TdGASA2, a cysteine-rich Snakin/GASA protein from Triticum turgidum, enhances stress-mediated inactivation of Listeria monocytogenes and improves the preservation performance of chitosan-based biodegradable active packaging films.

Abstract

The growing demand for natural, sustainable food preservation strategies has intensified research into plant-derived antimicrobial proteins with multifunctional bioactivities. This study reports the functional characterisation of TdGASA2, a cysteine-rich protein of the Snakin/GASA family isolated from Triticum turgidum ssp. durum, as a candidate bioactive ingredient for antimicrobial food packaging. TdGASA2 displayed broad-spectrum antibacterial activity against all tested strains, with the lowest minimal inhibitory concentration (MIC) values observed against Listeria monocytogenes and Pseudomonas aeruginosa (10.50 and 11.25 μg/mL, respectively), and retained a mean of 77.8% of its native antibacterial potency after autoclaving (121 °C, 20 min), reflecting the thermal resilience of its disulfide-stabilised Snakin/GASA scaffold. The protein additionally exhibited concentration-dependent α-amylase inhibition (IC₅₀ = 72.4 ± 0.31 μg/mL) and enhanced bactericidal inactivation of L. monocytogenes under oxidative stress. Incorporation of TdGASA2 into chitosan-based films improved tensile strength and reduced water vapor permeability and water solubility, consistent with protein-polysaccharide interactions inferred from established structure-property relationships in the literature; the films also maintained a high rate of soil-burial mass loss and macro-disintegration (>92% after 10 days), which reflects overall gravimetric mass reduction rather than confirmed biodegradation in the absence of an abiotic soil control. In a 10-day refrigerated chicken breast storage trial, TdGASA2-enriched films (2× MIC against L. monocytogenes) reduced total aerobic microbial growth by approximately 2.4 log CFU/g and attenuated lipid and protein oxidation relative to the unfunctionalised control, corresponding to an exploratory, Random Forest-predicted shelf-life extension of 3-4 days. Collectively, these results identify TdGASA2 as a promising, multifunctional bioactive candidate for biodegradable active food packaging; direct spectroscopic confirmation of the proposed protein-polymer interactions, migration behaviour in regulatory food simulants, toxicological and allergenicity profiling, sensory impact, and formal food-contact regulatory assessment remain to be established before its suitability for practical or commercial application can be confirmed.