Inhibitory Effects of Pinus Pinaster Aiton Subsp. Atlantica Bark Extracts Against Known Food Pathogens

Abstract

Pine bark extracts are a rich source of natural polyphenols that besides their known antioxidant activity may also exhibit significant antimicrobial properties. In the current study, the antimicrobial activity of Pinus pinaster Aiton subsp. atlantica extracts was screened against gram-positive bacteria (Bacillus cereus, Clostridium perfringens, Listeria monocytogenes, Staphylococcus aureus), gram-negative bacteria (Pseudomonas aeruginosa, Escherichia coli, Salmonella enterica serovar Enteritidis) and the fungus Aspergillus brasiliensis ATCC 16404, using the disk diffusion assay. The hydroethanolic extracts obtained by Microwave assisted extraction (MAE) at 1600 W and 110 ºC for 30 minutes, were compared to the Soxhlet extracts. The pine bark was dried to reach equilibrium humidity at 40 °C for 72 hours and milled to a particle’s size of 200-850 µm. The extraction yield was determined by the amount of extract recovered in mass compared to the initial amount of dry bark and the total phenolic content was determined by the Folin Ciocalteu colorimetric method at 725 nm. Hydroethanolic pine bark extracts were prepared at 0.25, 0.50 and 80.00 mg/mL concentrations. MAE attained higher extraction yield and higher total phenolic content (20.4±0.7% w/w and 71.1±3.4 mg of gallic acid equivalent (GAE) per g of sample, respectively) than Soxhlet extracts (17.6±0.2% w/w and 54.9±2.6 mg GAE/g sample, respectively). The pine bark extracts presented inhibition halos ranging from 7 to 17 mm against the gram positive bacteria, namely C. perfringens ATCC 13124 and B. cereus NCTC 11143. The MAE extracts presented higher inhibition halos than the Soxhlet ones. Regarding the tested concentrations, only C. perfringens ATCC 13124 and B. cereus NCTC 11143 were sensitive to the 0.25 and 0.50 mg/mL concentrations. At 80 mg/mL all the gram-positive bacteria were sensitive to the pine bark extracts obtained by both extraction methods. On the other hand, the extracts did not show inhibitory effect against the tested gram-negative bacteria and A. brasiliensis.This study reveals that MAE is a fast and efficient method for obtaining Pinus pinaster Aiton subsp. atlantica extracts with antimicrobial activities against Gram-positive bacteria.