## 4 Discussion
However, detecting the amount of the produced SEA toxin in the examined cheese samples is a limitation in the existing study. A quantitative ELISA kit with a much lower detection limit than 0.375 ng toxin/g is required be used in the future. So, it will be easier and more accurate to investigate the effect of the different treatments. On the other hand, small-sized NiO NPs (20 nm) were used in this study so that they can easily penetrate the bacterial cell membrane leading to a better antibacterial effect [43]. After examining the antibacterial effect of different concentrations of the studiednanoparticles, 35 \(\upmu\)g/mL was the minimum inhibitory concentration (MIC) that could inhibit the enterotoxicigenic _S. aureus_ strains. This result was lower than that obtained by [PERSON] et al. [28] who tested NiO NPs for their antibacterial activity against _S. aureus_ and reported that the MIC of NiO NPs was 60 \(\upmu\)g/mL. In contrast, a lower MIC value of NiO NPs (0.21 \(\upmu\)g/mL milk) was investigated by [PERSON] & [PERSON] [26]. Herein, we examined the cytotoxicity of NiO NPs on HECs for 48 h using MTT assays. Our findings indicated that the MIC used in this study is consider safe on the viability of HECs (93%) and it does not have IC\({}_{50}\) or IC\({}_{90}\). The obtained results are consistent with previous studies suggesting that the cytotoxicity of NiO NPs is mediated through ROS generation and oxidative stress [44,45]. [PERSON] et al. [46] reported that the cytotoxic effects of NiO NPs are studied in cultured human colorectal cancer cells (HCT-116), which have exhibited significant anticancer activity with 55 \(\upmu\)g/mL at IC\({}_{50}\). Hence, NiO NPs may offer a safe potential for many diseases management and can be applied to different medical and industrial applications [16,46]. Importantly, the cytotoxic effect of NiO NPs on intestinal epithelial cells is not clear. To confirm the anti-toxic effect of such particles, we had to examine their cytotoxicity through oral administration of lab animals besides MTT assay. This limitation in the current study will be avoided and compensated for in future studies. Domiati cheese that was manufactured from cow's milk formulated with 35 \(\upmu\)g/mL NiO NPs showed a drastic decrease in the number of bacterial cells (_S. aureus_) during the processing and ripening of cheees versus the control samples (\(p<0.05\)). It is clear from the present study that NiO NPs had a favorable effect on the inhibition of enterotoxicigenic _S. aureus_ strains in cheese and can be used to avoid the contamination of such products with _S. aureus_. Furthermore, the addition of NiO NPs to cheese could prevent enterotoxins production from _S. aureus_. Similarly, [PERSON] et al. [47] reported a strong antibacterial effect of NiO NPs against Gram-positive bacteria compared to Gram-negative bacteria. Importantly, the main effect of such particles may be due to the ability of NiO NPs to generate oxidative stress (ROS) that resulted in rapid membrane damage leading to bacterial cell death [47] beside the low pH and high NaCl concentration in Domiati cheese during ripening. Thus, these particles can be one of the most promising preventive measures that may be applied in dairy industry in the near future. However, there are some limitations in such part of the present study as the strong antibacterial mechanism of NiO NPs against _S. aureus_ in Domiati cheese that is not fully clear. Hence, the mechanism needs to be addressed in future research. Regarding the chemical examination of all types of cheees, there were not any significant changes in the examined parameters in comparison to control. However, NaCl % (in aqueous phase) increased gradually during ripening of cheese and reaching its highest level in the cheese with yoghourt starter culture. This can be attributed to the low pH in such kind of cheese as reported by [PERSON], [PERSON] & [PERSON] [48] who showed the decrease in the uptake of salt by the cheees during brining when the pH values increased.