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. 2025 Aug 27;30:102956. doi: 10.1016/j.fochx.2025.102956

Mitigative effects of carboxymethyl chitosan on the deterioration of gliadin tractility in frozen rice dough during frozen storage

Wangming Dong a,b,c, Yan Wu a,b,c, Ge Zhang d, Qi Wei a,b,c,⁎
PMCID: PMC12414905  PMID: 40927626

Abstract

Frozen storage deteriorates the texture and digestibility of frozen rice dough by damaging gliadin structure and starch integrity. This study investigated carboxymethyl chitosan (CMCh) and sodium carboxymethyl cellulose (CMCNa) as cry-oprotectants to mitigate these effects. Comprehensive analysis utilizing nuclear magnetic resonance (NMR), texture profile analysis (TPA), dynamic contact angle measurement (DCAT21), reversed-phase high-performance liquid chromatography (RP-HPLC), and circular dichroism (CD) demonstrated that 1.5 % CMCh-B optimally preserved gliadin integrity relative to both control and CMCNa treatments, it inhibited bound water conversion, enhanced protein cross-linking, minimized γ-prolamin loss (15.8 % less decline), stabilized secondary structure (reducing α-helix increase by 40.42 % and β-sheet decrease by 45.70 %), lowered surface hydrophobicity (75.17 %), and limited ice crystal damage. Consequently, texture significantly improved (hardness reduced 25.90 %, springiness reduced 38.24 %) and starch digestibility moderated (SDS increased 22.62 %, RS decreased 71.51 %). CMCh-B's hydroxyl-mediated hydrogen bonding forms a protective hydration layer, effectively inhibiting gliadin depolymerization and ice crystal growth, demonstrating significant potential for enhancing frozen rice dough quality.

Keywords: Carboxymethyl cellulose sodium, Carboxymethyl chitosan, Frozen rice dough, Tractility, Wheat gliadin

Highlights

  • •

    A method to mitigate the deterioration of frozen gliadin tractility was developed.

  • •

    Carboxymethyl chitosan (CMCh) attenuated the α-helix-to-β-sheet transition.

  • •

    CMCh reinforced the structural integrity of γ-gliadin within frozen rice dough.

  • •

    CMCh mitigated the age-related decline in gliadin surface hydrophobicity.

  • •

    CMCh with elevated degrees of substitution conferred superior cryoprotection.

1. Introduction

Glutinous rice serves as a traditional staple food in Asia (Fan et al., 2021), where its expansion capacity and masticatory properties constitute critical quality determinants (Wei, Zhang, and Xie, 2024). Frozen rice dough comprises glutinous rice starch and gluten protein, with their intermolecular interactions governing textural characteristics (Wei, Zhang, Ye, and Xie, 2024). Specifically, gliadin—a key gluten component—enhances dough extensibility and elasticity, thereby imparting desirable chewiness to the final product (Su et al., 2024). However, ice crystal formation during freezing disrupts the gluten network matrix, inducing protein denaturation and solubility reduction. These structural alterations ultimately compromise gliadin functionality, leading to extensibility deterioration (Wei et al., 2021; Moro, Gatti and Delorenzi, 2001). Concurrently, diminished water retention capacity further impairs textural and sensory attributes (Tao, Wang, Wu, Jin, and Xu, 2016; Tao et al., 2016; Luo, Sun, Zhu and Wang, 2018; Moro, Báez, Busti, Ballerini and Delorenzi, 2011). Consequently, maintaining quality stability in frozen rice dough has emerged as a pivotal challenge for the food industry.

Several approaches have been employed to improve the quality of frozen foods, including optimization of freezing processes and addition of cryoprotectants. Polysaccharides are commonly used food additives, such as sodium carboxymethyl cellulose (CMCNa) and chitosan, which enhance the delicate texture of frozen foods and provide effective solutions to the aforementioned problems. Research by Xin et al. (2018) demonstrated that CMCNa reduces ice crystal-induced damage to dough structure and improves dough quality. Additionally, El Sheikha et al. (2022) found that CMCNa-containing coatings can improve frozen food quality by forming oxygen barriers and reducing moisture loss. According to Chinese regulations, CMCNa is a widely used food additive primarily employed to enhance water retention capacity, with its maximum dosage depending on production requirements. Researchers continue to explore new resources to enrich these polysaccharides. Consequently, chitosan has become a focus of research. Both chitosan and CMCNa are polysaccharides mainly used to increase food viscosity and water absorption (Yousefi et al., 2025), with their maximum addition levels determined by production needs. Chitosan consists of N-acetyl-D-glucosamine and D-glucosamine units, exhibiting high reactivity and biocompatibility (Ali Akbar et al., 2025; Madanipour et al., 2019; Saeid et al., 2025). However, chitosan's poor water solubility limits its application in the food industry. Carboxymethyl chitosan (CMCh) has become a research hotspot. CMCh possesses strong moisture retention capacity and can significantly enhance the mechanical properties of food matrices through protein interactions, thereby improving gelation ability and stabilizing molecular structure. These characteristics are crucial for regulating ice crystal formation in frozen foods (Su et al., 2023; Zhao et al., 2023; Xiao et al., 2024; Yang, Guo, Zhang, Zhao, and Zhang, 2024; Yang et al., 2024). Despite these advantages, compared to CMCNa, CMCh's cryoprotective mechanisms in plant-protein systems require further investigation, particularly regarding structure-function relationships at different degrees of substitution (DS) and concentration gradients.

This study aims to elucidate the protective effects of CMCh and CMCNa on gliadin tractility in frozen rice dough. Distinct from existing studies focusing on plant-protein systems (Zhu et al., 2022) or single concentration effects (Xin et al., 2018), this investigation builds upon our established rice dough model (Wei, Zhang, and Xie, 2024) to examine the cryoprotective effects of CMCh and CMCNa on gliadin tractility following liquid nitrogen freezing (LF) treatment and subsequent storage at −18 °C for 70 days. Our central hypothesis posits that structural degradation of gluten proteins during frozen storage constitutes the primary pathway for quality deterioration, and that CMCh supplementation at specific parameter combinations (DS: 0.8, 1; concentration gradient: 0.5 %, 1 %, 1.5 %) can effectively mitigate this damaging process. Comparative analysis reveals that CMCh demonstrates superior cryoprotective efficacy in frozen rice dough relative to CMCNa, attributable to its optimized degree of substitution-concentration synergy and complex hydroxyl hydrogen-bonding networks. This work provides novel theoretical foundations for developing polysaccharide-based cry-oprotectants and demonstrates significant potential for industrial application in traditional Chinese frozen rice dough.

2. Materials and methods

2.1. Materials

Glutinous rice starch (≥ 95.1 % starch, dry basis) and wheat gluten (≥ 85 % protein) were obtained from Yihai Kerry Grain & Oil Co., Ltd. (Shanghai, China). CMCh (DS = 1.0 and 0.8), CMCNa (DS = 1.0), and chitosanase (specific activity ≥2.5 × 105 U·g−1) were procured from Haobo Biotechnology Co., Ltd. (Zhengzhou, Henan, China). The fluorescent probe 1-anilino-8-naphthalenesulfonate (ANS, ≥ 98 % purity) was acquired from Sigma-Aldrich Trading Co., Ltd. (Shanghai, China). All other chemicals were of analytical grade or higher purity.

2.2. Characterization of CMCh

The physicochemical properties of carboxymethyl chitosan variants (CMCh-A, CMCh-B) and CMCNa, as characterized by Wei, Zhang, and Xie, 2024, are compiled in Table 1. Key parameters for CMCh and CMCNa samples include degree of substitution (DS = 0.8 and 1.0 determined by potentiometric titration (Li and Xia, 2010)), weight-average molecular weight (Mw), dispersity index, and intrinsic viscosity - all quantified through gel permeation chromatography following standardized protocols (Ge & Luo, 2005). Hydrolysis was subsequently initiated by introducing chitosanase (10 U) into 2.0 wt% CMCh solutions maintained at 50 °C in 20 MM acetate buffer (pH 5.2).

Table 1.

Carboxymethylation extent, weight-average molar mass, polydispersity, and apparent viscosity (2 % w/v aqueous solution) of carboxymethyl chitosan and sodium carboxymethyl cellulose.

Sample DS Mw (Da) Mw/Mn Viscosity (m Pa. S)
CMCh-A 0.8 6.49 × 104 1.39 13.6 (2 wt%)
CMCh-B 1 6.73 × 104 1.58 10.6 (2 wt%)
CMCNa 1 6.66 × 104 1.47 10.1 (2 wt%)
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2.3. Preparation of frozen rice dough

Frozen rice doughs were prepared according to Wei et al. (2023) with minor modifications. CMCh replaced 0.5 %, 1.0 %, or 1.5 % of flour mass (w/w), while CMCh was incorporated at a fixed concentration of 1 %. An additive-free dough served as the control. After shaping, samples underwent cryogenic immersion freezing (2 min, low-frequency mode) prior to storage at −18.0 ± 0.5 °C. Specimens were retrieved at 10-day intervals throughout the 70-day storage period for analysis.

2.4. Water distribution

Water distribution patterns were delineated using the analytical framework established by Wei, Zhang, Ye, and Xie, 2024.

2.5. Texture properties analysis (TPA)

Texture profile analysis (TPA) parameters were determined following the methodology described by Li et al. (2022).

2.6. Starch-gluten separation

Gluten and starch were fractionated from the samples with minor refinements to the procedure outlined by Wei, Zhang, and Xie, 2024.

2.7. Changes of gluten protein

2.7.1. Gliadin isolation

Gliadin enrichment was performed using an optimized protocol adapted from Wang et al. (2014). Gluten (200 g) was delipidated through three sequential washings with dichloromethane (300 mL per wash, 1 h each, 20 °C), filtered through Whatman No. 1 filter paper (0.45 μm pore size), and air-dried. Twenty grams of defatted gluten underwent two sequential extractions with 60 % (v/v) ethanol (300 mL each) followed by a final extraction with deionized water (300 mL), with manual dispersion of cohesive glutenin aggregates prior to the second and third extraction cycles. Each 3-h extraction at 20 °C was terminated by centrifugation (3000 ×g, 10 min, 4 °C). Supernatants were pooled, concentrated via rotary evaporation at 30 °C, and lyophilized. Protein purity was verified through nitrogen quantification by the Kjeldahl method (N × 5.7).

2.7.2. Water solubility of gliadin

Water solubility was determined according to Itzhaki and Gill (1964) with minor modifications. Gliadin dispersions (0.3 % w/v) were prepared in deionized water and adjusted to pH 5.8 using 0.1 M NaOH/HCl to simulate wheat-dough conditions. After vortexing (30 s) and equilibration (30 min, 25 °C), samples were centrifuged at 10,000 ×g for 10 min at 20 °C. Soluble protein content in supernatants was quantified by the Biuret method against a bovine serum albumin standard curve. These supernatant fractions were subsequently utilized in downstream analyses.

2.7.3. Interfacial tension of gliadin dispersions

Interfacial tension measurements were performed at 25 °C using a DCAT21 tensiometer (DataPhysics Instruments, Filderstadt, Germany) equipped with a platinum Wilhelmy ring (40 mm circumference), following the methodology of Wang et al. (2014) with minor modifications. Prior to measurements, all glassware was rigorously cleaned through sequential rinsing with acetone and ultrapure water, followed by drying under ambient conditions. The platinum ring was flame-cleaned before each analysis cycle.

2.7.4. Reversed-phase high-performance liquid chromatography (RP-HPLC) profiling of gliadin sub-fractions

Gliadin subtypes (ω, α, γ) in native, dispersed, and foamed matrices were separated by reversed-phase HPLC according to Wang et al. (2016) with minor modifications. Lyophilized samples were dissolved in 70 % ethanol (5 mg mL−1), filtered (0.22 μm), and 20 μL was injected onto a Nucleosil 300–5 C8 column (Machery-Nagel, Düren, Germany) maintained at 25 °C. Separation was achieved using a linear gradient of 24–56 % acetonitrile in 0.1 % trifluoroacetic acid over 50 min at a flow rate of 1.0 mL min−1, with detection at 214 nm.

2.7.5. Circular dichroism (CD) spectroscopy

The secondary structure of gliadin was characterized by circular dichroism (CD) spectroscopy using a MOS-450 spectropolarimeter (Bio-Logic Science Instruments, Grenoble, France), adapting the methodology of Wang et al. (2014). Far-UV spectra (190–250 nm) were acquired at 25 °C using protein solutions (0.1 mg mL−1) in a 0.1 cm pathlength quartz cuvette. Spectral deconvolution via the CONTIN/LL algorithm quantified the relative proportions of α-helix, β-sheet, β-turn, and random coil structures.

2.7.6. Surface hydrophobicity

Hydrophobic surface exposure was quantified using ANS fluorescence, adapted from the method of Ryan et al. (2012). Gliadin solutions (0–0.09 % w/v) were prepared in 0.1 M phosphate buffer (pH 5.8). To 2 mL aliquots of these solutions, 10 μL of 8 mM ANS was added. After a 3-min equilibration period, fluorescence spectra were recorded using an F-7000 fluorometer (Hitachi, Japan) with excitation at 390 nm and emission at 470 nm (5 nm slit widths for both monochromators). Following baseline subtraction, fluorescence intensities were plotted versus protein concentration; the resultant slope was defined as the Surface Hydrophobicity Index (S0).

2.8. Changes of starch

2.8.1. In vitro digestibility

The in vitro digestibility of the samples was assessed according to the method described by Wang et al. (2018); Wang, Pei, Teng, and Liang (2018), with minor modifications. The procedure was as follows: One gram of freeze-dried sample was placed into a 100 mL conical flask. Thirty milliliters of 0.1 M acetate buffer (pH 5.1) was added, and the mixture was stirred at room temperature for 15 min. Subsequently, a mixed enzyme solution containing salivary α-amylase (5 mL, 290 U/mL) and pullulanase (15 U/mL) was added to the suspension. The mixture was then incubated in a water bath at 37 °C with continuous shaking at 150 rpm. Aliquots of 1 mL were withdrawn at 20 min (G20) and 120 min (G120) of incubation for the determination of hydrolyzed glucose, total glucose (Tgl), and free glucose (Fgo). Glucose content was quantified using the glucose oxidase-peroxidase method with a d-glucose assay kit (GOPOD, K-GLUX, Megazyme, Bray, Co. Wicklow, Ireland). Absorbance was measured at 420 nm using a UV-1601 spectrophotometer (Hitachi, Japan). Fgo represents the amount of free glucose released prior to enzymatic hydrolysis, while Tgl denotes the total glucose liberated after complete enzymatic hydrolysis. Finally, the contents of rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS) were calculated using the following equations:

RDS%=Gd−Go×0.9/Ts×100% (1-1)
SDS%=Gb−Gd×0.9/Ts×100% (1-2)
RS%=100%−SDS+RDS (1-3)

where, Go: Free glucose concentration in the untreated sample.

Gd: Glucose concentration released after 20 min of enzymatic digestion .

Gb: Glucose concentration released after 120 min of enzymatic digestion .

Ts: Total starch content of the sample.

0.9: Glucose-to-starch conversion factor.

2.8.2. Swelling power (SP) and solubility (SOL)

Swelling power (SP) and water solubility (SOL) were quantified gravimetrically using an adapted protocol derived from Chi et al. (2023). Sample suspensions were prepared by homogenizing freeze-dried material (1.0 g) with deionized water (100 mL), followed by incubation at 95 °C for 30 min in a thermostatic shaker. After centrifugation (4500 ×g, 20 min, 25 °C), the pellet (P) was collected and weighed. The supernatant was oven-dried (100 °C, 6 h) to constant mass.

Water solubility was calculated as:

SOL=MSMO×100% (1-4)

where, MS= mass of dried supernatant solids and MO= initial dry sample mass.

Swelling power was determined by:

SP=EPMO−MS×100g/g (1-5)

where, EP= the dried sediment after centrifugation.

2.9. Statistical analysis

Statistical analysis was performed using IBM SPSS Statistics 26.0 (IBM Corp., Armonk, NY, USA) after assessing normality and homogeneity of variance. Data are presented as mean ± standard deviation. Graphical representations were generated using OriginPro 8.0 (OriginLab Corporation, Northampton, MA, USA). All assays were performed in triplicate to ensure reproducibility.

3. Results and discussion

3.1. Changes in gliadin water solubility

To simulate the pH environment of traditional rice dough systems, distilled water adjusted to pH 5.8 was used as the solvent for evaluating gliadin water solubility (SOL). Experimental results demonstrated that the concentration of solubilized gliadin remained consistently low (approximately 0.1 % w/v) throughout storage, with no notable time-dependent variation. This observation is consistent with the study of Wang et al. (2014), who demonstrated that gliadin exhibits minimal SOL near neutral pH conditions owing to its isoelectric point (pI ≈ 7.8).

3.2. Changes in water distribution

Fig. 1 illustrates moisture redistribution in frozen rice dough fortified with either CMCNa or CMCh. Low-field 1H NMR T2 relaxation distributions (Fig. 1A) identified three distinct water populations: T21 (strongly bound), T22 (loosely bound), and T23 (free) (Jiang et al., 2023; Li et al., 2022). Fig. 1B–D displays the peak-area fractions of each water population throughout the storage period. Over 70 days of frozen storage, the T21 content in the control group decreased considerably by 36.87 %, while T22 and T23 increased by 4.52 % and 70.70 %, respectively. The data indicate a continuous decline in T21 and a pronounced rise in T22 and T23 over time. This shift is primarily attributed to ice-crystal formation and growth during freezing, which disrupts the gluten network and facilitates the conversion of bound water into free water (Wei, Zhang, Ye, and Xie, 2024). Relative to the control, T21 declined by 28.92 % (1 % CMCNa), 35.58 % (0.5 % CMCh-A), 31.89 % (1 % CMCh-A), 30.52 % (1.5 % CMCh-A), 34.46 % (0.5 % CMCh-B), 26.59 % (1 % CMCh-B), and 20.32 % (1.5 % CMCh-B). Conversely, T22 increased by 3.48 %, 4.34 %, 3.84 %, 3.68 %, 4.19 %, 3.18 %, and 2.33 % and T23 rose by 67.80 %, 70.61 %, 69.94 %, 68.89 %, 70.48 %, 66.83 %, and 65.04 %, respectively. These changes are primarily attributed to the superior water-holding capacity of CMCh and CMCNa and their inhibitory effects on ice-crystal growth. Consequently, CMCh and CMCNa delay the loss of T21 and considerably suppress the increase in T22 and T23. Notably, the present study clearly elucidates the crucial role of the degree of substitution (DS) in CMCh in promoting moisture retention in frozen rice dough, along with its underlying mechanism. High-DS CMCh-B forms a denser hydrogen-bond network, converting a portion of weakly bound water (T22) into strongly bound water (T21), thereby enhancing water retention. Notably, the carboxymethyl groups electrostatically cross-link gliadin, the hydroxyl groups anchor to the ice-crystal surface, and the CH2COONa segments provide additional noncovalent stabilization (Li et al., 2021; Zhu et al., 2022). This unique triple synergistic mechanism—electrostatic cross-linking, ice anchoring, and noncovalent stabilization—underpins the ability of 1.5 % CMCh-B to markedly inhibit ice-crystal growth and preserve the structural integrity of frozen rice dough. Therefore, the addition of 1.5 % CMCh-B considerably enhances the long-term storage stability of frozen rice dough via strengthening water molecule binding forces and effectively preventing its transition from bound to free water. However, the production cost of high-DS CMCh-B is typically higher than that of CMCNa and low-DS CMCh-A and its economic feasibility for industrial-scale application warrants further evaluation.

Fig. 1.

Fig. 1

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Spin-spin relaxation times (T2) of frozen rice doughs were measured by nuclear magnetic resonance (NMR) during frozen storage, following supplementation with sodium carboxymethyl cellulose (CMCNa), carboxymethyl chitosan-A (CMCh-A), and carboxymethyl chitosan-B (CMCh-B). (A) T2 relaxation distribution curve, (B) T21 peak proportions, (C) T22 peak proportions, and (D) T23 peak proportions (Image 1, Control; Image 2, 1 % CMCNa; Image 3, 0.5 % CMCh-A; Image 4, 1 % CMCh-A; Image 5, 1.5 % CMCh-A; Image 6, 0.5 % CMCh-B; Image 7, 1 % CMCh-B; Image 8, 1.5 % CMCh-B).

3.3. Changes in swelling power and SOL

A literature establishes an inverse correlation between swelling power (SP) and SOL and the mechanical properties of cereal doughs (Shu et al., 2022). In our investigation, over 70 days of storage, freezing induced pronounced increases in SP and SOL in rice dough (the control samples) (Fig. 2), demonstrating increases of 28.98 % and 18.34 %, respectively. These changes reflect progressive ice-crystal growth that disrupts the gluten network, promotes starch leaching, and ultimately deteriorates texture (Gomez et al., 2013; Yang et al., 2021). Over the 70-day storage period, SP and SOL increased in all additive-supplemented samples relative to the baseline. However, compared with the control, samples containing CMCh and CMCNa exhibited a notably attenuated rise in SP and SOL. This suggests that both the additives effectively mitigate the structural degradation typically associated with frozen storage. Notably, samples containing 1.5 % CMCh-B demonstrated the most effective suppression of structural degradation, with SP increasing by only 18.16 % and SOL by 10.57 %. Its superior performance was followed by 1 % CMCh-B, in which SP increased by 21.51 % and SOL by 15.11 % and 1 % CMCNa, in which SP increased by 22.89 % and SOL by 15.72 %. By contrast, low-substitution CMCh-A variants were observed to be less effective, as seen in the 1.5 % CMCh-A, in which SP increased by 24.43 % and SOL by 16.67 % (Fig. 2). This disparity is likely attributed to the progressive ice-crystal nucleation and its growth during frozen storage, which fractures the integrated starch–gluten matrix and precipitates a decline in mechanical integrity—manifested as reduced hardness and elasticity (Wang et al., 2021). The addition of CMCh and CMCNa facilitates the formation of hydrogen-bond networks via the carboxymethyl groups in their molecular structures, effectively retaining moisture and inhibiting ice-crystal growth and recrystallization. Among these, the highly substituted CMCh-B exhibits superior molecular chain hydration capacity, enabling the formation of a denser hydrogen-bond network. This structural advantage notably delays the increase in SP and SOL during frozen storage (Zhu et al., 2022; Wei, Zhang, Ye, and Xie, 2024). Accordingly, our findings clearly demonstrate that (1) CMCh-B outperforms CMCNa in preserving the structural integrity of frozen dough and (2) a higher DS in CMCh-B directly enhances its cryoprotective efficacy. Nevertheless, this study primarily investigates interactions within the starch–gluten–additive matrix and the potential roles of endogenous lipids or enzymes remain uncharacterized.

Fig. 2.

Fig. 2

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Swelling power (SP) and solubility (SOL) of frozen rice doughs were measured during frozen storage, following supplementation with sodium carboxymethyl cellulose (CMCNa), carboxymethyl chitosan-A (CMCh-A), and carboxymethyl chitosan-B (CMCh-B). (Image 9, Control; Image 10, 1 % CMCNa; Image 11, 0.5 % CMCh-A; Image 12, 1 % CMCh-A; Image 13, 1.5 % CMCh-A; Image 14, 0.5 % CMCh-B; Image 15, 1 % CMCh-B; Image 16, 1.5 % CMCh-B).

3.4. Texture property analysis

Fig. 3 illustrates the evolution of texture properties in frozen rice dough during low-temperature storage. Hardness and springiness are the two main indicators for assessing the quality of frozen rice dough. Over the 70-day storage period, both the parameters in the control sample showed a notable decline, whereas cohesiveness and resilience markedly. Increased. These changes are primarily attributed to the formation and growth of ice crystals during frozen storage, which disrupt the gluten protein network and compromise the structural integrity of the dough (Asghar et al., 2011; Wang et al., 2018; Wang, Pei, Teng, and Liang, 2018; Tao et al., 2018). Research indicates that the hardness of frozen rice dough is notably positively correlated with the integrity of the gluten protein network. Specifically, the mechanical action of ice crystals disrupts the three-dimensional structure of gluten proteins, leading to reductions in hardness and springiness and resulting in the softening of textural properties (Chi et al., 2023; Zhu et al., 2019). This finding aligns with the results reported by Zhang et al. (2021), further validating the impact of ice-crystal formation on the texture of frozen dough products. The incorporation of CMCNa or CMCh considerably mitigates the detrimental textural changes in frozen rice dough (Fig. 3), with all additive-treated samples demonstrating superior preservation of textural properties (p < 0.05) following 70 days of storage compared with the control. Notably, the sample containing 1.5 % CMCh-B showed a 25.90 % reduction in hardness, a 38.24 % decrease in springiness, a 53.19 % increase in cohesiveness, and a 31.48 % enhancement in resilience. The cryoprotective performance of the sample was significantly superior (p < 0.05) to that of 1 % CMCNa (32.36 % reduction in hardness and 40.30 % decrease in springiness) and 1.5 % CMCh-A (35.63 % reduction in hardness and 41.79 % decrease in springiness). Furthermore, the cryoprotective efficacy of CMCh shows a positive correlation with its DS. Our experimental results demonstrate that CMCNa and CMCh considerably inhibited the reduction in hardness and springiness while moderating the increase in cohesiveness and resilience of the frozen rice dough during storage. This protective effect is attributed to two synergistic mechanisms as follows: (i) the superior water-binding capacity of these polysaccharides suppresses ice nucleation and growth, thereby minimizing mechanical damage to the gluten network and (ii) electrostatic cross-linking between carboxylate groups (–COO−) on CMCh chains and charged residues on gluten proteins reinforces the protein matrix, enhancing its resistance to ice crystal–induced degradation (Wei et al., 2023, Wei, Zhang, and Xie, 2024). Notably, the higher density of carboxylate groups in 1.5 % CMCh-B facilitates the formation of a more extensive cross-linked network, which accounts for its superior cryoprotective performance. Supporting evidence from SP and SOL analyses (Fig. 2) further confirms that CMCh enhances the stability of the gluten protein network, thereby improving the textural properties of the frozen rice dough. These findings provide a theoretical foundation for quality control of frozen starch–based food products.

Fig. 3.

Fig. 3

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Texture properties analysis of frozen rice doughs were measured during frozen storage, following supplementation with sodium carboxymethyl cellulose (CMCNa), carboxymethyl chitosan-A (CMCh-A), and carboxymethyl chitosan-B (CMCh-B). (A) Hardness, (B) Springiness, (C) Cohesiveness, and (D) Resilience (Image 17, Control; Image 18, 1 % CMCNa; Image 19, 0.5 % CMCh-A; Image 20, 1 % CMCh-A; Image 21, 1.5 % CMCh-A; Image 22, 0.5 % CMCh-B; Image 23, 1 % CMCh-B; Image 24, 1.5 % CMCh-B).

3.5. Analysis of gluten

3.5.1. Analysis of the secondary structure of wheat gliadin

Fig. 4 illustrates the changes in the secondary structure of gliadin in the frozen rice dough stored for 70 days. In the control sample, the proportion of α-helices markedly increased, while β-sheets decreased; β-turns remained relatively stable, and unordered structures initially rose before tapering off. These results indicate that α-helices are the most susceptible ones to freeze-induced destabilization, with ice-crystal formation driving a conformational shift from α-helical to β-sheet and disordered structures. This observation aligns with the destabilization mechanism reported by Wang et al. (2018); Wang, Pei, Teng, and Liang (2018). The addition of either CMCNa or CMCh effectively alleviated the alterations in gliadin secondary structure during frozen storage (Fig. 4). After 70 days, all treatment groups demonstrated greater structural stability compared with the control. Notably, the group treated with 1.5 % CMCh-B showed a 40.42 % increase in its α-helix content and a 45.70 % reduction in β-sheet content while effectively suppressing fluctuations in random coil structures. In comparison, the group treated with 1 % CMCNa demonstrated a 41.58 % increase in its α-helix and a 48.49 % decrease in β-sheet content, whereas the group treated with 1.5 % CMCh-A showed a 41.54 % increase in its α-helix and a 54.07 % reduction in β-sheet content. These results clearly indicate that high degree–substituted CMCh-B outperformed CMCNa and low degree–substituted CMCh-A in preserving the secondary structure stability of gliadin (p < 0.05). The observed structural changes indicates that freezing induces gliadin conformational rearrangement, potentially mediated via disulfide bond breakage and reformation. Notably, CMCh-treated groups maintained considerably higher α-helix proportions and lesser β-sheet degradation than the control, demonstrating its efficacy in preserving protein secondary structure (Hong et al., 2021; Wei, Zhang, Ye, and Xie, 2024). This stabilizing effect likely arises due to the following reasons: (i) direct hydrogen bonding and electrostatic interactions between the carboxymethyl groups of CMCh and gliadin that stabilize protein conformation and (ii) indirect mechanical protection via polysaccharide-mediated water migration control and ice-crystal inhibition (Ooms and Delcour, 2019; Zhu et al., 2019). These results not only confirm the superior performance of CMCh-B in maintaining the structural integrity of protein but also elucidate the molecular mechanisms underlying polysaccharide-mediated protein stabilization. However, this study was conducted under constant temperature conditions (−18 °C) and further research is required to assess the impact of temperature fluctuations on protein structure.

Fig. 4.

Fig. 4

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Secondary structure of protein of frozen rice doughs were measured during frozen storage, following supplementation with sodium carboxymethyl cellulose (CMCNa), carboxymethyl chitosan-A (CMCh-A), and carboxymethyl chitosan-B (CMCh-B). (A) α-helix, (B) β-sheet, (C) β-turn, and (D) Unordered (Image 25, Control; Image 26, 1 % CMCNa; Image 27, 0.5 % CMCh-A; Image 28, 1 % CMCh-A; Image 29, 1.5 % CMCh-A; Image 30, 0.5 % CMCh-B; Image 31, 1 % CMCh-B; Image 32, 1.5 % CMCh-B).

3.5.2. The relationship between the distribution of gliadin types and frozen storage duration

Fig. S1 illustrates the compositional changes in gliadin fractions of the frozen rice dough during storage. Quantitative analysis using RP-HPLC with peak-area normalization revealed notable variations among gliadin subtypes (ω, α, and γ) under frozen conditions. In the control group, the relative content of γ-gliadin decreased considerably from 52.05 % ± 0.41 % to 41.29 % ± 0.56 %, while ω- and α-gliadin increased from 7.42 % ± 0.44 % to 8.79 % ± 0.53 % and from 40.53 % ± 0.49 % to 49.92 % ± 0.69 %, respectively. These differential changes are likely attributable to the molecular characteristics of each subtype—particularly the hydrophobic nature of γ-gliadin, which may render it more susceptible to conformational alterations during freezing (Guillerme et al., 1993; Wang et al., 2014). Peak-area analysis revealed a distinct hierarchy of cryoprotective efficacy: 1.5 % CMCh-B > 1 % CMCh-B > 1 % CMCNa >1.5 % CMCh-A > 1 % CMCh-A > 0.5 % CMCh-B > 0.5 % CMCh-A > control, with smaller compositional changes corresponding to greater gliadin stability. This gradient indicates that ice-crystal growth and recrystallization disrupt intra- and intermolecular protein cross-linking networks, while CMCh effectively mitigates such damage. Notably, no notable difference was observed in the intensity ratio between the 1 % and 1.5 % CMCh-B treatment groups, likely owing to their selective mechanism of action: although they exert minimal influence on gliadin composition, they remarkably enhance other quality attributes. From the perspective of molecular interactions, CMCh forms stable electrostatic complexes with gluten proteins via its carboxymethyl groups, whereas CMCNa primarily relies on noncovalent interactions. Notably, high-substitution CMCh demonstrates superior cryoprotective performance, attributed to its intricate hydrogen-bonding network and specific substitution pattern (Wei, Zhang, and Xie, 2024). Overall, the 1.5 % CMCh-B treatment group demonstrated optimal performance in maintaining gluten network integrity and inhibiting ice crystal–induced damage, offering an effective technological solution for preserving the quality of frozen rice dough. Herein, we did not investigate the effects of temperature fluctuations, which is a notable limitation and warrants further investigation.

3.5.3. Analysis of surface tension

Fig. 5 illustrates the changes in surface tension of rice dough during frozen storage. Over the 70-day storage period, the surface tension of the control samples increased by 17.68 %, a change closely associated with conformational alterations in gliadin. Low-temperature storage led to an increase in the α-helix content of protein molecules, rearrangement of β-sheet structures, and reduction in molecular flexibility. These structural changes diminished the adsorption efficiency of the proteins and their spreading capacity at the air–water interface, resulting in elevated interfacial tension. This phenomenon is likely attributable to the reduced adsorption of gliadin at the air–water interface. Following frozen storage, gliadin molecules became more rigid and compact than before, demonstrating a reduced ability to unfold at the interface, which consequently led to an increase in interfacial tension. Following 70 days of storage, compared with the control group, the samples supplemented with 1 % CMCNa, 0.5 % CMCh-A, 1 % CMCh-A, 1.5 % CMCh-A, 0.5 % CMCh-B, 1 % CMCh-B, and 1.5 % CMCh-B showed increased surface hydrophobicity by 15.59 %, 17.34 %, 16.54 %, 16.30 %, 17.03 %, 14.91 %, and 13.12 %, respectively. The experimental data indicate that samples containing CMCNa and CMCh demonstrated superior interfacial stability. Notably, the 1.5 % CMCh-B group showed the smallest increase in surface tension. The enhanced performance of the 1.5 % CMCh-B group can be attributed to the following synergistic mechanisms: (1) the dense hydroxyl groups on CMCh-B molecular chains formed a three-dimensional hydrogen-bonding network with water molecules, effectively inhibiting ice-crystal nucleation and growth, (2) hydrophilic groups created a protective hydration shell around protein molecules, helping preserve their native conformation and (3) intermolecular electrostatic forces hindered protein aggregation and denaturation. This multiscale interaction mechanism collectively preserved the adsorption capacity of interfacial proteins, thereby remarkably mitigating the rise in surface tension during frozen storage. However, a limitation of this study is that the influence of ice-crystal morphology on protein adsorption capacity was not examined.

Fig. 5.

Fig. 5

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Surface Tension of frozen rice doughs were measured during frozen storage, following supplementation with sodium carboxymethyl cellulose (CMCNa), carboxymethyl chitosan-A (CMCh-A), and carboxymethyl chitosan-B (CMCh-B). (Image 33, Control; Image 34, 1 % CMCNa; Image 35, 0.5 % CMCh-A; Image 36, 1 % CMCh-A; Image 37, 1 0.5 % CMCh-A; Image 38, 0.5 % CMCh-B; Image 39, 1 % CMCh-B; Image 40, 1.5 % CMCh-B).

3.5.4. Analysis of surface hydrophobicity

Fig. 6 illustrates the changes in surface hydrophobicity of frozen rice dough during storage. Over the 70-day period, the control group demonstrated a notable decrease in surface hydrophobicity—by 85.10 %—primarily due to ice-crystal growth, disrupting the secondary structure of gliadin. Structural analysis of gliadin reveals its amphiphilic nature: the N-terminal CA region is hydrophilic, whereas the C-terminal TA region is enriched with hydrophobic and ionizable amino acids. Among gliadin components, γ-gliadin exhibits the highest hydrophobicity, while ω-gliadin is the most hydrophilic. Based on the experimental results, the observed reduction in surface hydrophobicity is likely driven primarily by conformational changes in γ-gliadin (Wang et al., 2014). All treatment groups supplemented with CMCNa or CMCh demonstrated considerably higher hydrophobicity retention than the control (p < 0.05). After 70 days, compared with the control, samples containing 1 % CMCNa, 0.5 % CMCh-A, 1 % CMCh-A, 1.5 % CMCh-A, 0.5 % CMCh-B, 1 % CMCh-B, and 1.5 % CMCh-B demonstrated reduced hydrophobicity losses of 79.14 %, 84.77 %, 81.79 %, 80.46 %, 83.11 %, 77.48 %, and 75.17 %, respectively. Notably, the 1.5 % CMCh-B group demonstrated the most favorable outcome, with only 75.17 % reduction. This improvement is attributed to the abundance of hydroxyl groups in CMCh-B molecules, which competitively form hydrogen bonds with water molecules, thereby inhibiting ice-crystal growth. This dual mechanism reduces physical damage to gluten proteins while preserving their native conformation, ultimately delaying the loss of surface hydrophobicity. Herein, we explored the molecular mechanism via which hydrophilic colloids suppress ice crystallization through hydrogen-bond competition, providing a novel strategy for quality preservation in frozen rice products. However, a key limitation is the unexamined influence of CMCh structural variations on the hydrogen-bond competition efficiency.

Fig. 6.

Fig. 6

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Relative Surface hydrophobicity of frozen rice doughs were measured during frozen storage, following supplementation with sodium carboxymethyl cellulose (CMCNa), carboxymethyl chitosan-A (CMCh-A), and carboxymethyl chitosan-B (CMCh-B). (Image 41, Control; Image 42, 1 % CMCNa; Image 43, 0.5 % CMCh-A; Image 44, 1 % CMCh-A; Image 45, 1.5 % CMCh-A; Image 46, 0.5 % CMCh-B; Image 47, 1 % CMCh-B; Image 48, 1.5 % CMCh-B).

3.6. Analysis of starch

3.6.1. Changes in in vitro digestibility

Fig. 7 illustrates the evolution of rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS) fractions in frozen rice dough. In the control group, which lacked any improvers, 70 days of frozen storage resulted in a 17.86 % increase in RDS, a 29.27 % increase in SDS, and a dramatic 98.69 % decrease in RS. This result indicates that the formation of ice crystals and their recrystallization during freezing disrupted the starch–gluten composite network, leading to altered starch digestibility profiles. On the one hand, the mechanical action of ice crystals promoted the formation of new hydrogen bonds between starch molecules, thereby increasing the accessibility of digestive enzymes to starch. On the other hand, the physical entanglement between gluten proteins and starch molecules induced by freezing hindered the orderly reorganization of starch chains, leading to the disruption of crystalline structures and altered starch digestibility (Qadir and Wani, 2022; Zhang et al., 2021; Zeng et al., 2022). The experimental groups supplemented with CMCNa or CMCh demonstrated notable inhibition of starch digestibility deterioration (p < 0.05). Following 70 days of storage, the 1.5 % CMCh-B treatment resulted in a 12.11 % increase in RDS, 22.62 % increase in SDS, and 71.51 % reduction in RS compared with the control group. A clear hierarchy of protective efficacy was observed as follows: 1.5 % CMCh-B > 1 % CMCh-B > CMCNa > CMCh-A. These results indicate that the abundant hydroxyl groups in CMCNa and CMCh molecules competitively form hydrogen bonds with ice crystals, establishing a dual protective mechanism that reduces physical damage to starch granules and preserves the ordered molecular structure of starch. This mechanism effectively delays changes in starch digestibility. The findings demonstrate that high–degree-of-substitution by CMCh-B effectively regulates starch digestive stability, establishing a quantitative structure–activity relationship between substitution degree and protective efficacy while preserving the native digestive characteristics of starch in frozen rice dough. However, the potential effects of temperature fluctuations were not examined in this study, a limitation that warrants further investigation.

Fig. 7.

Fig. 7

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In vitro digestibility of frozen rice doughs were measured during frozen storage, following supplementation with sodium carboxymethyl cellulose (CMCNa), carboxymethyl chitosan-A (CMCh-A), and carboxymethyl chitosan-B (CMCh-B). (A) RDS, (B) SDS, and (C) RS (Image 49, Control; Image 50, 1 % CMCNa; Image 51, 0.5 % CMCh-A; Image 52, 1 % CMCh-A; Image 53, 1.5 % CMCh-A; Image 54, 0.5 % CMCh-B; Image 55, 1 % CMCh-B; Image 56, 1.5 % CMCh-B).

4. Conclusion

This study confirmed the reliability of the experimental data by analyzing the effects of adding CMCh and CMCNa on the prolamin structure of rice dough during frozen storage. The results demonstrate that extended frozen storage for 70 days significantly damaged the gliadin structure within the frozen rice dough. Notably, compared to the non-additive group (control), the addition of CMCNa and CMCh, particularly 1.5 % CMCh-B, effectively mitigated this structural deterioration. Specifically, 1.5 % CMCh-B exhibited the optimal protective effect after extended frozen storage: it significantly inhibited the conversion of bound water to weakly bound and free water, enhanced intermolecular cross-linking among prolamins, alleviated the decline in the relative content of γ-gliadin, and consequently better preserved the integrity of the gluten protein network. The key mechanism underlying this protection lies in the dense hydroxyl groups on the CMCh molecular chains, which form a three-dimensional hydrogen bond network with water molecules, constructing a stable protective hydration layer around the protein molecules. This layer helps maintain their native conformation and effectively suppresses gliadin aggregation and denaturation. Additionally, the hydroxyl groups of CMCh anchor ice crystals via hydrogen bonding, restricting their growth and mitigating mechanical damage to starch granules. Consequently, adding 1.5 % CMCh-B significantly improved the textural properties (25.90 % decrease in hardness, 38.24 % decrease in springiness) and starch digestibility characteristics of the rice dough (SP increased by only 18.16 %, SOL increased by only 10.57 %, RDS increased by 12.11 %, SDS increased by 22.62 %, and RS decreased by 71.51 %). At the protein secondary structure level, 1.5 % CMCh-B effectively inhibited the frozen storage-induced abnormal increase in α-helix content (40.42 %) and the substantial decrease in β-sheet content (45.70 %), while stabilizing the random coil structure. This ultimately manifested as a 13.12 % increase in gliadin surface tension and a significant 75.17 % decrease in surface hydrophobicity. In summary, this study clearly elucidates the positive role of adding 1.5 % CMCh-B in alleviating the structural deterioration of gliadin (including secondary structure, network structure, hydration status, and functional properties) in frozen rice dough during storage, along with its molecular mechanism.

However, this study also has certain limitations: (1) The observation period was limited to 70 days, which is insufficient to verify the sustained protective effect of 1.5 % CMCh-B over longer frozen storage durations. The maximum acceptable refrigerated shelf life of frozen rice dough with CMCh addition and the critical point of quality deterioration remain undetermined; (2) The impact of temperature fluctuations on protein structure was not systematically evaluated, as all experiments were conducted under constant temperature conditions (−18 °C); (3) The hydrogen bond competition efficiency of CMCh and the influence mechanism of ice crystal morphology on adsorption capacity were insufficiently explored; (4) Potential interference from endogenous lipids/enzymes was not investigated, as the study focused on the ternary system of starch-gluten protein-additive; (5) The production cost of high-degree substitution (DS) CMCh-B is significantly higher than that of CMCNa and low-DS CMCh-A, necessitating further validation of its industrial economic feasibility. Future research should extend storage periods to develop shelf-life prediction models, conduct temperature stress tests, elucidate multi-component interaction mechanisms, and reduce CMCh-B production costs through waste resource utilization.

CRediT authorship contribution statement

Wangming Dong: Writing – original draft, Validation, Software, Methodology, Formal analysis, Data curation, Conceptualization. Yan Wu: Supervision, Project administration. Ge Zhang: Software, Formal analysis, Conceptualization. Qi Wei: Writing – review & editing, Writing – original draft, Supervision, Project administration, Funding acquisition.

Funding

This work was supported by the Open Fund of Key Laboratory of Biodiversity Conservation and Characteristic Resource Utilization in Southwest Anhui (Wxn202408). All authors have read and agreed to the published version of the manuscript.

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.

Acknowledgments

We want to thank the anonymous reviewers for the helpful comments and suggestions.

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.fochx.2025.102956.

Contributor Information

Wangming Dong, Email: dwm18336905532@163.com.

Ge Zhang, Email: zhangge@cn.wilmar-intl.com.

Qi Wei, Email: 122408@aqnu.edu.cn.

Appendix A. Supplementary data

Supplementary material

mmc1.docx (331.8KB, docx)

Data availability

Data will be made available on request.

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Associated Data

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Supplementary Materials

Supplementary material

mmc1.docx (331.8KB, docx)

Data Availability Statement

Data will be made available on request.

Articles from Food Chemistry: X are provided here courtesy of Elsevier

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