## 4 Discussion
In addition, they tend to bind electrostatically to bacterial cell membranes, leading to the loss of membrane integrity and death, as reported by [PERSON] and [PERSON] [64]. [PERSON] et al. reported that CaOH\({}_{2}\) NP small size and greater surface area allowed higher interaction with bacteria and thus enhanced antibacterial activity [27]. We recently reported that C-CaOH\({}_{2}\) NP size was less than 100 nm (31.56-81.22 nm) [30], which may have contributed to their antibacterial effect. Additionally, scanning electron microscope findings have shown hexagonal-shaped C-CaOH\({}_{2}\) NPs [30], which may have influenced their effect. One study reported that the rough outer surface and corners of CaOH\({}_{2}\) NPs can cause mechanical damage to bacterial membranes and therefore induce an antibacterial effect [27]. Furthermore, the dissolution of CaOH\({}_{2}\) releases positively charged Ca\({}^{2+}\) ions which have been reported to attach, penetrate, and damage negatively charged bacterial cell membranes [27]. Moreover, studies have reported that CaOH\({}_{2}\) releases OH\({}^{-}\) ions, which contribute to its high alkalinity and block bacterial cell functions, jeopardizing their survival [4; 65]. [PERSON] et al. recently reported that OH\({}^{-}\) ions released from CaOH\({}_{2}\) NPs maximized their antibacterial efficacy by causing damage to cell walls, proteins, and DNA [28]. In our previous study [30], we reported the presence of hydroxyl groups and Ca element in C-CaOH\({}_{2}\) NPs, which were anticipated products of CaOH\({}_{2}\). Hence, we assume that they may have played a role in C-CaOH\({}_{2}\) NP activity. In addition, carob may have added to the effect via the binding of polyphenols present in carob to bacteria [66, 67]. Phenolic acids and flavonoids polyphenols have been reported to develop complexes with bacterial cell walls, leading to the disruption of bacterial walls and intracellular functions, as well as impaired growth [17, 33, 68]. Our previous characterization study confirmed the presence of C-C and C-O-OH groups in C-CaOH\({}_{2}\) NPs, which were linked to flavonoids and phenolic acid phytochemicals in carob [30]. Thus, their antibacterial role in C-CaOH\({}_{2}\) NP activity may also be implied. Numerous studies have affirmed the inhibitory activity of carob extracts against different strains, including _E. faecalis, S. aureus, S. epidermidis, P. aeruginosa, and E. coli_[15, 18, 19], which is consistent with our findings. However, the synergistic effect of carob in C-CaOH\({}_{2}\) NPs is uncertain, as the antibacterial activity of the C-CaOH\({}_{2}\) NPs presented in this study appeared similar to that reported for other CaOH\({}_{2}\) NPs. Importantly, the variations in C-CaOH\({}_{2}\) NP activity and intermediate inhibition effect may have been influenced by the culture media buffering ability, carob extract type and acidity, and NP vehicle, which can influence the extent of dissolution and alkalinity of C-CaOH\({}_{2}\) NPs that are crucial for their bactericidal potency [26, 28, 44, 69, 70, 71]. The current study aimed to establish baseline information on C-CaOH\({}_{2}\) NP antibacterial activity via standard in vitro assays. We concluded that within this study's parameters, C-CaOH\({}_{2}\) NPs demonstrated intermediate activity that was not superior to other CaOH\({}_{2}\) NPs. Therefore, future studies are encouraged to investigate the exact antibacterial mechanism of C-CaOH\({}_{2}\) NPs and their synergistic effect, and to address this study's limitations, including the duration-dependency effect, the type of vehicle used to disperse C-CaOH\({}_{2}\) NPs (e.g., aqueous or non-aqueous), and the type of carob extract used in synthesis (e.g., methanol or aqueous).