## 3 Results and Discussion
### Effect of Protein Application on the Fibre Water Content  
Water molecules play a vital role in keratinized tissues such as human hair by increasing the fibers' moisture, which affects hair shine, conditioning, manageability and physical properties [39, 40]. The water content and absorption profiles could be drastically altered by daily routines or cosmetic procedures that affect the keratin structure of hair [41, 42]. Despite water influencing several properties of hair, its effect is more notorious on the mechanical behavior of hair fibers [43]. The capacity of proteins to bind water is related to the proteins' composition and conformation. The interaction between the water  
Figure 5: Hair priming resistance across six wash cycles with commercial shampoo. The resistance values were obtained by comparing the perming efficiencies after a certain wash cycle with the perming efficiencies before any washing step. molecules and the polar hydrophilic groups of proteins occurs via hydrogen bonding, with the proteins' capacity to retain moisture being dependent on the type and number of these polar groups [44]. For this study, the water content in the hair fibers was expressed in terms of the percentage of water mass relative to the total hair fiber weight. This parameter was evaluated by thermogravimetric analysis with two heating steps to evaluate the fibers' loss of internal and external water [28]. As observed in Figure 6, there are significant differences (\(p\)-value \(\leq\) 0.0001) when comparing the results of the hair samples treated with the proteins and the \"Control\" sample relative to the internal and external water content. The \"Control\" sample corresponds to untreated Asian hair, where the BaByliss was applied for a total of 50 s. Considering external water, it was observed that all of the protein formulations were able to significantly increase (\(p\)-value \(\leq\) 0.0001) the fibers' external water content, with better results obtained when the proteins were applied in the ethanolic formulation prepared at pH 9. The increase in the fibers' external water content could be justified by the proteins' ability to interact and bind water molecules via hydrogen bonding [44; 45]. Figure 6: Percentage of external and internal water contents in the hair fibers after treatment with keratin, KP-UM, silk fibroin, BSA and KP-Cryst proteins prepared in phosphate buffer (pH 5, 7 and 9) and BaByliss application for 50 s. Hair treated with a commercial formulation for perming (“Chemical”) and hair curled with the BaByliss after treatment with the ethanolic formulation and phosphate buffers (“EtOH formulation”), or without any treatment (“Control”) were used as controls. The values represent the mean \(\pm\) SD of two measurements. The data were analyzed by [PERSON]’s multiple comparison test: \(p\)-value \(\leq\) 0.0001 (\(\blacksquare\)) compared to the “Control” data. Considering the internal water content, it was observed that the 'Control' sample lost around 2-3 times more internal water content than the other samples. These results could be due to the formation of a protein-based film around the hair fibers that protect the hair against the negative effects of the high temperature procedure. This behavior was already observed for KP-Cryst protein that formed a film-like structure over Asian and Caucasian hair and protected the hair fibers against the high temperature of straightening iron devices [24]. Significative differences (\(p\)-value \(\leq\) 0.0001) for internal and external water contents were also verified when compared to hair treated with the ethanolic formulations without the proteins and with the 'Control' sample, demonstrating that the ethanolic formulation used to resuspend the proteins also play an important role in the fibers' water content after BaByliss application. The results obtained for hair permed using the chemical method also demonstrate a decrease and an increase in the internal and external water contents, respectively, when compared with the \"Control\" data.