This article was written with support of the project IGA/FT/2017/007 of Tomas Bata University in Zlin.

Volunteers were recruited among employees and students of our university. The method of selecting was conducted according to &#34;International Ethical Guidelines for Biomedical Research Involving Human Subjects. Council for International Organizations of Medical Sciences, Geneva (2002).&#34;&#160;KH is a common cosmetic ingredient used in hair-care products (shampoos, conditioners, etc.) and hence approval from the institutional review board is not required.
1. Process Chicken Feathers into KH
<ol>
	<li>Collect chicken feathers from a poultry farm.</li>
	<li>Wash out any insoluble impurities and blood remnants from the chicken feathers with a sufficient excess of fresh running (cold) water. Place the feathers on a flat plate and dry overnight at 50 &#176;C. 
	NOTE: The protocol can be paused here.</li>
	<li>Grind 50 g dried feathers in a cutting mill (suitable for soft to medium-hard sample materials, and fibrous materials) into a final fineness of 1.0 mm. Alternatively, the final fineness of grinded feathers can be higher, but not more than 3.0 mm. 
	NOTE: The protocol can be paused here.</li>
	<li>Degrease feathers 
	NOTE: The most effective and economic method of degreasing poultry feather is using a commercial lipolytic enzyme.
	<ol>
		<li>In a stainless steel 27-L boiler container with temperature control, mix the feathers with water preheated up to 40 &#177; 2 &#176;C in a weight ratio 1:75. Add a lipolytic enzyme (activity 100 KLU/g) in a dose of 1.5 - 2.0% (related to weighed-in dry feathers) and gently stir the contents with an overhead stirrer for 5 min.</li>
		<li>Adjust the mixture pH to 9.0 &#177; 0.2, the value corresponding to the maximum activity of the lipolytic enzyme by adding 1% NaOH or 1% H3PO4 solution. Stir the mixture for 5 min with an overhead stirrer, and then check and re-adjust the pH level using a laboratory bench pH/mV meter.</li>
		<li>Gently stir the mixture with an overhead stirrer for 24 h at 40 &#177; 0.5 &#176;C. Alternatively, incubate the mixture at 40 &#177; 0.5 &#176;C, and during the first 6 h of incubation stir the contents at 1 h intervals.</li>
		<li>Filter the mixture through a fine sieve (100 &#181;m size) and wash the degreased feathers with a stream of fresh running (cold) water. Dry the feathers on a flat plate at 50 &#176;C in a drying chamber overnight. 
		NOTE: The protocol can be paused here.</li>
	</ol>
	</li>
	<li>Perform the first stage of the chicken feather hydrolysis. Mix the feathers with 0.3% KOH water solution in a weight ratio 1:50 and gently stir with an overhead stirrer at 60 &#177; 0.5 &#176;C for 24 h. The pH of the mixture decreases from approx. 12.5 at the start of incubation to approx. 11.0 at the end of incubation. After finishing the first stage of hydrolysis, adjust the pH of the mixture to the level corresponding to the maximum activity of the proteolytic enzyme with 10% H3PO4 (in this case, to a level of 9.0 &#177; 0.2) by adding 1% NaOH.</li>
	<li>Perform the second stage of chicken feather hydrolysis. Add to the mixture, the proteolytic enzyme in a dose of 5.0% (related to dry matter of quantity of feathers weighed-in at the beginning of the first stage of hydrolysis). Gently stir with an overhead stirrer at 60 &#177; 0.5 &#176;C for 8 h and then heat the mixture (in the same stainless steel 27-L boiler container) to a boiling point (100 &#176;C) and boil for 10 min to inactivate the enzyme.</li>
	<li>Separate the solution of KH (prepared in step 1.6) from the undissolved remnant by filtering it through low-density filter paper on a Buchner funnel with slight vacuum-pressure; alternatively, use a centrifuge. 
	NOTE: The protocol can be paused here for several days if a solution of KH is stored at 5 &#177; 1 &#176;C.</li>
	<li>In the plastic bucket (26 cm diameter x 26 cm height) dialyze the KH using 12 K MWCO membrane to remove small peptides and salts. Pour 400 mL of the KH solution into dialysis tubing and dialyze it against 4 L of distilled water for 80 h at room temperature; change the distilled water after 18, 36, and 60 h.</li>
	<li>Cast a dialyzed solution of KH on an anti-adhesive plate (e.g., silicone) on a ratio of 500 mL to 1,000 cm2 plate area, vacuum dry it on a thin film at 40 &#177; 0.5 &#176;C for overnight, grind to form a fine powder, and keep it in a closed vessel in a desiccator. 
	NOTE: The protocol can be paused here for several months if the KH powder is stored in a dry place.</li>
</ol>
2. Prepare Cosmetic Formulations with KH
NOTE: The OB used for testing was a commercial hydrophilic O/W cream base and comprised of aqua, paraffin, paraffin liquid, cetearyl alcohol, Laureth 4, sodium hydroxide, carbomer, methylparaben, and propylparaben.
<ol>
	<li>Prepare formulations containing 2, 4, and 6% KH (in accordance with the base weight of the ointment). Weigh the amount of KH powder into a polyethylene vessel (7 cm diameter x 10 cm height) and add the OB at an amount that ensures the total weight of the formulation equals 50 g; see recipe in Table 1.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cosmetic formulation</td>
			<td colspan="3">Weight of ointment base [g]</td>
			<td colspan="3">Weight of keratin hydrolysate [g]</td>
			<td colspan="2">Total weight [g]</td>
		</tr>
		<tr>
			<td>Ointment base</td>
			<td colspan="3">50</td>
			<td colspan="3">0</td>
			<td colspan="2" rowspan="4">50</td>
		</tr>
		<tr>
			<td>Ointment base + 2 % KH</td>
			<td colspan="3">49</td>
			<td colspan="3">1</td>
		</tr>
		<tr>
			<td>Ointment base + 4 % KH</td>
			<td colspan="3">48</td>
			<td colspan="3">2</td>
		</tr>
		<tr>
			<td>Ointment base + 6 % KH</td>
			<td colspan="3">47</td>
			<td colspan="3">3</td>
		</tr>
	</tbody>
</table>
Table 1: Weight-in quantities of ointment base and keratin hydrolysate to prepare cosmetic formulations.
<ol start="2">
	<li>Homogenize the mixture with a 3-bladed laboratory blender for 10 min at 134.16 x g and mix using a mechanical overhead stirrer. Maintain the prepared formulations at 5 &#177; 1 &#176;C and warm them at room temperature for 2 h prior to use. 
	NOTE: Homogenizing the mixture of OB with KH can be done with a non-digital stirrer as well. On a non-digital stirrer, there are scales with the approximate speed (in rpm) as well. Gentle stirring will work the best for this step. 
	NOTE: The protocol can be paused here for up to 5 months if the formulations are stored at 5 &#177; 1 &#176;C.</li>
</ol>
3. Test the Properties of KH by Measuring Skin Hydration, TEWL, and pH
NOTE: Perform all measurements in a conditioned room at 23 &#177; 2 &#176;C and the relative humidity of 56 &#177; 3%.
<ol>
	<li>Place 5 strips of filter paper (size 2 x 4 cm) into the physiological solution (0.90% NaCl) and leave them for approximately 1 min in the solution.</li>
	<li>Apply two strips to the inner side of the right forearm, and three to the inner side of the left forearm, and fix them for 4 h with adhesive plasters. This step is to degrease the skin and eliminate individual characteristics of the skin at the site. After 4 h, remove the strips and demark the areas with a permanent pen, see Figure 1.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56254/56254fig1.jpg" /> 
Figure 1: Method for location of test formulations on the forearm of the left and right upper limbs. <a href="//cloudfront.jove.com/files/ftp_upload/56254/56254fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="3">
	<li>Apply 0.1 mL of the tested formulations at each spot of the degreased forearm sites using syringes and spread it over the entire marked surface. On the left forearm, do not apply anything to the first site (it is the control), apply the OB to the second site, and the OB + 2% KH to the third. Apply the OB + 4% KH and OB + 6% KH to the right arm.</li>
	<li>Measure each sample at each site and each interval (1, 2, 3, 4, 24, and 48 h) and take 5 readings with the skin hydration meter probe for skin hydration, 15 readings with the TEWL meter probe for skin TEWL, and 1 reading with the skin pH meter probe for skin pH. Do not allow volunteers to apply any cosmetic product to the test sites and surrounding areas during the test period; they are permitted brief evening washes with running water. 
	NOTE: The protocol can be paused here.</li>
	<li>Process the resulting readings by basic numerical characteristics of the descriptive statistics, using spreadsheet software. From the 5 hydration readings measured for each sample, ignore the lowest and the highest readings, and calculate only 3 readings for the arithmetic mean and standard deviation. From the 15 TEWL readings measured for each sample, ignore the first 5, and calculate only 10 readings for arithmetic mean and standard deviation.</li>
</ol>

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The authors have nothing to disclose.

The advantage of alkaline-enzymatic hydrolysis is that it can be modified according to future applications of KH. For example, in hair-care cosmetics applications where a lightly brownish color of a product is not an obstacle, a higher temperature in the hydrolysis can be applied leading to a higher yield of KH. In addition, the longer processing time during both stages of the technological procedure significantly affects the overall process efficiency &#8211; yield of KH rises to 85%.
The fin...

Slaughterhouses, the food industry, and the tanning industry annually produce immense amounts of solid keratin by-products &#8211; wool, feathers, bristles, hooves, claws, horns, and the like. According to latest statistical data, the total live weight of chickens, turkeys, ducks, and other slaughtered poultry in the USA is 62.5 billion pounds per year1; in the EU it is approximately 28.7 billion pounds per year. Considering that feathers make up to 8.5% of the total poultry weight, the USA alone annually produces approx. 5.3 billion pounds of waste feathers2.
Keratin is a protein exhibiting high chemical resistance because it is strongly cross-linked with disulfide bridges that render its processing difficult. Obtaining soluble products requires cleaving cross-links and possibly carrying out hydrolysis of the peptide bonds3. Cleavage of the disulfide bridges may proceed through a reaction of thiol anion according to the following pattern4,5:
Sa- + &#8211;SbSc&#8211; &#8596; &#8211;Sb- + &#8211;SaSc-
With a very high pH level, hydrolysis of the disulfide bridges also appears, in accordance with the pattern6
&#8211;SS- + OH- &#8594; &#8211;S- + &#8211;SOH
Under mild conditions (pH approx. 8), even sulfitolysis takes place according to the following pattern:
&#8211;SS&#8211; + HSO3- &#8594; &#8211;SH + &#8211;SSO3-
The most economical way of degrading keratin is microbial breakdown, which is characterized by mild reaction conditions during processing and high breakdown efficiency (approx. 90%)7,8. Keratinases are produced by some bacteria isolated from soil and keratin waste9. Microbial keratinases hydrolyze rigid and strongly cross-linked keratin structures10 and the resulting KH prepared is rich in soluble proteins, with no loss in essential amino acids detected in it11.
In order to incorporate a protein in cosmetic preparations (e.g., emulsions, lotions, and gels), the requirements ensure that such proteins are soluble in water, the given systems are transparent, and that re-aggregation of the peptides is avoided due to hydrophobic interactions. Therefore, a common practice is to apply hydrolysates of proteins, such as hydrolyzed collagen, elastin, and keratin. When adding hydrolysates into cosmetic emulsions, steps are taken to ensure that the hydrolysate is first dissolved in water. In some cases, it is desirable that the protein (or the hydrolysate) is soluble in alcohol or other organic solvents12.
KH is normally featured in shampoos, conditioners, lotions, and nutritive serums for hair, as well as mascaras, nail polish, and eye make-up agents. The KH effects declared usually include forming a protective film, smoothing the hair or nail structure, heightened plasticity and appearance of the treated formation, regulating the consistency of products, and encouraging the formation of foam13,14. It has also been shown that KH reduces surface tension, hence supplementation in cosmetics can facilitate reduction in the amount of emulsifier added to stabilize creams. KH limit the effects of irritation triggered by cleaning agents (surfactants) to the skin, eyes, and hair, thus reducing any potential side effects of cleaning agents on tissue (e.g., dehydration of the skin, hardness, and decreased barrier function of the skin). The high buffering capability of hydrolysates is also exploited to stabilize the pH of cosmetics; peptides of shorter length have a greater buffering effect15,16. Although KHs have become established as standard components in hair and nail cosmetics as well as being utilized in products for skin care, studies on the moisturizing effects of KH do not appear in contemporary literature.
Alkaline-enzymatic technology has been developed for processing keratin by-products into KH, and active testing is in process on the effects of a number of cosmetic additives17,18,19,20,21,22. The advantage of two-stage alkaline-enzymatic hydrolysis using microbial proteases for chicken feathers achieves high efficiency under mild reaction conditions and the quality of KH is very high in contrast to hydrolysis employed in strong acids or alkalis. In the first stage, feathers are incubated at a higher temperature in an alkaline environment, which partially disrupts the keratin structure and swells the feathers; after adjusting the pH, the feathers are hydrolyzed with a proteolytic enzyme under mild conditions in the second stage. The dialyzed KH possesses a high content of proteins.
The purposes of the method described here are processing poultry feathers into a KH through alkaline-enzymatic hydrolysis and testing the effect of moisturizing properties of KH applied to O/W cosmetic emulsion. The moisturizing properties are investigated by instrumental non-invasive methods in vivo. The most frequent methods for measuring skin hydration and barrier function of SC include measuring electrical properties of the skin (conductance or capacitance). Different methods for investigating SC hydration include near infrared multispectral imagining method (NIM), nuclear magnetic resonance spectroscopy, optical coherence tomography, or transient thermal transfer23. Barrier function of SC correlates to the TEWL of SC and it is measured by the ventilated chamber method, unventilated chamber method, and open chamber method24.
Properties of the model formulations are determined using the Multi Probe adapter MPA 5 with three types of probes. The first one, corneometer CM 825, measures skin hydration by assessing changes in the electrical capacity of the skin&#39;s surface; the measuring capacitor shows changes in capacitance of the skin surface in corneometric units. The corneometer gives only a relative assessment of skin hydration25. For TEWL, the second probe, tewameter TM 300, is used for measuring the density gradient of water evaporation (in an open chamber instrument based on Fick&#39;s diffusion law) from the skin indirectly by the two pairs of sensors (temperature and relative humidity) indicating the quantity of water being transported per a defined area and period of time (g/m2/h). This method can detect even the slightest disruption of skin barrier function26. Skin pH is one indicator of barrier and anti-microbial function of the SC27. The acidity of the skin mantle was measured by a (third) skin PH 905 probe connected to the MPA 5 station. This specially designed probe consists of a flat-topped glass electrode for full skin contact, connected to a voltmeter. The system measures potential changes due to the activity of hydrogen cations surrounding the very thin layer of semi-solid forms measured at the top of the probe. The changes in voltage are displayed as pH28.
We present experiments divided into three sections: (1) Preparation of KH from chicken feathers by two-stage alkaline-enzymatic hydrolysis and its purification by dialysis (removing salts and low-molecular fractions), (2) Preparation of cosmetic formulations containing 2, 4, and 6% KH, and (3) Testing the properties of KH by measuring skin hydration, TEWL, and skin pH. Testing was carried out on 10 women with the mean age of 27.2 years and on 10 men with the mean age of 26.2 years. The method of selecting the volunteers and the testing itself were conducted in accordance with international ethical principles of bio-medical research utilizing human subjects29; all persons gave their informed consent prior to inclusion in the study. Before testing commenced, the volunteers were asked to complete a questionnaire on their health status. The volunteers committed to avoid applying any cosmetic product to the test sites and surrounding regions during the 24 h prior to and during the test period; furthermore, they were only permitted brief evening washes with running water.

<table>
	<tbody>
		<tr>
			<td>Material or chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LIPEX 100T</td>
			<td>Novozymes</td>
			<td>LJP30020</td>
			<td>Lipex - enzyme produced by submerged fermentation of a genetically-modified microorganism, activity 100 KLU/g</td>
		</tr>
		<tr>
			<td>Savinase Ultra 16L</td>
			<td>Novozymes</td>
			<td>PXN40001</td>
			<td>Savinase - enzyme produced by submerged fermentation of a genetically-modified microorganism, activity 16 KNPU-S/g</td>
		</tr>
		<tr>
			<td>Potassium hydroxide, KOH</td>
			<td>Sigma-Aldrich</td>
			<td>302510289</td>
			<td>Potassium hydroxide, KOH, 97,0 %, Mr 56,11</td>
		</tr>
		<tr>
			<td>Phosphoric acid solution, H3PO4</td>
			<td>Sigma-Aldrich</td>
			<td>W290017</td>
			<td>Phosphoric acid solution, H3PO4, 85 wt. % concentration in water, Mr 98,00</td>
		</tr>
		<tr>
			<td>Sodium chloride physiological solution</td>
			<td>Sigma-Aldrich</td>
			<td>52455</td>
			<td>Tablets of BioUltra NaCl physiological solution; 1 tablet in 1000 mL of water yields 0.9 % NaCl</td>
		</tr>
		<tr>
			<td>Sodium hydroxide, NaOH</td>
			<td>Penta s.r.o.</td>
			<td>40216</td>
			<td>Sodium hydroxide, NaOH, 97,0 %, Mr 40,00</td>
		</tr>
		<tr>
			<td>AmiFarm (Cremor base-A)</td>
			<td>Fagron</td>
			<td>608425</td>
			<td>Hydrophilic oil in water (O/W) cream base; the composition: aqua, paraffin, paraffin liquid, cetearyl alkohol, Laureth 4, sodium hydroxide, carbomer, methylparaben, propylparaben.</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IKA EUROSTAR POWER control-visc stirrers</td>
			<td>IKA-labortechnik</td>
			<td>Z404020</td>
			<td>Digital laboratory stirrer, for tasks up to the high viscosity range, 230V, 1/cs</td>
		</tr>
		<tr>
			<td>IKA Propeller stirrer, 3-bladed</td>
			<td>IKA-labortechnik</td>
			<td>R 1381</td>
			<td>Propeller stirrer, 3-bladed, stirrer &#216;: 45 mm, shaft &#216;: 8 mm, shaft length: 350 mm</td>
		</tr>
		<tr>
			<td>Dialysis tubing closures</td>
			<td>Sigma-Aldrich</td>
			<td>Z371017-10EA</td>
			<td>Dialysis tubing closures, red, size 110 mm</td>
		</tr>
		<tr>
			<td>Dialysis tubing cellulose membrane</td>
			<td>Sigma-Aldrich</td>
			<td>D9402-100FT</td>
			<td>Dialysis tubing cellulose membrane, average flat width 76 mm (3.0 in.)</td>
		</tr>
		<tr>
			<td>DOMO Pot with stailess, LCD</td>
			<td>DOMO Elektronic</td>
			<td>DO42325PC</td>
			<td>Preserving boiler stainless steel, 2000 W, 27-L container (diameter 37 cm, height 30 cm), temperature control 30-100 &#176; C, operation LCD display</td>
		</tr>
		<tr>
			<td>Hettich zentrifugen Universal 32</td>
			<td>Gemini bv</td>
			<td>2770 GS1R</td>
			<td>Mid bench centrifuge, speed 18000 rpm</td>
		</tr>
		<tr>
			<td>LT 3 shaking device</td>
			<td>Fischer Scientific</td>
			<td>6470.0002</td>
			<td>Orbital shaking device</td>
		</tr>
		<tr>
			<td>KERN 440-47N</td>
			<td>Kern</td>
			<td>440-47N</td>
			<td>Laboratory balance</td>
		</tr>
		<tr>
			<td>KERN 770</td>
			<td>Kern</td>
			<td>770 -N</td>
			<td>Laboratory analytical balance</td>
		</tr>
		<tr>
			<td>VENTICELL 222 - Komfort</td>
			<td>BMT, MMM Group</td>
			<td>C 131749</td>
			<td>Drying oven, temperature control 30-100 &#176; C, air circulation control</td>
		</tr>
		<tr>
			<td>Vacucell 55 - EVO</td>
			<td>BMT, MMM Group</td>
			<td>B 050328</td>
			<td>Vacuum drying oven, temperature control 30-100 &#176; C</td>
		</tr>
		<tr>
			<td>PULVERISETTE 19</td>
			<td>Fritsch</td>
			<td>19.1030.00</td>
			<td>Universal cutting mill, rotor with V-cutting edges and fixed knives</td>
		</tr>
		<tr>
			<td>Multi Probe Adapter System MPA 5</td>
			<td>Courage &#38; Kazaka Electronic</td>
			<td>10225237</td>
			<td>MPA 5 Station - equipment for measurement hydratation, TEWL and pH</td>
		</tr>
		<tr>
			<td>Skin pH-meter PH 905 probe</td>
			<td>Courage &#38; Kazaka Electronic</td>
			<td></td>
			<td>Probe to specifically measure the pH on the skin surface or the scalp</td>
		</tr>
		<tr>
			<td>Corneometer CM 825 probe</td>
			<td>Courage &#38; Kazaka Electronic</td>
			<td></td>
			<td>Probe to determine the hydration level of the skin surface (Stratum corneum).</td>
		</tr>
		<tr>
			<td>Tewameter TM 300</td>
			<td>Courage &#38; Kazaka Electronic</td>
			<td></td>
			<td>Probe for the assessment of the transepidermal water loss (TEWL)</td>
		</tr>
		<tr>
			<td>Heidolph RZR 2020</td>
			<td>Heidolph</td>
			<td>13-225-007-03-1</td>
			<td>Overhead stirrer, mechanical speed setting and stepless transmission; speed range 40-2000 rpm</td>
		</tr>
		<tr>
			<td>Heidolph mechanical stirrer BR 10</td>
			<td>Heidolph</td>
			<td>Z336688-1EA</td>
			<td>Blade impeller crossed stirrer</td>
		</tr>
		<tr>
			<td>Fagor FS 12</td>
			<td>Fagor</td>
			<td>BTT-138</td>
			<td>Laboratory refrigerator with freezer space</td>
		</tr>
		<tr>
			<td>WTW bench pH/mV meter</td>
			<td>WTW</td>
			<td>Z313165</td>
			<td>High-performance bench pH and pH/conductivity meters for routine and high precision laboratory measurements in research or quality control laboratories</td>
		</tr>
		<tr>
			<td>Container</td>
			<td>RPC Superfos</td>
			<td></td>
			<td>13-L plastic bucket, diameter 26 cm, height 26 cm</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microsoft Office 2010</td>
			<td>Microsoft</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>C+K software</td>
			<td colspan="2">Courage and Khazaka Electronic GmbH</td>
			<td>MPA 5 station operating software</td>
		</tr>
	</tbody>
</table>

preparation of keratin hydrolysate from chicken feathers and its application in cosmetics

Keratin hydrolysates (KHs) are established standard components in hair cosmetics. Understanding the moisturizing effects of KH is advantageous for skin-care cosmetics. The goals of the protocol are: (1) to process chicken feathers into KH by alkaline-enzymatic hydrolysis and purify it by dialysis, and (2) to test if adding KH into an ointment base (OB) increases hydration of the skin and improves skin barrier function by diminishing transepidermal water loss (TEWL). During alkaline-enzymatic hydrolysis feathers are first incubated at a higher temperature in an alkaline environment and then, under mild conditions, hydrolyzed with proteolytic enzyme. The solution of KH is dialyzed, vacuum dried, and milled to a fine powder. Cosmetic formulations comprising from oil in water emulsion (O/W) containing 2, 4, and 6 weight% of KH (based on the weight of the OB) are prepared. Testing the moisturizing properties of KH is carried out on 10 men and 10 women at time intervals of 1, 2, 3, 4, 24, and 48 h. Tested formulations are spread at degreased volar forearm sites. The skin hydration of stratum corneum (SC) is assessed by measuring capacitance of the skin, which is one of the most world-wide used and simple methods. TEWL is based on measuring the quantity of water transported per a defined area and period of time from the skin. Both methods are fully non-invasive. KH makes for an excellent occlusive; depending on the addition of KH into OB, it brings about a 30% reduction in TEWL after application. KH also functions as a humectant, as it binds water from the lower layers of the epidermis to the SC; at the optimum KH addition in the OB, up to 19% rise in hydration in men and 22% rise in women occurs.

The KH prepared according to procedure presented here (see Figure 2) is yellow in color, easily soluble in water with high protein content (inorganic solids represent &#60;2.0%); the pH of the 1.0% solution of KH is 5.3, and fulfils the requirements for cosmetic-grade hydrolysates. The yield of KH from 50 g raw material is approx. 30%. The molecular weight distribution of KH was determined by SDS-PAGE and is shown in Figure 3.</p...

Watch this Scientific Journal Video about Preparation of Keratin Hydrolysate from Chicken Feathers and Its Application in Cosmetics at JoVE.com

Preparation of Keratin Hydrolysate from Chicken Feathers and Its Application in Cosmetics

The goal of the protocol is to prepare keratin hydrolysate from chicken feathers by alkaline-enzymatic hydrolysis and to test whether adding keratin hydrolysate into a cosmetics ointment base improves skin barrier function (heightening hydration and diminishing transepidermal water loss). Tests are conducted on men and woman volunteers.

Keratin hydrolysates (KHs) are established standard components in hair cosmetics. Understanding the moisturizing effects of KH is advantageous for ...

preparation-keratin-hydrolysate-from-chicken-feathers-its-application

Research

JoVE Journal

Biochemistry

19.3K Views.  Tomas Bata University in Zlín. The goal of the protocol is to prepare keratin hydrolysate from chicken feathers by alkaline-enzymatic hydrolysis and to test whether adding keratin hydrolysate into a cosmetics ointment base improves skin barrier function (heightening hydration and diminishing transepidermal water loss). Tests are conducted on men and woman volunteers.

Video: Preparation of Keratin Hydrolysate from Chicken Feathers and Its Application in Cosmetics

Preparation and Delivery of Protein Microcrystals in Lipidic Cubic Phase for Serial Femtosecond Crystallography

Membrane proteins (MPs) are essential components of cellular membranes and primary drug targets. Rational drug design relies on precise structural information, typically obtained by crystallography; however MPs are difficult to crystallize. Recent progress in MP structural determination has benefited greatly from the development of lipidic cubic phase (LCP) crystallization methods, which typically yield well-diffracting, but often small crystals that suffer from radiation damage during traditional crystallographic data collection at synchrotron sources. The development of new-generation X-ray free-electron laser (XFEL) sources that produce extremely bright femtosecond pulses has enabled room temperature data collection from microcrystals with no or negligible radiation damage. Our recent efforts in combining LCP technology with serial femtosecond crystallography (LCP-SFX) have resulted in high-resolution structures of several human G protein-coupled receptors, which represent a notoriously difficult target for structure determination. In the LCP-SFX technique, LCP is recruited as a matrix for both growth and delivery of MP microcrystals to the intersection of the injector stream with an XFEL beam for crystallographic data collection. It has been demonstrated that LCP-SFX can substantially improve the diffraction resolution when only sub-10 &#181;m crystals are available, or when the use of smaller crystals at room temperature can overcome various problems associated with larger cryocooled crystals, such as accumulation of defects, high mosaicity and cryocooling artifacts. Future advancements in X-ray sources and detector technologies should make serial crystallography highly attractive and practicable for implementation not only at XFELs, but also at more accessible synchrotron beamlines. Here we present detailed visual protocols for the preparation, characterization and delivery of microcrystals in LCP for serial crystallography experiments. These protocols include methods for conducting crystallization experiments in syringes, detecting and characterizing the crystal samples, optimizing crystal density, loading microcrystal laden LCP into the injector device and delivering the sample to the beam for data collection.

We describe procedures for the preparation and delivery of membrane protein microcrystals in lipidic cubic phase for serial crystallography at X-ray free-electron lasers and synchrotron sources. These protocols can also be applied for incorporation and delivery of soluble protein microcrystals, leading to substantially reduced sample consumption compared to liquid injection.

Membrane proteins (MPs) are essential components of cellular membranes and primary drug targets. Rational drug design relies on precise structural ...

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

Activity-based protein profiling (ABPP) is a method for the identification of an enzyme of interest in a complex proteome through the use of a chemical probe that targets the enzyme&#39;s active sites. A reporter tag introduced into the probe allows for the detection of the labeled enzyme by in-gel fluorescence scanning, protein blot, fluorescence microscopy, or liquid chromatography-mass spectrometry. Here, we describe the preparation and use of the compound ARN14686, a click chemistry activity-based probe (CC-ABP) that selectively recognizes the enzyme N-acylethanolamine acid amidase (NAAA). NAAA is a cysteine hydrolase that promotes inflammation by deactivating endogenous peroxisome proliferator-activated receptor (PPAR)-alpha agonists such as palmitoylethanolamide (PEA) and oleoylethanolamide (OEA). NAAA is synthesized as an inactive full-length proenzyme, which is activated by autoproteolysis in the acidic pH of the lysosome. Localization studies have shown that NAAA is predominantly expressed in macrophages and other monocyte-derived cells, as well as in B-lymphocytes. We provide examples of how ARN14686 can be used to detect and quantify active NAAA ex vivo in rodent tissues by protein blot and fluorescence microscopy.

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

Here, we describe the preparation and use of an activity-based probe (ARN14686, undec-10-ynyl-N-[(3S)-2-oxoazetidin-3-yl]carbamate) that allows for the detection and quantification of the active form of the proinflammatory enzyme N-acylethanolamine acid amidase (NAAA), both in vitro and ex vivo.

Activity-based protein profiling (ABPP) is a method for the identification of an enzyme of interest in a complex proteome through the use of a chemical ...

Preparation of Giant Vesicles Encapsulating Microspheres by Centrifugation of a Water-in-oil Emulsion

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological materials. Such approaches have received considerable attention in research on the boundary between living and nonliving matter and have been used to construct artificial cells over the past two decades. In particular, Giant Vesicles (GVs) have often been used as artificial cell membranes. In this paper, we describe the preparation of GVs encapsulating highly packed microspheres as a model of cells containing highly condensed biomolecules. The GVs were prepared by means of a simple water-in-oil emulsion centrifugation method. Specifically, a homogenizer was used to emulsify an aqueous solution containing the materials to be encapsulated and an oil containing dissolved phospholipids, and the resulting emulsion was layered carefully on the surface of another aqueous solution. The layered system was then centrifuged to generate the GVs. This powerful method was used to encapsulate materials ranging from small molecules to microspheres.

Giant vesicles containing highly packed micrometer-sized components are useful cell models. The water-in-oil emulsion centrifugation method is a simple, powerful tool for the preparation of giant vesicles with encapsulated materials.

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological ...

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most cases, the action of noncoding RNAs is mediated by proteins whose functions are in turn regulated by these interactions with noncoding RNAs. Consistent with this, a growing number of proteins involved in nuclear functions have been reported to bind RNA and in a few cases the RNA-binding regions of these proteins have been mapped, often through laborious, candidate-based methods.
Here, we report a detailed protocol to perform a high-throughput, proteome-wide unbiased identification of RNA-binding proteins and their RNA-binding regions. The methodology relies on the incorporation of a photoreactive uridine analog in the cellular RNA, followed by UV-mediated protein-RNA crosslinking, and mass spectrometry analyses to reveal RNA-crosslinked peptides within the proteome. Although we describe the procedure for mouse embryonic stem cells, the protocol should be easily adapted to a variety of cultured cells.

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from the oxidation of reduced compounds such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2). The complete annotation of the Arabidopsis thaliana genome has established it as the most widely used plant model system, and thus the need to purify mitochondria from a variety of organs (leaf, root, or flower) is necessary to fully utilize the tools that are now available for Arabidopsis to study mitochondrial biology. Mitochondria are isolated by homogenization of the tissue using a variety of approaches, followed by a series of differential centrifugation steps producing a crude mitochondrial pellet that is further purified using continuous colloidal density gradient centrifugation. The colloidal density material is subsequently removed by multiple centrifugation steps. Starting from 100 g of fresh leaf tissue, 2 - 3 mg of mitochondria can be routinely obtained. Respiratory experiments on these mitochondria display typical rates of 100 - 250 nmol O2 min-1 mg total mitochondrial protein-1 (NADH-dependent rate) with the ability to use various substrates and inhibitors to determine which substrates are being oxidized and the capacity of the alternative and cytochrome terminal oxidases. This protocol describes an isolation method of mitochondria from Arabidopsis thaliana leaves using continuous colloidal density gradients and an efficient respiratory measurements of purified plant mitochondria.

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

As mitochondria are only a small percentage of the plant cell, they need to be purified for a range of studies. Mitochondria can be isolated from a variety of plant organs by homogenization, followed by differential and density gradient centrifugation to obtain a highly purified mitochondrial fraction.

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from ...

Genetic Incorporation of Biosynthesized L-dihydroxyphenylalanine (DOPA) and Its Application to Protein Conjugation

L-dihydroxyphenylalanine (DOPA) is an amino acid found in the biosynthesis of catecholamines in animals and plants. Because of its particular biochemical properties, the amino acid has multiple uses in biochemical applications. This report describes a protocol for the genetic incorporation of biosynthesized DOPA and its application to protein conjugation. DOPA is biosynthesized by a tyrosine phenol-lyase (TPL) from catechol, pyruvate, and ammonia, and the amino acid is directly incorporated into proteins by the genetic incorporation method using an evolved aminoacyl-tRNA and aminoacyl-tRNA synthetase pair. This direct incorporation system efficiently incorporates DOPA with little incorporation of other natural amino acids and with better protein yield than the previous genetic incorporation system for DOPA. Protein conjugation with DOPA-containing proteins is efficient and site-specific and shows its usefulness for various applications. This protocol provides protein scientists with detailed procedures for the efficient biosynthesis of mutant proteins containing DOPA at desired sites and their conjugation for industrial and pharmaceutical applications.

Genetic Incorporation of Biosynthesized L-dihydroxyphenylalanine DOPA and Its Application to Protein Conjugation

Here, we present a protocol for the genetic incorporation of L-dihydroxyphenylalanine biosynthesized from simple starting materials and its application to protein conjugation.

L-dihydroxyphenylalanine (DOPA) is an amino acid found in the biosynthesis of catecholamines in animals and plants. Because of its particular biochemical ...

Isolation of Lipoprotein Particles from Chicken Egg Yolk for the Study of Bacterial Pathogen Fatty Acid Incorporation into Membrane Phospholipids

Staphylococcus aureus and other Gram-positive pathogens incorporate fatty acids from the environment into membrane phospholipids. During infection, the majority of exogenous fatty acids are present within host lipoprotein particles. Uncertainty remains as to the reservoirs of host fatty acids and the mechanisms by which bacteria extract fatty acids from the lipoprotein particles. In this work, we describe protocols for enrichment of low-density lipoprotein (LDL) particles from chicken egg yolk and determining whether LDLs serve as fatty acid reservoirs for S. aureus. This method exploits unbiased lipidomic analysis and chicken LDLs, an effective and economical model for the exploration of interactions between LDLs and bacteria. The analysis of S. aureus integration of exogenous fatty acids from LDLs is performed using high-resolution/accurate mass spectrometry and tandem mass spectrometry, enabling the characterization of the fatty acid composition of the bacterial membrane and unbiased identification of novel combinations of fatty acids that arise in bacterial membrane lipids upon exposure to LDLs. These advanced mass spectrometry techniques offer an unparalleled perspective of fatty acid incorporation by revealing the specific exogenous fatty acids incorporated into the phospholipids. The methods outlined here are adaptable to the study of other bacterial pathogens and alternative sources of complex fatty acids.

This method provides a framework for studying incorporation of exogenous fatty acids from complex host sources into bacterial membranes, particularly Staphylococcus aureus. To achieve this, protocols for the enrichment of lipoprotein particles from chicken egg yolk and subsequent fatty acid profiling of bacterial phospholipids utilizing mass spectrometry are described.

Staphylococcus aureus and other Gram-positive pathogens incorporate fatty acids from the environment into membrane phospholipids. During infection, the ...

Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA is a novel transcription activator found specifically in the obligate intracellular bacterial pathogen Chlamydia. Protein pulldown assays using affinity beads have revealed that GrgA binds two &#963; factors, namely &#963;66 and &#963;28, which recognize different sets of promoters for genes whose products are differentially required at developmental stages. We have used BLI to confirm and further characterize the interactions. BLI demonstrates several advantages over pulldown: 1) It reveals real-time association and dissociation between binding partners, 2) It generates quantitative kinetic parameters, and 3) It can detect bindings that pulldown assays often fail to detect. These characteristics have enabled us to deduce the physiological roles of GrgA in gene expression regulation in Chlamydia, and possible detailed interaction mechanisms. We envision that this relatively affordable technology can be extremely useful for studying transcription and other biological processes.

Application of Biolayer Interferometry BLI for Studying Protein-Protein Interactions in Transcription

Interactions of transcription factors (TFs) with the RNA polymerase are usually studied using pulldown assays. We apply a Biolayer Interferometry (BLI) technology to characterize the interaction of GrgA with the chlamydial RNA polymerase. Compared to pulldown assays, BLI detects real-time association and dissociation, offers higher sensitivity, and is highly quantitative.

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA ...

Preparation of High-Quality Fermented Fish Product

This protocol provides a method for preparation of industrialized fermented fish product with sturgeon (Aquilaria sinensis) meat product. The procedures were: (1) pretreatment of farmed sturgeon including decapitation, evisceration, skinning-off, cleaning and cutting; (2) marinating fish cubes in 6-12% (w/v) salt solution (1:1, fish cube mass to solution volume); (3) drying fish cubes to a water content of 50-60% by hot air (40-60 &#176;C) or by vacuum; (4) fermentation involving inoculating fish cubes with 0.4-1.6% (w/w) S. cerevisiae in flavor solution to fish cubes and fermenting at 25-35 &#176;C for 6-10 h; (5) sealing fish cubes in vacuum packages with marinating and fermenting solutions; (6) sterilizing at 115-121 &#176;C for 10-20 min. The sturgeon meat product prepared by this method has delicious taste which is mellow and thick, has various types and large amounts of volatile flavor compounds such as alcohols and esters which could mask musty and unpleasant odor from fish, has moderate salt content but good texture properties such as high springiness, gumminess and chewiness, and has bright russet color and attractive appearance. This new technique could also be applied in the processing of other fish to provide convenient fish snack foods which could be stored at room temperature. It is appropriate for both marine and freshwater fish.

The goal of the protocol is to provide an industrialized fish fermentation technique based on inoculation of Saccharomyces cerevisiae.

This protocol provides a method for preparation of industrialized fermented fish product with sturgeon (Aquilaria sinensis) meat product. The procedures ...

JUMPn: A Streamlined Application for Protein Co-Expression Clustering and Network Analysis in Proteomics

With recent advances in mass spectrometry-based proteomics technologies, deep profiling of hundreds of proteomes has become increasingly feasible. However, deriving biological insights from such valuable datasets is challenging. Here we introduce a systems biology-based software JUMPn, and its associated protocol to organize the proteome into protein co-expression clusters across samples and protein-protein interaction (PPI) networks connected by modules (e.g., protein complexes). Using the R/Shiny platform, the JUMPn software streamlines the analysis of co-expression clustering, pathway enrichment, and PPI module detection, with integrated data visualization and a user-friendly interface. The main steps of the protocol include installation of the JUMPn software, the definition of differentially expressed proteins or the (dys)regulated proteome, determination of meaningful co-expression clusters and PPI modules, and result visualization. While the protocol is demonstrated using an isobaric labeling-based proteome profile, JUMPn is generally applicable to a wide range of quantitative datasets (e.g., label-free proteomics). The JUMPn software and protocol thus provide a powerful tool to facilitate biological interpretation in quantitative proteomics.

We present a systems biology tool JUMPn to perform and visualize network analysis for quantitative proteomics data, with a detailed protocol including data pre-processing, co-expression clustering, pathway enrichment, and protein-protein interaction network analysis.