Materials and methods

Materials

S. rugosoannulata samples at the commercial maturity stage (with unopened pileus and intact veil), uniform in size and shape, white in stipe color, and free from mechanical damage, were obtained from a regional farm in Wuhan (Hubei Province, China), and transported to the laboratory within 6 h of harvest. CS (at deacetylation degree 90%, of food grade) was provided by Yusuo Chemical Technology Co., Ltd. (Shandong, China). TiO2 (primary particle size 35–75 nm) was purchased from Shaoxing Lijie Chemical Technology Co., Ltd. (Zhejiang, China). NT, nisin, ε-PL and TP (food grade) were purchased from Shandong Yuantaibio Engineering Co., Ltd. (Shandong, China), Hangzhou Jinlimeidi Biotechnology Co., Ltd. (Zhejiang, China) and Zhengzhou Bainaifo Bioengineering Co., Ltd. (Henan, China), respectively.

Preparation of CS/N-TiO₂ nanohybrid films

CS/N-TiO₂ nanohybrid was synthesised via the co-precipitation method (Huang & Peng, 2021). Briefly, 1.00 g of CS was dispersed in 100 mL of 1% (v/v) aqueous acetic acid solution and stirred continuously until a transparent solution was obtained. Then, 1.00 g of TiO₂ and 0.30 g of urea were added and thoroughly blended. The mixture was subjected to ultrasonication for 30 min, followed by magnetic stirring for 2 h to obtain a homogeneous milky-white suspension. The pH of the solution was adjusted to 10.0 by adding 1.0 M NaOH, resulting in the formation of a white precipitate. Cross-linking was induced by adding 0.40 mL of 25% glutaraldehyde under stirring for 10 min. After the reaction, the precipitate was washed with distilled water until neutral.

For the preparation of antimicrobial composite films, 1.00 g of each antibacterial agent (NT, nisin, ε-PL, and TP) was separately added to the above neutralized precipitate, followed by thorough stirring and degassing via ultrasonication for 30 min. Subsequently, 20 mL of each coating solution was cast into 9 × 9 cm circular plastic molds and dried at 60 °C for 4 h. Films without antibacterial agents, namely 1% CS and CS/TiO₂, were prepared under the same conditions to serve as controls.

Effect of films on mycelial growth of spoilage fungi from S. rugosoannulata

Spoilage fungi were isolated and purified from S. rugosoannulata as described previously (Liu et al., 2024). Four predominant spoilage strains were selected and identified as F. pseudoanthophilum, A. niger, R. azygosporus, and T. purpureogenus based on morphological and molecular biological analyses. The effects of the composite films on mycelial growth were evaluated according to the method of Yang et al. (2016) with minor modifications. Briefly, 2.0 mL of each composite film-forming solution was mixed thoroughly with 18.0 mL of sterile potato dextrose agar (PDA) medium cooled to approximately 45 °C, and the mixture was poured into sterile Petri dishes. After solidification, mycelial discs (5 mm in diameter) taken from the margin of actively growing colonies of each test fungus were inoculated at the center of each plate. The plates were incubated at 28 °C for 72 h. Colony diameters were measured in two perpendicular directions using the cross-streak method, and the average values were used for inhibition rate calculation. The mycelial growth inhibition (MGI) rate was calculated using the following formula:

(1)MGI=dc−dtdc×100%

where dc is the mean colony diameter of the control sets and dt is the mean colony diameter after 72 h.

Characterisation and physicochemical properties of composite films

Characterisation

Fourier Transform Infrared (FT-IR) spectra were measured using a spectrometer (NEXUS-470, Nicoletnexus, Japan) in the wavelength range of 4000–400 cm−1. X-ray diffraction (XRD) patterns were determined using an X-ray diffractometer (D8 Advance, Bruker, Germany) and Cu—K as a radiation source. The surface microstructure was examined using Scanning Electron Microscopy (SEM, SU 8010, Tianmei, China).

Mechanical properties

Film thickness was determined using a digital micrometre (accuracy: 0.001 mm, n = 10) (Aladdin, Shanghai, China). Mechanical properties were tested according to the method of Ni et al. (2022). Tensile strength (TS) and elongation at break (EAB) were calculated using the following equations:where TS (MPa) is the tensile strength, F (N) is the maximum stress of stretching and S (mm2) is the sectional area of the films.where EAB(%) is the elongation at break, L__0 (mm) is the initial length of films, L (mm) is the length at break.

(2)TensilestrengthTS=FS

(3)ElongationatbreakEAB=L−L0L0×100%

Barrier properties

Water vapor permeability (WVP) was measured using a method described by Lin et al. (2020) and calculated as follows:where WVP (g·m−1·Pa−1·s−1) is the water vapor permeability, ∆m (g) is the weight difference, d (m) is the thickness of films, ∆t (s) is the permeation time, A (m2) is the permeation area of films and ∆P (Pa) is the pressure difference on both sides of films at 25 °C.

(4)WVP=∆m×d∆t×A×∆P

Oxygen permeability (OP) and carbon dioxide permeability (CDP) were determined according to the Chinese National Standard GB/T 1038.1–2022 using a differential pressure gas permeation instrument (VAC—V2, Jinan Labthink Electromechanical Technology Co., Ltd., Shandong, China) (Jiang et al., 2024).

Application of films to mushrooms

S. rugosoannulata samples with no mechanical damage, uniform maturity and consistent color were selected and randomly divided into six groups. They were soaked with CS/N-TiO2/NT composite film solution for 60 s and then removed and dried naturally (hereinafter referred to as the film-coated group). The mushrooms soaked in ultrapure water for 60 s were used as the control (hereinafter referred to as the blank group). The storage test was carried out at 4 °C, 10 °C and 25 °C, respectively.

Weight loss rate, color and decay rate

Weight loss rate of S. rugosoannulata was calculated using the following formula:where m__0 is the fresh weight (g) and m__1 is the weight after the sampling date (g).

(5)Weight loss late%=m0−m1m0×100

Color parameters were assessed using a colourimeter (DR-410, KONICA MINOLTA, Japan) by measuring the lightness (L*) values of the mushroom samples (Duan et al., 2024). Decay rate was expressed as the percentage of decayed mushrooms. Samples showing black spots, surface mycelial growth, or soft rot were considered decayed.

Total colony count, respiration rate, malondialdehyde (MDA) and enzyme activity tests

The total colony count was quantified using PCA plate medium (Wang et al., 2025). Incubation was conducted at 37 °C for 48 h, with results expressed as logarithmic values of colony-forming units per gram (log CFU/g).

MDA content was determined using a malondialdehyde content assay kit from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

The activity of antioxidative enzymes, such as superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) and polyphenol oxidase (PPO) was tested using respective assay kits according to the manufacture's instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Briefly, 4 g of samples was grounded on ice using 36 mL of 50 mM phosphate buffer saline (pH 7.8) and then homogenised at 10000 ×g for 20 min at 4 °C. The supernatant was collected for the enzyme activity test.

Statistical analysis

The experiments were repeated three times, and statistical analysis was carried out using analysis of variance (ANOVA) and Duncan's multiple range test (P < 0.05) with SPSS Statistics 20 software. Spearman's correlation coefficient between the spoilage bacteria and the key metabolites was analyzed and visualized using the Oebiotech tools at https://cloud.oebiotech.cn/task. Analyses of the random forest risk ratio and Cox regression model were conducted using Python 3.9.7.