Optimization of mangroveleaf extraction by mixture design and investigation of its antimicrobial effect on Listeria innocua, Enterococcus faecium and Escherichia coli

Avicennia marina, commonly known as gray or white mangrove, is a specie of mangrove tree classified in the plant family Acanthaceae. It is distributed along Africa's east coast, south-west, south and south-east Asia, and southern Iran along the Persian Gulf coast. It grows as a shrub or tree to a height of three to ten meters. Mixture design is one of the most popular smart systems which is based on simulation of linear and non-linear systems using mathematical and statistical techniques, and a useful tool for dealing with completely unknown systems. Chemical preservatives are commonly used for inhibition of pathogens in foods, people are concerned about the side effects of preservatives on their health. Replacement of chemical preservatives with natural substances have a great importance in food preservation. Natural preservatives, as well as, essential oils and plant extracts are suitable alternatives for chemical preservatives. The main purposes of this study are the evaluation of the effects of different combinations of four solvents (water, ethanol, methanol and glycerin) on the efficiency of mangrove leaf extraction using response surface method with mixture optimal design, the optimization of solvent formulation for mangrove leaf extraction, and, finally, the evaluation of the in vitro inhibitory and bactericidal effects of mangrove leaf extract on Listeria innocua ATTC33090 ¡ Enterococcus faecium ATTC 51559 and Escherichia coli ATTC 25992.
Fresh mangrove leaves were prepared from Qeshm Island, Persian Gulf, Iran, in August 2012. Water, ethanol, methanol and glycerin extracts were prepared by adding 50 g of powdered mangrove leaf to 250 mL of the solvent. Extraction was carried out for 48h, in ambient temperature. The mixture of extract and leaf powder was separated by Watman filter paper, then the filtrate was centrifuged in 3000g for 10 minutes and filtered using a 0.45 µm Millipore filter. Finally, in order to separate the solvent and concentrated extract, the solutions were evaporated using a rotary vacuum evaporator. The concentrated extract was stored in dark aluminum containers at 4°C. In this study, the effects of water, ethanol, methanol and glycerin at five levels (0, 31.25, 83.33, 125 and 250 ml) on efficiency of mangrove leaf extraction by mixture optimal design has been investigated. Modeling and optimization has been carried out by Scheffe polynomial. The antimicrobial activity of mangrove leaf extract was evaluated using disk diffusion method. The minimum inhibitory concentration (MIC) of mangrove leaf extract was determined using serial dilution tubes. For each extraction method (based on solvent, Water, Ethanol, Methanol and Glycerin), 8 serial concentrations (2, 4, 8, 16, 32, 64, 128, 256 mg/mL) and 1 control tube of mangrove leaf extract were prepared in Mueller-Hinton broth. The minimum bactericidal concentration (MBC) of mangrove leaf extract was determined using serial dilution tubes.
The Results indicated that Scheffe polynomial model was highly significant for prediction of efficiency of mangrove leaf extraction (R2 and R2adj values equal to 0.940 and 0.8447, respectively and The lack-of-fit tests did not result in a significant, also F-value (14.62) indicated that the model is sufficiently accurate). The optimum formulation was found as following: glycerin (0 ml), water (28.22 ml), methanol (59.83ml) and ethanol (161.95 ml) respectively. Maximum of antimicrobial effect on Listeria innocua and highest resistance against mangrove leaf extract on Escherichia coli were observed. Increasing concentration of mangrove extracts had a significant effect (p Finally, the results showed that mangrove leaf extract had a notable antimicrobial effect on the studied strains “in vitro”. More “in vivo” studies seem to be required in order to determine the best extract dosage which leads to inhibition of microbial infection.