CA, USA) prior to cryosectioning. Several hair cross-sections (5 μm) were collected for image analysis. Hair cross-section images were collected on a Leica DM8 (Leica biosystems, Wetzlar, Germany), equipped with the Leica DFC3000 G camera. Images were acquired using the Leica Application suite X (LAS X) software. Each image was recorded using two separate channels: green (Excitation:460–500 nm/DC:505 nm/Emis- sion:512–542 nm), exposure 150 ms, and red (Excitation:532– 558 nm/DC:565 nm/Emission:570–640 nm), exposure 2 ms. Scanning electron microscopy Scanning electron microscopy (SEM) images were obtained using a high-resolution field emission scanning electron microscope FEI XL30 FEG-SEM (Philips, The Netherlands). Random fibres were chosen from selected hair bundles. A total of 15 fibres from each control group were studied. Fibres were divided in half and fixed on a stud to assure approximately 5 cm hair length analysis. The hair fibres were attached to the surface of a 2.5 cm diameter stain- less steel disc using double sided carbon tape and coated with Irid- ium to approximately 7 nm. The SEM pictures were recorded at 10 KV accelerating voltage with a working distance of ~10 mm. Hair diameter and single-fibre tensile testing studies Relaxed hair bundles were soaked in 1% w/v aqueous solutions of L-Leucine, low-MW, mid- MW and high-MW keratin peptides for 24 h while constantly shaking (HY-5 orbital shaker, Zenith Lab Inc., CA, USA). Both relaxed and virgin hair controls were soaked in DI water for 24 h while shaking as well. It was thought that this duration, although prolonged, but still relevant to the hair type, would show the changes in mechanical hair property better. After 24 h, all hair samples were rinsed with DI water for 30 s and air-dried at 20 Æ 2°C and 60% relative humidity (RH). Fibres were mounted in brass crimps, and dimension measure- ments were performed at 20 Æ 2°C and 60% RH. The maximum and minimum diameters of the crimped fibres (150 per group) were measured at 5 locations using a laser scanning micrometre (FDAS770 Fibre Dimensional Analysis System, Diastron Ltd, UK). Cross-sectional area was calculated using UVWin 3.6 build 3 soft- ware (Diastron Ltd, UK). Stress-strain curves of fibres were recorded at 20% and 80% RH (≥15 h equilibration after water evaporated from the wet state) using a MTT690 Miniature Tensile Tester (Dias- tron Ltd, UK). A total of 50 fibres from each group were extended at 40 mm minÀ1 rate. Young’s modulus was calculated by analys- ing the slope in the 0.2–1% extension region to ensure that the analysis was done in the linear region. Differential scanning calorimetry Thermal properties of hair were tested using DSC2500 instrument (TA Instruments, DE, USA). Five replicates were prepared for each sample. For this study, the same hair and equilibration conditions were used as for the fibre dimension study. Approximately 10 mg of finely chopped hair was placed in a high-pressure hermetic DSC pan together with 50 μL of DI water. The heating rate was 5°C minÀ1. Prior to testing, the instrument was calibrated using an indium standard. Statistical analysis Pearson’s chi-squared test was used to evaluate whether the observed differences in premature breakage are dependent or inde- pendent of treatments. Statistical differences of all results were analysed using JMPTM 14.0.0 analytical software (SAS Institute Inc., NC, USA). Statistically significant differences were evaluated using a Dunnett’s test at 95% confidence level via comparing each treatment group to (a) relaxed hair treated with DI water (i.e. relaxed hair control) and (b) virgin hair treated with DI water (i.e. virgin hair control). The outliers were removed using the Tukey quartile method (includes median). Graphs are presented as means Æ the respective standard errors of mean. Results Effects of peptide treatments on fibre cross-sectional area The increase in fibre cross-sectional area at ambient humidity, or lateral hair swelling, can be used to investigate the effects of hair treatments on internal hair protein structures. In fact, significant swelling provides evidence that treatments have penetrated into the hair fibre. In this study, the cross-sectional area of 150 fibres Figure 1 Cross-sectional area of fibres as a function of treatment and hair condition (means +/- standard errors). Results that are statistically different in com- parison with relaxed hair control (DI water) are labelled with the letter a, and results that are significantly different from virgin hair control (DI water) are labelled with the letter b. © 2020 The Authors. International Journal of Cosmetic Science published by John Wiley & Sons Ltd on behalf of Society of Cosmetic Scientists and Societe Francaise de Cosmetologie 3 International Journal of Cosmetic Science, 1–12 Larger hydrolyzed keratins reduce hair breakage E. Malinauskyte et al. 3800.0 3600.0 3400.0 3200.0 3000.0 2800.0 ab ab Relaxed Relaxed Relaxed Relaxed Relaxed Hair DI Hair 1 % Hair 1 % Hair 1 % Hair 1 % Water Leucine Low Mw Mid Mw High Mw Keratin Keratin Keratin Peptide Peptide Peptide Virgin Hair DI Water