Extraction and characterization of bioactive keratin protein from chicken feathers and its applications /
Swati Sharma
- xv, 172 pages : illustrations (some color), charts ; 30 cm. + 1 CD-ROM
Faculty of Chemical and Natural Resources Engineering
Thesis (Doctor of Philosophy in Industrial Biotechnology) -- Universiti Malaysia Pahang – 2018
Includes bibliographical references
Keratin is a durable, fibrous protein which is mainly available in higher vertebrates (mammals, birds and reptiles) and human’s epithelial cells. The aim of this study was to extract, purify and characterize keratin from chicken feathers. The present study hypothesized that the protein rich waste biomass can be converted into value added products using chemical and enzymatic methods. The enzyme of microbial origin specifically proteases possess the property to hydrolyze the strong disulfide bridges in water insoluble proteins such as keratin. Thus, both chemical as well as enzymatic approaches to extract and separate the proteins from waste biomass were used. Prior to the extraction, the chicken feathers were cleaned by washing with Mili-Q water, detergents, ether and lastly with boiled water followed by treatment with surfactants i.e. ionic, non-ionic and cationic. The extraction was done using chemical and chemo-enzymatic method. Sodium sulfide was used as a reducing agent in the chemical method and protease enzyme was used in the chemo-enzymatic method. The precipitates obtained in the form of pellet were washed properly using Mili-Q water 2-3 times to remove foul smell and residues of sodium sulfide and hydrochloric acid. The soluble form of extracted keratin protein was prepared using sodium hydroxide. Keratin protein was purified by dialysis. Based on the SDS-PAGE analysis, the molecular weight of the keratin protein prepared using chemical and chemo-enzymatic methods were 16.5 and 14.8 KDa, respectively. The yield of keratin produced from 25 g (w/w) of feathers dry weight using chemical and chemo-enzymatic methods were 79.6 and 52.0 %, respectively. The extracted keratin was characterized using fourier transform infrared (FTIR) spectroscopy which confirms the secondary structure of keratin. Scanning electron microscopy (SEM) showed presence of spongy microporous particles. Thermo gravimetric analysis (TGA) demonstrated the 75-90% of weight loss. Differential scanning calorimetry (DSC) gave endothermic melting peak at 80 °C. Wide angle X-Ray diffraction (WAXD) illustrated that keratin is in crystalline form. The physiochemical characteristics of the feathers were compared with the keratin powder and its chemical composition, thermal strength and stability after chemical and chemo-enzymatic extraction was found to be preserved. The keratin powder was used to synthesize a hair treatment cream, biopolymeric film and nanoparticles. Two types of keratin microparticles which are KM1 and KM2 were synthesized under acidic conditions at pH 3.5 and 5.5, respectively. The microparticles were screened for their in vitro anticancer activities by SRB bioassay towards HeLa, SK-OV-3 and A549 cancer cell lines. Furthermore, their cytotoxicity towards healthy cell lines was analyzed with Malin Darby canine kidney (MDCK) cell lines along with in vitro antioxidant activity using DPPH and ABTS methods. KM1 and KM2 showed 214.16 ± 0.29 and 153.92 ± 0.61 of IC50 levels against SK-OV-3 cell lines. Moreover, KM1 and KM2 demonstrated significant antioxidant potency with IC50 levels of 13.15 and 9.02 g/mL, respectively for DPPH radical scavenging bioassay and 8.96 and 5.60 g/mL, respectively for ABTS radical scavenging bioassay. The hair cream based on chicken keratin showed a significant results when compared to the commercial cream. The calculated thickness of the biopolymeric film made from chicken keratin was 1.12×10-4 mm with tensile strength of 3.62 ± 0.6 MPa. Overall, this study presented a highly efficient method to convert the waste feather biomass into a hair treatment cream, biopolymeric film and nanoparticles which can be used in biopolymer, biomedical and pharmaceutical industry.
THE0007917(Local)
Faculty of Chemical & Natural Resources Engineering--Dissertations