Discussion

Enhanced antimicrobial activity of the CS/N-TiO₂/NT composite

The preservation of postharvest mushrooms is critically challenged by their susceptibility to microbial spoilage, particularly given the absence of a protective cuticle. The ternary CS/N-TiO₂/NT composite developed in this study addresses this challenge through a multi-tiered antifungal mechanism. The electrostatic interaction between protonated amino groups of CS and the negatively charged fungal cell surfaces constitutes the primary mode of action, disrupting membrane integrity and inducing intracellular leakage (Li & Zhuang, 2020; Luo et al., 2025). However, CS alone exhibited limited efficacy against key spoilage fungi such as A. niger and T. purpureogenus, necessitating enhancement strategies.

Nitrogen doping of TiO₂ extended its photocatalytic activity into the visible-light region (Qian et al., 2011; Salzano et al., 2026). Under typical storage illumination, N-TiO₂ generates reactive oxygen species (ROS) that inflict oxidative damage on fungal cells, synergistically complementing the membrane-compromising action of CS. This synergy explains the significantly enhanced inhibition observed for CS/N-TiO₂ against A. niger, R. azygosporus, and T. purpureogenus relative to CS alone. NT exhibited good inhibitory effects on fungi as a biosourced antifungal substance. It inhibited fungal growth by binding to sterols in the cell membrane (Zhang et al., 2022). Fig. 1(B) showed that CS/N-TiO2, when combined with NT, enhanced the inhibition rates against F. pseudoanthophyllum, T. purpureogenus and R. azygosporus to varying degrees.

Structural reinforcement and barrier enhancement

Nanoparticles typically existed in water as unstable aggregates. Loading N-TiO2 onto the CS film facilitates their uniform dispersion and increases the specific surface area. Due to the electrostatic repulsion between the inorganic nanoparticles and the polymer matrix, the flexible chains of CS molecules were fully extended, improving the dispersion of nanoparticles within the polymer matrix. This provided the composite film with a stable mesh structure and a larger working area (Li et al., 2016). NT tends to form droplets on the fruit peel surface, leading to uneven distribution that adversely affects its antibacterial performance. The combination of NT and CS could leverage the film-forming property of CS to enable a more uniform distribution of NT on the sample surface (Zhang et al., 2022). Furthermore, the cross-section of the CS/N-TiO2/NT composite film exhibited a rougher texture. Similar structures have been observed in CS films loaded with glycerol monolaurate and nano-TiO₂ (Chang et al., 2021).

The physicochemical characterisation of the composite film was conducted to evaluate its potential applications. Film thickness is crucial for food preservation, and also influences its mechanical and barrier properties (Sun et al., 2020). Tensile strength (TS) represents the maximum tensile stress a film can withstand, while elongation at break (EAB) denotes the maximum change in length before fracture (Sun et al., 2020). The CS/N-TiO2/NT composite film exhibited increased thickness and enhanced mechanical properties, particularly in TS. This notable enhancement is consistent with the findings of Kaewklin et al. (2017). The observed phenomenon may be attributed to the reinforcement of the film's network structure resulting from N-TiO2 nanoparticles incorporation. However, Zhu et al. (2019) reported a significant decrease in EAB after the addition of TiO₂ to CS, whereas He et al. (2016) observed an increase. This discrepancy likely arises from differences in TiO₂ content, as an inappropriate loading may induce self-aggregation of the nanoparticles, thereby compromising the mechanical integrity of the composite film (He et al., 2016). Additionally, the incorporation of NT has been shown to reduce EAB (Sun et al., 2020). In the present study, the EAB of the CS/N-TiO₂/NT composite exhibited no statistically significant change relative to the CS film. This result demonstrates that, under the formulation and preparation conditions employed herein, the incorporation of N-TiO₂ and NT did not appreciably compromise film ductility, an outcome that is advantageous for practical food packaging applications. Elucidating the precise factors governing EAB in these composite systems will require further systematic investigation, particularly through concentration-dependent studies of N-TiO₂ loading.

Barrier properties are crucial for preserving food quality and extending product shelf life (Chang et al., 2021). The WVP, OP and CDP levels represent the ability of packaged food to exchange water vapor, oxygen and carbon dioxide with the surrounding atmosphere, respectively. Water and gas molecules are distributed on one side of the film and move through the spaces between polymer chain segments, eventually transferring to the other side via desorption (Zhu et al., 2019). Ideally, WVP should be as low as possible. The hydrophobicity of NT along with the insoluble inclusion compounds formed upon its addition, enhances the moisture resistance of the film. N-TiO2 may influence the microscopic network of the film and provided a tortuous path for water vapor (Karthikeyan et al., 2017). It may also interact with the hydrophilic –OH and –NH groups of CS, thereby reducing water vapor adsorption on the film surface. Similar research results were reported by Chang et al. (2021), who found that WVP was significantly reduced due to the hydrophobic substances glycerol monolaurate and TiO₂. The concentrations of oxygen and carbon dioxide had a significant impact on freshness and quality, and lower permeability to oxygen and carbon dioxide regulates respiration rate and extends shelf life (Zhang et al., 2019). Previous studies have indicated that incorporating hydrophobic substances into CS films typically leads to reductions in both CDP and OP. However, our findings revealed no significant difference in CDP and a marked decrease in OP. The underlying reasons for this divergent behavior demand further investigation, potentially involving the interfacial reorganisation of hydrophobic additives within the CS matrix and anisotropic molecular interactions.

Temperature-dependent preservation mechanisms of the CS/N-TiO₂/NT composite coating

The preservation efficacy of the CS/N-TiO₂/NT coating exhibited a pronounced temperature dependence, with distinct mechanistic pathways dominating at different storage temperatures. This temperature-dependent behavior is consistent with the correlation analysis results (Fig. 6) and storage life predictions (Table 1), which collectively demonstrate optimal preservation at 4 °C, intermediate efficacy at 10 °C, and limited effectiveness at 25 °C.

Variable | HR | Sig. | 95% CId
PPO | 1.45 | <0.001 | 1.32–1.59
L* | 0.62 | <0.001 | 0.55–0.70
T₁₀°C | 2.10 | 0.002 | 1.52–2.90
T₂₅°C | 4.78 | <0.001 | 3.21–7.12
Coating | 0.55 | 0.008 | 0.39–0.77

At 25 °C, the coating failed to establish significant correlations between quality parameters and physiological indices (Fig. 6A, B), consistent with the minimal preservation observed. High-temperature stress accelerates senescence to such an extent that metabolic and oxidative regulatory networks become decoupled from quality attributes, overwhelming the protective capacity of the coating (Zheng et al., 2022). Although the coating still reduced decay rate and delayed browning compared to controls (Fig. 4A, C), the overall preservation effect was marginal, indicating that temperature dominates over coating treatment under these conditions.

At 10 °C, the coating shifted the primary quality-determining factor from oxidative enzyme activities to microbial control. In uncoated samples, quality parameters were significantly correlated with MDA content and the activities of CAT, POD, PPO, and SOD (Fig. 6C), indicating that oxidative stress and antioxidant enzyme regulation were the dominant determinants of quality. Following coating application, these correlations were substantially diminished, while total colony count became significantly correlated with quality (Fig. 6D). This transition suggests that the antimicrobial activity of the composite assumes a dominant role at this temperature, effectively substituting for the oxidative regulatory mechanisms that govern quality in uncoated samples (Chang et al., 2021). The activation of POD activity (Fig. 5C) and the retardation of SOD decline (Fig. 5A) further support the role of the coating in modulating oxidative responses at 10 °C.

At 4 °C, the coating achieved optimal preservation through selective decoupling of quality from multiple deteriorative pathways. The uncoated group exhibited the most extensive correlation network (Fig. 6E), reflecting sustained metabolic and oxidative activities during low-temperature storage. Coating treatment markedly reduced correlations for respiration rate, total colony count, and SOD activity, which no longer showed significant associations with quality (Fig. 6F). This selective decoupling indicates that the composite film mitigates quality deterioration through three complementary mechanisms: (i) suppression of respiratory activity, as evidenced by the delayed respiratory peak (Fig. 4D) and consistent with reduced oxygen permeability (Fig. 3E); (ii) inhibition of microbial growth, reflected in reduced total colony counts (Fig. 4E) and decay rates (Fig. 4C); and (iii) stabilization of SOD-mediated antioxidant defense, evidenced by maintained SOD activity (Fig. 5A). Unlike at 10 °C, where antimicrobial activity became the dominant factor, at 4 °C the coating exerts a multi-target effect that simultaneously addresses physical, microbial, and oxidative deteriorative pathways. This multi-target mechanism explains the superior preservation efficacy at 4 °C, where storage life was extended by 33.33–38.46% compared to uncoated controls (Table 2).

Condition | Predicted storage life (days) | Verified with actual measurement data
4 °C-coating | 18–20 | Decay rate was 16.5% and L* was 90.19 on the 15th day. (high freshness)
4 °C-CK | 13–15 | Decay rate reached 26.0% on day 15, approaching the critical value.
10 °C- coating | 10–12 | Decay rate on day 12 was 30%, consistent with model prediction.
10 °C-CK | 8–9 | Decay rate reached 26% on day 9 (approaching 30%) and exceeded 30% by day 12, demonstrating model effectiveness within acceptable risk control parameters.
25 °C- coating | ≤ 6 | Decay rate on day 6 was 25.5%
25 °C- CK | ≤ 5 | Decay rate on day 6 was 31.0%

The temperature-dependent hierarchy of preservation mechanisms, antimicrobial dominance at 10 °C versus multi-target action at 4 °C, reflects the interplay between coating functionality and temperature-modulated physiological responses. At moderate temperatures (10 °C), microbial proliferation becomes a primary driver of quality loss, and the coating's antimicrobial properties emerge as the dominant protective mechanism. At low temperatures (4 °C), microbial growth is inherently suppressed, allowing the coating's physical barrier properties (reduced WVP and OP) and antioxidant-stabilizing effects to contribute more substantially to quality retention. This mechanistic hierarchy underscores the importance of temperature management as a critical determinant of coating efficacy.