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

Summary

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.

Abstract

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.

Introduction

Slaughterhouses, the food industry, and the tanning industry annually produce immense amounts of solid keratin by-products – 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 h....

Protocol

Volunteers were recruited among employees and students of our university. The method of selecting was conducted according to "International Ethical Guidelines for Biomedical Research Involving Human Subjects. Council for International Organizations of Medical Sciences, Geneva (2002)." 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

1. Collect chicken feathers from a poultry farm.
2. Wash out any insoluble impurities and blood remnants from the chicken feathers with ....

Results

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 <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.

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Discussion

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 – yield of KH rises to 85%.

The fin.......

Materials

List of materials used in this article

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