NANOMETRIC NEEM OIL EMULSIFICATION THROUGH HIGH-PRESSURE HOMOGENIZATION AND ITS THERAPEUTIC POTENTIAL AGAINST AQUACULTURE PATHOGENS.

Increase in the mortality rate due to the disease outbreaks in this aquaculture sector leads to hindrance in the economic development of a nation. Several strategies has been developed to control the infections caused by the pathogenic microbes such as the use of antibiotics. The excessive application of these antibiotics against the pathogens have resulted in the development of resistance among the pathogens. These problems in the aquaculture sector urge for a better antimicrobial compound with more efficacy against the pathogens also with less eco-toxic property. An alternative biocontrol method that helps in the prevention of pathogenic infection is through application of nanoemulsion which is formulated using an essential oil. The bioactive compounds present in essential oils exhibits low toxicity towards the environment and have high potent inhibitory role against pathogenic bacterial and fungal pathogens.Among several strategies carried out for nano-emulsification process, high-pressure homogenization is one of the new and efficient methods. The present study emphasises on the neem oil nanoemulsion formulation that can act as an alternative to control the pathogenic infection in aquaculture using high-pressure homogenization. The effect of different concentrations of surfactant (tween 20, tween 80), the applied pressure and the number of cycles on the droplet size of neem oil nanoemulsion was investigated. The optimized pressure and cycle for the neem oil nanoemulsion formulation was found to be 20,000 psi at 25th cycle. A significant decrease in the particle size of neem oil nanoemulsion was observed as the pressure, cycle and concentration of surfactant increase. The stability studies of neem oil nanoemulsion formulated using both the surfactants were investigated based on the physicochemical parameters like turbidity, pH, different water matrices (sea water, lake water), and temperature. In all the physicochemical stability studies, the neem oil nanoemulsion prepared using the tween 80 (ratio 1:3) showed a good physicochemical stability with a droplet size of 20.1 ± 1.98 compared to all other formulated ratios. The concentration of the active ingredient, azadirachtin was checked using reverse phase high-performance chromatography and LC/MS technique, which was found to be 13.54 ± 1.23 mg/L and 21.187 µg/mL respectively. The stable ratio of 1:3 prepared using neem oil and tween 80, showed an efficient antibacterial activity against Aeromonas culicicola, Vibrio alginolyticus and Pseudomonas aeuroginosa, which was confirmed by MIC study. The SEM analysis and cell membrane integrity test confirmed the damage caused to the bacterial cells by the formulated nanoemulsion. Further, the In-vivo study of neem oil nanoemulsion on Penaeus monodon and Cyprinus carpio confirmed its antibacterial potential against Aeromonas culicicola, Vibrio alginolyticus and Pseudomonas aeuroginosa.