Antimicrobial activity of gallic acid against food-related Pseudomonas strains and its use as biocontrol tool to improve the shelf life of fresh black truffles

Refrigeration alone or in combination with other technologies represents the main tool used in the last decades to preserve the freshness of black truffles. This is principally due to the delicateness and vulnerability of this edible hypogeous fungus, so that other invasive preservation practices cannot be adopted. However, the proliferation of some microbial species during the cold storage still represents an unsolved problem. Pseudomonads are among the main spoiler bacteria responsible for the deterioration of refrigerated black truffles. Their growth ability at low temperatures requires the use of additional hurdles to prolong the shelf-life of truffles without altering their major features. The use of natural compounds may represent an alternative system for the bio-control of this kind of product. Specifically, gallic acid (GA) is a phenolic acid naturally present in different foods, whose effectiveness was in vitro demonstrated against Pseudomonas spp. In our study, we reported the antimicrobial activity expressed by GA not only in vitro, using as target bacteria Pseudomonas putida DSMZ 291(r), P. fluorescens DSMZ 50090(T), P. fragi DSMZ 3456(T) and Pseudomonas spp. P30-4, previously isolated from black truffles, but also in situ on fresh black truffles stored at 4 degrees C for 28 days. Our results showed Minimum Inhibitory Concentrations (MIC) of 2.5 mg/mL GA for all tested strains, except for P. fluorescens DSMZ 50090(T), having a MIC corresponding to 5 mg/mL GA. The Minimum Bactericidal Concentration (MBC) was 10 mg/mL for all strains. The analysis of kinetic parameters showed that the survival declined passing from 2.5 to 10 mg/mL GA concentrations, with P. fluorescens confirmed to be the most resistant strain. Moreover, images obtained from Scanning Electron Microscopy revealed that Pseudomonas cells were strongly injured by the treatment with GA at 2.5 mg/mL concentration, displaying visible pores on the cellular surfaces, absence of flagella and lysis with loss of cytoplasmic material. The storage test performed on fresh black truffles confirmed in situ the GA antimicrobial activity observed in vitro, with a drastic reduction not only of Pseudomonas spp., but also of the other assessed microbial groups, including Enterobacteriaceae and Eumycetes. Finally, sensory analysis established the absence of off-flavours and the preservation of positive features in black truffles treated with 2.5 mg/mL GA and stored for 28 stored at 4 degrees C. The results obtained in this study suggest that GA is a potential biocontrol tool to decontaminate and preserve fresh black truffles during refrigerated storage.