Identification and Mechanisms of Action of Antimicrobial Substance from Brown Algae

Abstract

The antimicrobial activties of 14 brown algae extract were measured using the disk diffusion method and the minimum inhibitory concentration test. Ishige okamurai, Ecklonia stolonifera, Sargassum siliquastrum, Sargassum thunbergii, Colpomenia bullosa, Myagropsis myagroides and Ecklonia cava ethanol extracts showed antimicrobial activity against gram-positive bacteria and some of the yeasts at 4 mg/mL. To isolate and identify the substances having strong antimicrobial activity from S. siliquastrum, M. myagroides and E. cava, silica gel column chromatography, high performance liquid chromatography (HPLC), electron-induced spectrometry (EI-MS) and nuclear magnetic resonance (NMR) analyses were conducted. In addtion, the mechanisms of antimicrobial action of these brown algae against Staphylococcus aureus, Escherichia coli and Listeria monoytogenes were investigated using electron microscopy, time-kill curves, the measurement of the loss of 260 nm absorbing material, the released beta-galactosidase, and the loss of ATP. Finally, toxicity testing and application of M. myagroides and E. cava to food system were carried out to evaluate their possible use as natural food preservatives. S. siliquastrum (SS) exhibited strong antimicrobial activity at 0.075 mg/mL against Bacillus subtilis and at 0.05 mg/mL against Clostridium perfringens. The SS ethanol extract was separated using silica gel column chromatography. The MIC test was conducted on each obtained fractions, and the ethyl acetate division possessed the lowest MIC against B. subtilis and C. perfringens ranging from 0.0125 mg/mL to 0.05 mg/mL. The ethyl acetate division demonstrating the greatest antimicrobial activity was separated by HPLC, and two fractions were obtained. Among these fractions, peak 1, which exhibited the highest activity, was verified as mojabanchromanol (a type of chromene) through EI-MS and NMR analysis. The ethanol extract of M. myagroides (MMEE) has substantial antimicrobial activity against gram-positive bacteria and some of the yeasts. The MIC of MMEE ranged from 0.0625 mg/mL to 0.125 mg/mL for B. subtilis, L. monocytogenes and C. perfringens. To isolate the antimicrobial substance from MMEE, liquid-liquid extraction was performed. Five divisions, including the n-hexane, chloroform, ethyl acetate, n-butanol and water division were obtained. Among these, the chloroform division had the strongest antimicrobial activity (MIC 0.03125 - 0.25 mg/mL), and was separated into 22 subfractions by using the silica gel column chromatography. The CH4 fraction possessed the strongest activity of the 22 subfractions, and showed antimicrobial activity against gram-negative as well as gram-positive bacteria. In the mechanisms experiments, leakage of the 260 nm absorbing material, beta-galactosidase and ATP were measured in the CH4 treated cells; in addition, altered morphology was observed under an electron microscopy. Given these results, CH4 is considered to act on the cytoplasmic membrane of the bacteria. The E. cava (EC), ethanol extract was more effective on gram-positive than gram-negative bacteria, and exhibited antimicrobial activity at the levels of 0.75 mg/mL - against B. subtilis, L. monocytogens and Lactobacillus plantarum, at 0.5 mg/mL against C. perfringens. The EC ethanol extract was partitioned into n-hexane, chloroform, ethyl acetate and butanol. Among these, the ethyl acetate division had the lowest MIC, ranging from 0.5 to 0.0625 mg/mL. This division was subjected to silica gel column chromatography, and 22 subfractions were obtained. Among them, EA7 exhibited the highest activity and was separated into five peaks by using HPLC. Of the five peaks, peak 5 was identified by GC-MS as D-homoandrostan-17a-one (C20H32O), a type of steroid, which has a molecular weight of 288.46748. To evaluate the possibility of using these compounds as natural food preservatives, the effects of food ingredients on the antimicrobial activity of CH4 from M. myagroides and EA7 from E. cava were determined. The efficacy of CH4 and EA7 were reduced in the presence of high concentration soluble starch, beef extract, and soybean oil. In particular, marked decreases in antimicrobial activity were observed in beef extract and soybean oil compared to soluble starch. Based on these results, the shelf-life of fruit juice was monitored after CH4 and EA7 were added. CH4 and EA7 inhibited the growth of food spoilage microbes, and the shelf-life of fruit juice increased. In the acute toxicity test, no mortalities occurred in mice administered 5 g/kg body weight of M. maygroides and E.cava ethanol extract over the 2-week observation period. Our results suggest that M. myagroides and E. cava can be used as natural food preservatives.