The Annealing of Acetylated Potato Starch with Various Substitution Degrees

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Article The Annealing of Acetylated Potato Starch with Various Substitution Degrees

Tomasz Zi˛eba 1 , Aleksandra Wilczak 2, Justyna Kobry´n 3 , Witold Musiał 3 , Małgorzata Kapelko-Zeberska˙ 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, Chełmo´nskiego37, 51-630 Wrocław, Poland; [email protected] (T.Z.); [email protected] (A.G.); [email protected] (M.M.) 2 Department of Physico-Chemistry of Microorganisms, University of Wroclaw, Przybyszewskiego 63-77, 51-148 Wrocław, Poland; [email protected] 3 Department and Chair of Physical Chemistry and Biophysics, Wrocław Medical University, Borowska 211A, 50-556 Wrocław, Poland; [email protected] (J.K.); [email protected] (W.M.) * Correspondence: [email protected]; 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-Zeberska,˙ 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 the control sample. Acetylated Potato Starch with Various 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 Molecules 2021, 26, x FOR PEER REVIEW 2 of 12

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 formulations to be used in the field of drug delivery [8]. The annealed native starch of various botanical origin has been broadly investigat- haveed and some described non-food [9,10 uses.]. In The turn, application few works of various are available modified regarding biopolymers changes may leadevoked to by theannealing development in the ofearlier new hydrogelchemically formulations-modified starch to be used [11– in13] the. Potato field of starch drug deliveryproves 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 granulesmodifications do not in containits properties other substances,[15]. Despite such a high as protein chemical and purity, fat [14 potato]. In the starch case is of not a potatohomogenous starch, smallmaterial. changes Its pasting in the chemicaltemperature structure depends of the on, polymer i.a., the cause size of significant its granules modifications[14], which ranges in its propertiesfrom a few [15 to]. over Despite 100 a µm. high In chemical the case purity, of its potatoannealing, starch th isis notproperty a homogenoustriggers differences material. in Its the pasting transformations temperature of depends granules on, having i.a., the various size of its sizes. granules To minimize [14], whichthe effect ranges of fromthis factor, a few to it overseems 100 reasonableµm. In the caseto use of itsthe annealing, fraction thisof granules property obtained triggers by differencessorting native in the potato transformations starch. of granules having various sizes. To minimize the effect of thisThe factor, aim it of seems this study reasonable was to usedete thermine fraction how of the granules annealing obtained of acetylated by sorting starch native with potatohomogeneous starch. granule size can affect its thermal pasting characteristics (DSC), resistance to amylases,The aim ofrheology this study of wasthe prepared to determine pastes, how swelling the annealing power of and acetylated drug release starch with dynam- homogeneousics. 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 PotatoPotato starch starch granules granules differ differ signiﬁcantly significantly in size; in theirsize; diameterstheir diameters can range can from range a few from a tofew over to over 100 µ 100m. Granulesµm. Granules of various of various sizes undergo sizes undergo pasting pasting at different at different temperatures temperatures and forand this for reason this reason the raw the material raw material used in used our study in our to producestudy to annealedproduce acetylatedannealed starchacetylated wasstarch ﬁrstly was homogenized firstly homogenized in terms ofin granule terms of size. granule Particle size. size Particle distribution size ofdistribution native starch of na- andtive ofstarch native and starch of native with homogenized starch with homogenized fraction size determined fraction size using determined a laser particle using size a laser diffractionparticle size analyzer diffraction (LSD) analyzer is presented (LSD) in is Figure presented1. in Figure 1.

Figure 1.1. ParticleParticle sizesize distribution distribution of of native native starch starch (NS) (NS) and and starch starch with with homogenous homogenous fraction fraction size size determined usingusing a a laser laser particle particle size size diffraction diffraction analyzer analyzer (LSD). (LSD).

TheThe meanmean granule granule diameter diameter of theof the non-homogenized non-homogenized starch, starch, expressed expressed as the volumet-as the volu- µ ricmetric diameter diameter D[4,3], D[4, reached3], reache 42.3d 42.3m, and μm, that and measured that measured after separating after separating small granules 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 (Table1). 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 acetylated with various doses of an acetic acid anhydride.

Acetic Acid Anhydride Dose Table 1. Degree of substitution and initialAcetylation temperature Degree of pasting [] ofInitial large granulesTemperature of starch of acetylatedPasting [°C] [mL] with various doses of an acetic acid anhydride. 6.5 0.05 ± 0.001 53.8 ± 0.01 Acetic Acid Anhydride13 Dose [mL] Acetylation0.11 ± 0.001 Degree [] Initial Temperature49.2 ± of 0.02 Pasting [◦C] 26 6.50.18 0.05 ± ±0.0010.001 53.844.9± 0.01± 0.01 LSD13 0.110.01± 0.001 49.2 ±0.030.02 Mean values from 26three replications ± standard 0.18 ±deviations,0.001 LSD-least significant 44.9 ± 0.01 differences. LSD 0.01 0.03 MeanThe values dependency from three replications between± standardstarch deviations,acetylation LSD-least and significantthe resulting differences. decrease in pasting temperatures, including the greater decrease along with an increasing degree of starch substitutionThe dependency with acetic between acid residues, starch acetylation has been andextensive the resultingly documented decrease inin pastingliterature [16temperatures,–19]. For this including reason, thethe greatermodified decrease starch along preparations with an increasing were subjected degree of to starch the anneal- sub- ingstitution treatment with at acetic various acid temperature, residues, has beenhowever extensively in each documented case the annealing in literature temperature [16–19]. wasFor only this reason,slightly the lower modified than starchtheir initial preparations pasting were tempe subjectedrature. 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 (Figure2) peraturesand final and temperature the lower of heat pasting of this (Figure process3). It also(Figure caused 4). The the narrowerobserved range changes of tempera- were pro- motedtures andby an the increasing lower heat acetylation of this process degree (Figure of 4the). The annealed observed starch changes preparations. were promoted Accord- ingby to an Adebowale increasing acetylationet al. [6], an degree increase of thein pasting annealed temperatures starch preparations. reflected According the melting to of crystallites,Adebowale which et al. [ 6are], an formed increase during in pasting interactions temperatures of amylose reflected- theamylo meltingse and of crystal-amylose- amylopectinlites, which arechains formed as a during result interactionsof heating induced of amylose-amylose by native starch and amylose-amylopectin structure reorganization,chains and as which a result cause of heatingd its crystallinity induced by to native increase. starch It structure needs to reorganization, be remembered and that which in the presentcaused study its crystallinity we deal not to increase. with natural It needs starch to be but remembered with acetyla thatted in starch, the present in which study bonds we betweendeal not chains with natural were weakened starch but due with to acetylated the replacement starch, in of which hydroxyl bonds groups between with chains acetyl groupswere weakened imparting due the to amp thehiphilic replacement character of hydroxyl to starch groups [20]. with Acetylation acetyl groups of starch imparting with an aceticthe amphiphilic anhydride proceeded character to mainly starch [in20 the]. Acetylation outer layers of of starch starch with granules an acetic [21]. anhydride Therefore, probably,proceeded its mainlyinterior in underwent the outer layerssimilar of processes starch granules during [ 21annealing]. Therefore, as the probably, native starch its did.interior Hence, underwent the changes similar in processesthe thermal during pasting annealing characteristics as the native determined starch did. with Hence, DSC 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 C] o

30 initial temperature of pasting [ pasting of temperature initial 20

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.

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80 80 acetylated starch LSD = 0.02 acetylated starch acetylated and annealed starch LSD = 0.02 acetylated and annealed starch 70 70

60 60 C] o C] o 50 50

40 40

30 30 end temperature of pasting [ end temperature of pasting [ 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 Figureannealed 3. End starch. temperature of pasting of preparations of acetylated starch and of acetylated and annealedannealed starch. starch.

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

6 heat of phase transition [J/g] transition [J/g] phase of heat 4 heat of phase transition [J/g] transition [J/g] phase of heat 4 2

2 0 6.5 13 26

0 anhydride dose [mL] 6.5 13 26 Figure 4. Heat of the phase transition of preparationsanhydride dose [mL] of acetylated starch and of acetylated and Figure 4. Heat of the phase transition of preparations of acetylated starch and of acetylated and annealedannealed starch. starch. Figure 4. Heat of the phase transition of preparations of acetylated starch and of acetylated and Figure5 presents flow curves of starch pastes made from acetylated starch and from annealedFigure starch. 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 jected to the annealing process in the entire course of the flow curve, the highest viscosity subjectedFigure to 5 the presents annealing flow process curves in ofthe starch entire pastescourse madeof the flow from curve, acetylated the highest starch vis- and was noted for the paste prepared from acetylated starch with the highest degree of substitu- fromcosity acetyl was atednoted and for thermally the paste- annealedprepared starch.from acetylated Among the starch preparations with the highestwhich weredegree not tion (DS). Starch esters with a lower DS formed less viscous pastes. Analogous tendencies subjectedof substitution to the (DS).annealing Starch process esters inwith the a entire lower course DS formed of the less flow viscous curve, pastes.the highest Analo- vis- were observed in the values of rheological coefficients (Table1). Similar dependencies were cositygous tendencieswas noted werefor the observed paste prepared in the values from of acetylated rheological starch coefficients with the(Table highest 1). Similar degree ofpreviously substitution reported (DS). inStarch acetylated esters potato with starcha lower by DS many formed authors less [19 viscous,22,23]. Thepastes. annealing Analo- dependenciesof acetylated starchwere contributedpreviously toreported opposite in dependencies.acetylated potato In the starch whole by course many of authors the flow gous tendencies were observed in the values of rheological coefficients (Table 1). Similar [19curve,,22,23]. the The highest annealing viscosity of wasacetylated noted forstarch the pastecontributed prepared to fromopposite starch dependencies. acetylated with In dependencies were previously reported in acetylated potato starch by many authors thethe whole lowest course dose of of the the acetic flow acid curve, anhydride, the highest whereas viscosity the lowest was viscositynoted for was the for paste the pastepre- [19,22,23]. The annealing of acetylated starch contributed to opposite dependencies. In paredmade from of starch starch acetylated acetylated with with the the highest lowest anhydride dose of the dose. acetic The acid reversal anhydride, of tendencies whereas 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

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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 anhydride dose. The reversal of tendencies also applied to the values of rheological coefficients. In the case of native starch, annealing increases paste viscosity [5]. At a low de- greeincreases of esterification, paste viscosity the viscosity [5]. At aof low the degreestarch ofpaste esterification, increased due the to viscosity hydrothermal of the starch hardeningpaste increased the same due as to in hydrothermal the case of natural hardening starch. the In samethe case as inof thestarch case esterified of natural with starch. In thethe highest case of dose starch of esterified acetic anhydride, with the it highest can be dose assumed of acetic that anhydride,the greatest ithydrolytic can be assumed changesthat the in greatest the starch hydrolytic structure changes occurred. in The the consequence starch structure of these occurred. changes The was consequence proba- of blythese the changes flowing of was more probably starch substance the flowing into of the more solution starch during substance annealing into thecompared solution to during theannealing other preparations. compared toLarge the otherchanges preparations. in the structure Large of this changes starch in could the structure reduce the of vis- this starch cositycould of reduce the prepared the viscosity starch paste. of the prepared starch paste.

25 shear stress [Pa]

15 Acetylated starch/anhydride dose [mL]: DGA6,56.5 DGA1313 DGA2626

10 Acetylated and thermally-annealed starch/anhydride dose [mL] DGA6,5H6.5 DGA13H13 DGA26H26

0 0 50 100 150 200 250 300 shear rate [1/ ] s FigureFigure 5. 5. FlowFlow curves curves of starch of starch pastes pastes prepared prepared from acetylated from acetylated starch and starch from andacetylated from acetylatedand and thermallythermally-annealed-annealed starch. starch.

TheThe resistance resistance of ofstarch starch acetylated acetylated with with various various doses doses of acetic of aceticacid anhydride acid anhydride to to amylolysisamylolysis increased increased from from a afew few to to dozen dozen per per cents cents along along with with an an increasing increasing DS DS of of starch starchacetates acetates (Figure (Figure6). The 6). annealing The annealing caused caused ca. a twofold ca. a twofold increase increase in starch in resistance.starch re- Starch sistance.acetylation Starch contributes acetylationto contributes its partial to resistanceits partial resistance to amylolysis to amyloly [24]sis because [24] because the attached theester attached groups ester hamper groups enzyme’s hamper accessenzyme’s to theaccess bond to beingthe bond hydrolyzed being hydrolyzed [25]. Hydrothermal [25]. Hydrothermalmodifications, modifications, like e.g., heat-moisture like e.g., heat- treatmentmoisture treatment (HMT) or(HMT) annealing, or annealing, also increase also the increasecontent the of resistantcontent of starch resistant in astarch starch in granulea starch [granule26–29]. [26 The–29 earlier]. The earlier described 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 anfrom increased the amorphous content of regionthe resistant of starch fractions granules, in a starch which granule. leads to an increased content of the 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 (Figure7). 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 starch granules increase granules’ porosity [37] and, consequently, the swelling power5 of the modified preparation. Molecules 2021, 26, x FOR PEER REVIEW 6 of 12

30 acetylated starch LSD = 1.11 acetylated and annealed starch

15 resistance [g/100g] resistance 10

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0 6.5 13 26 30 acetylatedanhydride starch dose [mL] LSD = 1.11 acetylated and annealed starch Figure25 6. Resistance to amylolysis of preparations of acetylated starch and of acetylated and annealed starch.

20 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 pa- rameters15 [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 resistance [g/100g] resistance of the10 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 starch6.5 preparations could13 have been the homogenized26 granule size of

starch. The simultaneous swellinganhydride during dose [mL] heating in water and the release of low- 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- Figureconsequently, 6. Resistance the to swelling amylolysis power of preparations of the modified of acetylated preparation. starch and of acetylated and annealed starch. nealed starch.

6 The annealing of native starch inducesacetylated starch changes in its swelling powers.LSD = 0.09 Trends and acetylated and annealed starch magnitude of these changes depend on the botanical origin of starch and process pa- rameters5 [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 the4 modified preparations (Figure 7). Values of this parameter increased along with an increasing acetylation degree of the double-modified starch preparations. This in-

crease3 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 [g/g] swelling power practical 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- molecular1 -weight chains from all starch granules increase granules’ porosity [37] and, consequently, the swelling power of the modified preparation.

0 6.5 13 26 6 acetylatedanhydride starch dose [mL] LSD = 0.09 acetylated and annealed starch Figure5 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 preparations. The modified properties of annealed acetylated starch prompted us to perform a pilot4 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 drug3 delivery [35]. Figure8 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

caused2 a relatively small change in the drug release dynamics compared to the gel produced without [g/g] swelling power practical 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 difference0 in drug extraction rate was 26.2 g/100 g compared to the control hydrogel (H0) and maintained6.5 at a similar level until13 the end of the experiment,26 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 preparations.

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The modified properties of annealed acetylated starch prompted us to perform a 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 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 caused 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 to 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 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 forthe the use controlled of modiﬁed local starch drug can delivery. enable developing This will, however, new formulations require further for the controlledspecialized local research.drug delivery. This will, however, require further specialized research.

released amount [%] starch addition [%]

H00 H2020 H4040 10

0 0 60 120 180 240 300 360 420 time [min] FigureFigure 8. 8. DynamicsDynamics of of a a model model drug drug release release from from a a hydrogel hydrogel containing containing modified 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 PEPEESExperimental Łomza˙ S.A. materials Acetic anhydrideincluded: Superior was purchased Standard at POCH potato SAstarch Gliwice manufactured company. by PEPEES Łomża S.A. Acetic anhydride was purchased at POCH SA Gliwice company. 3.2. Production of Modified Preparations 3.2. ProductionA fraction of ofM largeodified granules Preparations was separated from native potato starch with the sedi- mentationA fraction method. of large A suspension granules was was separated made from from 5.3 gnative of starch potato per starch 100 g ofwith the the solutions sedi- mentationin a 15-L vessel,method. with A suspension the height ofwas the made liquid from column 5.3 g reaching of starch 19.5 per cm. 100 After g of thoroughthe solu- tionsstirring, in a the 15- suspensionL vessel, with was the left height for 3 min. of the Then, liquid the column precipitate reaching was decanted 19.5 cm. and After the thor- miss- oughing volume stirring, was the completed suspension with was distilled left for 3 water. min. Then, Another the sedimentation precipitate was and decanted decantation and thecycle missing was repeated volume threewas completed times more. with The distilled precipitate water. of largeAnother granules sedimentation was air-dried and de- at a ◦ cantationtemperature cycle of was 30 Crepeated for 48 h. three Native times starch more. and The starch precipitate left after of the large separation granules of was the large air- driedgranule at a fraction temperature were analyzedof 30 °C for using 48 h. a laserNative particle starch sizeandanalyzer starch left (Malvern after the Instruments 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 starch7 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 calorimeter (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 analyses. 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: 0.5 0.5 τ = τ0C + (ηC × γ) (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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