## 3 Results and Discussion  
### Perming efficiency  
In this study, we evaluated the ability of different proteins (keratin, KP-UM, silk fibroin, BSA and KP-Cryst) to curl straight Asian hair. To maximize the binding of the proteins to the hair fiber, their penetration into the hair cortex and the curling effect, the proteins were incorporated into a phosphate buffer with different pH values (5, 7 and 9). To promote the swelling of the hair fibers and the lifting of the cuticles [29; 30], the hair tresses were pretreated with an urea solution followed by the ethanolic formulation prepared at pH 5, 7 and 9 prior to treatment with the proteins. To curl and fix the new shape, the hair tresses were thermally treated using a hot BaByliss for 50 s.  
With the goal of understanding if and how the proteins' conformation structure influence the protein capacity to curl Asian hair, we selected five proteins with different structural conformations: \(\beta\)-sheets (silk fibroin) [31], \(\alpha\)-helix (keratin) [15], globular (BSA) [32], \(\beta\)-barrel (KP-UM) [23] and Greek-key motifs (KP-Cryst) [24]. Protein's purity was assessed  
Figure 1: Protein purity analysis by SDS-PAGE in a 12.5% polyacrylamide gel. The tested proteins included keratin, KP-UM, BSA, KP-Cryst and silk fibroin (SF) proteins. by SDS-PAGE (Figure 1) prior to application to the hair. Analyzing the gel, high purity can be observed for the KP-UM and KP-Cryst fusion proteins. The higher molecular weight bands detected for these two proteins correspond to the respective dimmers. The presence of two cysteine residues on the KP sequence might promote the formation of these structures. The BSA presents an intense band with the expected size (66.5 kDa); however, this protein has a lower degree of purity since other lower molecular weight bands can also be observed. Both keratin and silk fibroin present diffuse patterns through the gel [18; 21], with more intense bands around 43 and 58 kDa for the keratin and around 29 kDa and higher than 209 kDa for the silk fibroin. After perming, the hair shape was analyzed relative to the number of loops and hair length (Figure 2) for the different proteins before and after each wash cycle (six cycles in total). Figure 2: Effect of hair washing with a commercial shampoo on the curliness of Asian hair. The hair tresses were treated with keratin, KP-UM, silk fibroin, bovine serum albumin (BSA) and KP-Cryst proteins dissolved in phosphate buffer (pH7) before curling using a hot BaByliss. Hair treated with a commercial formulation for perming (“Chemical”), treated with the ethanolic formulation and phosphate buffers (“EOH”), or without any treatment (“Control”) were used as controls for the proteins’ capacity to curl the Asian hair. The number of loops and the hair length were used to calculate the perming efficiency as a measure of the capacity of the keratin, silk fibroin, BSA, KP-UM and KP-Cryst proteins to curl hair and to maintain hair shape throughout wash cycles [27]. The permeing efficiency of the hair treated with the different proteins was compared with the perming efficiency of the Asian hair without treatment (Control) (Figure 3) and with the perming efficiency of the hair treated with the respective ethanolic formulation/phosphate buffer (EtOH formulation) (Figure 4). The results presented on both figures were determined using the number of loops and the fiber length after six wash cycles with shampoo. Despite the good results obtained for keratin, KP-UM and BSA proteins at pH 5 and 9, lower perme efficiencies were observed for the samples tested at pH 7. However, these seem to be related to the effect of pH on the hair fibers. Comparing the results of the hair treated with the ethanolic formulations at pH 5 and 9 with the results of the hair treated with the ethanolic formulation at pH 7, decreases of about 34% and 37%, respectively, for this sample was observed.