Abstract
Lignin, a major component of lignocellulosic biomass, has gained attention as a potential antimicrobial agent. However, lignin's limited solubility often requires the use of harsh or non-eco-friendly solvents, which can hinder its applications and pose environmental concerns. This work aimed to evaluate the antimicrobial capacity of two lignin samples extracted from grape stalks using different methods: alkaline extraction and deep eutectic solvents. The antimicrobial activity of lignin was evaluated in three formulations: micronized, dissolved, and nanoparticles, against Escherichia coli , Pseudomonas aeruginosa , and Staphylococcus aureus . Results showed that dissolved lignin exhibited higher antimicrobial activity with expressive log reductions and total inhibition of bacterial growth at low concentrations (0.026-0.228 mg/mL). Micronized lignin demonstrated antimicrobial properties against S. aureus but only at higher concentrations (10-30 mg/mL). Lignin nanoparticles showed antimicrobial activity against S. aureus at much lower concentrations (0.250-0.468 mg/mL) than micronized lignin assays. Flow cytometry analysis revealed that dissolved lignin caused extensive damage to the bacterial cell membrane, leading to cell death. While dissolved lignin demonstrated the highest antimicrobial activity, micronized lignin and lignin nanoparticles also showed potential for specific applications. The development of sustainable extraction methods and green solvents is crucial for the successful utilization of lignin as an antimicrobial agent. This study highlights the potential of lignin from grape stalks as a natural antimicrobial alternative and discusses the importance of lignin's physical form in optimizing its antimicrobial performance and applications.