| D-limonene,4-isopropenyl-l-methylcyclohexene, belongs to the class of natural monoterpenes, which is extensively found in several citrus essential oils. It is endowed with a strong antimicrobial activity to a wide gamut of Gram-negative and Gram-positive microorganisms. It is generally recognized as safe according to the United States code of federal regulation. In nature, it is a transparent liquid with a pleasurable citrus odor. Those positive characteristics have made this compound one of the most popular natural antimicrobial agent who is widely used in food, cosmetic and pharmaceutical industries. However, D-limonene is a hydrophobic compound (oily nature), and this will restrict its application in water rich surfaces, which are favorable cites for the food-borne pathogens’growth. Besides that, it oxidative nature under normal storage conditions is another limitation. The oxidation of D-limonene will transform it to alcohols and epoxides resulting in the loss of its proprieties. To overcome these drawbacks, encapsulation of D-limonene into an optimal nano-dispersion delivery system might be a good choice to increase its solubility, stability and antimicrobial efficiency. In addition, its combination with some other strong antimicrobial agents can be very helpful to enhance its antimicrobial activity in food. The aim of the current research was the encapsulation of D-limonene into novel nano-scale systems in order to overcome its above cited limitations.D-limonene organogel was prepared and the gel formation as well as the incorporation efficiency of D-limonene into the gel clusters was confirmed by:the evaluation of the critical gelation concentration, organoleptic test investigation,and the application of (XRD, FTIR, and microscopic visualization). The formed organogel was used as the oil phase, and the nano-dispersion delivery system was prepared by high pressure homogenization. A number of parameters such as the organogelator and the surfactant type and amount, homogenization conditions (the pressure value, and the number of passes), stability of the prepared formulation over 2 weeks of storage at 28 and 4℃ were investigated. The different results have shown that D-limonene was successfully incorporated into the organogel networked structure. It was also found that 5%(w/w) stearic acid,10%(w/w) Tween 80,30 Mpa and 10 cycles were the adequate conditions for the production of D-limonene organogel-based nanoemulsion with a smallest mean droplet diameter d^ 112 nm. In addition, the developed formulation has shown a good stability throughout the period of storage at two distinct temperatures.An ameliorate D-limonene organogel-based nanoemulsion in terms of the mean droplet diameter size was developed by changing the loading amount D-limonene into the organogel and the homogenization conditions. A study on the formulation’s antimicrobial efficiency was assessed by broth dilution method against four food-borne pathogens (Escherichia coli, Bacillus subtilis, Staphylococcus aureus, and Saccharomyces cerevisiae). In addition, the mechanism of antimicrobial action was determined by SEM analysis, and the evaluation of the cell release constituent. The antimicrobial activity of D-limonene alone and following its incorporation into the nanoemulsion was compared in order to study the effect of the delivery system on the compound antimicrobial efficiency. The obtained data revealed that at a D-limonene organogel-based nanoemulsion (d≈;36 nm) with a good stability and narrow structure can be easily formed at the following optimal conditions 5%(w/w) monostearin,10%(w/w) Tween 80,10 cycles and 100 Mpa. Furthermore, The MICs data exhibited that, the incorporation of D-limonene into the formulation has contributed towards the increase of its antimicrobial efficiency. SEM and cell constituent release has shown that the formulation caused a detrimental effect on cell membrane integrity of the tested microorganisms.D-limonene nanoemulsion with the inclusion of ε-polylysine was developed by taking the advantages of smart combination and nanoemulsion technologies in one system. The antimicrobial effects of both D-limonene and ε-polylysine were firstly checked against the above cited microorganisms, and then investigations of any synergism, addition, antagonism or indifferent effect between these two compounds were established by the use of the checkerboard method. Afterwards, the formulation was prepared by high pressure homogenization technology and the effect of the inclusion of a different amount of ε-Polylysine on the nanoemulsions oil droplets diameter was investigated. The antimicrobial effect of the developed delivery system was studied by MICs measurements, and the obtained results were compared with those of D-limonene nanoemulsion without the inclusion of ε-Polylysine. In addition, the mechanism of the antimicrobial action was determined by SEM and cell leakage measurements. The results have displayed that the nano- disperion has merged the profit of nanoemulsion technology and the synergistic effect of D-limonene and ε-Polylysine. It was found also that this nanoemulsion has contributed to a wide increase on the two compounds’antimicrobial efficiency by reducing their respective MICs. Furthermore, the SEM visualizations and the cell leakage determination revealed that cell treated with D-limonene nanoemulsion with the inclusion of ε-Polylysine suffered more shape deteriorations comparing to those treated with D-limonene nanoemulsion. |
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