Enhancement of Hair Fiber Strength and Surface Morphology by Saccharomyces Lysate Assessed Using Tensile Testing and μ-CT

Abstract

Consumer demand for sustainable solutions to protect against hair damage is growing, yet quantitative studies linking molecular interactions to mechanical strengthening and structural changes remain limited. Here, we investigated the effectiveness of biotechnologically obtained Saccharomyces Lysate as a formulated active ingredient for hair care. Molecular modeling was used to explore the interactions between peptides in the lysate and keratin and suggested a network of intermolecular interactions at multiple sites on the proteins. Based on these observations, the strength and structural integrity of hair fibers treated with Saccharomyces Lysate were assessed using tensile measurements. We observed an improvement in the strength of bleached hair tresses, with an increased Young’s modulus compared to tresses treated only with water along with a significantly increased break stress. To visualize the hair fibers and their surface roughness after treatment with the lysate, we employed micro-computed tomography (µ-CT) offering high-resolution visualization of hair fibers. We introduce this method to qualitatively highlight surface appearance following application of a cosmetic product and complemented it with combing force measurements. Our results demonstrate the potential of this complex mixture of small peptides to strengthen hair integrity and we propose a hypothesis for its putative mode of action at the molecular level.

1. Introduction

In all hair types, the hair shaft comprises two components that affect hair structure and appearance, the cuticle and cortex. Large and thick hairs also have a third component called the medulla, which is the innermost layer of the hair shaft [1]. The cuticle is the outermost layer, comprising 6–8 layers of flat overlapping cells that each have a layered structure of amorphous proteins and is involved in hair surface formation [2]. The cortex is the major site of keratinization, an essential process which gives the hair shaft its rigidity via the presence of cortical cells rich in keratin filaments. There are two types of keratin fibers in hair: type I, characterized by its acidic amino acid residues, and type II, which features a high proportion of basic amino acid residues. When a type I fiber and a type II fiber are arranged in an $\alpha$-helical conformation, a dimer is formed based on ionic forces, hydrogen bonds, van der Waals interactions, hydrophobic interactions, and disulfide bonds. These dimers coil together in an antiparallel direction to form tetramers called intermediate filaments (IFs). They are oriented parallel to the long axis of the hair shaft and are embedded in an amorphous matrix with a high content of sulfur proteins called intermediate filament-associated proteins (IFAPs) [3,4,5].

The overall behavior of keratin fibers is commonly attributed to the presence of these different molecular interactions. The action of physicochemical agents used during various cosmetic treatments is considered to be the result of an interaction with these forces. Hair damage can affect these interactions, for example by altering the relative balance of disulfide and hydrogen bonds as well as the contribution of hydrophobic interactions, thereby changing the perceived hair behavior [6,7,8]. Damaged hair is a primary concern for hair care consumers and can be caused by a combination of physical, chemical, and environmental factors. Physical damage can be caused by the mechanical stresses of frequent brushing, combing, or aggressive styling, which can cause cuticle abrasion and breakage. Heat exposure from the use of hair dryers, straighteners, or curling irons at high temperatures, which weaken keratin and dehydrate hair fibers, is also a cause of physical damage. Chemical treatments such as hair coloring and bleaching are another significant reason for hair damage. These processes break down the hair’s natural pigment and alter the hair structure, making it porous and fragile [9]. Perming and relaxing chemicals also disrupt disulfide bonds in keratin, reducing elasticity and strength, while harsh shampoos strip natural oils, leaving hair dry and prone to breakage [10]. In many countries, these potentially damaging treatments are part of regular hair care routines linked to new beauty trends. Yet at the same time, today’s consumers want hair that feels truly restored—strong, resilient and radiant—with minimal effort [8,11,12].

Recently, several categories of products have emerged claiming to be bond builders. This term can be defined as an ingredient in a formulation that is able to penetrate into the hair to improve or restore its internal structure. Such products typically aim to repair the hair via cross-linking keratin fibers on a molecular level to increase macroscopic tensile strength and smoothen the hair. They are frequently associated with terms such as bond repair or hair repair [13,14,15]. Proteins, peptides, as well as small molecules have been studied as functional ingredients for hair products in this area [11,16,17]. Common metrics to quantify the effectiveness of such products include Young’s modulus, breaking force as well as combing force measurements [11,16,17]. Fluorescence microscopy was used to demonstrate that these materials can penetrate hair depending on their molecular weight [18]. An analysis of peptide interactions with keratin proteins found differences in the binding affinity based on their chemical nature. The results point to the formation of hydrophobic interactions and disulfide bonds between small peptides and human hair keratins as the main driving forces for the interactions [19]. These investigations have been done on keratin extracted from non-damaged human hair. However, chemical bleaching is a very aggressive procedure for human hair, known to cause irreversible damage to both its internal protein structure and external cuticle layers. The underlying damage results from oxidative degradation of amino acids and disruption of disulfide bonds leading to irreversible oxidized cysteine groups. Several compounds based on amino acids or peptides have been tested for their properties to restore the strength of hair. Their mode of action is explained by re-establishing disulfide bonds with smaller organic acids, amino acids, or peptides [7,20].

Inspired by previous work on the benefits of Saccharomyces Lysate in skin care [21] and the growing demand for sustainable and natural cosmetic ingredients [22,23], we examined its potential benefits in hair care. An additional rationale for selecting Saccharomyces Lysate is its fermentative origin, which provides bioactive molecules without reliance on animal-derived sources, aligning with current trends toward vegan cosmetic formulations [24,25,26]. Saccharomyces Lysate is a biotechnological product derived from Saccharomyces cerevisiae prepared by an autolysis process that results in a complex mixture of small peptides. The production of cosmetic ingredients through fermentative processes has gained traction in recent years due to their ability to provide complex compositions of active compounds, inherent naturality, sustainability, and formulation compatibility [22,23,27]. In skin care, yeast-derived matrices have been applied for their antioxidant potential, their potential for skin barrier repair, and their moisturizing properties [27,28]. In keratinocytes, Saccharomyces Lysate restored mitochondrial gene expression and energetic impairment after UV-induced stress [21]. In hair care, fermentation products derived from Saccharomyces cerevisiae and other yeasts were shown to relieve subjects suffering from sensitive scalp symptoms by restoring microbial homeostasis [29]. Vesicles derived from Saccharomyces cerevisiae have been shown to promote hair growth through enhancement of dermal papilla cell viability and metabolic activity [30], while other yeast-derived ingredients improve it by ameliorating oxidative stress in dermal papilla cells [31]. Altogether, knowledge on the effects of Saccharomyces Lysate in hair applications, especially in the area of hair fiber strengthening and morphological changes, remains elusive.

Despite widespread claims of hair bond repair in the cosmetic industry, scientific studies linking molecular interactions to mechanical and structural hair properties remain limited. Our work contributes to closing this gap by examining a complex mixture of peptides in Saccharomyces Lysate using molecular simulations, tensile testing, combing force measurements, and 3D imaging. The results suggest a strengthening effect in bleached hair. In addition, we demonstrate the potential of µ-CT as a visualization tool to support the assessment of hair surface condition in response to cosmetic treatment.

2. Materials and Methods

2.1. Peptide Analysis and Molecular Modeling

To better understand the mode of action of the Saccharomyces Lysate tested, a detailed peptide analysis was conducted. The LC instrument setup consisted of a Thermo Scientific Vanquish Horizon UHPLC system (Thermo Fisher Scientific, Waltham, MA, USA). Chromatographic separation was performed using a Waters ACQUITY UPLC BEH C18 column (1.7 µm, 2.1 mm × 150 mm) (Waters, Wilmslow, UK). The mobile phase was composed of 0.1% formic acid in water (v/v) (A) and 0.1% formic acid in acetonitrile (v/v) (B). The column temperature was maintained at 40 °C, and linear gradient elution was performed at 0.4 mL/min starting at 0% B for 3 min, increasing to 18% B over 12 min, then increasing to 100% B in 2 min, and subsequently staying there for another 1 min. The column equilibration time was 2 min.

MS detection was performed with a Thermo Fisher Orbitrap Exploris 240 (Thermo Fisher, Waltham, MA, USA) using electrospray ionization (ESI) in positive-ion mode, applying ddMS2 and AcquireX with background exclusion. PEAKS studio (Bioinformatics Solutions Inc., Waterloo, ON, Canada) was used for the identification of the proteins present in the lysate.

Molecular modeling comprises computational methods and theoretical techniques such as molecular mechanics or quantum mechanics used to simulate, visualize, and predict the physical structures, properties, and behaviors of molecules [32]. Here, from the peptides identified in the lysate, we selected those containing at least one cysteine residue and showing a quantifiable chromatographic area for analysis on the molecular level. The atomic structure of the keratin heterodimer consisting of K35 and K85 was obtained from the literature [33]. The protein structure was treated with the default settings of the protein preparation module in the Schrödinger Small-Molecule Drug Discovery Suite [34]. The peptide sequences were built as three-dimensional molecular models in extended conformation. The structures were protonated at physiological pH and optimized using the PROPKA algorithm and the OPLS4 force field in the LigPrep protocol before they were docked to the complete model of keratin using the Glide SP [35] docking protocol.

2.2. Tensile Testing

Tensile measurements on human hair assess the strength, elasticity, and structural integrity of hair fibers and are among the most fundamental mechanical tests in hair fiber science. Tensile strength is the maximum force a hair fiber can withstand before breaking. It reflects the integrity of the cortex (the main load-bearing structure), the degree of chemical damage (bleaching, dyeing, perming), and any loss of structural proteins. The higher tensile strength is, the healthier and less damaged hair is, while lower tensile strength is a sign of weakened fibers. Human hair behaves like a composite elastic material. Tensile testing produces a stress–strain curve, comprising the elastic region, where hair stretches and returns to original length, the plastic region, where permanent deformation begins, and the break point. Tensile tests are extremely sensitive to chemical modifications. Bleaching, for example, drastically reduces tensile strength and modulus, oxidative dyes cause moderate weakening, and perming/relaxing changes disulfide bond structure. In this way, tensile measurements act as a quantitative damage indicator [36,37,38].

For this test, virgin, brown, European hair (20 cm long, colour 6/0, Mischung 745 from Kerling) was used. All hair samples were obtained as commercially available standardized European human hair tresses without follicular tissue, which are pooled from multiple anonymous donors and sorted by the supplier to ensure uniformity in color and length. The hair was bleached twice with a commercial bleaching product, Wella Blondor Powder/9% Welloxon, used in a ratio of 1:2 in accordance with the product description. The mixture was homogeneously applied to dry tresses of hair that had not been washed beforehand. The hair tresses were covered with aluminum foil for 45 min and rinsed afterwards with 39 °C tap water until the water was clear. The hair tresses were then kept for 50 min in demineralized water to remove all chemicals and stop chemical reactions. Afterwards, the hair tresses were lightly dried with paper towels and left to dry overnight in a climate-controlled room at 20 °C/60% relative humidity (RH). Bleaching was then carried out for a second time following the same procedure.

Different test solutions were prepared by diluting the test substances in demineralized water at different concentrations. The test solutions were applied directly to the dried hair tresses at an amount of 0.12 g/g hair. Again, the hair tresses were dried overnight in a climate-controlled room at 20 °C/60% RH. The following samples were tested: demineralized water (placebo), a 1% formulated active ingredient in aqueous solution as supplied, 1% hydrolyzed keratin in aqueous solution, and a commercial leave-on hair mask as marketed to reflect consumer-relevant use conditions. The commercial hydrolyzed keratin product was supplied as an aqueous stock containing 15–25% hydrolysate. Saccharomyces Lysate was formulated with the following composition: Aqua, Glycerin, Saccharomyces Lysate, Valine, Threonine, Glutamic Acid, 1,2-Hexanediol, Caprylyl Glycol, Glycine, and Disodium Succinate. The commercial mask contained the following ingredients: Aqua, Alcohol denat., Propylene Glycol, Cetearyl Alcohol, Dicaprylyl Ether, Cetyl Esters, Behentrimonium Chloride, Polysorbate 20, SH-oligopeptide-78, Hydrolyzed Wheat Protein, Hydrolyzed Wheat Starch, Isopropyl Alcohol, Tocopherol, Phenoxyethanol, Potassium Sorbate, Citric Acid, Fragrance, Geraniol, Linalool, Hexyl Cinnamal, and Benzyl Alcohol.

Tensile testing was conducted at the Normec–Schrader Institute in Germany with 50 ± 1 hair fibers for each sample. The test was performed at 22 °C and 55% RH. To determine the tensile strength of hair samples, the cross-sectional areas of the 50 single hairs per sample were analyzed. Afterwards these hairs were extended until they broke. To compare the hair samples, the test parameters used were Young’s modulus of the elastic phase and break stress. As the study aimed to quantify strengthening effects within bleached, chemically damaged hair, comparisons were performed against untreated bleached hair and a placebo control to account for handling and drying effects.

2.3. µ-CT Investigation

Micro-computed tomography (µ-CT) is a high-resolution 3D X-ray imaging technique, akin to hospital CT scans but designed for smaller samples with much finer detail. In radiography, X-ray microtomography (high-resolution X-ray tomography) uses X-rays to create cross-sections of a physical object that can be used to recreate a virtual model (3D model) without destroying the original object. The prefix micro is used to indicate that the pixel sizes of the cross-sections are in the micrometer range.

µ-CT operates by transmitting a micro-focused X-ray beam through a specimen and measuring the spatially varying attenuation of the radiation as it exits the material. As the sample is rotated incrementally over 180° or 360°, a series of two-dimensional projection images is acquired, each representing the attenuation pattern from a different angle. These projections contain the integrated X-ray absorption information along the beam paths. After acquisition, the complete set of projections is processed using mathematical reconstruction algorithms to compute the three-dimensional distribution of X-ray attenuation coefficients. The result is a volumetric dataset composed of voxels whose gray values are proportional to local attenuation, thereby enabling visualization and quantitative analysis of the internal microstructure at micrometer-scale resolution without physically sectioning the material. Unlike scanning electron microscopy (SEM), µ-CT shows not only a small part of one single hair but a full bundle of hair. Although the overall resolution of µ-CT is lower than the one of SEM, it provides an overview of hair condition with a 3D visualization of the full bundle of hair in a single picture [39,40].

For this test, European, bleached hair (Nr. 814010-E-D from Kerling) was used. The hair was bleached with a commercial bleaching product, Wella Blondor Powder/9% Welloxon, used in a ratio of 1:2 in accordance with the product description. The mixture was homogeneously applied to dry tresses of hair that had not been washed beforehand. The hair tresses were covered with aluminum foil for 45 min and rinsed afterwards with 39 °C tap water until the water was clear. The hair tresses were then kept for 50 min in demineralized water to remove all chemicals and stop chemical reactions. Afterwards, the hair tresses were lightly dried with paper towels and left to dry overnight in a climate-controlled room at 20 °C/60% RH. Next, the hair tresses were soaked in the aqueous solution of Saccharomyces Lysate for 15 min and were left to dry overnight at 20 °C/60% humidity. Because µ-CT was used as a qualitative visualization method, the active was applied undiluted (aqueous solution as supplied) to maximize the detectability of surface features on the fibers and was not intended to be directly comparable to the quantitative tensile and combing experiments. These treated hair fibers were imaged using µ-CT. For this, approximately 100 to 150 hair fibers were selected for each measurement and embedded in resin glue at the outer ends. This was done to prevent any movement of the hair during the scans. The µ-CT system used for this study was a SkyScan 2214 (Bruker, Billerica, MA, USA), performed at RJL Micro & Analytic GmbH in Germany. The SkyScan 2214 system is equipped with a high-performance nano focus tube with acceleration voltages ranging from 20 to 160 kV and a maximum tube power of 16 W, which produces a cone-shaped X-ray beam that images the samples onto a fully digital detector. The CT system can scan objects with dimensions of up to 400 mm (H) × 300 mm (W), and voxel resolutions down to 60 nm. For high-resolution scans, the minimum spot size of the source can be reduced to below 500 nm. The following parameters were used during the scans: a voltage of 70 kV, a current of 150 μA, a voxel size of 0.35 μm, a rotation of 360° in 0.17° steps, an X-ray exposure time of 4950 ms, frame averaging of 3, and a scan time of 10 h.

Semi-quantitative roughness calculations were conducted for the hair tresses with one tress per treatment. To that end, the 3D datasets were loaded into Bruker’s CTAnalyser software and manipulated using the tool’s erosion/dilation algorithm. Hair fibers were first segmented from the background using a gray-value threshold to generate a binary mask. The erosion step removes pixels/voxels from the edges of selected objects, effectively shrinking each object’s boundary inward. In contrast, dilation adds pixels/voxels to the edges, expanding the objects outward by a layer. For both erosion and dilation, 5 pixels/voxels were removed and added, respectively. The roughness ratio was calculated as the ratio between the processed (smoothed) and the original segmented dataset, and the surface enlargement by hair damage was derived from the corresponding relative difference. In the current case, erosion was used to treat all datasets until a visually smooth surface was obtained. As these metrics are derived from images encompassing multiple hair fibers within a single tress, they reflect pooled surface properties at the tress level rather than isolated measurements of individual fibers. This reduces the reliance on comparisons between single hair fibers, whose surface properties can vary considerably within the same tress, and supports the qualitative interpretation of surface smoothing despite the absence of replicate tress level scans.

2.4. Combing Force Measurement: Shampoo/Tonic Treatment

Combing force measurements on human hair are a standard analytical method in cosmetic science and hair fiber research. They show how much mechanical resistance hair presents when it is combed and provide insights into the condition, surface properties, and manageability of the hair. Because combing force measurements quantify how easy or difficult it is to comb hair, they are a reflection of damage level, friction, and the performance of hair care treatments. For example, bleaching, styling, or UV radiation can cause the hair surface to become rougher due to more lifted or broken cuticles. Combing force is directly related to friction between hair fibers, with higher forces indicating rougher, higher-friction surfaces and lower forces indicating smoother, more aligned cuticles. In wet hair the adhesion of water molecules to the hair surface leads to increased fiber–fiber contact and, consequently, increased surface roughness. Wet combing forces are therefore generally higher than dry combing forces, but both values give a strong indication of a formulation’s potential to protect hair. In addition, high combing forces correlate with an increased likelihood of fiber breakage, more split ends, and greater hair loss during grooming. So, the measurements also help predicting damage during real life combing [41,42,43].

For this test, European, bleached hair (colour 10/0, 20 cm, from Kerling) was used. The hair was bleached with a commercial bleaching product, Wella Blondor Powder/9% Welloxon, used in a ratio of 1:2 in accordance with the product description. The mixture was homogeneously applied to dry tresses of hair that had not been washed beforehand. The hair tresses were covered with aluminum foil for 45 min and rinsed afterwards with 39 °C tap water until the water was clear. The hair tresses were then kept for 50 min in demineralized water to remove all chemicals and stop chemical reactions. Afterwards, the hair tresses were lightly dried with paper towels and left to dry overnight in a climate-controlled room at 20 °C/60% RH.

In the second step, the hair tresses were treated with the test materials incorporated into a shampoo formulation and a tonic formulation (

Table 1. Test shampoo formulations.

INCI	(1a) Weight-%	(1b) Weight-%	(1c) Weight-%
Aqua	57.65	56.65	56.65
Aqua, Polyquaternium-10, Sodium Acetate, Sodium Chloride, Isopropyl Alcohol	0.10	0.10	0.10
Aqua, Sodium Laureth Sulfate	35.00	35.00	35.00
Aqua, Cocamidopropyl Betaine	5.00	5.00	5.00
Sodium Benzoate	0.50	0.50	0.50
Lactic Acid, Aqua	0.25	0.25	0.25
Sodium Chloride	1.50	1.50	1.50
Aqua, Saccharomyces Lysate, Valine, Threonine, Glutamic Acid, Glycine, Glycerin, Disodium Succinate, 1,2-Hexandiol, Caprylyl Glycol	–	1.00	–
Hydrolyzed Keratin	–	–	1.00

and

Table 2. Test tonic formulations.

INCI	(2a) Weight-%	(2b) Weight-%	(2c) Weight-%
Aqua	64.85	63.85	63.85
Pentylene Glycol	5.00	5.00	5.00
Polyquaternium-37	0.15	0.15	0.15
Alcohol	30.0	30.0	30.0
Aqua, Saccharomyces Lysate, Valine, Threonine, Glutamic Acid, Glycine, Glycerin, Disodium Succinate, 1,2-Hexandiol, Caprylyl Glycol	–	1.00	–
Hydrolyzed Keratin	–	–	1.00

). For combing force experiments, the active ingredient as well as the benchmark were incorporated at 1% in both the shampoo and tonic formulations, matching the defined nominal inclusion level used in tensile testing. While tensile testing used a direct application from an aqueous solution to isolate intrinsic strengthening effects under controlled conditions, combing was conducted using finished formulations to mimic consumer-relevant use conditions, which can influence deposition and surface friction.

The hair tresses were washed with their respective test shampoos at an amount of 0.12 g shampoo/g hair for 30 s with gentle movement between the fingertips. Then, they were rinsed for 30 s with tap water at 37 °C. This procedure was repeated once more. Next, the test tonic was applied to the wet hair at an amount of 0.12 g tonic/g hair and the hair tresses were placed in the climate chamber for 4 h at 40 °C/60% RH. The full process of treating twice with shampoo and once with tonic was repeated 4 times (4 cycles). After the last treatment with the test tonic, the hair tresses were dried slightly with paper towels and combing force measurements on wet hair were taken immediately. After the wet-hair combing force measurements, the hair tresses were dried overnight in a climate chamber at 20 °C/60% RH and combing force measurements on dry hair were then taken.

Combing force measurements were taken using an INSTRON 5542 texture analyzer equipped with a pneumatic clamp and the INSTRON Bluehill 2 software. For combing force measurement, a special rack with an 80 mm dust comb with 10 teeth/cm was applied to the machine. The hair swatch was fixed in the pneumatic clamp and pulled through the comb at a velocity of 500 cm/min. The routine was set to 10 measurement cycles. Combing force measurements using the Instron system were taken in a climate chamber at 20 °C/60% humidity.

2.5. Treatment Selection and Statistical Analysis

The experimental setups in this work probe different hair attributes and therefore use method-appropriate application formats and vehicle-matched placebos. Tensile testing was conducted using aqueous solutions to quantify intrinsic single-fiber strengthening under controlled conditions and, accordingly, demineralized water served as the placebo control (with untreated bleached hair additionally included to account for handling/drying). Combing force testing is governed by surface friction and is affected by deposition effects; therefore, it was conducted using finished shampoo and tonic formulations to reflect consumer-relevant use conditions. Hence, the corresponding formulation bases without active served as placebo controls. µ-CT was applied as a qualitative proof-of-concept visualization method. Therefore, Saccharomyces Lysate was applied as supplied to maximize detectability of micrometer-scale surface features, and the µ-CT data are not intended for direct quantitative comparison with tensile or combing outcomes.

Regarding the statistical evaluation, linear mixed-effects models were fitted to the wet and dry combing data using the lmer function from the lmerTest package [44]. The models included product and measurement cycle as fixed effects and hair tress as a random intercept to account for repeated measurements on the same tresses. As combing force reflects the collective frictional behavior of fibers within a tress, the hair tress was considered the experimental unit, and repeated combing cycles were not treated as independent observations. For dry combing, tests were performed on two different days; therefore, the test date was also included as a fixed effect. In our combing experiments, a substantial proportion of the total variability arose from repeated measurements within the same tress rather than from differences between tresses, supporting the use of repeated measurements to improve estimation precision.

Estimated marginal means (EMMs) for each product were obtained using the em-means package. Pairwise comparisons between products were performed with Tukey adjustment to control for multiple testing. Model assumptions, including homoscedasticity, normality of residuals, and the distribution of random effects, were assessed using diagnostic plots (residuals versus fitted values, Q–Q plots, and random-effects diagnostics). No violation of the model assumptions was detected. Model-based 95% confidence intervals were computed for EMMs, and compact letter displays were generated to visualize overlapping and distinct treatment means.

For Young’s modulus and break stress, products were compared using pairwise Wilcoxon rank-sum (Mann–Whitney U) tests. To account for multiple pairwise comparisons, p-values were adjusted using the Bonferroni correction. This non-parametric approach was selected due to the potentially non-Gaussian distribution of the tensile measurements. Tensile testing was performed on 50 ± 1 independent hair fibers per treatment, with the individual hair fiber considered the experimental unit, as tensile properties are measured on single fibers and are known to exhibit substantial inter-fiber variability. This sample size is consistent with established practice in hair fiber mechanics and cosmetic science [18,45] and allows robust estimation of Young’s modulus and break stress. All statistical tests were two-sided, and a significance level of 5% was used. All statistical analyses in this work were conducted in R.

3. Results

3.1. Peptide Analysis and Molecular Modeling

Saccharomyces Lysate is an aqueous solution with an overall protein content of 0.8% and a clear to light-yellow color. It is obtained biotechnologically from Saccharomyces cerevisiae yeast cultures. Its INCI is Aqua, Glycerin, Caprylyl Glycol, 1,2-Hexanediol, Saccharomyces Lysate, Valine, Threonine, Glutamic acid, Glycine, and Disodium Succinate. The natural origin content of the product is 98% according to ISO16128. Peptide analysis was performed with a UPLC/HR-MS system. Characterization and quantification using proteomics revealed 725 different peptides of different chain lengths, all with a molecular weight below 2570 Dalton. The number of peptides per peptide length is given in

Table 3. Number of peptides of different lengths.

Peptide Length	No. of Peptides
Di-, tripeptides	76
Tetrapeptides	88
Penta-, hexapeptides	156
>Heptapeptides	405

. Further, we identified single amino acids including phenylalanine, glutamine, proline, arginine, isoleucine, leucine, and tryptophan in addition to the ones present in the formulated active ingredient in the UPLC-MS dataset.

From the peptides identified in the lysate, 19 peptides containing at least one cysteine residue were selected. The peptides selected for modeling have a molecular weight between 807 and 1315 Dalton and a length between 7 and 12 amino acids. Computational modeling was used to explore the putative mode of action of these peptides at molecular level. Molecular docking was used to predict their binding orientation to a keratin heterodimer in crystallized form.

In the simulation we identified two major findings. As we had chosen only peptides with a cysteine residue, we expected the docking simulation to predict that these peptides preferably attach to areas of the keratin with free cysteine residues. However, this was not the case and no direct interaction between cysteine residues was observed. On the other hand, the simulations showed three specific hotspots where the peptides could potentially associate with the keratin fibers. The peptides underwent various molecular interactions such as hydrogen bonds, salt bridges, aromatic interactions, and van der Waals contacts (Figure 1a). Together, the predictions suggest the formation of a network of intermolecular interactions (Figure 1b).

3.2. Tensile Test

Two parameters were measured to describe hair strength in our study: Young’s modulus and break stress. Young’s modulus (elastic modulus) measures the stiffness of the hair fiber—how much it resists deformation under stress in the elastic region (before permanent deformation). The typical range for human hair is between 2–6 GPa (gigapascals), depending on factors such as moisture, hair type, and treatment. The higher the modulus, the stiffer the hair. Break stress (ultimate tensile strength) also includes break extension and is the maximum stress a hair can withstand before breaking. For healthy human hair, the typical range is between 0.15–0.25 GPa. These endpoints give insight into the health of the cuticle and cortex and the structural damage of the keratin. In our tensile test study, the results for the test substances were compared to untreated bleached hair and to bleached hair treated with demineralized water only (placebo) as shown in Figure 2.

The aqueous solution of 1% Saccharomyces Lysate increased the strength of the hair tresses in the elastic region with a Young’s modulus 7% higher than for hair tresses treated with water only. Break stress increased significantly, with a 22% higher value compared to the placebo (p < 0.001). Notably, the optimized finished market leave-on mask, which was tested as is, showed a much lower impact on break stress. This result shows that a mixture of peptides, such as those in the Saccharomyces Lysate solution, can improve the strength of hair in a statistically significant way.

3.3. µ-CT

The use of µ-CT in this study to visualize hair fibers and differences on the surface following treatments was intended as proof of concept for the method. Figure 3a shows hair that was purchased as standardized bleached hair and in Figure 3b this hair was treated again with commercially available bleaching agents. The images qualitatively highlight that the surface of the additionally bleached hair fibers is damaged in comparison to the initial hair tresses and has rough areas.

Following treatment with Saccharomyces Lysate these hair fibers appeared noticeably smoother on the surface, consistent with reduced micrometer-scale surface irregularities (Figure 4).

In addition to the above visualization, calculations were carried out on the 3D dataset of the images to examine potential changes in the hair’s surface roughness upon treatment. The roughness ratio shown in

Table 4. Surface enlargement calculated via µ-CT analysis.

Tress No.	Treatment	Roughness Ratio	Surface Enlargement by Hair Damage
Hair tress 1	European bleached hair	0.9900	1.00%
Hair tress 2	European bleached hair, 1× bleached	0.9652	3.48%
Hair tress 3	European bleached hair, 1× bleached and treated with Saccharomyces Lysate	0.9863	1.37%

The given values are semi-quantitative and not intended as statistically validated roughness measurements. Each measurement was done for a single hair tress (n = 1) with the respective treatment.

is the ratio between the eroded and original datasets. Due to the single sample that was analyzed, the results of this numerical analysis were not subjected to statistical inference. The data shows that the additional bleaching step increased the surface area/roughness of the hair fibers. After treatment with Saccharomyces Lysate, the apparent increase in surface damage was markedly reduced, based on the semi-quantitative surface enlargement. This effect could also be visualized using random cross-sections from the recorded data cubes as shown in Figure 5. The outer surface of the hair fibers of the tress treated with Saccharomyces Lysate appears less tattered compared to the bleached hair with no further treatment. The surface-smoothing properties of Saccharomyces Lysate are visibly demonstrated by comparing the surface roughness of the different hair fibers. In analogy to SEM work in other studies on hair surface properties [18,46], the µ-CT analysis presented here is intended as a qualitative proof-of-concept visualization and does not aim to provide statistically validated or absolute quantitative surface roughness measurements.

3.4. Combing Force Measurement

Our results show that after four treatments with a shampoo and tonic containing 1% Saccharomyces Lysate, the combing force on wet hair was significantly reduced by 22% compared to the placebo (Figure 6). Additionally, on dry hair, a statistically significant reduction in combing force of 18% compared to placebo was observed. The 1% hydrolyzed keratin treatment also showed a reduction in combing force, although the effect was slightly less pronounced than that observed for the 1% Saccharomyces Lysate.

4. Discussion

4.1. Mechanical Hair Damage and Compensatory Strengthening Mechanisms

The weight of hair fibers consists of 65–95% proteins and up to 32% water, depending on the humidity, with the rest accounting for lipids, pigments, and other components. Chemically, therefore, the properties of human hair are dominated by $\alpha$-keratin. It has been demonstrated that the tensile properties of hair are mostly produced by the cortex, not the cuticle [13,47,48]. This is why products that address hair damage including Saccharomyces Lysate mainly target keratin proteins in the cortex [20,49]. In our work, we focused on damage in bleached hair. During the bleaching process, the disulfide bonds are split and oxidized by hydrogen peroxide. Because the oxidation of the sulfur groups to cysteic acid is an irreversible process, hair repair products cannot restore oxidized disulfide bonds. The bleaching process also changes the overall conditions in the keratin matrix, and hydrogen bonds play a greater role in this environment [50,51]. Overall, this means that other modes of action are required to restrengthen the weakened hair fibers. Evaluating virgin unbleached hair as an additional reference could provide further insight into the extent to which treatment effects restore mechanical as well as surface properties and could be considered in future studies beyond this work.

Wortmann et al. concluded from their DSC investigations that the thermal stability of helical structures, i.e., the denaturation temperature, is controlled by the amount and the cross-linked density of the surrounding non-helical matrix material [52]. Furthermore, in the literature, the models for the mechanics of $\alpha$-keratin fibers mention the contributions of hydrogen bonds, ionic interactions, and disulfide bonds. When looking at the role of the matrix on fiber elasticity, the contributions of various forces mainly relate to Young’s modulus of the matrix. Although all models mention the three types of interactions as dictating fiber mechanical behavior, there is no direct assay to quantify them [6]. Breakspear et al. reported that when tensile tests were conducted under wet conditions, Young’s modulus appeared to be dependent entirely on disulfide bonds, but under dry conditions this dependence was less pronounced or other bonds contributed under these conditions. Further, it has been estimated that in keratin fibers there is an approximate ratio of nine hydrogen bonds to one disulfide bond. This ratio means that hydrogen bonds may have a significant effect on the mechanical behavior of hair fibers [6]. Quantitatively, our observed increase in break stress of 22% relative to the placebo at 55% RH is comparable to literature reports in which keratin peptides resulted in tensile strength improvements of 16–19% at 20% RH and 32–40% at 80% RH in one study [18], as well as in the prevention of a UV-induced tensile strength loss of 14% in a different study [17]. As hair mechanics are strongly humidity-dependent, the absolute magnitude of tensile effects observed at 55% RH may differ under other environmental conditions, warranting validation across a broader humidity range for a comprehensive comparison.

4.2. Molecular and Structural Basis of the Observed Effects

Based on the observed improvement in mechanical properties, combing behavior, and macroscopic smoothing, Saccharomyces Lysate appears to have a strengthening and conditioning effect on bleached hair fibers. Because there are many peptides present in the lysate, it is likely that several contribute to the observed benefits.

Several computational models have already been developed to perform molecular simulations either to describe the influence of hydrogen bonds on the interaction between keratin fibers or to predict the binding affinity of peptides [45,46,49,53]. Modeling enables the study of atomic-level interactions and has been used here to investigate the potential interactions of the peptides present in Saccharomyces Lysate with keratin. Penetration of peptides into the hair cortex is a prerequisite for interactions with keratin. Different techniques have been used to analyze the penetration of actives into hair fibers [18,54]. According to E. Malinauskyte et al. peptides can penetrate the cortex of hair fibers up to a molecular weight of 2570 Dalton. Another study showed several peptides with a molecular weight below 3000 Dalton to penetrate the hair cortex using fluorescent microscopy [45]. The peptides we analytically characterized in our study fall within the molecular weight range reported to be compatible with penetrating the amorphous matrix of hair fibers [18].

Hence, our proposed molecular hypothesis provides a plausible explanation of how a compensatory effect takes place, leading to the measured effects on the hair fiber. Along with the molecular interactions such as hydrogen bonds, ionic and hydrophobic interactions, the displacement of water molecules from the matrix through peptide binding could possibly increase the strength of the hair [13,55]. This is supported by the tensile measurements for the complex peptide mixture of Saccharomyces Lysate in our study. In accordance with our computational analysis, recent work reported the discovery of a tripeptide and showed non-covalent interactions such as hydrogen bonds to be the main driver for the observed hair-strengthening effect [46]. The results of the molecular analysis may also be relevant to the IFAP region, which is not accessible to structural modeling. However, to substantiate the proposed mode of action, future work will be needed to provide analytical evidence of the penetration of peptides from Saccharomyces Lysate into the cortex and the binding events to keratin proteins. Importantly, the computational work presented here is not intended to provide experimental proof of causality as direct binding would have to be validated biochemically or through structure elucidation.

Because the test material represents a formulated cosmetic active ingredient, rather than the isolated lysate, the observed effects should be interpreted at the ingredient level and cannot be uniquely assigned to a single component. The additional low-molecular-weight components are commonly used in cosmetic formulations as auxiliary ingredients and were not reported to induce measurable hair fiber strengthening when applied alone except for certain amino acids [56] which are present in the lysate as shown in our analytical characterization. Further, while glycerol can even decrease tensile strength [57], disodium succinate is typically used to adjust the pH or as surfactant [58], 1,2-hexanediol is used as a multifunctional humectant [59], and caprylyl glycol is used as an emollient [60]. Nevertheless, the effects reported here are attributed to treatment with the formulated Saccharomyces Lysate ingredient.

4.3. Surface Smoothing, Functional Performance, and Methodological Implications

For consumers, ease of combing wet and dry hair after washing is an important parameter and they tend to correlate ease of combing with smooth and healthy hair [42]. Although hydrolyzed keratin was the gold standard for hair repair, it has become less desirable for consumers as it is derived from animals [24,25]. Since Saccharomyces Lysate is produced biotechnologically, it offers an attractive alternative for significantly improving combability in wet and dry conditions as shown in our data, providing consumers with a tangible feeling of repaired hair [61]. This consumer benefit is largely dependent on the surface properties of hair, especially its smoothness [43], which can be assessed with imaging techniques such as µ-CT which offers high-resolution 3D visualization. Accordingly, our conclusions regarding functional surface smoothing are primarily supported by the statistically evaluated wet and dry combing force measurements, with µ-CT serving as complementary visual evidence. Treatment effects are interpreted within each method relative to its matched placebo or vehicle and, therefore, effect magnitudes are not intended to be quantitatively compared across tensile testing, combing force measurements, and µ-CT visualization. Future work will be needed to quantify numerical readouts with a larger number of hair samples and samples from donors with different ethnicity. Previously, the method has been used to produce high-resolution 3D images of low-curled, medium-curled and high-curled hair fibers in their natural form [62]. We have applied this technique for the first time, to the best of our knowledge, to visualize hair fibers before and after treatment with a cosmetic active ingredient for hair care. As proof of concept for the method, we used µ-CT to visualize hair fibers and differences in surface roughness. The use of µ-CT was intended as a qualitative method for visualization. Based on the images taken, we determined that the hair fibers appeared notably smoother on the surface after treatment with Saccharomyces Lysate and the calculation of the surface roughness supported the result in accordance with our combing force measurements on bleached hair. The combability improvement is in accordance with SEM-based studies reporting that cuticle lifting and surface roughness are associated with increased friction and tangling, implying increased combing force, and show that peptide-containing treatments can improve cuticle integrity and smoothness [43,46].

Consequently, µ-CT provides non-destructive 3D visualization of the hair and could offer additional options to analyze internal and external hair structures. Compared to studies showing improved surface properties after peptide treatment using SEM as a visualization tool [18,46], our use of µ-CT allows the unbiased visualization of a complete hair tress across many fibers simultaneously, providing a more representative overview of surface condition than SEM albeit at a lower spatial resolution [40]. We were thus able to show that µ-CT is a promising method suitable for testing the performance of new hair care products.

5. Conclusions

Hair damage caused by physical, chemical, and environmental stressors is an ongoing concern for many consumers. In this study we highlight the effectiveness of a biotechnologically derived hydrolysate as an active ingredient for improving hair integrity after chemical damage. By combining molecular modeling with tensile strength tests, combing force measurements, and µ-CT, we provide a comprehensive assessment linking molecular interactions to macroscopic hair behavior. Tensile measurements presented an improved strength compared to placebo, indicating a compensatory strengthening in chemically damaged hair. Although our simulations support a plausible molecular hypothesis, direct experimental evidence of peptide penetration and binding to the cortex will be required to fully substantiate this mode of action. Qualitative µ-CT imaging revealed a visibly smoother hair surface after treatment and a semi-quantitative analysis supported the reduction of surface roughness that we measured in separate combing measurements. Albeit µ-CT was applied primarily as a proof-of-concept visualization technique, the results highlight its potential as a non-destructive tool for three-dimensional evaluation of hair surface condition in response to hair care products. The combined experimental and computational work presented here contributes to a more mechanistic understanding of peptide-based hair care products and provides a foundation for future studies.