Extracting keratin from chicken feathers by using a ...

Process Biochemistry 47 (2012) 896–899

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Extracting keratin from chicken feathers by using a hydrophobic ionic liquid Yun-Xian Wang, Xue-Jun Cao ∗ State Key Laboratory of Bioreactor Engineering, Department of Bioengineering, East China University of Science and Technology, Shanghai 200237, China

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Article history: Received 4 September 2011 Received in revised form 5 January 2012 Accepted 14 February 2012 Available online 22 February 2012 Keywords: Ionic liquid 1-hydroxyethyl-3-methylimidazolium bis(trifluoromethanesulfonyl)amide Keratin Chicken feathers Separation

a b s t r a c t Keratin was extracted from chicken feathers by using a hydrophobic ionic liquid (IL), 1-hydroxyethyl-3methylimidazolium bis(trifluoromethanesulfonyl)amide ([HOEMIm][NTf2 ]). Extracted keratin has good solubility in water while the ionic liquid is immiscible with water, and therefore the extracted keratin could be easily separated from the reaction system by water. The effects of ionic liquid, NaHSO3 , reaction temperature and time were investigated and extracting conditions were optimized. The maximum yield of keratin was up to about 21% with mass ratio of feathers to NaHSO3 1:1 and mass ratio of feathers to ionic liquid 1:40 at 80 ◦ C for 4 h. Moreover, there was no obvious loss in the yield after ionic liquid was reused for five batches under optimized conditions. In addition, the recovery of ionic liquid was about 95% each time. The results indicated that [HOEMIm][NTf2 ] was very efficient as catalyst and solvent for dissolving feathers and could be easily recovered due to its hydrophobicity. © 2012 Elsevier Ltd. All rights reserved.

1. Introduction The poultry industry produces a great amount of waste feathers each year, e.g. 1.8 million tons in US [1], which causes an environmentally difficult disposal problem. Therefore, from both an economic and environmental point of view, it is quite desirable to develop effective and profitable process to use these resources. However, feathers are mainly used as low nutritional value animal feed. Currently, researchers have been searching for new applications of feathers and many publications and patents proposing applications of feathers have been issued. Polyethylene-based composites can be prepared using keratin fibers obtained from chicken feathers [2]. Keratin fibers from chicken feathers were used as a short-fiber reinforcement for a poly(methyl methacrylate) matrix [3]. Feather keratin and polyurethane were combined to synthesize hybrid synthetic-natural membranes which could be applied to separation process [4]. Keratin is the major component of feathers, which is a structural protein characterized by a high cystine content and a significant amount of hydroxyl amino acids, especially serine (about 15%) [5,6]. It contains a range of noncovalent interactions (electrostatic forces, hydrogen bonds, hydrophobic forces) and covalent interactions (disulfide bonds), which must be destroyed in terms of dissolution of feathers. Keratin is insoluble in polar solvents like water, weak acids and bases, as well as in apolar solvents while it is active because cystine can be reduced, oxidized and hydrolyzed [7–11].

∗ Corresponding author. Tel.: +86 21 6425 2695; fax: +86 21 6425 2695. E-mail address: [email protected] (X.-J. Cao). 1359-5113/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2012.02.013

However, one of the most serious shortcomings concerning these methods for extracting keratin is that a large quantity of reagents such as acids or reductants is consumed and cannot be recycled. Thus, researchers have focused on finding simple and eco-friendly processing methods to dissolve feather keratin. Recently, ionic liquids (ILs) have received recognition as green and promising materials for potential applications in various fields because they are typically non-volatile, non-flammable, chemical and thermal stability and remarkable solubility [12–16]. They have also been termed as ‘designer solvents’ as their properties can be manipulated by a careful choice of cation/anion according to the requirements. Ionic liquids exhibit excellent solubility characteristics because of their special structures compared to the traditional molecular solvents. Some recent works have been reported regarding the dissolution and regeneration of keratin fibers in ionic liquids. Xie et al. [17] reported the dissolution and regeneration of wool keratin fibers in 1-butyl-3methylimidazolium chloride (BMIMCl) ionic liquid. Hameed et al. [18] prepared natural wool/cellulose blends in BMIMCl and the films were formed subsequently from the coagulated solutions. In fact, the costs of ionic liquids are obviously higher than that of inorganic reagents, but they can be reused and improve the efficiency of whole process, leading to lower the overall cost. Separation of keratin from hydrophilic ionic liquids after reaction is an inconvenient and troublesome problem. In this study, a hydrophobic ionic liquid ([HOEMIm][NTf2 ]) was used to dissolve chicken feathers to obtain keratin. The aim of our research work was to extract keratin with hydrophobic ionic liquids instead of acids and bases. The effects of mass ratio of feathers and ionic liquid, mass ratio of feathers and NaHSO3 ,

Y.-X. Wang, X.-J. Cao / Process Biochemistry 47 (2012) 896–899

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Table 1 Water content of equilibrated and dried IL; solubility in water of IL. Ionic liquid

Water equilibrateda (g/100 ml)

Driedb (g/100 ml)

Water solubility (g/100 ml)

[HOEMIm][NTf2 ]

4.890

0.453

7.941

a b

Water equilibrated refers to IL that has been stored in contact with water, which incorporates vigorous agitation and mixing. Dried IL is water equilibrated IL that has been dried at 70 ◦ C for 6 h on a vacuum line.

reaction temperature and time on the extraction yield of keratin were investigated and the reusability of ionic liquid was also studied. Furthermore, the extracted keratin was analyzed by infrared spectrometry (IR) to confirm its structure and determined by gel permeation chromatography (GPC) to get its molecular weight. 2. Materials and methods 2.1. Water content and solubility in water The water content of [HOEMIm][NTf2 ] was determined using a volumetric Aquastar Karl Fischer titrator (Super Scientech, Shanghai, China) with composite 5 solution as the titrant and anhydrous methanol as the solvent. Each sample was at least 0.5 g and duplicate measurements were performed. The absorption spectrum of [HOEMIm][NTf2 ] was measured by full wavelength scan with UV–vis spectrophotometer (PerkinElmer Lamda 900). In a 10 ml polypropylene centrifuge tube, 1.0 g of the ionic liquid and 2.5 ml of deionized water were shaken on a vortex mixer for 30 min and centrifuged for 15 min at 4000 rpm. A 2 ␮l aliquot of the aqueous phase was taken with a microsyringe and diluted to 5 ml with deionized water. The absorbance of this solution at maximum absorption peak was measured and compared with that obtained from dissolving a weighed amount (0.1–0.6 mg) of the ionic liquid in 5 ml of deionized water [19]. 2.2. Preparation of feather keratin solution Before dissolution, chicken feathers were washed with water, filtered, dried and cut into small pieces or milled. Reactions were performed in a round flask containing 1.0 g chicken feathers, 40 g ionic liquid, 1.0 g NaHSO3 and 500 ␮l water. The mixture was agitated at 80 ◦ C for 4 h. After the process was finalized, water (20 g, 50% wt, based on the weight of ionic liquid) was added to the mixture and tri-phase systems containing ionic liquid/parts of insolubilized feathers/keratin solution were formed after centrifugation. Then the system of keratin solution was filtered, and the filtrate was transferred to dialysis bag (MWCO 3500–5000 Da) and extensively dialyzed against distilled water for 48 h. The protein concentration of the extracts was measured by the Bradford protein assay method (Bio–Rad), using bovine serum albumin as standard. The amount of protein dissolved was expressed as a percentage of the total weight of dried feathers used. In addition, the other two systems were vacuumized for 6 h (70 ◦ C) and introduced into the fresh substrates for the next batch. The recovery of ionic liquid was expressed as a percentage of the total weight of it used. 2.3. Molecular weight of feather keratin The molecular weight of extracted keratin was determined by gel permeation chromatography. It was carried out on a PerkinElmer Series 200 system at 35 ◦ C (TSKgel PWXL 10 ␮m, 7.8 × 300 mm column). 0.05 M NaNO3 was used as the eluent and the flow rate was 0.8 ml/min.

3. Results and discussion 3.1. Properties of [HOEMIm][NTf2 ] According to full wavelength scan, [HOEMIm][NTf2 ] existed maximum absorption peak at 217.5 nm, which mainly attributes to imidazole ring in the ionic liquid. As shown in Table 1, the water content of equilibrated [HOEMIm][NTf2 ] was 4.89%(w/v) and the solubility of [HOEMIm][NTf2 ] in water was 7.941%(w/v), which indicated that [HOEMIm][NTf2 ] was slightly soluble in water and maximum recovery of it could be up to 96% (50% wt water, based on the weight of ionic liquid). Moreover, [HOEMIm][NTf2 ] possesses unexpected “hyperpolarity” close to protic ILs and water [20], which could greatly enhance the dissolution of feathers. Therefore, [HOEMIm][NTf2 ] was a proper ionic liquid for extracting keratin from feathers.

3.2. Solubility of chicken feathers in ionic liquid 3.2.1. Effect of mass ratio of feathers and NaHSO3 on the yield of keratin To reduce the disulfide bonds, the reaction was carried out with various mass ratio of feathers to NaHSO3 (1:0, 1:0.3, 1:0.5, 1:0.75, 1:1, 1:1.25 and 1:1.5) under identical conditions. As shown in Fig. 1a, the extraction yield of keratin increased obviously with the increasing of mass ratio from 1:0 to 1:1 and then very slowly from 1:1.25 to 1:1.5. It suggested that more disulfide bonds of feather keratin were destroyed with higher mass ratio. Nevertheless, it also showed that the yield did not have significant increase when the mass ratio surpassed 1.0. This effect could be because all the disulfide bonds of feather keratin were reduced when excessive NaHSO3 was supplied. In the reaction system without NaHSO3 , the yield of extracted keratin was up to 7.86% only relying on the hyperpolarity of ionic liquid [HOEMIm][NTf2 ]. Therefore, the optimal mass ratio was 1:1 considering both the yield of keratin and the production costs. 3.2.2. Effect of mass ratio of feathers and ionic liquid on the yield of keratin The dissolution of feathers can be divided into two stages, including swelling and solubilization. The first stage is that solvent molecules gradually permeate into feathers so that the physical interactions of feather keratin are taken place by interactions between solvent molecules and feather keratin. The second stage is that chains of feather keratin unfold from aggregation due to salvation effects and reduction of disulfide bonds. Therefore, solubility of feathers is significantly related to polarity of ionic liquid and the yield of keratin is increased with the increasing polarity of ionic liquid. Reactions were conducted with different mass ratio of feathers to ionic liquid (1:20, 1:25, 1:30, 1:40, 1:45 and 1:50) under identical conditions. As a result (Fig. 1b), although the highest yield of 21.75% was obtained when the mass ratio was 1:45, the yield of keratin was increased slowly with mass ratio from 1:40 to 1:45 and declined with further increase of mass ratio. This effect could be attributed to that ionic liquid [HOEMIm][NTf2 ] could produce electrostatic and hydrogen-bond interactions between itself and feather keratin [20]. It also indicated that part of peptide bond of feather keratin was disrupted when excessive amounts of ionic liquid were applied. From these results, the mass ratio of feathers to ionic liquid 1:40 was adopted in the subsequent experiments considering the cost of ionic liquid. 3.2.3. Effects of reaction temperature and time on the yield of keratin Temperature is an important factor affecting the yield of keratin, and the rate of dissolution of feather keratin at various temperatures (70–100 ◦ C) was determined while keeping other conditions constant. From the Fig. 1c, it can be seen that the yield increased obviously with temperature from 70 ◦ C to 80 ◦ C and approached flat stage from 80 ◦ C to 90 ◦ C. It was because higher reaction temperature can provide much energy to accelerate physical and chemical change of feather keratin and thus promote the dissolution of feathers. However, when the temperature was over 90 ◦ C, the yield decreased markedly. Peptide bond scission of feather keratin

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Fig. 1. (a) Effect of mass ratio of feathers and NaHSO3 on the extraction yield of keratin. Reaction conditions: mass ratio of feathers to ionic liquid 1:20, 500 ␮l water, 80 ◦ C, 4 h. (b) Effect of mass ratio feathers and ionic liquid on the extraction yield of keratin. Reaction conditions: mass ratio of feathers to NaHSO3 1:1, 1.0 g NaHSO3 , 500 ␮l water, 80 ◦ C, 4 h. (c) Effect of reaction temperature on the extraction yield of keratin. Reaction conditions: mass ratio of feathers to NaHSO3 1:1, mass ratio of feathers to ionic liquid 1:40, 500 ␮l water, 4 h. (d) Effect of reaction time on the extraction yield of keratin. Reaction conditions: mass ratio of feathers to NaHSO3 1:1, mass ratio of feathers to ionic liquid 1:40, 500 ␮l water, 80 ◦ C.

occurred at the higher temperature. Therefore, 80 ◦ C was selected as the optimal reaction temperature. The effect of reaction time on the dissolution of feathers keratin was studied and the results were shown in Fig. 1d. It was found that the optimum reaction time was 4 h and the maximum yield was up to 21.50%.

3.3. Recycle of ionic liquid Even though previous works have demonstrated the dissolution of feathers in ionic liquids, there is yet no effective way to separate the protein from ionic liquids. Therefore, the reusability of ionic liquid [HOEMIm][NTf2 ] was investigated under the optimum conditions mentioned above. In our study, extracted keratin has good solubility in water, while [HOEMIm][NTf2 ] is immiscible with water. Consequently, keratin could be easily separated from the reaction mixture by water, and the extracted keratin could be purified by dialysis and precipitated by ethanol according to requirement of final product. The remaining ionic liquid could be used directly in the next time. As shown in Fig. 2, the ionic liquid was used for five batches without essential loss in the process of dissolving feathers. It was also found that the recovery of ionic liquid was about 95% in each cycle. This indicated that [HOEMIm][NTf2 ] is an effective hydrophobic ionic liquid for extracting keratin from chicken feathers.

Fig. 2. The reusability of ionic liquid for dissolving feathers. Reaction conditions: mass ratio of feathers to NaHSO3 1:1, mass ratio of feathers to ionic liquid 1:40, 500 ␮l water, 80 ◦ C, 4 h.

3.4. FTIR The FTIR spectra of extracted keratin in the region 400–4000 cm−1 are given in Fig. 3, where the characteristic absorption bands are mainly assigned to the peptide bonds


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keratin were 10,240 and 10,000 respectively. This GPC data agrees with published data that feather keratin consists of 96 amino acid residues and has a molecular weight of 10,206 [6]. The polydispersion degree was at the level of 1.024, which shows that the extracted keratin was uniform in its molecular weight. However, the yield of extracted keratin was less 25%, probably because keratin was degraded to amino acids and small peptides and not separated from reaction mixture completely. 4. Conclusions The eco-friendly ionic liquid [HOEMIm][NTf2 ] could be a suitable solvent and catalyst for dissolving feathers. To our knowledge, this should be the first report concerning the dissolution of feathers in a hydrophobic ionic liquid and an easy separation of extracted keratin. References Fig. 3. IR spectrum of extracted keratin.

Fig. 4. The GPC data of extracted keratin.

( CONH ). The vibrations in the peptide bonds originate bands known as amides I–III [21–23]. The amide I band is connected mainly with the C O stretching vibration and it occurs in the range of 1700–1600 cm−1 . The amide II band is related to N H bending and C H stretching vibration and it falls at 1520 cm−1 region. The amide III band occurs in the range of 1220–1300 cm−1 and it results from in phase combination of C N stretching and in N H in plane bending, with some contribution from C C stretching and C O bending rations. It can be seen from Fig. 3 that these bands exist in extracted keratin. 3.5. Molecular weight The molecular weight of feather keratin solution was examined by using GPC. There was only one sharp peak in the molecular weight distribution of extracted keratin (Fig. 4). The weightaveraged and number-averaged molecular weights of extracted

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