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Supramolecular and molecular structures of potato starches and their digestion features
Journal Pre-proofs Supramolecular and molecular structures of potato starches and their digestion features Dongling Qiao, Zhong Wang, Hao Li, Binjia Zhang, Huayin Pu, Fatang Jiang, Siming Zhao PII: DOI: Reference:
S0141-8130(19)37227-7 https://doi.org/10.1016/j.ijbiomac.2019.10.214 BIOMAC 13715
To appear in:
International Journal of Biological Macromolecules
Received Date: Revised Date: Accepted Date:
7 September 2019 4 October 2019 24 October 2019
Please cite this article as: D. Qiao, Z. Wang, H. Li, B. Zhang, H. Pu, F. Jiang, S. Zhao, Supramolecular and molecular structures of potato starches and their digestion features, International Journal of Biological Macromolecules (2019), doi: https://doi.org/10.1016/j.ijbiomac.2019.10.214
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© 2019 Published by Elsevier B.V.
1
Supramolecular and molecular structures of potato starches
2
and their digestion features
3 4
Dongling Qiaoa, Zhong Wanga, Hao Lia, Binjia Zhangb,*,[email protected], Huayin Puc, Fatang
5
Jianga, Siming Zhaob
6 7
aGlyn
8
Engineering, Hubei University of Technology, Wuhan 430068, China
9
bGroup
O. Phillips Hydrocolloid Research Centre at HBUT, School of Food and Biological
for Cereals and Oils Processing, College of Food Science and Technology, Key Laboratory
10
of Environment Correlative Dietology (Ministry of Education), Huazhong Agricultural University,
11
Wuhan 430070, China
12
cSchool
13
710021, China
14 15
*Corresponding
of Food and Biological Engineering, Shaanxi University of Science and Technology, Xi’an
author.
Highlights
16
CP2 starch had a lower ratio of starch branching enzyme to soluble starch synthase.
17
CP2 starch showed a higher proportion of long amylopectin chains.
18
CP2 starch showed increased crystallites and thickened lamellae.
19
CP2 starch showed increased resistance to enzyme digestion and hydrothermal effects.
20
CP2 starch showed increased paste stability under heating.
21
1
22
Abstract
23
This work inspects the supramolecular/molecular structures and digestion rate of potato starches
24
(BEM, C7H, CP2 and CP4) as affected by starch biosynthetic enzymes. Among the starches, CP2
25
had a lower digestion rate with a higher paste heating stability. Regarding this, predominantly
26
enzyme-sets (i) and (ii) were revealed to produce amylopectin chains. For CP2, the reduced activity
27
ratio of starch-branching enzymes to soluble starch synthases allowed more long amylopectin chains
28
(polymerization degree ≥ 34). Such molecular features tended to increase the crystallites and thicken
29
the lamellae. With similar surface morphology and amylose content, the bulk density of chain
30
packing in CP2 supramolecular structures could be increased. Then, there were an increase in the
31
resistance of starch structures to hydrothermal effects, and a reduction in the enzyme hydrolysis rate.
32
Also, the increased long amylopectin chains played roles in increasing the paste stability during
33
heating with shearing and in reducing the digestion rate.
34
Keywords: starch; supramolecular structure; digestion rate
35 36 37 38 39
2
40 41
1. Introduction Starch, as a storage carbohydrate in green plants, is normally a food stuff that offers energy for
42
humans. The properties of starch have close relations to the quality of related food products. In
43
particular, the digestion of starch in foods can release glucose components; this food stuff digestion
44
event has been found to affect the occurrence of potential metabolic diseases, e.g., the cardiovascular
45
diseases, and the Type II diabetes [1, 2]. The pasting of starch displays effects on the gelling and
46
thickening characteristics of foods [3]. Thus, great efforts have been made to rationally develop
47
starch resources with related digestion and pasting features.
48
In green plants such as potato and maize, starch is primarily biosynthesized by four classes of
49
biosynthetic enzymes, which include the ADP-glucose pyrophosphorylase (viz., AGPase), the starch
50
branching enzymes (viz., SBEs), the starch synthases (viz., SSs), and the starch debranching enzymes
51
(viz., DBEs) [4]. Mainly two kinds of starch molecules, amylose and amylopectin, are produced. The
52
glucan chains of starch molecules can assemble on different scales to form the multi-scale
53
supramolecular structures; these structures involve the granule, the growth rings, the lamellae, the
54
polymorphs, and the helices [5-8]. The supramolecular and molecular structures of starch have been
55
confirmed to affect its physicochemical properties. For instance, the supramolecular structures of
56
unprocessed starch contain tightly assembled chains and are more resistant to the enzyme hydrolysis;
57
this fact can make the digestion rate of untreated starch several times lower than that of fully cooked
58
counterpart [9-11]. Hence, it necessitates understanding the properties of starch on its structural
59
features resulting from the actions of starch biosynthetic enzymes.
60 61
Potato tuber is an important agro-product for foods consumed worldwide. Starch, as the main component of potato tuber, shows versatile applications for food and other industries. Researches 3
62
have explored the supramolecular features, involving granule morphology, semicrystalline lamellae,
63
crystallites and double-helices [12, 13], as well as the molecular features such as the molar mass and
64
the molecule size distribution [14]. Also, earlier findings reported the changes in the supramolecular
65
and molecular characteristics of starch and the evolutions in its properties such as pasting [12].
66
However, though the biosynthetic enzymes can govern starch structures and thus the properties, there
67
is still limited study on the digestion and pasting features of potato starches from a view of
68
supramolecular/molecular structures as tailored by the biosynthetic enzymes. This prevents us from
69
well establishing the biosynthesis-structure-property links for potato starch.
70
Hence, four kinds of potato tubers (namely BEM, C7H, CP2 and CP4) cultivated in Hubei
71
province of China were used as the raw materials. The potato starches were isolated from the four
72
tubers, and then series of techniques spanning different scales were used to inspect the
73
supramolecular/molecular structures and digestion rate of potato starches as tailored by starch
74
biosynthetic enzymes. The results affirmed that CP2, among those four starches, displayed a reduced
75
digestion rate and an increased paste stability under heating and shearing conditions. Thereafter, how
76
the digestion and pasting performance of CP2 differ from the other three starches was discussed by
77
establishing the related biosynthesis-structure-property links. The present results are valuable for the
78
rational screening and usage of potato starches for foods with demanded pasting and digestion
79
features.
80 81 82
2. Materials and methods
83
2.1 Materials 4
84
Four cultivars of potatoes, namely BEM15-6 (BEM), C7H005-6 (C7H), Huashu-2 (CP2),
85
Huashu-4 (CP4), cultivated in Hubei province of China were used. All of the potatoes were
86
harvested in 2018. The A-3176 α-Amylase from porcine pancreas with 25 unit/mg activity, and the
87
10115 amyloglucosidase from Aspergillus niger with 65 unit/mg activity were commercially
88
acquired from Sigma-Aldrich. A YLS16A moisture analyzer (Techcomp Ltd., China) was applied to
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measure the moisture contents of the starches.
90 91
2.2 Isolation of starches
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A method [15] with proper modifications was used to isolate starch samples from the potatoes.
93
The tubers were peeled and cut, and were added into excess aqueous sodium metabisulphite (0.3 %
94
w/v), followed by blending using a blender under ambient conditions. A screen mesh of 106 μm was
95
used to filter the puree. The acquired filtrate was kept at 4°C overnight to allow the sediment of
96
starch. Then, the slurry underwent centrifugation at 8000 rpm for 30 min to obtain starch precipitate;
97
the precipitate was washed thrice with 0.1 mol/L NaCl solution, thrice with pure water, and twice
98
with absolute ethanol. The collected starch was air-dried at 35 °C for 48 h to obtain dried starch. The
99
moisture contents of BEM, C7H, CP2 and CP4 were 11.97%, 10.07%, 9.19% and 10.62%,
100
respectively.
101 102 103
2.3 Scanning electron microscopy (SEM) The microscopic morphology of starch granules were observed by a JEOL-Model 6390
104
scanning electron microscope under 15.0 kV voltage. Each of the starch powders was mounted on a
105
metal stage covered with conductive tape, and then coated with a gold layer. The starches were 5
106
observed at 1000×magnification.
107 108 109
2.4 Laser diffraction analysis A Mastersizer 2000 laser-diffraction analyzer (Malvern, UK) was applied to measure the starch
110
granule size distributions. The starch powder was gradually placed into the reservoir containing
111
distilled water at 26 ± 2 °C. The measurement would be started when an obscuration value higher
112
than 10% was acquired. The Mastersizer 2000 software (Version 5.60) was used to acquire the
113
related parameters of granule size distributions.
114 115 116
2.5 Small angle X-ray scattering (SAXS) The SAXS experiments were conducted on the BL19U2 SAXS beamline at Shanghai
117
Synchrotron Radiation Facility (Shanghai, China). The slurries containing 20% starch were prepared
118
and kept at room temperature for two hours; then, the slurries were used as the starch samples for
119
measurements. The data were recorded by a Pilatus 1 M detector. The background was the scattering
120
from the empty sample cell with water. The data were background subtracted and normalized. The
121
scattering data of ca. 0.008 < q < 0.30 Å-1 were used. The scattering vector q was equal to 4πsinθ/λ
122
in which 2θ is the scattering angle.
123
The average thickness (d) of semicrystalline lamellae and those of their crystalline (dc)
124
and amorphous (da) lamellar components were acquired based on the linear correlation
125
function f(r) in Eq. (1) [16].
126
f (r )
0
I (q )q 2 cos(qr )dq
0
I (q )q 2 dq
(1)
6
127 128
In this equation, r (nm) indicates the distance in real space.
129 130
2.6 X-ray diffraction (XRD)
131
The starch crystalline structure was inspected by a D8 Advance X-ray powder diffractometer
132
(Bruker, USA), operated at 40 kV and 30 mA. The XRD data in 2θ range of 4°-40° were acquired,
133
under 0.02° step size and 0.5 s per step. The PeakFit software was applied to obtain the relative
134
degree of crystallinity Xc (%) for the starches [17, 18] based on Eq. (2).
135 136
Xc
n i 1
At
Aci
(2)
137 138
Here, At is the total area of the pattern; Aci is the area under the diffraction peak with index i.
139 140 141
2.7 Size exclusion chromatography (SEC) The chain features of debranched starch molecules were evaluated based on a method with
142
modifications [19]. Each starch was dissolved in DMSO/LiBr solvent with 0.5% (w/w) LiBr at 80°C
143
overnight. The concentration of starch in DMSO/LiBr was about 2 mg/mL. Then, the starch
144
molecules were debranched by isoamylase using a reported method [20] and were used for the
145
measurements for chain length distributions (CLDs) of debranched starch samples. An Agilent 1100
146
Series SEC system was applied, and the GRAM precolumn, GRAM 100 and GRAM 1000 columns
147
(PSS, Germany) under a flow rate of 0.6 mL/min were used. For the debranched starch molecules
7
148
with linear glucans, the hydrodynamic volume (Vh) could be transformed into the degree of
149
polymerization (DP) according to the Mark–Houwink equation [21].
150
Also, the number CLDs of debranched starch were fitted using a model to obtain the activity
151
ratios of three categories of biosynthetic enzymes for starch, which includes SBE, DBE and SSs [22].
152
Note that the term “enzyme set” is used to represent a group of the three enzymes (SS, SBE, and
153
DBE) irrespective of the specific informs. Here, primarily two enzyme-sets, (i) and (ii), took part in
154
the synthesis of amylopectin CLDs.
155 156 157
2.8 Pasting properties The pasting properties of the starches were evaluated using an RVA4500 rapid visco analyzer
158
(RVA) (Perten, Sweden). 3 g of the starch was added into a sample canister with 25 g of distilled
159
water in advance. The impeller of RVA was rotated in the sample canister to make the starch
160
granules suspended in the water. The canister and impeller were positioned to begin the trial. The test
161
involved six stages with a total testing time of 23 min, as detailed in an earlier study [23].
162 163 164
2.9 Digestion behaviors The in vitro digestion of the starches was conducted using a reported method [16] with
165
modifications. A tube with starch (90.0 mg), deionized water (6.0 mL) and sodium acetate buffer at
166
pH 6.0 (10.0 mL) was incubated for 10 min at 37 °C, followed by addition of enzyme buffer solution
167
containing 42 unit/mL α-amylase and 42 unit/mL amyloglucosidase. At specific time points, the
168
concentration of glucose in the digestion system was determined by glucose oxidase/peroxidase
169
reagent (Megazyme). A standard glucose solution at 1 mg/mL glucose concentration was used. The 8
170
amount of digested starch (SD) could be calculated with Eq. (3).
171
SD(%) Asample
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100L / mL 100% 162 (3) 10 210 90mg 180 Aglucose
173 174
In this equation, Asample or Aglucose, the absorbance for the digestion solution of starch or the glucose
175
standard; 10 × 210, the multiple from 100 μL aliquots to 21.0 mL of the whole solution; 162/180, the
176
ratio of glucose to starch in weight. The digestion rate of starch was obtained with the logarithm of the slope (LOS) plot in Eq. (4)
177 178
and the non-linear curve fitting [16, 24]. The LOS plot, shown by the slope for digestion pattern
179
(ln(dCt/dt)) versus time (t), was used to show the number of digestion stages with changed digestion
180
rates during the whole digestion. And the non-linear curve fitting with first-order kinetics (Eq. (5))
181
was used to generate the rate coefficient of starch digestion (kfitting).
182
dC t k t In(C k ) (4) dt
183
In
184
Ct C 1 e k t (5)
185
Here, Ct (%) indicates the digested starch amount at a time t (min); C∞ (%) is the estimated amount
186
of starch hydrolyzed at the end of digestion; k (min−1) is the digestion rate coefficient of starch.
187 188
2.10 Statistical analysis
189
Data were presented as means ± standard deviations. A statistical difference of P < 0.05 was
190
termed to be significant. Statistical analysis was carried out in Microsoft excel 2016 (Redmond, WA, 9
191
USA).
192 193 194
3. Results and discussion
195
3.1 Granule characteristics
196
Fig. 1 shows the SEM photographs for the four kinds of starch granules. The starch granules
197
exhibited predominantly in oval, spherical and olive shapes, as well as a smooth exterior surface
198
without any micropores. Among those four starches, no substantial differences in the morphological
199
characteristics could be seen. The present results are consistent with earlier findings regarding potato
200 201 202
starch microscopic features [25].
203 204 205 206
The granule size distributions for the four starches are presented in Fig. S1 in the supplementary
207
material. The starches showed similar distribution profiles having one peak ranging from 10 to 100
208
μm. Table 1 summarizes the particle size parameters for starches. And those parameters values were
209
relatively larger than that for water chestnut starch published in our previous work [26]. This
210
indicates that the granules for four potato starches are larger than those for water chestnut starch. In
211
Table 1, the particle size parameters (D[4, 3], and d(0.5)) revealed that BEM, C7H and CP4 had a
212
larger granule size than did CP2. The width of the granule size distribution can be indicated by the
213
span value [27]. The span values for the starches were in an order of BEM C7H CP4 CP2,
214
affirming that BEM and CP2 exhibited the narrowest and the broadest size distributions,
215
respectively. 10
216 217 218
3.2 Lamellar structural characteristics Fig. 2 includes the synchrotron SAXS patterns for the four starches. A visible scattering peak
219
emerged at around 0.065 Å-1, ascribed to the semicrystalline lamellae of starch with an average
220
thickness (interlamellar repeat distance) of about 9 nm [14]. The average thicknesses of the
221
semicrystalline (d), crystalline (dc) and amorphous (da) lamellae were calculated based on the linear
222
correlation function, and the results are recorded in Table 1. The results show that d was between
223
about 9.01 nm and 9.08 nm, close to the average lamellar thickness for other starches, such as water
224
chestnut starch [23, 26, 28]. Among the starches, CP2 possessed the largest d, with relatively large
225
dc and da values. In addition, the lamellar peak intensity is positively related to the electron density
226
difference between the crystalline and amorphous lamellae [23]. In Fig. 2, C7H showed a peak
227
intensity similar to those for BEM and CP4 but somewhat higher than that for CP2. This result
228
indicates a larger difference in the compactness between the crystalline and amorphous lamellae for
229
C7H as well as BEM and CP4.
230 231 232 233 234 235
3.3 Crystalline structural characteristics The main kinds, A-, B- and V-types, of starch polymorphs can be clearly distinguished by XRD
236
[29, 30]. Fig. 3 shows the XRD curves for the BEM, C7H, CP2 and CP4. The starches exhibited a
237
B-type polymorphic structure, as affirmed by a characteristic diffraction peak at ca. 5.6°, an intense 11
238
diffraction peak at around 17°, and several weaker diffraction peaks at approximately 15°, 20°, 22°,
239
and 24°. Fig. 3 also includes the relative crystallinity degree (Xc) for the starches. The Xc was in the
240
range of ca. 44.8% to 47.7%, comparative to the crystallinity for rice starches [31] and water
241
chestnut starch [26]. Also, CP2 and C7H had the largest Xc, followed by the smallest Xc for BEM and
242
an intermediate Xc for CP4.
243 244 245 246
3.4 Features of whole and debranched starch molecules To evaluate the features of whole starch molecules, we inspected the weight size distributions
247
(w (logVh)) of branched starch molecules (Vh, hydrodynamic volume; Rh, the corresponding
248
hydrodynamic radius). The molecule size distribution profiles are detailed in Fig. 4a. The four
249
starches displayed mainly two size distribution regions, i.e., the amylose fractions at smaller Rh
250
values and the amylopectin fractions at larger Rh values. A size parameter, the average Rh of amylose
251
molecules (Rh, amylose) [32], was calculated (Table 2). It is noted that C7H had a Rh, amylose value
252
close to that for BEM and CP4, but larger than that of CP2. And Rh, amylose values for those potato
253
starches were slightly smaller than that for water chestnut starch and apparently smaller than cassava
254
starch [26].
255
Fig. 4b shows the chain length distributions (CLDs) of the debranched starch molecules. The
256
CLDs were shown as weight distributions w (logVh). In Fig. 4b, two peaks existed for amylopectin
257
chains with DP < 100, and multiple smaller bumps were observed for amylose chains with DP ≥ 100
258
[19, 33]. The two peaks of amylopectin chains were related to the branches confined to a single
259
lamella range (Ap1) and those spanning more than one single lamella range (Ap2). Table 2 records 12
260
the ratio of Ap2 chains to Ap1 chains, i.e., the height ratio (hAp2/Ap1) for the Ap2 peak maximum to
261
that of Ap1 peak. Apparently, BEM and C7H had a hAp2/Ap1 value smaller than that for CP2 and
262
larger than that for CP4. That is, BEM and C7H contained an intermediate amount of amylopectin
263
long chains (Ap2) somewhere between those for CP4 and CP2. Additionally, the amylose contents
264
for the starches were acquired from the weight CLDs by calculating the ratio of the area under the
265
curve of whole amylose range to the area under the curve of whole starch distribution and are shown
266
in Table 2. The amylose content for the starches was 19.46%-21.15% without statistical differences.
267 268 269 270 271 272
3.5 Parameterized biosynthetic enzyme activities The weight CLDs were transformed into the number distributions Nde(DP) (Fig. 5), according to
273
the equation wde(DP) = DP2 Nde(DP) [21]. Then, a method was used to fit the number CLDs to yield
274
the relative activities of core starch biosynthetic enzymes, i.e., SS, SBE and DBE [22]. In Fig. 5, the
275
starches had two distinct peaks, corresponding to short amylopectin chains with DPs of 6-33 (Ap1 in
276
Fig. 4b) and long amylopectin chains with DPs of 34-67. Fitting to the number CLDs of amylopectin
277
chains confirmed mainly two enzyme-sets, i.e., enzyme-set (i) fitted from short amylopectin chains
278
(shown as an orange fit pattern) and enzyme-set (ii) fitted from long amylopectin chains (shown as a
279
pink fit pattern).
280
The fitting generated six parameters within enzyme-sets (i) and (ii), viz., γ(i) and γ(ii), activity
13
281
ratio of DBE to SS; β(i) and β(ii), activity ratio of SBE to SS; h(i) and h(ii), relative contribution of
282
specific enzyme-set to whole CLDs. The results are shown in Table 2. Among the starches, CP2
283
displayed lower β(i), β(ii), γ(i) and γ(ii), indicating lower activity ratios of SBE:SS and DBE:SS within
284
enzyme-sets (i) and (ii). For these four parameters, there were relatively larger values for CP4 and
285
intermediate values for BEM and C7H. In addition, CP2 and BEM had the lowest and the highest h(i)
286
respectively, indicating the corresponding contribution of chains from enzyme-set (i) to whole
287
amylopectin chains. CP4 exhibited lowest h(ii) values which suggested the lowest contribution of
288
chains from enzyme-set (ii) to whole amylopectin chains.
289 290 291 292 293 294 295
3.6 Pasting properties The pasting parameters of the four starches are collected in Table 3. CP2 possessed the highest
296
pasting temperature (Tps), with the lowest value for CP4 and the intermediate values for the other two
297
starches. This reveals that CP2 starch granules were most resistant to the swell and rupture effects in
298
water during pasting [14]. The peak viscosity (ηpk) presented a similar value for the four starches.
299
CP2 and C7H displayed a lower breakdown viscosity (Δηbd) than did BEM and CP4; this indicates
300
that the former two starches had a higher paste stability under heating and shearing [34].
301
Additionally, the setback viscosity (Δηsb) was in an order of CP2 > C7H > BEM > CP4. This trend
302
confirmed that CP4 had the highest paste stability under cooling conditions with shearing, since Δηsb 14
303
is negatively correlated with the paste cooling stability [23]. Hence, CP4 had a relatively high paste
304
stability during cooling with shearing, while CP2 showed a relatively high paste stability under
305
heating and shearing.
306 307 308
3.7 Digestion behaviors
309
The digestion plots, LOS plots and their fit curves for the starches are shown in Fig. 6. The
310
digestion rate and the digested starch amount at 12 h (C12) are listed in Table 3. The starches showed
311
one linear range on LOS plot curves, revealing a single-phase digestion manner under first-order
312
kinetics. Note that starch digestion is pseudo-first-order, as the digestion rate of starch can be
313
affected by the enzyme concentrations used [24]. The resulting digestion rate for starch from the
314
LOS plot is inherently inaccurate, because LOS plot uses the numerical derivative of discrete rate
315
data points. The non-linear curve fitting under first-order kinetics was used to generate the digestion
316
rate coefficient kfitting [26, 35]. In Table 3, no significant variations could be observed among BEM,
317
C7H and CP4, whose digestion rates were apparently higher than that of CP2 and were comparative
318
with water chestnut starch [26]. When the digestion time prolonged to 12 h, CP2 displayed a
319
proportion of digested starch evidently lower than those for BEM, C7H and CP4. The present results
320
confirmed that CP2 showed reduced susceptibility to the enzyme hydrolysis.
321 322 323 324 15
325 326 327
3.8 Discussion on biosynthesis-structure-property relationship for starch The biosynthesis of starch and its chain assembly in the supramolecular structures are related to
328
different kinds of biosynthetic enzymes [36, 37]. More specifically, SSs transfer ADP-glucose units
329
to the non-reducing ends of original glucans via new α-(1,4)-bonds to gradually form starch chains
330
[36, 38]. SBEs lead to new branch chains by cleaving α-(1,4)-bonds and transferring the reducing
331
ends of released chains to the glucose residues of pristine or other chains via new α-(1,6)-bonds [39,
332
40]. DBEs remove unsuitably located branch chains that hinder chain clustering and crystallization
333
[41, 42].
334
In the present work, enzyme-set (i) mainly produced short amylopectin chains with DP ≤ 33,
335
aligned in single crystalline lamella regions to form the semicrystalline lamellae, and part of long
336
amylopectin chains of DP 34-67, protruding from single lamella regions to enter the adjacent
337
amorphous lamella regions (and probably the following crystalline lamellae) [22]. The rest of long
338
amylopectin chains were mainly produced by enzyme-set (ii); these chains could protrude from the
339
single crystalline lamella regions and remain in the adjacent amorphous lamella space [22, 43]. Also,
340
the amylopectin chains formed helical components via intra-molecular hydrogen bonding; the helices
341
encapsulated thirty-six water molecules to form hexagonal crystal units via hydrogen bonding; then,
342
the unit cells assembled to construct B-type polymorphs as affirmed by XRD.
343
We suggest that the variations in starch pasting behaviors resulted from the changes in the
344
multi-scale structures as regulated by the biosynthetic enzymes (Fig. 7). In particular, for CP2 with
345
reduced SBE:SS activity ratio, the suppressed SBE, with SSs elongating amylopectin glucan chains,
346
contributed to the formation of long amylopectin chains (shown by the increased hAp2/Ap1). This fact 16
347
allowed the formation of starch helices with increased lengths, increasing the amount of starch
348
crystallites (see the increased Xc) and the thicknesses of semicrystalline, and crystalline lamellae (see
349
the increased d and dc). These structural features enhanced starch structure resistance to
350
hydrothermal effects (reflected by the increased Tps). Then, as the temperature rose, the paste
351
viscosity gradually increased to ηpk, which was similar with the starches related to the swelling
352
degree of swollen granules [14]. Furthermore, as the pasting proceeded, the fully swollen starch
353
granules were disrupted gradually, leading to the occurrence of Δηbd. Note that the swollen granules
354
are the granule “ghosts” with an amorphous shell containing physically entangled chains [23]. The
355
increased long amylopectin chains for CP2 probably enhanced its chain entanglement in the ghost
356
shell, increasing the paste stability during heating with shearing (see the reduced Δηbd). Also, such
357
chain features promoted the chain reassembly during cooling, reducing the paste stability under
358
cooling (see the increased Δηsb).
359 360 361 362
For the starch digestion, mainly two enzymes, α-amylase and amyloglucosidase, exist during
363
the digestion. The digestion of starch is a reaction associated with the enzymes’ diffusion towards the
364
substrate, followed by the absorption and subsequent hydrolysis [44]. Like the discussion on pasting,
365
among those starches, CP2 possessed the increased amount of long amylopectin chains, the
366
thickened semicrystalline and crystalline lamellae, the increased proportion of crystallites, and no
367
granule surface pores. Such structural characteristics on different scales probably increased the bulk
368
compactness of molecule chain alignment in starch structures; this change could restrict the diffusion 17
369
and permeation of the enzymes within the matrices of starch. Then, this event tended to retard the
370
absorption of the enzymes to starch glucan chains at the molecular scale, and eventually reduce the
371
rate of chain hydrolysis induced by the enzymes. In contrast, when the surface micropores of starch
372
granules reduce their bulk density, the diffusion of enzymes into the granule matrices and eventually
373
the digestion of starch can be accelerated [45]. In addition, the increased ratio (hAp2/Ap1) of long
374
amylopectin chains to the short ones for CP2 played some role in suppressing the starch hydrolysis
375
by the enzymes, as hAp2/Ap1 is found to be negatively related to the digestion rate of starch [35, 46].
376 377 378
4. Conclusions
379
The present work concerns the supramolecular/molecular structures and digestion behaviors of
380
potato starches as tailored by their biosynthetic enzymes. Two starch biosynthetic enzyme-sets, viz.,
381
enzyme-set (i) and enzyme-set (ii), were confirmed to primarily synthesize the amylopectin chains.
382
Among the starches, CP2 showed reduced activity ratios of SBE:SS and DBE:SS, contributing to
383
increasing the amount of long amylopectin chains. This molecular feature should enhance the
384
formation of starch crystallites and thicken the lamellar structure. Such structural features, with
385
comparative surface morphology and amylose content, could increase the chain packing bulk density
386
in the CP2 supramolecular structures. Then, the water or enzyme permeation in starch structure
387
matrices could be suppressed, enhancing the resistance of starch structures to hydrothermal effects as
388
reflected by the elevated pasting temperature and slowing the enzyme absorption and hydrolysis.
389
Again, the increased ratio of long amylopectin chains played roles in increasing the paste stability
390
under heating and shearing for CP2 and in reducing its enzyme digestion rate. The results from this 18
391
investigation enable a better understanding of the digestion rate for potato starches based on the
392
variations in starch supramolecular and molecular structure as tailored by starch biosynthetic
393
enzymes. This understanding is of value for the rational screening and application of potato starches
394
for foods with related pasting and digestion performance.
395 396 397 398
Acknowledgments The authors acknowledge the National Natural Science Foundation of China (31801582 and
399
31601509), and the Project funded by China Postdoctoral Science Foundation (2019T120708). The
400
authors also thank Dr. Cheng Li, and Dr. Enpeng Li from Prof. Robert Gilbert’s lab at Yangzhou
401
University for their assistance on SEC experiment and analysis. Also, we thank the staffs from
402
BL19U2 beamline of National Facility for Protein Science in Shanghai (NFPS) at Shanghai
403
Synchrotron Radiation Facility, for their assistance during data collection. B. Zhang thank the Young
404
Elite Scientists Sponsorship Program by China Association for Science and Technology
405
(2018QNRC001).
406 407 408
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742-749.
528 529 530
Fig.1 SEM images for BEM, C7H, CP2 and CP4 granules. Fig. 2 Synchrotron SAXS patterns for BEM, C7H, CP2 and CP4.
531
Fig. 3 XRD patterns for BEM, C7H, CP2 and CP4.
532 533 534 535 536 537 538 539
Fig. 4 Weight size distributions of branched molecules (a), and weight chain length distributions of debranched molecules (b) from BEM, C7H, CP2 and CP4. Fig. 5 Number chain-length distributions and their fit curves for debranched molecules from BEM, C7H, CP2 and CP4. Fig. 6 Digestion plots, LOS plots and nonlinear fitting patterns for BEM, C7H, CP2 and CP4. , , fit curve of non-linear curve fitting; , linear fit curve experimental data; , LOS plot data; for LOS plot data.
540
Fig 7 Schematic representation for the variations in pasting and digestion features for CP2.
541 542 543
Table 1 Particle size distributions and lamellar parameters of BEM, C7H, CP2 and CP4 granules A Sample
BEM
C7H
CP2
CP4
D[4, 3]
38.56±0.12bB
39.46±0.42b
35.83±0.10a
37.26±0.62a,b
d(0.5)
36.11±0.10b
36.84±0.42b
33.09±0.10a
34.55±0.65a,b
span
0.77±0.02a
1.06±0b
1.19±0c
1.13±0.02b
d (nm)
9.03±0.01b
9.01±0.01a
9.08±0.01c
9.07±0.01c
da (nm)
2.47±0.00a
2.48±0.00b
2.49±0.00b
2.47±0.00a
dc (nm)
6.56±0.01b
6.53±0.01a
6.59±0.01c
6.60±0.01c
25
D[4, 3], volume weight mean diameter; d(0.5), 50% of overall granules had a size below this value (μm); span,
544
A
545
a value equal to (d(0.9) – d(0.1)) / d(0.5); d, the semicrystalline lamella thickness; da, the amorphous lamellar
546
thickness; dc, the crystalline lamellar thickness.
547
B
Values with different lowercase letters in a row have significant difference at P 0.05.
548 549
Table 2 Parameterized biosynthetic enzyme parameters of BEM, C7H, CP2 and CP4 A BEM
C7H
CP2
CP4
Rh, amylose(nm)
25.85±0.11bB
26.18±0.36b
24.52±0.03a
26.07±0.30b
hAp2/Ap1
0.993±0.006b
1.006±0.001b
1.048±0.006c
0.919±0.004a
Amylose content (%)
20.82±0a
21.15±0.01a
20.55±0.01a
19.46±0.01a
β(i)
0.0921±0.0001a,b
0.0932±0.0007a,b
0.0908±0.0005a 0.0955±0.0009b
β(ii)
0.0500±0.0001b
0.0499±0.0004a,b,c 0.0453±0.0010a 0.0511±0.0002c
(i)
0.0661±0.0001c
0.0640±0.0003b
0.0606±0.0002a 0.0662±0.0010b,c
(ii)
0.0428±0a,b
0.0427±0.0003b,c
0.0390±0.0004a 0.0436±0.0001c
h(i)
1.0806±0.0022c
1.0283±0.0063b
0.9880±0.0069a 1.0610±0.0276a,b,c
h(ii)
0.0632±0.0004b
0.0643±0.0005b
0.0655±0.0011b 0.0565±0.0003a
550
A
551
β(ii), activity ratio of SBE:SS from enzyme-set (i) or (ii); γ(i) or γ(ii), activity ratio of DBE:SS from
552
enzyme-set (i) or (ii); h(i) or h(ii), relative contribution of enzyme-set (i) or (ii) to the whole chain
553
length distributions.
554
B
Rh, amylose, average Rh of amylose molecules; hAp2/Ap1, height ratio of Ap2 peak to Ap1 peak; β(i) or
The different lowercase letters within a row indicate significant difference at P < 0.05.
555 556
Table 3 Pasting and digestion parameters for BEM, C7H, CP2 and CP4 A 26
Samples
BEM
C7H
CP2
CP4
Tps (°C)
68.6±0.25b
68.7±0.20b
71.2±0.03c
67.2±0.03a
ηpk (cP)
10630±17b
9751±104a
10243±109a,b
10265±177a,b
Δηbd (cP)
6365±38b
4943±184a
5238±157a
6743±174b
Δηsb (cP)
717±37a
1076±15b
1357±32c
566±11a
kfitting (min-1)
0.0020±0b
0.0020±0b
0.0008±0a
0.0018±0b
C12 (%)
72.0±1.2b
68.8±2.2b
56.0±0.9a
67.3±1.2b
557
A
558
kfitting, digestion rate from non-linear curve fitting; C12, digested starch amount at 12 h.
559
B
Tps, pasting temperature; ηpk, peak viscosity; Δηbd, breakdown viscosity; Δηsb, setback viscosity;
Values with the different lowercase letter in a row have significant difference at P 0.05.
560
27
S0141-8130(19)37227-7 https://doi.org/10.1016/j.ijbiomac.2019.10.214 BIOMAC 13715
To appear in:
International Journal of Biological Macromolecules
Received Date: Revised Date: Accepted Date:
7 September 2019 4 October 2019 24 October 2019
Please cite this article as: D. Qiao, Z. Wang, H. Li, B. Zhang, H. Pu, F. Jiang, S. Zhao, Supramolecular and molecular structures of potato starches and their digestion features, International Journal of Biological Macromolecules (2019), doi: https://doi.org/10.1016/j.ijbiomac.2019.10.214
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1
Supramolecular and molecular structures of potato starches
2
and their digestion features
3 4
Dongling Qiaoa, Zhong Wanga, Hao Lia, Binjia Zhangb,*,[email protected], Huayin Puc, Fatang
5
Jianga, Siming Zhaob
6 7
aGlyn
8
Engineering, Hubei University of Technology, Wuhan 430068, China
9
bGroup
O. Phillips Hydrocolloid Research Centre at HBUT, School of Food and Biological
for Cereals and Oils Processing, College of Food Science and Technology, Key Laboratory
10
of Environment Correlative Dietology (Ministry of Education), Huazhong Agricultural University,
11
Wuhan 430070, China
12
cSchool
13
710021, China
14 15
*Corresponding
of Food and Biological Engineering, Shaanxi University of Science and Technology, Xi’an
author.
Highlights
16
CP2 starch had a lower ratio of starch branching enzyme to soluble starch synthase.
17
CP2 starch showed a higher proportion of long amylopectin chains.
18
CP2 starch showed increased crystallites and thickened lamellae.
19
CP2 starch showed increased resistance to enzyme digestion and hydrothermal effects.
20
CP2 starch showed increased paste stability under heating.
21
1
22
Abstract
23
This work inspects the supramolecular/molecular structures and digestion rate of potato starches
24
(BEM, C7H, CP2 and CP4) as affected by starch biosynthetic enzymes. Among the starches, CP2
25
had a lower digestion rate with a higher paste heating stability. Regarding this, predominantly
26
enzyme-sets (i) and (ii) were revealed to produce amylopectin chains. For CP2, the reduced activity
27
ratio of starch-branching enzymes to soluble starch synthases allowed more long amylopectin chains
28
(polymerization degree ≥ 34). Such molecular features tended to increase the crystallites and thicken
29
the lamellae. With similar surface morphology and amylose content, the bulk density of chain
30
packing in CP2 supramolecular structures could be increased. Then, there were an increase in the
31
resistance of starch structures to hydrothermal effects, and a reduction in the enzyme hydrolysis rate.
32
Also, the increased long amylopectin chains played roles in increasing the paste stability during
33
heating with shearing and in reducing the digestion rate.
34
Keywords: starch; supramolecular structure; digestion rate
35 36 37 38 39
2
40 41
1. Introduction Starch, as a storage carbohydrate in green plants, is normally a food stuff that offers energy for
42
humans. The properties of starch have close relations to the quality of related food products. In
43
particular, the digestion of starch in foods can release glucose components; this food stuff digestion
44
event has been found to affect the occurrence of potential metabolic diseases, e.g., the cardiovascular
45
diseases, and the Type II diabetes [1, 2]. The pasting of starch displays effects on the gelling and
46
thickening characteristics of foods [3]. Thus, great efforts have been made to rationally develop
47
starch resources with related digestion and pasting features.
48
In green plants such as potato and maize, starch is primarily biosynthesized by four classes of
49
biosynthetic enzymes, which include the ADP-glucose pyrophosphorylase (viz., AGPase), the starch
50
branching enzymes (viz., SBEs), the starch synthases (viz., SSs), and the starch debranching enzymes
51
(viz., DBEs) [4]. Mainly two kinds of starch molecules, amylose and amylopectin, are produced. The
52
glucan chains of starch molecules can assemble on different scales to form the multi-scale
53
supramolecular structures; these structures involve the granule, the growth rings, the lamellae, the
54
polymorphs, and the helices [5-8]. The supramolecular and molecular structures of starch have been
55
confirmed to affect its physicochemical properties. For instance, the supramolecular structures of
56
unprocessed starch contain tightly assembled chains and are more resistant to the enzyme hydrolysis;
57
this fact can make the digestion rate of untreated starch several times lower than that of fully cooked
58
counterpart [9-11]. Hence, it necessitates understanding the properties of starch on its structural
59
features resulting from the actions of starch biosynthetic enzymes.
60 61
Potato tuber is an important agro-product for foods consumed worldwide. Starch, as the main component of potato tuber, shows versatile applications for food and other industries. Researches 3
62
have explored the supramolecular features, involving granule morphology, semicrystalline lamellae,
63
crystallites and double-helices [12, 13], as well as the molecular features such as the molar mass and
64
the molecule size distribution [14]. Also, earlier findings reported the changes in the supramolecular
65
and molecular characteristics of starch and the evolutions in its properties such as pasting [12].
66
However, though the biosynthetic enzymes can govern starch structures and thus the properties, there
67
is still limited study on the digestion and pasting features of potato starches from a view of
68
supramolecular/molecular structures as tailored by the biosynthetic enzymes. This prevents us from
69
well establishing the biosynthesis-structure-property links for potato starch.
70
Hence, four kinds of potato tubers (namely BEM, C7H, CP2 and CP4) cultivated in Hubei
71
province of China were used as the raw materials. The potato starches were isolated from the four
72
tubers, and then series of techniques spanning different scales were used to inspect the
73
supramolecular/molecular structures and digestion rate of potato starches as tailored by starch
74
biosynthetic enzymes. The results affirmed that CP2, among those four starches, displayed a reduced
75
digestion rate and an increased paste stability under heating and shearing conditions. Thereafter, how
76
the digestion and pasting performance of CP2 differ from the other three starches was discussed by
77
establishing the related biosynthesis-structure-property links. The present results are valuable for the
78
rational screening and usage of potato starches for foods with demanded pasting and digestion
79
features.
80 81 82
2. Materials and methods
83
2.1 Materials 4
84
Four cultivars of potatoes, namely BEM15-6 (BEM), C7H005-6 (C7H), Huashu-2 (CP2),
85
Huashu-4 (CP4), cultivated in Hubei province of China were used. All of the potatoes were
86
harvested in 2018. The A-3176 α-Amylase from porcine pancreas with 25 unit/mg activity, and the
87
10115 amyloglucosidase from Aspergillus niger with 65 unit/mg activity were commercially
88
acquired from Sigma-Aldrich. A YLS16A moisture analyzer (Techcomp Ltd., China) was applied to
89
measure the moisture contents of the starches.
90 91
2.2 Isolation of starches
92
A method [15] with proper modifications was used to isolate starch samples from the potatoes.
93
The tubers were peeled and cut, and were added into excess aqueous sodium metabisulphite (0.3 %
94
w/v), followed by blending using a blender under ambient conditions. A screen mesh of 106 μm was
95
used to filter the puree. The acquired filtrate was kept at 4°C overnight to allow the sediment of
96
starch. Then, the slurry underwent centrifugation at 8000 rpm for 30 min to obtain starch precipitate;
97
the precipitate was washed thrice with 0.1 mol/L NaCl solution, thrice with pure water, and twice
98
with absolute ethanol. The collected starch was air-dried at 35 °C for 48 h to obtain dried starch. The
99
moisture contents of BEM, C7H, CP2 and CP4 were 11.97%, 10.07%, 9.19% and 10.62%,
100
respectively.
101 102 103
2.3 Scanning electron microscopy (SEM) The microscopic morphology of starch granules were observed by a JEOL-Model 6390
104
scanning electron microscope under 15.0 kV voltage. Each of the starch powders was mounted on a
105
metal stage covered with conductive tape, and then coated with a gold layer. The starches were 5
106
observed at 1000×magnification.
107 108 109
2.4 Laser diffraction analysis A Mastersizer 2000 laser-diffraction analyzer (Malvern, UK) was applied to measure the starch
110
granule size distributions. The starch powder was gradually placed into the reservoir containing
111
distilled water at 26 ± 2 °C. The measurement would be started when an obscuration value higher
112
than 10% was acquired. The Mastersizer 2000 software (Version 5.60) was used to acquire the
113
related parameters of granule size distributions.
114 115 116
2.5 Small angle X-ray scattering (SAXS) The SAXS experiments were conducted on the BL19U2 SAXS beamline at Shanghai
117
Synchrotron Radiation Facility (Shanghai, China). The slurries containing 20% starch were prepared
118
and kept at room temperature for two hours; then, the slurries were used as the starch samples for
119
measurements. The data were recorded by a Pilatus 1 M detector. The background was the scattering
120
from the empty sample cell with water. The data were background subtracted and normalized. The
121
scattering data of ca. 0.008 < q < 0.30 Å-1 were used. The scattering vector q was equal to 4πsinθ/λ
122
in which 2θ is the scattering angle.
123
The average thickness (d) of semicrystalline lamellae and those of their crystalline (dc)
124
and amorphous (da) lamellar components were acquired based on the linear correlation
125
function f(r) in Eq. (1) [16].
126
f (r )
0
I (q )q 2 cos(qr )dq
0
I (q )q 2 dq
(1)
6
127 128
In this equation, r (nm) indicates the distance in real space.
129 130
2.6 X-ray diffraction (XRD)
131
The starch crystalline structure was inspected by a D8 Advance X-ray powder diffractometer
132
(Bruker, USA), operated at 40 kV and 30 mA. The XRD data in 2θ range of 4°-40° were acquired,
133
under 0.02° step size and 0.5 s per step. The PeakFit software was applied to obtain the relative
134
degree of crystallinity Xc (%) for the starches [17, 18] based on Eq. (2).
135 136
Xc
n i 1
At
Aci
(2)
137 138
Here, At is the total area of the pattern; Aci is the area under the diffraction peak with index i.
139 140 141
2.7 Size exclusion chromatography (SEC) The chain features of debranched starch molecules were evaluated based on a method with
142
modifications [19]. Each starch was dissolved in DMSO/LiBr solvent with 0.5% (w/w) LiBr at 80°C
143
overnight. The concentration of starch in DMSO/LiBr was about 2 mg/mL. Then, the starch
144
molecules were debranched by isoamylase using a reported method [20] and were used for the
145
measurements for chain length distributions (CLDs) of debranched starch samples. An Agilent 1100
146
Series SEC system was applied, and the GRAM precolumn, GRAM 100 and GRAM 1000 columns
147
(PSS, Germany) under a flow rate of 0.6 mL/min were used. For the debranched starch molecules
7
148
with linear glucans, the hydrodynamic volume (Vh) could be transformed into the degree of
149
polymerization (DP) according to the Mark–Houwink equation [21].
150
Also, the number CLDs of debranched starch were fitted using a model to obtain the activity
151
ratios of three categories of biosynthetic enzymes for starch, which includes SBE, DBE and SSs [22].
152
Note that the term “enzyme set” is used to represent a group of the three enzymes (SS, SBE, and
153
DBE) irrespective of the specific informs. Here, primarily two enzyme-sets, (i) and (ii), took part in
154
the synthesis of amylopectin CLDs.
155 156 157
2.8 Pasting properties The pasting properties of the starches were evaluated using an RVA4500 rapid visco analyzer
158
(RVA) (Perten, Sweden). 3 g of the starch was added into a sample canister with 25 g of distilled
159
water in advance. The impeller of RVA was rotated in the sample canister to make the starch
160
granules suspended in the water. The canister and impeller were positioned to begin the trial. The test
161
involved six stages with a total testing time of 23 min, as detailed in an earlier study [23].
162 163 164
2.9 Digestion behaviors The in vitro digestion of the starches was conducted using a reported method [16] with
165
modifications. A tube with starch (90.0 mg), deionized water (6.0 mL) and sodium acetate buffer at
166
pH 6.0 (10.0 mL) was incubated for 10 min at 37 °C, followed by addition of enzyme buffer solution
167
containing 42 unit/mL α-amylase and 42 unit/mL amyloglucosidase. At specific time points, the
168
concentration of glucose in the digestion system was determined by glucose oxidase/peroxidase
169
reagent (Megazyme). A standard glucose solution at 1 mg/mL glucose concentration was used. The 8
170
amount of digested starch (SD) could be calculated with Eq. (3).
171
SD(%) Asample
172
100L / mL 100% 162 (3) 10 210 90mg 180 Aglucose
173 174
In this equation, Asample or Aglucose, the absorbance for the digestion solution of starch or the glucose
175
standard; 10 × 210, the multiple from 100 μL aliquots to 21.0 mL of the whole solution; 162/180, the
176
ratio of glucose to starch in weight. The digestion rate of starch was obtained with the logarithm of the slope (LOS) plot in Eq. (4)
177 178
and the non-linear curve fitting [16, 24]. The LOS plot, shown by the slope for digestion pattern
179
(ln(dCt/dt)) versus time (t), was used to show the number of digestion stages with changed digestion
180
rates during the whole digestion. And the non-linear curve fitting with first-order kinetics (Eq. (5))
181
was used to generate the rate coefficient of starch digestion (kfitting).
182
dC t k t In(C k ) (4) dt
183
In
184
Ct C 1 e k t (5)
185
Here, Ct (%) indicates the digested starch amount at a time t (min); C∞ (%) is the estimated amount
186
of starch hydrolyzed at the end of digestion; k (min−1) is the digestion rate coefficient of starch.
187 188
2.10 Statistical analysis
189
Data were presented as means ± standard deviations. A statistical difference of P < 0.05 was
190
termed to be significant. Statistical analysis was carried out in Microsoft excel 2016 (Redmond, WA, 9
191
USA).
192 193 194
3. Results and discussion
195
3.1 Granule characteristics
196
Fig. 1 shows the SEM photographs for the four kinds of starch granules. The starch granules
197
exhibited predominantly in oval, spherical and olive shapes, as well as a smooth exterior surface
198
without any micropores. Among those four starches, no substantial differences in the morphological
199
characteristics could be seen. The present results are consistent with earlier findings regarding potato
200 201 202
starch microscopic features [25].
203 204 205 206
The granule size distributions for the four starches are presented in Fig. S1 in the supplementary
207
material. The starches showed similar distribution profiles having one peak ranging from 10 to 100
208
μm. Table 1 summarizes the particle size parameters for starches. And those parameters values were
209
relatively larger than that for water chestnut starch published in our previous work [26]. This
210
indicates that the granules for four potato starches are larger than those for water chestnut starch. In
211
Table 1, the particle size parameters (D[4, 3], and d(0.5)) revealed that BEM, C7H and CP4 had a
212
larger granule size than did CP2. The width of the granule size distribution can be indicated by the
213
span value [27]. The span values for the starches were in an order of BEM C7H CP4 CP2,
214
affirming that BEM and CP2 exhibited the narrowest and the broadest size distributions,
215
respectively. 10
216 217 218
3.2 Lamellar structural characteristics Fig. 2 includes the synchrotron SAXS patterns for the four starches. A visible scattering peak
219
emerged at around 0.065 Å-1, ascribed to the semicrystalline lamellae of starch with an average
220
thickness (interlamellar repeat distance) of about 9 nm [14]. The average thicknesses of the
221
semicrystalline (d), crystalline (dc) and amorphous (da) lamellae were calculated based on the linear
222
correlation function, and the results are recorded in Table 1. The results show that d was between
223
about 9.01 nm and 9.08 nm, close to the average lamellar thickness for other starches, such as water
224
chestnut starch [23, 26, 28]. Among the starches, CP2 possessed the largest d, with relatively large
225
dc and da values. In addition, the lamellar peak intensity is positively related to the electron density
226
difference between the crystalline and amorphous lamellae [23]. In Fig. 2, C7H showed a peak
227
intensity similar to those for BEM and CP4 but somewhat higher than that for CP2. This result
228
indicates a larger difference in the compactness between the crystalline and amorphous lamellae for
229
C7H as well as BEM and CP4.
230 231 232 233 234 235
3.3 Crystalline structural characteristics The main kinds, A-, B- and V-types, of starch polymorphs can be clearly distinguished by XRD
236
[29, 30]. Fig. 3 shows the XRD curves for the BEM, C7H, CP2 and CP4. The starches exhibited a
237
B-type polymorphic structure, as affirmed by a characteristic diffraction peak at ca. 5.6°, an intense 11
238
diffraction peak at around 17°, and several weaker diffraction peaks at approximately 15°, 20°, 22°,
239
and 24°. Fig. 3 also includes the relative crystallinity degree (Xc) for the starches. The Xc was in the
240
range of ca. 44.8% to 47.7%, comparative to the crystallinity for rice starches [31] and water
241
chestnut starch [26]. Also, CP2 and C7H had the largest Xc, followed by the smallest Xc for BEM and
242
an intermediate Xc for CP4.
243 244 245 246
3.4 Features of whole and debranched starch molecules To evaluate the features of whole starch molecules, we inspected the weight size distributions
247
(w (logVh)) of branched starch molecules (Vh, hydrodynamic volume; Rh, the corresponding
248
hydrodynamic radius). The molecule size distribution profiles are detailed in Fig. 4a. The four
249
starches displayed mainly two size distribution regions, i.e., the amylose fractions at smaller Rh
250
values and the amylopectin fractions at larger Rh values. A size parameter, the average Rh of amylose
251
molecules (Rh, amylose) [32], was calculated (Table 2). It is noted that C7H had a Rh, amylose value
252
close to that for BEM and CP4, but larger than that of CP2. And Rh, amylose values for those potato
253
starches were slightly smaller than that for water chestnut starch and apparently smaller than cassava
254
starch [26].
255
Fig. 4b shows the chain length distributions (CLDs) of the debranched starch molecules. The
256
CLDs were shown as weight distributions w (logVh). In Fig. 4b, two peaks existed for amylopectin
257
chains with DP < 100, and multiple smaller bumps were observed for amylose chains with DP ≥ 100
258
[19, 33]. The two peaks of amylopectin chains were related to the branches confined to a single
259
lamella range (Ap1) and those spanning more than one single lamella range (Ap2). Table 2 records 12
260
the ratio of Ap2 chains to Ap1 chains, i.e., the height ratio (hAp2/Ap1) for the Ap2 peak maximum to
261
that of Ap1 peak. Apparently, BEM and C7H had a hAp2/Ap1 value smaller than that for CP2 and
262
larger than that for CP4. That is, BEM and C7H contained an intermediate amount of amylopectin
263
long chains (Ap2) somewhere between those for CP4 and CP2. Additionally, the amylose contents
264
for the starches were acquired from the weight CLDs by calculating the ratio of the area under the
265
curve of whole amylose range to the area under the curve of whole starch distribution and are shown
266
in Table 2. The amylose content for the starches was 19.46%-21.15% without statistical differences.
267 268 269 270 271 272
3.5 Parameterized biosynthetic enzyme activities The weight CLDs were transformed into the number distributions Nde(DP) (Fig. 5), according to
273
the equation wde(DP) = DP2 Nde(DP) [21]. Then, a method was used to fit the number CLDs to yield
274
the relative activities of core starch biosynthetic enzymes, i.e., SS, SBE and DBE [22]. In Fig. 5, the
275
starches had two distinct peaks, corresponding to short amylopectin chains with DPs of 6-33 (Ap1 in
276
Fig. 4b) and long amylopectin chains with DPs of 34-67. Fitting to the number CLDs of amylopectin
277
chains confirmed mainly two enzyme-sets, i.e., enzyme-set (i) fitted from short amylopectin chains
278
(shown as an orange fit pattern) and enzyme-set (ii) fitted from long amylopectin chains (shown as a
279
pink fit pattern).
280
The fitting generated six parameters within enzyme-sets (i) and (ii), viz., γ(i) and γ(ii), activity
13
281
ratio of DBE to SS; β(i) and β(ii), activity ratio of SBE to SS; h(i) and h(ii), relative contribution of
282
specific enzyme-set to whole CLDs. The results are shown in Table 2. Among the starches, CP2
283
displayed lower β(i), β(ii), γ(i) and γ(ii), indicating lower activity ratios of SBE:SS and DBE:SS within
284
enzyme-sets (i) and (ii). For these four parameters, there were relatively larger values for CP4 and
285
intermediate values for BEM and C7H. In addition, CP2 and BEM had the lowest and the highest h(i)
286
respectively, indicating the corresponding contribution of chains from enzyme-set (i) to whole
287
amylopectin chains. CP4 exhibited lowest h(ii) values which suggested the lowest contribution of
288
chains from enzyme-set (ii) to whole amylopectin chains.
289 290 291 292 293 294 295
3.6 Pasting properties The pasting parameters of the four starches are collected in Table 3. CP2 possessed the highest
296
pasting temperature (Tps), with the lowest value for CP4 and the intermediate values for the other two
297
starches. This reveals that CP2 starch granules were most resistant to the swell and rupture effects in
298
water during pasting [14]. The peak viscosity (ηpk) presented a similar value for the four starches.
299
CP2 and C7H displayed a lower breakdown viscosity (Δηbd) than did BEM and CP4; this indicates
300
that the former two starches had a higher paste stability under heating and shearing [34].
301
Additionally, the setback viscosity (Δηsb) was in an order of CP2 > C7H > BEM > CP4. This trend
302
confirmed that CP4 had the highest paste stability under cooling conditions with shearing, since Δηsb 14
303
is negatively correlated with the paste cooling stability [23]. Hence, CP4 had a relatively high paste
304
stability during cooling with shearing, while CP2 showed a relatively high paste stability under
305
heating and shearing.
306 307 308
3.7 Digestion behaviors
309
The digestion plots, LOS plots and their fit curves for the starches are shown in Fig. 6. The
310
digestion rate and the digested starch amount at 12 h (C12) are listed in Table 3. The starches showed
311
one linear range on LOS plot curves, revealing a single-phase digestion manner under first-order
312
kinetics. Note that starch digestion is pseudo-first-order, as the digestion rate of starch can be
313
affected by the enzyme concentrations used [24]. The resulting digestion rate for starch from the
314
LOS plot is inherently inaccurate, because LOS plot uses the numerical derivative of discrete rate
315
data points. The non-linear curve fitting under first-order kinetics was used to generate the digestion
316
rate coefficient kfitting [26, 35]. In Table 3, no significant variations could be observed among BEM,
317
C7H and CP4, whose digestion rates were apparently higher than that of CP2 and were comparative
318
with water chestnut starch [26]. When the digestion time prolonged to 12 h, CP2 displayed a
319
proportion of digested starch evidently lower than those for BEM, C7H and CP4. The present results
320
confirmed that CP2 showed reduced susceptibility to the enzyme hydrolysis.
321 322 323 324 15
325 326 327
3.8 Discussion on biosynthesis-structure-property relationship for starch The biosynthesis of starch and its chain assembly in the supramolecular structures are related to
328
different kinds of biosynthetic enzymes [36, 37]. More specifically, SSs transfer ADP-glucose units
329
to the non-reducing ends of original glucans via new α-(1,4)-bonds to gradually form starch chains
330
[36, 38]. SBEs lead to new branch chains by cleaving α-(1,4)-bonds and transferring the reducing
331
ends of released chains to the glucose residues of pristine or other chains via new α-(1,6)-bonds [39,
332
40]. DBEs remove unsuitably located branch chains that hinder chain clustering and crystallization
333
[41, 42].
334
In the present work, enzyme-set (i) mainly produced short amylopectin chains with DP ≤ 33,
335
aligned in single crystalline lamella regions to form the semicrystalline lamellae, and part of long
336
amylopectin chains of DP 34-67, protruding from single lamella regions to enter the adjacent
337
amorphous lamella regions (and probably the following crystalline lamellae) [22]. The rest of long
338
amylopectin chains were mainly produced by enzyme-set (ii); these chains could protrude from the
339
single crystalline lamella regions and remain in the adjacent amorphous lamella space [22, 43]. Also,
340
the amylopectin chains formed helical components via intra-molecular hydrogen bonding; the helices
341
encapsulated thirty-six water molecules to form hexagonal crystal units via hydrogen bonding; then,
342
the unit cells assembled to construct B-type polymorphs as affirmed by XRD.
343
We suggest that the variations in starch pasting behaviors resulted from the changes in the
344
multi-scale structures as regulated by the biosynthetic enzymes (Fig. 7). In particular, for CP2 with
345
reduced SBE:SS activity ratio, the suppressed SBE, with SSs elongating amylopectin glucan chains,
346
contributed to the formation of long amylopectin chains (shown by the increased hAp2/Ap1). This fact 16
347
allowed the formation of starch helices with increased lengths, increasing the amount of starch
348
crystallites (see the increased Xc) and the thicknesses of semicrystalline, and crystalline lamellae (see
349
the increased d and dc). These structural features enhanced starch structure resistance to
350
hydrothermal effects (reflected by the increased Tps). Then, as the temperature rose, the paste
351
viscosity gradually increased to ηpk, which was similar with the starches related to the swelling
352
degree of swollen granules [14]. Furthermore, as the pasting proceeded, the fully swollen starch
353
granules were disrupted gradually, leading to the occurrence of Δηbd. Note that the swollen granules
354
are the granule “ghosts” with an amorphous shell containing physically entangled chains [23]. The
355
increased long amylopectin chains for CP2 probably enhanced its chain entanglement in the ghost
356
shell, increasing the paste stability during heating with shearing (see the reduced Δηbd). Also, such
357
chain features promoted the chain reassembly during cooling, reducing the paste stability under
358
cooling (see the increased Δηsb).
359 360 361 362
For the starch digestion, mainly two enzymes, α-amylase and amyloglucosidase, exist during
363
the digestion. The digestion of starch is a reaction associated with the enzymes’ diffusion towards the
364
substrate, followed by the absorption and subsequent hydrolysis [44]. Like the discussion on pasting,
365
among those starches, CP2 possessed the increased amount of long amylopectin chains, the
366
thickened semicrystalline and crystalline lamellae, the increased proportion of crystallites, and no
367
granule surface pores. Such structural characteristics on different scales probably increased the bulk
368
compactness of molecule chain alignment in starch structures; this change could restrict the diffusion 17
369
and permeation of the enzymes within the matrices of starch. Then, this event tended to retard the
370
absorption of the enzymes to starch glucan chains at the molecular scale, and eventually reduce the
371
rate of chain hydrolysis induced by the enzymes. In contrast, when the surface micropores of starch
372
granules reduce their bulk density, the diffusion of enzymes into the granule matrices and eventually
373
the digestion of starch can be accelerated [45]. In addition, the increased ratio (hAp2/Ap1) of long
374
amylopectin chains to the short ones for CP2 played some role in suppressing the starch hydrolysis
375
by the enzymes, as hAp2/Ap1 is found to be negatively related to the digestion rate of starch [35, 46].
376 377 378
4. Conclusions
379
The present work concerns the supramolecular/molecular structures and digestion behaviors of
380
potato starches as tailored by their biosynthetic enzymes. Two starch biosynthetic enzyme-sets, viz.,
381
enzyme-set (i) and enzyme-set (ii), were confirmed to primarily synthesize the amylopectin chains.
382
Among the starches, CP2 showed reduced activity ratios of SBE:SS and DBE:SS, contributing to
383
increasing the amount of long amylopectin chains. This molecular feature should enhance the
384
formation of starch crystallites and thicken the lamellar structure. Such structural features, with
385
comparative surface morphology and amylose content, could increase the chain packing bulk density
386
in the CP2 supramolecular structures. Then, the water or enzyme permeation in starch structure
387
matrices could be suppressed, enhancing the resistance of starch structures to hydrothermal effects as
388
reflected by the elevated pasting temperature and slowing the enzyme absorption and hydrolysis.
389
Again, the increased ratio of long amylopectin chains played roles in increasing the paste stability
390
under heating and shearing for CP2 and in reducing its enzyme digestion rate. The results from this 18
391
investigation enable a better understanding of the digestion rate for potato starches based on the
392
variations in starch supramolecular and molecular structure as tailored by starch biosynthetic
393
enzymes. This understanding is of value for the rational screening and application of potato starches
394
for foods with related pasting and digestion performance.
395 396 397 398
Acknowledgments The authors acknowledge the National Natural Science Foundation of China (31801582 and
399
31601509), and the Project funded by China Postdoctoral Science Foundation (2019T120708). The
400
authors also thank Dr. Cheng Li, and Dr. Enpeng Li from Prof. Robert Gilbert’s lab at Yangzhou
401
University for their assistance on SEC experiment and analysis. Also, we thank the staffs from
402
BL19U2 beamline of National Facility for Protein Science in Shanghai (NFPS) at Shanghai
403
Synchrotron Radiation Facility, for their assistance during data collection. B. Zhang thank the Young
404
Elite Scientists Sponsorship Program by China Association for Science and Technology
405
(2018QNRC001).
406 407 408
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supramolecular structures and pasting features of adlay seed starches, Food Hydrocolloid, 83 (2018)
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using plots for first-order kinetics, Carbohydr. Polym., 87 (2012) 2189-2197.
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monoester on aggregation structures of heat-moisture treated potato starches, Carbohydr. Polym.,
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103 (2014) 228-233.
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multi-scale structure and digestion rate of water chestnut starch, Food Hydrocolloid, 91 (2019)
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311-318.
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crosslinked starch microspheres and their drug loading and releasing properties, Carbohydr. Polym.,
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74 (2008) 379-384. 22
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and amylopectin fine structure for textural properties of cooked rice grains, Food Chem., 196 (2016)
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526
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527
742-749.
528 529 530
Fig.1 SEM images for BEM, C7H, CP2 and CP4 granules. Fig. 2 Synchrotron SAXS patterns for BEM, C7H, CP2 and CP4.
531
Fig. 3 XRD patterns for BEM, C7H, CP2 and CP4.
532 533 534 535 536 537 538 539
Fig. 4 Weight size distributions of branched molecules (a), and weight chain length distributions of debranched molecules (b) from BEM, C7H, CP2 and CP4. Fig. 5 Number chain-length distributions and their fit curves for debranched molecules from BEM, C7H, CP2 and CP4. Fig. 6 Digestion plots, LOS plots and nonlinear fitting patterns for BEM, C7H, CP2 and CP4. , , fit curve of non-linear curve fitting; , linear fit curve experimental data; , LOS plot data; for LOS plot data.
540
Fig 7 Schematic representation for the variations in pasting and digestion features for CP2.
541 542 543
Table 1 Particle size distributions and lamellar parameters of BEM, C7H, CP2 and CP4 granules A Sample
BEM
C7H
CP2
CP4
D[4, 3]
38.56±0.12bB
39.46±0.42b
35.83±0.10a
37.26±0.62a,b
d(0.5)
36.11±0.10b
36.84±0.42b
33.09±0.10a
34.55±0.65a,b
span
0.77±0.02a
1.06±0b
1.19±0c
1.13±0.02b
d (nm)
9.03±0.01b
9.01±0.01a
9.08±0.01c
9.07±0.01c
da (nm)
2.47±0.00a
2.48±0.00b
2.49±0.00b
2.47±0.00a
dc (nm)
6.56±0.01b
6.53±0.01a
6.59±0.01c
6.60±0.01c
25
D[4, 3], volume weight mean diameter; d(0.5), 50% of overall granules had a size below this value (μm); span,
544
A
545
a value equal to (d(0.9) – d(0.1)) / d(0.5); d, the semicrystalline lamella thickness; da, the amorphous lamellar
546
thickness; dc, the crystalline lamellar thickness.
547
B
Values with different lowercase letters in a row have significant difference at P 0.05.
548 549
Table 2 Parameterized biosynthetic enzyme parameters of BEM, C7H, CP2 and CP4 A BEM
C7H
CP2
CP4
Rh, amylose(nm)
25.85±0.11bB
26.18±0.36b
24.52±0.03a
26.07±0.30b
hAp2/Ap1
0.993±0.006b
1.006±0.001b
1.048±0.006c
0.919±0.004a
Amylose content (%)
20.82±0a
21.15±0.01a
20.55±0.01a
19.46±0.01a
β(i)
0.0921±0.0001a,b
0.0932±0.0007a,b
0.0908±0.0005a 0.0955±0.0009b
β(ii)
0.0500±0.0001b
0.0499±0.0004a,b,c 0.0453±0.0010a 0.0511±0.0002c
(i)
0.0661±0.0001c
0.0640±0.0003b
0.0606±0.0002a 0.0662±0.0010b,c
(ii)
0.0428±0a,b
0.0427±0.0003b,c
0.0390±0.0004a 0.0436±0.0001c
h(i)
1.0806±0.0022c
1.0283±0.0063b
0.9880±0.0069a 1.0610±0.0276a,b,c
h(ii)
0.0632±0.0004b
0.0643±0.0005b
0.0655±0.0011b 0.0565±0.0003a
550
A
551
β(ii), activity ratio of SBE:SS from enzyme-set (i) or (ii); γ(i) or γ(ii), activity ratio of DBE:SS from
552
enzyme-set (i) or (ii); h(i) or h(ii), relative contribution of enzyme-set (i) or (ii) to the whole chain
553
length distributions.
554
B
Rh, amylose, average Rh of amylose molecules; hAp2/Ap1, height ratio of Ap2 peak to Ap1 peak; β(i) or
The different lowercase letters within a row indicate significant difference at P < 0.05.
555 556
Table 3 Pasting and digestion parameters for BEM, C7H, CP2 and CP4 A 26
Samples
BEM
C7H
CP2
CP4
Tps (°C)
68.6±0.25b
68.7±0.20b
71.2±0.03c
67.2±0.03a
ηpk (cP)
10630±17b
9751±104a
10243±109a,b
10265±177a,b
Δηbd (cP)
6365±38b
4943±184a
5238±157a
6743±174b
Δηsb (cP)
717±37a
1076±15b
1357±32c
566±11a
kfitting (min-1)
0.0020±0b
0.0020±0b
0.0008±0a
0.0018±0b
C12 (%)
72.0±1.2b
68.8±2.2b
56.0±0.9a
67.3±1.2b
557
A
558
kfitting, digestion rate from non-linear curve fitting; C12, digested starch amount at 12 h.
559
B
Tps, pasting temperature; ηpk, peak viscosity; Δηbd, breakdown viscosity; Δηsb, setback viscosity;
Values with the different lowercase letter in a row have significant difference at P 0.05.
560
27