INTRODUCTION:

Chicken feathers are profusely out there as poultry waste. Thus, it becomes another advantage to provide the environmentally friendly product [1]. Chicken feathers are the renewable and natural stuff that is produced in abundance. Million tons of these chicken feather biomass is being generated and are wasted every year by the poultry farms. The feeding of biological waste to placental is associate in nursing accepted follow and has arisen due to the requirement to scale back prices each in terms of waste disposal and meat production from farm animal [2]. These chicken feathers are difficult to dispose, however there are methods like burying and burning the feathers but these methods are not environmental friendly. Keratins are a family of enormous structural proteins found profusely in hair and fingernails. It will self-assemble into nanofilaments once placed in resolution [3]. Keratin contain many amide binding motifs that support the attachment of a large sort of cell types, almost like alternative well know proteins of living thing matrix, together with fibronectin and albuminoid [4].

The scleroprotein composed of structural proteins and together with natural products, these become simpler to soak up the wetness or wound exudates [5]. Keratin will function staple within the production of biocompatible proteinous materials, that are environmentally friendly and might be applied in textile production, building materials, films, microcapsules, sponges, bioplastics, edible materials, compostable packaging and therefore the components of composites [6]. Keratin is extracted in many ways and different techniques like oxidation, processing in ionic liquids and reduction. The economically favourable and efficient method for extraction of protein is sodium sulfide based extraction method [7]. Wasted chicken feathers are collected from the nearby poultry shop and it is pre-treated and processed. After the pre-treatment, the feathers are dried and grinded then the extraction procedure is initiated and protein precipitation and purification is carried out and confirmatory test is done for protein. As the chicken feathers are rich in keratin this is used for many purposes and also used as the protein rich dietary supplement for animals so that it may protect from the diseases which spreads through foods and feeds [8]. As this keratin is naturally extracted it can also replace the biological products which are artificially made, artificially made bioproducts has many disadvantages such as degrading very soon, it may be the pathway for some other diseases, it may get infected and it may cause some damages. So, the keratin extracted from chicken feathers are useful and it can also be environmental friendly to use. This work mainly concentrates on the nanoparticle synthesis and the characterization of chicken feathers.

Application of Keratin:

Keratin which is prepared from the wasted chicken feathers has many applications and which are listed below,

· The keratin which is extracted from chicken feathers is used as the biomaterials in tissue engineering process, and mainly used in bone grafting techniques where it is the main composition of the bone graft.

· The keratin is used for the wounds to heal quickly, where the keratin has the ability to maintain the moisture and which protects the damaged tissue. The dressing materials are made with this keratin and used for wound dressing [9].

· The keratin-based products are used in the production of cosmetics. The hair and skin products uses keratin. In hair care products the keratin is mainly used which enhances the growth of hair and strengthens the hair follicles and softens the hair.

· In skin care products such as face creams it increases the elasticity of skin and maintains the hydration of the skin.

· The products like diaper uses the absorbent materials to absorb the body fluids. These absorbents has the ability to absorb the moisture content and to keep dry and these are made artificially has the beneficial activity of rashes. So, the keratin is made into the solid dry powdery form and used in the diapers. The diapers has the different layers in which the keratin can be coated in the upper layer or the layer which is next to the outer layer. So that this keratin will prevent the area from rashes and other infections [10].

· Bio-sorbents are nothing but, In the process of bio-sorption the ability of a materials to accumulate heavy metals from the waste water. The bio material which is used in bio-sorption process is called bio-sorbents. The keratin fibers are used as the bio-sorbents for treating the waste water, where the contaminants present in the waste water will bind to the cellular surface of biomaterials [1].

· In pharmaceutical industries, the keratin plays a major role as drug delivering system. The solid keratin is coated with the nonwoven film and used, when active pharmaceutical agents are incorporated it can form a drug delivery system. These are few applications of keratin.

MATERIALS AND METHODS:

Collection of Sample:

1 kg of wasted chicken feather is weighted and collected from the neighbouring chicken shop. The feather must be collected as soon as the feather removed from the chicken. Now the bloody mixed feathers where carried into the polythene cover [8].

Pre-treatment:

The chicken feather is thoroughly washed with running tap water now the chicken feather is let it dry for about 24 hours. Now the partially dried chicken feather is washed with distilled water (H₂O) for about 15minutes and again let it dry for the whole night. Now the sample is less free from the dust particle but still the blood and greases are remains in the sample so the sample is further treated with detergent using soap solution and washed twice a time. Finally, the sample is treated with alcohol (ethanol) and wait till it get evaporated [11].

Solvent used:

Petroleum ether is used as the solvent here. Petroleum ethers are highly consist of hydrocarbon. Among all the solvent petroleum ether plays the best result. This solvent removes all the remaining blood and grease from the sample that is collected chicken feather [12]. The chicken feather is dumped into the petroleum ether for 24hours and the sample is allowed to dry for 5 days in direct sunlight.

Chemicals used:

Sodium sulphide, Ammonium sulphate, sodium hydroxide, copper sulphate, potassium hydroxide.

Mixture of solvents:

Once the sample is dried thoroughly and free from dust particles and then it’s time to grind the sample. The sample is grinded in the mixer grinder. Don’t not grind the sample for longer time make a time interval to grind a sample. The sample should be in semisolid in form. Grind it for 1min make an interval and again grind it. The sample now look like a cotton candy carefully transfer the mixture into the zip lock cover. Store it for the future.

Sample treated with sodium sulfide:

Samples were treated with sodium sulfide for a purification purpose to get a purified keratin. 10g of sodium sulfide is weighed using weighing machine.12.5g of chicken feather is weighed accurately. The weighed sodium sulfide is dissolved in 250 mL of distilled water. Now weighed chicken feather is immersed into the conical flask containing sodium sulfide mixture. Both the solution and sample should mixed thoroughly using stirrer until the feather get immersed. Now the mixture is heated in 30 ℃ for 10mins. pH is tested and pH should flow between 12 to 13. Now the mixture is kept into the shaker for 6hours with minimal rpm. The mixture is filtered using what Mann filter paper. The filtrate is centrifuged at 10,000 rpm for 5mins the centrifugation is done to remove the unwanted particle present in the sample so through centrifugation the particles trends to settle down it is considered as a debris. The supernatant is collected in a breaker separately and the debris are discarded. Hence the chicken filtrate is obtained [9].

Preparation of Aluminium sulfate solution:

The ammonium sulfate is prepared separately. This method is done for the further purification of the sample. 175g of ammonium sulphate is weighed using weighing machine. Ammonium sulfate should be crystal white in colour. No yellowish crystals are collected hence they already losing its property so collect only pure white crystals of ammonium sulfate. Now 500mL of distilled water is measured using 100mL measuring flask and transfer it into the sterile conical flask. Mix the weighed ammonium crystals with 500mL of distilled water mix them vigorously till it get dissolved completely. Incubate it for 10minutes under the room temperature [13]. Now the mixture is ready to filter, the filtration is done for sure because some impurities may present in the ammonium sulfate during the collection so this process has to done to remove all the impurities from the mixture. The filtration has to be done using what man filter paper. The filter paper is set inside the funnel so that the liquid passes through the funnel and reaches the conical flask. It takes some time to get completely filtered.

Precipitation of protein:

In order to get protein precipitation, the equal volume of filtered ammonium sulfate and chicken feather filtrate was taken in a 500mL of conical flask. So, the concentration was taken by the method of 1:1 ratio. Now 100mL of chicken feather filtrate is given and same equal volume of ammonium sulfate is also taken. The chicken feather filtrate is first taken into the 500mL of conical flask and the ammonium sulfate is added drop wise. Simultaneously rotate the flask in clock wise and continue the stirring until all the ammonium sulfate get into the flask. Now the precipitation starts occurring and its visible too it appears as light greenish colour and wait for about 10minutes till the protein precipitation settles down. The protein precipitated completely. The precipitations were transferred to do centrifuge [14]. The mixture is centrifuged at 10,000rpm for 5mins. The protein precipitation automatically settles down hence they are dense particle. The pellet was collected separately and the supernatant was discarded. The pelleted was collected separately and the distilled water is added in to it washed well again same quantity of distilled water is added and then led for centrifugation at 10,000rpm for 5minutes.

Protein purification:

Now again the pellet is collected and mixed with 2M of sodium hydroxide. To prepare 2M sodium hydroxide (NaOH), 8 g of sodium hydroxide pellets is weighed and dissolved in 100mL of distilled water (H₂O). Hence, they are exothermic in nature. Now the prepared sodium hydroxide (NaOH) is mixed with the pellets of protein precipitation. The mixture is centrifuged at 10,000 rpm for 5minutes. Finally, the supernatant was stored and the pellets were discarded. Now the supernatant is suspected to have a keratin protein hence to prove that the confirmatory test has to be done.

Confirmatory test for proteins:

Preparation of chemicals:

Both Cuso₄and KOH are taken in an equal ratio. 1g of copper sulphate is weighed and dissolved in 100mL of distilled water similarly 1g of potassium hydroxide is weighed and dissolved in 100mL of distilled water. So, both the solutions are equal in concentration.

Method:

The final supernatant of the mixture is taken from that 5mL of sample is taken in a test tube and equal volume of potassium hydroxide is mixed that is 5mL of potassium hydroxide is added into the test tube, now slowly 3 drops of copper sulphate is added slowly to the test tube shake the test tube and check the colour change.

Indication:

The sample solution is turned into violet colour which indicates the presence of protein in the sample.

Synthesis of keratin Nanoparticle using Silver nitrate and Zinc oxide:

1mM of silver nitrate was dissolved in 50mL of distilled water i.e., 0.008g of silver nitrate is dissolved in 50mL of de-ionized water. Similarly, 1mM of zinc oxide was dissolved in 50mL of distilled water i.e., 0.004g of zinc oxide is dissolved in 50mL of de-ionized water. The indication for Silver nitrate: change of colourless to reddish brown colour [10] and for Zinc oxide: change of colourless to yellow colour.

Procedure:

· Both the solution of silver nitrate and zinc oxide were taken in a different test tubes

· 5mL of each sample is added into the different test tubes followed by 5mL of silver nitrate and zinc oxide were added into the respective test tubes

· Both the test tubes were kept under the dark conditions

· Observe the colour change

Synthesis of Keratin nanoparticle using different concentrations of Silver nitrate:

The concentration of silver nitrate was varied (0.05mM, 0.10mM, 0.15mM, 0.20mM, 0.25mM). Each concentration of the silver nitrate was dissolved in 10mL distilled water containing 5 test tubes.

Procedure:

· The test tube-1 contains 5mL of sample and (0.05mM) of silver nitrate is added in 10mL of distilled.

· The test tube-2 contains 5mL of sample and (0.10mM) of silver nitrate is added in 10mL of distilled water.

· The test tube-3 contains 5mL of sample and (0.15mM) of silver nitrate is added in 10mL of distilled water.

· The test tube-4 contains 5mL of sample and (0.20mM) of silver nitrate is added in 10mL of distilled water

· The test tube-5 contains 5mL of sample and 10mL of silver nitrate(0.25mM)is added 10mL of distilled water

· Then the test tubes were covered using aluminium foil and kept under dark for 24 hours.

RESULTS AND DISCUSSION:

This work is carried out to extract the keratin proteins. Since chicken feathers are made from 90% crude protein, it is an ideal material for the production of keratin protein due to environmental problems due to long-term decomposition. Chicken feathers is first dissolved by the reducing agent, and the protein is precipitated from the solution in ammonium sulphate as shown in Fig. 1 and it is highly dependent on the pH of the solution, which is controlled by the amount of sodium hydroxide added to the solution.

Fig. 1: Precipitation of Keratin Protein

It is assumed that ammonium sulfate, precipitated protein, will be used simultaneously for protein purification. The presence of protein was first identified by a biuret test, in which the sample turns violet in the presence of a peptide bond as shown in Fig. 2 [15].

Fig. 2: Presence of Keratin Protein

Synthesis of nanoparticle:

The extracted keratin was proceeded for nanoparticle synthesis withboth silver nitrate and zinc oxide.The change of colour was observed in silver nitrate as shown in Fig. 3 which confirms the synthesis of keratin nanoparticle [16] but in zinc chloride, there is no colour change was observed. So, the silver nitrate is proceeded for synthesis of silver nanoparticle with different concentration and the concentration of keratin at 8mL and silver nitrate at 2mL showed better result while comparing to other concentrations.

Fig. 3: Presence of Silver Nanoparticle

Then, characterization of keratin nanoparticle was done by FT-IR analysis and FT-IR spectrum was recorded for silver nitrate nanoparticles. Fig. 4 shows that the bands seen at 3782 cm⁻¹ and 2279 cm⁻¹ were assigned to the stretching vibrations of primary and secondary amines respectively. The two bands observed at 1379 cm⁻¹ and 824 cm⁻¹ can be assigned to the C–N stretching vibrations of aromatic and aliphatic amines, respectively [17].

CONCLUSION:

In this work, we have concluded that the keratin was extracted from the wasted chicken feathers by various steps such as pre-treatment of feathers, then dissolving the feathers in sodium sulfide solution and protein precipitation was done using ammonium sulfate. For the confirmation of keratin, biuret test was done with the sample solution which turns to violet colour and this indicates the presence of protein. Further the nanoparticles were synthesised using silver nitrate and zinc oxide. Silver nitrate synthesised keratin nanoparticle and the characterization was done using FTIR with absorbance range 200-600nm, the peak at 415nm confirms the synthesis of silver nanoparticles.

CONFLICTS OF INTERESTS:

All authors have none to declare.

REFERNCES:

1. Priyaah K, Arun G, Swati S. Synthesis of Wound-Healing Keratin hydrogels using Chicken feathers Proteins and its properties. Int J Pharm Pharm Sci 2016;9:171-178.

2. El-Boushi, A.R.Y., van der Poel, F.B., 2000. Handbook of Poultry Feed from Waste. Processing and Use. Kluwer Academic Publishers, Dordrecht.

3. Thomas H, Conrads A, Phan KH, van de Locht M, Zahn H. In vitro reconstitution of wool intermediate filaments. Int J Biol. Macromol.1986;8:258–265.

4. Tachibana A, Furuta Y, Takeshima H, Tanabe T, Yamauchi K. Fabrication of wool keratin sponge scaffolds for long-term cell cultivation. J Biotechnol2002;93:165–170.

5. Barone JR. Lignocellulosic fiber-reinforced keratin polymer composites. J Polym Environ 2009;17:143-51.

6. Tanabe T, Okitsu N, Yamauchi Y. Fabrication and characterization of chemically crosslinked keratin films. Mat. Sci. Eng.2004;24:441–446.

7. Yin XC, Li FY, He YF, Wang Y, Wang RM. Study on effective extraction of chicken feather keratins and their films for controlling drug release. Biomater. Sci. 2013;1(5):528-536.

8. Gupta A, Kumar P, Bin Mohd Yunus R, Binti Kamarudin N. Extraction of keratin protein from chicken feather. Chemeca2011;2200:18-21.

9. Sharma S, Gupta A, Chik SM, Kee CG, Mistry BM, Kim DH, Sharma G. Characterization of keratin microparticles from feather biomass with potent antioxidant and anticancer activities. Int J Biol. Macromol. 2017;104:189-196.

10. Martin JJ, Cardamone JM, Irwin PL, Brown EM. Keratin capped silver nanoparticles–Synthesis and characterization of a nanomaterial with desirable handling properties. Colloids Surf B Biointerfaces. 2011;88(1):354-361.

11. Ji Y, Chen J, Lv J, Li Z, Xing L, Ding S. Extraction of keratin with ionic liquids from poultry feather. Sep. Purif. Technol. 2014; 132:577-583.

12. Eslahi N, Dadashian F, Nejad NH. An investigation on keratin extraction from wool and feather waste by enzymatic hydrolysis. Prep Biochem Biotechnol. 2013;43(7):624-648.

13. Bertsch A, Coello N. A biotechnological process for treatment and recycling poultry feathers as a feed ingredient. Bioresour. Technol. 2005;96(15):1703-1708.

14. Moore GR, Martelli SM, Gandolfo C, do Amaral Sobral PJ, Laurindo JB. Influence of the glycerol concentration on some physical properties of feather keratin films. Food Hydrocoll. 2006;20(7):975-982.

15. Tran HQ, Nguyen QV, Le TA. Silver nanoparticles: Synthesis, properties, toxicology, applications and perspectives. Adv Nat Sci NanosciNanotechnol2013;4:1-20.

16. Krithiga J, Briget MM. Synthesis of silver nanoparticles of Momordica charantia leaf extract, characterization and antimicrobial activity. Pharm Anal Acta 2015;6:1-7.

17. Gomathi M, Rajkumar VP, Prakasam A, Ravichandran K. Green synthesis of silver nanoparticles using Datura stramonium leaf extract and assessment of their antibacterial activity. Resour. Effic. Technol. 2017;3:280-284.

Received on 07.02.2019 Modified on 28.03.2019

Research J. Pharm. and Tech. 2019; 12(6): 2664-2668.

DOI: 10.5958/0974-360X.2019.00445.1