubility as compared to the native starches The storage temperature did not affect the crystallinity and functional properties of the debranched starches but sig-nificantly influenced in RS formation The RS contents of both cassava and potato debranched starches were the
potato starch is considered to be a good starting material
to produce high amount of RS by debranching and
auto-Table 3 Solubility (%) of native and debranched cassava
and potato starches at different storage
a) The same letter in the same column is not significantly different
188C
NS
DS+25
DS+4
DS-18
Cassava
Potato 0
10
20
30
40
50
60
70
80
Figure 4 Paste clarity (%T) of native and debranched
cassava and potato starches at different storage
Trang 8clave treatment However, both debranched cassava and
potato starches can be used for functional food processing
and pharmaceutical and cosmetic industries
The authors thank the National Foundation for Science
and Technology Development, Vietnam (NAFOSTED),
research grant no 106.99-2010.66 for the financial
support
The authors have declared no conflict of interest
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