molecules

Article
The Annealing of Acetylated Potato Starch with Various
Substitution Degrees
Tomasz Zi˛eba 1 , Aleksandra Wilczak 2 , Justyna Kobryń 3 , Witold Musiał 3 , Małgorzata Kapelko-Żeberska 1, * ,
Artur Gryszkin 1 and Marta Meisel 1

1 Department of Food Storage and Technology, Faculty of Biotechnology and Food Science,
Wroclaw University of Environmental and Life Sciences, Chełmońskiego 37, 51-630 Wrocław, Poland;
tomasz.zieba@upwr.edu.pl (T.Z.); artur.gryszkin@upwr.edu.pl (A.G.); marta.meisel@upwr.edu.pl (M.M.)
2 Department of Physico-Chemistry of Microorganisms, University of Wroclaw, Przybyszewskiego 63-77,
51-148 Wrocław, Poland; aleksandra.wilczak@uwr.edu.pl
3 Department and Chair of Physical Chemistry and Biophysics, Wrocław Medical University, Borowska 211A,
50-556 Wrocław, Poland; justyna.kobryn@umed.wroc.pl (J.K.); witold.musial@umed.wroc.pl (W.M.)
* Correspondence: malgorzata.kapelko@upwr.edu.pl; Tel.: +48-71-320-7765

Abstract: This study aimed to determine the effect of “annealing” acetylated potato starch with a
homogenous granule size and various degrees of substitution on the thermal pasting characteristics
(DSC), resistance to amylases, rheology of the prepared pastes, swelling power and dynamics of drug
release. A fraction of large granules was separated from native starch with the sedimentation method
and acetylated with various doses of acetic anhydride (6.5, 13.0 or 26.0 26 cm3 /100 g starch). The
starch acetates were then annealed at slightly lower temperatures than their pasting temperatures.



The annealing process caused an almost twofold increase in the resistance to amylolysis and a


threefold increase in the swelling power of the modified starch preparations. The heat of phase
Citation: Zi˛eba, T.; Wilczak, A.;
transition decreased almost two times and the range of starch pasting temperatures over two times,
Kobryń, J.; Musiał, W.;
but the pasting temperature itself increased by ca. 10 ◦ C. The 40 g/100 g addition of the modified
Kapelko-Żeberska, M.; Gryszkin, A.;
starch preparation decreased the rate of drug release from a hydrogel by ca. one-fourth compared to
Meisel, M. The Annealing of
Acetylated Potato Starch with Various
the control sample.
Substitution Degrees. Molecules 2021,
26, 2096. https://doi.org/10.3390/ Keywords: annealing; acetylation; starch particle size distribution
molecules26072096

Academic Editor: Drago Šubarić

1. Introduction
Received: 11 March 2021 The process of native starch heating in a water suspension having a temperature that
Accepted: 5 April 2021
fits within the range of glass transition temperatures and the initial pasting temperatures
Published: 6 April 2021
is called annealing. The goal of this thermal modification is to bring starch temperature
to such a value that would excite the motility of starch molecules but prevent starch
Publisher’s Note: MDPI stays neutral
transformation into a paste. It makes the crystalline structure of starch homogeneous,
with regard to jurisdictional claims in
thereby protecting the structure of granules [1–3]. Annealing causes an increase in pasting
published maps and institutional affil-
temperatures and, at the same, makes the temperature range narrower. In addition, it has
iations.
a little effect on process enthalpy. If starch retains its grainy structure after modification,
its swelling power decreases with an increase in pasting temperatures [4]. In turn, the
viscosity of pastes obtained from annealed starch depends on starch origin. For example,
the annealing process was reported to increase the viscosity of pastes made of wheat
Copyright: © 2021 by the authors. and potato starches, and to decrease the viscosity of those obtained from lentil and oat
Licensee MDPI, Basel, Switzerland.
starches [5]. Starch modified using this type of heat treatment has some special applications
This article is an open access article
in the food industry. Due to its thermal stability and reduced tendency for retrogradation,
distributed under the terms and
it is used in food preserves and frozen foods [6]. In turn, Hormdok R. and Noomhorm
conditions of the Creative Commons
A. [7] showed the hydrothermally-modified starch exhibited properties desirable in pasta
Attribution (CC BY) license (https://
making. The annealing can also contribute to an increased content of resistant starch,
creativecommons.org/licenses/by/
especially when coupled with other modifications [4]. The double-modified starch can also
4.0/).

Molecules 2021, 26, 2096. https://doi.org/10.3390/molecules26072096 https://www.mdpi.com/journal/molecules

 increased content of resistant starch, especially when coupled with other modifications
[4]. The double-modified starch can also have some non-food uses. The application of
Molecules 2021, 26, 2096 2 of 11
various modified biopolymers may lead to the development of new hydrogel formula-
tions to be used in the field of drug delivery [8].
The annealed native starch of various botanical origin has been broadly investigat-
ed and
have described
some non-food [9,10].
uses. In Theturn, few works
application are available
of various modified regarding
biopolymers changes
may leadevokedto by
the development
annealing in the of new hydrogel
earlier formulations starch
chemically-modified to be used in thePotato
[11–13]. field ofstarch
drug delivery
proves to [8].
be an
The annealed native starch of various botanical origin has been broadly investigated
excellent raw material for chemical modifications because, unlike cereal starches, its
and described [9,10]. In turn, few works are available regarding changes evoked by
granules do not contain other substances, such as protein and fat [14]. In the case of po-
annealing in the earlier chemically-modified starch [11–13]. Potato starch proves to be
tato starch, small changes in the chemical structure of the polymer cause significant
an excellent raw material for chemical modifications because, unlike cereal starches, its
modifications
granules do not incontain
its properties [15]. Despite
other substances, a high
such chemical
as protein andpurity, potato
fat [14]. starch
In the caseisofnot a
homogenous material. Its pasting temperature depends on, i.a.,
potato starch, small changes in the chemical structure of the polymer cause significantthe size of its granules
[14], which ranges
modifications from a few[15].
in its properties to over 100aµm.
Despite highIn the casepurity,
chemical of its potato
annealing,
starchthis property
is not a
triggers differences in the transformations of granules having various
homogenous material. Its pasting temperature depends on, i.a., the size of its granules [14],sizes. To minimize
the effect
which of from
ranges this factor,
a few toitoverseems
100reasonable to use
µm. In the case theannealing,
of its fraction this
of granules
property obtained
triggers by
sorting native
differences potato
in the starch.
transformations of granules having various sizes. To minimize the effect
of thisThe aimitof
factor, this study
seems reasonablewas to
to use
determine howofthe
the fraction annealing
granules of acetylated
obtained by sortingstarch
nativewith
homogeneous granule size can affect its thermal pasting characteristics (DSC), resistance
potato starch.
to amylases,
The aim ofrheology
this study of was
the prepared
to determine pastes,
how swelling power
the annealing of and drug release
acetylated starch withdynam-
ics.
homogeneous granule size can affect its thermal pasting characteristics (DSC), resistance to
amylases, rheology of the prepared pastes, swelling power and drug release dynamics.
2. Results
2. Results
Potatostarch
Potato starchgranules
granules differ
differ significantly
significantly in their
in size; size; diameters
their diameters canfrom
can range range from a
a few
few to over 100 µm. Granules of various sizes undergo pasting at different
to over 100 µm. Granules of various sizes undergo pasting at different temperatures and temperatures
andthis
for forreason
this reason
the rawthe raw material
material used in used in our
our study study toannealed
to produce produce acetylated
annealed starch
acetylated
starch was firstly homogenized in terms of granule size. Particle size distribution
was firstly homogenized in terms of granule size. Particle size distribution of native starch of na-
tive of
and starch and
native of native
starch starch with homogenized
with homogenized fraction size
fraction size determined determined
using usingsize
a laser particle a laser
particle size
diffraction diffraction
analyzer (LSD)analyzer (LSD)
is presented in is presented
Figure 1. in Figure 1.

Figure1.1. Particle
Figure Particlesize
sizedistribution
distributionofofnative
native starch
starch (NS)
(NS) andand starch
starch with
with homogenous
homogenous fraction
fraction sizesize
determinedusing
determined usingaalaser
laserparticle
particle size
size diffraction
diffraction analyzer
analyzer (LSD).
(LSD).

Themean
The meangranule
granule diameter
diameter of the
of the non-homogenized
non-homogenized starch,starch, expressed
expressed as the volu-
as the volumet-
ric diameter D[4,3], reached 42.3 µm, and that measured after separating small granules
metric diameter D[4,3], reached 42.3 μm, and that measured after separating small gran-
reached 61.7 µm. In the present study, the acetylation of large starch granules with var-
ules reached 61.7 μm. In the present study, the acetylation of large starch granules with
ious doses of acetic acid anhydride resulted in the production of starch acetates which
various doses of acetic acid anhydride resulted in the production of starch acetates
differed significantly in the degree of substitution and, consequently, in the initial pasting
which differed significantly in the degree of substitution and, consequently, in the initial
temperatures (Table 1).
pasting temperatures (Table 1).

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Molecules 2021, 26, 2096 3 of 11

Table 1. Degree of substitution and initial temperature of pasting of large granules of starch acety-
lated with various doses of an acetic acid anhydride.

Acetic Acid Anhydride Dose

Acetylation
Table 1. Degree of substitution and initial temperatureDegree []
of pasting Initial
of Temperature
large granules of acetylated
of starch Pasting [°C]
[mL]
with various doses of an acetic acid anhydride.
6.5 0.05 ± 0.001 53.8 ± 0.01
13
Acetic Acid Anhydride Dose [mL] 0.11 ± 0.001
Acetylation Degree [] 49.2 ±of0.02
Initial Temperature Pasting [◦ C]
26 6.5 0.18
0.05± ±
0.001
0.001 44.9
53.8 ± 0.01
± 0.01
LSD13 0.01
0.11 ± 0.001 0.03
49.2 ± 0.02
Mean values from 26three replications ± standard deviations,
0.18 ± 0.001 LSD-least significant differences.
44.9 ± 0.01
LSD 0.01 0.03
The
Mean dependency
values between
from three replications starch deviations,
± standard acetylation and significant
LSD-least the resulting decrease
differences. in pasting
temperatures, including the greater decrease along with an increasing degree of starch
substitution with aceticbetween
The dependency acid residues, has been and
starch acetylation extensively documented
the resulting decrease in in pasting
literature
[16–19]. For this reason, the modified starch preparations were subjected to the anneal-
temperatures, including the greater decrease along with an increasing degree of starch sub-
stitution
ing treatmentwithat acetic acid temperature,
various residues, has been extensively
however in each documented in literature
case the annealing [16–19].
temperature
For this reason, the modified starch preparations
was only slightly lower than their initial pasting temperature. were subjected to the annealing treatment
at various temperature, however in each case the annealing temperature was only slightly
The annealing of acetylated starch led to a significant increase in the initial (Figure
lower than their initial pasting temperature.
2) and final temperature of pasting (Figure 3). It also caused the narrower range of tem-
The annealing of acetylated starch led to a significant increase in the initial (Figure 2)
peratures
and final and the lower
temperature of heat of this
pasting process
(Figure 3). It (Figure 4). The
also caused observedrange
the narrower changes were pro-
of tempera-
moted by an increasing acetylation degree of the annealed starch
tures and the lower heat of this process (Figure 4). The observed changes were promoted preparations. Accord-
ing
bytoanAdebowale et al. [6], andegree
increasing acetylation increase in pasting
of the annealedtemperatures reflectedAccording
starch preparations. the melting to of
crystallites, which are formed during interactions of amylose-amylose
Adebowale et al. [6], an increase in pasting temperatures reflected the melting of crystal- and amylose-
amylopectin
lites, which arechains as aduring
formed result interactions
of heating induced by native starch
of amylose-amylose structure reorganiza-
and amylose-amylopectin
tion, andaswhich
chains a resultcaused its crystallinity
of heating induced by to increase.
native starchItstructure
needs toreorganization,
be remembered andthat in the
which
present
causedstudy we deal not
its crystallinity with natural
to increase. starch
It needs to bebut with acetylated
remembered that in starch, in which
the present studybonds
we
between chains were weakened due to the replacement of hydroxyl groups with acetyl
deal not with natural starch but with acetylated starch, in which bonds between chains
groups impartingdue
were weakened thetoamphiphilic
the replacementcharacter to starch
of hydroxyl [20].with
groups Acetylation of starch
acetyl groups with an
imparting
acetic anhydride proceeded
the amphiphilic character tomainly in theAcetylation
starch [20]. outer layers of of starch
starch granules
with [21].
an acetic Therefore,
anhydride
proceeded
probably, itsmainly
interiorinunderwent
the outer layers
similarofprocesses
starch granules
during[21]. Therefore,
annealing as theprobably, its
native starch
interior underwent similar processes during annealing as the
did. Hence, the changes in the thermal pasting characteristics determined with DSC native starch did. Hence,
the changes in the thermal pasting characteristics determined with DSC were due to the
were due to the effects of acetylation and annealing, and their magnitude depended on
effects of acetylation and annealing, and their magnitude depended on conditions of both
conditions of both these processes.
these processes.

80
acetylated starch LSD = 0.04
acetylated and annealed starch
70

60
initial temperature of pasting [oC]

50

40

30

20

10

0
6.5 13 26
anhydride dose [mL]

Figure 2. Initial temperature of pasting of preparations of acetylated starch and of acetylated and
Figure 2. Initial temperature of pasting of preparations of acetylated starch and of acetylated and
annealed starch.
annealed starch.

3
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Molecules 2021, 26,26,
2021, x xFOR
FORPEER
PEERREVIEW
REVIEW 4 of 1212
4 of
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80
80 acetylated starch LSD = 0.02
acetylated starch LSD = 0.02
acetylated and annealed starch
acetylated and annealed starch
70
70

60
60

end temperature of pasting [ C]

end temperature of pasting [ C]o
o 50
50

40
40

30
30

20
20

10
10

0
6.5 13 26
0
6.5 anhydride13
dose [mL] 26
anhydride dose [mL]

Figure 3. End temperature of pasting of preparations of acetylated starch and of acetylated and
Figure 3. End temperature of pasting of preparations of acetylated starch and of acetylated and
Figure 3. End
annealed temperature of pasting of preparations of acetylated starch and of acetylated and
starch.
annealed starch.
annealed starch.

14
acetylated starch LSD = 0.07
14
acetylated and annealed starch
acetylated starch LSD = 0.07
12
acetylated and annealed starch

12

10
heat of phase transition [J/g]

10
heat of phase transition [J/g]

8
6

6
4

4
2

2
0
6.5 13 26
0 anhydride dose [mL]
6.5 13 26

anhydride dose [mL]

Figure 4. Heat of the phase transition of preparations of acetylated starch and of acetylated and
Figure 4. Heat of the phase transition of preparations of acetylated starch and of acetylated and
annealed starch.
annealed starch.
Figure 4. Heat of the phase transition of preparations of acetylated starch and of acetylated and
annealed starch.
Figure
Figure
5 presents flow curves of starch pastes made from acetylated starch and from
5 presents flow curves of starch pastes made from acetylated starch and
acetylated and thermally-annealed starch. Among the preparations which were not sub-
from acetylated and thermally-annealed starch. Among the preparations which were not
Figure
jected
subjected
to the5 annealing
presents
to the annealingflow
processcurves
in theof
process in thestarch
entire pastes
entire course
course made
of the flowfrom
curve,acetylated
the higheststarch
of the flow curve, and
viscosity
the highest vis-
from
was
cosityacetylated
noted for
was noted and
the
forthermally-annealed
paste
the paste prepared starch.
prepared from acetylated Among the
starch
from acetylated
with preparations
the
starch
highest which were
degree
with the highest
of not
substitu-
degree
subjected
tion (DS).toStarch
the annealing
esters with process
a lowerinDS the entire
formed course
less of
viscous the flow
pastes. curve,
Analogous
of substitution (DS). Starch esters with a lower DS formed less viscous pastes. Analo- the highest vis-
tendencies
were was
cosity observed
notedin for
the values of rheological coefficients (Table 1). Similar
with dependencies were
gous tendencies werethe paste
observed prepared from
in the values ofacetylated
rheologicalstarch
coefficients the highest
(Table degree
1). Similar
previously
ofdependencies reported
substitution were in acetylated
(DS). previously
Starch esters potato starch
with ainlower by many
DS formedauthors [19,22,23].
less viscous The annealing
pastes. Analo-
of acetylated starch contributed to reported
opposite acetylated
dependencies. potato
In thestarch
wholeby many
course of authors
the flow
gous tendencies
[19,22,23]. The were observed
annealing of in the values
acetylated starch ofcontributed
rheologicaltocoefficients
opposite (Table 1). Similar
dependencies. In
curve, the highest viscosity was noted for the paste prepared from starch acetylated with
dependencies
the were previously reported in acetylated potato starch by many authors
the whole
lowest course
dose of ofthethe flow
acetic curve,
acid the highest
anhydride, whereasviscosity was viscosity
the lowest noted forwas thefor
paste pre-
the paste
[19,22,23].
pared from The annealing
starch of acetylated
acetylated with the starchdose
lowest contributed
of the to opposite
acetic acid dependencies.
anhydride, whereas In
made of starch acetylated with the highest anhydride dose. The reversal of tendencies
the whole course of the flow curve, the highest viscosity was noted for the paste pre-
also applied to the values of rheological coefficients. In the case of native starch, annealing
pared from starch acetylated with the lowest dose of the acetic acid anhydride, whereas

4
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Molecules 2021, 26, 2096 5 of 11

the lowest viscosity was for the paste made of starch acetylated with the highest anhy-
dride dose. The reversal of tendencies also applied to the values of rheological coeffi-
cients. In the case of native starch, annealing increases paste viscosity [5]. At a low de-
gree of esterification,
increases the viscosity
paste viscosity [5]. At aoflow
thedegree
starch of
paste increased due
esterification, thetoviscosity
hydrothermal
of the starch
hardening the same
paste increased dueastoinhydrothermal
the case of natural starch.the
hardening In same
the case of the
as in starch
caseesterified with
of natural starch. In
the
the highest
case of dose
starchofesterified
acetic anhydride,
with the ithighest
can bedose
assumed that anhydride,
of acetic the greatestithydrolytic
can be assumed
changes
that thein the starch
greatest structurechanges
hydrolytic occurred.inThe
the consequence
starch structureof these changes
occurred. was
The proba-
consequence of
bly thechanges
these flowing ofwasmore starch substance
probably the flowingintoofthe solution
more starchduring annealing
substance compared
into the to during
solution
the other preparations.
annealing compared to Large changes
the other in the structure
preparations. Large of this starchincould
changes reduce the
the structure ofvis-
this starch
cosity of the prepared starch paste.
could reduce the viscosity of the prepared starch paste.

50

45

40

35
shear stress [Pa]

30

25

20

15 Acetylated starch/anhydride dose [mL]:

6.5
DGA6,5 13
DGA13 26
DGA26

10 Acetylated and thermally-annealed starch/anhydride dose [mL]

6.5
DGA6,5H 13
DGA13H 26
DGA26H

0
0 50 100 150 200 250 300
shear rate [1/s]

Figure
Figure5.5.Flow curves
Flow of starch
curves pastes
of starch prepared
pastes from acetylated
prepared starch and
from acetylated from and
starch acetylated and
from acetylated and
thermally-annealed
thermally-annealed starch.
starch.

The
Theresistance
resistance of of
starch acetylated
starch withwith
acetylated various dosesdoses
various of acetic acid anhydride
of acetic to
acid anhydride to
amylolysis increased from a few to dozen per cents along with an increasing
amylolysis increased from a few to dozen per cents along with an increasing DS of starch DS of
starch
acetatesacetates
(Figure(Figure
6). The6).annealing
The annealing
causedcaused ca. a twofold
ca. a twofold increase
increase in resistance.
in starch starch re- Starch
sistance. Starch acetylation contributes to its partial resistance to
acetylation contributes to its partial resistance to amylolysis [24] because amylolysis [24] because
the attached
the attached ester groups hamper enzyme’s access to the bond being
ester groups hamper enzyme’s access to the bond being hydrolyzed [25]. Hydrothermal hydrolyzed [25].
Hydrothermal modifications, like e.g., heat-moisture treatment (HMT)
modifications, like e.g., heat-moisture treatment (HMT) or annealing, also increase or annealing, also the
increase the content of resistant starch in a starch granule [26–29]. The earlier described
content of resistant starch in a starch granule [26–29]. The earlier described interactions
interactions of amylose and amylopectin impair the enzymatic hydrolysis [4,30,31].
of amylose and amylopectin impair the enzymatic hydrolysis [4,30,31]. Starch resistance
Starch resistance can also be increased by the elution of amylolysis-susceptible low-
can also be increased by the elution of amylolysis-susceptible low-molecular-weight chains
molecular-weight chains from the amorphous region of starch granules, which leads to
from
an the amorphous
increased content of region of starch
the resistant granules,
fractions which
in a starch leads to an increased content of the
granule.
resistant fractions in a starch granule.
The annealing of native starch induces changes in its swelling powers. Trends and
magnitude of these changes depend on the botanical origin of starch and process param-
eters [32–35]. The heating of large granules of acetylated starch at temperatures below
its pasting temperature led to ca. a threefold increase in the practical swelling power of
the modified preparations (Figure 7). Values of this parameter increased along with an
increasing acetylation degree of the double-modified starch preparations. This increase
could have been due to a higher number of acetyl groups incorporated into the starch chain,
which decreased the strength of binding between starch molecules, thereby increasing their
swelling power [35,36]. An additional factor that increased the swelling power of the starch
preparations could have been the homogenized granule size of starch. The simultaneous
swelling during heating in water and the release of low-molecular-weight chains from all
5
starch granules increase granules’ porosity [37] and, consequently, the swelling power of
the modified preparation.
 re
10

5
Molecules 2021,
Molecules 26,26,
2021, x FOR
2096 PEER REVIEW 6 of611
of 12

0
6.5 13 26
30
anhydride
acetylated dose
starch [mL] LSD = 1.11
acetylated and annealed starch

Figure
25 6. Resistance to amylolysis of preparations of acetylated starch and of acetylated and an-

nealed starch.
20
The annealing of native starch induces changes in its swelling powers. Trends and
resistance [g/100g]

magnitude of these changes depend on the botanical origin of starch and process pa-
rameters
15
[32–35]. The heating of large granules of acetylated starch at temperatures be-
low its pasting temperature led to ca. a threefold increase in the practical swelling power
of the
10 modified preparations (Figure 7). Values of this parameter increased along with
an increasing acetylation degree of the double-modified starch preparations. This in-
crease5
could have been due to a higher number of acetyl groups incorporated into the
starch chain, which decreased the strength of binding between starch molecules, thereby
increasing their swelling power [35,36]. An additional factor that increased the swelling
0
power of the starch 6.5
preparations could13 have been the homogenized 26
granule size of
starch. The simultaneous swellinganhydride during heating in water and the release of low-
dose [mL]
molecular-weight chains from all starch granules increase granules’ porosity [37] and,
Figure 6. Resistance to amylolysis of preparations of acetylated starch and of acetylated and an-
consequently,
Figure thetoswelling
6. Resistance power
amylolysis of the modified
of preparations preparation.
of acetylated starch and of acetylated and an-
nealed starch.
nealed starch.
6
The annealing of native starch induces changes in its swelling powers.
acetylated starch LSD = 0.09 Trends and
acetylated and annealed starch
magnitude of these changes depend on the botanical origin of starch and process pa-
rameters
5
[32–35]. The heating of large granules of acetylated starch at temperatures be-
low its pasting temperature led to ca. a threefold increase in the practical swelling power
of the modified preparations (Figure 7). Values of this parameter increased along with
practical swelling power [g/g]

an increasing acetylation degree of the double-modified starch preparations. This in-

crease
3
could have been due to a higher number of acetyl groups incorporated into the
starch chain, which decreased the strength of binding between starch molecules, thereby
increasing their swelling power [35,36]. An additional factor that increased the swelling
2
power of the starch preparations could have been the homogenized granule size of
starch. The simultaneous swelling during heating in water and the release of low-
molecular-weight
1 chains from all starch granules increase granules’ porosity [37] and,
consequently, the swelling power of the modified preparation.
0
6
6.5 13 26
acetylated starch
anhydride dose [mL] LSD = 0.09
acetylated and annealed starch

Figure
5 7. Practical swelling power of acetylated and of acetylated and annealed starch preparations.
Figure 7. Practical swelling power of acetylated and of acetylated and annealed starch prepara-
tions. The modified properties of annealed acetylated starch prompted us to perform a
practical swelling power [g/g]

4
pilot experiment to identify possibilities of the practical application of starch preparations
modified in this way. Many authors work on bioderivative polymers used in the controlled
drug
3 delivery [35]. Figure 8 presents the dynamics of a model drug release from a hydrogel
containing modified starch. The 20 g/100 g addition of the modified starch preparation 6
caused
2
a relatively small change in the drug release dynamics compared to the gel produced
without starch. At the first stage of the process, differences in the amount of extracted
drug were negligible, whereas after 420 min–the difference reached 5.2 g/100 g compared
to1 the control sample. Significantly greater differences were observed for the hydrogel
produced with 40 g/100 g addition of the modified starch. As soon as after 30 min, the
difference
0 in drug extraction rate was 26.2 g/100 g compared to the control hydrogel
6.5 13 26
(H0) and maintained at a similar level until the end of the experiment, to finally reach
anhydride dose [mL]
27.5 g/100 g after 420 min. Results of this pilot experiment allowed us to conclude that

Figure 7. Practical swelling power of acetylated and of acetylated and annealed starch prepara-
tions.
 amount of extracted drug were negligible, whereas after 420 min–the difference reached
5.2 g/100 g compared to the control sample. Significantly greater differences were ob-
served for the hydrogel produced with 40 g/100 g addition of the modified starch. As
soon as after 30 min, the difference in drug extraction rate was 26.2 g/100 g compared to
Molecules 2021, 26, 2096
the control hydrogel (H0) and maintained at a similar level until the end of the experi- 7 of 11

ment, to finally reach 27.5 g/100 g after 420 min. Results of this pilot experiment allowed
us to conclude that the use of modified starch can enable developing new formulations
for
thethe
usecontrolled local
of modified drug
starch candelivery. This will, however,
enable developing require further
new formulations specializedlocal
for the controlled re-
search.
drug delivery. This will, however, require further specialized research.

35

30

25
released amount [%]

20

15
starch addition [%]

H0
0 H20
20 H40
40
10

0
0 60 120 180 240 300 360 420
time [min]

Figure
Figure8.8.Dynamics
Dynamicsof
ofaamodel
modeldrug
drugrelease
releasefrom
fromaahydrogel
hydrogelcontaining
containingmodified
modified starch.
starch.

3. Materials and Methods

3. Materials and Methods
3.1. Materials
3.1. Materials
Experimental materials included: Superior Standard potato starch manufactured by
Experimental
PEPEES Łomża S.A.materials included: Superior
Acetic anhydride Standard
was purchased potatoSA
at POCH starch manufactured
Gliwice company. by
PEPEES Łomża S.A. Acetic anhydride was purchased at POCH SA Gliwice company.
3.2. Production of Modified Preparations
3.2. Production
A fractionofof
Modified Preparations
large granules was separated from native potato starch with the sedi-
A fraction
mentation of large
method. granules was
A suspension wasseparated
made from from
5.3 gnative potato
of starch per starch
100 g ofwith
the the sedi-
solutions
mentation method.
in a 15-L vessel, withA suspension
the height of was
themade
liquidfrom 5.3 greaching
column of starch percm.
19.5 100After
g of thorough
the solu-
tions in athe
stirring, 15-L vessel, with
suspension wastheleftheight of the
for 3 min. liquid
Then, the column reaching
precipitate 19.5 cm.and
was decanted After
thethor-
miss-
ough stirring,was
ing volume thecompleted
suspensionwith wasdistilled
left for 3water.
min. Then,
Anotherthesedimentation
precipitate was anddecanted and
decantation
the missing volume was completed with distilled water. Another sedimentation and de-
cycle was repeated three times more. The precipitate of large granules was air-dried at a
◦ C for 48 h. Native starch and starch left after the separation of the large
cantation cycle was repeated three times more. The precipitate of large granules was air-
temperature of 30
dried
granuleat afraction
temperature of 30 °C for
were analyzed 48 h.
using Native
a laser starchsize
particle andanalyzer
starch left after theInstruments
(Malvern separation
LTD, UK) with a Hydro 2000 attachment.
The large granule fraction was acetylated with the basic dose of acetic acid anhydride
used for potato starch modification under industrial conditions (13 mL/100 g starch) [38]
and with its 0.5-fold and 2-fold doses (6.5 or 26 mL/100 g starch). The acetylated starch 7
was rinsed with distilled water until reagent residues were removed, dried at 30 ◦ C for
24 h, ground, and sieved through a screen with mesh size of 400 µm.
The pasting characteristics (DSC) of acetylated starch was used to establish the initial
pasting temperature, which was further used to selected annealing temperatures. A
suspension from 10 g of starch/100 g of the solutions was prepared in a 2-L vessel from
starch acetylated with three doses of the acetic acid anhydride (6.5, 13 or 26 mL/100 g
starch). Under continuous stirring, the suspension was heated (water bath with stirrer) to
the temperatures of 52 ◦ C, 48 ◦ C or 43 ◦ C (for respective anhydride doses) with stirring
speed 240 rpm for 8 h and left for 48 h. Afterward, it was rinsed three times with 5-L
portions of distilled water. Starch precipitate was separated each time from the suspension
Molecules 2021, 26, 2096 8 of 11

using a Stratos flow centrifuge (Heraeus, Sepatech, Germany), then air-dried at 30 ◦ C for
24 h, ground in a mortar, and sieved through a screen with mesh size of 400 µm.

3.3. Determination of the Degree of Acetylation of Starch Preparations

A suspension of 10 g of starch was prepared in 65 cm3 of distilled water and stirred.
Then, phenolphthalein was added and a 0.1 M NaOH solution was instilled to obtain a
light-pink color of the mixture that maintained for 1 min. Next, 25 cm3 of a 0.5 N NaOH
solution were added and the reaction mixture was stirred on a shaker for 35 min. Afterward,
it was titrated with a 0.5 M HCl solution with a known titer [8].

3.4. Determination of the Characteristics of Phase Transitions of Starch Preparations with

Differential Scanning Calorimetry (DSC)
This determination was conducted using a DSC 822E differential scanning calorime-
ter (Mettler Toledo, Giessen, Germany). Before the measurement, the calorimeter was
calibrated using and indium sample and a zinc sample. Pre-tests were performed in a
temperature range of 0–100 ◦ C. Aluminum crucibles (100 µL) with lids were used for anal-
yses. A 10-g starch sample weighed exact to ± 0.02 mg was placed on crucible’s bottom,
and then distilled water was added in the ratio of 3:1 respective to sample weight. The
measuring crucible was covered with a lid, conditioned at 25 ◦ C for 30 min, transferred to
an oven chamber having a temperature of 25 ◦ C, and heated to a temperature of 100 ◦ C at
the heating rate of 4 ◦ C/min. The initial and final temperatures of pasting, and the heat of
this transition were determined from thermograms [39,40].

3.5. Determination of the Flow Curves of Pastes Made of Starch Preparations Using a Haake
Oscillating-Rotating Viscosimeter
Analyses were carried out using an RS 6000 oscillating-rotating viscosimeter Haake
(Karlsruhe, Germany) for 5 g/100 g starch suspensions that were heated at 96 ◦ C for 30 min
under continuous stirring [41].
Flow curves were plotted for the pastes at a measurement temperature of 50 ◦ C and
shear rate of 1–300 s−1 . A hot paste was placed in a system of coaxial cylinders (Z38AL
type) of an RS 6000 rheometer, then cooled, and relaxed at the measurement temperature
for 15 min. The flow curves plotted were described using the following equations:
Oswald de Waele:
τ = K × γn (1)
Casson:
τ = τ0C 0.5 + (ηC × γ)0.5 (2)
where: τ–shear stress (Pa), K–consistency coefficient (Pa·sn), γ–shear rate (s−1 ), n–flow
index, τ0C –yield point (Pa), ηc –Casson’s plastic viscosity (Pa·s).

3.6. Determination of the Resistance of Starch Preparations to Amyloglucosidase Action

A 0.72 g/100 g suspension was prepared from 38 g of starch that was heated to the
boiling point and then cooled. The volume of evaporated water was completed, and then
34 mL of an acetate buffer and 4 mL of an amyloglucosidase solution (Genecor) were added.
Thus prepared sample was hydrolyzed in a water bath at a temperature of 37 ◦ C. Enzyme
concentration was adjusted to ensure the complete saccharification of native starch after
120 min of the process. The amount of released glucose was controlled every hour. To
this end, 1-mL samples were collected, centrifuged and transferred in 10-µL portions to
2-mL cuvettes containing 1 mL of the reagent for glucose concentration determination
(BIOSYSTEM), stirred and incubated at 20 ◦ C for 15 min. Then, absorbance was measured
at a wavelength of λ = 500 nm using a CECIL CE 2010 colorimeter (Cecil Instruments,
Cambridge, United Kingdom). Glucose content was read out from the standard curve
plotted as above with glucose solutions. Three results that did not differ from each other
were acknowledged as the moment of complete saccharification of starch [41].
Molecules 2021, 26, 2096 9 of 11

3.7. Determination of Practical Swelling Power of Starch Preparations in Water Having a

Temperature of 20 ◦ C
Starch (300 mg) was weighed into a 4-mL test tube, then 2.5 mL of distilled water
were added and the sample was conditioned at a temperature of 20 ◦ C for 10 min. Next,
the sample was centrifuged using an MPW-55 laboratory centrifuge (MPW Instruments,
Warsaw, Poland) at 5000 rpm for 10 min. Afterward, supernatant was collected and the
precipitate left in test tubes was dried with a filter paper and weighed. The swelling power
was expressed in grams of water absorbed by one gram of starch dry matter.

3.8. Determination of the Dynamics of Model Drug Release from a Hydrogel with the Addition of a
Modified Starch Preparation
These analyses were conducted with starch acetylated using the basic dose of acetic
acid anhydride (13 mL/100 g starch) and annealed. Selected modified starch batches
(20 g/100 g or 40 g/100 g) were used to produce hydrogels with 1.5 g/100 g of polyacrylate
crosspolymer 11 (PC-11, Aristoflex Velvet, Clariant, Muttenz, Switzerland) as the vehicle,
and with 12 g/100 g of a dense extract of chestnut seeds (extractum spissum hippocastani,
EH, WZZ Herbapol SA, Wrocław, Poland) as the model drug. The formulation contained
water was the completive component. The hydrogels shortened, respectively, as H20 and
H40 were used to evaluate the drug release from formulations containing modified starch.
Hydrogel of the same composition, containing water instead of starch, was established as
the control–H0. The release study for the selected modified starch batches was performed
acc. to the pharmacopoeial method with extraction cells, and purified water applied as
a dissolution medium (Drug Dissolution Tester Erweka GmbH DT 700, Heusenstamm,
Germany) [Ph. Eur. 9.0, 20903 (01/2016)]. The released drug amount was assessed via
spectrophotometry (Spectrophotometer UV/VIS Jasco V-530, Tokyo, Japan) according to
the available bibliography [42,43].

3.9. Statistical Analysis

Results were statistically analyzed using Statistica 13.3 PL software package. Based
on statistical computations (from at least three parallel replications), values of the least
significant differences and standard deviation were calculated and equations of flow curves
were determined. For statistical evaluation, the results were subjected to one-way analysis
of variance at a significance level of 0.05. Values of the least significant difference (LSD)
between the means were computed using the Duncan’s test at a significance level of 0.05
(StatSoft, Inc., Tulsa, OK, USA, 2011).

4. Conclusions
The annealing of large granules of acetylated starch caused an increase in pasting
temperatures, starch resistance to amylolysis and swelling power, but also a decrease in
the heat of modified starch pasting. The magnitude of the observed changes depended
on starch acetylation degree. The viscosity of pastes made from double-modified starch
was determined mainly by the degree of starch acetylation. The annealing of starch with a
lower DS caused an increase, whereas that of starch with the highest DS caused a decrease
in the viscosity of the pastes. The 40% addition of modified starch decreased the rate of
drug release from the hydrogel by 25% compared to the control sample. This promising
discovery deserves in-depth investigations to identify its potential applicability in the
pharmaceutical industry.

Author Contributions: Conceptualization, T.Z. and W.M.; methodology, A.W. and J.K.; software,
A.G.; validation, M.K.Z.; formal analysis, J.K.; investigation, M.K.Z.; resources, M.M.; data curation,
A.G.; writing—original draft preparation, T.Z.; writing—review and editing, W.M.; visualization,
A.W.; supervision, A.G.; project administration, M.K.Z.; funding acquisition, A.W. All authors have
read and agreed to the published version of the manuscript.
Molecules 2021, 26, 2096 10 of 11

Funding: The publication is co-financed under the Leading Research Groups support project from
the subsidy increased for the period 2020–2025 in the amount of 2% of the subsidy referred to Art.
387 (3) of the Law of 20 July 2018 on Higher Education and Science, obtained in 2019. This research
was funded by Josip Juraj Strossmayer University of Osijek, under the research project Potential
of application a distillery wastewater from the production of apple brandy for the production of
naturally modified starches (UNIOS-ZUP 2018-26).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data is contained within the article.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are not available from the authors.

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