# Neem Oil (Azadirachta indica L.) Response Surface Methodology (RSM)-Optimized Nanoemulsions for Sensory Quality Preservation of Oreochromis niloticus Fillets

## TL;DR

Neem oil nanoemulsions were developed as a natural, eco-friendly preservative to maintain the quality and safety of fish fillets during storage.

## Contribution

The study introduces RSM-optimized neem oil nanoemulsions (<200 nm) as an effective and safe green preservative for fish.

## Key findings

- Neem oil nanoemulsions inhibited Staphylococcus aureus by increasing bacterial membrane permeability.
- Fish fillets treated with the nanoemulsions retained pH, moisture, and protein content during storage.
- Sensory attributes like flavor, color, and aroma remained acceptable in treated fish fillets.

## Abstract

Synthetic preservatives in food products have raised toxicity concerns; hence, they should be replaced with safer alternatives. Neem oil nanoemulsions (NO NEs) have shown promise as natural antibacterial agents. Here, NO NEs effectively inhibited Staphylococcus aureus via DNA and protein leakage due to the increased permeability of bacterial membranes caused by NEs <200 nm in size. NO NEs were made with an ultrasonicator. The main compounds found, aside from nimbiol, were nimbandiol, 6-deacetyl nimbinene, and azadirachtin. NE dip coatings on fish fillets of Oreochromis niloticus maintained their pH (6.65 ± 0.43), moisture (8.23 ± 0.32%), and protein content (38 ± 7%) during the storage period. Interestingly, sensory attributes such as flavor, color, and aroma were in an acceptable range. These findings suggest that NO NEs can be a safe, effective, and ecofriendly preservative for fish and other meat products.

Neem oil nanoemulsions (NO NEs) have gained attention as natural antibacterial agents due to toxicity concerns surrounding synthetic preservatives. This study aimed to prepare a response surface methodology (RSM)-optimized NO NE < 200 nm to achieve a stable dip solution to maintain the sensory quality of Oreochromis niloticus fillets. The NO NE achieved a stable formulation with a particle size of 160.2 ± 0.04 nm on average. The polydispersity index (PDI) was 0.1 ± 0.05, and the zeta potential was found to be 18.2 ± 0.09 mV. Gas chromatography confirmed the presence of nimbiol, nimbandiol, 6-deacetyl nimbinene, and azadirachtin in NO after ultrasonic homogenization for 10 min (alternating between 30 s rest and 30 s work time). The NE had a spherical shape with a smooth surface, as was evident from transmission electron microscopy (TEM). Furthermore, NO:PM (neem oil–potassium metabisulphite) had an MIC (minimum inhibitory concentration) value of 150 ppm, compared to 210 ppm for the NO NE alone, against Staphylococcus aureus. Time–kill dynamics revealed the more effective control of S. aureus until 72 h with NO:PM. Moreover, DNA and protein leakage also increased from 0.145 ± 0.001 to 0.769 ± 0.002 OD (optical density) and from 0.142 ± 0.002 to 0.740 ± 0.001 OD, respectively, with the co-formulation of NO:PM. Conclusively, NO:PM inhibited S. aureus at a lower dose compared to the NO NE alone. Time–kill dynamics revealed complete inhibition of S. aureus in vitro for a period of 72 h. On the other hand, a proximate analysis of O. niloticus fillets showed no alteration in pH, no protein loss, and juiciness/moisture retention during 30 days of storage (4 °C). Sensory panelists reported that O. niloticus fillets treated with NE NO had improved color, flavor, juiciness, aroma, and overall quality. These results show that NE NO is a suitable green preservative for fish and possibly other meat-based products.