Impact of Acid Hydrolysis on Morphology, Rheology, Mechanical Properties, and Processing of Thermoplastic Starch

Academic Editors: Tiago Linus Silva Coêlho, Luiz Brito de Souza Filho and Darlisson Slag Neri Silva Received: 7 April 2025 Revised: 5 May 2025 Accepted: 9 May 2025 Published: 11 May 2025 Citation: Kouka, S.; Gajdosova, V.; Strachota, B.; Sloufova, I.; Kuzel, R.; Stary, Z.; Slouf, M. Impact of Acid Hydrolysis on Morphology, Rheology, Mechanical Properties, and Processing of Thermoplastic Starch. Polymers 2025,17, 1310. https://doi.org/ 10.3390/polym17101310 Copyright: © 2025 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/ licenses/by/4.0/). Article Impact of Acid Hydrolysis on Morphology, Rheology, Mechanical Properties, and Processing of Thermoplastic Starch Saffana Kouka 1, Veronika Gajdosova 1, Beata Strachota 1, Ivana Sloufova 2, Radomir Kuzel 3, Zdenek Stary 1 and Miroslav Slouf 1,* 1Institute of Macromolecular Chemistry of the Czech Academy of Sciences, Heyrovsky Sq. 2, 16206 Prague, Czech Republic; [email protected] (S.K.); [email protected] (V.G.); [email protected] (B.S.); [email protected] (Z.S.) 2 Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, Hlavova 2030, 12840 Prague, Czech Republic; [email protected] 3Faculty of Mathematics and Physics, Charles University, Ke Karlovu 5, 12116 Prague, Czech Republic; radomir[email protected] *Correspondence: [email protected] Abstract: We modified native wheat starch using 15, 30, and 60 min of acid hydrolysis (AH). The non-modified and AH-modified starches were converted to highly homogeneous thermoplastic starches (TPSs) using our two-step preparation protocol consisting of solution casting and melt mixing. Our main objective was to verify if AH can decrease the processing temperature of TPS. All samples were characterized in detail by microscopic, spectroscopic, diffraction, thermomechanical, rheological, and micromechanical methods, including in situ measurements of torque and temperature during the final melt mixing step. The experimental results showed that (i) AH decreased the average molecular weight preferentially in the amorphous regions, (ii) the lower-viscosity matrix in the AH-treated starches resulted in slightly higher crystallinity, and (iii) all AH-modified TPSs with a less viscous amorphous phase and higher content of crystalline phase exhibited similar properties. The effect of the higher crystallinity predominated at a laboratory temperature and low deformations, resulting in slightly stiffer material. The effect of the lower viscosity dominated during the melt mixing, where the shorter molecules acted as a lubricant and decreased the in situ measured processing temperature. The AH-induced decrease in the processing temperature could be beneficial for energy savings and/or possible temperature-sensitive admixtures for TPS systems. Keywords: thermoplastic starch; low viscosity; melt mixing; processing temperature 1. Introduction Starch is the primary carbohydrate reserve in plants, composed of two polysaccharides: amylose and amylopectin [ 1 ]. Amylose is a linear polymer linked by α -1,4 glycosidic bonds, while amylopectin is formed by highly branched molecules with α -1,4 linkages in the backbone and α -1,6 linkages at the branching points [ 2 ]. Molecular weights of both polymers are very high: ca. 10 6 g/mol for amylose and ca. 10 8 g/mol for amylopectine [3–6] . In the native starch granules, amylose is localized in the amorphous regions, whereas amylopectin is in semicrystalline regions [ 7 , 8 ]. Due to its high molecular weight, the native starch cannot be processed by melt mixing like other common polymers, because it decomposes before melting [ 9 ]. To overcome this limitation, starch is typically mixed with low-molecular-weight plasticizers such as water, glycerol, citric acid, and/or urea [ 10 , 11 ]. Polymers 2025,17, 1310 https://doi.org/10.3390/polym17101310 Polymers 2025,17, 1310 2 of 21 In our previous work, we demonstrated that oligomers such as maltodextrin can be added as lubricants that decrease the starch processing temperature [2]. When starch is heated in the presence of plasticizers and subjected to shear forces, it undergoes gelatinization—a process in which the starch granules are partially disrupted and merged [ 8 , 12 ]. This transforms the native starch to thermoplastic starch (TPS), which can be processed similarly to conventional thermoplastic polymers. TPS can be prepared by solution casting in the excess of water (SC; [ 13 ]), by melt mixing (MM; [ 14 ]), or by the combination of both protocols (SC+MM; [ 15 ]). TPS must be prepared at elevated temperatures, as starch gelatinization generally occurs above 100 ◦ C, regardless of the water content [ 16 ]. However, even a moderate reduction in the processing temperature would be advantageous for energy-saving reasons. One of possible strategies to decrease the processing temperature is the lowering of starch molecular weight, M. Polymers with lower Mexhibit lower viscosity and require lower temperatures to flow [17]. The reduction in the starch molecular weight can be achieved through physical methods, such as hydrothermal treatment [ 18 ] or ultrasonication [ 19 , 20 ], by means of chemical processes (typically acid hydrolysis [ 21 ]), or with enzymatic methods [ 22 ]. Acid hydrolysis (AH) is widely employed in the food industry to tailor starch’s physicochemical properties [ 23 , 24 ]. It is often conducted below the gelatinization temperature to preserve the granular structure of starch [ 25 ]. The AH proceeds in two phases: an initial rapid hydrolysis targeting the amorphous regions, followed by a slower phase where crystalline regions are hydrolyzed [ 26 ]. Consequently, acid-hydrolyzed starch often exhibits higher crystallinity than native starch [ 23 , 25 ]. The acid hydrolysis of starch also affects the rheological properties of the TPS: mild acid hydrolysis can enhance gel strength and stiffness, while prolonged hydrolysis reduces molecular weight, producing weaker gels [ 27 ]. Although the acid hydrolysis of starch is a well-established industrial process, particularly in the context of food applications [ 23 , 28 ], limited information is available on its role in plasticization and the resulting processing properties of thermoplastic starch prepared by melt mixing [ 29 , 30 ]. In this contribution, we subjected native wheat starch to acid hydrolysis (AH) for 0, 15, 30, and 60 min. The AH starch was thermoplasticized using single-step solution casting (SC) and a two-step protocol combining solution casting with melt mixing (SC+MM), which yields highly homogeneous thermoplastic starch [ 15 ]. The prepared materials were characterized thoroughly by numerous microscopic, spectroscopic, diffraction, rheological, thermomechanical, and micromechanical measurements. The first question we asked was how the AH treatment influences the morphology, homogeneity, and mechanical performance of thermoplastic starch. The second question was if the acid hydrolysis can decrease the starch viscosity and, as a result, its processing temperature during melt mixing. Highly homogeneous starch with a lower processing temperature would be a promising material for both technical applications in packaging and agriculture (energy savings during the processing) and medical applications in pharmaceutics (the high homogeneity to secure reproducibility and lower processing temperature to improve the stability of sensitive admixtures, such as antibiotics [31]). 2. Materials and Methods 2.1. Materials The wheat starch powder used in this study (starch type A, amylose content ca. 25%) was supplied by Škrobárny Pelhˇrimov a.s. (Pelhˇrimov, Czech Republic). Anhydrous glycerol (C 3 H 8 O 3 ; >99%), hydrochloric acid, (HCl; 35%), and sodium bromide (NaBr; >99%) reagents were bought from Lach-Ner s.r.o. (Neratovice, Czech Republic). Polymers 2025,17, 1310 3 of 21 2.2. Preparation of Hydrolyzed Starch Acid hydrolysis was performed following a previously described method [ 32 ] with a few minor modifications. A 40% starch slurry was prepared by dispersing starch in an aqueous 1M hydrochloric acid solution. The reaction was conducted under mechanical stirring and mild heating in a water bath maintained at 45 ◦ C. Hydrolysis durations were varied (15 min, 30 min, and 60 min) to evaluate the effects of reaction time. To terminate the reaction, the mixture’s pH was adjusted to 7 using a 1 M sodium hydroxide solution. The neutralized slurry was then washed thoroughly with distilled water, followed by filtration. The resulting material was dried in an oven at 45 ◦ C for 24 h and subsequently cooled to room temperature and milled into a fine powder. 2.3. Preparation of Thermoplastic Starch The thermoplastic starch (TPS) was prepared from the native starch powders (S) with various acid hydrolysis (AH) times. TPS was prepared by both a single-step solution casting protocol (SC; Section 2.3.1) and two-step protocol comprising solution casting followed by melt mixing (SC+MM; Section 2.3.2). This approach is based on our previous work [ 15 ], with slight modifications described below. The TPS samples prepared in this study are summarized in Table 1. Table 1. List of prepared samples. Native Starches * Thermoplastic Starches ** AH Time (min) *** S-AH-00min TPS-AH-00min 0 S-AH-15min TPS-AH-15min 15 S-AH-30min TPS-AH-30min 30 S-AH-60min TPS-AH-60min 60 * Native starches were all based on wheat starch type A, differing only in AH time. ** Thermoplastic starches were prepared by both single-step SC and two-step SC+MM protocols. *** Acid hydrolysis (AH) was applied to original native starches, from which TPSs were prepared. 2.3.1. TPS Prepared by Single-Step Solution Casting The starch powders (70 wt.%) were premixed with glycerol (30 wt.%) and distilled water (6 parts of water per 1 part of starch) with a magnetic stirrer in a beaker for 30 min at room temperature. The pre-mixed suspension was transferred to a mechanical stirrer, where it was heated to initiate the starch gelatinization. A significant increase in viscosity was observed at temperatures between 63 ◦ C and 70 ◦ C. The viscosity increase indicated the onset of gelatinization, and the mixture was stirred continuously for about 15 min until a homogeneous pudding-like consistency was obtained. Then, the solution was cast onto a polyethylene (PE) foil to form a film with a thickness of approximately 2 mm. The thin film was left to dry at room temperature for three days to allow for the evaporation of residual water. 2.3.2. TPS Prepared by Two-Step Protocol: Solution Casting Followed by Melt Mixing The solution-casted and dried TPS films from the previous steps were processed by melt mixing using a twin-screw laboratory kneader (Brabender Plasti-Corder, Duisburg, Germany) to further increase their homogeneity [ 15 , 33 ]. The samples were mixed in the chamber pre-heated to 120 ◦ C, using a rotation speed of 60 rpm for at least 8 min while recording the real processing temperature and torque moments. Subsequently, the sample was compression molded into plaques with a thickness of 2 mm. This was achieved using a laboratory hot press (Fontijne Grotnes; Vlaardingen, The Netherlands) in the following multi-step process: Initially, the material was pressed at 130 ◦ C under a pressure of 50 kN for 2 min to deaerate. This was followed by pressing at the same temperature under 100 kN for Polymers 2025,17, 1310 4 of 21 2 min. Finally, the press with the molded plaques was cooled with water while maintaining a pressure of 100 kN for approximately 10 min, until room temperature was reached. 2.3.3. Storing of the Samples in Defined Conditions The final TPS plaques were stored in defined conditions: at room temperature in a desiccator over a supersaturated solution of sodium bromide, which yielded a relative humidity = 57%. The samples were in the desiccator all the time, being removed just before the measurements of their properties by the characterization methods described below. Our experience showed that storing the samples at a well-defined humidity leads to more reproducible results of mechanical and rheological measurements, even if storage at ambient conditions is possible as well. 2.4. Characterization Methods 2.4.1. Light and Electron Microscopy The morphology and homogeneity of TPS samples were checked by light microscopy (LM), polarized light microscopy (PLM), and scanning electron microscopy (SEM). The thin sections (thickness 5 µ m) for LM and PLM were prepared with a rotary microtome RM 2255 (RM 2255; Leica, Vienna, Austria). The sections were placed in a thin oil layer between the microscopic glasses and observed in transmitted light (LM) or polarized transmitted light (PLM) under a Nikon Eclipse 80i microscope (supplied by Laboratory Imaging, Prague, Czech Republic). The fracture surfaces for SEM observations were prepared in liquid nitrogen. The specimens were fixed on a metallic support with a conductive adhesive carbon tape (Plano GmbH, Wetzlar, Germany), sputter-coated with a thin platinum layer (vacuum sputter coater SCD 050; Leica, Austria; thickness of the Pt layer: approx. 4 nm), and observed under a MAIA3 SEM microscope (Tescan, Brno, Czech Republic) using secondary electron imaging at an accelerating voltage of 3 kV. 2.4.2. Vibrational Spectroscopy Fourier-transform infrared (FTIR) spectra were recorded on a Thermo Fisher Scientific Nicolet iS50 FTIR spectrometer (Nicolet CZ s.r.o.; Prague, Czech Republic) using a 4 cm −1 resolution in the 400–4000 cm −1 region (with KBr beamsplitter and Happ-Genzel apodization) by means of the ATR (diamond crystal) technique. Standard ATR correction was applied. Raman spectra were collected on a dispersive micro Raman system, MonoVista CRS+ (Spectroscopy & Imaging GmbH; Warstein, Germany), interfaced to an Olympus microscope (50 × objective) equipped with a 785 nm excitation laser, grating of 150 g/mm, spectrograph aperture with a 50 µ m slit, and laser power of 10.5 mW on the sample. The wavelength and intensity calibration of the spectrometer was performed by the software-controlled auto alignment procedure using mercury and Ne-Ar lamps. The total amount of 150–300 spectra for each sample with an exposure time of 5 s per spectrum was collected. All spectra were subsequently baseline corrected according to the singular value decomposition method [34], averaged, and normalized (max–min). 2.4.3. Wide-Angle X-Ray Scattering Wide-angle X-ray scattering (WAXS) patterns were obtained with a Panalytical MPD system (Panalytical; Almelo, the Netherlands) with a vertical goniometer, CoK α radiation, variable divergence slits (mostly fixed irradiated length of 10 mm), and 1D Pixcel detector. The specimens (powders, films, bulk samples) were always placed on so-called non-diffracting Si substrates, giving little background. The ranges of 4–70 deg 2 θ (CoK α ) were taken with the total measurement time of 1 hr. For comparison with the other literature, the final diffractograms were recalculated so that they corresponded to more common CuK α radiation (the wavelengths of CoK α and CuK α were taken as 1.79 and 1.54 Å, re- Polymers 2025,17, 1310 5 of 21 spectively). The crystallinity (weight fraction of crystalline phase) was calculated by means of Fityk software [35]. 2.4.4. Dynamic Mechanical Thermal Analysis The thermomechanical properties of the TPS systems were measured in torsion by dynamic mechanical thermal analysis (DMTA) on specimens of a rectangular platelet shape (40 mm × 10 mm × 2 mm), using an ARES G2 (TA Instruments, New Castle, DE, USA) in oscillatory mode, at a deformation frequency of 1 Hz. The deformation amplitude ranged from 0.01 to 3% (regulated automatically by the auto-strain function, in response to sample resistance). The investigated temperature range was from − 90 to 140 ◦ C, while the heating rate was 3 ◦ C/min. The temperature dependences of the storage shear modulus (G ′ ), =the loss modulus (G′′), and the loss factor tan (δ) were recorded. 2.4.5. Rheology Rheological properties of the TPS systems were measured in shear on a straincontrolled ARES G2 rheometer (TA Instruments, New Castle, DE, USA) using a parallel plate fixture with a diameter of 30 mm (plates with cross-hatched surface to prevent slipping). The thickness of the specimens was 2 mm. At first, the linear viscoelasticity region (LVER) was determined in view of the dependence of the storage modulus on the strain amplitude, which was measured at 120 ◦ C at a frequency of 1 Hz. Next, the frequency sweep experiments were performed in a frequency range from 0.1 to 100 rad/s at a strain amplitude of 0.05% (always well within the LVER) and at a constant temperature of 120 ◦ C. To ensure a uniform temperature in the specimen, all samples were equilibrated for 2 min prior to the start of each type of experiment. The frequency sweep was performed twice for each TPS specimen. 2.4.6. Microindentation Hardness Testing Micromechanical properties were measured with an instrumented microindentation hardness tester (MCT tester; CSM, Switzerland). The microindentation hardness testing (MHI) experiments were carried out using a Vickers method: a diamond square pyramid (with an angle between non-adjacent faces of 136 ◦ ) was forced against the flat surface of a specimen. The flat smooth surfaces for the testing were prepared by cutting from the 2 mm thick plates with a rotary microtome RM 2255 (Leica, Vienna, Austria). The micromechanical properties were deduced from the loading force, which was measured as a function of penetration depth. From each sample, three independent cut surfaces were prepared. For each measured surface, at least 10 independent measurements/indentations were performed and the final results were averaged. As we repeated the measurement for each sample three times to verify the reproducibility, the final averaged values of all micromechanical properties represent more than 90 measurements (3 cut surfaces per sample × at least 10 indentation per surface × 3 repetitions = more than 90). The parameters of MHI measurements were as follows: maximal loading force Fmax = 500 mN , dwell time (time of maximal load) 60 s, and linear loading and unloading rates 15,000 mN/min (i.e., ~2 s to achieve and release F max ). The evaluated micromechanical properties were as follows: an indentation modulus (E IT ) proportional to the macroscopic elastic modulus, an indentation hardness (H IT ) proportional to the macroscopic yield stress, Martens hardness (H M ) also referred as universal hardness, indentation creep (C IT ) related to the macroscopic creep, and the elastic part of the indentation work ( ηIT ) defined as the ratio of elastic deformation to total deformation. The calculations of E IT and H IT ,E IT were based on the theory of Oliver and Pharr [ 36 ], while the values of H M ,C IT , and ηIT were independent of the O&P theory [ 37 ]. The exact definitions of the above-listed micromechanical properties can be Polymers 2025,17, 1310 6 of 21 found in textbooks on micromechanical properties [ 38 , 39 ], and more detailed descriptions of the MHI measurements are also given in our recent studies [40–42]. 2.4.7. In Situ Measurements During Melt Mixing During the melt mixing of TPS in a laboratory kneader (Section 2.3.2), we recorded the values of torque (TQ, moment of force; in Nm) and real processing temperature (T; in ◦ C) as a function of processing time (t; in seconds). To ensure the maximal reproducibility of the measurements, the kneading chamber was filled with the same amount of material (75 g) at the same time (2 min). Moreover, the final values of TQ and Twere evaluated from the final part of the TQ-tand T-tcurves, after ca. 6 min, when the curves reached a plateau indicating that the mixed system achieved a steady state. 3. Results and Discussion 3.1. Morphology and Homogeneity 3.1.1. Light and Electron Microscopy Figure 1displays representative polarized-light micrographs (PLMs) of thermoplastic starches prepared by two different protocols (one-step SC vs. two-step SC+MM) with or without acid hydrolysis (AH). In the PLMs, the bright areas indicate anisotropic material. In the case of TPS materials, the bright spots correspond to non-fully plasticized starch granules that kept their semicrystalline structure [2]. Polymers 2025, 17, 1310 6 of 21 theory of Oliver and Pharr [36], while the values of HM, CIT, and ηIT were independent of the O&P theory [37]. The exact definitions of the above-listed micromechanical properties can be found in textbooks on micromechanical properties [38,39], and more detailed descriptions of the MHI measurements are also given in our recent studies [40–42]. 2.4.7. In Situ Measurements During Melt Mixing During the melt mixing of TPS in a laboratory kneader (Section 2.3.2), we recorded the values of torque (TQ, moment of force; in Nm) and real processing temperature (T; in °C) as a function of processing time (t; in seconds). To ensure the maximal reproducibility of the measurements, the kneading chamber was filled with the same amount of material (75 g) at the same time (2 min). Moreover, the final values of TQ and T were evaluated from the final part of the TQ-t and T-t curves, after ca. 6 min, when the curves reached a plateau indicating that the mixed system achieved a steady state. 3. Results and Discussion 3.1. Morphology and Homogeneity 3.1.1. Light and Electron Microscopy Figure 1 displays representative polarized-light micrographs (PLMs) of thermoplastic starches prepared by two different protocols (one-step SC vs. two-step SC+MM) with or without acid hydrolysis (AH). In the PLMs, the bright areas indicate anisotropic material. In the case of TPS materials, the bright spots correspond to non-fully plasticized starch granules that kept their semicrystalline structure [2]. Figure 1. PLM micrographs showing four thermoplastic starch samples: (a) TPS-AH-0min after SC, (b) TPS-AH-60min after SC, (c) TPS-AH-0min after SC+MM, and (d) TPS-AH-60min after SC+MM. The samples without the AH treatment (a,c) contain fewer anisotropic inhomogeneities (bright Figure 1. PLM micrographs showing four thermoplastic starch samples: (a) TPS-AH-0min after SC, (b) TPS-AH-60min after SC, (c) TPS-AH-0min after SC+MM, and (d) TPS-AH-60min after SC+MM. The samples without the AH treatment (a,c) contain fewer anisotropic inhomogeneities (bright spots) than the corresponding samples after AH (b,d). The complete list of the prepared TPS samples is given in Table 1. Polymers 2025,17, 1310 7 of 21 The PLM results confirmed our previous findings [ 2 , 15 , 31 ] that single-step SC does not yield highly homogeneous starch. The non-fully plasticized granules could be observed in TPS without AH treatment (Figure 1a), and even more in the TPS after 60 min of AH (Figure 1b). The most homogeneous starch was obtained with the two-step SC+MM preparation protocol without AH treatment (Figure 1c). The two-step preparation after 60 min of AH resulted in less homogeneous material (Figure 1d). This could be explained by combining two facts known from the previous studies: (i) the amorphous regions of starch granules are more susceptible to AH than the crystalline regions [ 23 , 43 ], and (ii) if the amorphous matrix is more degraded and less viscous, the disintegration of starch granules is less complete due to the lower shear forces during SC+MM processing [ 2 ]. Interestingly, an analogous trend is observed in immiscible polymer blends: if the viscosity of the matrix decreases, the disintegration of minor phase droplets during the melt mixing is less complete and the structure coarsens [44,45]. The morphological changes were monitored by SEM as well. The SEM/SE micrographs of TPS fracture surfaces (Figure A1 in Appendix A) confirmed the results of PLM (Figure 1), but the differences among the samples could not be observed so clearly. The not-fully plasticized starch granules exhibited higher contrast in polarized light (where they could be distinguished clearly as bright spots due to their anisotropic nature) than in SEM/SE micrographs (where they could be observed only in the form of blunt, rounded asperities on fracture surfaces). 3.1.2. Vibrational Spectroscopy Figure 2summarizes the vibrational spectroscopy results for all 12 studied samples, i.e., the three starch types (original powder, TPS after SC, and TPS after SC+MM) with four AH times (0, 15, 30, and 60 min). Both infrared spectroscopy (Figure 2a) and Raman scattering (Figure 2b) were in agreement that the dominant chemical change was the incorporation of glycerol into the starch structure during SC, while AH and MM did not alter the starch molecular structure significantly (see also Figure A2 in Appendix A). The peak positions in TPS and their assignment to characteristic vibrations of starch and glycerol corresponded to those in the detailed study of Almeida et al. [46]. Polymers 2025, 17, 1310 7 of 21 spots) than the corresponding samples after AH (b,d). The complete list of the prepared TPS samples is given in Table 1. The PLM results confirmed our previous findings [2,15,31] that single-step SC does not yield highly homogeneous starch. The non-fully plasticized granules could be observed in TPS without AH treatment (Figure 1a), and even more in the TPS after 60 min of AH (Figure 1b). The most homogeneous starch was obtained with the two-step SC+MM preparation protocol without AH treatment (Figure 1c). The two-step preparation after 60 min of AH resulted in less homogeneous material (Figure 1d). This could be explained by combining two facts known from the previous studies: (i) the amorphous regions of starch granules are more susceptible to AH than the crystalline regions [23,43], and (ii) if the amorphous matrix is more degraded and less viscous, the disintegration of starch granules is less complete due to the lower shear forces during SC+MM processing [2]. Interestingly, an analogous trend is observed in immiscible polymer blends: if the viscosity of the matrix decreases, the disintegration of minor phase droplets during the melt mixing is less complete and the structure coarsens [44,45]. The morphological changes were monitored by SEM as well. The SEM/SE micrographs of TPS fracture surfaces (Figure A1 in Appendix A) confirmed the results of PLM (Figure 1), but the differences among the samples could not be observed so clearly. The not-fully plasticized starch granules exhibited higher contrast in polarized light (where they could be distinguished clearly as bright spots due to their anisotropic nature) than in SEM/SE micrographs (where they could be observed only in the form of blunt, rounded asperities on fracture surfaces). 3.1.2. Vibrational Spectroscopy Figure 2 summarizes the vibrational spectroscopy results for all 12 studied samples, i.e., the three starch types (original powder, TPS after SC, and TPS after SC+MM) with four AH times (0, 15, 30, and 60 min). Both infrared spectroscopy (Figure 2a) and Raman scattering (Figure 2b) were in agreement that the dominant chemical change was the incorporation of glycerol into the starch structure during SC, while AH and MM did not alter the starch molecular structure significantly (see also Figure A2 in Appendix A). The peak positions in TPS and their assignment to characteristic vibrations of starch and glycerol corresponded to those in the detailed study of Almeida et al. [46]. Figure 2. Vibrational spectroscopy results: (a) infrared spectra measured in ATR (attenuated total reflectance) mode and (b) Raman scattering measured at 785 nm excitation. Each of the two plots Figure 2. Vibrational spectroscopy results: (a) infrared spectra measured in ATR (attenuated total reflectance) mode and (b) Raman scattering measured at 785 nm excitation. Each of the two plots shows, from top to bottom, the following spectra: pure glycerol, original starch powders with various AH times, TPS after single-step SC preparation with various AH times, and TPS after two-step SC+MM preparation with various AH times. Polymers 2025,17, 1310 8 of 21 There was a clear difference between the spectra of all original starch powders (glycerol-free materials) and the spectra of all TPSs (with glycerol added during SC). The change in the spectra could be attributed to the fact that the glycerol molecules penetrated the granules and interacted with starch molecules, forming new intraand intermolecular hydrogen bonds [ 47 ]. Within each group of materials (powders, TPS after SC, and TPS after SC+MM), negligible variations were observed with the AH time. We conclude that AH caused starch chain scissions, which changed both the morphology (as discussed above) and properties (as discussed below), but the concentration of the newly formed end groups was below the detection limit of IR and Raman spectroscopy. This is in accordance with the literature [ 48 ], even if Chung et al. [ 49 ] have demonstrated that near-infrared (NIR) spectra are more sensitive in this case, enabling the monitoring of the extent of AH. The differences between spectra of all TPSs after SC and after SC+MM were insignificant, although there were some local variations (we note that each spectrum in Figure 5 is a normalized average of >150 individual spectra, as described in the Experimental Section). 3.1.3. Wide-Angle X-Ray Scattering Figure 3shows the results of wide-angle X-ray scattering (WAXS) of all investigated samples as a function of increasing acid hydrolysis time. The native starch powders (Figure 3, upper row) possessed the highest crystallinity. TPS after the one-step SC protocol (Figure 3, middle row) exhibited slightly lower crystallinity due to partial disintegration of native starch granules after gelatinization. TPS after the two-step SC+MM protocol (Figure 3, lower row) showed the lowest crystallinities due to (almost) complete destruction of the starch granules after the melt mixing. With an increasing acid hydrolysis time, the crystallinity slightly increased (see all three rows of Figure 3from left to right). This is in accordance with the literature, which documents that the AH of starch preferentially targets the amorphous regions, enhancing crystallinity and double-helical content [ 23 , 25 ]. The crystallinity values for our investigated samples, ranging from 14 to 30%, are largely in line with other available reports [ 29 , 50 ]. The increase in crystallinity after chain scissions in amorphous region is a general trend that has been observed also for other semicrystalline polymers, such as ultra-high-molecular-weight polyethylene (UHMWPE) after oxidative degradation [51,52]. Polymers 2025, 17, 1310 8 of 21 shows, from top to bottom, the following spectra: pure glycerol, original starch powders with various AH times, TPS after single-step SC preparation with various AH times, and TPS after two-step SC+MM preparation with various AH times. There was a clear difference between the spectra of all original starch powders (glycerolfree materials) and the spectra of all TPSs (with glycerol added during SC). The change in the spectra could be attributed to the fact that the glycerol molecules penetrated the granules and interacted with starch molecules, forming new intraand intermolecular hydrogen bonds [47]. Within each group of materials (powders, TPS after SC, and TPS after SC+MM), negligible variations were observed with the AH time. We conclude that AH caused starch chain scissions, which changed both the morphology (as discussed above) and properties (as discussed below), but the concentration of the newly formed end groups was below the detection limit of IR and Raman spectroscopy. This is in accordance with the literature [48], even if Chung et al. [49] have demonstrated that near-infrared (NIR) spectra are more sensitive in this case, enabling the monitoring of the extent of AH. The differences between spectra of all TPSs after SC and after SC+MM were insignificant, although there were some local variations (we note that each spectrum in Figure 5 is a normalized average of >150 individual spectra, as described in the Experimental Section). 3.1.3. Wide-Angle X-Ray Scattering Figure 3 shows the results of wide-angle X-ray scattering (WAXS) of all investigated samples as a function of increasing acid hydrolysis time. The native starch powders (Figure 3, upper row) possessed the highest crystallinity. TPS after the one-step SC protocol (Figure 3, middle row) exhibited slightly lower crystallinity due to partial disintegration of native starch granules after gelatinization. TPS after the two-step SC+MM protocol (Figure 3, lower row) showed the lowest crystallinities due to (almost) complete destruction of the starch granules after the melt mixing. With an increasing acid hydrolysis time, the crystallinity slightly increased (see all three rows of Figure 3 from left to right). This is in accordance with the literature, which documents that the AH of starch preferentially targets the amorphous regions, enhancing crystallinity and double-helical content [23,25]. The crystallinity values for our investigated samples, ranging from 14 to 30%, are largely in line with other available reports [29,50]. The increase in crystallinity after chain scissions in amorphous region is a general trend that has been observed also for other semicrystalline polymers, such as ultra-high-molecularweight polyethylene (UHMWPE) after oxidative degradation [51,52]. Figure 3. WAXS diffraction patterns and crystallinities of all investigated samples. The crystallinity values are printed in the upper right corner of each subplot. The rows show, from top to bottom, original starch powder, TPS after solution casting (SC), and TPS after solution casting and melt Figure 3. WAXS diffraction patterns and crystallinities of all investigated samples. The crystallinity values are printed in the upper right corner of each subplot. The rows show, from top to bottom, original starch powder, TPS after solution casting (SC), and TPS after solution casting and melt mixing (SC+MM). The columns show, from left to right, the studied materials after 0, 15, 30, and 60 min of acid hydrolysis. Polymers 2025,17, 1310 9 of 21 All starch powder diffractograms (Figure 3, upper row, all AH times) exhibited prominent diffraction peaks around 2 θ≈ 15 ◦ , 17 ◦ , 18 ◦ , and 23 ◦ at the CuK α wavelength. Moreover, several lower-intensity peaks could be observed at higher angles around 27 ◦ , 31 ◦ , 33 ◦ , 38 ◦ , and 41 ◦ . All above-listed peaks are characteristic of the starch type-A crystallinity, which is typical of cereals [ 25 , 29 , 30 , 53 ]. Therefore, the results documented that AH alone attacked mostly the amorphous regions and did not change the starch crystallinity type. Nevertheless, after SC, the crystallinity started to change. Some diffractions decreased and/or disappeared due to partial destruction of the original amylopectin A-type crystallinity. Some new diffraction appeared, corresponding to newly formed amylose V-type crystals [ 2 , 53 ] and amylopectin B-type crystals formed due to A-to-B-type crystallinity transition [ 53 , 54 ]. After SC+MM, the crystallinity changed substantially. The diffractions of the original starch powder either disappeared completely or merged into broad peaks in the regions of 15–25 ◦ and 30–45 ◦ . The only sharp diffractions at 13.5 ◦ , 20 ◦ , and 21 ◦ corresponded to V-type crystallinity [ 2 , 50 ]. A table summarizing all observed diffraction peaks together with a figure showing selected diffraction patterns with annotated diffraction are given in Appendix A(Table A1 and Figure A3). 3.2. Mechanical and Rheological Properties The final TPS samples after SC+MM with various degrees of AH were characterized in detail by thermomechanical (Figure 4), rheological (Figure 5), and micromechanical measurements (Figures 6and 7). The three methods were in agreement that the properties of all samples were quite similar, although a slight increase in stiffness with increasing AH time was observed. The similar properties of the final TPS samples could be explained as follows: (i) the AH treatment caused chain scissions in the amorphous region, (ii) the chain scissions led to a softer and less viscous amorphous phase, (iii) the lower viscosity of the amorphous phase resulted in less complete disintegration of the crystalline phase during solution casting and melt mixing as documented by the PLM and WAXS results above, and (iv) the two contradictory effects—the softer amorphous phase with shorter molecules and the higher volume fraction of the less disintegrated crystalline phase—tended to cancel out, although the impact of increased crystallinity prevailed moderately at the end. The increase in TPS stiffness after AH was observed in previous studies as well [30,55]. Polymers 2025, 17, 1310 9 of 21 mixing (SC+MM). The columns show, from left to right, the studied materials after 0, 15, 30, and 60 min of acid hydrolysis. All starch powder diffractograms (Figure 3, upper row, all AH times) exhibited prominent diffraction peaks around 2θ ≈ 15°, 17°, 18°, and 23° at the CuKα wavelength. Moreover, several lower-intensity peaks could be observed at higher angles around 27°, 31°, 33°, 38°, and 41°. All above-listed peaks are characteristic of the starch type-A crystallinity, which is typical of cereals [25,29,30,53]. Therefore, the results documented that AH alone attacked mostly the amorphous regions and did not change the starch crystallinity type. Nevertheless, after SC, the crystallinity started to change. Some diffractions decreased and/or disappeared due to partial destruction of the original amylopectin A-type crystallinity. Some new diffraction appeared, corresponding to newly formed amylose Vtype crystals [2,53] and amylopectin B-type crystals formed due to A-to-B-type crystallinity transition [53,54]. After SC+MM, the crystallinity changed substantially. The diffractions of the original starch powder either disappeared completely or merged into broad peaks in the regions of 15–25° and 30–45°. The only sharp diffractions at 13.5°, 20°, and 21° corresponded to V-type crystallinity [2,50]. A table summarizing all observed diffraction peaks together with a figure showing selected diffraction patterns with annotated diffraction are given in Appendix A (Table A1 and Figure A3). 3.2. Mechanical and Rheological Properties The final TPS samples after SC+MM with various degrees of AH were characterized in detail by thermomechanical (Figure 4), rheological (Figure 5), and micromechanical measurements (Figures 6 and 7). The three methods were in agreement that the properties of all samples were quite similar, although a slight increase in stiffness with increasing AH time was observed. The similar properties of the final TPS samples could be explained as follows: (i) the AH treatment caused chain scissions in the amorphous region, (ii) the chain scissions led to a softer and less viscous amorphous phase, (iii) the lower viscosity of the amorphous phase resulted in less complete disintegration of the crystalline phase during solution casting and melt mixing as documented by the PLM and WAXS results above, and (iv) the two contradictory effects—the softer amorphous phase with shorter molecules and the higher volume fraction of the less disintegrated crystalline phase—tended to cancel out, although the impact of increased crystallinity prevailed moderately at the end. The increase in TPS stiffness after AH was observed in previous studies as well [30,55]. Figure 4. Thermomechanical properties of all investigated TPS samples after solution casting and melt mixing, measured by DMTA in rectangular torsion mode in the temperature range from −90 to 140 °C: (a) storage modulus, G′, (b) loss modulus, G″, and (c) damping factor, tan(δ). As the properties of all four samples were quite similar and hard to differentiate on a logarithmic scale, the insets in (a,b) show the properties at the laboratory temperature (20 °C) in greater detail. Note that G′ and Figure 4. Thermomechanical properties of all investigated TPS samples after solution casting and melt mixing, measured by DMTA in rectangular torsion mode in the temperature range from −90 to 140 ◦C : (a) storage modulus, G ′ , (b) loss modulus, G ′′ , and (c) damping factor, tan( δ ). As the properties of all four samples were quite similar and hard to differentiate on a logarithmic scale, the insets in (a,b) show the properties at the laboratory temperature (20 ◦ C) in greater detail. Note that G ′ and G ′′ are plotted with the same y-axis limits in order to facilitate the direct comparison of the two quantities. Polymers 2025,17, 1310 16 of 21 0 and 60 min of AH) with annotated diffraction peaks. The assignment of the diffraction peaks together with the relevant references and brief descriptions is given in Table A1. Polymers 2025, 17, 1310 16 of 21 Table A1 summarizes all diffraction peaks observed in the 12 measured samples (i.e., powders, SC samples, and SC+MM samples after 0, 15, 30, and 60 min of AH). Figure A3 shows selected diffraction patterns (i.e., all powders, SC samples, and SC+MM samples after 0 and 60 min of AH) with annotated diffraction peaks. The assignment of the diffraction peaks together with the relevant references and brief descriptions is given in Table A1. Figure A1. SEM micrographs showing fracture surfaces of TPS samples: (a) TPS-AH-0min after SC, (b) TPS-AH-60min after SC, (c) TPS-AH-0min after SC+MM, and (d) TPS-AH-60min after SC+MM. The list of the prepared TPS samples is given in Table 1; SC = solution casting; SC+MM = solution casting followed by melt mixing. The samples were observed in the secondary electron mode at the accelerating voltage of 3 keV. Figure A2. Raman spectra of four studied samples: pure glycerol, wheat starch powder, TPS after single-step SC and 60 min of AH, and TPS after two-step SC+MM and 60 min of AH. Table A1. Summary of WAXS diffraction peaks. WAXS Diffraction Observation of the Diffraction Peaks Starch Structure Types 2θ (°) Intensity 1 S (Powder) TPS (SC) TPS (SC+MM) Structure 2 References 5.3 w no yes no B [2] 13.5 m no no yes V [50,53] Figure A1. SEM micrographs showing fracture surfaces of TPS samples: (a) TPS-AH-0min after SC, (b) TPS-AH-60min after SC, (c) TPS-AH-0min after SC+MM, and (d) TPS-AH-60min after SC+MM. The list of the prepared TPS samples is given in Table 1; SC = solution casting; SC+MM = solution casting followed by melt mixing. The samples were observed in the secondary electron mode at the accelerating voltage of 3 keV. Polymers 2025, 17, 1310 16 of 21 Table A1 summarizes all diffraction peaks observed in the 12 measured samples (i.e., powders, SC samples, and SC+MM samples after 0, 15, 30, and 60 min of AH). Figure A3 shows selected diffraction patterns (i.e., all powders, SC samples, and SC+MM samples after 0 and 60 min of AH) with annotated diffraction peaks. The assignment of the diffraction peaks together with the relevant references and brief descriptions is given in Table A1. Figure A1. SEM micrographs showing fracture surfaces of TPS samples: (a) TPS-AH-0min after SC, (b) TPS-AH-60min after SC, (c) TPS-AH-0min after SC+MM, and (d) TPS-AH-60min after SC+MM. The list of the prepared TPS samples is given in Table 1; SC = solution casting; SC+MM = solution casting followed by melt mixing. The samples were observed in the secondary electron mode at the accelerating voltage of 3 keV. Figure A2. Raman spectra of four studied samples: pure glycerol, wheat starch powder, TPS after single-step SC and 60 min of AH, and TPS after two-step SC+MM and 60 min of AH. Table A1. Summary of WAXS diffraction peaks. WAXS Diffraction Observation of the Diffraction Peaks Starch Structure Types 2θ (°) Intensity 1 S (Powder) TPS (SC) TPS (SC+MM) Structure 2 References 5.3 w no yes no B [2] 13.5 m no no yes V [50,53] Figure A2. Raman spectra of four studied samples: pure glycerol, wheat starch powder, TPS after single-step SC and 60 min of AH, and TPS after two-step SC+MM and 60 min of AH. Polymers 2025,17, 1310 17 of 21 Table A1. Summary of WAXS diffraction peaks. WAXS Diffraction Observation of the Diffraction Peaks Starch Structure Types 2θ(◦)Intensity 1S (Powder) TPS (SC) TPS (SC+MM) Structure 2References 5.3 w no yes no B [2] 13.5 m no no yes V [50,53] 15.1 s yes no no A/B [30] 17.0 s yes yes no A/B [2,30] 18.1 s yes no no A [30] 19.7 s no no yes V [50,53] 20.0 s yes yes no A/B [2,30] 20.8 w no no yes V [50,53] 21.8 m no yes no V [2] 23.2 w yes no no A [30] 26.6 m yes yes no A/B [30] 30.6 w yes yes no r [29,66,67] 33.4 w yes yes yes r [29,66,67] 38.4 w yes yes yes r [29,66] 43.1 w yes no no r [29,66] 1 WAX diffraction intensity notation: w = weak; m = medium; s = strong. 2 Starch structure type: A = residual Atype amylopectin crystallinity from the original starch powder; B = newly formed B-type amylopectin crystallinity due to intensive hydration that occurs during SC; V = newly formed V H -type amylose crystallinity according to [53], r = residual crystallinity from the original starch powder, lower-intensity peaks at higher angles. Polymers 2025, 17, 1310 17 of 21 15.1 s yes no no A/B [30] 17.0 s yes yes no A/B [2,30] 18.1 s yes no no A [30] 19.7 s no no yes V [50,53] 20.0 s yes yes no A/B [2,30] 20.8 w no no yes V [50,53] 21.8 m no yes no V [2] 23.2 w yes no no A [30] 26.6 m yes yes no A/B [30] 30.6 w yes yes no r [29,66,67] 33.4 w yes yes yes r [29,66,67] 38.4 w yes yes yes r [29,66] 43.1 w yes no no r [29,66] 1 WAX diffraction intensity notation: w = weak; m = medium; s = strong. 2 Starch structure type: A = residual A-type amylopectin crystallinity from the original starch powder; B = newly formed B-type amylopectin crystallinity due to intensive hydration that occurs during SC; V = newly formed VHtype amylose crystallinity according to [53], r = residual crystallinity from the original starch powder, lower-intensity peaks at higher angles. Figure A3. Selected WAXS diffraction patterns with annotated diffraction peaks. The diffractions are marked according to Table A1 and references therein. Appendix B. More Details About TPS Micromechanical Properties Figure A4 summarizes the principle of microindentation measurement, shows raw experimental data in the form of representative F-h curves, and gives exact definitions of all micromechanical properties (EIT, HIT, HM, CIT, and ηIT) employed in this study. Complete results of micromechanical measurements are given in the Supplementary Materials. Figure A3. Selected WAXS diffraction patterns with annotated diffraction peaks. The diffractions are marked according to Table A1 and references therein. Appendix B. More Details About TPS Micromechanical Properties Figure A4 summarizes the principle of microindentation measurement, shows raw experimental data in the form of representative F-hcurves, and gives exact definitions of all micromechanical properties (E IT ,H IT ,H M ,C IT , and ηIT ) employed in this study. Complete results of micromechanical measurements are given in the Supplementary Materials. Polymers 2025,17, 1310 18 of 21 Polymers 2025, 17, 1310 18 of 21 Figure A4. Representative real F-h curves from microindentation hardness testing for TPS-AH-0min and TPS-AH-60min samples and brief description of principle of MHI measurements. (a) Micromechanical properties were deduced from the F-h curves (F = indenter loading force, h = indenter penetration depth) by means of the formulas from (b) and the relationships from (c) containing experimental parameters, such as the maximum loading force (Fmax), slope at the beginning of the unloading curve (S), penetration depths at the beginning and end of the maximal load (h1 and h2), and areas under the loading and unloading curves (Welast and Wtotal). The additional parameter, contact depth (hc), was calculated in terms of the Oliver and Pharr theory and employed in the calculation of EIT and HIT. References 1. He, Z.; Chi, C.; Huang, S.; Li, X. A novel method for obtaining high amylose starch fractions from debranched starch. Curr. Res. Food Sci. 2023, 7, 100589. https://doi.org/10.1016/j.crfs.2023.100589. 2. Rana, L.; Kouka, S.; Gajdosova, V.; Strachota, B.; Konefał, M.; Pokorny, V.; Pavlova, E.; Stary, Z.; Lukes, J.; Patocka, M.; et al. Thermoplastic Starch with Maltodextrin: Preparation, Morphology, Rheology, and Mechanical Properties. Materials 2024, 17, 5474. https://doi.org/10.3390/ma17225474. 3. Buléon, A.; Colona, P.; Planchot, V.; Ball, S. Starch granules: Structure and biosynthesis. Int. J. Biol. Macromol. 1998, 23, 85–112. https://doi.org/10.1016/S0141-8130(98)00040-3. 4. Ai, Y.; Jane, J. Gelatinization and rheological properties of starch. Starch-Stärke 2015, 67, 213–224. https://doi.org/10.1002/star.201400201. 5. Suortti, T.; Gorenstein, M.V.; Roger, P. Determination of the molecular mass of amylose. J. Chromatogr. A 1998, 828, 515–521. https://doi.org/10.1016/S0021-9673(98)00831-0. 6. Cui, C.; Ji, N.; Wang, Y.; Xiong, L.; Sun, Q. Bioactive and intelligent starch-based films: A review. 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Synergistic effects in mechanical properties of PLA/PCL blends with optimized composition, processing, and morphology. RSC Adv. 2015, 5, 98971–98982. https://doi.org/10.1039/C5RA21178F. 11. Thakur, R.; Pristijono, P.; Scarlett, C.J.; Bowyer, M.; Singh, S.P.; Vuong, Q.V. Starch-based films: Major factors affecting their properties. Int. J. Biol. Macromol. 2019, 132, 1079–1089. https://doi.org/10.1016/j.ijbiomac.2019.03.190. Figure A4. Representative real F-hcurves from microindentation hardness testing for TPS-AH-0min and TPS-AH-60min samples and brief description of principle of MHI measurements. 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