Quaternized chitosan-based hollow salts as a novel curing strategy for dry-cured meat: enhancing NaCl diffusion, protein hydrolysis, and sensory quality in air-dried duck

Abstract

Traditional dry-cured meats typically contain excessive amounts of NaCl. This study introduces a novel technique using quaternized chitosan-based hollow salt to cure duck for air-dried production, aiming to reduce NaCl usage while improving product quality. Compared with traditional NaCl curing, substitution with hollow salt significantly enhanced Na⁺ diffusion in muscle and accelerated its release during mastication, thereby reducing overall NaCl usage and enhancing early saltiness perception. A moderate substitution level (particularly 60%) optimally activated endogenous muscle protease activity, promoted myofibrillar protein degradation, and increased the accumulation of free amino acids. A total of 62 volatile flavour compounds were identified in air-dried duck, with results indicating that hollow salt substitution slightly diminished the aromatic intensity. However, sensory attributes such as colour, texture, taste, and overall acceptability were significantly improved. This study provides a promising strategy for developing healthier and more palatable dry-cured meat products.

Highlights

• •The developed hollow salt reduces the usage of NaCl in air-dried duck while keeping the saltiness.
• •Moderate substitution activated endogenous proteases, promoted myofibrillar degradation, and increased free amino acid content.
• •Hollow salt substitution in curing enhances the overall sensory quality of air-dried duck.

1. Introduction

Sodium chloride (NaCl), often referred to as the “king of flavouring agents,” has played a fundamental role in food processing and sensory enhancement for thousands of years (Jia et al., 2024). In traditional dry-cured meats (e.g., ham, bacon, and sausages), NaCl lowers water activity, suppresses spoilage microorganisms, modulates proteolysis and lipolysis, and supports flavour and colour development (Wang et al., 2023). However, despite its essential role, global sodium intake consistently exceeds the World Health Organisation's recommended limit of 5 g per person per day (Wang et al., 2023). Notably, several traditional products (e.g., Harbin sausages, Jinhua ham, Iberian ham, and salami) often contain more than 10% NaCl (w/w). Increasing clinical and epidemiological evidence indicates a strong positive association between high sodium intake and elevated risks of hypertension, cardiovascular disease, and stroke (Allison & Fouladkhah, 2018; Leyvraz et al., 2018). This creates a critical nutritional and public health dilemma: NaCl is necessary for ensuring the safety and quality of dry-cured meats, yet its excessive use poses major health risks. Therefore, a key challenge is to reduce NaCl in meat processing without compromising perceived saltiness or product quality.

Current strategies for reducing sodium in meat products include three approaches: salt substitution, physical processing to enhance diffusion, and NaCl crystal modification (Yang et al., 2024). These approaches aim to lower sodium while maintaining quality, but implementation is constrained by sensory, technical, and economic limitations. Sodium substitutes—such as potassium chloride, phosphate salts, and “plant-based salts”—have been widely studied. While effective in reducing sodium content, they often produce off-flavours, metallic bitterness, and discolouration in cured meats (Jia et al., 2024; Wang et al., 2023). Physical processes (e.g., ultrasound, high-pressure processing, and pulsed electric fields) can enhance salt diffusion by disrupting muscle microstructure or increasing tissue permeability (Bhat et al., 2020; Garcia-Perez et al., 2019; Picouet et al., 2012). However, high capital costs and limited scalability hinder their widespread adoption in industrial applications. An emerging sodium reduction strategy focuses on altering the physical properties of NaCl, particularly by reducing salt crystal particle size (Rios-Mera et al., 2021). Smaller, fine salt particles with larger surface area dissolve faster in food matrices. This promotes more uniform diffusion of Na⁺ throughout the food matrix (Rios-Mera et al., 2021). During prolonged air-drying, muscle tissue hardens and salt diffusion slows, which can increase the amount of NaCl needed to achieve adequate penetration in traditional products. Therefore, using fine or micro-sized salt during meat curing may provide an effective strategy to reduce NaCl usage.

Nanoemulsion and hollow salts are two engineered small-scale NaCl systems designed to enhance saltiness while reducing sodium content (He & Tan., 2024). Nanoemulsion salts are produced by dispersing NaCl into oil-in-water (O/W) or water-in-oil (W/O) emulsions using high-energy methods like high-shear homogenization or high-pressure microfluidization. These emulsions contain nanoscale droplets with large interfacial areas, which enables more uniform distribution of Na⁺ in the dispersed phase. Upon ingestion, nanodroplets adhere to the tongue and rapidly release Na⁺ onto taste receptors (Azooz et al., 2025; Lee et al., 2025; Ramezani et al., 2024; Wang et al., 2021). This results in a faster and more intense saltiness perception than conventional crystalline salt. However, because they are liquid systems, nanoemulsion salts are mainly suitable for liquid or semi-solid foods and are difficult to apply to dry-cured or solid products such as meat. Hollow salt is produced by encapsulating NaCl within a biopolymer shell (e.g., chitosan, gum arabic, or carrageenan) using spray drying, steam-assisted drying, or mechanical milling (He & Tan., 2024). Spray drying is attractive because it is scalable, controllable, and suitable for continuous production. In this process, a homogeneous salt–biopolymer solution is atomized into fine droplets. Rapid evaporation drives internal water migration and generates vapour pressure, which expands the droplets and forms a shell. Simultaneously, NaCl and biopolymers concentrate at the droplet surface, forming a salt-rich shell around a hollow core (He & Tan., 2024). The resulting particles are smaller and have a higher surface-area-to-mass ratio, which improves dissolution and sensory efficiency. Recent studies have begun to explore the functional versatility of hollow salts. For example, Ma et al. developed capsaicin-loaded hollow salt microparticles via nanoemulsion-assisted spray drying, achieving antioxidant activity and enhanced saltiness (Ma et al., 2025). Despite these advances, it remains unclear whether hollow salt can penetrate meat tissue during dry curing and thereby improve Na⁺ delivery, perceived saltiness, and overall quality.

Therefore, we replaced NaCl with previously prepared quaternized chitosan–based hollow salt at different ratios during air-dried duck curing and evaluated NaCl penetration and diffusion in duck muscle. Sensory evaluation, electronic nose, and electronic tongue analyses were conducted to assess the impact of different curing conditions on the sensory quality of air-dried duck. Additionally, to clarify the impact of hollow salt curing on the edible quality of air-dried duck meat, multiple key biochemical indicators were monitored, including protein hydrolysis degree, protein oxidation degree, endogenous proteases activity, free amino acid (FAA) profile, and volatile flavour compound profile. This study provides theoretical guidance for the processing of low-salt dry-cured meat products.

2. Materials and methods

2.1. Materials

Quaternized chitosan (Qac), succinic acid (SA), KBr, 5,5-Dithiobis(2-nitrobenzoic acid) (DTNB), Tris(hydroxymethyl)aminomethane-Glycine (Tris-Gly), NaOH, HClO₄, methanol, phosphoric acid, ethanol, ethyl acetate, hydrochloric acid guanidine, Ethylene diamine tetraacetic acid (EDTA), trichloroacetic acid (TCA), and NaCl were purchased from Shanghai Macklin Co., Ltd. (Shanghai, China). Slaughtered ducks (4 Kg ±0.2 Kg) were purchased from Ningbo Langde Food Co., Ltd. (Zhejiang, China). Qac exhibited an average degree of substitution (DS) of 98%, a molecular weight of 150,000 Da, and a deacetylation degree (DD) of 35%.

2.2. Preparation of hollow salt

Hollow salt powder was prepared with slight modifications to a previously reported method (Shi et al., 2025). Briefly, 1.0 g of Qac, 11.2 g of SA, and 15.0 g of NaCl were fully dissolved in 72.8 mL of deionised water under magnetic stirring for 30 min to ensure complete dissolution of all components. The pH of the resulting solution was then adjusted to 7.0 using NaOH. Subsequently, the solution was subjected to ultrasonic treatment at 500 W, 40 kHz, and 60 °C for 30 min. Upon completion of sonication, the solution was cooled to room temperature and immediately processed using a spray dryer (Shanghai Yacheng Instrument Co., Ltd., Shanghai, China). Spray drying was carried out at an inlet temperature of 140 °C and an outlet temperature of 75 °C, with a peristaltic pump flow rate set at 10 rpm. The resulting hollow salt powder was collected and stored at 25 °C and 30% relative humidity in a controlled desiccation environment for subsequent experimental use.

2.3. Preparation of hollow salt-cured air-dried duck

Post-slaughtered ducks were immersed in water for 3 h to facilitate the removal of residual blood, after which they were drained to eliminate surface moisture. Subsequently, 6% (w/w) NaCl was evenly applied to both sides of each duck, followed by a 24 h curing period. This group was named the control group. To evaluate the effect of hollow salt substitution, five experimental groups were prepared by replacing NaCl with hollow salt at substitution ratios of 20%, 40%, 60%, 80%, and 100%, respectively. These groups were named as groups A, B, C, D, and E. Following the curing process, tap to remove any undissolved salt powder, all duck samples were subjected to air-drying in a controlled chamber (Hangzhou Ouyi Electric Co., Hangzhou, Zhejiang) maintained at 16 °C and 60% relative humidity for 7 days (Zhao et al., 2025). Upon completion of drying, the duck breast meat was excised from each sample for subsequent physicochemical and sensory analyses.

2.4. Evaluation of the effect of different curing methods on the NaCl content in air-dried duck

Air-dried duck breast meat was cut into 30 × 30 × 20 mm pieces. The pieces were then cut into rectangular blocks along the direction of NaCl penetration, with a length of 2 cm, a width of 2 cm, and thicknesses of 2 mm, 4 mm, 6 mm, 4 mm, 2 mm, and 2 mm. Each sample was placed into a crucible and subsequently incinerated in a muffle furnace at 500 °C for 3 h until complete ashing was achieved. The resulting ash was dissolved in 10 mL of deionised water and filtered through a 0.22 μm membrane. The NaCl content in the filtrate was then quantified at 589 nm using a ZEEnit 700P atomic absorption spectrometer (Analytik Jena GmbH Co., Ltd., Jena, Germany). All measurements were performed in triplicate on independently prepared samples. Meanwhile, take another 10 g of air-dried duck meat, treat it with ash, and determine the total NaCl content in the sample using the same method.

2.5. Scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy (SEM-EDS)

The microstructural characteristics and Na element distribution of air-dried duck samples from different treatment groups were examined using SEM-EDS. Cubic meat specimens measuring 5 mm × 5 mm × 5 mm were excised and fixed in a 3% glutaraldehyde solution for 48 h. Following fixation, samples were rinsed three times with 0.1 mol/L phosphate buffered saline (PBS) and then dehydrated through a graded ethanol series of 30%, 50%, 70%, 80%, 90%, and 100%. Dehydrated samples were subsequently subjected to vacuum freeze-drying for 48 h. Gold sputter coating was then applied to the surface of each sample to enhance conductivity. Surface morphology was imaged using a field-emission scanning electron microscope (Zeiss Sigma 300, Germany), and elemental mapping of C, N, O and Na was performed using the integrated Ametek EDAX TEAM analysis system.

2.6. Determination of NaCl release rate during the chewing process of air-dried duck

According to the method described by Konitzer et al. (2013), 10 sensory evaluators were selected. Each evaluator chewed 2 g of air-dried duck meat in their mouths for 15, 30, 45, and 60 s, respectively. Subsequently, at each time point, the food bolus was expelled into a 50 mL centrifuge tube. The assessor then ingested 10 mL of ddH₂O to rinse their mouth for 30 s, ensuring all residual salt dissolved in the water. This rinse water was also collected into the centrifuge tube. The tubes were then centrifuged at 4000 rpm and 4 °C for 15 min, and the NaCl content in the supernatant was determined using atomic flame absorption spectroscopy. The NaCl naturally present in the saliva were deducted during the calculation process.

2.7. Determination of endogenous protease activity in air-dried duck

The enzymatic activities of cathepsin B, cathepsin L, and calpain in air-dried duck were determined using Z-Arg-Arg-AMC, Z-Phe-Arg-AMC, and Suc-LLVY-AMC as respective substrates (Zhang et al., 2025). Briefly, 1 g of duck meat was homogenised with 5 mL of 50 mmol/L sodium citrate buffer (pH 5.0), containing 0.2% Triton X-100 and 1 mmol/L EDTA, at 10000 rpm for 30 s at 4 °C. The homogenate was then centrifuged at 12000 rpm for 20 min under the same temperature conditions, and the resulting supernatant was collected as the crude enzyme extract. Protein concentrations in the supernatant were quantified using a bicinchoninic acid (BCA) protein assay kit. Subsequently, the activities of cathepsin B, cathepsin L, and calpain were assayed according to the method described by Zhou, Wu, et al. (2024).

2.8. Determination of the degree of protein hydrolysis in air-dried duck

The degree of proteolysis in air-dried duck samples from various treatment groups was evaluated following the protocol described by Zhou, Wu, et al. (2024). Briefly, 1 g of duck muscle was homogenised with 10 mL of deionised water at 10000 rpm for 30 s. The total protein concentration in the homogenate was measured using a BCA protein assay kit. Subsequently, 300 μL of the homogenate was mixed with 1200 μL of 12.5% TCA and incubated at 4 °C for 15 min to precipitate the proteins. The mixture was centrifuged at 12000 rpm for 10 min, and the supernatant was collected. The pH of the supernatant was adjusted to 9.0 using NaOH. Next, 1 mL of the pH-adjusted supernatant was mixed with 360 μL of fluorescamine solution (0.6 mg/mL) and incubated in the dark at 25 °C for 1 h. Fluorescence intensity was measured using a microplate reader with an excitation wavelength of 375 nm and an emission wavelength of 475 nm. To quantify proteolytic activity across groups, a glycine standard curve was constructed under identical conditions using glycine solutions ranging from 5 to 50 mmol/L in place of the sample supernatant. The resulting calibration equation was used to convert the fluorescence intensities of duck samples into glycine-equivalent concentrations, enabling estimation of protein hydrolysis. Additionally, myofibrillar proteins were extracted and their degradation profiles were analysed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE).

2.9. Determination of FAA content in air-dried duck

The determination of FAA was carried out with slight modifications based on the method described by Li et al. (2025). Briefly, 1 g of duck meat was homogenised in 25 mL of 5% TCA solution at 10000 rpm for 30 s. The homogenate was then centrifuged at 10000 rpm for 10 min, and the resulting supernatant was collected. After filtration through a 0.22 μm membrane filter, the filtrate was subjected to quantification using a fully automated amino acid analyser.

2.10. Determination of the degree of protein oxidation in air-dried duck

2.10.1. Determination of carbonyl content

The method for extracting myofibrillar protein and the determination of carbonyl content were consistent with the approach described by Cheng et al. (2024). Briefly, 1 mL of 2 mol/L HCl containing 0.1% (w/v) 2,4-dinitrophenylhydrazine (DNPH) was mixed with 1 mL of myofibrillar protein solution (5 mg/mL) in a 10 mL centrifuge tube. To exclude interference from DNPH, 1 mL of a 2 mol/L HCl solution free of DNPH was mixed with myofibrillar protein solution (5 mg/mL) as a control. All samples were incubated in the dark for 1 h, with vortexing performed every 10 min to ensure thorough mixing. Following the reaction, 1 mL of 20% (w/v) TCA was added to each tube to precipitate the proteins, followed by incubation for 20 min. The mixtures were then centrifuged at 8000 rpm for 15 min, and the supernatants were discarded. The resulting precipitates were washed three times with an equal-volume mixture of ethanol and ethyl acetate to remove unreacted DNPH and other impurities. Finally, the pellets were redissolved in 3 mL of 20 mmol/L PBS containing 6 mol/L guanidine hydrochloride. The absorbance of the resulting solution was measured at 370 nm using a spectrophotometer. The carbonyl content (nmol/mg protein) was calculated using the following equation:

$$\text{Carbonyl content}=\frac{3\times \mathrm{A}\times {10}^6}{\mathrm{C}\times \mathrm{V}\times 22000}$$ (1)

where A represents the absorbance at 370 nm, C is the protein concentration (mg/mL), V is the volume of the protein solution (mL), and 22,000 M⁻¹·cm⁻¹ is the molar extinction coefficient of the protein-hydrazone complex.

2.10.2. Determination of total mercaptan content

The total sulfhydryl content was determined according to the method reported by Cheng et al. (2024). Briefly, 1 mL of myofibrillar protein solution (5 mg/mL) was mixed with 9 mL of 50 mmol/L PBS and used as the control. For the experimental group, 1 mL of the same protein solution was thoroughly mixed with 9 mL of a denaturing buffer containing 8 mol/L urea, 4 mmol/L EDTA, 0.09 mol/L glycine, and 0.086 mol/L Tris–gly. The mixtures were then centrifuged at 7000 rpm for 10 min, and the resulting supernatants were collected. Subsequently, 0.5 mL of Ellman's reagent—comprising 4 mg/mL of DTNB—was added to 4 mL of each supernatant. After vortex mixing, the solutions were incubated at 25 °C for 30 min. The absorbance was measured at 412 nm, and the total sulfhydryl content was calculated using a molar extinction coefficient of 13,600 M⁻¹·cm⁻¹.

2.11. Determination of the flavour profile of air-dried duck

Headspace solid-phase microextraction (HS-SPME) was employed for the extraction of volatile compounds from air-dried duck, followed by identification using gas chromatography–triple quadrupole mass spectrometry (GC–MS; Shimadzu, Japan). Specifically, 5 g of duck meat was placed in a 20 mL extraction vial, and 10 ppm of o-dichlorobenzene was added as an internal standard. The SPME fibre must be inserted into the GC–MS inlet for 15 min prior to use to remove residual flavour compounds from previous experiments. A preconditioned SPME fibre was inserted into the vial, which was then transferred to a metal bath and heated at 55 °C for 50 min to allow the volatiles to equilibrate and be adsorbed onto the fibre. Upon completion of the extraction, the fibre was immediately inserted into the GC–MS injection port and desorbed at 230 °C for 5 min. The GC was programmed as follows: initial temperature of 40 °C held for 5 min; ramped to 120 °C at 8 °C/min and held for 2 min; then increased to 200 °C at 5 °C/min with a 2-min hold; and finally ramped to 230 °C at 10 °C/min and held for an additional 5 min. Helium was used as the carrier gas at a constant flow rate of 1.0 mL/min under splitless injection conditions. Mass spectrometric detection was performed in electron ionisation (EI) mode at 70 eV, with the ion source temperature set to 230 °C. Full-scan acquisition was conducted over the mass-to-charge ratio (m/z) range of 33–650, and the emission current was maintained at 100 μA. Data processing was conducted using MassHunter B.07.00 software (Agilent Technologies, USA).

2.12. Sensory quality of air-dried duck analysed by human-machine interaction evaluation

A total of twenty professionally trained panellists (ten male and ten female, aged between 20 and 28 years) were recruited to assess the gustatory attributes of air-dried duck samples across different treatment groups. All participants were fully informed of the sensory experiment's content and potential risks prior to the experiment and voluntarily agreed to evaluate the air-dried duck. The duck meat was cut into uniform cubes measuring 2 cm × 2 cm × 1 cm and placed individually in clean glass dishes. Prior to tasting, each assessor was instructed to rinse their mouth thoroughly with purified water to eliminate residual flavours. Subsequently, the judges were asked to focus on detecting differences in the colour, texture, taste, flavour, and acceptability of the products. A 10-point structured quantitative scale was used for scoring. After each assessment, the tongue was cleaned with standard mouthwash before moving on to the next sample to minimise taste interference between samples. Each judge conducted the assessment independently under controlled conditions to avoid mutual influence or communication.

Based on the method developed by Zhu et al. (2022), the taste of air-dried duck from different groups was measured using an SA402B electronic tongue (E-tongue) (Insent Inc., Atsugi-shi, Japan). The samples (40 g) were mixed with 200 mL of pure water and homogenised, then centrifuged at 5000 rpm for 10 min. The supernatant was collected and analysed using the electronic tongue to assess sourness, umami, saltiness, and richness. Before each test, the sensor was rinsed with ddH₂O for 60 s, then immersed in the sample solution for 30 s to equilibrate, followed by a 30 test.

The PEN3 electronic nose system was used to analyse the flavour characteristics of different groups of air-dried duck. The PEN3 electronic nose system is equipped with ten sensitive gas sensors, included: W1C (aromatic components, benzene), W3C (aromatic components, ammonia), W5C (short-chain alkane aromatic components), W1S (methyl components), W2S (alcohols, aldehydes, ketones), W3S (long-chain alkanes), W5S (nitrogen oxides), W6S (hydrino compounds), W1W (sulfides), and W2W (aromatic components, organic sulfides). 5 g of duck meat sample was placed in a 20 mL sealed extraction bottle, sealed and equilibrated at 50 °C for 1 h, then the injection needle was inserted into the extraction bottle. Analysis was performed at a carrier gas flow rate of 400 mL/min and an injection flow rate of 200 mL/min for 60 s.

2.13. Statistical analysis

All experiments were conducted independently in triplicate, and the results are presented as mean ± standard deviation. Statistical analysis was performed using one-way analysis of variance (ANOVA) and Duncan in SPSS version 25.0, and differences were considered statistically significant at p < 0.05. Graphical representations of the data were generated using Origin 2024.

3. Results and discussion

3.1. Hollow salt curing promotes the penetration of NaCl into the air-dried duck

Fig. 1A shows the effects of various curing methods on NaCl content in air-dried duck. Conventional curing resulted in 4.47 ± 1.41 g/kg NaCl. A low hollow-salt substitution (Group A) showed no significant difference (5.91 ± 0.54 g/kg). With increasing substitution ratios, NaCl content rose to 8.11, 9.41, 12.05, and 15.21 g/kg. As reported previously, 1 kg of hollow salt contained 306.87 ± 2.36 g NaCl (Shi et al., 2025). Under conventional curing, NaCl diffusion efficiency was only 4.47%. In contrast, hollow salt greatly enhanced NaCl penetration, peaking at 49.56% with higher substitution ratios. These results suggest that smaller hollow-salt particles facilitate NaCl diffusion and may reduce the total salt needed to achieve comparable curing. Notably, a 29.38% hollow salt substitution was sufficient to replicate the curing effect of traditional NaCl.

Fig. 1.

Diffusion of NaCl during the substitution of hollow salt for curing and chewing in air-dried duck. A: Total NaCl content in air-dried duck of different groups. B: NaCl content in air-dried duck meat of varying thicknesses. C: NaCl release during oral chewing of air-dried duck meat from different groups. Significant differences (p < 0.05) between groups are indicated by a, b, c, d, and e.

To further assess NaCl diffusion, samples of varying thickness were directly analysed. Fig. 1B depicts NaCl concentration–distance relationships under different curing treatments. All groups showed a V-shaped NaCl profile, indicating non-linear diffusion within muscle tissue. Compared with the control group (Con, 100% NaCl), samples cured with hollow salt consistently exhibited higher NaCl concentrations at corresponding distances. This suggests that hollow salt significantly enhances NaCl penetration into muscle tissue. The enhanced diffusion likely reflects hollow salt's structural features—smaller particle size, greater specific surface area, and faster dissolution—which together accelerate NaCl release early in curing (He & Tan, 2024). Additionally, hollow salt may alter local ionic strength or modify muscle microstructure, further changing NaCl penetration and distribution (Astruc et al., 2018).

• •SEM-EDS was used to visualise tissue structure and Na distribution under different curing conditions (Zhou, Liu, et al., 2024; Zhou, Wu, et al., 2024). Fig. 2 shows that moisture loss during air drying caused noticeable tissue shrinkage. The control showed marked contraction, consistent with the curing of NaCl. In contrast, the hollow salt substitution groups showed less shrinkage, likely reflecting differences in ionic strength during salt penetration (Zhou et al., 2020). Elevated ionic strength promotes protein denaturation and myofibrillar contraction, which in turn mitigates tissue collapse due to water loss. Moreover, NaCl can regulate endogenous enzyme activity in meat, promoting proteolysis and disrupting muscle structure. EDS spectra showed a clear Na gradient, increasing from 1.43% in the control (Con) to 4.35% in group E; intermediate values were 1.68% (A), 2.26% (B), 2.77% (C), and 3.40% (D). This confirms enhanced Na penetration consistent with Fig. 1B diffusion profiles. Nevertheless, excessive hollow salt substitution may lead to incomplete surface dissolution and overly high NaCl concentrations, potentially impairing the sensory quality of the final product. Therefore, optimising the substitution ratio is crucial to balance NaCl diffusion efficiency and overall product quality.

Fig. 2.

Muscle tissue status and microscopic distribution of NaCl within the muscle tissue of six groups of air-dried duck. Con, A, B, C, D, and E represent air-dried duck produced through different curing treatments, respectively.

3.2. Analysis of Na⁺ release characteristics during chewing of air-dried duck meat from different groups

The oral cavity is the main site for salt perception, where rapid Na⁺ release from food quickly activates salt receptors, reducing the time to reach the taste threshold and potentially lowering sodium intake (Yang et al., 2024). The curing method markedly affected Na release kinetics during mastication (Fig. 1C). Although all groups showed a gradual increase in NaCl content, their release rates differed substantially. Group E released the most sodium (2.76 g/kg at 60 s) compared with the control (1.93 g/kg). Notably, clear differences in NaCl release rates emerged during the initial mastication phase (0–30 s). At 30 s, groups D and E had released 1.52 and 1.59 g/kg of NaCl, respectively, compared to only 0.96 g/kg in the control group. These results indicate that hollow salt curing enhances early-stage Na bioavailability. Accelerated sodium release may promote quicker activation of salt receptors, enhance saltiness perception at lower concentrations, and support Na reduction strategies (Ye et al., 2023).

3.3. Hollow salt replacement in curing promotes protein degradation in air-dried duck meat

Cathepsin B, cathepsin L, and calpains are major endogenous muscle proteases that drive myofibrillar degradation and influence texture in processed meat. Their activities are affected by temperature, ionic strength, and moisture. Figs. 3A–3C show their activity profiles under different curing conditions in air-dried duck. Significant differences (p < 0.05) in enzyme activity were observed among groups for all three proteases. Enzyme activity showed a non-linear response to salt concentration, increasing at moderate NaCl levels but decreasing at higher levels. Notably, Group C (moderate salt) showed the highest activities: cathepsin B, 12.66 U/g; cathepsin L, 32.15 U/g; and calpain, 36.73 U/g. These values were significantly higher than in other groups, indicating that a moderate salt level optimally activates proteolysis. The simultaneous peak activities of cathepsin B, L, and calpain in Group C suggest a potential synergistic effect in the muscle proteolytic system. Cathepsin B functions mainly in early proteolysis, while cathepsin L is active in later degradation stages. In contrast, calpains are critical for degrading cytoskeletal proteins. Their coordinated activation may facilitate muscle fibre disintegration and enhance tenderization. Groups B and D showed slightly lower activities than Group C but remained above the control. In Group B, activities were 12.66 (cathepsin B), 21.34 (cathepsin L), and 32.36 U/g protein (calpain). In Group D, the corresponding values were 9.99, 14.08, and 31.70 U/g protein; both groups remained significantly higher than the control (Con). Enzyme activities in Groups A and E were moderate, suggesting a limited ability to stimulate proteolysis under either low or excessively high salt conditions. Group E, which had the highest NaCl level, showed reduced activity: cathepsin B and cathepsin L decreased to 6.39 and 10.57 U/g protein, respectively, and were both lower than in Group C (p < 0.05). The reduced activity observed in both low-salt (Group A) and high-salt (Group E) conditions highlights the biphasic regulatory role of NaCl on endogenous proteolytic activity. Low-salt environments may stabilize lysosomal membranes, inhibit Ca²⁺-dependent activation, and reduce enzyme flexibility, thereby limiting protease expression and activity. Conversely, moderate NaCl concentrations may enhance lysosomal membrane permeability, promoting the cytosolic release of proteases like cathepsins B and L (Zhang et al., 2024). Under these conditions, proteases may adopt more catalytically favourable conformations, with improved active-site exposure and substrate accessibility (Zhang et al., 2020). At higher NaCl concentrations, both cathepsin B and L showed significant activity loss, possibly due to hyperosmotic stress-induced membrane disruption and ionic-strength-related protein destabilisation. High salinity may promote enzyme denaturation and may also suppress expression or hinder cytosolic release, thereby limiting post-mortem proteolysis. Excessive NaCl levels have also been associated with increased oxidative stress in muscle tissue, which may indirectly impair enzyme stability (Gheisari et al., 2010). Collectively, these results suggest that moderate salt levels enhance protease activity and myofibrillar degradation, which may improve sensory quality in cured meat.

Fig. 3.

Effects of hollow salt substitution curing at varying ratios on myofibrillar protein hydrolysis and oxidation in air-dried duck. A, B, and C: Effects of hollow salt substitution curing on cathepsin B, cathepsin L and calpain activity in air-dried duck. D: Protein electrophoresis analysis of myofibrillar protein degradation degree following hollow salt substitution curing. E: Proteolytic indices of air-dried duck in different groups. F: Results of carbonyl group content in myofibrillar proteins of air-dried duck in different groups. G: Results of sulfhydryl group content in myofibrillar proteins of air-dried duck in different groups. Significant differences (p < 0.05) are represented by a, b, c, d and e.

To determine whether enzymatic variations led to measurable proteolysis, SDS-PAGE and protein hydrolysis assays were conducted. As shown in Fig. 3D, myofibrillar protein degradation varied significantly among the treatment groups. All hollow salt-treated groups exhibited greater myofibrillar protein degradation than the control group. Group C showed the most extensive protein degradation, with marked breakdown of MHC, actin, tropomyosin (Tm), troponin-T (TnT), MLC, and troponin-C (TnC), consistent with its high protease activity. Groups A and B also showed marked MHC degradation, suggesting that moderately low salt levels can still support proteolysis. These findings align with Li, who reported that extensive MHC and actin hydrolysis accelerates the release of FAA and peptides, contributing to flavour development in dry-cured meats (Li et al., 2025).

The proteolysis index (P.I.) is a key indicator of the extent to which endogenous proteases degrade myofibrillar proteins in post-mortem muscle. An elevated P.I. indicates a higher concentration of non-protein nitrogen (NPN), including FAA and small peptides, which are key contributors to flavour development (Abellán et al., 2018). As shown in Fig. 3E, P.I. values varied significantly among air-dried duck samples processed with different curing methods. Group C showed the highest P.I. (7.8%), indicating significantly enhanced proteolytic activity. This pattern likely reflects optimal activation of endogenous proteases at moderate salt levels. As previously discussed, Group C showed peak activity of all three major endogenous proteases, suggesting effective enzymatic stimulation that accelerated muscle protein degradation. Groups A, B, and D showed moderately higher PI values (6.77%, 6.71%, and 6.57%, respectively), possibly reflecting partial protease activation. In contrast, Group E, exposed to high NaCl concentrations, showed a lower P.I. (5.7%) than the control group. This decline is consistent with reduced protease activity under high-salt conditions and suggests that excess salt may inhibit proteolysis by lowering enzyme activity, increasing osmotic pressure, or stabilizing protein structures. Overall, salt concentration showed a biphasic effect on protein hydrolysis: moderate levels promoted degradation, whereas both low and high levels inhibited it (Schivazappa & Virgili, 2020). This pattern highlights the importance of optimising salt levels to enhance proteolysis during dry-cured meat processing.

3.4. Hollow salt curing can moderately delay protein oxidation

Protein carbonylation is an irreversible, non-enzymatic modification and a widely used marker of protein oxidation. As shown in Fig. 3F, the Con group exhibited the highest carbonyl content (8.76 μmol/g), followed by groups B (8.13 μmol/g) and A (7.61 μmol/g). Conversely, groups C, D, and E showed lower carbonyl levels of 6.93, 7.32, and 6.72 μmol/g, respectively. Together, these results suggest that moderate hollow-salt substitution attenuates protein oxidation. Sulfhydryl group loss is also a well-established indicator of protein oxidation in meat products. As oxidation progresses, sulfhydryl content decreases due to intra- and intermolecular disulfide bond formation. Fig. 3G shows that the Con group had relatively low sulfhydryl content (6.59 μmol/g). However, sulfhydryl concentrations gradually increased with higher hollow salt substitution levels, peaking at 13.65 μmol/g in group E. This trend indicates that hollow salt curing suppresses protein oxidation, with higher substitution ratios enhancing antioxidant efficacy. These effects may be attributed to the intrinsic properties of the hollow salt formulation. Protein oxidation in muscle can be catalysed by transition metals (e.g., Fe³⁺ and Cu²⁺) and can also be initiated by lipid peroxidation (Wen et al., 2019). In this study, the hollow salt primarily comprised quaternized chitosan and succinic acid. Amino and hydroxyl groups in quaternized chitosan can chelate metal ions to form stable coordination complexes, which may reduce radical formation and overall oxidative stress (López-Maldonado et al., 2024). Succinic acid, a dicarboxylic acid, may enhance metal binding under mildly acidic conditions and thereby further limit metal-catalysed oxidation. Previous research by our group corroborates that hollow salt curing mitigates oxidative degradation in meat products, thereby reinforcing the current findings (Shi et al., 2025).

3.5. Analysis of the effect of different curing methods on the flavour quality of air-dried duck

The flavour composition of six groups of air-dried duck samples was characterised using gas chromatography–mass spectrometry (GC–MS). As shown in Fig. 4A, PC1 and PC2 explained 28.9% and 23.8% of the variance, respectively, and separated the six groups. Notably, Group A was distinctly separated from the other five groups, suggesting a marked divergence in its volatile profile. In contrast, Groups B, C, and the control group (Con) were closely clustered, indicating substantial similarity in their volatile compound compositions. Groups D and E also clustered together, indicating comparable volatile profiles. Fig. 4B presents a detailed profile of the 62 volatile compounds identified across all six sample groups. Key volatiles were identified with VIP >1.0 and p < 0.05 (Fig. 5). We compared the control (Con; NaCl curing) with three hollow-salt groups in which NaCl was replaced at 20% (A), 60% (C), and 100% (E). The results revealed pronounced differences in the profiles of key volatile constituents, primarily comprising aldehydes, alcohols, and alkanes—compounds closely associated with lipid oxidation and fatty acid degradation pathways. As shown in Fig. 5A, comparison between the Con and A groups indicated that a 20% substitution with hollow salt moderately reduced the concentrations of lipid oxidation-derived volatiles such as heptanal, nonanal, 1-octen-3-ol, 1-hexanol, decane, and dodecane. These aldehydes typically arise from oxidative degradation of oleic and linoleic acids and contribute fatty, green, and slightly rancid notes in dry-cured meat (Gao et al., 2024). A similar pattern was observed when the Con group was compared with the C and E groups (Fig. 5B and C). Many of the same discriminant volatiles were detected, but their abundances were lower than in the control, suggesting a gradual reduction in overall flavour intensity with increasing substitution. This phenomenon is most likely attributable to the suppression of lipid oxidative degradation during curing. Specifically, the chitosan component of the hollow salt wall material possesses radical-scavenging activity, which inhibits the propagation of lipid peroxidation chains, while the succinic acid moiety may slightly reduce the pH of the meat matrix, thereby slowing oxidation kinetics (Shi et al., 2025). Comparisons across low, moderate, and high substitution levels (Figs. 5D–F) showed a progressive decline in lipid-oxidation–derived volatiles as the proportion of hollow salt increased. These results confirm that complete substitution of NaCl with hollow salt effectively suppresses lipid oxidation pathways, leading to a simplified volatile profile dominated by low-odour-intensity alkanes. Consequently, the flavour of dry-cured duck tends to become less rich and more subdued. Therefore, a moderate substitution level of approximately 60% appears to achieve the optimal balance between sodium reduction and the preservation of acceptable sensory quality.

Fig. 4.

Analysis of flavour substances in air-dried duck across different groups. A: Principal component analysis of flavour substance composition in air-dried duck from different groups. B: Heatmap analysis of flavour substances in air-dried duck from different groups.

Fig. 5.

VIP score analysis of key volatile compounds differentiating air-dried duck samples under varying levels of hollow salt substitution. The red squares indicate that the flavour compound is high in this group, while green squares mean the opposite. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

3.6. Analysis of FAA composition in air-dried duck under different curing methods

FAA is a crucial contributor to taste development in dry-cured meats. Table 1 shows the variations in FAA profiles among air-dried duck samples subjected to different curing treatments. The total FAA concentration in the control (Con) group was 79.66 mg/100 g. Notably, progressive substitution of NaCl with hollow salt resulted in a substantial increase in FAA concentrations. Group C exhibited the highest FAA concentration (151.31 mg/100 g), followed by groups B (139.64 mg/100 g) and D (130.72 mg/100 g), corresponding to 1.89-, 1.75-, and 1.64-fold increases relative to the Con group. The elevated FAA levels in groups B, C, and D are likely attributable to enhanced endogenous protease activity, stimulated by hollow salt substitution, which in turn accelerated the degradation of myofibrillar proteins, particularly myosin. This interpretation is supported by SDS-PAGE analysis. Moreover, umami amino acids—such as glutamic acid and aspartic acid—were significantly elevated in these groups compared to the Con group. These compounds are known to enhance saltiness perception via taste synergy and are thus considered key flavour enhancers in salt-reduced meat products. Additionally, elevated levels of sweet-tasting (e.g., proline, glycine, tryptophan, phenylalanine) and bitter-tasting amino acids (e.g., histidine, tyrosine, arginine, valine, lysine, isoleucine, leucine) contributed to the enhanced sensory complexity of the air-dried duck (Wang et al., 2025).

Table 1.

Contents of free amino acids in air-dried duck meat from different groups. Note: a, b, c, d, and e represent significant differences in the same amino acid in different groups of air-dried duck.

Free amino acids	Free amino acids content (mg/100 g)
	Con	A	B	C	D	E
Asp	4.44 ± 0.25d	5.25 ± 0.14c	7 ± 0.66a	7.55 ± 0.34a	6.27 ± 0.43b	6 ± 0.18b
Glu	11.88 ± 1.61c	14.61 ± 1.57bc	18.47 ± 0.93a	19.49 ± 0.68a	19.51 ± 1.82a	18.01 ± 3.51ab
Ser	1.05 ± 0.09d	1.35 ± 0.04cd	1.86 ± 0.17ab	2.09 ± 0.11a	1.7 ± 0.24b	1.68 ± 0.31bc
His	2.51 ± 0.46b	4.34 ± 2.02a	4.78 ± 0.24a	5.12 ± 0.27a	4.21 ± 0.38a	4.27 ± 0.35a
Gly	6.15 ± 1.16b	7.46 ± 0.29b	9.29 ± 1.16a	10.41 ± 1.00a	8.99 ± 0.47a	9.03 ± 0.41a
Thr	5.78 ± 0.95d	7.38 ± 0.74c	10.87 ± 0.19a	11.32 ± 0.46a	10.25 ± 0.12ab	9.37 ± 0.96b
Arg	3.97 ± 1.78b	5.34 ± 1.67ab	7.29 ± 5.02ab	9.27 ± 0.79a	5.62 ± 0.7ab	6.18 ± 3.04ab
Ala	13.26 ± 2.44bc	16.22 ± 0.65b	20.61 ± 0.89a	21.47 ± 0.58a	21.72 ± 0.29a	12.99 ± 0.11c
Tyr	1.94 ± 0.75c	2.42 ± 0.67bc	4.26 ± 0.15ab	4.89 ± 0.76a	2.89 ± 0.37bc	2.18 ± 0.21c
Cys	0.08 ± 0.01b	0.11 ± 0.02b	0.18 ± 0.04a	0.18 ± 0.01a	0.18 ± 0.04a	0.18 ± 0.02a
Val	3.77 ± 0.61b	4.81 ± 0.2b	6.87 ± 0.52a	7.47 ± 0.54a	6.59 ± 0.5a	6.47 ± 1.02a
Met	2.05 ± 0.27b	1.9 ± 0.96b	3.73 ± 0.45a	4.03 ± 0.25a	3.54 ± 0.31a	3.35 ± 0.43a
Phe	3.25 ± 0.49d	4.31 ± 0.28c	6.58 ± 0.28ab	7.05 ± 0.56a	6.04 ± 0.7b	5.91 ± 0.56b
Ile	2.59 ± 0.35d	3.39 ± 0.14c	5.06 ± 0.36ab	5.45 ± 0.42a	4.69 ± 0.45b	4.64 ± 0.55b
Leu	5.81 ± 0.89d	7.63 ± 0.39c	11.65 ± 0.64ab	12.6 ± 1.10a	10.35 ± 0.93b	10.24 ± 0.89b
Lys	5.71 ± 1.44c	7.59 ± 1.67bc	12.06 ± 0.49a	13.11 ± 0.58a	9.19 ± 0.81b	9.31 ± 2.01b
Pro	5.4 ± 0.66c	6.86 ± 0.64b	9.08 ± 0.52a	9.75 ± 0.41a	8.95 ± 0.33a	8.31 ± 1.72a
Total	79.66 ± 11.53e	100.97 ± 5.09d	139.64 ± 5.02ab	151.31 ± 5.67a	130.72 ± 6.33bc	118.11 ± 8.44c

3.7. Analysis of the effects of different curing methods on the sensory quality of air-dried duck

Fig. 6A presents the sensory evaluation scores of air-dried duck samples in five attributes: colour, taste, flavour, texture, and overall acceptability. All groups received relatively high scores for colour, with group C showing the highest rating, suggesting that partial substitution of NaCl with hollow salt positively influences the visual appeal of the product. In taste evaluation, group C again obtained the highest score, while groups B, D, and E showed similar ratings, all slightly higher than that of the Con group. These results suggest that moderate replacement of NaCl with hollow salt enhances taste perception. Although flavour scores varied minimally among groups, the Con group exhibited a slight advantage. This finding suggests that traditional NaCl may better preserve the duck's intrinsic flavour profile. A plausible explanation is that quaternized chitosan, used as the wall material in hollow salt, imparts a subtle marine-like odour that may interfere with the desired flavour complexity of air-dried duck. Texture scores showed considerable variation among groups. The Con group received the lowest ratings, while groups with hollow salt substitution exhibited improved textural properties. This improvement may result from the activation of endogenous muscle proteases during curing, which subsequently facilitates protein degradation. Consequently, moderate softening of muscle fibres occurred, potentially improving mouthfeel. Moreover, proteolysis likely facilitated the release of FAA and other taste-active compounds, further enhancing the taste profile. Taken together, these findings indicate that partial substitution of NaCl with hollow salt improves the colour, texture, and palatability of air-dried duck, thereby enhancing overall consumer acceptability.

Fig. 6.

Sensory evaluation (A), electronic tongue (B) and electronic nose (C) results for air-dried duck from different groups.

Due to the potential subjectivity of human sensory evaluation, intelligent sensory technologies such as the electronic tongue and electronic nose were employed to simulate human perception and analyse the taste and flavour of air-dried duck samples from different treatment groups. As shown in Fig. 6B, all six groups exhibited similar taste richness but consistently low sourness, which may be attributed to the presence of succinic acid in the hollow salt that remains on the meat surface after curing. Electronic tongue data confirmed the sensory trends, with the hollow salt groups showing significantly higher intensities of umami and saltiness compared to the control group. This may be due to increased concentrations of umami-related FAA and enhanced Na⁺ diffusion during the curing process. According to the electronic nose results shown in Fig. 6C, no significant differences were observed among the six groups for sensors W1C, W5S, W3C, W6S, W5C, W1S, and W1W, suggesting similar levels of broad compound classes such as methylated compounds and nitrogen oxides. In contrast, the Con group showed higher response intensities than the hollow salt substitution groups for sensors W1C, W3S, W2W, and W2S, indicating greater levels of aromatic compounds, long-chain alkanes, organosulfur compounds, and alcohols, aldehydes, and ketones. These findings are consistent with the GC–MS results.

4. Conclusion

This study demonstrates that partially replacing traditional curing methods with quaternised chitosan-based hollow salt significantly enhances NaCl diffusion efficiency and edible quality in air-dried duck meat. As the substitution ratio increases, NaCl diffuses more rapidly within the meat, with 100% hollow salt replacement achieving the highest Na + content of 15.21 g/kg. Moreover, air-dried duck meat cured with hollow salt substitution exhibited markedly increased NaCl release during chewing, thereby enhancing perceived saltiness. Hollow salt substitution also moderately enhanced endogenous protease activity. At a 60% substitution level, activities of cathepsin B, cathepsin L, and calpain peaked at 12.66, 32.15, and 36.73 U/g, respectively, promoting intensive myofibrillar degradation. Consequently, the FAA content increased significantly (151.31 mg/100 g vs. 79.66 mg/100 g). Oxidative markers, including carbonyl and sulfhydryl content, were significantly reduced in high hollow salt substitution groups, indicating notable antioxidant benefits. Sensory evaluation confirmed significant improvements in texture, taste, and overall acceptability. These findings provide practical validation for using hollow salt as an effective sodium-reduction strategy in dry-cured meats.

CRediT authorship contribution statement

Zihang Shi: Writing – review & editing, Writing – original draft, Methodology, Investigation, Formal analysis, Data curation. Yangyang Hu: Methodology, Investigation. Qiang Xia: Visualization, Resources, Methodology. Changyu Zhou: Validation, Resources, Methodology, Investigation, Data curation. Yangying Sun: Validation, Methodology, Investigation, Formal analysis, Conceptualization. Daodong Pan: Writing – review & editing, Project administration, Funding acquisition, Conceptualization.

Ethics statement

The sensory evaluation experiment was approved by the Ethics Review Committee of Ningbo University. The experiment was conducted in accordance with the personal wishes of the participants. All participants were fully informed of the sensory experiment's content and potential risks prior to the experiment and voluntarily agreed to evaluate the air-dried duck. All subjects' personal information is strictly confidential. The results of this experiment will not disclose any personal information of the subjects.

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.