Microwave-induced complexation between sorghum starch and caffeic acid: Structural modulation and its impact on digestive and retrogradation properties

Abstract

Microwave-assisted technology offers an efficient and eco-friendly method for starch modification. In this study, sorghum starch (SS) was treated under varying microwave durations and complexed with caffeic acid (CA) to form SS-CA complexes. Microwave treatment significantly reduced crystallinity (24.07 % to 3.51 %), disrupted short-range order, and caused granule swelling, aggregation, and decreased thermal stability. Complexation induced a V-type inclusion structure. The SS-CA (120 s) complex showed a 9.17 % reduction in rapidly digestible starch (RDS) and a 14.40 % increase in resistant starch (RS). Molecular docking and DFT analyses confirmed that hydrogen bonding and van der Waals forces dominated SS–CA interactions. During 0–7 days of storage, free water content in the SS-CA gel increased only 1.58 % compared to 5.93 % in native SS, indicating reduced retrogradation. The SS-CA complex displayed lower gelatinization enthalpy and improved phase stability. These findings suggest the potential of microwave-assisted SS-CA complexes in developing low-glycemic starch-based foods.

Highlights

• •Microwave treatment effectively disrupted sorghum starch (SS) crystallinity and short-range ordering.
• •Microwave-assisted complexation promoted V-type inclusion between SS and caffeic acid (CA).
• •SS-CA complexes exhibited enhanced resistant starch content and antioxidant activity.
• •Retrogradation and water migration in SS-CA gels were significantly reduced during storage.

1. Introduction

With the continuous advancement of the modern food industry and the growing awareness of health among consumers, the synergistic application of green processing technologies and natural functional compounds has emerged as a promising approach to enhance the functional properties of traditional food matrices (Chi et al., 2022). Starch, as a natural and renewable carbohydrate resource, plays a vital role in the formation of food structures. It is also widely utilized in functional starch products, food ingredient modification, and nutritional regulation (Li et al., 2025). However, native starches—particularly those derived from cereal sources—often exhibit undesirable characteristics such as rapid digestibility, poor thermal stability, and a strong tendency toward retrogradation, which limit their application in advanced functional food systems (Yu et al., 2021). Consequently, structural modification and functional regulation of starch have become focal points of research in the field of food science and technology.

Sorghum (Sorghum bicolor) is one of the most important drought-tolerant coarse cereal crops in China, characterized by abundant starch content, a high proportion of amylose, and a relatively high degree of crystallinity (Zhang, Ran, Jiang, & Dou, 2021). However, native sorghum starch (SS) exhibits a certain susceptibility to enzymatic hydrolysis, i.e., low resistance to digestion, as well as a strong tendency toward retrogradation, which to some extent limits its application in the development of functional foods (Zhu, 2014). Although SS is widely available and cost-effective, its inherent physicochemical properties often fail to meet the dual demands of the modern food industry for enhanced health attributes (e.g., slower digestibility) and improved storage stability (e.g., anti-retrogradation behavior). Therefore, enhancing the digestibility resistance and modulating the retrogradation behavior characteristics of SS has become essential for its functional improvement and high-value utilization.

In recent years, plant-derived polyphenolic compounds—particularly phenolic acids such as caffeic acid (CA)—have garnered increasing attention due to their remarkable antioxidant properties, free radical scavenging activity, as well as antimicrobial and anti-inflammatory effects (Zheng et al., 2020). Emerging evidence suggests that polyphenols can interact with starch molecules through non-covalent forces such as hydrogen bonding, hydrophobic interactions, and electrostatic attractions, thereby forming stable starch–polyphenol complexes. These interactions can modulate the crystalline domains, short-range ordering, and enzymatic degradation behavior of starch at the microstructural level (Zhu, 2015). For instance, polyphenol-assisted modification has been shown to enhance starch resistance to digestion (i.e., increase resistant starch, RS) and suppress retrogradation, thereby improving its applicability and stability in functional food systems (Chai, Wang, & Zhang, 2013). CA, which contains both phenolic hydroxyl and carboxyl functional groups, exhibits strong reactivity and a high capacity to bind with starch chains. This makes it a promising modifier for improving the thermodynamic stability and retrogradation behavior of starch-based matrices (Yu et al., 2021). Simultaneously, microwave treatment has emerged as a green and efficient processing technology in the food industry. Compared with conventional heating methods, microwave irradiation offers advantages such as rapid heating, energy efficiency, uniform treatment, and reduced dependence on heat conduction (Yuan, Ding, Li, Huang, & Zhang, 2020). Specifically, microwave energy can directly disrupt the hydrogen bond network within starch granules via high-frequency electromagnetic fields, thereby promoting chain depolymerization and rearrangement. This facilitates alterations in crystalline structure and short-range ordering and enhances the degree of interaction between starch and small molecules, contributing to improved structural stability and functional performance of the resulting complexes (Fan et al., 2014). However, the multi-scale structural modulation of microwave-assisted SS–CA complexes, along with the structure–function relationships related to digestibility and retrogradation behavior, remains insufficiently explored and warrants systematic investigation.

Based on this, the present study aims to construct starch–phenolic acid complexes using SS and CA as raw materials, assisted by microwave treatment. The effects of microwave duration on the structural and physicochemical properties of starch and its complexes were systematically investigated, including microstructural features (e.g., crystalline type, short-range ordering, and granule morphology), thermal and physical characteristics (e.g., gelatinization enthalpy, transparency, and rheological behavior), as well as digestive behavior—reflected by the proportions of rapidly digestible starch (RDS), slowly digestible starch (SDS), and RS—and retrogradation properties (e.g., water-holding capacity, free water content, and degree of recrystallization). A combination of analytical techniques, including scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FT-IR), Raman spectroscopy, in vitro simulated digestion, and density functional theory (DFT) calculations, was employed to elucidate the interaction mechanisms between SS and CA under microwave induction and to uncover the correlations between structural modifications and functional performance. This study not only provides a theoretical basis for understanding the structure–function relationships of starch–phenolic complexes, but also offers practical guidance for the development of functional starch materials using green and energy-efficient microwave-assisted processing. Furthermore, it delivers scientific support for the design and development of low-glycemic-response foods and high-value-added starch products, while laying a foundation for the future application of green processing technologies in cereal processing and nutritional regulation.

2. Materials and methods

2.1. Materials

Sorghum starch (SS) was prepared by the laboratory with a starch content of 90.56 %. Caffeic acid (CA) was purchased from Aladdin Co., Ltd. (Shanghai, China). A 2,2-diphenyl-1-picrylhydrazyl (DPPH)-based total antioxidant capacity assay kit was obtained from Comin Biotechnology Co., Ltd. (Suzhou, China). Porcine pancreatic α-amylase (EC 3.2.1.1, ≥ 5 U/mg) and Aspergillus niger α-glucosidase (EC 3.2.1.3, ≥ 100 U/mg) were supplied by Yuanye Biotechnology Co., Ltd. (Shanghai, China). All other chemicals were of analytical grade.

2.2. Sample preparation

2.2.1. Preparation of microwave-treated sorghum starch (SS)

A total of 5 g (dry basis) of SS was accurately weighed into a 100 mL microwave-safe vessel, mixed with 50 mL distilled water for 20 s, and subjected to intermittent microwave treatment using a microwave oven (Model G70F20CN1L-DG, Galanz Electrical Appliance Mfg. Co., Ltd., Guangdong, China) at 350 W. The treatment involved 30 s of heating followed by 10 s of stirring, repeated 1–4 times to obtain samples with microwave durations of 30 s, 60 s, 90 s, and 120 s. After treatment, samples were immediately frozen and freeze-dried. The final products were labeled as “SS-X", where “X” represented the microwave time. Untreated starch (SS) served as the control.

2.2.2. Preparation of SS-CA complex

A mixture containing 5 g SS, 0.5 g CA (dissolved in anhydrous ethanol), and 50 mL distilled water was prepared in a 100 mL microwave-safe vessel. Subsequent microwave treatment was performed following the procedure described in Section 2.2.1. After treatment, free caffeic acid was removed by washing the mixture with 50 % ethanol solution, followed by centrifugation. The resulting precipitate was freeze-dried to obtain the SS-CA complex, designated as “SS-CA-X", where “X” indicated the microwave treatment time.

2.2.3. Preparation of retrograded gels of SS-CA complexes

SS and SS-CA complexes gelatinized by microwave treatment (350 W for 120 s) were stored at 4 °C for 1, 3, 5, 7, and 14 days to simulate the retrogradation process. The samples were partially freeze-dried for further analysis.

2.3. Particle size distribution analysis

The particle size distribution of the samples was determined using a laser diffraction particle size analyzer (Mastersizer 2000, Malvern Instruments Ltd., UK). A measured amount of sample was dispersed in distilled water until the appropriate obscuration level was reached. The suspension was then introduced at a pump speed of 2000 r/min for measurement.

2.4. Determination of water absorption index (WAI)

Approximately 0.1 g of sample was accurately weighed and placed into a 50 mL centrifuge tube. Then, 20 mL of distilled water was added, and the mixture was stirred thoroughly in a water bath at room temperature for 20 min. Afterward, the suspension was centrifuged at 4000 r/min for 10 min. The supernatant was decanted, and the water absorption index (WAI) was calculated using the following equation:

$$\mathrm{WAI}\left(\%\right)=\frac{m_2}{m_1}\times 100.$$

Where $m_1$ is the sample mass (g) and $m_2$ is the sample mass after centrifugation (g).

2.5. Transparency measurement

A starch suspension was prepared by mixing 50 mg of starch with 4.95 mL of distilled water in a 10 mL centrifuge tube. After vortexing, the mixture was heated in a boiling water bath for 30 min to ensure complete gelatinization, then cooled to room temperature. The transparency of the starch paste was evaluated by measuring the transmittance at 620 nm using a UV–Vis spectrophotometer (DU-730, Beckman Coulter Corporation, USA), with distilled water as the blank.

2.6. SEM

A small amount of sample was collected using a cotton swab and evenly applied onto double-sided adhesive tape. After sputter-coating with gold for 30 s, the samples were examined under a Flex1000II tungsten filament scanning electron microscope (Hitachi, Tokyo, Japan) at appropriate magnifications to observe starch granule morphology.

2.7. X-ray diffraction (XRD)

Freeze-dried samples were analyzed by XRD using an X-ray diffractometer (Rigaku, Japan). The test conditions were as follows: a copper-targeted X-ray tube was used with an operating voltage of 40 kV and a current of 25 mA, and the scanning range was set to 5–40° with a scanning rate of 10.0°/min and a step size of 0.05° (Ji et al., 2021).

2.8. FT-IR and Raman spectroscopy

Approximately 2 mg of freeze-dried sample was accurately weighed and thoroughly mixed with 200 mg of dried KBr. The mixture was ground uniformly and pressed into a transparent pellet. FT-IR spectra were collected using a Nicolet 6700 spectrometer (Thermo Scientific, USA) within the wavenumber range of 4000–400 cm⁻¹, with a resolution of 4 cm⁻¹ and 32 scans per sample. The deconvoluted spectra were used to calculate the ratio of 1047/1022 and 1022/995 cm⁻¹ using Omnic software. For Raman analysis, the Raman spectra of SS–CA complexes were acquired using a Raman spectroscopy (Horiba Scientific, France) equipped with a 785 nm laser source. Spectra were recorded in the range of 3200–100 cm⁻¹ at room temperature (Hao et al., 2025). The full width at half maxima (FWHM) at 480 cm⁻¹ was obtained by using Peakfit software.

2.9. Differential scanning calorimetry (DSC)

Approximately 2 mg of starch sample was accurately weighed into a standard aluminum pan, mixed with distilled water at a 1:3 (w/v) ratio, sealed, and equilibrated at 4 °C for 24 h. Thermal analysis was performed using a DSC8000 (PerkinElmer Corporation, USA) from 30 to 110 °C at a heating rate of 10 °C/min under a nitrogen atmosphere. The degree of retrogradation (DR) was calculated using the following equation:

$$\mathrm{DR}\left(\%\right)=\frac{{\mathit{\Delta H}}_2}{{\mathit{\Delta H}}_1}\times 100$$

where ΔH₁ is the gelatinization enthalpy of the freshly prepared sample, and ΔH₂ is the enthalpy of the retrograded sample after storage.

2.10. Antioxidant activity determination

The total antioxidant capacity of samples was measured using a DPPH assay kit. Briefly, 0.1 g of sample was mixed with 1 mL of extraction solvent, thoroughly vortexed, and kept on ice. The mixture was then centrifuged at 10,000 ×g and 4 °C for 10 min, and the supernatant was collected. The DPPH radical scavenging rate (%) was calculated using the following formula:

$$\text{DPPH scavenging rate}\left(\%\right)=\frac{A_1-A_2}{A_1}\times 100$$

Where A₁ is the absorbance of the control solution without a sample, and A₂ is the absorbance of the solution containing the sample.

2.11. Complexing index (CI) value determination

Approximately 0.4 g (dry basis) of the starch sample was accurately weighed and dispersed in 5 mL of distilled water. The suspension was heated in a water bath at 95 °C for 15 min with continuous agitation to ensure complete gelatinization. After cooling to 25 °C, 25 mL of distilled water was added, and the mixture was vortexed thoroughly to obtain a homogeneous dispersion. Subsequently, 0.5 mL of this dispersion was transferred into a test tube, mixed with 15 mL of distilled water and 2 mL of iodine reagent (containing 2.0 % KI and 1.3 % I₂). The CI was determined by measuring the absorbance at 690 nm using a UV–Vis spectrophotometer (UV-2600i, Shimadzu, Japan) according to the following equation.

$$\mathrm{CI}\left(\%\right)=\left[\left(\mathrm{A}0-\mathrm{AS}\right)\times 100\right]/\mathrm{A}0$$

Where A₀ indicates the absorbance value of the SS; As indicates the absorbance value of the SS complex.

2.12. In vitro digestibility

A total of 200 mg starch sample was mixed with 20 mL sodium acetate buffer (pH 5.2) in a flask and heated at 95 °C for 30 min. Then, 5 mL of a pre-activated enzyme mixture (14,500 U α-amylase and 850 U amyloglucosidase, 37 °C for 30 min) was added. The mixture was incubated at 37 °C, and 0.5 mL aliquots were taken at 20 and 120 min. Each aliquot was immediately mixed with 4.5 mL absolute ethanol to stop the reaction, followed by centrifugation (4000 rpm, 10 min). Glucose concentration in the supernatant was determined using a glucose assay kit (Englyst, Kingman, & Cummings, 1992). The fractions of RDS, SDS, and RS were calculated as:

$$\mathrm{RDS}\left(\%\right)=\left[\left(\mathrm{G}20-\mathrm{G}0\right)\times 0.9\right]/\mathrm{TS}\times 100$$

$$\mathrm{SDS}\left(\%\right)=\left[\left(\mathrm{G}120-\mathrm{G}20\right)\times 0.9\right]/\mathrm{TS}\times 100$$

$$\mathrm{RS}\left(\%\right)=\left(1-\mathrm{RDS}-\mathrm{SDS}\right)\times 100$$

Where G₀, G₂₀, and G₁₂₀ refer to glucose content at 0, 20, and 120 min, respectively, and TS is the total starch mass (mg, dry basis).

2.13. Moisture distribution measurement

Starch gel samples (2–3 g) were placed in 15 mm glass tubes and analyzed by LF-NMR (NMI20-015 V-I, Newmai Electronics, Suzhou). Measurements were performed at 18.397 MHz. Data were processed with Multi-ExpInv software. The transverse relaxation time (T₂) was calculated as:

$$\mathrm{M}\left(\mathrm{t}\right)=\sum _i{A}_i\exp \left(-\frac{t}{T_{2i}}\right)$$

Where M(t) is the signal amplitude at time t, T_2i are relaxation times, and A_i are proton-related coefficients.

2.14. DFT calculations

To elucidate the potential noncovalent interaction sites between SS and CA, molecular docking and DFT analyses were conducted. The three-dimensional structure of CA was obtained from the PubChem database. Considering the large molecular weight and multiscale structural complexity of starch granules, the entire starch molecule could not be simulated directly. Therefore, a starch fragment with a degree of polymerization of 10 (DP = 10) was selected as the representative model for calculation. The molecular geometry of the starch fragment was optimized using the B3LYP/6-311G++(d, p) basis set within Gaussian 16 software (Gaussian Inc., Wallingford, CT, USA), and the electrostatic potential (ESP) map was visualized using Multiwfn 3.8 in combination with Visual Molecular Dynamics (VMD) (Zheng, Liu, Chen, Qiu, & Li, 2022). Molecular docking between SS and CA was performed using AutoDock 4.2 (The Scripps Research Institute, La Jolla, CA, USA) under a flexible docking mode, allowing conformational changes of both ligand and receptor. Fifty docking conformations were generated, and the complex with the lowest binding energy was selected as the optimal configuration. The resulting docking poses and interaction sites were visualized using PyMOL 2.5 (Schrödinger, LLC, New York, USA) (Hao et al., 2024).

2.15. Statistical analysis

All experiments were conducted in triplicate, and Statistix 8.0 (Analytical Software, USA) was used for analysis of variance (ANOVA) (p < 0.05).

3. Results and discussion

3.1. Effects of microwave treatment on the structural and physicochemical properties of SS

3.1.1. Particle size distribution analysis

Distinct differences in morphology and particle size distribution among starches from various cereals influence their gelatinization, retrogradation, and enzymatic digestibility (Tan et al., 2015). As illustrated in Fig. 1A, untreated SS displayed a multimodal particle size distribution predominantly between 2 and 35 μm, with uniform dispersion. Prolonged microwave treatment caused a clear shift toward larger particle sizes, reflecting an overall increase in granule dimensions. Particle size parameters at different microwave durations are summarized in Table 1. Notably, after 30 s of treatment, D(0.1), D(0.5), and D(0.9) decreased to 0.98, 16.51, and 23.78 μm, respectively, likely due to fragmentation of surface-impaired granules yielding smaller particles. Conversely, extending the microwave time to 120 s resulted in significant increases in D(0.1), D(0.5), and D(0.9) values (29.47, 54.05, and 91.27 μm, respectively), indicating swelling and aggregation. The mechanism underlying this phenomenon can be ascribed to the extensive disruption of intra- and intermolecular hydrogen bonds in starch molecules under prolonged microwave treatment, which loosens the internal granule structure and facilitates water infiltration, thereby promoting starch gelatinization. With increasing gelatinization, enhanced van der Waals interactions and hydrophobic forces among starch chains reinforce intergranular adhesion, further promoting aggregation (Kang et al., 2022). Moreover, extended microwave exposure may induce partial cleavage of starch chains, increasing chain flexibility and the potential for entanglement, thereby generating additional binding sites at the microscopic level and strengthening intergranular interactions.

Fig. 1.

Effects of different microwave treatment times on sorghum starch granule size distribution (A), water absorption index (B), transparency (C), and microstructure (D). Different letters represent significant differences (p < 0.05) between groups.

Table 1.

Particle size parameters of SS with different microwave treatment times.

Sample	D (4,3) /(μm)	D (3,2) /μm	D (0.1) /μm	D (0.5) /μm	D (0.9) /μm
SS	16.76 ± 3.76c	18.18 ± 0.53b	12.46 ± 0.47c	18.68 ± 0.19c	29.26 ± 0.43d
SS-30	14.99 ± 0.01d	3.70 ± 0.01c	0.98 ± 0.01d	16.51 ± 0.01c	23.78 ± 0.02e
SS-60	39.68 ± 0.20b	20.45 ± 0.07b	18.79 ± 0.09b	37.87 ± 0.06b	64.78 ± 0.10c
SS-90	55.04 ± 2.96a	43.86 ± 3.36a	29.59 ± 1.66a	51.82 ± 1.49a	86.26 ± 1.54b
SS-120	57.31 ± 2.96a	44.02 ± 3.65a	29.47 ± 3.11a	54.05 ± 2.99a	91.27 ± 2.90a

Different letters represent significant differences (p < 0.05) between groups.

3.1.2. Water absorption index and transmittance analysis

Water absorption index (WAI) reflects the ability of starch granules to bind water molecules and is influenced by starch structure and granule state. As shown in Fig. 1B, microwave treatment significantly increased the WAI of SS, rising from 0.75 g/g in the untreated sample to 9.52 g/g after 120 s of treatment. This enhancement is likely due to dielectric polarization induced by microwaves, which causes rapid oscillation of water molecules within the starch system, promoting granule swelling and leaching of amylose. The disruption of the original compact structure and formation of a gel network strengthened starch–water interactions. Moreover, microwave-induced cleavage of intramolecular hydrogen bonds exposed more hydrophilic hydroxyl groups, further improving water binding capacity. Starch paste transparency, which reflects swelling and dispersion states, is commonly measured by transmittance at 620 nm. Fig. 1C shows a significant decrease in transmittance of SS after microwave treatment, with a further decline as treatment time increased, indicating reduced transparency. This reduction likely results from microwave-induced disruption of starch granule structure and degradation of amylopectin into short amylose chains, which tend to retrograde and aggregate, thereby decreasing light transmittance (Zailani, Kamilah, Husaini, & Sarbini, 2021).

3.1.3. SEM analysis

Fig. 1D presents the SEM images of SS subjected to varying microwave treatment durations, observed at magnifications of 1000× and 2500×. Native SS granules exhibited irregular polygonal shapes with smooth surfaces and well-defined edges, although a few showed minor surface depressions. As microwave exposure time increased, progressive structural degradation of the granules was observed, including blurred edges, swelling, and an increasing number of fractured particles. After 60 s of treatment, significant granule expansion and fragmentation occurred, accompanied by the formation of visible aggregates due to adhesion among starch fragments. At 120 s, the original granule morphology was almost completely lost, and the degree of gelatinization was markedly enhanced, resulting in a high degree of fragmentation after freeze-drying. These morphological changes are likely driven by the intense oscillation and polarization of water molecules under microwave irradiation, which promotes the disruption of intermolecular hydrogen bonds, facilitating water uptake, granule swelling, and partial gelatinization. This in turn leads to the collapse of granule integrity and the formation of gel-like agglomerates (Wang et al., 2022). These observations are in agreement with the particle size distribution results, which showed a shift toward larger size fractions and an increased tendency for particle aggregation following prolonged microwave treatment.

3.1.4. XRD analysis

XRD technology is employed to characterize the crystal structure of starch samples. Typically, starch exhibits A-type, B-type, or C-type crystalline features in XRD patterns, reflecting the ordering of starch molecular chains and their degree of crystallinity (Warren, Gidley, & Flanagan, 2016). As shown in Fig. 2A, native SS and samples treated with microwaves for 30 and 60 s displayed characteristic A-type diffraction peaks at 15.3°, 17.2°, 18.2°, and 23°, indicating that short-term microwave exposure did not alter the crystalline structure. However, with prolonged treatment (90 and 120 s), the intensities of these peaks markedly decreased or disappeared, suggesting substantial disruption of the crystalline regions. Correspondingly, the relative crystallinity (Table 2) decreased significantly from 24.07 % (control) to 3.51 % after 120 s of microwave treatment. These changes indicate that microwave irradiation disrupts the ordered arrangement of starch molecules, promoting the transformation from crystalline to amorphous regions. The mechanism is likely attributed to intense molecular polarization and localized overheating under microwave fields, leading to glycosidic bond cleavage and hydrogen bond dissociation, thereby damaging the crystalline domains and facilitating amorphization (Liu, Jiang, Liu, Li, & Li, 2021).

Fig. 2.

Effects of different microwave treatment times on the XRD patterns (A), FT-IR spectra (B, C), and Raman spectra (D) of sorghum starch.

Table 2.

Relative crystallinity, R_1047/1022, R_1022/995 and FWHM of microwave preparation of SS.

Sample	Crystallinity (%)	R1047/1022	R1022/995	FWHM at 480−1
SS	24.07 ± 2.51a	0.81 ± 0.00a	0.79 ± 0.07c	16.01 ± 0.03c
SS-30	16.38 ± 0.73b	0.81 ± 0.01a	0.78 ± 0.11c	16.22 ± 0.18c
SS-60	11.77 ± 1.81c	0.80 ± 0.00ab	1.04 ± 0.12b	16.86 ± 0.02b
SS-90	6.43 ± 0.99d	0.76 ± 0.04ab	1.32 ± 0.15a	17.06 ± 0.04b
SS-120	3.51 ± 0.54e	0.77 ± 0.02b	1.31 ± 0.15a	17.87 ± 0.26a

Different letters represent significant differences (p < 0.05) between groups.

3.1.5. FT-IR and Raman spectroscopic analysis

FT-IR was employed to investigate the short-range molecular order of SS. As shown in Fig. 2B, no new absorption peaks were observed after different durations of microwave treatment, indicating that microwave irradiation did not induce chemical bond cleavage or the formation of new functional groups. The primary molecular structure of starch remained intact. The deconvoluted FT-IR spectra (Fig. 2C) exhibited characteristic absorption peaks at 1047 cm⁻¹, 1022 cm⁻¹, and 995 cm⁻¹, corresponding to crystalline and amorphous regions of starch. The absorbance ratio of 1047/1022 cm⁻¹ (R_1047/1022) is commonly used to assess short-range molecular order, while the 1022/995 cm⁻¹ ratio (R_1022/995) reflects the integrity of the double helical structure (Liu et al., 2021). As shown in Table 2, native SS exhibited R_1047/1022 and R_1022/995 values of 0.81 and 0.79, respectively. With increasing microwave treatment time up to 120 s, R_1047/1022 decreased to 0.77, while R_1022/995 increased to 1.31, suggesting disruption of the double helices and a reduction in molecular order within the crystalline regions (Xie, Liu, & Cui, 2006). These structural changes were further supported by Raman spectroscopy. As illustrated in Fig. 2D, the skeletal vibration band at 480 cm⁻¹ did not exhibit peak shifting or the appearance of new bands, confirming that no substantial changes in chemical bonding occurred. However, a significant decrease in peak intensity and an increase in full width at half maximum (FWHM) from 16.01 to 17.87 were observed following 120 s of microwave exposure. This implies a loss of crystallinity, likely due to the cleavage of glycosidic and hydrogen bonds under localized overheating and dipole-induced molecular motion. These effects may promote chain relaxation and rearrangement, leading to a broader energy distribution among molecular bonds. These results are consistent with FT-IR findings, collectively demonstrating that microwave treatment disrupts the short-range ordered structure of starch and enhances amorphous characteristics.

3.1.6. DSC analysis

DSC was utilized to analyze the thermal properties of starch, particularly its gelatinization behavior. As shown in Table 3, microwave treatment significantly increased the onset temperature (T_o), peak temperature (T_p), and conclusion temperature (T_c) of gelatinization. These parameters shifted from 65.58 °C, 70.67 °C, and 80.78 °C in the native starch to 78.41 °C, 82.36 °C, and 90.55 °C, respectively, after 120 s of microwave exposure. The upward shift in the gelatinization temperature range indicates that microwave treatment promotes molecular chain rearrangement, resulting in the formation of more thermally stable structures and elevating the thermal sensitivity threshold (Li et al., 2018). Meanwhile, the enthalpy change (ΔH) exhibited a decreasing trend with prolonged microwave treatment, suggesting that less energy was required for gelatinization. This reduction in ΔH is likely attributed to the partial disruption of crystalline regions due to microwave-induced cleavage of intra- and intermolecular hydrogen bonds, which leads to a more disordered and less compact granular structure, enhancing the starch's responsiveness to thermal input. These observations suggest that microwave treatment not only disrupts the native double helical structures but may also facilitate the formation of new, more thermally stable arrangements of amylose chains. Together, these structural transformations contribute to the observed alterations in gelatinization behavior.

Table 3.

Thermal characteristics of SS at different microwave treatment times.

Sample	To/°C	Tp/°C	Tc/°C	ΔH/(J/g)
SS	65.58 ± 0.03c	70.67 ± 0.01c	80.78 ± 1.84b	10.79 ± 0.15a
SS-30	64.85 ± 0.37d	70.35 ± 0.45c	81.00 ± 3.54b	7.81 ± 0.40b
SS-60	67.57 ± 0.14b	71.62 ± 0.03b	80.02 ± 0.10b	4.30 ± 0.44c
SS-90	65.30 ± 0.13c	70.60 ± 0.17c	86.58 ± 2.93a	4.13 ± 0.20c
SS-120	78.41 ± 0.02a	82.36 ± 0.38a	90.55 ± 0.02a	0.94 ± 0.01d

Different letters represent significant differences (p < 0.05) between groups.

3.2. Structural and digestive properties of microwave-prepared SS–CA complexes

3.2.1. Antioxidant activity analysis

As shown in Table 4, the DPPH radical scavenging activity of the starch-CA complexes was significantly higher than that of the control group (p < 0.05). With increasing microwave treatment time, the radical scavenging rate increased from 31.92 % to 80.73 %. This enhancement can be primarily attributed to the strong free radical scavenging ability of CA itself (Shiozawa, Inoue, Murata, & Kanamoto, 2018). In addition, microwave treatment disrupted the double-helical structures within the starch molecules, leading to their disintegration and exposing more hydrophobic cavities (As shown in Table 4, the CI of SS-CA significantly increased with prolonged microwave treatment. Notably, after 120 s of microwave exposure, the CI value rose from 18.11 % to 40.97 %, indicating enhanced complex formation). These structural changes facilitated the non-covalent encapsulation of a greater amount of CA within the starch matrix. Moreover, microwave-induced short-range structural changes in starch can delay the release of phenolic compounds, thereby providing more sustained antioxidant protection under oxidative stress conditions (Mude, Maroju, Balapure, Ganesan, & Ray Dutta, 2022).

Table 4.

Free radical scavenging activity, complexing index, relative crystallinity, R_1047/1022, R_1022/995 and FWHM of microwave-prepared SS-CA complexes.

Sample	Radical scavenging activity (%)	Complexing index (%)	Crystallinity (%)	R1047/1022	R1022/995	FWHM at 480−1
SS-CA	31.92 ± 0.06d	18.11 ± 0.22d	19.37 % ± 1.91a	0.811 ± 0.008a	0.815 ± 0.113c	16.09 ± 0.23d
SS-CA-30	42.46 ± 0.01c	20.08 ± 1.91d	16.97 % ± 0.54ab	0.805 ± 0.006a	0.844 ± 0.066bc	16.36 ± 0.22cd
SS-CA-60	77.88 ± 0.18b	24.90 ± 1.33c	10.49 % ± 1.43d	0.799 ± 0.004ab	0.944 ± 0.033b	16.68 ± 0.38c
SS-CA-90	78.30 ± 0.25b	30.76 ± 1.91b	11.47 % ± 2.32cd	0.795 ± 0.003ab	1.063 ± 0.007a	17.60 ± 0.33b
SS-CA-120	80.73 ± 0.97a	40.97 ± 2.33a	14.05 % ± 1.49bc	0.775 ± 0.029b	1.144 ± 0.018a	18.16 ± 0.14a

Different letters represent significant differences (p < 0.05) between groups.

3.2.2. SEM analysis

The effect of microwave-assisted CA modification on the surface morphology of SS was investigated by SEM (Fig. 3). The results revealed that microwave treatment significantly altered the surface structure of SS-CA complexes, with the degree of morphological change increasing progressively with extended microwave exposure time. Native starch granules exhibited smooth and intact surfaces with regular shapes. After 30 s of microwave treatment, slight surface depressions and a small number of fractured fragments appeared. Additionally, partial coating or embedding of CA on the starch granules was observed, indicating the initial formation of the SS-CA complex. When the microwave time was extended to 60–90 s, the number of surface pits and cracks increased noticeably, accompanied by visible aggregation and local deformation of the starch granules. These changes suggest that microwave irradiation promoted partial disruption and rearrangement of starch molecular chains, thereby enhancing the interaction and binding between starch and CA. At 120 s of microwave treatment, the starch granules were severely disrupted, showing large porous structures and extensive aggregation between granules and CA molecules, eventually forming bulky agglomerates. This phenomenon could be attributed to the complete destruction of crystalline regions under prolonged microwave exposure, leading to increased molecular chain exposure and more intensive interactions with CA via hydrogen bonding or hydrophobic forces. Moreover, some small aggregated particles observed in the SEM images may result from starch fragmentation and CA reassembly during the drying process, potentially induced by temperature fluctuations (Gao, Tian, Pan, & Sun, 2025).

Fig. 3.

Effects of different microwave treatment times on the microstructure of SS–CA complexes.

3.2.3. XRD analysis

Starch can form V-type inclusion complexes with polyphenolic compounds through non-covalent interactions, and their crystalline structural transitions are typically characterized by XRD (Hao et al., 2024). Fig. 4A presents the XRD patterns of the physical mixture and SS-CA complexes prepared under different microwave treatment times. The physical mixture exhibited typical A-type starch diffraction peaks at 2θ = 5.6°, 17.0°, 19.0°, 22.0°, and 24.0°, indicating that mechanical mixing with CA did not alter the native crystalline structure of starch. After 30 s of microwave treatment, the diffraction pattern remained largely unchanged; however, at 60 s, new peaks appeared around 2θ = 13.0°, 17.0°, and 20.0°, characteristic of V-type inclusion complexes. The intensity of these peaks progressively increased with longer treatment times, suggesting a gradual formation of V-type structures between SS and CA under microwave assistance. As shown in Table 4, the crystallinity of the complex reached its lowest value (10.49 %) at 60 s, then gradually increased with prolonged treatment, reaching 14.05 % at 120 s. This trend indicates that initial microwave exposure disrupted the ordered structure, while subsequent release of short-chain starch and its interaction with CA facilitated partial recrystallization. Moreover, the overall crystallinity of the microwave-treated complexes was higher than that of native starch, implying that the incorporation of CA may help stabilize the crystalline structure, likely due to the formation of stable complex units within the helical cavities of starch molecules (Zhang et al., 2024).

Fig. 4.

Effects of different microwave treatment times on the XRD patterns (A), FTIR spectra (B, C), Raman spectra (D), and thermal properties (E, F) of SS-CA complexes.

3.2.4. FT-IR and Raman spectroscopic analysis

Fig. 4B–C shows the FT-IR spectra and corresponding deconvolution profiles of physical mixtures and SS-CA complexes subjected to varying microwave treatment times. The broad absorption band in the 3000–3700 cm⁻¹ region corresponds to O—H stretching vibrations and reflects the hydrogen bonding network within the system. With prolonged microwave exposure, this band gradually broadens, indicating a weakening of both intra- and intermolecular hydrogen bonds and a reduction in hydroxyl group density. Compared to the control, the SS-CA complex exhibits more pronounced peak broadening, suggesting the formation of additional weak hydrogen bonds during complexation. The peak at 2940 cm⁻¹ is attributed to C—H stretching vibrations, while the band at 1645 cm⁻¹ corresponds to H–O–H bending vibrations of amorphous water. Peaks in the 1450–1600 cm⁻¹ region are characteristic of the aromatic skeletal vibrations of CA, confirming its incorporation into the complex structure (Yin et al., 2024). No new absorption bands associated with functional group transformations were observed, indicating that the complexation is primarily governed by non-covalent interactions. To assess short-range molecular order in starch, the ratios R_1047/1022 and R_1022/995 were calculated from the FT-IR spectra (Table 4). In the physical mixture, these ratios were 0.811 and 0.815, respectively, suggesting negligible disruption to the native crystalline structure. After microwave treatment, R_1047/1022 decreased to 0.775 and R_1022/995 increased to 1.144, indicating a disturbance in the ordered arrangement of starch chains and a reduction in crystalline stability. However, in the SS-CA complex, the change in these ratios was less significant, implying potential structural rearrangement or partial recrystallization during complexation, which contributed to the preservation of molecular order through stabilized hydrogen bonding networks. Raman spectroscopy (Fig. 4D) further supports these findings. Characteristic starch vibrational bands were observed at 480 cm⁻¹ (C–O–C), 940 cm⁻¹ (C—C), and 1460 cm⁻¹ (CH₂). The reduced intensity of the 940 cm⁻¹ peak suggests limited backbone mobility and increased chain rigidity, possibly due to CA insertion into the amorphous regions, promoting interchain crosslinking. The aromatic peak of CA at 1610 cm⁻¹ underwent a red shift and peak broadening, indicative of π–hydrogen bonding or other non-covalent interactions with the hydrophobic cavities of starch. Additionally, alterations at 1280 cm⁻¹, corresponding to phenolic –OH vibrations, further confirm complex formation (Wang, Jiang, Guo, Zheng, & Zhang, 2021). The FWHM of the main Raman band at 480 cm⁻¹ increased from 16.09 to 18.16 upon microwave treatment (Table 4), suggesting reduced short-range order. However, compared to microwave-treated starch alone, the SS-CA complex showed a more pronounced peak shift at 1280 cm⁻¹ and a smaller increase in FWHM, indicating that the complexation mitigated microwave-induced structural disruption. These observations are consistent with the FT-IR results, collectively suggesting that microwave irradiation promotes the formation of a more stable SS-CA configuration through enhanced non-covalent interactions such as hydrogen bonding and hydrophobic effects.

3.2.5. TGA analysis

Thermogravimetric analysis (TGA) was employed to evaluate the thermal stability of SS-CA composites prepared under microwave treatment (Fig. 4E). The thermal decomposition process can be divided into three stages: the initial weight loss stage (30–150 °C), corresponding to the evaporation of free and bound water; the main decomposition stage (250–350 °C), attributed to the breakdown of starch molecular chains and thermal cleavage of glycosidic bonds; and the carbonization stage (>400 °C) (Fan et al., 2013). As shown in Fig. 4F, the derivative thermogravimetric (DTG) curves—obtained by the first derivative of the TGA curves—exhibit two prominent peaks for SS: a dehydration peak before 110 °C, and a decomposition peak within 250–390 °C. The peak in DTG indicates the maximum weight loss rate, and its corresponding temperature (T_max) reflects the thermal stability of the sample (Turola Barbi, Teixeira, Hornung, Ávila, & Hoffmann-Ribani, 2018). The T_max of the SS-CA-60 composite was 299.93 °C, which was 11.21 °C lower than that of the control (311.14 °C), indicating reduced thermal stability upon short-term microwave treatment. Interestingly, prolonging the microwave duration to 120 s increased the T_max to 301.4 °C, suggesting a slight enhancement in thermal stability. This may be attributed to the reorganization of disrupted starch chains with CA via hydrogen bonding and van der Waals forces, as well as interactions between CA and liberated linear/branched starch fractions, which enhance the interfacial binding between crystalline and amorphous regions (Wang et al., 2020).

3.2.6. In vitro digestion analysis

Table 5 summarizes the variations in RDS, SDS, and RS contents in SS-CA complexes under different treatment conditions. The results demonstrated that microwave-assisted CA treatment significantly increased the RS content of the complexes while decreasing their overall digestibility. Compared with the SS-CA physical mixture, microwave-treated samples (30 s) exhibited an 8.07 % increase in RS content, suggesting that microwave irradiation facilitated the complexation between CA and starch, resulting in a denser and more stable structure that enhances resistance to enzymatic hydrolysis. Notably, after 120 s of microwave treatment, the RDS content decreased from 33.82 % to 24.65 %, whereas RS content increased markedly to 67.17 %, indicating a substantial enhancement in resistance to enzymatic digestion. This effect can be ascribed to the dual function of CA during complex formation: firstly, CA molecules adsorb onto the surface of starch granules, creating a physical barrier that limits the accessibility of α-amylase to its substrate; secondly, CA inherently exerts inhibitory activity against starch-hydrolyzing enzymes. On the other hand, CA can interact non-covalently with starch chains via hydrogen bonding, van der Waals forces, and CH–π interactions, potentially inserting into the hydrophobic helical cavities of starch molecules to form inclusion complexes resembling V-type structures (i.e., RS5-type resistant starch), thereby enhancing structural stability and delaying enzymatic hydrolysis (Cai et al., 2024; Xu, Chen, Luo, & Lu, 2019). Furthermore, during the digestion process, part of the CA may gradually be released and bind to α-amylase or glucosidase, inducing conformational changes in their secondary structure, which further impairs their catalytic activity (Zheng et al., 2020). This synergistic mechanism—combining physical barrier formation with enzymatic inhibition—is considered a key factor in the enhanced digestibility resistance of the SS-CA complexes.

Table 5.

The digestibility characteristics (RDS, SDS, and RS) of microwave-prepared SS-CA complexes.

Sample	RDS/%	SDS/%	RS/%
SS-CA	33.82 ± 1.10a	13.41 ± 0.67a	52.77 ± 0.60a
SS-CA-30	27.88 ± 0.64ab	11.28 ± 1.76b	60.84 ± 0.97a
SS-CA-60	26.91 ± 2.13ab	10.22 ± 0.75c	62.87 ± 0.54ab
SS-CA-90	26.06 ± 1.25ab	8.99 ± 0.38d	64.95 ± 1.05ab
SS-CA-120	24.65 ± 0.59b	8.17 ± 0.91e	67.17 ± 0.42b

Different letters represent significant differences (p < 0.05) between groups.

3.2.7. DFT calculations

To elucidate the molecular interaction mechanism of the SS-CA composite system, density functional theory (DFT) calculations were employed to investigate potential binding sites and interaction types. As shown in Fig. 5A-B, the electrostatic potential (ESP) distributions of the starch model and CA were computed to identify active regions on the molecular surface that may participate in intermolecular interactions. In the ESP maps, red regions indicate areas of positive electrostatic potential, while blue regions represent negative potential, reflecting variations in electron density. The results revealed that the extreme potential values were predominantly located around hydroxyl and carboxyl groups in both starch and CA molecules, suggesting that these polar functional groups play key roles in molecular recognition and binding. Specifically, hydroxyl groups enriched on the surface of the starch cavity exhibited strong positive electrostatic potential, whereas the carboxyl group of CA tends to ionize and exhibit negative potential. Under electrostatic attraction, CA preferentially interacts with the positively charged regions of the starch cavity, indicating this site as a potential binding hotspot (Xu et al., 2021). Further molecular docking and non-covalent interaction analyses (Fig. 5C-D) demonstrated that the CA molecule could be embedded into the helical cavity of starch via hydrophobic interactions, accompanied by van der Waals forces and several hydrogen bonds at the binding interface. The calculated binding energy of the optimal complex was −5.39 kcal/mol, indicating favorable binding stability between the ligand and receptor. Based on these results, it is proposed that CA is stabilized within the hydrophobic channel of the starch helix primarily through hydrophobic insertion, which may promote the formation of helical structures. In addition, multiple hydrogen bonds can be formed between the hydroxyl and carboxyl groups of CA and starch molecules, enhancing the stability of the double-helical structure and potentially inducing local crystallization. The formation of this composite structure strengthens intermolecular interactions and increases aggregate compactness, thereby hindering enzymatic recognition and hydrolysis of starch chains. These findings partially explain the molecular mechanism underlying the increased resistant starch content in the SS-CA system (Hao, Xu, et al., 2024).

Fig. 5.

Electrostatic potential (ESP) distributions on the molecular surfaces of the starch model (A) and CA monomer (B), optimized stable conformations of the SS–CA complex (C), and IGM analysis of the SS-CA complex system (D).

3.3. Evaluation of the retrogradation properties of SS–CA complexes

3.3.1. Moisture distribution analysis

Moisture distribution plays a pivotal role in the retrogradation behavior of starch gels. Based on differences in transverse relaxation times (T₂), water in the starch matrix can be classified as bound water (T₂₁), immobilized water (T₂₂), and free water (T₂₃) (Hu, Li, Tan, McClements, & Wang, 2022). To investigate moisture dynamics during storage, low-field nuclear magnetic resonance (LF-NMR) and water mobility analysis were employed to monitor T₂ shifts and corresponding signal amplitudes (P₂₁, P₂₂, P₂₃) in microwave-gelatinized SS gels and their CA complexes (SS-CA) over time. As shown in Fig. 6A-B, both SS and SS-CA exhibited a single water population (monomodal peak) at day 1, indicating a homogeneous moisture environment. However, with prolonged storage, a bimodal distribution emerged, implying the gradual release of immobilized water into the free phase as the gel network deteriorated. Quantitative data (Table 6) further revealed that both T₂₁ and T₂₂ values in the SS–CA gels gradually decreased, indicating increasingly restricted water mobility and enhanced hydrogen bonding or spatial confinement between water molecules and starch chains (Chen et al., 2020). Notably, the SS-CA system exhibited minimal changes in peak area distribution, with free water content increasing by only 1.58 % after 7 days, markedly lower than the 5.93 % increase observed in the SS control. This suggests that caffeic acid effectively retards water migration and gel aging. However, a significant increase in free water was observed in SS-CA after 14 days, potentially due to partial rearrangement of starch chains disrupting CA–starch interactions, resulting in compromised network integrity and the release of previously constrained water molecules. Microwave-induced gelatinization is known to promote the leaching of short-chain starch fragments, which act as mobile structural units, facilitating the formation of a continuous gel phase and enhancing gel integrity (Vicente et al., 2024). In the SS-CA system, molecular docking simulations suggest that caffeic acid aligns well with the starch helical structure, and its carboxyl and phenolic hydroxyl groups establish multivalent non-covalent interactions with both starch chains and water molecules. This hierarchical bridging not only strengthens water retention but also contributes to the stabilization of the three-dimensional gel framework under microwave treatment.

Fig. 6.

Transverse relaxation curves (A, B), XRD patterns (C, D), and Raman spectra (E, F) of microwave-treated SS and SS–CA complexes during retrogradation.

Table 6.

Moisture distribution of microwave-treated SS and SS–CA complex during retrogradation.

Sample		T21 (ms)	T22 (ms)	T23 (ms)	P21 (%)	P22 (%)	P23 (%)
SS	1d	2.09 ± 0.76bcd	54.79 ± 1.47e	n.d.	1.62 ± 1.42abc	98.38 ± 1.43a	n.d.
	3d	1.12 ± 0.38cd	39.64 ± 1.61f	1937.97 ± 151.61a	0.66 ± 0.83bc	98.57 ± 0.61a	0.77 ± 0.10e
	5d	0.73 ± 0.01d	33.70 ± 1.23g	1245.88 ± 32.45b	0.07 ± 0.01c	95.79 ± 0.21bc	4.14 ± 0.23d
	7d	n.d.	27.36 ± 0.90h	1367.45 ± 55.43b	n.d.	94.07 ± 0.40d	5.93 ± 0.40c
	14d	n.d.	19.37 ± 1.34i	1534.37 ± 40.85ab	n.d.	83.41 ± 0.88e	16.59 ± 0.88b
SS-CA	1d	5.23 ± 0.91a	166.38 ± 4.28a	n.d.	2.49 ± 0.32a	97.51 ± 0.33ab	n.d.
	3d	3.64 ± 1.54b	138.34 ± 5.61b	n.d.	2.63 ± 1.09a	97.37 ± 1.20ab	n.d.
	5d	2.61 ± 0.50bc	135.10 ± 3.33b	n.d.	2.10 ± 1.23ab	97.90 ± 1.31a	n.d.
	7d	1.62 ± 0.41cd	120.40 ± 4.88c	1956.07 ± 643.32a	3.22 ± 0.28a	95.20 ± 1.49cd	1.58 ± 1.22e
	14d	2.13 ± 0.41bcd	77.53 ± 3.19d	1500.07 ± 59.41ab	1.93 ± 0.38ab	74.04 ± 0.97f	24.03 ± 0.24a

Different letters represent significant differences (p < 0.05) between groups.

3.3.2. Crystalline structure analysis

XRD analysis revealed distinct differences in crystallization behavior between SS and its complex with CA during retrogradation (Fig. 6C–D). With prolonged storage, the diffraction peak intensities of native SS increased progressively, indicating that starch chains realigned through hydrogen bonding to form more ordered crystalline regions, a hallmark of amylose retrogradation. Correspondingly, the relative crystallinity of SS increased from 8.04 % to 21.12 % (Table 7), confirming the enhancement of crystallization over time. In contrast, the SS-CA complex exhibited markedly lower peak intensities and a broad peak centered around 19.5°, suggesting a more disordered internal structure. Its relative crystallinity increased only modestly from 6.35 % to 16.47 %, significantly lower than that of native SS. This indicates that the incorporation of CA effectively disrupted the rearrangement and alignment of starch chains, thereby inhibiting the formation of long-range ordered structures. This inhibitory effect is likely attributed to the rigid aromatic ring and conjugated system of CA, which interact with starch chains via hydrogen bonding and hydrophobic interactions, occupying intermolecular spaces and impeding close packing. Moreover, the broadened peak at 19.5° may be associated with structural disorder within the composite, implying that CA molecules may partially embed into the helical cavities of starch, disturbing the regular chain alignment (Cerruti et al., 2011).

Table 7.

Relative crystallinity and FWHM at 480 cm⁻¹ of microwave-treated SS and SS–CA complex during retrogradation.

Retrogradation time (d)	Relative crystallinity		FWHM at 480 cm−1
	SS (%)	SS-CA (%)	SS (%)	SS-CA (%)
1	8.04 ± 0.57d	6.35 ± 0.50c	18.26 ± 0.42a	18.05 ± 0.07a
3	11.78 ± 2.05cd	7.05 ± 0.50c	17.67 ± 0.17ab	17.62 ± 0.03ab
5	13.33 ± 2.1bc	8.10 ± 1.34c	17.15 ± 0.54ab	17.38 ± 0.17bc
7	16.48 ± 0.80b	12.03 ± 2.58b	16.89 ± 0.37b	17.02 ± 0.24cd
14	21.12 ± 2.95a	16.47 ± 2.13a	15.37 ± 1.19c	16.73 ± 0.50d

Different letters represent significant differences (p < 0.05) between groups.

3.3.3. Raman spectroscopic analysis

To further investigate the differences in molecular chain rearrangement between SS and SS-CA complex during retrogradation, Raman spectroscopy was employed to analyze changes in their short-range ordered structures (Fig. 6E–F). In Raman spectroscopy, which is sensitive to the short-range ordering conformations of starch, the band at 480 cm⁻¹ corresponds to the deformation vibration of the C–O–C bond, while the band at 940 cm⁻¹ is associated with the C—O stretching vibration of the α-1,4 glycosidic bond. Both peaks can be used to characterize the molecular ordered structure of starch. With prolonged retrogradation time, the peak intensities at these two wavenumbers in SS gradually increased, indicating the formation of more crystalline regions and enhanced molecular ordering due to the reorganization of starch chains into double helical structures (Galvis, Bertinetto, Holopainen, Tamminen, & Vuorinen, 2015). In contrast, the corresponding peak intensities for SS-CA increased only slightly, accompanied by peak overlapping and broadening, suggesting that CA effectively inhibited the reformation of ordered helical structures. The characteristic peak of CA (such as the aromatic ring vibration at 1600 cm⁻¹) remained stable in the complex system, indicating that CA primarily interacts non-covalently with starch chains via hydrogen bonding and hydrophobic interactions, maintaining its stability within the matrix. Further analysis from Table 7 shows that the FWHM of SS decreased more significantly during retrogradation compared to SS-CA, suggesting that the helical structure of SS becomes more ordered, while molecular chain rearrangement in the complex is relatively limited. This inhibitory effect is likely attributed to the rigidity of the CA molecule, whose aromatic ring and conjugated double bonds restrict conformational flexibility and thus suppress chain helicity upon binding to starch (Meinhart et al., 2019; Wellner, Georget, Parker, & Morris, 2011). The introduction of CA not only disrupts the reconstruction of starch chain short-range order but also delays molecular rearrangement and crystallization at multiple structural scales, effectively suppressing starch retrogradation.

3.3.4. Thermal property analysis

Starch retrogradation refers to the gradual reorganization of amylose and amylopectin chains into ordered crystalline structures through intermolecular hydrogen bonding during low-temperature storage (Wu, Li, Bai, Yu, & Zhang, 2019). The associated enthalpy change (ΔH) directly reflects the quantity and perfection of crystalline regions. As shown in Table 8, both the ΔH and the degree of retrogradation (DR) increased over time for SS and SS-CA. However, the ΔH values of SS-CA remained consistently lower than those of SS, indicating that the incorporation of CA effectively suppressed the molecular rearrangement and recrystallization of starch chains. After 14 days of storage, the ΔH value of SS increased from 2.42 J/g to 3.36 J/g, whereas that of the SS-CA complex rose from 0.59 J/g to 3.04 J/g. Although the ΔH of SS-CA also increased, its initial enthalpy was markedly lower than that of SS, and the overall increment was smaller, indicating that the incorporation of CA partially inhibited the reformation of crystalline structures. Combined with the XRD and LF-NMR results, it can be inferred that CA interfered with the reorganization of starch molecular chains through hydrogen bonding and hydrophobic interactions (Strapasson et al., 2021), leading to the formation of looser and less stable crystalline regions during storage. Consequently, CA effectively retarded the reassociation of starch chains and the accumulation of structural energy during retrogradation. Second, the rigid aromatic ring of CA introduces steric hindrance, restricting the linear extension and ordered alignment of starch chains, thereby delaying the formation of short-range crystalline structures. Additionally, the hydrophobic phenyl groups of CA may interact with the branching points of amylopectin, limiting side-chain mobility and disrupting parallel alignment, ultimately reducing the crystallization tendency and gelatinization enthalpy. In a word, the changes in ΔH not only reflect the adjustment of the system's energy state but also confirm the effectiveness of CA in inhibiting starch retrogradation.

Table 8.

Thermal characteristics of microwave-treated SS and SS–CA complex during retrogradation.

Sample		To (°C)	Tp (°C)	Tc (°C)	ΔH (J/g)	DR (%)
native SS		65.58 ± 0.03a	70.67 ± 0.01a	80.78 ± 1.84a	10.79 ± 0.15a	–
SS	1d	47.33 ± 0.55cd	52.61 ± 0.47bc	57.48 ± 0.43b	2.42 ± 0.28de	22.47 ± 2.61cd
	3d	46.80 ± 0.19de	52.81 ± 0.27bc	56.46 ± 0.03bc	2.64 ± 0.27cde	24.44 ± 2.46bcd
	5d	48.38 ± 0.80cd	53.16 ± 0.75bc	56.88 ± 0.25bc	3.00 ± 0.06bcd	27.84 ± 0.56abc
	7d	46.86 ± 1.54de	52.95 ± 0.06bc	56.80 ± 0.27bc	3.09 ± 0.26bc	28.68 ± 2.38ab
	14d	48.50 ± 0.18cd	52.98 ± 0.02bc	56.50 ± 0.06bc	3.36 ± 0.09b	31.18 ± 0.85a
SS-CA	1d	47.55 ± 1.52cd	48.50 ± 2.01d	54.62 ± 2.15d	0.59 ± 0.77f	5.48 ± 0.71e
	3d	53.30 ± 1.53b	54.10 ± 1.38b	55.87 ± 0.85bcd	2.15 ± 0.45e	19.93 ± 0.41d
	5d	45.27 ± 0.03e	52.97 ± 0.01bc	55.46 ± 0.02cd	2.51 ± 0.15cde	23.29 ± 1.43bcd
	7d	49.00 ± 0.02c	52.38 ± 0.05c	56.37 ± 0.02bc	2.80 ± 0.17bcd	25.98 ± 1.43abc
	14d	47.94 ± 1.15cd	53.25 ± 0.43bc	56.72 ± 0.06bc	3.04 ± 0.29bcd	28.22 ± 1.62abc

Different letters represent significant differences (p < 0.05) between groups.

4. Conclusions

In this study, SS was used as a raw material to prepare SS-CA complexes via microwave modification and microwave-assisted complexation. The effects of microwave treatment on the structural, physicochemical, antioxidant, digestibility, and gel retrogradation properties were systematically investigated. Results showed that microwave treatment disrupted the native crystalline structure of starch, with the relative crystallinity significantly decreasing from 24.07 % to 3.51 %. The short-range ordering, indicated by a reduction in the R_1047/1022 ratio (from 0.81 to 0.77) and an increase in FWHM (from 16.09 to 18.16), was also impaired, providing structural conditions favorable for complexation with small molecules. For the SS-CA complexes, prolonged microwave treatment induced granule aggregation and localized deformation, while the DPPH radical scavenging activity increased by 48.81 %, indicating a significant enhancement in antioxidant capacity. XRD analysis confirmed the formation of a stable V-type inclusion structure, and the relative crystallinity decreased by 8.88 %, demonstrating that microwave-assisted complexation effectively inhibited the reorganization of crystalline regions. Thermal analysis showed that the introduction of CA initially increased the gelatinization temperature, but extended microwave irradiation slightly reduced thermal stability. Digestibility tests revealed that, compared with the control group, the complex prepared at 120 s exhibited a 9.17 % reduction in RDS and a 14.40 % increase in RS, demonstrating improved resistance to enzymatic hydrolysis. Storage tests revealed that the free water proportion of the complexes increased by only 1.58 % over 7 days, significantly lower than that of the control (5.93 %), indicating that CA enhanced water-binding capacity through hydrogen bonding and hydrophobic interactions, effectively delaying gel network rearrangement and retrogradation. This study provides both theoretical insights and practical guidance for constructing functional starch–phenolic complexes via green microwave-assisted processing, offering potential for the development of low-glycemic and high-value-added starch-based products.

CRediT authorship contribution statement

Zongwei Hao: Writing – original draft, Methodology, Investigation, Data curation, Conceptualization. Jiameng Wang: Methodology. Yixuan Xu: Visualization. Huijun Sun: Formal analysis. Zongjun Wu: Data curation. Yibin Zhou: Writing – review & editing, Funding acquisition. Yiqun Du: Writing – review & editing. Zhenyu Yu: Supervision, Resources, Project administration, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

Data will be made available on request.