Structure and properties of ozone-induced Schiff-base crosslinked starch-chitosan complex under ozone duration

Abstract

This study investigates the regulatory effect of ozone-induced Schiff base crosslinking on the structure and properties of a chitosan/waxy rice starch composite system. By varying the ozone treatment duration (0–60 min), changes in chemical structure, crystalline characteristics, thermal stability, and gel properties were systematically analyzed. Structural characterizations including FTIR and NMR confirmed the formation of Schiff base bonds and structural rearrangement. SEM and rheological analysis indicated that ozone optimized the pore structure and rheological properties of the gel, endowing it with excellent extensibility and coating performance. Results indicate that short-term ozonation (≤30 min) promotes crosslinking between carbonyl and amino groups, significantly enhancing the composite’s molecular weight, crystallinity, and thermal stability. Conversely, excessive oxidation (≥45 min) causes chain scission and performance degradation. This study elucidates the structural evolution mechanism of chitosan/starch composites under ozone treatment, providing theoretical foundations for designing green, controllable crosslinking and edible functional materials.

Introduction

Bio-based polymer materials have garnered significant attention due to their renewability, biocompatibility, and environmental friendliness. Starch is a naturally occurring polymer carbohydrate found in nature in relatively large quantities. Due to its rich hydroxyl groups, as well as the particles can form hydrogels after heating and water absorption and pasting, starch exhibits unique advantages such as uniformly distributed network structure and biodegradability, biocompatibility, water absorption and water retention, etc. Thus, starch-based materials have been widely used in the fields of excipients, delivery of functional factors¹, tissue engineering², and other food and pharmaceutical fields. Waxy rice starch (WRS) has a high branched amylose content (≥98%), a property that enables it to form dense gels and flexible matrices³, which gives it unique competitiveness in the system of natural polymer materials. However, natural WRS-formed gel material exhibits relatively low strength, toughness, and has limited. In addition, WRS is also prone to rupture or deformation. These limitations seriously restrict its practical applications.

To address these issues, current researches focus on compositing starch with other functional polymers. Chitosan (a deacetylated derivative of chitin) has emerged as a prime candidate for improving the functional properties of starch due to its biocompatibility and ease of processing⁴. Specifically, its abundant amine group (–NH₂) in chitosan can interact with the hydroxyl group (–OH) on the starch chain to modulate the properties of starch gels⁵. The complexation between chitosan and WRS is expected to enhance the gel properties. Previous studies have explored various crosslinking approaches between chitosan and starch, demonstrating that synergistic interactions and the incorporation of new functional groups enhance the structural stability and functional performance of the composite system. Many studies have focused on the cross-linking of chitosan with modified starch (de-branched starch⁶, oxidized starch⁷, carboxymethyl starch⁸, esterified starch⁹, etc.), and the complexation was achieved through non-covalent interactions¹⁰ (hydrogen bonding, ionic bonding, etc.) and covalent cross-linking¹¹ (esterification, Schiff base reaction, etc.). However, physical crosslinking methods often face challenges in achieving sufficient structural stability, while conventional chemical crosslinking, although effective in reinforcing the composite networks, may introduce potential toxicity or compromise biocompatibility.

In this context, ozone oxidation represents a promising green alternative for polysaccharide modification. Unlike conventional chemical agent, ozone introduces carbonyl/carboxyl groups through controlled depolymerization without generating toxic residues, which is crucial for food contact materials. It has been reported that ozone treatment usually increases the solubility and acidity of starch, but prolonged ozone treatment breaks glycosidic bonds, reduces molecular weight and alters its thermal stability¹², which in turn affects the pasting and ageing properties of starch¹³. In contrast, a moderate ozonation time reduces starch crystallinity and balances the dissociation and cross-linking of starch chains. Therefore, controlling the reaction time of ozone oxidation is essential to optimize its performance in food processing¹⁴. However, most of the existing studies have focused on the ozone effect of single biopolymers rather than composite systems, while the cross-linking reaction occurring between the resultant –CHO and –OH has been rarely utilized to constructing new starch derived. Based on the past research, the ozonation has been used as an interesting method for chitosan modification, and the treating time was also closely related to the molecular weight of chitosan and its biological activity¹⁵. Here, we propose that ozone-induced aldolization of starch hydroxyl groups and acid regulation probably could provide a reaction environment and substrate for some cross-linking reactions, such as the starch/chitosan Schiff-based reaction which has been proved in our previous report¹⁶. This method does not involve starch pre-oxidation steps or the introduction of chemical reagents, and is relatively green. Remarkably, this gap is particularly critical because intermolecular interactions such as hydrogen bonding between chitosan amino groups and starch hydroxyl groups might be both affected by ozone. The key was found that short-term ozone exposure can enhance product properties, whereas prolonged treatment may lead to excessive degradation. Optimizing the parameters for ozone oxidation is essential to enhance the potential of chitosan/waxy starch composites, as evidenced by studies demonstrating the controlled degradation and functionalization of chitosan through this process.

This work systematically investigates the effect of ozone oxidation time (0–60 min) on chitosan/WRS composite products. Fourier transform infrared (FTIR), X-ray diffraction (XRD) and nuclear magnetic resonance (NMR) were used to characterize the chemical and crystal structure evolution. The structural changes of the gel network were investigated by rheology, Low-field nuclear magnetic resonance (LF-NMR) and hydration characterization to provide theoretical support for the preparation and performance regulation of chitosan/WRS composite gels.

Results and discussion

Structure and hydration properties of OCS with different degrees of deacetylation

FTIR was utilized to characterize the structural changes of three chitosan with different degrees of deacetylation/complexed with WRS under the effect of ozone. As shown in Fig. 1A, the absorption peaks at 3300–3500 cm⁻¹ in the FTIR curves corresponded to the –OH stretching vibration. The peaks at 1417 cm⁻¹ and 2928 cm⁻¹ were assigned to the bending vibration of the C–H bond and the stretching vibration of the CH₂ bond, respectively. The peaks in the range of 920–960 cm⁻¹ were attributed to the vibration of α-1,4-glycosidic bond. Moreover, the peaks at 761 cm⁻¹, 849 cm⁻¹, and 1081 cm⁻¹ should be related to the C–C bond stretching vibration, CH₂ bond deformation vibration, and C–OH bending vibration¹⁷, respectively. Compared with FTIR curve of OS, OCS showed a new absorption peak at 1560 cm⁻¹, which was attributed to the C=N stretching vibration formed by the ozone-induced Schiff base reaction. It was also found that the intensity was enhanced with the increasing chitosan deacetylation due to the increase of free amino groups¹⁸, which raised the reaction possibility to the formation of C=N increased accordingly.

Fig. 1. Structure and hydration properties of OCS with different degrees of deacetylation.

A FTIR spectra of samples prepared using composite systems of chitosan with varying degrees of deacetylation and waxy rice starch after 30 min of ozonation. B RVA spectra of samples prepared using composite systems of chitosan with varying degrees of deacetylation and waxy rice starch after 30 min of ozonation. C Solubility (S) and Swelling power (SP) of samples prepared using composite systems of chitosan with varying degrees of deacetylation and waxy rice starch after 30 min of ozonation, mean ± standard deviation; variables with the same letter within the same group are not significantly different (p < 0.05). D Water holding capacity (WHC) and Water absorption index (WAI) of samples prepared using composite systems of chitosan with varying degrees of deacetylation and waxy rice starch after 30 min of ozonation, mean ± standard deviation; variables with the same letter within the same group are not significantly different (p < 0.05).

The RVA spectra of different samples was shown in Fig. 1B. Deacetylation degree affects the properties of chitosan, which in turn affects the pasting properties of OCS. Chitosan with high deacetylation degree has a more rigid molecular chain, which forms a more complex and stable network structure in combination with WRS. When the temperature changes, the OCS formed by low-deacetylated chitosan has weak interactions between molecular chains, and the viscosity may decrease rapidly with the increase of temperature, while the OCS formed by high-deacetylated chitosan has strong interactions between molecular chains, and the change of viscosity was relatively stable.

Figure 1C, D shows the solubility & swelling (C) and water holding & water absorption index (D) of the samples. As can be seen from Fig. 1C, the introduction of chitosan significantly reduced the solubility of oxidized starch. Chitosan and starch formed a three-dimensional network through electrostatic interaction or covalent cross-linking (Schiff base reaction), which increased the spatial site resistance and inhibited solubilization. And the higher the degree of deacetylation, the more amino (–NH₂) starch molecules crosslinked on the molecular chain of chitosan to form a dense network structure, resulting in an increase in the rigidity of the molecular chain and a decrease in flexibility. Therefore, the swelling capacity of starch decreases with increasing chitosan deacetylation. In Fig. 1D, it is observed that the water holding capacity (WHC) of starch shows the same trend as the water absorption index. This is attributed to the dense cross-linking network limiting the hydration of starch and hindering the penetration and retention of water molecules. This result suggested that the ozone treatment as well as the degree of deacetylation of chitosan together affect the functional group interactions of the complexes, which ultimately led to the differences in the properties such as solubility, swelling power, water holding and water absorption. Therefore, to achieve the highest possible degree of crosslinking between chitosan and starch, chitosan with a deacetylation degree of 95% was used in the subsequent analyzes.

Structure characterization of OCS with different degrees of oxidation

FTIR spectroscopy was employed to characterize the chemical groups and short-range ordered structures in starch-based materials. As illustrated in Fig. 2A, characteristic absorption peaks at 3419 cm⁻¹ and 2930 cm⁻¹ were observed across all samples, corresponding to O–H stretching vibrations and C–H stretching vibrations of CH₂ groups in glucose units¹⁹, respectively. The spectral feature at 1637 cm⁻¹ arises from H-O-H bending vibrations of bound water molecules. A distinct absorption band at 1458 cm⁻¹ can be assigned to CH₂ scissoring vibrations. The spectral region between 1155 and 1065 cm⁻¹ displays characteristic C–O–C stretching vibrations typical of starch polysaccharides²⁰. Comparative analysis revealed significant structural differences between control sample (CS) and ozonated starch-chitosan complexes (OCS). Specifically, OCS samples exhibited a novel absorption band at 1560 cm⁻¹, absent in CS, corresponding to C=N stretching vibrations of imine groups. This spectroscopic evidence confirmed the formation of covalent bonds between starch and chitosan through Schiff base reactions between carbonyl groups (generated via ozone oxidation) and amine groups from chitosan²¹. In Fig. 2A, the appearance and gradual enhancement of the 1560 cm⁻¹ band confirmed the progressive formation of imine linkages through the ozone-mediated Schiff base reaction. However, excessive ozonation (≥45 min) weakened this peak, likely due to overoxidation of carbonyls into carboxyls, thereby decreasing available reaction sites.

Fig. 2. Structure characterization of OCS with different ozonation times.

A FTIR of samples obtained by blending chitosan with 95% deacetylation degree and waxy rice starch, subjected to varying ozonation durations. B The absorbance ratio at 1047/1022 cm⁻¹ in FTIR spectroscopy was calculated to characterize the short-range ordered structure of the sample, mean ± standard deviation; variables with the same letter within are not significantly different (p < 0.05). C XRD spectra of samples obtained by blending chitosan with 95% deacetylation degree and waxy rice starch, subjected to varying ozonation durations. D Relative crystallinity of the sample was obtained by performing fitting calculations on the XRD curve, mean ± standard deviation; variables with the same letter within are not significantly different (p < 0.05).

In addition to considering the chemical changes in the starch structure, the FTIR absorbance ratio at 1047/1022 cm⁻¹ was calculated to characterize the short-range ordered structure of the samples. The relatively high FTIR absorbance ratio at 1047/1022 cm⁻¹ indicated that the starch exhibits superior short-range order²². Ozone treatment resulted in a significant decrease in the 1047/1022 cm⁻¹ ratio, indicating reduced short-range order in the modified samples. This structural disruption may originate from the introduction of oxygen-containing functional groups, particularly carbonyl (C=O) and carboxyl (–COOH) moieties, through ozone-mediated oxidation. Notably, the duration of ozonation exhibited no statistically significant influence on the short-range ordering parameters.

XRD analysis was employed to investigate potential modifications in the crystalline architecture of starch. Figure 2C presents the XRD patterns and relative crystallinity data for all samples. The patterns of CS and OCS displayed characteristic diffraction peaks at 2θ angles of 15.1°, 17.2°, 18°, and 23°, confirming the preservation of A-type crystalline structure post-ozonation. This proved that the ozonation did not change the crystal structure type of CS. In Fig. 2D, it could be observed that the relative crystallinity of OCS increased continuously at 15 min and 30 min of ozonation, which was attributed to the destruction of amorphous area²³. However, after 30 min of ozone treatment, the relative crystallinity of OCS decreased due to ozone attacking its semi-crystalline regions. This oxidation induced localized structural changes in the molecular chains, disrupting their original order and thereby weakening crystal integrity and reducing crystallinity²⁴.

The resonance peaks observed in the NMR spectra of the samples were determined by the chemical shifts of the substances. Figure 3A, B shows the hydrogen spectrum (¹H-NMR). As in previous results, there was a significant peak near the chemical shift of about 2 ppm, which was attributed to the resonance of acetylamino groups in chitosan^25,26. The group corresponding to this peak might originate from the cross-linking of chitosan with starch molecules²⁷. Upon comparing the NMR spectra of CS and OCS at different ozonation times, distinct peak shape alterations were observed in OCS-15 relative to CS. These spectral differences provided evidence that ozonation induced structural modifications in the chitosan/starch complex. As the time was extended to 30 min (OCS-30), 45 min (OCS-45) and 60 min (OCS-60), subtle changes in the peaks continued to appear. This peak shape alteration coupled with a slight shift in chemical shift indicates a change in the local chemical environment, suggesting the rearrangement of new chemical bonds and intermolecular forces.

Fig. 3. Nuclear magnetic resonance spectrum of OCS obtained from different ozonation times.

A, B ¹H-NMR spectrum in the δ rang of 0–13 ppm and 1.6–2.1 ppm. C, D ¹³C-NMR spectrum in the δ rang of 20–120 ppm and 20–23 ppm. E Mechanism and reaction process of ozone-induced Schiff base crosslinking between chitosan and waxy rice starch.

In the ¹³C NMR spectra shown in Fig. 3C, D, a new resonance peak appeared at ~21 ppm. This peak corresponds to the methylene groups (–CH₂–) in –O–CH₃ or OCO-CH₃ moieties that are not part of the glucose ring structure²⁸. The intensity and shape of this peak varied with the ozonation duration. The enhanced signal observed after short-term ozonation (15 min) supports that brief ozone exposure not only oxidized hydroxyl groups of starch into carbonyl groups but also promoted condensation reactions with amino groups of chitosan, thereby generating new substituted chemical environments. With prolonged ozonation (≥30–45 min), this peak and other related resonances exhibited further broadening or attenuation, which is consistent with molecular chain scission, excessive oxidation, and the increasing complexity of the chemical environment.

Thus, peak broadening and signal intensity changes commonly observed in NMR spectra correlate with molecular transformations. Short-term ozone oxidation (OCS-15) typically induces peak broadening and the emergence of new resonance peaks, indicating partial molecular cross-linking or polymerization. Conversely, as ozone oxidation time increases, fine spectral features gradually weaken, disappear, or become absent, suggesting ongoing chain scission. This interpretation aligns with findings from FTIR, which reveals the appearance of a C=N bond absorption peak at 1560 cm⁻¹ and a subsequent weakening of this peak’s intensity under excessive oxidation conditions.

Hydration properties, carbonyl content and molecular weight

Figure 4A, B shows the effect of ozone oxidation time on the solubility (S), swelling (SP), WHC, and water absorption index (WAI) of the samples. In Fig. 4A, it was found that the solubility (S) of OCS decreased significantly with the increase of ozonation time, which was attributed to the reduction of hydrophilic groups due to the oxidation of hydroxyl groups (–OH) in starch molecules to carbonyl groups and induced cross-linking with amino groups (NH₂) in chitosan by ozone. After ozonation of CS, SP, WHC, and WAI showed a tendency to increase and then decrease, which might be due to ozone attacked the chitosan-starch molecules, resulting in the appearance of many tiny pits and bumps on the originally smooth surface. Therefore, locally charged regions appear on the surface of the complex. Positively charged areas attract the negatively charged end of water molecules, while negatively charged areas attract the positively charged end. This mutual attraction between charges, compared with weak interactions such as van der Waals forces alone when the surface was smooth, could adsorb the water molecules on the surface of the complex more strongly and promote the dissolution process. After 30 min of ozonation, SP, WHC, and WAI decreased. This might be the result of ozone-induced structural disintegration within the starch granules or granule weakening^29,30. When ozone interacted with chitosan starch complexes, it caused changes in the molecular structure. This oxidation might also cut the macromolecular chains of chitosan and starch into many small molecular fragments. Therefore, it was difficult to form an effective three-dimensional network structure to accommodate water molecules. The space that could have been formed by cross-linking between the molecular chains was destroyed, and water molecules can’t be effectively immobilized.

Fig. 4. Investigation of hydration behavior and distribution of oxygen-containing functional groups and molecular chain lengths of OCS prepared obtained from different ozonation times.

A Solubility (S) and Swelling power (SP) of samples obtained by blending chitosan with 95% deacetylation degree and waxy rice starch, subjected to varying ozonation durations, mean ± standard deviation; variables with the same letter within are not significantly different (p < 0.05). B Water holding capacity (WHC) and Water absorption index (WAI) of samples obtained by blending chitosan with 95% deacetylation degree and waxy rice starch, subjected to varying ozonation durations, mean ± standard deviation; variables with the same letter within are not significantly different (p < 0.05). C Carbonyl content of samples obtained by blending chitosan with 95% deacetylation degree and waxy rice starch, subjected to varying ozonation durations, mean ± standard deviation; variables with the same letter within are not significantly different (p < 0.05). D Molecular weight of samples obtained by blending chitosan with 95% deacetylation degree and waxy rice starch, subjected to varying ozonation durations.

Figure 4C shows the process of carbonyl content change of the sample. Many hydroxyl groups exist on the molecular chains of starch. In the oxidation process, hydroxyl groups, especially those on C2, C3 and C6, were first oxidized to carbonyl groups and then converted to carboxyl groups¹⁴. The content of carbonyl groups gradually increased with the ozonation time. This result was consistent with previous results on the ozonation of cassava and buckwheat starches^31,32.

Gel permeation chromatography was applied to determine the molecular weight distribution of the samples. As shown in Fig. 4D and Table 1, the number average molecular weight (Mn) and weight average molecular weight (Mw) of OCS were significantly higher after 15 min ozonation (Mn = 671 kDa, Mw = 3103 kDa), which was much higher than that of the untreated sample (CS). This indicated that ozone treatment induced cross-linking reactions (oxidative cross-linking of the amino groups of chitosan with the hydroxyl groups of starch) of chitosan and starch molecular chains to form larger molecular aggregates. It was noteworthy that Mn and Mw gradually falling to 203 kDa (Mn) and 986 kDa (Mw). This was attributed to the persistent oxidative action of ozone causing molecular chain breakage (particularly the oxidative cleavage of glycosidic bonds)³³, whereby degradation gradually outweighed cross-linking, leading to a significant reduction in molecular weight. The polydispersity coefficient (PD) of OCS was lower than that of CS, indicating that the ozone treatment narrowed the molecular weight distribution, and the cross-linking or degradation process was relatively homogeneous. The PD value of OCS exhibited a tendency of decreasing and then increasing, which indicated that the prolonged ozone treatment caused the molecular chain of the complexes to be broken³⁴, generating more low molecular weight fragments and leading to a re-broadening of the distribution. The Rz (131.6 nm) of OCS-15 was the largest, indicating that the cross-linking reaction resulted in molecular stretching and increase in size. Similarly, the Rz gradually decreased with ozonation time (OCS-60 to 115.7 nm), approaching CS (114 nm), suggesting that the molecular chains of the complexes began to degrade under ozonation, which promoted the production of more short molecular chains.

Table 1.

Molecular weight of OCS prepared obtained from different ozonation times

Samples	Mn (kDa)	Mw (kDa)	PD (Mw/Mn)	Rz (nm)
CS	89.359	447.674	5.010	113.999
OCS-15	671.222	3102.719	4.622	131.584
OCS-30	394.204	1666.589	4.228	125.453
OCS-45	303.265	1408.342	4.644	123.022
OCS-60	203.025	985.996	4.857	115.737

Mn, number-average molecular weight; Mw, weight-average molecular weight; PD (Mw/Mn), polydispersity coefficient; Rz, z-average radius of gyration.

Micromorphology and surface properties

Figure 5A–E illustrates the cross-sectional microstructures of starch-chitosan gels subjected to different ozonation times. The unozonized CS gel exhibited uneven pore sizes, characterized by numerous large cavities and loose pore walls. This was attributed to the dominance of intra- and intermolecular hydrogen bonding in the absence of covalent crosslinking, resulting in a less uniform network. Upon ozonation, a structural reorganization occurred as ozone preferentially reacted with surface functional groups, inducing local deformation and rearrangement of the pore architecture. With prolonged ozonation, the pore uniformity first improved and then deteriorated. The OCS-15 sample still showed slight heterogeneity, while OCS-30 exhibited the most homogeneous and moderately sized pores. Further oxidation (OCS-45 and OCS-60) led to irregular pores and partial collapse of the network. These observations indicate that ozone oxidation generated reactive groups, promoting covalent crosslinking between starch and chitosan molecules. At short ozonation times (15–30 min), increasing numbers of reactive sites enhanced network density and uniformity, whereas excessive oxidation (45–60 min) caused chain scission or over-crosslinking, disrupting the structural integrity. This balance can be attributed to ozone-mediated interfacial chemistry, where synergistic hydrogen bonding and electrostatic interactions, reinforced by Schiff-base crosslinking, collectively stabilize the network through covalent anchoring and multidimensional noncovalent interactions.

Fig. 5. Micromorphology and surface properties of OCS prepared obtained from different ozonation times.

A–E Scanning Electron Microscope (SEM) of samples obtained by blending chitosan with 95% deacetylation degree and waxy rice starch, subjected to varying ozonation durations. F Water contact angle of samples obtained by blending chitosan with 95% deacetylation degree and waxy rice starch, subjected to varying ozonation durations.

As shown in Fig. 5F, these results provide insight into the evolution of surface characteristics during ozonation. Compared with CS, the water contact angle (θ) of OCS-15 increased, indicating enhanced surface hydrophobicity. This enhancement can be attributed to the selective oxidation of reactive sites along polysaccharide chains, leading to the introduction of hydrophobic carbonyl groups such as ketones and aldehydes. However, as the ozonation time was further prolonged, hydrophobicity decreased, and the contact angle systematically declined. Prolonged oxidation induced structural reorganization characterized by two concurrent processes: (1) chain scission exposed previously embedded hydrophilic groups, and (2) generation of polar carboxyl functionalities via advanced oxidation. These effects accounted for the observed increase in hydrophilicity, as evidenced by the continuous reduction in θ with ozonation time. The temporal modification pattern aligned with the proposed mechanism of ozone-mediated surface restructuring, in which initially formed hydrophobic moieties are progressively converted into hydrophilic structures through cumulative oxidative transformations.

Figure 6A displays the particle size distribution curves of chitosan–starch composites under different ozone treatment durations. All samples exhibit bimodal distributions, indicating that particles predominantly aggregate within two characteristic size ranges. Notably, the curve of CS is broad with an indistinct unimodal feature, stemming from severe agglomeration caused by strong intermolecular hydrogen bonding. As ozone treatment time increased, the distribution peaks of OCS gradually narrowed and shifted to the left, indicating that moderate ozone oxidation and cross-linking disrupted large particle aggregates, enhancing system dispersion. When treatment exceeded 30 min, the curve broadened again with pronounced unimodal characteristics, suggesting that excessive oxidation caused molecular chain breakage and secondary aggregation of small particles, thereby reducing system uniformity. Table 2 further corroborates this trend: Compared to CS, OCS exhibits a significantly reduced volume-weighted average particle size (D_4,3), with the most pronounced decrease (38.7% reduction) observed in the 30-min ozone-treated sample. This phenomenon might be attributed to ozone-induced polysaccharide chain breakage, which diminished the average particle size and increased the proportion of smaller particles.

Fig. 6. Microstructural dimensions and surface charge of OCS prepared obtained from different ozonation times.

A Particle size distribution of samples obtained by blending chitosan with 95% deacetylation degree and waxy rice starch, subjected to varying ozonation durations. B Zeta potential of samples obtained by blending chitosan with 95% deacetylation degree and waxy rice starch, subjected to varying ozonation durations, mean ± standard deviation; variables with the same letter within are not significantly different (p < 0.05).

Table 2.

Particle size distribution of ozonated chitosan/waxy rice starch complex (OCS) obtained from different ozonation times

Samples	D4,3 (µm)	D10 (µm)	D50 (µm)	D90 (µm)	Specific surface area (m²/kg)
CS	54.00 ± 0.38d	5.60 ± 0.05b	146 ± 1.00c	15.22 ± 0.22b	475.3 ± 4.27a
OCS-15	52.20 ± 1.61d	4.85 ± 0.03a	154 ± 4.16d	16.24 ± 0.16c	576.4 ± 6.19c
OCS-30	43.70 ± 0.35c	4.81 ± 0.02a	125 ± 1.00b	17.37 ± 0.14d	579.4 ± 0.76c
OCS-45	29.20 ± 1.65a	4.84 ± 0.02a	78.5 ± 6.55a	15.07 ± 0.20b	621.1 ± 6.21d
OCS-60	32.30 ± 0.71b	5.76 ± 0.03c	74.4 ± 2.54a	11.46 ± 0.23a	498.8 ± 1.87b

D_4,3 represents the average particle diameter by volume; D₁₀ and D₉₀ refer to the content of particles smaller than this size as 10% and 90% of all particles, respectively; D₅₀ refers to the content of particles smaller and larger than this size as 50% of all particles. Mean ± standard deviations; variations followed by the same small letters (a-d) within a column do not differ significantly (p < 0.05).

Intriguingly, the D₅₀ and D₉₀ values of OCS-15 were significantly elevated (154 ± 4.16 μm and 16.24 ± 0.16 μm, respectively), suggesting that short-duration ozonation primarily facilitates cross-linking reactions between chitosan and starch molecules, leading to formation of larger aggregates. Conversely, prolonged ozonation induced excessive particle degradation, as evidenced by increased specific surface area (from 475.3 m²/kg to 621.1 m²/kg) and surface charge imbalance caused by carboxyl group accumulation (7.16 ± 0.45 mV zeta potential). These physicochemical alterations promoted secondary aggregation of fragmented particles through hydrogen bonding and van der Waals interactions. The significant decrease in D₅₀ and D₉₀ with prolonged ozonation from 15 to 60 min demonstrated the significant time-dependent structural modification of the complexes, suggesting that oxidative restructuring critically influences aggregation behavior and ultimate particle size characteristics³⁵.

Figure 6B illustrates the zeta potential variation profiles of all investigated samples. It was found that the chitosan/starch complexes showed a decrease in zeta potential following 15-min ozonation treatment. This was attributed to the oxidation of amino and hydrogen groups. Specifically, positively charged amino groups on the molecular chain formed electrostatic interactions with oxidized carbonyl groups possessing negative charges, thereby reducing the surface positive charge density and consequently diminishing the zeta potential values. Notably, prolonged ozonation duration (30 min), the cross-linking reaction and molecular depolymerization increased the number of surface amino groups, manifesting as an observable increase in positive charge density. Progressive oxidation facilitated molecular chain cleavage, which produced the additional anionic functional groups while simultaneously decreasing the absolute surface charge magnitude through charge neutralization effects.

Thermogravimetric analysis and Paste property

The thermogravimetric analysis (TGA) and derivative thermogravimetric (DTG) curves characterizing the thermal degradation processes of various samples are presented in Fig. 7A. DTG profiles, derived through differentiation of TG curves, quantitatively illustrate mass loss rates during thermal decomposition. Three distinct degradation stages were identified across all specimens: The first stage (30–150 °C) corresponds to water evaporation; the second stage (150–420 °C) arises from depolymerization and pyrolysis of molecular chains in the complexes; while the third stage (420–600 °C) reflects polymer carbonization processes³⁶. Figure 7B demonstrated a progressive increase in weight retention with ozonation duration, reaching stabilization after 45 min. This phenomenon originated from ozone-induced inter-molecular cross-linking between chitosan and starch chains. Enhanced cross-linking density facilitated the formation of stabilized three-dimensional networks that restrict molecular chain mobility and enhance char residue formation at elevated temperatures. The temperatures of CS, OCS-15, OCS-30, OCS-45, and OCS-60 at maximum weight loss rate were found to be 318.8 °C, 320.3 °C, 319.4 °C, 318.7°C, and 317.4 °C, respectively, from Fig. 7C. Cross-linking of molecular chains due to Schiff base reaction led to the increase in thermal decomposition temperature and thermal stability of OCS-15 and OCS-30. However, prolonged ozone exposure induced depolymerization of molecular architectures, consequently reducing thermal stability in OCS-45&60 specimens³⁷.

Fig. 7. Thermal and Paste properties of OCS prepared obtained from different ozonation times.

A TGA of samples obtained by blending chitosan with 95% deacetylation degree and waxy rice starch, subjected to varying ozonation durations. B By continuously monitoring the mass changes of the sample during the programmed temperature ramping process and combining this with the initial mass reference, the weight retention is calculated. C DTG curve was obtained by performing first-order differentiation on the raw mass-temperature data from TG (thermogravimetric analysis). D RVA curves of samples obtained by blending chitosan with 95% deacetylation degree and waxy rice starch, subjected to varying ozonation durations.

The pasting curves of all the samples are presented in Fig. 7D. In addition, the peak viscosity (PV), trough viscosity (TV), final viscosity (FV), disintegration value (BD), retrogradation value (SB), and pasting temperature (PT) of the samples are summarized in Table S2. Consistent with findings from Castanha et al. regarding ozonated potato starch¹⁴, ozone significantly reduced the PV of the CS system, and quantitative analysis revealed a progressive decrease in PV values from 7761.5 ± 6.36 mPa·s to 3643.0 ± 147.08 mPa·s with extended ozonation duration. This phenomenon could be attributed to the alteration of surface properties in CS through ozonation, resulting in increased exposure of hydrophilic functional groups (particularly carboxyl groups). The enhanced hydration capacity facilitates stronger water molecule binding, thereby improving system fluidity while concurrently reducing viscosity in ozone-treated complexes (OCS). Additionally, the BD value reflects the heat resistance of starch pastes, with lower values indicating better heat resistance³⁸. Notably, OCS-15 exhibited the lowest BD value (201.0 ± 86.27 mPa·s) among treated samples, indicating optimal thermal stability. This observation aligned with TGA results, confirming the structural modifications induced by ozone treatment.

Rheological property and moisture distribution

As shown in Fig. 8A, B, the energy storage modulus (G’) and loss modulus (G”) of all samples increased with increasing frequency, exhibiting frequency dependence. For starch gels, the G’ values always dominate their G” values (tan δ < 1.0) (Fig. 8C), which indicates their gel-like nature. The G’ of OCS-15 was slightly higher than that of CS in the high frequency phase, and G” was higher than that of CS in the full frequency range. higher than CS in the full frequency range, which was attributed to the ozone-promoted cross-linking between the molecular chains of chitosan and starch. Specifically, the amino group on the molecular chain of chitosan reacts with the hydroxyl group on the molecular chain of starch under the action of ozone to form a covalent bond connection. This cross-linking makes the connection between the molecular chains tighter and more solid, which leads to a rise in the energy storage modulus. However, with the ozonation time, the G’ and G” of OCS gradually decreased, especially OCS-60 decreased sharply, but significantly elevated the loss angle tangent (tan δ). This was due to the strong oxidizing property of ozone, and a short time of ozonation might induce the cross-linking between the molecular chains of chitosan-starch complexes, resulting in a rise in the energy storage modulus. However, with the prolongation of ozonation time, the reactive groups such as amino and hydroxyl groups on the molecular chains were over-oxidized, leading to chemical bond breakage. The ability of the gel to store energy elastically was weakened and the energy storage modulus decreased. The above results indicate that the gels prepared in this paper have low viscosity and high flowability, present excellent spreading and coating properties, and were therefore high-quality materials for the preparation of edible films and tablet coatings³⁹.

Fig. 8. Rheological properties of OCS at different ozonation times and water migration patterns within the gel network.

A Changes in the storage modulus (G’) and loss modulus(G”) of composite systems under dynamic shear loading. B The ratio of the storage modulus (G’, representing elasticity) to the loss modulus (G”, representing viscosity). C Shearing properties of samples obtained by blending chitosan with 95% deacetylation degree and waxy rice starch, subjected to varying ozonation durations. D Moisture distribution of samples obtained by blending chitosan with 95% deacetylation degree and waxy rice starch, subjected to varying ozonation durations. E Gels appearance of samples obtained by blending chitosan with 95% deacetylation degree and waxy rice starch, subjected to varying ozonation durations.

Figure 8C shows the variation of apparent viscosity with shear rate for CS and OCS gels. It could be observed that all the samples exhibit the typical shear-thinning property of pseudoplastic fluids. CS demonstrated the highest apparent viscosity, and the apparent viscosity of the gels decreased with the increase of ozonation time. This suggested that the decrease in viscosity and the weakening of the pseudoplastic behavior of OCS were interdependent, and they were basically caused by the ozone-induced partial depolymerization of the amorphous region of starch molecules³⁹.When chitosan-starch complexes form gels, they rely on interactions between molecular chains to build up a three-dimensional network structure. Prolonged ozonation altered the structure of the molecular chains, resulting in the destruction of the sites that originally interacted to form the network. Therefore, the integrity and cohesion of the gel decrease, making it more prone to flow under shear forces and other influences, thereby reducing its apparent viscosity. In addition, starch with a higher carbonyl content also exhibited a greater reduction in viscosity, as observed by Sangseethong et al. ⁴⁰.

The state and distribution of water molecules inside the gel system and their interaction with the polymer network were analyzed by LF-NMR. For starch-based hydrogels, the lower transverse relaxation time (T₂) of hydrogen protons usually indicates the tight binding of water molecules to polysaccharides and the relative reduction of their mobility⁴¹. In general, T₂₁ (1–10 ms) represents bound water, T₂₂ (10–100 ms) represents immobilized water in the gel structure, and T₂₃ (100–10,000 ms) represents free water in the system⁴². As shown in Fig. 8D, three signal peaks (T₂₁, T₂₂ and T₂₃) were present in the plots of all gel samples. Compared with CS, the three signal peaks of OCS were shifted to the right, and the mobility of water molecules in the system was elevated, which indicated that more water molecules underwent tighter binding with other components⁴³. As shown in Table 3, the proportions of T₂₃ peak areas of OCS were all significantly lower than those of CS, indicating that the free water within the composite gel system decreased and the water-holding capacity of the gel system increased⁴⁴. This was due to the cross-linking of chitosan with WRS in the presence of ozone, forming a denser gel network. In this environment, the space for free movement of water molecules was restricted, leading to a decrease in free water. The gradual increase in T₂₁, T₂₂ peak area with ozonation time was attributed to the gel network intercepted some of the free water and converts it into bound or immobilized water.

Table 3.

Peak area percentages of T₂₁, T₂₂ and T₂₃

Samples	T21 (%)	T22 (%)	T23 (%)
CS	0.79 ± 0.13a	3.30 ± 0.42ab	95.92 ± 0.29 d
OCS-15	1.26 ± 0.02b	3.52 ± 0.03abc	95.23 ± 0.06c
OCS-30	1.54 ± 0.05c	3.68 ± 0.02bc	94.78 ± 0.03ab
OCS-45	1.94 ± 0.00d	4.02 ± 0.02c	94.04 ± 0.02a
OCS-60	2.06 ± 0.14d	3.04 ± 0.10a	94.89 ± 0.04bc

Mean ± standard deviations, variations followed by the same small letters (a-d) within a column do not differ significantly (p < 0.05).

As the ozonation time increased from 0 to 60 min, the appearance of the composite gel gradually changed. As shown in Fig. 8E, the gel prepared from unozonized CS appeared relatively transparent, with a few small bubble-like structures trapped inside. When the ozonation time was extended to 15 and 30 min, the gels remained transparent but became visually more homogeneous, with fewer or less distinct bubbles or particulate domains, suggesting internal structural rearrangement or partial dissolution and fusion of microdomains. At 45 min, a slight yellowish tint appeared, accompanied by a noticeable reduction in transparency, which became more pronounced at 60 min. This color change was likely caused by side reactions associated with excessive oxidation, which altered the chemical composition and optical properties of the gel.

Textural properties

The textural properties of the composite gels were shown in Table 4. The hardness showed a gradual decrease from CS to OCS-60. CS had the highest hardness of 4.09 g, while OCS-60 had the lowest hardness of 2.06 ± 0.01 g. This indicates that the hardness of the gels was decreasing with the increase of ozonation time. This might be due to the Schiff base reaction of chitosan with WRS to form a more pliable network structure. With the extension of ozonation time, the molecular chains of chitosan and starch were broken, which weakened the network structure of the gel and led to a decrease in its resistance to external forces. The elasticity of CS and OCS-30 and OCS-45 was relatively close to each other, with 1.90 ± 0.10, 1.90 ± 0.03, and 2.07 ± 0.03, respectively; and the elasticity of OCS-15 was 1.56 ± 0.11, which was relatively low; The elasticity of OCS-60 was 1.66 ± 0.09, which was also at a low level. Concurrently, the overall adhesiveness of the gels exhibited a decreasing trend. This indicates that excessive oxidation leads to molecular degradation and disruption of the three-dimensional network structure. These findings are highly consistent with rheological and thermal analysis results, confirming that controlled ozone treatment enhances network density and gel strength through limited crosslinking, whereas prolonged oxidation causes chain scission and weakens the gel matrix. Therefore, in the starch-chitosan system, selecting the optimal ozone treatment duration is crucial to balance crosslinking efficiency and structural integrity.

Table 4.

The textural properties of ozonated chitosan/waxy rice starch complex (OCS) obtained different ozonation times

Samples	Hardness (g)	Adhesiveness (g*s)	Cohesiveness	Springiness	Gumminess (g)
CS	4.09 ± 0.00e	0.30 ± 0.01a	0.25 ± 0.01ab	1.90 ± 0.10b	1.02 ± 0.03c
OCS-15	2.84 ± 0.10 d	0.33 ± 0.00b	0.23 ± 0.02a	1.56 ± 0.11a	0.64 ± 0.02b
OCS-30	2.44 ± 0.07c	0.33 ± 0.01bc	0.27 ± 0.00b	1.90 ± 0.03b	0.66 ± 0.02b
OCS-45	2.22 ± 0.01b	0.33 ± 0.01b	0.29 ± 0.00b	2.07 ± 0.03b	0.65 ± 0.01b
OCS-60	2.06 ± 0.01a	0.35 ± 0.00c	0.25 ± 0.00 b	1.66 ± 0.09c	0.52 ± 0.01a

Mean ± standard deviations, variations followed by the same small letters (a-e) within a column do not differ significantly (p < 0.05).

The present study systematically reveals the regulation of the structure, physicochemical properties and functional behavior of chitosan/waxy rice starch complexes (OCS) by ozone oxidation time. The results demonstrated that ozone promotes intermolecular cross-linking and modifies the network structure of the complexes by inducing the Schiff base reaction between amino groups of chitosan and hydroxyl groups of starch. Chitosan with a high deacetylation degree exhibited more cross-linking sites due to its elevated amino content, forming a denser and more rigid three-dimensional network. Ozone treatment time exerted dual effects on structural evolution: short-term treatment (≤30 min) predominantly enhanced cross-linking, increasing molecular weight, crystallinity, thermal stability, and paste stability; whereas prolonged treatment (≥45 min) triggered molecular chain degradation and reduced crystallinity. Microstructural analysis (SEM) revealed that ozone treatment gradually modulated pore distribution through surface group oxidation and internal penetration. Initial treatment (15 min) increased hydrophobicity (evidenced by elevated contact angles) due to hydrophobic carbonyl group formation, while extended oxidation exposed hydrophilic carboxyl groups, enhancing surface hydrophilicity. Ozone further optimized gel water-holding capacity by disrupting starch amorphous regions and reorganizing molecular chains. Rheological characterization indicated that the composite gels exhibited shear-thinning behavior with low viscosity and high fluidity, demonstrating excellent extensibility and coating potential. These effects highlight the importance of controlling the ozonation time to achieve a balance between crosslinking efficiency and molecular stability, thereby realizing optimal material performance.

Materials and method

Materials

WRS (Moisture, % ≥12.5, Protein, %≤2.0, Mw = 1,657,264 Da, Mn = 74,098 Da, Mp = 262,8421 Da) was obtained from Jiangsu Baby Suqian Natural Biotechnology Co. Ltd (Jiangsu, China). Chitosan (C, Purity: Degree of deacetylation ≥95%, viscosity 100–200 mpa.s) was obtained from Shanghai McLean Biochemical Technology Co. Ltd (Shanghai, China). The ozone generator was produced from Shenzhen Feili Electric Technology Co. Ltd (Shenzhen, China) (Ozone output: 3000 mg/h Rated power: 80 W). All other chemicals were of analytical grade in purity.

Ozonized waxy rice starch preparation (OS)

50 g of WRS was dispersed in acetic acid solution (1%) and magnetically stirred at 25 °C for 1 h. The reaction was carried out in the presence of an ozonizer. An aeration stone was connected to an ozone generator through a silica tube and ozone was passed into the already prepared composite solution for 30 min. reaction time. It was then dried in an oven at 50 °C and crushed through a 100-mesh sieve.

Chitosan/waxy rice starch complex (CS) preparation

Referring to the existing studies and adapting them, 1.5 g of chitosan powder was weighed and mixed with 200 mL of 1% acetic acid solution at 25 °C with continuous stirring until the chitosan powder was completely dissolved⁴⁵. Subsequently, 48.5 g of WRS was added to the prepared chitosan solution and stirred for 1 h until the starch granules were completely dispersed in the solution. The mixture was then dried at 50 °C. After drying, the product was grinded and passed through a 100-mesh sieve.

Preparation of ozonized chitosan/starch complex (OCS) with different degrees of deacetylation and different ozonization times

Chitosan with deacetylation degrees of 50%, 70%, and 95% was dissolved in acetic acid solutions, respectively. WRS was added to each solution, and composite solutions were prepared according to the method described in OS preparation. An aeration stone was connected to the ozone generator via a silica gel tube, and ozone was introduced into the composite solutions. The reaction was allowed to proceed for 30 min before termination. Subsequently, the ozonated solution was dried at 50 °C, ground, and sieved through a 100-mesh screen to obtain samples 50%-OCS, 70%-OCS, and 95%-OCS.

The degree of deacetylation (DD) of the chitosan used in this step of sample preparation is 95%. The aeration stone was connected to an ozone generator through a silica tube, and ozone was passed into the already prepared complex solution (as described in OS preparation) and ozonized for 15 min (OCS-15), 30 min (OCS-30), 45 min (OCS-45), and 60 min (OCS-60). The ozone-treated solution was dried at 50 °C, ground into powder and passed through a 100-mesh sieve.

Scanning electron microscopy (SEM)

The section morphology of the samples was observed using a scanning electron microscope (Hitachi SU8600, JP). 5 g of fresh starch gel (refer to 2.3.7 for preparation method) was weighed and placed in a refrigerator at 4 °C for 24 h. The stabilized gel was freeze-dried and the sections were obtained by brittleness in liquid nitrogen. Subsequently, each sample was evenly distributed on a sample stage and observed under an accelerating voltage of 2 kV.

Contact angle

The water contact angle of the sample was measured using an interfacial rheometer (DSA30R, DE). The samples were molded into circular flakes with a thickness of 2 mm and a diameter of 13 mm. Subsequently, the contact angle of each flake was recorded by the interfacial rheometer, and the value was calculated based on the Laplace-Young equation.

Zeta potentials and particle size distribution measurement

The particle size and zeta potential of starch were measured using a Mastersizer instrument (Malvern Instruments Ltd, UK). The starch sample was homogeneously dispersed in deionized water at a concentration of 0.1 mg/mL as the sample to be tested. The prepared sample was dispersed into the dispersed water medium of the automatic sampling system and connected to the analyzing unit of the laser particle size analyzer equipped with the instrument. The measurement parameters were set as follows: the refractive index parameter was set to 1.52, and the absorbance value was set to 0.01. During the measurement process, the shading ratio was adjusted to a range of 10% to 20%, so that the actual shading ratio was about 12% when the samples were finally injected.

Determination of carbonyl content

The testing method was slightly modified based on previous studies^14,29. The starch suspension was prepared by dispersing 3 g of the sample in 100 mL of water. The suspension was then heated in an oscillating water bath at 90 °C for 30 min to fully pasturize and subsequently cooled to 40 °C. Subsequently, the pH of the suspension was adjusted to 3.2 using HCl (0.1 mol/L). 15 mL of hydroxylamine solution was added, and the mixture was subsequently placed in a water bath at 40 °C with continuous stirring for 4 h. Then, HCl (0.1 mol/L) was added to adjust the pH to 3.2 again and the volume of HCl added was recorded. The pH was adjusted to 3.2 and the volume of HCl added was recorded. Hydroxylamine solution was used as a blank sample. The results were expressed as the number of carbonyls per 100 glucose units (CO/100 GU), and the carbonyl content was calculated by Eq. 1.

$$\frac{\mathrm{CO}}{100\mathrm{GU}}=\frac{({\mathrm{V}}_0-{\mathrm{V}}_1)\times \mathrm{c}\times 0.028\times 100}{\mathrm{m}}$$ 1

Where, V₀ is the volume consumed for HCl titration of blank (mL);

V₁ is the volume consumed for HCl titration of sample (mL);

c is the concentration of the calibration HCl solution (mol/L);

m is the mass of sample (g);

0.028 is the conversion factor from chemical equivalent to g carbonyl (28) and from L to mL (1/1000).

Determination of hydration properties

A 1% (w/v) suspension of the sample was prepared and heated by shaking in a water bath at 90 °C for 30 min, then cooled to room temperature and centrifuged at 4000 rpm for 30 min. The supernatant was transferred to a constant weight aluminum box and dried at 105 °C for 6 to 10 h until the box reached a constant weight. Based on the above measurements, the solubility, swelling power, WHC and water absorption index of the samples were calculated by Eqs. 2–5:

$$\mathrm{Solubility}\left(\mathrm{S}\right)(\%)=\frac{W_2}{W_1}\times 100\%$$ 2

$$\mathrm{Swelling\; power}\left(\mathrm{SP}\right)(\mathrm{g}/\mathrm{g})=\frac{W_3}{W_1-W_2}$$ 3

$$\mathrm{Water\; holding\; capacity}\left(\mathrm{WHC}\right)(\mathrm{g}/\mathrm{g})=\frac{W_3}{W_1}$$ 4

$$\mathrm{Water\; absorption\; index}\left(\mathrm{WAI}\right)(\mathrm{g}/\mathrm{g})=\frac{W_3-\left(W_2-W_1)\right)}{W_2-W_1}$$ 5

Where, W₁ is the dry weight of the sample;

W₂ is the dry weight of the sample in the supernatant;

W₃ is the weight of the wet sample after centrifugation.

Gel permeation chromatography (GPC)

Gel permeation chromatography was used to measure the molecular mass distribution of starch. A 60 mg sample was weighed and dissolved in 20 mL of dimethyl sulfoxide (HPLC grade) and stirred in a water bath at 55 °C for 1 h. The sample was allowed to stand until it was completely dissolved and able to pass through a 0.45 μm filter membrane. A gel chromatography-scattering detector equipped with a gel exclusion chromatography column (Ohpak SB-805 HQ (300 × 8 mm), an Optilab T-rex (Wyatt technology, CA, USA) for the oscillometric detector and a DAWN HELEOS II (Wyatt technology, CA, USA) for the laser light scattering detector was used. DMSO was used as the eluent at a flow rate of 0.3 mL min⁻¹, and the column temperature and injection volume were 60 °C and 200 μL, respectively. The instrument was calibrated with standard glucose.

Pasting properties

The pasting characteristics of the samples were tested using a Rapid Viscosity Analyzer (RVA-Super4, SE). Samples (3 g) were weighed and added to deionized water (25 mL) in the RVA tank. Viscosity changes were recorded using a predetermined temperature profile: a constant temperature of 50 °C was maintained for 1 min, the temperature was gradually increased from 50 to 95 °C in 3.7 min, maintained for 2.5 min, and finally the samples were cooled down to 50 °C in 3.8 min. The total time for each sample to undergo this process was 13 min.

Thermogravimetric analyses (TGA)

The thermal degradation process of the samples was determined by thermosgravimetric analyzer (TGA/DSC/1100SF, USA). The samples were weighed 3 mg into a crucible and heated at a rate of 10 °C/min over a temperature range of 30–600 °C with a flow rate of 20 mL/min of nitrogen.

Fourier transform infrared spectroscopy (FTIR)

The samples were equilibrated at 50 °C for 24 h, mixed with KBr at a ratio of 1:100 and thoroughly ground, and measured in transmission mode using an FTIR spectrometer (IRTracer-100, JPN) with a scanning range of 4000–400 cm⁻¹, a resolution of 4 cm⁻¹, and scans of 32, with a pure potassium bromide background.

Nuclear magnetic resonance (NMR)

The samples were dissolved in 99.9% pure DMSO-d₆ (with tetramethylsilane (TMS) as an internal standard) at a concentration of 100 mg/mL. The clear solution was obtained by shaking in a water bath for 2 h at 55 °C. Spectra were recorded on a nuclear magnetic resonance spectrometer (Bruker Avance III HD 400 MHz NB). Data were analyzed using MestReNova software.

X-ray diffraction analysis (XRD)

The crystal structure of the samples was analyzed by X-ray diffractometer (Rigaku Smart Lab SE, JPN) using Cu-Ka rays under the test conditions of tube pressure of 40 kV and tube current of 30 mA, scanning area in the 2θ range of 5–55° and scanning speed of 2° min^-1. The data were analyzed using Jade 6 software.

Apparent viscosity

The apparent viscosity of the starch paste was measured as a function of shear rate using a rheometer (TA Instruments Ltd, UK). The experiment was carried out using 40 mm plate with a gap of 1 mm. Shear rates ranged from 0.1 to 300 s⁻¹ and the test temperature was maintained at 25 °C.

Dynamical frequency scanning

Based on the experimental results obtained from the linear viscoelastic range, the strain was consistently maintained at 1%. Additionally, the scanning frequency range was consistently maintained between 0.1 and 10 Hz. The measurement temperature was set to 25 °C. Furthermore, the energy storage modulus (G’), loss modulus (G”), and loss factor (tan δ) of the samples were determined to vary according to the scanning frequency.

Low-field nuclear magnetic resonance (LF-NMR)

The transverse (T₂) relaxation of hydrogen protons in starch gels was determined using low-field NMR measurements. 3 g of gel was weighed (refer to 3.3.12 for preparation method) and wrapped in plastic wrap to prevent water loss. The LF-NMR relaxation curves were fitted and then inverted to obtain and T₂ relaxation curve and the time of peak apex.

Determination of texture

Structural analyzes were carried out on starch gels using a texturometer (TMS-Pro, USA). 5 g of fresh starch gel (for preparation refer to 3.3.11) was weighed and placed in a refrigerator at 4 °C for 24 h. The mass spectrometer used was equipped with a P6 probe and the test speed was 1 mm/s. The strain was 50% and the trigger force was set at 5 g. Each sample was tested 5 times and the data were recorded.

Statistical analysis

The experiments were replicated thrice in each group, and statistical significance was analyzed using IBM SPSS Statistics 19 software. ANOVA and Duncan’s t-test were employed to assess statistical significance at a p-value threshold of 0.05.

Author contributions

Xiao Fang: Software, Validation, Formal analysis, Data curation, Writing - original draft. Lei Chen: Conceptualization, Methodology, Writing - review & editing, Funding acquisition. Wenzhou Zhao: Writing - original draft Yuehui Wang: Resource. Xi Chen: Writing - review & editing. Kun Zhuang: Resource. Wenping Ding: Supervision, Funding acquisition, Project administration.

Data availability

All data generated or analysed during this study are included in this published article and its supplementary information files.

Competing interests

The authors declare no competing interests.

Supplementary information

The online version contains supplementary material available at 10.1038/s41538-025-00674-7.