## 4 Discussion
The authors found that chitosan interfered with the demineralization process by inhibiting the release of phosphorus in vitro and that demineralization was influenced by the concentration and exposure time of the biopolymer to the enamel. They also suggested that chitosan can serve as a barrier due to its adsorption to tooth enamel, impeding the penetration of acids from microbial fermentation or diet and contributing to the inhibition of the demineralization of the enamel. Foaming capacity is an important factor in any formulation intended for oral use, as it enables the compound to widely reach in the oral cavity during hygiene activities [43]. According to [PERSON] et al. [22], formulations with biosurfactants are effective as a good detergent for oral use. These observations corroborate the results obtained in this present work for the proposed formulations, which showed good emulsification activity and foaming capacity. The occurrence of a large quantity of micronuclelei in cells may be related to mutagenic events or be evidence of a carcinogenic process, which may be influenced by the contact between the plant and toxic compounds [23]. The macroscopic examination of the formulations prepared in this current study revealed no necrotic tissues or anomalies. The absence of micronuclelei indicates that the molecules of the biosurfactants have no mutagenic or carcinogenic effects [23]. Bioassay with _Allium cepa_ is a system accepted by international agencies as a tool for analyzing the cytotoxic effects of different products [44]. Plants have self-defense systems that become weakened when submitted to stress, which can be a triggering factor of disease [23]. Using the genotoxicity test, it was possible to test the safety of mouthwash formulations. Due to the absence of micronuclelei in cells exposed to the formulations, we can conclude that the biosurfactants used in this study are reliable and harmless to human cells as they do not cause cellular changes. In contrast to natural biosurfactants, the synthetic surfactants tested, Tween 20 and SDS, induced alterations in onion cells, contributing to the formation of micronuclelei, which are indicators of cellular toxicity. Other studies have highlighted the toxic potential of chemical surfactants, such as the presence of micronuclelei in samples treated with Tween 80 and Tween 20, which are examples of synthetic surfactants widely used in cosmetic products [45]. The essential oil from spearmint (_Mentha spicata_) was used in our formulations, as the literature states that this essential oil has antimicrobial activity against _S. aureus_, _Pseudomonas aeruginosa_, _Candida albicans_, and other target microorganisms [46]. Moreover, spearmint essential oil gives the mouthwash a fresh taste and aroma characteristic of oral hygiene products; in mouthwash formulations with biosurfactants and chitosan hydrochloride, the essential oil (_Mentha spicata_) was in a sub-inhibitory concentration, thus being considered a flavoring agent, rather than an active antimicrobial agent. Minimum inhibitory concentrations were obtained for all microorganisms tested, with the best antimicrobial action for Gram-positive bacteria and _C. albicans_. The antimicrobial action of plant-derived biosurfactants has previously been described in the literature [22]. This test is extremely important to find the exact amount of the test substance that causes the inhibition of a certain microorganism. In the case of this present study, the substances tested were mouthwash formulations containing vegetable biosurfactants, which were exposed to different microorganisms including _Streptococcus mutans_, the main etiological agent of dental cavitation. In this respect, the formulations showed excellent effectiveness against the microorganisms tested, especially _S. mutans_, whose growth was significantly inhibited even when using them in small amounts.