Sustainable valorization of tannery hair waste into keratin by an acoustic cavitation-assisted process using a green solvent

Sustainable valorization of tannery hair waste into keratin by an acoustic cavitation-assisted process using a green solvent | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Sustainable valorization of tannery hair waste into keratin by an acoustic cavitation-assisted process using a green solvent Prasath Loganathan, Nitin prakash Lobo, Surianarayanan Mahadevan, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7252336/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Keratin, a valuable biomaterial from tannery hair waste, has potential for biomedical applications due to its inherent biocompatibility, and biodegradability. This investigation deals with eco-efficient acoustic cavitation assisted method for extraction of keratin from tannery animal hair waste using ionic liquid [BMIM]Cl. Here, ionic liquid has been used as a solvent for better keratin recovery. Optimizing parameters such as solid-to-liquid ratio, acoustic irradiation time, and power, achieved a 71% keratin yield and a 95% reduction in reaction time, mitigating environmental impact and facilitating a cleaner, faster and more sustainable process. The extracted keratin's structural integrity and intactness were substantiated by ATR-FTIR and solid-state ¹³C NMR spectroscopy. Additionally, CD analysis revealed the presence of α-helix and β-sheet structures of keratin. XRD analysis confirmed the keratin's crystallinity, while DSC and TGA thermograms proved its thermal stability. FE-SEM studies elucidated the morphological features. Furthermore, the ionic liquid was successfully recovered from the effluent, and NMR studies confirmed its intact chemical structure, suggesting its potential for industrial applications. The process contributes to resource efficiency, waste minimization, and the development of sustainable materials from industrial residues. Acoustic cavitation [BMIM]Cl Dissolution Hair waste Keratin Tannery Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Introduction Keratin is composed of protein component of various biological materials, such as hair, wool, and horns [1]. The tanning industry generates approximately 40 kilotons of animal hair waste annually [2]. This waste poses environmental problems and disposal challenges associated with hair generated during the unhairing process of the skins/hides, typically being landfilled, contributing to pollution [3]. However, this waste material can be valorized for obtaining valuable keratin-based products. Unlike other natural materials, keratin is difficult to dissolve and extract due to its unique polypeptide chain composition, featuring extensive inter- and intramolecular interactions, including disulphide cross-linking between cysteine residues, which impart remarkable stability and insolubility in various solvents [4]. The presence of cysteine disulphide bonds plays a vital role in determining the physico-chemical properties of keratin [5]. Several methods have been followed to extract keratin which includes reduction, alkaline hydrolysis, oxidation, sulphitolysis and ionic liquids [6]. In the reduction process, keratin is extracted through the dissolution of wool using urea and 2-mercaptoethanol, resulting in a lower yield of keratin hydrolysate (45%). However, this method produces toxic and harmful by products [7]. Furthermore, the alkaline hydrolysis method compromises keratin's structure, leading to considerable amino acid degradation and low keratin recovery [1]. Among the given literature, in the oxidation method, although is notable for its ease of extraction, it employs per-acetic or per-formic acid leading to the oxidation of cystine residues to cysteic acid, which not only altering keratin's chemical structure but also results in a relatively lower keratin yield of 6% [8]. In the sulphitolysis method, red sheep hair is treated with a mixture of urea and sodium metabisulfite to extract keratin in a hydrolysate form, with a poor yield of 29% [9]. In a recent study, deep eutectic solvent (DES) has been used to viable option for keratin extraction from sheep wool. However, it resulted in lower yield of keratin (23%) at optimal conditions [10]. The studies are given in the literature are not suitable for commercial applications due to poor yield (20-45%) of keratin and generation of toxic waste. Ionic liquids (ILs) have recently gained attention as a sustainable solvent over deep eutectic solvents in various applications, including biomass extraction [11]. Further it was also used in electrochemistry [12], ion-conductive media, and catalysis [13], owing to their exceptional physicochemical properties. Li et al. [14] demonstrated the effectiveness of acidic IL (type of protic IL) in hydrolysing keratin from wool under microwave radiation, with 1-propylsulfonic-3-methylimidazolium hydrogen sulphate ([PSMIM]HSO₄) showing better results for keratin hydrolysis. Studies on the effect of different IL for dissoluting the wool was conducted by Xie et al. [15] and Idris et al. [16]. However, the studies reported a poor yield. Ji et al. [17] investigated with three primary ILs: 1-Butyl-3-methylimidazolium bromide [BMIM]Br, 1-Allyl-3-methylimidazolium chloride [AMIM]Cl and 1-Butyl-3-methylimidazolium chloride [BMIM]Cl, and found that Cl-containing ILs exhibited higher keratin dissolution rates, achieving a 75% keratin yield from feathers. This approach underscores the potential of chloride containing ILs in effective extraction and producing keratin in its native form (non-hydrolysate). Despite the demonstrated potential of [BMIM]Cl in keratin recovery from wool and poultry feathers, its application for the sustainable valorization of keratin-rich tannery hair waste has not been investigated yet. On the other hand, ultrasonic-assisted processing has been widely explored for its potential to enhance chemical reactions and mass transfer of biomolecules such as proteins, enzymes, and polymers [18]. Studies have shown that probe-type sonication generates acoustic streaming and cavitation, thereby enhancing mass transfer and facilitating chemical reactions by mechanical vibration effect. This physical phenomenon of acoustic cavitation has significant implications for various applications such as cleaning, biomass extraction, industrial and biomedical applications [19]. Recently, Feroz et al. [5] introduced a novel approach combining sonication with protic ionic liquids Tetra butyl phosphonium hydroxide (TBPH) and choline hydroxide (CH), facilitate wool dissolution to produce keratin hydrolysate. Similarly, Azmi et al. [20] demonstrated that the low frequency high power sonication is effective in extraction of keratin by dissolving the feather using [BMIM]Cl. Although the keratin was extracted in its native structure, the yield was relatively low (~10%). The keratin extracted from different waste biomaterials are provided in Table 1. Table 1 Extraction of keratin by sonication assisted methods Methods Bio-materials Treatment conditions Keratin Yields (%) References Non-Hydrolysate Hydrolysate Probe sonication assisted with ionic liquid Wool Tetra butyl phosphonium hydroxide (TPBH) at optimized conditions acoustic power 40%, acoustic time 20 min. with initial loading of 20%. - 95% [5] Turkey Feathers Aprotic Ionic liquid [BMIM]Cl at sonication at 20 kHz with lower sonication power at 200 W for 52 min. 10% - [20] Chicken feathers 8M Urea and 15 % Cysteine with pH 10.5 at 130 W for 2.7 h - 63.2% [21] This work advances the principles of green chemistry and biomass valorization by demonstrating a sustainable and efficient strategy for the recovery of native keratin from tannery hair waste. The advantage lies in the significant reduction of dissolution time compared to conventional methods. Furthermore, optimizing sonication time and power parameters demonstrates its potential as a scalable and effective technique for keratin extraction. This study presents a novel eco-friendly approach for keratin extraction from tannery animal hair waste using an aprotic ionic liquid ([BMIM]Cl) in combination with acoustic cavitation. This method overcomes key limitations of conventional approaches by significantly reducing dissolution time, enhancing keratin yield, and preserving the native protein structure. Based on previous studies, the chloride-containing imidazolium salt [BMIM]Cl was selected as the solvent for its efficacy in dissolving keratin. The primary objective of this study is to optimize the acoustic cavitation assisted dissolution process while extracting keratin in its native form with enhanced yield. To the best of our knowledge, this is the first report to demonstrate the integration of acoustic cavitation with a chloride-based aprotic IL for keratin extraction from tannery hair waste. This innovative combination enables the sustainable valorization of a low-value industrial by-product into a high-value biomaterial, aligning with circular economy and sustainable waste management principles. The method developed in this work is scalable, energy-efficient, and avoids the use of harsh chemicals, aligning with industrial sustainability targets . Systematic studies were conducted to evaluate the operating parameters, paving the way for sustainable keratin extraction and promoting a circular economy. By utilizing the tannery animal hair waste as a raw material, this study contributes to waste reduction and resource recovery, enabling the efficient valorization of tannery animal hair waste into a valuable biomedical product. The extracted keratin was characterized using various techniques, including Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR), X-ray Diffraction (XRD), solid-state 13 C Nuclear Magnetic Resonance (NMR), Circular Dichroism (CD), Differential Scanning Calorimetry (DSC) and Thermogravimetric Analysis (TGA) to determine its physical, chemical, and thermal properties. Additionally, morphological studies were carried out by Field Emission Scanning Electron Microscopy (FE-SEM) to examine the surface and structural features of the obtained keratin. Experimental Materials and Methods In this study, sheep hair waste, obtained from the post-unhairing unit operation (lime-sulphide process) at the Leather Processing Division, CSIR-CLRI, was used as raw material. To remove the impurities the hair was washed with distilled water and dried. It was then defatted with a hexane: DCM mixture (1:1) in a Soxhlet extractor for 48 hours to remove fats and oils. After defatting, the hair was dried at 70 °C for 48 hours in vacuum oven. The dried hair was size-reduced to 10-12 mm in length and used for dissolution studies. To improve keratin yield, the hair sample was pre-soaked in a 2% thioglycolic acid (TGA) solution for 15 minutes prior to dissolution. For pure keratin, raw hair obtained from the tannery of CSIR-CLRI, washed and cleaned, was used as a reference and standard to compare with the extracted keratin. In this study the chemicals used are 1-Butyl-3-methylimidazolium chloride ([BMIM]Cl) and thioglycolic acid (TGA), procured from Sigma Aldrich, India. Hexane, dichloromethane (DCM), dimethyl sulphoxide (DMSO) and deuterated dimethyl sulfoxide (DMSO-d 6 ) were purchased from SRL Chemicals, India. Acoustic cavitation assisted hair dissolution The dissolution of hair samples was conducted using [BMIM]Cl as a solvent, followed by acoustic irradiation with a probe-type sonicator (Ultrasonic Vibracell, 50 Hz, Model VCX1500, Sonics & Materials, Inc., USA) operating in pulse mode of five seconds. The ultrasonic probe was immersed to a depth of approximately one third of the total height in a 25 mL beaker containing the hair and IL mixture. To prevent overheating and potential degradation of keratin the beaker was surrounded by ice packs to dissipate the heat generated during the acoustic irradiation process. In order to confirm complete dissolution, required volume of DMSO was added to the mixture, and visually inspected for any undissolved hair particles in the solution. A homogeneous solution indicated complete dissolution. The addition of DMSO also reduced the viscosity of the mixture, facilitating the movement of keratin molecules and enhancing precipitation. Subsequently, the solution was stirred at 250 rpm and centrifuged at 5000 rpm for 25 min., at 22 °C to facilitate keratin extraction. The resulting mixture was then regenerated using distilled water via centrifugation at 5000 rpm for 25 minutes at 22 °C. To remove residual IL and DMSO, the precipitate was subjected to repeated washings with distilled water, and centrifuged at 12000 rpm for 10 min. Finally, the precipitated insoluble keratin was then lyophilized to obtain a keratin powder. The sequence of unit operations for keratin extraction by an acoustic cavitation assisted dissolution is shown in Fig. 1. The keratin yield was calculated by using mathematical expression Characterization studies CHNS analysis Elemental composition was determined by CHNS analysis using an Elementar UNICUBE analyser (Serial No: 0400BD1009). The protein content was estimated from the nitrogen content using a conversion factor of 6.25 [8]. ATR-FTIR analysis FTIR spectra were recorded using ATR-FTIR spectrometer (Bruker Alpha II-Platinum) with a platinum-diamond sample module and a deuterated triglycine sulphate (DTGS) detector, over a wavenumber range of 4000-600 cm −1 , spectral resolution of 4 cm −1 , averaging 24 scans and subsequently baseline-corrected [16]. XRD analysis Powder X-ray diffraction (XRD) patterns were registered at 25 °C using X-ray diffractometer (Rigaku Mini Flex-II) with Cu Kα radiation (λ = 1.5418 Å) in Bragg-Brentano geometry. The finely ground sample (~5 mg) was kept on a custom-designed sample holder made of flat brass with an O-ring seal and Mylar sheet cover to maintain airtight atmosphere. XRD parameters included a voltage of 15 kV, current of 30 mA, 2θ range of 5-50°, step size of 0.02°, and scan rate of 1° min −1 [16]. NMR analysis NMR spectra were obtained from 400 MHz spectrometer (Bruker Avance). Solid-state 13 C NMR spectra were recorded with 976 scans at 400 MHz. Solution-state 1 H and 13 C NMR spectra were obtained at 400 MHz with 16 scans, using deuterated dimethyl sulfoxide (DMSO-d 6 ) as solvent. Chemical shifts are reported in parts per million (ppm) on the δ scale [16]. DSC analysis Differential scanning calorimetry (DSC) measurements were conducted using a TA Instruments Discovery DSC 25 series instrument under a nitrogen atmosphere. Samples (2-3 mg) sealed in Tzero aluminium pans underwent a thermal protocol involving cooling to -40 °C for 5 minutes, and then by heating from 20 °C to 350 °C at 10 °C min −1 . Prior to sample testing the instrument was calibrated (using indium; melting point 156.6 °C) to ensure thermal accuracy [22]. TGA analysis Thermogravimetric analysis (TGA) was carried out using thermal analyser (NETZSCH STA 449F3) to assess the thermal stability of the sample. This was done by heating the sample (2-5 mg) from 30 °C to 600 °C at an increment of 10 °C/min under nitrogen atmosphere [5]. CD analysis Circular dichroism (CD) spectroscopy was done by using Jasco J-715 spectrometer. The analysis was conducted under a nitrogen atmosphere (5 LPM) in a quartz cuvette, 1 mm path length, scanning from 190 to 260 nm at the rate of 100 nm/min [9]. FE-SEM analysis The morphology of extracted keratin and raw hair were examined using scanning electron microscope (SEM, make TESCAN CLARA). The samples were coated using gold-palladium alloy (80:20), analysed at an acceleration voltage of 10 kV under nitrogen atmosphere [9]. Results and Discussions Optimizing the process parameters for hair dissolution using [BMIM]Cl To maximize keratin extraction efficiency, the dissolution of hair samples was optimized with respect to three key parameters, i.e. solid-to-liquid ratio, acoustic cavitation irradiation time and acoustic power. These parameters were systematically varied to determine their impact on keratin yield. Effect of solid to liquid ratios on keratin yield Fig. 2 illustrates the impact of solid-to-liquid ratio on keratin yield using [BMIM]Cl, where ratios of 1:20, 1:40, and 1:60 were evaluated under constant acoustic cavitation conditions (450 W, 30 min). The corresponding keratin yields were 54% ± 0.9%, 71% ± 1.6%, and 74% ± 1.6%, respectively. Notably, the higher yields observed at 1:40 and 1:60 ratios can be attributed to the optimal ionic liquid volume, facilitating effective propagation of ultrasonic waves and maximizing cavitation intensity. This, in turn, enhances the disruption of disulphide bonds and penetration of the ionic liquid, ultimately leading to improved keratin extraction. Conversely, the lower yield at 1:20 ratio (54% ± 0.9%) is likely due to insufficient solvent volume, resulting in reduced cavitation efficiency and incomplete dissolution. Considering the marginal difference in yields between the 1:40 and 1:60 ratios, the 1:40 ratio was determined as the optimal solid-to-liquid ratio, offering a balance between maximizing keratin yield and preserving its native structure Effect of acoustic cavitation irradiation time on keratin yield A critical parameter influencing the acoustic cavitation assisted dissolution process is the acoustic irradiation time, which significantly affects both energy consumption and keratin extraction efficiency. Fig. 3 shows the impact of varying acoustic irradiation times (20, 25, and 30 min) on hair dissolution in [BMIM]Cl at a constant acoustic power of 450 W. The corresponding keratin yields were 28% ± 2.5%, 47% ± 1.62%, and 71% ± 1.35%, respectively, demonstrated a notable increase in yield with prolonged irradiation time. The optimal irradiation time was determined to be 30 min., as further extension beyond this duration yielded only marginal improvements in keratin extraction. This suggested that prolonged exposure to acoustic cavitation is not necessary. Effect of acoustic power on keratin yield The acoustic power on hair dissolution in IL was examined by varying power levels such as 300, 450, and 600 W at a constant acoustic irradiation time of 30 min. The results, showed in Fig. 4, reveal the keratin yields of 33% ± 1%, 70.5% ± 1.5%, and 65% ± 1% for the respective power levels. Acoustic power plays a crucial role in modulating cavitation intensity, thereby significantly impacting the dissolution process. Particularly, an acoustic power of 450 W yielded the highest keratin recovery of 70.5% ± 1.5%, suggesting optimal cavitation conditions for efficient keratin extraction. In contrast, the decreased yield at 600 W (65% ± 1%) can be attributed to excessive energy input (∼52,683 kJ/kg), potentially leading to keratin degradation or fragmentation into smaller peptide chains. The degradation compromises the overall keratin yield and structural integrity, highlighted the importance of optimizing acoustic power for efficient keratin extraction preserving its structural integrity. Temperature variation with time during hair dissolution Fig. 5 depicts the temperature profile of different acoustic powers showing a significant increase in temperature over time. At acoustic power of 300, 450 and 600 W, corresponding energy input as in fig. 5, the temperature gradually rises to maximum values of 62, 76, and 90 °C, respectively, after 30 min. of acoustic irradiation. This temperature increase is attributed to the conversion of acoustic energy into thermal energy, resulting from the acoustic cavitation phenomenon. The elevated temperature is believed to enhance ionic mobility and reduce mixture viscosity, facilitating efficient hair dissolution by promoting better solvent penetration and interaction with the keratin structure. Specifically, the increased ionic mobility and reduced viscosity enable the IL to more effectively disrupt the keratin disulphide bonds, leading to improved dissolution. However, beyond 130 °C, keratin's structural integrity will lead to degradation and reduce the keratin yield, as reported in previous studies. 1 Based on the experimental findings, the optimized parameters for efficient keratin extraction were determined to be a solid-to-liquid ratio of 1:40, acoustic irradiation of 30 min. and power of 450 W, corresponding to a specific energy input of approximately 39,512 kJ/kg. Under these optimized conditions, the extracted keratin was subsequently characterized. Influence of reducing agent on keratin extraction The addition of reducing agent, TGA, prior to acoustic cavitation assisted dissolution process significantly enhanced keratin extraction yield up to 75.1±0.7%, representing 6.5% increase compared to the yield without using reducing agent. This improvement can be attributed to the partial reduction of inter- and intramolecular disulphide bonds in keratin, which disrupted its rigid structure and increased its solubility. The disruption of these bonds facilitated the extraction of keratin, resulting in a higher yield. This observation is consistent with previous studies, such as those reported by Idris et al. [16], which emphasize the crucial role of disulphide bond reduction in enhancing protein solubility and extraction efficiency. The substantial increase in keratin yield suggests effective dissolution of hair, enabling efficient extraction and potentially preserving the protein's structural integrity. Characterization of extracted keratin CHNS analysis The chemical composition and protein content of extracted keratin and raw hair were evaluated by CHNS elemental analysis. It is observed that, between the two samples, there is a marginal difference in the composition of elements. Raw hair comprised 43.9% carbon, 6.8% hydrogen, 14.6% nitrogen, and 3% sulphur, whereas extracted keratin consisted of 45.4% carbon, 7.67% hydrogen, 14.72% nitrogen, and 2% sulphur. The slightly higher nitrogen content in extracted keratin (14.72%) compared to raw hair (14.6%) is consistent with the high protein yield of 92%, indicating a purity level exceeding 90% [8]. This increase in nitrogen content suggests that during the extraction process the non-proteinaceous components have been effectively removed. Thermogravimetric analysis (TGA) further supported the high purity of the extracted keratin, with minimal weight loss observed during the initial stage due to the evaporation of bound water. ATR-FTIR analysis The ATR-FTIR spectra of raw hair and extracted keratin shown in Fig. 6(a) and 6(b) exhibit characteristic absorption peaks associated with peptide bonds (-CONH-), providing insights into their secondary structure. In raw hair, the amide I peak is observed at 1627 cm -1 , attributed to C=O stretching vibrations, while amide II peak appears at 1534 cm -1 , resulting from C-N stretching and N-H bending vibrations. Similarly, the extracted keratin exhibits a strong amide I peak at 1633 cm -1 and an amide II peak at 1522 cm -1 . These vibrations are sensitive to protein secondary structure, enabling the determination of conformational changes in α-helix and β-sheet structures. The slight peak shifts in the extracted keratin suggest interactions between the ionic liquid and polypeptide chains. The amide A peak, corresponding to N-H stretching vibrations, is observed at 3266 cm -1 in the extracted keratin and 3271 cm -1 in raw hair. Additionally, the amide III peak, indicative of C-N stretching and N-H bending vibrations, appears as a weak peak at 1232 cm -1 in the extracted keratin and 1233 cm -1 in raw hair. This similarity in peak positions between raw hair and extracted keratin indicates that the keratin was successfully extracted in its native form (non-hydrolysate), retaining its secondary structure (α-helix and β-sheet). XRD analysis Fig. 7 shows the crystallinity of the raw hair and the extracted keratin. The patterns of raw hair revealed characteristic peaks at 2θ values of 9.38° (0.94 nm) and 16.58° (0.54 nm), corresponding to α-helix structures, and a peak at 9.38° (0.94 nm) and 21.7° (0.42 nm), characteristic of β-sheet structures. However, the extracted keratin shows the peak at 9.38° was disappeared, suggesting a significant reduction in crystallinity during the dissolution process. The extracted keratin showed a similar diffraction pattern with a peak at 23.8° (0.38 nm) but slightly higher intensity (77%) compared to raw hair (75%), which suggested an increased β-sheet content and reduced crystallinity [5,16,19]. DSC-TGA analysis Fig.8 shows the thermal properties of raw hair and extracted keratin, investigated using DSC and TGA analysis. DSC analysis revealed endothermic peaks around 100 °C corresponded to the evaporation of water in both raw hair and extracted keratin, while peaks above 230 °C indicated the denaturation of α-helix [22]. TGA curves showed two distinct stages of decomposition, the first stage around 100 ℃ corresponded to moisture evaporation with weight losses of approximately 7.5% and 10.5% for extracted keratin and raw hair respectively. The lower weight loss in extracted keratin suggests higher purity, potentially exceeding 90%, based on nitrogen content determined using CHNS analysis [9]. The second stage, attributed to keratin's helical structure (α-helix) degradation and disulfide bond breakage above 230 ℃. During this stage, the extracted keratin exhibited a weight loss of about 14.8% and whereas raw hair showed a weight loss of approximately 16.2%. These results showed that the extracted keratin is more stable than the raw hair and the extracted keratin possesses high thermal stability make it a promising biomaterial for various biomedical applications. Solid state 13 C NMR analysis Fig. 9(a) and 9(b) shows the NMR spectra of raw hair and extracted keratin, revealing its structural characteristics of the keratin molecule. Compared to raw hair (Fig. 9(a)), the extracted keratin spectrum (Fig. 9(b)) exhibited an asymmetric peak at 172-174 ppm, attributed to amide carbonyl carbons found in keratin's protein backbone. The peak at 172-175 ppm provides insight into α-helix and β-sheet conformations. Aromatic amino acids are indicated by a peak at ~127 ppm, while α-carbons and β-carbons found in leucine and cross-linked cysteine residues of the protein structure are detected at 50-53 ppm and 39-40 ppm, respectively [23]. The retention of newly formed disulfide bridges (-S-S-) found in cysteine residues is suggested by the presence of peak at 39-40 ppm, indicating stability of the extracted keratin. Peaks between 29-40 ppm corresponded to proline, glutamic acid, and glutamine residues, while the peak at ~20 ppm indicates alanine residues. The α-carbon peak (50-53 ppm) exhibits slight broadening and shifting to the lower frequency of the chemical shift, indicating disruption of hydrogen bonding in the hair due to dissolution process and formation of a more ordered β-sheet structure in the extracted keratin, consistent with XRD results. FE-SEM analysis FE-SEM images in Figure 10 reveals the morphological transformation of raw hair (Fig. 10(a)) to extracted keratin (Fig. 10(b)). The raw hair tubular structure is transformed into a non-uniform, hollow honeycomb-like keratin structure, characterized by disruption of cuticle layer, exposing cortex and medulla. This structural change, facilitated by the acoustic cavitation assisted ionic liquid dissolution process, indicates efficient keratin extraction while preserving its structural integrity. The resulting porous morphology retains key features of native keratin structure due to mild processing conditions. CD analysis The CD spectrum of extracted keratin is given in Fig. 11. The characteristic features of α-helix and β-sheet conformations, providing insight into its of its secondary structure. The negative ellipticity bands at 208-215 nm and a positive band at approximately 190 nm indicates α-helical structures, while the negative band at 218-220 nm and positive band at 192-195 nm suggests the presence of β-sheets. These spectral characteristics are consistent with previous reports [8], confirming that the extraction process preserves the native structural integrity of keratin. Table 2 Physicochemical and structural properties of raw hair and extracted keratin Analysis Raw hair (reference) Extracted keratin ATR-FTIR Amide A (cm -1 ) 3271 3266 Amide I (cm -1 ) 1627 1633 Amide II (cm -1 ) 1534 1522 Amide III (cm -1 ) 1233 1232 CD α-helix (positive and negative bands) in nm - 190 & 208-215 β-sheet (positive and negative bands) in nm - 192-195 & 218-220 XRD d-spacing value (2q; nm) 21.7°; 0.42 23.8°; 0.38 DSC and TGA Water loss temperature (℃)/ Weight loss (%) Around 100 & 10.5 Around 100 & 7.5 Denaturation temperature (℃)/ Weight loss (%) Above 250 & 16.2 Above 250 & 14.8 Solid-state 13 C NMR C=O group (ppm) 172-175 172-175 α-carbon (ppm) 52 53 β-carbon (ppm) 39 40 Alky side chains (ppm) 19-30 19-30 SEM Morphology No damage in hair Damage in hair fragments CHNS analysis Protein yield (%) 91 92 Recovery of IL from the effluent Following dissolution studies, IL recovery is essential for ensuring the economic viability of the keratin extraction process. The successful recovery of IL was achieved via evaporation method, and the observed yield is 77%, indicating a relatively efficient recovery process. However, further optimization is needed to achieve higher yields and minimize the losses due to strong interactions between the IL and DMSO. In order to confirm the purity, the recovered IL was subjected to NMR studies, and got confirmed through ¹H and ¹³C NMR spectroscopy. The spectra are given in Fig. 12 and Fig. 13. It shows that identical proton and carbon signals when compared to standard [BMIM]Cl procured from M/s Sigma-Aldrich, indicating retention of the molecular structure. This spectral matching confirmed the preservation of the IL chemical structure and purity, suitability for its reuse. In order to further substantiate, ATR-FTIR spectroscopy of the recovered IL is given in Fig. 14. It is well corroborated and these findings exhibiting identical vibration frequencies to the standard IL. Interpretations of NMR spectra The recovered ionic liquid [BMIM]Cl was characterized by 1 H-NMR (400 MHz, DMSO-d 6 , δ 3.5 ppm for solvent peak) and 13 C-NMR (400 MHz, DMSO-d 6 , δ 39.8 ppm for solvent peak) spectroscopy. The 1 H-NMR spectrum (Fig. 12) exhibited the following chemical shifts (δ, ppm) and multiplicities: δ 9.3 (1H, s), 7.8 (1H, s), 7.7 (1H, s), 4.2 (2H, t), 3.8 (3H, s), 2.5 (1H, s), 1.7 (2H, m), 1.3 (2H, m), 0.9 (3H, t). The 13 C-NMR spectrum (Fig. 13) displayed the following chemical shifts (δ, ppm): δ 136.8, 123.9, 122.7, 48.9, 36.0, 31.9, 19.2, 13.9. Comparison of ATR-FTIR spectra of standard and recovered [BMIM]Cl Fig. 14 shows the ATR-FTIR spectra of standard and recovered [BMIM]Cl, confirming the structural integrity of the recovered IL through characteristic peaks corresponding to specific bond vibrations. The absorption peaks at 2951-2961 cm −1 and 2864-2873 cm −1 are attributed to asymmetric and symmetric (C–H) aliphatic stretching of methyl groups, respectively. The HC-C and H-C-N bond vibrations peak located at 1165-1172 cm −1 , while the C-N vibration appears at 750-764 cm −1 in the imidazole ring. A peak observed at 3382 cm −1 is due to the quaternary amine salt formation with chlorine. These results are consistent with previous literature reported by Borges et al. [24]. The results confirm the successful recovery and structural integrity of [BMIM]Cl, indicating its potential for reuse in the hair dissolution process and contributing to waste reduction. Studies on the recovery and reuse of the ionic liquid are currently in progress. Conclusion As compared to our previous studies without the application of acoustic cavitation (submitted for review elsewhere), acoustic cavitation-assisted keratin extraction from tannery animal hair waste using [BMIM]Cl achieved a yield of up to 71% keratin in its native form. By optimizing key parameters and utilizing a reducing agent, this approach significantly enhanced keratin yield while reducing environmental impact. The acoustic cavitation process reduced dissolution time by 95% compared to conventional methods, demonstrating its potential for scalable, eco-friendly production. Furthermore, 77% of [BMIM]Cl was recovered from the spent solution, enhancing the process environmental and economic viability through solvent recovery and waste minimization. While further optimization is needed, this study lays the groundwork for scaling up keratin extraction, potentially facilitating the development of novel biomedical applications. Overall, the process supports circular economy initiatives by valorizing low-value industrial tannery hair waste into high-value biomaterials, thereby promoting sustainable resource utilization and advancing the field of biomass valorization. Declarations This work was supported by Anusandhan National Research Foundation (ANRF), Government of India. (Grant number. EEQ/2021/000148). Dr. Lajapathi Rai Chockalingam has received research support. Authors contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Prasath Loganathan, Nitin Prakash Lobo, Surianarayanan Mahadevan, and Lajapathi Rai Chockalingam. The first draft of the manuscript was written by Prasath Loganathan and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Data Availability The datasets generated during and/or analysed during the current study are not publicly available due to privacy constraints but are available from the corresponding author on reasonable request. Acknowledgement The authors thank the Anusandhan National Research Foundation (ANRF)/ Science and Engineering Research Board (SERB), Government of India, for funding this project (File No. EEQ/2021/000148). We acknowledge the Centre for Analysis, Testing, Evaluation & Reporting Services (CATERS), CSIR-CLRI, for providing analytical services and data that facilitated our research. We acknowledge the support of the Knowledge Resource Centre (KRC), CSIR-CLRI, in assigning Communication Number 2129 to this manuscript for publication. References Shavandi, A., Silva, T.H., Bekhit, A.A., Bekhit, A.E.A.: Keratin: dissolution, extraction and biomedical application. Biomater. Sci. 5, 1699–1735 (2017). https://doi.org/10.1039/C7BM00411G. 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Zhang, X., Feng, Y., Yang, X.: Extraction of Keratin from Poultry Feathers with Choline Chloride-Oxalic Acid Deep Eutectic Solvent. Fibers and Polymers. 22, 3326–3335 (2021). https://doi.org/10.1007/s12221-021-0255-z. Yamauchi, K., Yamauchi, A., Kusunoki, T., Kohda, A., Konishi, Y.: Preparation of stable aqueous solution of keratins and physiochemical and biodegradational properties of films. J. Biomed. Mater. Res. 31, 439–44 (1996). Shavandi, A., Carne, A., Bekhit, A.A., Bekhit, A.E.A.: An improved method for solubilisation of wool keratin using peracetic acid. J. Environ. Chem. Eng. 5, 1977–1984 (2017). https://doi.org/10.1016/j.jece.2017.03.04. Ramya, K.R., Thangam, R., Madhan, B.: Comparative analysis of the chemical treatments used in keratin extraction from red sheep’s hair and the cell viability evaluations of this keratin for tissue engineering applications. Process Biochem. 90, 223–232 (2019). https://doi.org/10.1016/j.procbio.2019.11.015. Paakkonen, I., Rissanen, T., Ora, A., Anugwom, I.: Valorization of Waste Wool to Keratin with a Green Solvent Based on a Deep Eutectic Mixture of Choline Chloride and Lactic Acid. Waste Biomass Valorization. 16, 1045–1055 (2025). https://doi.org/10.1007/s12649-024-02733-8. Wang, H., Gurau, G., Rogers, R.D.: Dissolution of Biomass Using Ionic Liquids. Structure and Bonding 151, 79–105 (2013). https://doi.org/10.1007/978-3-642-38619-0-3. Shiddiky, M.J.A. Torriero, A.A.J.: Application of ionic liquids in electrochemical sensing systems. Biosensors and Bioelectronics 26, 1775–1787 (2011). Matsumoto, K., Endo, T.: Synthesis of Ion Conductive Networked Polymers Based on an Ionic Liquid Epoxide Having a Quaternary Ammonium Salt Structure. Macromolecules 42, 4580–4584 (2009). Li, X., Guo, Z., Li, J., Yang, M., Yao, S.: Swelling and microwave-assisted hydrolysis of animal keratin in ionic liquids. J. Mol. Liq. 341, 117306 (2021). https://doi.org/10.1016/j.molliq.2021.117306. Xie, H., Li, S., Zhang, S.: Ionic liquids as novel solvents for the dissolution and blending of wool keratin fibers. Green Chem. 7, 606–608 (2005). https://doi.org/10.1039/B502547H. Idris, A., Vijayaraghavan, R., Rana, U.A., Patti, A.F., MacFarlane, D.R.: Dissolution and regeneration of wool keratin in ionic liquids. Green Chem. 16, 2857 (2014). Ji, Y., Chen, J., Lv, J., Li, Z., Xing, L., Ding, S.: Extraction of keratin with ionic liquids from poultry feather. Separation and Purification Technology 132, 577–583 (2014). Riesz, P., Kondo, T.: Free Radical Formation Induced by Ultrasound and Its Biological Implications. Free Radical Biology & Medicine 13, 247–270 (1992). Kerboua, K.: Acoustic Cavitation and Ionic Liquid Combined: A Modelling Investigation of the Possible Promises in Terms of Physico-Chemical Effects. Eng. Proc. 56, 237 (2023). Azmi, N.A., Idris, A., Yusof, N.S.M.: Ultrasonic Technology for Value Added Products from Feather Keratin. Ultrasonics Sonochemistry 47, 99–107 (2018). https://doi.org/10.1016/j.ultsonch.2018.04.016. Qin, X., Yang, C., Guo, Y., Liu, J., Bitter, J.H., Scott, E.L., Zhang, C.: Effect of ultrasound on keratin valorization from chicken feather waste: Process optimization and keratin characterization. Ultrasonics Sonochemistry 93, 106297 (2023). https://doi.org/10.1016/j.ultsonch.2023.106297. Kakkar, P., Madhan, B., Shanmugam, G.: Extraction and characterization of keratin from bovine hoof: A potential material for biomedical applications. Springer Plus 3, 596 (2014). https://doi.org/10.1186/2193-1801-3-596. Zhang, Z., Nie, Y., Zhang, Q., Liu, X., Tu, W., Zhang, X., Zhang, S.: Quantitative Change in Disulfide Bonds and Microstructure Variation of Regenerated Wool Keratin from Various Ionic Liquids. ACS Sustainable Chem. Eng. 5, 2614–2622 (2017). https://doi.org/10.1021/acssuschemeng.6b02963. Borges, M.S., Barbosa, R.S., Rambo, M.K.D., Rambo, M.C.D., Scapin, E.: Evaluation of residual biomass produced in Cerrado Tocantinense as potential raw biomass for biorefinery. Biomass Conv. Bioref. 12, 3055–3066 (2022). https://doi.org/10.1007/s13399-020-00892-x. Supplementary Files Highlightsforreview.docx Graphicalabstract.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 06 Sep, 2025 Reviewers invited by journal 06 Sep, 2025 Editor invited by journal 17 Aug, 2025 Editor assigned by journal 30 Jul, 2025 First submitted to journal 30 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7252336","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":511277553,"identity":"26b89c62-eca6-4dfc-b9f7-5523ff10ef7a","order_by":0,"name":"Prasath Loganathan","email":"","orcid":"","institution":"CSIR-CLRI: Central Leather Research Institute CSIR","correspondingAuthor":false,"prefix":"","firstName":"Prasath","middleName":"","lastName":"Loganathan","suffix":""},{"id":511277554,"identity":"b45bf242-62d0-43c0-89dc-68c7f9a0cfb6","order_by":1,"name":"Nitin prakash Lobo","email":"","orcid":"","institution":"CSIR-CLRI: Central 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14:41:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":170002,"visible":true,"origin":"","legend":"\u003cp\u003eSequence of unit operations carried out for keratin extraction by an acoustic cavitation assisted method\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7252336/v1/e205fa267c3a9ecf0dd31e9d.png"},{"id":91194009,"identity":"a5bf0185-936d-41bd-8754-234694e329e2","added_by":"auto","created_at":"2025-09-12 14:49:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":22074,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of solid-to-liquid ratios on keratin yield using acoustic cavitation-assisted dissolution 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14:41:58","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":17002,"visible":true,"origin":"","legend":"","description":"","filename":"Highlightsforreview.docx","url":"https://assets-eu.researchsquare.com/files/rs-7252336/v1/6d3dca9272e43e94d945f1fb.docx"},{"id":91195389,"identity":"26544d89-6735-4db3-9023-6898f916af43","added_by":"auto","created_at":"2025-09-12 14:57:58","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":123873,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.docx","url":"https://assets-eu.researchsquare.com/files/rs-7252336/v1/4ad51642bb2cc18f6cfed910.docx"}],"financialInterests":"","formattedTitle":"Sustainable valorization of tannery hair waste into keratin by an acoustic cavitation-assisted process using a green solvent","fulltext":[{"header":"Introduction","content":"\u003cp\u003eKeratin is composed of protein component of various biological materials, such as hair, wool, and horns [1]. The tanning industry generates approximately 40 kilotons of animal hair waste annually [2]. This waste poses environmental problems and disposal challenges associated with hair generated during the unhairing process of the skins/hides, typically being landfilled, contributing to pollution [3]. However, this waste material can be valorized for obtaining valuable keratin-based products. Unlike other natural materials, keratin is difficult to dissolve and extract due to its unique polypeptide chain composition, featuring extensive inter- and intramolecular interactions, including disulphide cross-linking between cysteine residues, which impart remarkable stability and insolubility in various solvents [4]. The presence of cysteine disulphide bonds plays a vital role in determining the physico-chemical properties of keratin [5].\u003c/p\u003e\n\u003cp\u003eSeveral methods have been followed to extract keratin which includes reduction, alkaline hydrolysis, oxidation, sulphitolysis and ionic liquids [6]. In the reduction process, keratin is extracted through the dissolution of wool using urea and 2-mercaptoethanol, resulting in a lower yield of keratin hydrolysate (45%). However, this method produces toxic and harmful by products [7]. Furthermore, the alkaline hydrolysis method compromises keratin\u0026apos;s structure, leading to considerable amino acid degradation and low keratin recovery [1].\u003c/p\u003e\n\u003cp\u003eAmong the given literature, in the oxidation method, although is notable for its ease of extraction, it employs per-acetic or per-formic acid leading to the oxidation of cystine residues to cysteic acid, which not only altering keratin\u0026apos;s chemical structure but also results in a relatively lower keratin yield of 6% [8]. In the sulphitolysis method, red sheep hair is treated with a mixture of urea and sodium metabisulfite to extract keratin in a hydrolysate form, with a poor yield of 29% [9].\u003c/p\u003e\n\u003cp\u003eIn a recent study, deep eutectic solvent (DES) has been used to viable option for keratin extraction from sheep wool. However, it resulted in lower yield of keratin (23%) at optimal conditions [10]. The studies are given in the literature are not suitable for commercial applications due to poor yield (20-45%) of keratin and generation of toxic waste.\u003c/p\u003e\n\u003cp\u003eIonic liquids (ILs) have recently gained attention as a sustainable solvent over deep eutectic solvents in various applications, including biomass extraction [11]. Further it was also used in electrochemistry [12], ion-conductive media, and catalysis [13], owing to their exceptional physicochemical properties. Li et al. [14] demonstrated the effectiveness of acidic IL (type of protic IL) in hydrolysing keratin from wool under microwave radiation, with 1-propylsulfonic-3-methylimidazolium hydrogen sulphate ([PSMIM]HSO₄) showing better results for keratin hydrolysis. Studies on the effect of different IL for dissoluting the wool was conducted by Xie et al. [15] and Idris et al. [16]. However, the studies reported a poor yield. Ji et al. [17] investigated with three primary ILs: 1-Butyl-3-methylimidazolium bromide [BMIM]Br, 1-Allyl-3-methylimidazolium chloride [AMIM]Cl and 1-Butyl-3-methylimidazolium chloride [BMIM]Cl, and found that Cl-containing ILs exhibited higher keratin dissolution rates, achieving a 75% keratin yield from feathers. This approach underscores the potential of chloride containing ILs in effective extraction and producing keratin in its native form (non-hydrolysate). Despite the demonstrated potential of [BMIM]Cl in keratin recovery from wool and poultry feathers, its application for the sustainable valorization of keratin-rich tannery hair waste has not been investigated yet.\u003c/p\u003e\n\u003cp\u003eOn the other hand, ultrasonic-assisted processing has been widely explored for its potential to enhance chemical reactions and mass transfer of biomolecules such as proteins, enzymes, and polymers [18]. Studies have shown that probe-type sonication generates acoustic streaming and cavitation, thereby enhancing mass transfer and facilitating chemical reactions by mechanical vibration effect. This physical phenomenon of acoustic cavitation has significant implications for various applications such as cleaning, biomass extraction, industrial and biomedical applications [19].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRecently, Feroz et al. [5] introduced a novel approach combining sonication with protic ionic liquids Tetra butyl phosphonium hydroxide (TBPH) and choline hydroxide (CH), facilitate wool dissolution to produce keratin hydrolysate. Similarly, Azmi et al. [20] demonstrated that the low frequency high power sonication is effective in extraction of keratin by dissolving the feather using [BMIM]Cl. Although the keratin was extracted in its native structure, the yield was relatively low (~10%). The keratin extracted from different waste biomaterials are provided in Table 1.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e Extraction of keratin by sonication assisted methods\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"600\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBio-materials\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTreatment conditions\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 184px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;Keratin\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eYields (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eReferences\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNon-Hydrolysate\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHydrolysate\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eProbe sonication assisted with ionic liquid\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 98px;\"\u003e\n \u003cp\u003eWool\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eTetra butyl phosphonium hydroxide (TPBH) at optimized conditions acoustic power 40%, acoustic time 20 min. with initial loading of 20%.\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003e95%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003e[5]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eTurkey Feathers\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003eAprotic Ionic liquid\u0026nbsp;[BMIM]Cl at sonication at 20\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;kHz with lower sonication power at 200 W for 52 min.\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e10%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e[20]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003eChicken feathers\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e8M Urea and 15 % Cysteine with pH 10.5 at 130 W for 2.7 h\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e63.2%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e[21]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThis work advances the principles of green chemistry and biomass valorization by demonstrating a sustainable and efficient strategy for the recovery of native keratin from tannery hair waste. The advantage lies in the significant reduction of dissolution time compared to conventional methods. Furthermore, optimizing sonication time and power parameters demonstrates its potential as a scalable and effective technique for keratin extraction.\u003c/p\u003e\n\u003cp\u003eThis study presents a novel eco-friendly approach for keratin extraction from tannery animal hair waste using an aprotic ionic liquid ([BMIM]Cl) in combination with acoustic cavitation. \u0026nbsp;This method overcomes key limitations of conventional approaches by significantly reducing dissolution time, enhancing keratin yield, and preserving the native protein structure. Based on previous studies, the chloride-containing imidazolium salt [BMIM]Cl was selected as the solvent for its efficacy in dissolving keratin.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe primary objective of this study is to optimize the acoustic cavitation assisted dissolution process while extracting keratin in its native form with enhanced yield. To the best of our knowledge, this is the first report to demonstrate the integration of acoustic cavitation with a chloride-based aprotic IL for keratin extraction from tannery hair waste. This innovative combination enables the sustainable valorization of a low-value industrial by-product into a high-value biomaterial, aligning with circular economy and sustainable waste management principles.\u003c/p\u003e\n\u003cp\u003eThe method developed in this work is scalable, energy-efficient, and avoids the use of harsh chemicals, aligning with industrial sustainability targets\u003cstrong\u003e.\u003c/strong\u003e Systematic studies were conducted to evaluate the operating parameters, paving the way for sustainable keratin extraction and promoting a circular economy. By utilizing the tannery animal hair waste as a raw material, this study contributes to waste reduction and resource recovery, enabling the efficient valorization of tannery animal hair waste into a valuable biomedical product. The extracted keratin was characterized using various techniques, including Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR), X-ray Diffraction (XRD), solid-state \u003csup\u003e13\u003c/sup\u003eC Nuclear Magnetic Resonance (NMR), Circular Dichroism (CD), Differential Scanning Calorimetry (DSC) and Thermogravimetric Analysis (TGA) to determine its physical, chemical, and thermal properties. Additionally, morphological studies were carried out by Field Emission Scanning Electron Microscopy (FE-SEM) to examine the surface and structural features of the obtained keratin.\u003c/p\u003e"},{"header":"Experimental","content":"\u003cp\u003e\u003cstrong\u003eMaterials and Methods\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, sheep hair waste, obtained from the post-unhairing unit operation (lime-sulphide process) at the Leather Processing Division, CSIR-CLRI, was used as raw material. To remove the impurities the hair was washed with distilled water and dried. It was then defatted with a hexane: DCM mixture (1:1) in a Soxhlet extractor for 48 hours to remove fats and oils. After defatting, the hair was dried at 70 \u0026deg;C for 48 hours in vacuum oven. The dried hair was size-reduced to 10-12 mm in length and used for dissolution studies.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo improve keratin yield, the hair sample was pre-soaked in a 2% thioglycolic acid (TGA) solution for 15 minutes prior to dissolution.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor pure keratin, raw hair obtained from the tannery of CSIR-CLRI, washed and cleaned, was used as a reference and standard to compare with the extracted keratin.\u003c/p\u003e\n\u003cp\u003eIn this study the chemicals used are 1-Butyl-3-methylimidazolium chloride ([BMIM]Cl) and thioglycolic acid (TGA), procured from Sigma Aldrich, India. \u0026nbsp;Hexane, dichloromethane (DCM), dimethyl sulphoxide (DMSO) and deuterated dimethyl sulfoxide (DMSO-d\u003csub\u003e6\u003c/sub\u003e) were purchased from SRL Chemicals, India.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcoustic cavitation assisted hair dissolution\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe dissolution of hair samples was conducted using [BMIM]Cl as a solvent, followed by acoustic irradiation with a probe-type sonicator (Ultrasonic Vibracell, 50 Hz, Model VCX1500, Sonics \u0026amp; Materials, Inc., USA) operating in pulse mode of five seconds. The ultrasonic probe was immersed to a depth of approximately one third of the total height in a 25 mL beaker containing the hair and IL mixture. To prevent overheating and potential degradation of keratin the beaker was surrounded by ice packs to dissipate the heat generated during the acoustic irradiation process.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn order to confirm complete dissolution, required volume of DMSO was added to the mixture, and visually inspected for any undissolved hair particles in the solution. A homogeneous solution indicated complete dissolution. The addition of DMSO also reduced the viscosity of the mixture, facilitating the movement of keratin molecules and enhancing precipitation. Subsequently, the solution was stirred at 250 rpm and centrifuged at 5000 rpm for 25 min., at 22 \u0026deg;C to facilitate keratin extraction. The resulting mixture was then regenerated using distilled water via centrifugation at 5000 rpm for 25 minutes at 22 \u0026deg;C. To remove residual IL and DMSO, the precipitate was subjected to repeated washings with distilled water, and centrifuged at 12000 rpm for 10 min. Finally, the precipitated insoluble keratin was then lyophilized to obtain a keratin powder. The sequence of unit operations for keratin extraction by an acoustic cavitation assisted dissolution is shown in Fig. 1.\u003c/p\u003e\n\u003cp\u003eThe keratin yield was calculated by using mathematical expression\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCharacterization studies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCHNS analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eElemental composition was determined by CHNS analysis using an Elementar UNICUBE analyser (Serial No: 0400BD1009). The protein content was estimated from the nitrogen content using a conversion factor of 6.25 [8].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eATR-FTIR analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFTIR spectra were recorded using ATR-FTIR spectrometer (Bruker Alpha II-Platinum) with a platinum-diamond sample module and a deuterated triglycine sulphate (DTGS) detector, over a wavenumber range of 4000-600 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e, spectral resolution of 4 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e, averaging 24 scans and subsequently baseline-corrected [16].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eXRD analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePowder X-ray diffraction (XRD) patterns were registered at 25 \u0026deg;C using X-ray diffractometer (Rigaku Mini Flex-II) with Cu K\u0026alpha; radiation (\u0026lambda; = 1.5418 \u0026Aring;) in Bragg-Brentano geometry. The finely ground sample (~5 mg) was kept on a custom-designed sample holder made of flat brass with an O-ring seal and Mylar sheet cover to maintain airtight atmosphere. XRD parameters included a voltage of 15 kV, current of 30 mA, 2\u0026theta; range of 5-50\u0026deg;, step size of 0.02\u0026deg;, and scan rate of 1\u0026deg; min\u003csup\u003e\u0026minus;1\u003c/sup\u003e [16].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNMR analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNMR spectra were obtained from 400 MHz spectrometer (Bruker Avance). Solid-state \u003csup\u003e13\u003c/sup\u003eC NMR spectra were recorded with 976 scans at 400 MHz. Solution-state \u003csup\u003e1\u003c/sup\u003eH and \u003csup\u003e13\u003c/sup\u003eC NMR spectra were obtained at 400 MHz with 16 scans, using deuterated dimethyl sulfoxide (DMSO-d\u003csub\u003e6\u003c/sub\u003e) as solvent. Chemical shifts are reported in parts per million (ppm) on the \u0026delta; scale [16].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDSC analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDifferential scanning calorimetry (DSC) measurements were conducted using a TA Instruments Discovery DSC 25 series instrument under a nitrogen atmosphere. Samples (2-3 mg) sealed in Tzero aluminium pans underwent a thermal protocol involving cooling to -40 \u0026deg;C for 5 minutes, and then by heating from 20 \u0026deg;C to 350 \u0026deg;C at 10 \u0026deg;C min\u003csup\u003e\u0026minus;1\u003c/sup\u003e. Prior to sample testing the instrument was calibrated (using indium; melting point 156.6 \u0026deg;C) to ensure thermal accuracy [22]. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTGA analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThermogravimetric analysis (TGA) was carried out using thermal analyser (NETZSCH STA 449F3) to assess the thermal stability of the sample. This was done by heating the sample (2-5 mg) from 30 \u0026deg;C to 600 \u0026deg;C at an increment of 10 \u0026deg;C/min under nitrogen atmosphere [5].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCD analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCircular dichroism (CD) spectroscopy was done by using Jasco J-715 spectrometer. The analysis was conducted under a nitrogen atmosphere (5 LPM) in a quartz cuvette, 1 mm path length, scanning from 190 to 260 nm at the rate of 100 nm/min [9].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFE-SEM analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe morphology of extracted keratin and raw hair were examined using scanning electron microscope (SEM, make TESCAN CLARA). The samples were coated using gold-palladium alloy (80:20), analysed at an acceleration voltage of 10 kV under nitrogen atmosphere [9].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eResults and Discussions\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOptimizing the process parameters for hair dissolution using [BMIM]Cl\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo maximize keratin extraction efficiency, the dissolution of hair samples was optimized with respect to three key parameters, i.e. solid-to-liquid ratio, acoustic cavitation irradiation time and acoustic power. These parameters were systematically varied to determine their impact on keratin yield.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of solid to liquid ratios on keratin yield\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFig. 2 illustrates the impact of solid-to-liquid ratio on keratin yield using [BMIM]Cl, where ratios of 1:20, 1:40, and 1:60 were evaluated under constant acoustic cavitation conditions (450 W, 30 min). The corresponding keratin yields were 54% \u0026plusmn; 0.9%, 71% \u0026plusmn; 1.6%, and 74% \u0026plusmn; 1.6%, respectively. Notably, the higher yields observed at 1:40 and 1:60 ratios can be attributed to the optimal ionic liquid volume, facilitating effective propagation of ultrasonic waves and maximizing cavitation intensity. This, in turn, enhances the disruption of disulphide bonds and penetration of the ionic liquid, ultimately leading to improved keratin extraction. Conversely, the lower yield at 1:20 ratio (54% \u0026plusmn; 0.9%) is likely due to insufficient solvent volume, resulting in reduced cavitation efficiency and incomplete dissolution. Considering the marginal difference in yields between the 1:40 and 1:60 ratios, the 1:40 ratio was determined as the optimal solid-to-liquid ratio, offering a balance between maximizing keratin yield and preserving its native structure\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of acoustic cavitation irradiation time on keratin yield\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA critical parameter influencing the acoustic cavitation assisted dissolution process is the acoustic irradiation time, which significantly affects both energy consumption and keratin extraction efficiency. Fig. 3 shows the impact of varying acoustic irradiation times (20, 25, and 30 min) on hair dissolution in [BMIM]Cl at a constant acoustic power of 450 W. The corresponding keratin yields were 28% \u0026plusmn; 2.5%, 47% \u0026plusmn; 1.62%, and 71% \u0026plusmn; 1.35%, respectively, demonstrated a notable increase in yield with prolonged irradiation time. The optimal irradiation time was determined to be 30 min., as further extension beyond this duration yielded only marginal improvements in keratin extraction. This suggested that prolonged exposure to acoustic cavitation is not necessary.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of acoustic power on keratin yield\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe acoustic power on hair dissolution in IL was examined by varying power levels such as 300, 450, and 600 W at a constant acoustic irradiation time of 30 min. The results, showed in Fig. 4, reveal the keratin yields of 33% \u0026plusmn; 1%, 70.5% \u0026plusmn; 1.5%, and 65% \u0026plusmn; 1% for the respective power levels. Acoustic power plays a crucial role in modulating cavitation intensity, thereby significantly impacting the dissolution process. Particularly, an acoustic power of 450 W yielded the highest keratin recovery of 70.5% \u0026plusmn; 1.5%, suggesting optimal cavitation conditions for efficient keratin extraction. In contrast, the decreased yield at 600 W (65% \u0026plusmn; 1%) can be attributed to excessive energy input (\u0026sim;52,683 kJ/kg), potentially leading to keratin degradation or fragmentation into smaller peptide chains. The degradation compromises the overall keratin yield and structural integrity, highlighted the importance of optimizing acoustic power for efficient keratin extraction preserving its structural integrity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTemperature variation with time during hair dissolution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFig. 5 depicts the temperature profile of different acoustic powers showing a significant increase in temperature over time. At acoustic power of 300, 450 and 600 W, corresponding energy input as in fig. 5, the temperature gradually rises to maximum values of 62, 76, and 90 \u0026deg;C, respectively, after 30 min. of acoustic irradiation. This temperature increase is attributed to the conversion of acoustic energy into thermal energy, resulting from the acoustic cavitation phenomenon. The elevated temperature is believed to enhance ionic mobility and reduce mixture viscosity, facilitating efficient hair dissolution by promoting better solvent penetration and interaction with the keratin structure. Specifically, the increased ionic mobility and reduced viscosity enable the IL to more effectively disrupt the keratin disulphide bonds, leading to improved dissolution. However, beyond 130 \u0026deg;C, keratin\u0026apos;s structural integrity will lead to degradation and reduce the keratin yield, as reported in previous studies.\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eBased on the experimental findings, the optimized parameters for efficient keratin extraction were determined to be a solid-to-liquid ratio of 1:40, acoustic irradiation of 30 min. and power of 450 W, corresponding to a specific energy input of approximately 39,512 kJ/kg. Under these optimized conditions, the extracted keratin was subsequently characterized.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInfluence of reducing agent on keratin extraction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe addition of reducing agent, TGA, prior to acoustic cavitation assisted dissolution process significantly enhanced keratin extraction yield up to 75.1\u0026plusmn;0.7%, representing 6.5% increase compared to the yield without using reducing agent. \u0026nbsp;This improvement can be attributed to the partial reduction of inter- and intramolecular disulphide bonds in keratin, which disrupted its rigid structure and increased its solubility. The disruption of these bonds facilitated the extraction of keratin, resulting in a higher yield. This observation is consistent with previous studies, such as those reported by Idris et al. [16], which emphasize the crucial role of disulphide bond reduction in enhancing protein solubility and extraction efficiency. The substantial increase in keratin yield suggests effective dissolution of hair, enabling efficient extraction and potentially preserving the protein\u0026apos;s structural integrity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCharacterization of extracted keratin\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCHNS analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe chemical composition and protein content of extracted keratin and raw hair were evaluated by CHNS elemental analysis. It is observed that, between the two samples, there is a marginal difference in the composition of elements. Raw hair comprised 43.9% carbon, 6.8% hydrogen, 14.6% nitrogen, and 3% sulphur, whereas extracted keratin consisted of 45.4% carbon, 7.67% hydrogen, 14.72% nitrogen, and 2% sulphur. The slightly higher nitrogen content in extracted keratin (14.72%) compared to raw hair (14.6%) is consistent with the high protein yield of 92%, indicating a purity level exceeding 90% [8].\u003csup\u003e\u0026nbsp;\u003c/sup\u003eThis increase in nitrogen content suggests that during the extraction process the non-proteinaceous components have been effectively removed. Thermogravimetric analysis (TGA) further supported the high purity of the extracted keratin, with minimal weight loss observed during the initial stage due to the evaporation of bound water.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eATR-FTIR analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe ATR-FTIR spectra of raw hair and extracted keratin shown in Fig. 6(a) and 6(b) exhibit characteristic absorption peaks associated with peptide bonds (-CONH-), providing insights into their secondary structure. In raw hair, the amide I peak is observed at 1627 cm\u003csup\u003e-1\u003c/sup\u003e, attributed to C=O stretching vibrations, while amide II peak appears at 1534 cm\u003csup\u003e-1\u003c/sup\u003e, resulting from C-N stretching and N-H bending vibrations. Similarly, the extracted keratin exhibits a strong amide I peak at 1633 cm\u003csup\u003e-1\u003c/sup\u003e and an amide II peak at 1522 cm\u003csup\u003e-1\u003c/sup\u003e. These vibrations are sensitive to protein secondary structure, enabling the determination of conformational changes in \u0026alpha;-helix and \u0026beta;-sheet structures. The slight peak shifts in the extracted keratin suggest interactions between the ionic liquid and polypeptide chains. The amide A peak, corresponding to N-H stretching vibrations, is observed at 3266 cm\u003csup\u003e-1\u003c/sup\u003e in the extracted keratin and 3271 cm\u003csup\u003e-1\u003c/sup\u003e in raw hair. Additionally, the amide III peak, indicative of C-N stretching and N-H bending vibrations, appears as a weak peak at 1232 cm\u003csup\u003e-1\u003c/sup\u003e in the extracted keratin and 1233 cm\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003ein raw hair. This similarity in peak positions between raw hair and extracted keratin indicates that the keratin was successfully extracted in its native form (non-hydrolysate), retaining its secondary structure (\u0026alpha;-helix and \u0026beta;-sheet).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eXRD analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFig. 7 shows the crystallinity of the raw hair and the extracted keratin. The patterns of raw hair revealed characteristic peaks at 2\u0026theta; values of 9.38\u0026deg; (0.94 nm) and 16.58\u0026deg; (0.54 nm), corresponding to \u0026alpha;-helix structures, and a peak at 9.38\u0026deg; (0.94 nm) and 21.7\u0026deg; (0.42 nm), characteristic of \u0026beta;-sheet structures. However, the extracted keratin shows the peak at 9.38\u0026deg; was disappeared, suggesting a significant reduction in crystallinity during the dissolution process. The extracted keratin showed a similar diffraction pattern with a peak at 23.8\u0026deg; (0.38 nm) but slightly higher intensity (77%) compared to raw hair (75%), which suggested an increased \u0026beta;-sheet content and reduced crystallinity [5,16,19].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDSC-TGA analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFig.8 shows the thermal properties of raw hair and extracted keratin, investigated using DSC and TGA analysis. DSC analysis revealed endothermic peaks around 100 \u0026deg;C corresponded to the evaporation of water in both raw hair and extracted keratin, while peaks above 230 \u0026deg;C indicated the denaturation of \u0026alpha;-helix [22]. TGA curves showed two distinct stages of decomposition, the first stage around 100 ℃ corresponded to moisture evaporation with weight losses of approximately 7.5% and 10.5% for extracted keratin and raw hair respectively. The lower weight loss in extracted keratin suggests higher purity, potentially exceeding 90%, based on nitrogen content determined using CHNS analysis [9]. The second stage, attributed to keratin\u0026apos;s helical structure (\u0026alpha;-helix) degradation and disulfide bond breakage above 230 ℃. During this stage, the extracted keratin exhibited a weight loss of about 14.8% and whereas raw hair showed a weight loss of approximately 16.2%. These results showed that the extracted keratin is more stable than the raw hair and the extracted keratin possesses high thermal stability make it a promising biomaterial for various biomedical applications.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSolid state \u003csup\u003e13\u003c/sup\u003eC NMR analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFig. 9(a) and 9(b) shows the NMR spectra of raw hair and extracted keratin, revealing its structural characteristics of the keratin molecule. Compared to raw hair (Fig. 9(a)), the extracted keratin spectrum (Fig. 9(b)) exhibited an asymmetric peak at 172-174 ppm, attributed to amide carbonyl carbons found in keratin\u0026apos;s protein backbone. The peak at 172-175 ppm provides insight into \u0026alpha;-helix and \u0026beta;-sheet conformations. Aromatic amino acids are indicated by a peak at ~127 ppm, while \u0026alpha;-carbons and \u0026beta;-carbons found in leucine and cross-linked cysteine residues of the protein structure are detected at 50-53 ppm and 39-40 ppm, respectively [23].\u003csup\u003e\u0026nbsp;\u003c/sup\u003eThe retention of newly formed disulfide bridges (-S-S-) found in cysteine residues is suggested by the presence of peak at 39-40 ppm, indicating stability of the extracted keratin. Peaks between 29-40 ppm corresponded to proline, glutamic acid, and glutamine residues, while the peak at ~20 ppm indicates alanine residues. The \u0026alpha;-carbon peak (50-53 ppm) exhibits slight broadening and shifting to the lower frequency of the chemical shift, indicating disruption of hydrogen bonding in the hair due to dissolution process and formation of a more ordered \u0026beta;-sheet structure in the extracted keratin, consistent with XRD results.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFE-SEM analysis \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFE-SEM images in Figure 10 reveals the morphological transformation of raw hair (Fig. 10(a)) to extracted keratin (Fig. 10(b)). The raw hair tubular structure is transformed into a non-uniform, hollow honeycomb-like keratin structure, characterized by disruption of cuticle layer, exposing cortex and medulla. This structural change, facilitated by the acoustic cavitation assisted ionic liquid dissolution process, indicates efficient keratin extraction while preserving its structural integrity. The resulting porous morphology retains key features of native keratin structure due to mild processing conditions. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCD analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe CD spectrum of extracted keratin is given in Fig. 11. The characteristic features of \u0026alpha;-helix and \u0026beta;-sheet conformations, providing insight into its of its secondary structure. The negative ellipticity bands at 208-215 nm and a positive band at approximately 190 nm indicates \u0026alpha;-helical structures, while the negative band at 218-220 nm and positive band at 192-195 nm suggests the presence of \u0026beta;-sheets. These spectral characteristics are consistent with previous reports [8], confirming that the extraction process preserves the native structural integrity of keratin.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e Physicochemical and structural properties of raw hair and extracted keratin\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 202px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAnalysis\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 199px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRaw hair\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(reference)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 200px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eExtracted keratin\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 202px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eATR-FTIR\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 199px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 200px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 202px;\"\u003e\n \u003cp\u003eAmide A (cm\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 199px;\"\u003e\n \u003cp\u003e3271\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 200px;\"\u003e\n \u003cp\u003e3266\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 202px;\"\u003e\n \u003cp\u003eAmide I (cm\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 199px;\"\u003e\n \u003cp\u003e1627\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 200px;\"\u003e\n \u003cp\u003e1633\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 202px;\"\u003e\n \u003cp\u003eAmide II (cm\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 199px;\"\u003e\n \u003cp\u003e1534\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 200px;\"\u003e\n \u003cp\u003e1522\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 202px;\"\u003e\n \u003cp\u003eAmide III (cm\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 199px;\"\u003e\n \u003cp\u003e1233\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 200px;\"\u003e\n \u003cp\u003e1232\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 202px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 199px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 200px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 202px;\"\u003e\n \u003cp\u003e\u0026alpha;-helix (positive and negative bands) in nm\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 199px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 200px;\"\u003e\n \u003cp\u003e190 \u0026amp; 208-215\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 202px;\"\u003e\n \u003cp\u003e\u0026beta;-sheet (positive and negative bands) in nm\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 199px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 200px;\"\u003e\n \u003cp\u003e192-195 \u0026amp; 218-220\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 202px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eXRD\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 199px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 200px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 202px;\"\u003e\n \u003cp\u003ed-spacing value (2q;\u0026nbsp;nm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 199px;\"\u003e\n \u003cp\u003e21.7\u0026deg;; 0.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 200px;\"\u003e\n \u003cp\u003e23.8\u0026deg;; 0.38\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 202px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDSC and TGA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 199px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 200px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 202px;\"\u003e\n \u003cp\u003eWater loss temperature (℃)/ Weight loss (%)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 199px;\"\u003e\n \u003cp\u003eAround 100 \u0026amp; 10.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 200px;\"\u003e\n \u003cp\u003eAround 100 \u0026amp; 7.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 202px;\"\u003e\n \u003cp\u003eDenaturation temperature (℃)/ Weight loss (%)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 199px;\"\u003e\n \u003cp\u003eAbove 250 \u0026amp; 16.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 200px;\"\u003e\n \u003cp\u003eAbove 250 \u0026amp; 14.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 202px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSolid-state \u003csup\u003e13\u003c/sup\u003eC NMR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 199px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 200px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 202px;\"\u003e\n \u003cp\u003eC=O group (ppm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 199px;\"\u003e\n \u003cp\u003e172-175\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 200px;\"\u003e\n \u003cp\u003e172-175\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 202px;\"\u003e\n \u003cp\u003e\u0026alpha;-carbon (ppm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 199px;\"\u003e\n \u003cp\u003e52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 200px;\"\u003e\n \u003cp\u003e53\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 202px;\"\u003e\n \u003cp\u003e\u0026beta;-carbon (ppm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 199px;\"\u003e\n \u003cp\u003e39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 200px;\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 202px;\"\u003e\n \u003cp\u003eAlky side chains (ppm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 199px;\"\u003e\n \u003cp\u003e19-30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 200px;\"\u003e\n \u003cp\u003e19-30\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 202px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSEM\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eMorphology\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 199px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eNo damage in hair\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 200px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eDamage in hair fragments\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 202px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCHNS analysis\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eProtein yield (%)\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 199px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 200px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e92\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eRecovery of IL from the effluent\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing dissolution studies, IL recovery is essential for ensuring the economic viability of the keratin extraction process. The successful recovery of IL was achieved via evaporation method, and the observed yield is 77%, indicating a relatively efficient recovery process. However, further optimization is needed to achieve higher yields and minimize the losses due to strong interactions between the IL and DMSO.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn order to confirm the purity, the recovered IL was subjected to NMR studies, and got confirmed through \u0026sup1;H and \u0026sup1;\u0026sup3;C NMR spectroscopy. The spectra are given in Fig. 12 and Fig. 13. It shows that identical proton and carbon signals when compared to standard [BMIM]Cl procured from M/s Sigma-Aldrich, indicating retention of the molecular structure. This spectral matching confirmed the preservation of the IL chemical structure and purity, suitability for its reuse. In order to further substantiate, ATR-FTIR spectroscopy of the recovered IL is given in Fig. 14. It is well corroborated and these findings exhibiting identical vibration frequencies to the standard IL. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInterpretations of NMR spectra\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe recovered ionic liquid [BMIM]Cl was characterized by \u003csup\u003e1\u003c/sup\u003eH-NMR (400 MHz, DMSO-d\u003csub\u003e6\u003c/sub\u003e, \u0026delta; 3.5 ppm for solvent peak) and \u003csup\u003e13\u003c/sup\u003eC-NMR (400 MHz, DMSO-d\u003csub\u003e6\u003c/sub\u003e, \u0026delta; 39.8 ppm for solvent peak) spectroscopy.\u003c/p\u003e\n\u003cp\u003eThe \u003csup\u003e1\u003c/sup\u003eH-NMR spectrum (Fig. 12) exhibited the following chemical shifts (\u0026delta;, ppm) and multiplicities: \u0026delta; 9.3 (1H, s), 7.8 (1H, s), 7.7 (1H, s), 4.2 (2H, t), 3.8 (3H, s), 2.5 (1H, s), 1.7 (2H, m), 1.3 (2H, m), 0.9 (3H, t).\u003c/p\u003e\n\u003cp\u003eThe\u0026nbsp;\u003csup\u003e13\u003c/sup\u003eC-NMR spectrum (Fig. 13) displayed the following chemical shifts (\u0026delta;, ppm):\u003cbr\u003e\u0026nbsp;\u0026delta; 136.8, 123.9, 122.7, 48.9, 36.0, 31.9, 19.2, 13.9.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComparison of ATR-FTIR spectra of standard and recovered [BMIM]Cl\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFig. 14 shows the ATR-FTIR spectra of standard and recovered [BMIM]Cl, confirming the structural integrity of the recovered IL through characteristic peaks corresponding to specific bond vibrations. The absorption peaks at 2951-2961 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e and 2864-2873 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e are attributed to asymmetric and symmetric (C\u0026ndash;H) aliphatic stretching of methyl groups, respectively. The HC-C and H-C-N bond vibrations peak located at 1165-1172 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e, while the C-N vibration appears at 750-764 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e in the imidazole ring. \u0026nbsp;A peak observed at 3382 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e is due to the quaternary amine salt formation with chlorine. These results are consistent with previous literature reported by Borges et al. [24].\u003c/p\u003e\n\u003cp\u003eThe results confirm the successful recovery and structural integrity of [BMIM]Cl, indicating its potential for reuse in the hair dissolution process and contributing to waste reduction. Studies on the recovery and reuse of the ionic liquid are currently in progress.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eAs compared to our previous studies without the application of acoustic cavitation (submitted for review elsewhere), acoustic cavitation-assisted keratin extraction from tannery animal hair waste using [BMIM]Cl achieved a yield of up to 71% keratin in its native form. By optimizing key parameters and utilizing a reducing agent, this approach significantly enhanced keratin yield while reducing environmental impact. The acoustic cavitation process reduced dissolution time by 95% compared to conventional methods, demonstrating its potential for scalable, eco-friendly production. Furthermore, 77% of [BMIM]Cl was recovered from the spent solution, enhancing the process environmental and economic viability through solvent recovery and waste minimization. While further optimization is needed, this study lays the groundwork for scaling up keratin extraction, potentially facilitating the development of novel biomedical applications. Overall, the process supports circular economy initiatives by valorizing low-value industrial tannery hair waste into high-value biomaterials, thereby promoting sustainable resource utilization and advancing the field of biomass valorization.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThis work was supported by Anusandhan National Research Foundation (ANRF), Government of India. (Grant number. EEQ/2021/000148). Dr. Lajapathi Rai Chockalingam has received research support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Prasath Loganathan, Nitin Prakash Lobo, Surianarayanan Mahadevan, and Lajapathi Rai Chockalingam. \u0026nbsp;The first draft of the manuscript was written by Prasath Loganathan and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analysed during the current study are not publicly available due to privacy constraints but are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank the Anusandhan National Research Foundation (ANRF)/ Science and Engineering Research Board (SERB), Government of India, for funding this project (File No. EEQ/2021/000148). We acknowledge the Centre for Analysis, Testing, Evaluation \u0026amp; Reporting Services (CATERS), CSIR-CLRI, for providing analytical services and data that facilitated our research. We acknowledge the support of the Knowledge Resource Centre (KRC), CSIR-CLRI, in assigning Communication Number 2129 to this manuscript for publication.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eShavandi, A., Silva, T.H., Bekhit, A.A., Bekhit, A.E.A.: Keratin: dissolution, extraction and biomedical application. Biomater. Sci. 5, 1699\u0026ndash;1735 (2017). https://doi.org/10.1039/C7BM00411G.\u003c/li\u003e\n \u003cli\u003eShavandi, A., Bekhit, A.E.A., Carne, A., Bekhit, A.A.: Evaluation of keratin extraction from wool by chemical methods for bio-polymer application. Journal of Bioactive and Compatible Polymers. 32, 1\u0026ndash;15 (2016). https://doi.org/10.1177/0883911516662069.\u003c/li\u003e\n \u003cli\u003eMidolo, G., Porto, S.M.C., Cascone, G., Valenti, F.: Sheep Wool Waste Availability for Potential Sustainable Re-Use and Valorization: A GIS-Based Model. Agriculture. 14, 872 (2024). https://doi.org/10.3390/agriculture14060872.\u003c/li\u003e\n \u003cli\u003eSinkiewicz, I., Śliwińska, A., Staroszczyk, H., Kołodziejska, I.: Alternative Methods of Preparation of Soluble Keratin from Chicken Feathers. Waste Biomass Valor. 8, 1043\u0026ndash;1048 (2017). https://doi.org/10.1007/s12649-016-9678-y.\u003c/li\u003e\n \u003cli\u003eFeroz, S., Muhammad, N., Dias, G., Alsaiari, M.A.: Extraction of keratin from sheep wool fibres using aqueous ionic liquids assisted probe sonication technology. J. Mol. Liq. 350, 11859 (2022). https://doi.org/10.1016/j.molliq.2022.118595.\u003c/li\u003e\n \u003cli\u003eZhang, X., Feng, Y., Yang, X.: Extraction of Keratin from Poultry Feathers with Choline Chloride-Oxalic Acid Deep Eutectic Solvent. Fibers and Polymers. 22, 3326\u0026ndash;3335 (2021). https://doi.org/10.1007/s12221-021-0255-z.\u003c/li\u003e\n \u003cli\u003eYamauchi, K., Yamauchi, A., Kusunoki, T., Kohda, A., Konishi, Y.: Preparation of stable aqueous solution of keratins and physiochemical and biodegradational properties of films. J. Biomed. Mater. Res. 31, 439\u0026ndash;44 (1996).\u003c/li\u003e\n \u003cli\u003eShavandi, A., Carne, A., Bekhit, A.A., Bekhit, A.E.A.: An improved method for solubilisation of wool keratin using peracetic acid. J. Environ. Chem. Eng. 5, 1977\u0026ndash;1984 (2017). https://doi.org/10.1016/j.jece.2017.03.04.\u003c/li\u003e\n \u003cli\u003eRamya, K.R., Thangam, R., Madhan, B.: Comparative analysis of the chemical treatments used in keratin extraction from red sheep\u0026rsquo;s hair and the cell viability evaluations of this keratin for tissue engineering applications. Process Biochem. 90, 223\u0026ndash;232 (2019). https://doi.org/10.1016/j.procbio.2019.11.015.\u003c/li\u003e\n \u003cli\u003ePaakkonen, I., Rissanen, T., Ora, A., Anugwom, I.: Valorization of Waste Wool to Keratin with a Green Solvent Based on a Deep Eutectic Mixture of Choline Chloride and Lactic Acid. Waste Biomass Valorization. 16, 1045\u0026ndash;1055 (2025). https://doi.org/10.1007/s12649-024-02733-8.\u003c/li\u003e\n \u003cli\u003eWang, H., Gurau, G., Rogers, R.D.: Dissolution of Biomass Using Ionic Liquids. Structure and Bonding 151, 79\u0026ndash;105 (2013). https://doi.org/10.1007/978-3-642-38619-0-3.\u003c/li\u003e\n \u003cli\u003eShiddiky, M.J.A. Torriero, A.A.J.: Application of ionic liquids in electrochemical sensing systems. Biosensors and Bioelectronics 26, 1775\u0026ndash;1787 (2011).\u003c/li\u003e\n \u003cli\u003eMatsumoto, K., Endo, T.: Synthesis of Ion Conductive Networked Polymers Based on an Ionic Liquid Epoxide Having a Quaternary Ammonium Salt Structure. Macromolecules 42, 4580\u0026ndash;4584 (2009).\u003c/li\u003e\n \u003cli\u003eLi, X., Guo, Z., Li, J., Yang, M., Yao, S.: Swelling and microwave-assisted hydrolysis of animal keratin in ionic liquids. J. Mol. Liq. 341, 117306 (2021). https://doi.org/10.1016/j.molliq.2021.117306.\u003c/li\u003e\n \u003cli\u003eXie, H., Li, S., Zhang, S.: Ionic liquids as novel solvents for the dissolution and blending of wool keratin fibers. Green Chem. 7, 606\u0026ndash;608 (2005). https://doi.org/10.1039/B502547H.\u003c/li\u003e\n \u003cli\u003eIdris, A., Vijayaraghavan, R., Rana, U.A., Patti, A.F., MacFarlane, D.R.: Dissolution and regeneration of wool keratin in ionic liquids. Green Chem. 16, 2857 (2014).\u003c/li\u003e\n \u003cli\u003eJi, Y., Chen, J., Lv, J., Li, Z., Xing, L., Ding, S.: Extraction of keratin with ionic liquids from poultry feather. Separation and Purification Technology 132, 577\u0026ndash;583 (2014).\u003c/li\u003e\n \u003cli\u003eRiesz, P., Kondo, T.: Free Radical Formation Induced by Ultrasound and Its Biological Implications. Free Radical Biology \u0026amp; Medicine 13, 247\u0026ndash;270 (1992).\u003c/li\u003e\n \u003cli\u003eKerboua, K.: Acoustic Cavitation and Ionic Liquid Combined: A Modelling Investigation of the Possible Promises in Terms of Physico-Chemical Effects. Eng. Proc. 56, 237 (2023).\u003c/li\u003e\n \u003cli\u003eAzmi, N.A., Idris, A., Yusof, N.S.M.: Ultrasonic Technology for Value Added Products from Feather Keratin. Ultrasonics Sonochemistry 47, 99\u0026ndash;107 (2018). https://doi.org/10.1016/j.ultsonch.2018.04.016.\u003c/li\u003e\n \u003cli\u003eQin, X., Yang, C., Guo, Y., Liu, J., Bitter, J.H., Scott, E.L., Zhang, C.: Effect of ultrasound on keratin valorization from chicken feather waste: Process optimization and keratin characterization. Ultrasonics Sonochemistry 93, 106297 (2023). https://doi.org/10.1016/j.ultsonch.2023.106297.\u003c/li\u003e\n \u003cli\u003eKakkar, P., Madhan, B., Shanmugam, G.: Extraction and characterization of keratin from bovine hoof: A potential material for biomedical applications. Springer Plus 3, 596 (2014). https://doi.org/10.1186/2193-1801-3-596.\u003c/li\u003e\n \u003cli\u003eZhang, Z., Nie, Y., Zhang, Q., Liu, X., Tu, W., Zhang, X., Zhang, S.: Quantitative Change in Disulfide Bonds and Microstructure Variation of Regenerated Wool Keratin from Various Ionic Liquids. ACS Sustainable Chem. Eng. 5, 2614\u0026ndash;2622 (2017). https://doi.org/10.1021/acssuschemeng.6b02963.\u003c/li\u003e\n \u003cli\u003eBorges, M.S., Barbosa, R.S., Rambo, M.K.D., Rambo, M.C.D., Scapin, E.: Evaluation of residual biomass produced in Cerrado Tocantinense as potential raw biomass for biorefinery. Biomass Conv. Bioref. 12, 3055\u0026ndash;3066 (2022). https://doi.org/10.1007/s13399-020-00892-x.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"waste-and-biomass-valorization","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wave","sideBox":"Learn more about [Waste and Biomass Valorization](http://link.springer.com/journal/12649)","snPcode":"12649","submissionUrl":"https://submission.nature.com/new-submission/12649/3","title":"Waste and Biomass Valorization","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Acoustic cavitation, [BMIM]Cl, Dissolution, Hair waste, Keratin, Tannery","lastPublishedDoi":"10.21203/rs.3.rs-7252336/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7252336/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eKeratin, a valuable biomaterial from tannery hair waste, has potential for biomedical applications due to its inherent biocompatibility, and biodegradability. This investigation deals with eco-efficient acoustic cavitation assisted method for extraction of keratin from tannery animal hair waste using ionic liquid [BMIM]Cl. Here, ionic liquid has been used as a solvent for better keratin recovery. Optimizing parameters such as solid-to-liquid ratio, acoustic irradiation time, and power, achieved a 71% keratin yield and a 95% reduction in reaction time, mitigating environmental impact and facilitating a cleaner, faster and more sustainable process. The extracted keratin's structural integrity and intactness were substantiated by ATR-FTIR and solid-state \u0026sup1;\u0026sup3;C NMR spectroscopy. Additionally, CD analysis revealed the presence of α-helix and β-sheet structures of keratin. XRD analysis confirmed the keratin's crystallinity, while DSC and TGA thermograms proved its thermal stability. FE-SEM studies elucidated the morphological features. Furthermore, the ionic liquid was successfully recovered from the effluent, and NMR studies confirmed its intact chemical structure, suggesting its potential for industrial applications. The process contributes to resource efficiency, waste minimization, and the development of sustainable materials from industrial residues.\u003c/p\u003e","manuscriptTitle":"Sustainable valorization of tannery hair waste into keratin by an acoustic cavitation-assisted process using a green solvent","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-12 14:41:53","doi":"10.21203/rs.3.rs-7252336/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-09-07T02:25:09+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-06T22:14:49+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Waste and Biomass Valorization","date":"2025-08-17T06:34:57+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-30T20:26:31+00:00","index":"","fulltext":""},{"type":"submitted","content":"Waste and Biomass Valorization","date":"2025-07-30T07:35:16+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"waste-and-biomass-valorization","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wave","sideBox":"Learn more about [Waste and Biomass Valorization](http://link.springer.com/journal/12649)","snPcode":"12649","submissionUrl":"https://submission.nature.com/new-submission/12649/3","title":"Waste and Biomass Valorization","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b6aa42df-9147-4045-93d4-c5ece41dfa26","owner":[],"postedDate":"September 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-16T14:46:28+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-12 14:41:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7252336","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7252336","identity":"rs-7252336","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}