Antibacterial Activity of Korean Pine (Pinus densiflora) Needle Extracts on Escherichia coli and Staphylococcus aureus: Mechanism and Properties of Membrane

Abstract

Korean red pine (Pinus densiflora) is one of the major Pinus species in Korea. The inner needle extracts contain high levels of phenols and flavonoid compounds and exhibit strong antioxidative, antimicrobial, inhibiting lipid peroxidation, and lipid synthesis activities in the membrane. So the use of pine needle extracts can inhibit or stop the spread of the pathogenic bacteria in foods. However, the possible action mechanism and functional food activities of the extract have not yet been fully elucidated. This study aimed to investigate the antibacterial effect of aqueous steam distilled pine needle extract (SDPNE) on the growth of both Escherichia coli and Staphylococcus aureus and compare the dynamic diffusion and enzymatic biomarkers through lipid membrane. Results showed that the content of total phenolic (TPC) and total flavonoid (TFC) in SDPNE were 3.61±0.002 mg gallic acid/g extract and 24.97±1.55 mg quercetin equivalent/100g extract; ABTS and FRAP value of 0.1% SDPNE were 3.82±1.39% and 15.78±2.25 μM FeSO4/ml, respectively. This data confirmed that SDPNE has antioxidant components and activity. Five food-borne pathogenic bacteria were tested to determine SDPNE's antibacterial activity and mechanism. As a result, Gram-negative bacteria (Salmonella enteritidis and E. coli) were more sensitive to SDPNE than Gram-positive bacteria (Listeria monocytogenes, S. aureus, and Bacillus subtilis). Cell membrane fluidity analysis showed that with time, the fluidity of E. coli and S. aureus cell membrane gradually approached the control group and returned to normal. A combination of gas chromatography-mass spectrometry analysis revealed that E. coli and S. aureus cells increased membrane fluidity by altering the composition of membrane fatty acids (FAs) against the SDPNE exposure. With the increase in treatment time with 0.1% SDPNE, the SFAs in E. coli gradually increased from 57.37% at 6 h to 61.19% at 12 h. The SFAs in S. aureus decreased from 69.77% at 6 h to 59.21% at 12 h. The trans-C18:1n-9 increased from 13.95% at 6 h to 23.03% at 12 h. Real-time qPCR analysis showed a significant decrease in FA biosynthesis-related genes in bacteria (E. coli and S. aureus) after exposure to 0.1% SDPNE for 6 h. However, the expression levels of the fabI gene, known for the rate-limiting step of FA biosynthesis in E. coli, significantly decreased at 9 h. In agreement with experimental results, this study demonstrates that SDPNE modifies membrane properties and FA synthesis-related genes in bacteria, thus decreasing bacterial growth. Taken together, I might emphasize that SDPNE could be used as a natural oxidant and/or preservative against various pathogenic bacteria such as E. coli, S. aureus, and other food-borne pathogens to control food poisoning.