Feather keratin hydrolysates obtained from microbial keratinases: effect on hair fiber
© Villa et al.; licensee BioMed Central Ltd. 2013
Received: 4 August 2012
Accepted: 11 February 2013
Published: 18 February 2013
Hair is composed mainly of keratin protein and a small amount of lipid. Protein hydrolysates, in particular those with low molecular weight distribution have been known to protect hair against chemical and environmental damage. Many types of protein hydrolysates from plants and animals have been used in hair and personal care such as keratin hydrolysates obtained from nails, horns and wool. Most of these hydrolysates are obtained by chemical hydrolysis and hydrothermal methods, but recently hydrolyzed hair keratin, feather keratin peptides, and feather meal peptides have been obtained by enzymatic hydrolysis using Bacillus spp in submerged fermentation.
Keratin peptides were obtained by enzymatic hydrolysis of keratinases using Bacillus subtilis AMR. The microorganism was grown on a feather medium, pH 8.0 (1% feathers) and supplemented with 0.01% of yeast extract, for 5 days, at 28°C with agitation. The supernatant containing the hydrolysates was colleted by centrifugation and ultra filtered in an AMICON system using nano–membranes (Millipore – YC05). The Proteins and peptides were analyzed using HPTLC and MALDI-TOF-MS. Commercial preparations of keratin hydrolysates were used as a comparative standard. After five days the feather had been degraded (90-95%) by the peptidases and keratinases of the microorganism. MALDI-TOF mass spectrometry showed multiple peaks that correspond to peptides in the range of 800 to 1079 Daltons and the commercial hydrolysate was in the range of 900 to 1400 Da. HPTLC showed lower molecular mass peptides and amino acids in the enzymatic hydrolysate when compared with the commercial hydrolysate . A mild shampoo and a rinse off conditioner were formulated with the enzymatic hydrolysate and applied to hair fibers to evaluate the hydration, with and without heat, using a Corneometer® CM 825. The hydration was more efficient with heat, suggesting a more complete incorporation of hydrolysates into the fibers. Scanning Electron Microscopy showed deposits of organic matter in the junction of the cuticles that probably collaborates to the sealing of the cuticles, increasing the brightness and softness.
These results show that the enzymatic method to produce keratin peptides for hair care products is an attractive and eco- friendly method with a great potential in the cosmetic industry.
The major function of keratin cuticle is to protect the cortex of the hair from damage caused by several factors including heat, chemicals and daily maintenance. Keratin is a fibrous and insoluble protein with excellent mechanical properties. The chemistry of hair can also be modified by aging and by environmental factors such as pollution and sunlight . Permanent waving, straightening or relaxing, bleaching during hair coloring processes and brushing can also cause damage to hair [6, 7]. High concentrations of amino acid cysteine are responsible for its unique structure due to the strong chemical bond known as a disulfide bridge . Keratin is found in other epidermic structures such as feathers, nails, scales and horns of mammals, reptiles and birds .
Protein hydrolysates are efficient restorers in hair care processes. These active peptides are reparative and conditioning agents and provide benefits for the hair such as strengthening hair fibers and reducing fiber breakages. Oligopeptides with a molecular mass <1,000 Daltons are able to penetrate the cortex. In permanent waving and bleaching, proteins have a substantial protecting effect on the hair structure. The addition of protein hydrolysates to hair coloring sprays and toners enables hair to absorb the dyes more uniformly. In the leave-on products, a natural conditioning effect of protein hydrolysates is reported [9, 10]. Many types of proteic hydrolysates from plants and animals had been used in hair repair products and in also in skin cosmetics such as wheat protein  and keratin from nails, horns and wool . Some commercial protein hydrolysates include Nutrilan H and Crotein A (hydrolyzed collagen), Elastin P (hydrolyzed elastin) and Crotein HKP S/F (keratin aminoacids) . Most of them are obtained through chemical hydrolysis and hydrothermal methods. Recently hydrolyzed hair keratin , feather keratin peptides [15, 16], and feather meal peptides  were obtained by our lab via enzymatic hydrolysis with Bacillus spp in submerged fermentation. The use of the enzymatic process to produce hydrolysates has grown immensely in recent years because it is more successful in preserving amino acids, and it is safer for the environment than the other methods and also it is a relatively gentle process .
The aim of this study was to produce keratin hydrolysates from feathers using microbial peptidases and then incorporate them into a cosmetic formulation for hair and investigate the effect of the treatment on hair fiber previously submitted to coloration, bleaching, relaxation and highlights.
Yeast extract was obtained from Oxoid Ltd. (Hampshire, England). Reagents used in electrophoresis were purchased from Amersham Life Science (Little Chalfont, England). All other reagents were of analytical grade. The molecular mass SDS–PAGE standards were obtained from Pharmacia Biotech. A commercial hydrolysate (KH1) obtained from chemically hydrolyzed animal keratin (hair and horns) was used as control.
This was determined in the culture supernatants according to Lowry et al. , using albumin bovine serum as the standard. Readings were carried out in a spectrophotometer at 660 nm.
Feather keratin hydrolysates: enzymatic method
Bacillus subtilis AMR was inoculated in a medium containing yeast extract 0.5%, peptone 0.5%, KCl 2.0% and sucrose 2.0%. After 48 hours at 28°C the bacteria were washed three times with saline 0.85%. The inoculums (108cell/ml) were added to 1 L of feather medium (yeast extract 0.01%, KCl 2.0% and supplemented with chicken feather 1%). The feathers were obtained from poultry waste and were previously washed with water and detergent, delipidated with chloroform: methanol (1:1, v/v) and dried at 60°C before use. The culture was grown for 5 days at 28°C/ rev min−1 in an orbital shaker. The crude feather keratin hydrolysate was obtained by centrifugation for 20 min at 2000 g. The supernatant containing the hydrolysates was concentrated further by ultrafiltration (Millipore) in an Amicon YC 05 system (1000 Daltons, NMWL, Nominal Molecular Weight Limit). The hydrolysates recovered corresponded to the enzymatic keratin hydrolysates.
Keratinases and gelatinase assays
Keratinase assay was done as described by Mazotto et al. . One unit of keratinolytic activity was defined as the amount of enzyme required to produce an increase of 0.01 absorbance unit, at 280 nm, under standard assay conditions (1 h at 37°C). Gelatinases were analyzed according the method described by Cedrola et al  One unit of gelatinase activity was defined as the amount of enzyme required to produce 1 lg of peptides under the described assay conditions.
High-performance thin-layer chromatography (HPTLC)
The commercial and enzymatic keratin hydrolysates were placed on high-performance thin-layer chromatography plates (Merck silica gel 60 HPTLC). The development was carried out in butanol/acetic acid/distilled water (4:1:1 v/v/v) until the solvent front reached the top of the plate. The HPTLC plate was stained in ninhydrin reagent (7.5% in butanol/acetone 1:1 v/v). Commercial amino acids were used as the standard .
Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS)
The commercial and the enzymatic keratin hydrolysates obtained from feather fermentation by B. subtilis were identified using matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). Immediately prior to mass spectrometry, acetonitrile/water (5:95 v:v) and trifluoroacetic acid were added to the samples. The sample was loaded using a hydrated Zip tip C18, after which it was washed with water. The sample was eluted three times with acetonitrile/water (60:40) containing 0.1% trifluoroacetic acid. An equal volume of α-cyano-4-hydroxycinnamic acid (CCA) matrix was added to the sample, and 1 μl of the sample mixture was spotted directly on a MALDI target for analysis. Peptide mass mapping was carried out with a Voyager DE PRO (Applied Biosystems) mass spectrometer .
Cosmetic formulation with microbial keratin hydrolysates
Mild Shampoo composition - enzymatic hydrolysates from chicken feathers
Sodium lauryl sulfate
Laureth Decyl polyglucose
Coconut fatty acid diethanolamide
Phenochem or Phenova
Essence of anise
Distilled water qs
Rinse-conditioner composition - enzymatic hydrolysates from chicken feathers
Essence of anis
Distilled water qs
Cosmetic evaluation scheme
The treatment (mild shampoo and a rinse-conditioner containing the enzymatic keratin hydrolysate) was applied over a 5 week period to locks of virgin and chemically treated hair. The hair had been washed and defatted previous to the treatment with the shampoo and rinse-conditioner prepared with the microbial keratin hydrolysate. Then the samples underwent hair-drying and hair straightening at 180°C, in order to assess the degree of capillary fiber hydration.
The hydration measurement assays were carried out every 7 days using a Corneometer® CM 825 (Courage and Khazaka, Germany), which was mounted on a Multi Probe Adapter® MPA 5 (Courage and Khazaka, Germany). The measurement was made using the capacitance method. This method makes use of the relatively high dielectric constant of water (εr =81C2/Nm2) compared to other substances in the skin (εr <7C2/Nm2). The front surface of the sensor contains a measuring condenser. The capacitances change depending on the water content and these differences can be measured and converted into a digital value that is proportional to the skin or hair humidity. Because of the short measurement time, errors due to skin deformation or hair malleability or evaporative build-up were excluded. Ten measurements were made at different points of the hair treated with or without the peptides. The measurements were performed at a room temperature of 18-20°C and at a relative humidity ranging from 25% to 40%. The results are given in “arbitrary units” (A.U.). The Analysis of Variance ANOVA was used to statistically evaluate the final hydration of the hair structures.
Scanning electron microscopy (SEM)
To characterize the effect of the keratin hydrolysate applied onto normal and chemically treated hair the samples were observed in a scanning electron microscope before and after the treatment. Samples were put on stubs and gold sputtered and then observed in a JEOL JSM 5310 scanning electron microscope operating at 15 kV .
Results and discussion
Some reports have described the production of keratinases by Bacillus species such as B. subtilis KD-N2 ; B. pumilus KS12 , B. megaterium SN1 . However these works describe the isolation of new strains, mutant production and the characterization of keratinases suggesting its potential applications. In our study the focus was on the feather keratin hydrolysate produced by B. subtilis, specifically the peptides, and the aim of our work was to analyze the effect of the hydrolysate on hair fiber. Different methodologies for keratinases analysis have been used by other authors and this great variability makes it difficult to compare results. However the native strain of Bacillus subtilis used in the present manuscript showed an excellent proteolytic (gelatinase) activity with a production of 350 U/ml and 400 U/ml of keratinases and proteases respectively.
After fermentation, the enzymatic hydrolysate had a protein concentration of 3,5 mg/ml. After filtration the protein content went to1,5 mg/ml, corresponding to a percentage of 42, 8% relative to the total protein. The enzymatic hydrolysate was applied to the hair locks at a concentration of 10% as described in Materials and Methods and in Figure 2.
Hydration effect of the enzymatic hydrolysates on hair fiber
Types of Treatment (average – 5 weeks in AU)
1. Untreated hair
2. Colored hair
3 Colored hair with highlights
4 Colored hair with relaxer
Protein hydrolysates, in particular those with a low molecular weight distribution-i.e., < 1,000 Daltons are known to provide efficient protection and care to hair. Various sources of proteins have been used to produce hydrolysates. Wheat protein , wool keratin  and collagenous hydrolysates  are examples that have been used in skin and personal hair care products and are known to confer improved compatibility, feel, moisturization and help maintain the natural structure [12, 29]. In hair care products, the lower molecular weight peptides have two effects: 1) They are capable of penetrating the cortex of the hair fiber and 2) They can promote a surface coating. The penetration appears to be deeper with longer treatments. Besides this, bleached hair shows a higher level of penetration of hydrolysates when compared with non-damaged control hair . These properties have beneficial effects on the hair structure replacing lost keratin and also have an antiaging effect . The effect of wool keratin peptide on the skin in an aqueous or in liposome formulation was investigated by  and an increase in hydration and elasticity as a result of the keratin peptide application was observed.
When hair chemistry is modified, some of the natural properties of hair are compromised. Several mechanisms can cause damage to hair fiber. For example, environmental stresses and UV radiation photo-oxidizes proteins. Protein photo-oxidation leads to the cleavage of disulfide bonds which cross-link the proteins, and breaking of thioester bonds, which results in the release of bound surface lipids and loss of hair structure. These reactions lead to a deterioration of hair properties, noticeable to consumers in the form of poor manageability, dryness and brittleness, loss of shine and, in extreme cases, decreased strength . Some cosmetic treatments like permanent curling, permanent coloring, bleaching and relaxing/straightening are known to alter hair properties [9, 31]. Even cosmetic handling such as daily combing and brushing can damage hair . Recently, Cao et al  used different concentrations of the fermentation broth (chicken feathers) obtained from Stenotrophomonas maltophilia, in the hair. The supernatant was incubated for 30 min. The broth was found to be protective to hair, as evidenced by the improved flexibility and strength for both normal and damaged hair.
Sionkowska, et al  using UV–Vis spectroscopy, Fourier transform, infrared spectroscopy (FTIR) and fluorescence spectroscopy, evaluated the influence of UV irradiation on keratin hydrolysates. New photoproducts were formed during UV irradiation of keratin hydrolysates and a slight increase in oxidized sulfur species was also observed. The authors proposed that photodegradation of keratin hydrolysates could be a useful method for the preparation of hydrolysates with lower molecular weight. In the present work an increase in the hydration, brightness and softness was observed in the different types of hair after the treatment with 10% of keratin peptides obtained by the enzymatic hydrolysis process. The use of feathers, an industrial waste generated by poultry as a biomass source for the process is very interesting because this raw material is cheap and it is bio transformed into a new product with an aggregate value. Currently, keratin hydrolysates are usually prepared from keratin-containing animal parts, such as feathers, horns, hoofs, hair and wool. Aromatic amino acids (tryptophan, tyrosine, and phenylalanine) and cystine (amino acid containing sulfur) play a pivotal role in the photochemistry of keratin . Some industries have developed products which use a complex of 18 free amino acids derived from wheat, corn and soy proteins to mimic the natural composition of keratin. The high sulfur amino acid content of the soy is similar to that of human hair and wool . However, keratin is an irreplaceable protein in respect to its mechanical and protective properties.
The enzymatic method described in the present work can be used for industrial wastes/residues in general to produce value added products. Previous studies in the literature have described the use of keratinases /peptidases for recycling feather keratin discarded by the poultry industry [34–36]. The present work reports for the first time on the use of keratin peptides in the cosmetic industry, specially focused on the hair care segment. Taking into consideration all these factors, the enzymatic method for keratin peptide production for hair care products is an attractive and eco- friendly method with great potential within the cosmetic industry.
Our results demonstrate that the keratin hydrolysates obtained enzimatically are peptides with a molecular mass of 800 to 1079 Daltons. The keratin peptides increased the hydration of hair fiber and scanning electron microscopy analysis showed sealed cuticles in the fibers treated with the hydrolysates, which also presented a significative increase of the brightness and softness.
The enzymatic hydrolysis is an attractive method for biotechnological applications in the cosmetic industry, taking into consideration that it is an eco-friendly method, based on the sustainability and in the biotransformatiom of a protein rich biomass.
This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação Carlos Chagas Filho de Amparo á Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (MCT ⁄ CNPq). The authors are grateful to Dr. Ulysses Lins by the support with the electronic micrographs and to Denise da Rocha de Souza for technical support.
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