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Research Article
Distinct properties and structures among B-crystalline starch granules†
Varatharajan Vamadevan1,4, Andreas Blennow2, Alain Buléon3, Avi Goldstein1,4, Eric
Bertoft1,5
1
Department of Food Science and Nutrition, University of Minnesota, St Paul, MN,
U.S.A.
2
Department of Plant and Environmental Sciences, University of Copenhagen,
Frederiksberg C, Denmark.
3
UR1268 Biopolymères Interactions Assemblages, INRA, Nantes, France.
4
Present address: Cargill, Minneapolis, MN, U.S.A.
5
Present address: Bertoft Solutions, Turku, Finland.
Correspondence: Dr Eric Bertoft, Bertoft Solutions, Gamla Sampasvägen 18, 20960
Turku, Finland.
E-mail: eric.bertoft@abo.fi
†
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: [10.1002/star.201700240].
This article is protected by copyright. All rights reserved.
Received: September 14, 2017 / Revised: October 16, 2017 / Accepted: October 17, 2017
Abstract
Starch granules derived from certain tuber or root crops exhibit a B-type polymorphic
pattern and amylopectin with a high content of long B-chains and comparatively long
segments between the building blocks. In this investigation, four B-crystalline
starches were selected to study their morphology and molecular composition
including lintnerization conducted at two different temperatures. The structure of the
granules suggested that the B-type crystalline starches could be divided into two
distinct groups, with potato and edible canna forming one group having large granules
with typical "growth rings" and the molecular structure of the lintners being
dependent on the temperature of lintnerization. The other group consisted of shoti and
lesser yam starches possessing granules with alternating "granular slices" instead of
rings or shells and the molecular composition of the lintners was not dependent on the
temperature. We found that the former group of starch possessed lower gelatinization
temperatures and swelled at lower temperatures than the latter group, suggesting a
more labile granular structure of potato and canna starches compared to shoti and
lesser yam. As the properties were not related to the amylose or phosphate content of
the granules, the result suggests that B-crystalline starch granules are found as at least
two distinct structural types with unique architectures.
Keywords:
B-type crystalline starch / Granule structure / Thermal properties / Starch composition
/ Lintnerization
1 Introduction
The main components of starch, amylose (essentially linear) and amylopectin
(extensively branched), are packed into semi-crystalline granules. In the granules,
semi-crystalline shells appear as rings, often called "growth rings", when observed in
light microscope and they are embedded in a softer, amorphous matrix [1]. The
amylopectin component contributes to the formation of stacks of thin, amorphous and
crystalline lamellae in the semi-crystalline rings [2]. The internal chains of
amylopectin build up the amorphous lamellae together with most of the branched
glucosyl units [3], which essentially connects to short external chains with a length
(CL) of 10–15 glucosyl units through flexible spacer arms [4]. The external chains
unite into double-helices that crystallize into either A- or B-type crystalline
polymorph patterns [5, 6], of which the latter is found in several starches from tubers,
roots and rhizomes. A-type polymorphs are generally found in cereal starches and
some starches (e.g. from pulses) exhibit C-polymorphic pattern that is a mixture of Aand B-type unit cells [7].
The internal unit chain profile of amylopectin, as analysed by highperformance anion-exchange chromatography (HPAEC), has been found to have
distinct patterns in plants from different genetic backgrounds [8]. The patterns were
classified into four types, of which type 4 amylopectin structure appears to confine to
most B-crystalline starches. Type 4 is characterized by a high amount of long chains
(CL > 38) compared to amylopectins representing type 1, 2, or 3. Clusters, defined as
groups of chains with < 9 glucosyl units between branches [9], are small in type 4
amylopectins [10, 11]. The clusters are essentially composed of groups of very small,
densely branched units known as building blocks [12] and, generally, these units are
interconnected by interblock segments with a length (IB-CL) of 5–8 glucose residues.
Type 4 amylopectins possess the longest IB-CL (7–8), whereas IB-CL in the other
structural types are shorter [11].
In an earlier investigation [13], we analysed the thermal properties of a range
of starches representing all four types of amylopectin structures. Interestingly, we
found a strong, positive correlation between the gelatinization onset temperature (To)
and IB-CL. This was remarkable as the samples were randomly selected and
represented a wide selection of genotypes and tissues. This suggested that starch
granules share a common and universal principle of their architechture, in which IBCL of the amylopectin component plays a key role: The longer the inter-block
segment, the more flexible it becomes and the better it adapts to stabilise the parallel
arrangement of the double-helices in the crystalline lamella [14], as many of the interblock segments make up the connection of the amorphous and crystalline lamellae
[3]. This much resembles the model of starch by Waigh et al. [4], who described the
behaviour of amylopectin as a side-chain liquid crystal. As we found that the
correlation of To and IB-CL included all four types of amylopectin internal structures,
it also included the different crystalline polymorphs of the granules. However, only
two B-type crystalline starches were included in the investigation (edible canna and
lesser yam), of which edible canna starch possessed a considerably lower
gelatinization temperature than expected based on its molecular structure [13]. From
our and others previous works [15-19], we noted that potato starch granules have a
low To comparable to that of edible canna. The question arose, therefore, whether
potato and canna starch granules, with their apparently "abnormal" gelatinization
behaviour, represent an unusual architechture giving rise to properties different from
what would be expected based on their molecular structure, which is similar to that of
lesser yam [8, 11, 12]. This would be of special interest as potato starch, in particular,
has been considered as a model substance for the typical B-crystalline structure. In
this work we therefore reinvestigated the thermal properties of all three B-crystalline
starches (potato, edible canna and lesser yam) and included one additional B-type,
namely shoti starch. This starch, deposited in rhizomes of turmeric, Curcuma
zedoaria, is also known under the names of zedoary, white turmeric, and kentjur
(Wikipedia, 2017), and has been shown to contain a very high phosphate content –
even higher than potato [20]. We also analyzed the crystalline polymorphic pattern
and the swelling capacity of the granules. Furthermore, their morphology and
molecular composition was analysed prior to and after treatment in dilute
hydrochloric acid (lintnerization). We found that these B-type crystalline starches
essentially can be divided into two distinct groups based on their structural and
thermal properties.
2 Experimental
2.1 Materials
Starch was extracted from potato (Solanum tuberosum, variety Dianella) as described
previously [21]. Shoti (Curcuma zedoaria) starch was kindly provided by Dr K.
Yamamoto, Japan, and edible canna (Canna edulis) and lesser yam (Dioscorea
esculenta) starches were generous gifts from Dr K. Sriroth, Thailand. Isoamylase (EC
3.2.1.68) from Pseudomonas sp., 1000 U/mL, and pullulanase (EC 3.2.1.41) from
Klebsiella planticola, 700 U/mL, were purchased from Megazyme International
(Bray, Wicklow, Ireland). All other chemicals and solvents were of ACS certified
grade.
2.2 Microscopy
Native and acid hydrolysed starch suspensions were observed under bright field light
and cross polarized light using a binocular microscope (Nikon Microscope, Eclipse
80i) equipped with real time viewing (Q-capture Pro™). A QImaging digital camera
(QICAM fast 1394, Canada) was used for the image capture.
2.3 Wide angle X-ray scattering (WAXS)
X-ray diffractograms of starch samples were obtained with a Rigaku Ultima IV X-ray
diffractometer. The native starches were kept in a desiccator over saturated K2SO4
solutions (aw 0.97) at room temperature for 14 days prior to X-ray diffraction
measurements. The hydrated native starches were then packed tightly into a round
aluminium holder and WAXS patterns were recorded with operating conditions of
target voltage 40 kV, current 44 mA, scanning range 3–30°, scan speed 1.00°/min,
step time 0.95, divergence slit width 0.5°, scatter slit width 0.5°, sampling width
0.03°, and receiving slit width 0.3 mm. Jade software (Material Data Inc. CA, USA)
was employed for the calculation of the relative crystallinity (RC).
2.4 Differential scanning calorimetry (DSC)
TA Instruments, Q2000 differential scanning calorimeter (DSC) equipped with a
thermal analysis data station and data recording software (TA Instruments, Universal
Analysis 2000) was used to measure the gelatinization parameters of the B-type
starches. Starch dispersions in excess water (starch/water ratio = 1:3) were
equilibrated for 3 h at room temperature before DSC analysis. The scanning
temperature range and the heating rates were 10 °C to 110 °C and 5 °C/min,
respectively, and the thermogram was recorded with deionized water as a reference.
The transition temperatures reported are the onset (To), peak (Tm), and conclusion (Tc)
temperatures. The enthalpy of gelatinization (ΔH) was estimated by integrating the
area between the thermogram and a base line under the peak and was expressed as J/g
of dry starch.
2.5 Swelling factor
The swelling factor of the native B-type crystalline starches was determined
according to the method of Tester and Morrison [22]. Starch (50 mg, db) dispersions
in deionized water (5 mL) were heated (55–95 °C) in screw cap tubes in a shaking
water bath for 30 min. The sample tubes were then cooled rapidly to 20 °C in an ice
water bath and 0.5 mL of Blue Dextran (Pharmacia MW 2×106, 5 mg/mL) was added
and the contents were mixed gently by inverting tubes. The tubes were centrifuged at
1500 × g for 10 min and the absorbance of the supernatant obtained from samples and
the reference tube that contained no starch was measured at 620 nm. The swelling
factor was reported as the ratio of the volume of the swollen starch granules to the
volume of the dry starch.
2.6 Lintnerization
Lintnerization was conducted following the method of Robin et al. [23]. Starch
granules were mixed into 2.2 N HCl (25 mg/mL) at 25 or 45 °C and the suspesion
was gently stirred daily. Aliquots (15 µL) were diluted, centrifuged (15 min, 1800 ×
g), and the supernatant was analysed for total carbohydrate content with the phenolsulfuric acid reagent [24] to follow the hydrolysis of the granules. At 80% hydrolysis,
the residual starch granules were centrifuged, washed twice with 0.1 M NaOAc to
neutralize the solution and then washed five additional times with water. The final
starch residues (lintners) were lyophilized.
2.7 Unit chain profile of amylopectin and lintners
Lyophilized amylopectin, previously separated from amylose in butanol-isoamyl
alcohol mixture essentially as described by Klucinec and Thompson [25] but with
minor modifications [11, 26], or lyophilized lintners were dissolved in 90% DMSO
(50 mg/mL) by stirring on a boiling water bath. The samples were then diluted with
0.01 M NaOAc buffer, pH 5.5, to a final concentration of 5 mg/mL. An aliquote (0.5
mL) was treated with isoamylase and pullulanase (1 µL each) overnight at room
temperature (~22 °C). The debranched samples were then analysed by highperformance anion-exchange chromatography coupled to pulsed amperometric
detection (HPAEC-PAD) as decribed previously [11]. Untreated lintners were also
analysed by HPAEC using the same gradient as for debranched samples. The results
are given as average of duplicate analyses.
2.8 Amylose composition
The composition of amylose was analysed by debranching whole starch as described
for the debranching of amylopectin. The samples were then analysed on a column of
Sepharose CL 6B [8]. Long chains of amylose were defined as chains eluting in the
void volume and short chains of amylose were chains eluting between the long chains
and chains of amylopectin. All results are average values of duplicate analyses.
2.9 Composition of phosphate esters
Glucose 3-phosphate and glucose 6-phosphate were analysed with HPAEC following
the method of Blennow et al. [27].
3 Results
3.1 Granular characteristics
Microscopy images in polarized light of the four starches analyzed in this
investigation showed that all starches possessed birefringence with the well-known
"Maltese cross" extending from a distal hilum (Fig. 1). The birefringence pattern is an
effect of the directional organisation of the double-helices in the crystalline lamellae
towards the granule surface [28]. The granules of canna starch were large with a long
axis up to 100 µm, which was about the same size as the potato starch granules in
accordance with previous reports [29-31]. Shoti granules were smaller with
approximate lengths ≤ 45 µm. Leonel [32] found a similar size distribution between
15–45 µm, and Jane el al. [29] showed that the granules of shoti are flat with a
thickness of 8–12 µm at the center of the lenticularly shaped granules and only about
1–3 µm at the edges. The sizes of the granules of lesser yam were comparable with
shoti granules, but they possessed a different morphology. Many of these granules had
shapes resembling three-sided pyramidals. The "Maltese cross" extended from the top
of the pyramidals. Some granules had clearly irregular "Maltese crosses", some even
with zig-zag patterns (examples are highlighted by arrows in Fig. 1).
All four starch granules possessed the typical B-type crystalline pattern [33,
34] when analysed by WAXS (Fig. 2). However, minor differences in the peak
patterns were apparent. Thus, the typical doublet at 22–24° 2θ was less pronounced in
shoti and lesser yam starch compared to potato and canna starch granules. The same
was true for the doublets at 10.0–11.5° and 14.5–15.0° 2θ. Furthermore, the peak at
19–20° 2θ was weaker in shoti and lesser yam starch. The relative crystallinity of the
starches demonstrated a crystallinity ranging 26–32% (Table 1), which agreed with
previous data on potato, canna and lesser yam [30, 31, 35].
The thermal properties of the starch granules were analysed by DSC (Table 1).
Potato and canna starch granules possessed similar thermal characteristics. Thus, the
onset gelatinization temperature (To) occurred at 57.5 and 60.4 °C for potato and
canna, respectively. The peak gelatinization temperature (Tp) was 61.5 and 65.7 °C,
respectively, and the gelatinization range (Tc–To) was 10.5 and 10.8 °C, respectively.
Generally, these values were similar to those found elsewhere using similar conditions
for the gelatinization [35-37]. Shoti and lesser yam were similar to each other, but
possessed considerably higher To (74.5–74.8 °C) and Tp (77.1–77.5 °C) than potato
and canna starch. In addition, Tc–To was much narrower (6.5–6.6 °C). In contrast, all
four starches possessed rather similar gelatinization enthaphy values (15.1–16.5 J/g).
Swelling of the starch granules over the temperature range 55–95 °C
illustrated that potato starch granules swelled most extensively of all samples (Fig. 3).
Already at 55 °C, the swelling factor (defined as swollen volume/initial volume of
dried starch [22]) approached 20. Maximum swelling was achieved at 85 °C, after
which the granules collapsed. A high swelling capacity at comparatively low
temperatures compared to other starches is a well known property of potato starch
granules [38-40]. Canna granules swelled considerably less than potato, but the
temperature range of the swelling was the same, i.e., swelling started at 55 °C and
increased up to 85 °C, after which the granules disintegrated. At the maximum
temperature the swelling factor of canna granules was 47 as compared to 78 for potato
granules. Shoti and lesser yam starch granules were more stable than potato and
canna; they only started to swell at a temperature above 65 °C. The maximum
temperature was 95 °C, at which the swelling factor of shoti and lesser yam granules
was 37 and 44, respectively.
3.2 Molecular composition
The covalently linked phosphate ester content of the starch granules was analysed by
HPAEC and is presented in Table 2. Potato starch granules contained a total of 21.2
nmol phosphate/mg starch, of which 15.2 nmol/mg represented glucose 6-phosphate
(G6P) and 6.0 nmol/mg glucose 3-phosphate (G3P), giving a ratio of G6P/G3P of 2.5.
Gomand et al. [35] found that the content of G6P varied between 9.2–20.4 nmol/mg
in different potato varieties. Shoti starch granules contained about three times more
phosphate than potato and almost all surplus was in the form of G6P. This result was
in accordance with a previous report [20]. Edible canna and lesser yam starch
contained considerably less phosphate than potato starch, 13.7 and 14.7 nmol/mg,
respectively, and these values were also of the same order of magnitude as found in
the literature [41-43]. In canna starch, the ratio of G6P/G3P was similar to that in
potato, whereas in lesser yam it was only 1.2, mostly due to a relatively large presence
of G3P (Table 2).
The composition of polysaccharide components in the starch granules was
investigated by analysing the unit chain composition of debranched starch by GPC on
Sepharose CL 6B. With this technique the long chains typical of amylose elute in
front of the shorter chains of amylopectin origin (Fig. 4). The chromatograms
suggested fairly similar amylose levels in all samples between 18.6–24.8% (Table 3).
Amylose levels in potato starch varies greatly with the cultivar [37, 40] and the
concentration obtained here is well within reported values. The amylose content of
canna was also in the order of that described by others (23–28%)[30, 41]. However,
Ren [31] recently reported a lower content of amylose (19.23%) in edible canna
starch. Amylose content in shoti starch was previously found to be between 28 and
41.8% [44-47], i.e. higher than for the sample analysed here (24.3%). On the other
hand, Leonel et al. [32] reported 20.53% amylose in shoti starch. The amylose content
of starches from different varieties of yam (D. esculenta) was found to be 20~24% by
Jayakody et al. [48], whereas Srichuwong et al. [30] reported the amylose content in
lesser yam to be 14.2%, as compared to 18.6% in this report. However, it is doubtful
if the samples from different varieties of lesser yam can be compared as the size of the
granules was much smaller in the lesser yams analysed previously [30, 48]. In
addition, the morphology was different and in the sample analysed by Srichuwong et
al. [30] the crystalline polymorph was of the C-type (mixture of A- and B-types)
rather than B-type found in this work.
The structure of amylose can be partly characterized from the proportion of
long and short chains (LCAM and SCAM, respectively) as obtained by analysing the
debranched starch by gel-permeation chromatography (GPC) on Sepharose CL 6B
and defined in Fig. 4. Potato amylose contained the largest amount of LCAM (15.4%),
but the smallest amount of SCAM (7.1%), thus giving the highest ratio of LCAM:SCAM
of 2.2 (Table 3). Canna also possessed much LCAM, but intermediate amounts of
SCAM resulting in a ratio of 1.3. Shoti and lesser yam amylose was different. They
contained a lower amount of LCAM than SCAM and, thus, the ratio was below one in
these starches.
The unit chain profile of amylopectin was analysed by HPAEC (Fig. 5).
Potato and canna amylopectin possessed very similar chain profiles (Fig. 5a) with
only very small differences in the quantity of short (DP 6–38) and long chains (DP ≥
39). On the other hand, the unit chain profiles of shoti and lesser yam amylopectin
were different from potato and canna but were similar between theselves (Fig. 5b).
Difference plots between shoti and potato and between lesser yam and potato,
respectively (Fig. 5c), demonstrate that shoti amylopectin contained less of the
shortest chains with DP between 6–15, and similarly yam had less of chains with DP
6–13. Of these short chains, those with DP 6–8 have been named "fingerprint" Afpchains, because of their characteristic profile in different plants [8]. The rest of the
short chains with DP 9–14 is here depicted as "S1". The remaining part of the short
chains, depicted "S2", were found in larger amounts than the corresponding chains in
potato. Difference plot for potato and canna showed very minor difference (data not
shown).
The relative number of the chain categories is given in Table 4. Afp-chains
were present in about double amount in potato and canna compared to shoti and yam
amylopectins. S1-chains represented 31.0 and 32.4% of the chains in canna and
potato, respectively, whereas only 25.1 and 27.6% in shoti and lesser yam. As S2chains were more abundant in shoti and yam, the overall content of short chains was
rather similar in the four amylopectin samples. The largest difference was found
between potato and shoti amylopectin with 88.8 and 85.1% short chains, respectively,
and instead, therefore, shoti amylopectin had the largest amount of long chains. The
average chain lengths of the short (SCL) and long chains (LCL) are also given in
Table 4. As the lengths of both chain types were longer in shoti and lesser yam, the
average chain length (CL) for the entire amylopectin in these two starches was longer
(24.8 and 23.0, respectively) than in potato and canna (21.4 and 21.9, respectively).
3.3 Lintnerization
Lintnerization of the starch granules was performed at two temperatures, 25 and 45
°C (Fig. 6). It is well known that the solubilisation rate is very sensitive to the
temperature [49, 50]. Thus, at 25 °C the solubilisation took days rather than hours as
at 45 °C. The inital rate of solubilisation at 25 °C up to roughly 30% was similar
among the samples, after which the solubilisation rate became diverse for the different
samples (Fig. 6a). Potato starch granules reached about 80% solubilisation after 53
days, lesser yam reached this level after more than 80 days. The time to reach the
same solubilisation level at 45 °C was only 25 h for potato and canna starch. In
contrast, lesser yam reached 80% solubilisation after about 74 hours. At this time
shoti still had not reached the same level and the solubilisation continued slowly until
80% solubilisation finally was achieved after 145 h (not shown in Fig. 6b).
Srichuwong et al. [51] linterized starch at 35 °C, at which potato starch was
hydrolyzed somewhat faster than edible canna, i.e. in line with what our result
suggests.
The size-distributions of the dextrins in the residual granules (lintners) at 80%
solubilisation are shown in Fig 7. The well established fact that the composition of the
dextrins in lintners of potato starch depends on the temperature at which the
lintnerization is performed [15, 50, 52] was confirmed in this work. Similarily, the
composition of the the lintners of canna starch granules depended on the temperature.
At 45 °C the lintner possessed considerably more of large dextrins with DP ≥ 30 than
at 25 °C, whereas the amount of smaller dextrins with a peak approximately at DP
15–16 predominated at 25 °C. Interestingly, the dextrin composition in the lintners of
shoti and lesser yam was not notably affected by the temperature, as in both starches
the small dextrins predominated regardless the temperature (Fig. 7). The average DP
of the lintners was approximately 20–21 in all samples, except in potato and canna
lintners made at 45 °C, in which DP was 26.6 and 27.0, respectively (Table 5). The
lintners were also debranched (not shown) and the apparent chain length (CL) of the
dextrins in potato and canna lintners was higher at 45 °C than at 25 °C (Table 5). A
slight increase of the average number of chains (NC = DP/CL) at the higher
temperature was also found, especially in canna. In contrast, CL and NC were not
affected by the temperature in shoti and lesser yam lintners. The CL of shoti was
higher (~18) than in lesser yam (~15). However, it should be noted that the CL values
(and therefore also NC) are only apparent, as the debranching of the dextrins with
pullulanase and isoamylase is not complete, due to the fact that certain branches in the
lintners are resistant to the enzymes [50, 52].
Albeit the phosphate levels generally were lower in the lintners (Table 5) than
in the original starch granules (Table 2), suggesting that the phosphate groups to a
major part was found in the amorphous layers, in agreement with previous findings
[15, 20], details in the phosphate contents were different and suggested rather
complex distributions. Potato and canna lintners retained more of the phosphate when
lintnerized at 45 °C compared to 25 °C (Table 5). In fact, in the canna lintner the
phosphate concentration was even slightly higher (14.7 nmol/mg) than in the original
starch (13.7 nmol/mg), due to a high remaining concentration of G6P, which resulted
in a high ratio of G6P/G3P. In shoti starch the decrease in phosphate concentration
after lintnerization was for the most part due to the removal of G6P, whereas the
remaining concentration of G3P even increased, which rendered the ratio of G6P/G3P
to decrease from 8.3 (Table 2) to 1.9 and 2.8 at 25 and 45 °C, respectively. In
contrast, the decrease of phosphate in the yam lintner was more moderate and did not
affect the distribution of G6P and G3P as the ratio largely remained unchanged.
The morphology of the residual starch granules was largely unchanged after
the lintnerization (Fig. 8), despite the fact that only 20% of the original starch
remained in these granules. The granules also possessed the "Maltese cross" in
polarised light, which was in agreement with a previous report for potato lintners [15]
and as summarized by Kainuma and French [53]. This showed that the orientation of
short chains in the crystalline lamellae remained intact in all starches. The "growth
rings" of potato and canna granules were clearly distinguished after lintnerization
(Fig. 8a and 8b). However, in shoti and, especially, in lesser yam, these layers
appeared to resemble "slices" rather than "rings" (Figs. 8c-f). In fact, lesser yam
appeared to contain two distinct types of granules, of which one type possessed the
granular "slices" being partly separated from each other at this stage of lintnerization
(Figs. 8e and 8f), whereas the other type did not have "slices" but were fragmenting
into smaller pieces. The former type of granules were associated with the smooth
"Maltese crosses", whereas the latter granules had the irregular crosses and appeared
more dense with sharper birefringence in the images (Fig. 8d). Whether shoti
possessed granular "slices" or "rings" remained somewhat unclear, but some of the
layers appeared to reach the edge of the granules, suggesting more flat structure that
possibly were a kind of "slices" (Fig. 8c). All granules were very fragile at this stage
of solubilisation and fragmented extensively after freeze-drying.
4 Discussion
In this investigation we followed up our earlier finding that potato and canna starch
granules are exceptions from the general trend that the gelatinization onset
temperature (To) of any kind of normal or waxy starch granules correlates positively
with the inter-block chain length (IB-CL) in the amylopectin component [13]. We
now extended the analyses of thermal properties with shoti starch granules and found
that this starch possessed very similar gelatinization temperature (To, Tm and Tc) as
lesser yam (Table 1). The temperature range for the melting (Tc–To) was also similar
to yam. This indicated that potato and canna formed one pair and shoti and lesser yam
formed another pair with respect to their gelatinization properties, which was
unexpected as they all had amylopectin of type 4 molecular structure and, thus,
similar IB-CL values between 7–8 [9, 11], suggesting no correlation with the
gelatinization temperature. This was interesting because, like potato starch, shoti
starch contains very high content of phosphate, whereas both yam and canna has
much lower, and between themselves similar, phosphate levels (Table 2). The
influence of phosphate on gelatinization of starch granules remains unclear, as
contradicting results have been reported [15, 17, 37, 54-56]. Moreover, the
distribution of G3P and G6P differed in the lintners in an unpredicted way in the
different starches, i.e. was not correlated to any thermal effects. More data is required
to discern such variation. Hence, our data suggest that phosphate content had less
influence on the thermal properties of starch granules than other molecular structures.
However, it should here be noted that once gelatinised, the phosphoester groups
clearly affects starch properties due to a higher degree of hydration, which results in
more viscous and clearer solutions or gels [47, 57].
The enthalphy of gelatinization (∆H) is a measure of the unwinding of the
double-helices in the crystalline lamellae and the melting of the lamellae. This was
shown to correlate positively with the length of the external chains (ECL) [13]. ECL
was not measured in this investigation, but according to previous works, ECL of
potato, canna and yam is fairly similar (13.5–15.0) [8, 58], which agreed with the
similar ∆H-values, and also with the rather similar relative crystallinities (Table 1).
Using molecular modeling techniques, G3P, but not G6P, has been shown to interfere
with the double-helix structure by introducing local amorphization of the crystallites
[57], but the present result did not detect any destabilising effect (i.e. ∆H) by G3P, nor
G6P, on the crystallites. Indeed, shoti starch, with the by far highest content of
phosphate, also possessed the highest ∆H (Table 1). Possibly, the destabilising effect
of G3P is below the detectable limit of DSC.
The swelling properties of starch granules has also been considered highly
dependent on the phosphorylation of amylopectin [37, 39, 59], based on the well
known fact that potato starch granules swell extensively and at a comparatively low
temperature. Indeed, this was once again confirmed in this work (Fig. 3). However,
we could not confirm that this property of potato granules is defined by its high
phosphate content, because shoti starch granules swelled considerably less, and at
higher temperature, despite a three times higher content of phosphate. Instead, canna
granules behaved similar to potato starch with respect to the temperature interval at
which they swelled, but their degree of swelling remained lower. In contrast, lesser
yam behaved as shoti starch and, in fact, the swelling factor of yam was slightly
higher than for shoti at 95 °C, despite more than four times lower phosphate content.
Therefore, one can conclude that also swelling, which is closely related to the
gelatinization properties (cf. Table 1 and Fig. 3), was unaffected by the phosphate
content in the granules.
If phosphate does not exert an influence on the thermal properties of the Bcrystalline starch granules, then some other factor, or factors, must be responsible,
such as the packing of the starch components in the granules or the molecular
structure of the components, or possibly both, as the structure would affect the
packing. As shown in Fig. 2, all starch samples possessed the typical B-crystalline
polymorph with signature peaks at 5.6°, 22.0° and 24.0° 2θ [34]. Minor differences
were detected, however. For example, the double peak at 22° and 24° 2θ was sharper
in potato and canna starch granules. Further, B-type crystalline starches tend to
possess a rather poorly resolved doublet peak at 14.5–15° 2θ , but lesser yam
possessed a more unusually shaped emerging single peak at this position, which was
in common with other D. esculenta varieties [48]. As some Dioscorea starches were
reported to have C-type crystalline structures [30, 48], and A-type crystallinity is
associated with a single peak at about 15° 2θ [34], we can not exclude a small
proportion of A-type unit cells in lesser yam. Nevertheless, the detected small
differences in WAXS patterns were largely confined to the two apparent groups of Bcrystalline samples (potato and canna versus shoti and yam), but the significance with
regards to the crystalline structure is more difficult to unravel.
Overall, the relative crystallinity (RC) based on the WAXS results did not
obey any special pattern among the samples (Table 1). Muhr et al. [60] concluded that
∆H of gelatinization as measured by DSC is not related to the degree of crystallinity
in the granules and Cook and Gidley [61] found that ∆H primarily measures the
melting of helical structures, but many other physical events induced by heating play
also important roles. This was confirmed here, as the measured ∆H did not reflect the
RC based on WAXS. However, the gelatinization temperature can be considered as a
measure of the stability of the crystallites, and the temperature range (Tc–To) is an
indication of the homogeneity of the crystallites [19, 38, 62]. For shoti and yam
starch, Tp was high and Tc–To was narrow showing that the crystallites in these
granules were more stable and homogenous in structure in comparison to potato and
canna.
Even though the crystallites generally are formed by the major group of short
chains of amylopectin, the properties of the crystallites are affected by the chains in
the amorphous lamella [63] and especially the length of the inter-block segments (IBCL) have been shown to be important [13, 14]. In addition to amylopectin, amylose is
assumed to be found to a large part in the amorphous lamellae and, if interacting with
inter-block segments – or even more likely, with the longer inter-cluster segments [3]
– it might possess an indirect effect on the melting property of the amylopectin
crystallites. Indeed, amylose was suggested to be mixed with amylopectin in potato
starch, as opposed to maize starch where it is separated from amylopectin and no or
only weak interactions between the two components exist [64, 65]. It is also possible
that amylose segments can form double helical segments with the short amylopectin
chains if amylose transverses the stacks of lamellae [66, 67]. The amylose content in
the B-crystalline starch granules in this work was fairly similar (Table 3) and did not
suggest any correlation with the thermal properties or the swelling of the granules. On
the other hand, the structure of the amylose component was different in the starches
and appeared to correlate with the gelatinization properties. Thus, potato and canna
starch contained considerably more of long amylose chains, so that the ratio of
LCAM:SCAM was about two to three times higher than in shoti and yam (Table 3, Fig.
4). It is well known that amylose exist both as linear and branched molecules and the
proportion varies between types of starches [68]. Branched amylose has generally
larger molecular weight than linear amylose, but the chains are comparatively short
[68-71]. Thus, a high amount of short chains (low ratio of LCAM:SCAM) implies a
higher proportion of branched amylose and vice versa. Along with this line, shoti and
yam starch had more branched amylose, whereas potato and canna were enriched in
linear amylose. At this time, however, a discussion of how and to what extent these
differences will affect the properties of the granules rests solely on speculation.
Details in the unit chain distribution of the amylopectin component revealed
that the amount of Afp-chains (DP 6–8) was considerably larger in potato and canna
compared to shoti and yam (Fig. 5, Table 4). As these chains are too short to
effectively participate in the double-helical structures [72], they have been considered
to introduce defects into the crystallites rather than contributing to their structure [73,
74]. Therefore, this could explain at least partially the weaker crystal structure in
potato and canna starch. In addition, also other short chains with DP up to ~14 (here
designated "S1") were more common in potato and canna and appeared therefore to
have a negative effect on the crystal stability. Many investigators have found the same
effect, i.e. amylopectin chains with DP ≤ 12 tend to be associated with low
gelatinization temperature [30, 35, 75, 76] and high swelling power [77] and a
possible explanation is that these chains are shorter than the optimal length to fill up
the crystalline lamella [78]. This explanation is not immediately clear, however,
especially as chains with DP around 12 are commonly found in lintnerized starches
[19, 51, 52, 79] and corresponds fairly well to the thickness of the crystalline
lamellae, which is 4–6 nm (or DP 12~17) [66, 74]. A considerable number of chains
with DP ≤ 12 are probably A-chains, which – if correctly defined – are chains not
carrying other chains at the C6-positions [80], but others are B-chains (which carry
other chains) and some A-chains are, in fact, longer than DP 12 [8]. It is possible that
some A-chains form double helices with other A-chains along the backbone of
amylopectin [3]. In contrast to A-B double-helices, such A-A double-helices have
been very little considered hitherto, if at all. In the A-B chain union the double-helix
is stabilised by the branching point [81], but this is not the case in the A-A doublehelix and, especially if in addition being comparatively short, it would be less stable
than the majority of the double-helices, contributing to a lower gelatinization
temperature in potato and canna.
Shoti and yam starches had more S2-chains (DP 15–38) than potato and canna
(Fig. 5, Table 4). These chains correspond mostly to the major part of the short,
internal chains (so called BSmajor-chains) [8] and they were suggested to be involved
in the interconnection of building blocks along the backbone, but probably they are
also to a large extent forming short branches to the backbone [82]. I.e., these chains
possess the comparatively long inter-block segments with DP 7–8 (typically found in
type 4 starches) and more of these chains will therefore stabilise the crystals in line
with the finding that IB-CL correlates positively with To [13].
The starch granules reacted very different when treated in dilute hydrochloric
acid and, again, they could be divided into two groups. As expected, the rate of
hydrolysis was much slower at 25 °C compared to 45 °C (Fig. 6). Initially, all starches
were hydrolysed by approximately the same rate at the respective temperature. During
this period, most of the amorphous regions are removed by the acid [23] and it
appeared that the constitution of these parts, which are formed mostly by the internal
chains of the amylopectin and a large part of amylose, were similar in the starches.
The second, slower stage was different, however. Potato and canna were hydrolyzed
with a higher rate at 45 °C and the difference to shoti and yam was large. It has been
suggested that during this stage both amorphous and crystalline structures are
simultaneously degraded, as the relative crystallinity in the granules do not increase
anymore [83]. As the molecular distribution of the single chain fraction found around
the peak at DP 15~17 [23, 84] do not gradually change into shorter chains by time, it
was suggested that the double-helices slowly dissolves into the solution and finally
degrades further in the solution rather than as being part of the residual granules [15].
As this step was clearly different in the two groups of starches, it suggested that the
crystallites – and the residual amorphous areas – possessed different resistance
towards the solubilisation of the helices at the higher temperature. As all starches
were B-crystalline, the difference could not depend on the organisation of the doublehelices into the crystalline pattern per se, but other factors must be involved, such as
the degree of defects caused by short chains, amylose tie chains, etc.
Another striking result was the fact that the molecular composition of potato
and canna lintners was strongly dependent on the temperature, whereas shoti and yam
lintners were not (Fig. 7). The apparent chain length (CL) also increased at the higher
temperature in potato and canna, and the number of chains per molecule (NC)
increased especially in canna, showing that some branches escaped the acid
hydrolysis (Table 5). This phenomenon has been described earlier for potato starches
with different amylose contents as well as phosphate contents and was suggested to
depend on a reorganisation of the double-helices in a fashion similar to what happens
during annealing [15, 50, 52]. Apparently, the double-helices in shoti and yam starch
granules were more fixed into their positions, which prevented similar movements in
these starches. Indeed, the gelatinisation temperature interval was considerably
narrower for shoti and lesser yam (Table 1), suggesting that these granules already
possessed a well optimised crystalline structure, which would make them more stiff in
comparison to the crystallites in potato and canna.
Interestingly, an optimised crystalline structure did not necessarily lead to an
optimal organisation of the crystallites in a radial fashion inside starch granules. Fig. 8
shows that the granules generally retained the polarisation cross after lintnerization. In
lesser yam, many granules possessed a very irregular cross (Fig. 8d), suggesting
poorer orientation of the crystallites. Indeed, in these granules there were no sign of
the "granular rings" even after lintnerisation, suggesting a lack of higher-range order
as well. Yet, the size-distribution of the dextrins in yam linters was not different from
shoti (or the other starches at 25 °C) (Fig. 7). Another remarkable finding was that the
other part of the yam granules, i.e. those with a regular polarization cross, possessed
"granular slices" instead of the common rings (Figs. 8e-f). This suggested a very well
developed organisation inside these granules with the crystallites found in one
direction from the distal hilum at the top of the pyramide-like granules to the base of
the pyramidals, instead of a radial organisation. A quite similar organisation of the
helices was described for the giant starch granules from the pseudo-bulbs of the
orchid Phajus grandifolius [85]. Some of these granules, which are B-crystalline,
have protruberances extending from an ogival body. The protruberances have almost
planar "growth rings", much resembling slice-like structures, that at the edges turn
and appear to fuse together [85]. Possibly the "slices" in lesser yam granules also fuse
together at the ends, but these parts were eventually removed by the acid. The internal
structure of shoti starch granules was not as clearly observed as in yam, but several
granules appeared to possess layers that ended at the edges (Fig. 8c), thus resembling
the structures in lesser yam or Phajus grandifolius granules.
5 Conclusions
The results in this investigation, which included four starch samples, showed
that the B-crystalline starch granules can be divided into two types based on their
structure and granular properties. However, as more samples are investigated a
broader diversity might be found. Nevertheless, one structural type is represented by
potato and edible canna starch granules with growth rings, or shells, and radially
organised double-helices. The amylose component in these starches is comparatively
little branched, whereas the amylopectin component has much short chains, especially
Afp-chains, which possibly contribute to defected, less organised crystallites
characterised by low melting temperatures as well as low swelling temperatures. The
other structural type is represented by shoti and lesser yam granules, which appear to
possess more planar internal structures with double-helices oriented in one direction
from a proximal hilum to a distal, more flattened end. In this group amylose is more
branched and amylopectin contains less short chains. This results in more organised
and rigid crystallites with high melting and swelling temperatures, as well as a higher
resistance to acid hydrolysis. It is noted that all starches in this investigation have
amylopectin with an internal structure classified as type 4, for which one of the
structural signatures is long inter-block segments, resulting in expected high
gelatinization temperatures. In this aspect, potato and canna starch are exceptions
possessing unexpectedly low gelatinization temperatures. While this work have
highlighted a correlation between the molecular and granular structure with the
thermal properties of B-crystalline starch granules, the actual relationship between the
crystalline polymorph, thermal properties and branch configuration and the proposed
higher “blocklet” structure remains to be understood.
Acknowledgement
Ms Jeanette Wikman is acknowledged for skillful laboratory assistance.
Authors declare no conflict of interest.
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Legends to figures
Figure 1. Microscopy in polarized light of starch granules from potato, edible canna,
shoti and lesser yam. Irregular birefringence patterns in the latter are highlighted by
white arrows. Bar = 50 µm.
Figure 2. Wide-angle X-ray scattering pattern of B-crystalline starch granules.
Figure 3. Swelling factor of B-crystalline starch granules at 55–95 °C.
Figure 4. Gel permeation chromatography on Sepharose CL 6B of debranched
starches of potato () and canna () (upper figure), and of shoti () and lesser yam
() (lower figure). Long chains of amylose (LCAM), short chains of amylose (SCAM),
and chains of amylopectin are indicated.
Figure 5. (a) Unit chain profiles obtained by HPAEC of amylopectin from potato ()
and canna (), and (b) from shoti () and lesser yam (). (c) Difference plots of
shotii () and lesser yam () versus potato amylopectin. Chain categories are
indicated.
Figure 6. Lintnerization of B-crystalline starch granules at 25 °C (a) and 45 °C (b):
Potato (); canna (); shoti (); lesser yam (). Note the different time scales in (a)
and (b). Note also that 80% solubilisation with shoti starch, in fact, was obtained after
145 h (not shown in this figure).
Figure 7. HPAEC analysis of the composition of dextrins in lintners after about 80%
solubilisation at 25 °C (----) and 45 °C (–––––).
Figure 8. Microscopy in polarized light of lintnerized starch granules from potato,
edible canna, shoti and lesser yam. (f) shows a magnification of granules in (e).
Arrows in (c), (e) and (f) highlight granules that appears to have "slices" rather than
granular "rings". Arrows in (d) highlight granules with irregular "Maltese cross" and
no "slices". Bar = 50 µm.
Table 1. Relative crystallinity and gelatinization parameters of starch granulesa
RC
To
Tp
Tc
Tc–To
∆H
(%)
(°C)
(°C)
(°C)
(°C)
(J/g)
Potato
32
57.3
61.5
67.8
10.5
15.1
Canna
26
60.4
65.7
71.2
10.8
16.1
Shoti
30
74.5
77.1
81.1
6.6
16.5
Lesser yam
30
74.8
77.5
81.3
6.5
15.7
Sample
a
RC = Relative crystallinity; To, Tp, and Tc = Onset, peak, and conclusion
temperature of gelatinization, respectively; ∆H = Enthalpy of gelatinization.
Values shown are average of duplicates.
Table 2. Phosphate content in starcha
Sample
Total P
G6P
G3P
(nmol/mg)
(nmol/mg)
(nmol/mg)
Potatob
21.2
15.2
6.0
2.5
Canna
13.7
9.6
4.1
2.3
Shoti
63.2
56.4
6.8
8.3
Lesser yam
14.7
8.0
6.7
1.2
a
G6P/G3P
P = Phosphate; G6P = Glucose 6-phosphate; G3P = Glucose 3-phosphate.
From [15].
Values are average of duplicates.
b
Table 3. Amylose content and structure based on debranched starches analysed by
GPC on Sepharose CL 6Ba
Sample
Amylose
LCAM
SCAM
(%)
(%)
(%)
Potato
22.5
15.4
7.1
2.2
Canna
24.8
14.0
10.8
1.3
Shoti
24.3
8.9
15.4
0.6
Lesser yam
18.6
8.4
10.2
0.8
a
LCAM:SCAM
LCAM = Long chains of amylose; SCAM = Short chains of amylose.
Values are average of duplicates.
Table 4. Relative amount of chains (mol%) and chain lengths in amylopectin
Sample
Afp
S1
S2
S
L
CL
SCL
LCL
Potato
7.0
32.4
49.4
88.8
11.2
21.4
16.7
58.5
Canna
6.5
31.0
50.0
87.5
12.5
21.9
16.7
58.6
Shoti
3.9
25.1
56.1
85.1
14.9
24.8
18.4
61.9
Lesser yam
3.5
27.6
56.2
87.3
12.7
23.0
18.0
59.4
DP-division of chain segments based on Fig. 5: Afp = 6–8, S1 = 9–14, S2 = 15–38, S
= ≤ 38, L = ≥ 39, CL = chain length, SCL = CL of S-chains, LCL = CL of L-chains.
Values are average of duplicates.
Table 5. Molecular structure of lintnerized starches
Lintner
Temp.
DP
CLa
NCb
(°C)
Potato
Canna
Shoti
Lesser yam
a
b
Total P
G6P/G3P
(nmol/mg)
25
21.0
14.9
1.4
11.3
1.9
45
26.6
17.7
1.5
15.9
1.9
25
20.3
14.8
1.4
10.4
2.1
45
27.0
16.1
1.7
14.7
3.7
25
21.3
17.9
1.2
36.3
1.9
45
20.2
18.3
1.1
27.0
2.8
25
19.9
15.0
1.3
9.2
1.1
45
20.9
15.1
1.4
7.5
1.0
Apparent average chain length after debranching with isoamylase and pullulanase.
Average number of chains per molecule = DP/CL.
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