Chicken feather dissolution to obtain keratin

Chicken feather dissolution to obtain keratin | 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 Chicken feather dissolution to obtain keratin Vahidesadat Zebhi-Ashkezari1, Mohammad Hassan Fazaelipoor This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5105662/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 22 You are reading this latest preprint version Abstract The dissolution of chicken feathers was investigated for the purpose of reducing solid waste and obtaining keratin. For the first part, the effects of sodium sulfite (0.05–0.3 M), urea (0–4 M), temperature (50–85 °C), and incubation time (2–4 h) on feather dissolution were investigated via a factorial design. Another factorial design was applied to investigate the effects of sodium dodecyl sulfate (SDS) (0–0.02 M), and lower ranges of urea (0.5–2 M), and incubation time (0.5–2 h) on feather dissolution. Keratin was precipitated from the solutions by ammonium sulfate and characterized via FTIR and SDS‒PAGE methods. The results of the first part demonstrated the feasibility of feather dissolution in the presence of sodium sulfite, and the effectiveness of urea and temperature on feather dissolution. For this part, a maximum feather dissolution of 45.35% was obtained with sodium sulfite and urea concentrations of 0.05 M and 4 M, respectively, at 85 °C for 4 hours. The results of the second part revealed a positive effect of SDS on feather dissolution. A maximum feather dissolution of 57.63% was obtained with sodium sulfite, urea, and SDS concentrations of 0.05 M, 2 M, and 0.02 M, respectively, at 85 °C for 2 hours. FTIR analysis confirmed the keratinous nature of the precipitated protein, and SDS‒PAGE revealed that the mass of the protein molecules was less than 15 kDa. This work confirms the feasibility of chemical dissolution of chicken feathers to obtain keratin as a useful product. chicken feather dissolution keratin urea sodium dodecyl sulfate sodium sulfite Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Chicken feathers are waste produced in poultry slaughterhouses in large amounts. It consists mainly of a protein named keratin (approximately 90%). Keratin is one of the most abundant structural proteins in animals and one of the many proteins found in the bodies of reptiles, birds and mammals. This protein is a structural component of nails, wool, feathers, hooves, etc. [ 1 ]. Keratin has high strength and hardness and is insoluble in polar and nonpolar solvents. Its stability is due to intra- and extramolecular disulfide cross-links, hydrogen bonds and crystallinity. These properties distinguish it from other fibrous proteins, such as myofibrillar and collagen [ 2 ]. Keratin is a protein rich in cysteine, which is connected to each other through intermediate strands with a diameter of 8–10 nm and is available in two forms: α-keratin and β-keratin [ 3 ]. The presence of cysteine in high amounts makes keratin susceptible to hydrolytic reactions and oxidation [ 2 ]. This polymer is composed of different amino acids that are able to increase intra- and extramolecular connections, and this feature allows the resulting materials to have diverse functional properties [ 4 ]. In recent years, attempts have been made to extract keratin via chemical and enzymatic methods. The methods of extracting keratin from biomass include acid hydrolysis, alkaline hydrolysis [ 5 ], enzymatic hydrolysis [ 6 – 7 ], ionic liquid hydrolysis [ 4 , 8 – 9 ], alkaline-enzymatic hydrolysis, and sulfitolysis [ 10 ]. Acid hydrolysis creates severe conditions that damage some useful amino acids during hydrolysis. In contrast, enzymatic hydrolysis results in fewer species changes, but the process is slow and expensive, which makes commercialization difficult [ 11 ]. Ionic liquids are also expensive, and their protein recovery is very low compared with that of other methods. In the sulfitolysis method, a reducing agent such as sodium sulfite is used to break disulfide bonds in the keratin structure. Urea is also used to denature protein structure, which leads to the extraction of keratin from keratin containing materials [ 12 ]. The extraction of keratin via sulfitolysis has been the subject of several studies. Isarankura et al. reported 87.6% keratin extraction from feathers in a solution containing 0.2 M sodium metabisulfite and 8 M urea [ 13 ]. Kamarudin et al. reached a maximum extraction yield of 80.2% in 6 h at 500 mM sodium sulfide and 50°C [ 14 ]. Khumalo et al. conducted an optimization study for the extraction of keratin from feathers. Sodium bisulfite at three concentrations of 0.2, 0.3, and 0.4 M; sodium dodecyl sulfate at three concentrations of 0, 0.035 and 0.07 M, temperature at two levels of 50, 70°C, and the reaction times at three levels 2, 3 and 4 h, were examined in a factorial experimental design. The concentration of urea was 1.5 M in all the runs. Maximum keratin extraction was reported at 0.3 M sodium bisulfite, 0.07 M SDS, 70°C, and 4 h [ 12 ]. Sinkiewicz et al. examined sulfitolysis to extract keratin in the presence of different reducing agents. 2-Mercaptoethanol and sodium bisulfite resulted in extraction yields ranging from 82–84% in 2 hours. Further studies revealed that with sodium bisulfite as the reducing agent, the same range can be obtained in 1 hour [ 11 ]. Zeng and Qi performed a sulfitolysis reaction with sodium bisulfite and sodium dodecyl sulfate in the presence of lithium bromide to extract keratin from wool. Lithium bromide helps break hydrogen bonds in wool, which results in easier dissolution of wool. Zeng and Qi reported a keratin extraction yield of 50.2% from wool with sodium bisulfite, lithium bromide, and SDS concentrations of 0.5 M, 0.1 M, and 0.02 M, respectively, at 90°C for 4 h [ 15 ]. In this study, sulfitolysis was studied for the extraction of keratin from chicken feathers, with an emphasis on minimizing the required chemicals, temperature, and time needed for feather dissolution. For this purpose, feather dissolution data were obtained in a series of factorial experimental designs with sodium sulfite, urea, SDS, incubation time, and temperature as effective factors. 2. Materials and methods 2.1 Materials Fresh chicken feathers were obtained from a chicken slaughterhouse in Yazd. Sodium sulfite and sodium dodecyl sulfate were purchased from Mojallali Chemical Company. Urea and ammonium sulfate were of industrial grade and were purchased from a local market. Standard proteins for SDS‒PAGE were obtained from Sigma Aldrich. 2.2 Factorial Experimental Design A full factorial experimental design was employed to determine the effects of sodium sulfite, urea, temperature, and incubation time on feather dissolution. Four factors, namely, sodium sulfite at two concentrations of 0.05 and 0.3 M; urea at two concentrations of 0 and 4 M; temperature at two levels of 50 and 85°C; and incubation time at two levels of 2 and 4 h, were used in the design. The feather to solution ratio was 1:60 (kg:L) for all the experiments. To investigate the effects of urea at concentrations less than 2 M, incubation times ranging from 0–2 h, and the presence of SDS, a separate full factorial experimental design was employed. For urea, factor levels of 0.5, 1, and 2 M, for time, factor levels of 30, 60, and 120 min, and for SDS, factor levels of 0 and 0.02 M were selected. For all runs of this experiment, the concentration of sodium sulfite, temperature, and feather to solution ratio were 0.05 M, 85°C, and 1:60 (kg:L), respectively. Minitab 18 was used for data analysis, and plotting the normal and mean effect plots. 2.3 Determination of feather dissolution After dissolution, the solid residues were separated from the solution using a filter cloth, washed with distilled water, and dried to a constant weight at ambient temperature. The dissolution percentage was calculated using the following relation: $$\:\%Feather\:dissolution=\left(\frac{initial\:weight\:of\:feather-weight\:of\:dried\:residues}{initial\:weight\:of\:feather}\right)100$$ 2.5 Precipitation of keratin from solutions Salting out with ammonium sulfate was used to precipitate keratin from the solutions. For this purpose, 500 g ammonium sulfate was added to each liter of the filtrate obtained with a filter cloth. The resulting mixture was centrifuged at 3000 rev/min for 5 min. The precipitated protein was washed with distilled water, dried at 50°C, and stored at 4°C for later use. 2.6 FTIR and SDS‒PAGE tests FTIR analysis was carried out to characterize the extracted keratin. This analysis was performed by Equinox manufactured by Bruker Company in attenuated reflectance mode in the range of 4000 − 650 cm − 1 . KBr pellets were made from powdered keratin. Polyacrylamide gel electrophoresis test (SDS‒PAGE) was used to obtain the molecular weight distribution of the extracted keratin. 3. Results and Discussions 3.1 Effects of urea, sodium sulfite, temperature, and incubation time on feather dissolution Table 1 shows the results of the factorial experimental design for the effects of sodium sulfite concentration (in the range of 0–4 M), urea concentration (in the range of 0.05–0.3 M), temperature (in the range of 50–80°C), and incubation time (in the range of 2–4 h) on feather dissolution. Figure 1 shows the normal plot for the effects of the factors. In factorial analysis, on the basis of the experimental results, numerical values (called effects) are calculated for the factors and interactions among them. An effect is a quantity that shows how variations in a factor’s level affect the response. More effective factors produce larger effects, and a negative effect means that an increase in the factor level causes a decrease in the response. The normal probability plot is drawn for the effect values, and the significant factors are determined statistically [ 16 ]. The normal plot for the effects shows that sodium sulfite, urea, temperature, and time are all effective factors affecting feather dissolution in the investigated ranges, and among the interactions, only the interaction between urea concentration and temperature is statistically significant. Table 1 Factorial design for the effects of the sodium sulfite and urea concentrations, temperature, and incubation time on feather dissolution Sodium sulfite (M) urea (M) Temp (°C) time (h) Feather dissolution percentage 1 0.05 0 50 2 14.13 2 0.05 0 50 4 16.53 3 0.05 0 85 2 20.66 4 0.05 0 85 4 23.49 5 0.05 4 50 2 17.15 6 0.05 4 50 4 19.54 7 0.05 4 85 2 44.25 8 0.05 4 85 4 45.35 9 0.3 0 50 2 10.53 10 0.3 0 50 4 12.73 11 0.3 0 85 2 16.55 12 0.3 0 85 4 19.08 13 0.3 4 50 2 13.53 14 0.3 4 50 4 15.86 15 0.3 4 85 2 43.35 16 0.3 4 85 4 44.27 Table 1 : Factorial design for the effects of the sodium sulfite and urea concentrations, temperature, and time on feather dissolution Figure 1 : Normal plot of the effects of sodium sulfite, urea, temperature, and time on feather dissolution Among the investigated factors, temperature had the greatest effect on feather dissolution. The average feather dissolution in the experiment increased from 15–32.12% when the temperature increased from 50°C to 80°C (Fig. 2 ). Higher temperatures facilitate the diffusion of urea and sodium sulfite into the rigid structure of the feather, increase the reaction rate of disulfide reduction, and promote protein denaturation. The addition of urea had a major effect on feather dissolution. The average degree of feather dissolution increased from 16.71% when there was no urea to 30.41% when 4 M urea was applied (Fig. 2 ). The use of urea as a protein denaturant disrupts the three dimensional structure of proteins formed by hydrogen bonding of the polar residues of the protein [ 17 ]. Sodium sulfite, as a reducing agent, plays an important role in feather dissolution. Protein fibers in keratin are cross-linked by disulfide bridges. Cleaving these bridges is essential for keratin extraction. Disulfide bonds in keratin are electrophile and react easily with sulfite anions as nucleophiles, resulting in the cleavage of disulfide bonds [ 18 ]. The average feather dissolution decreased from 25.13–21.98% when the sodium sulfite concentration increased from 0.05 M to 0.3 M (Fig. 2 ). The reason for this reduction might be explained by the fact that higher concentrations of sodium sulfite result in higher pH and lower SO 3 2− available for disulfide cleavage. The average feather dissolution percentage increased from 22.52% when the incubation time was 2 h to 24.61% when the incubation time was 4 h. This finding implies that extending the incubation time from 2 h to 4 h caused only a marginal increase in feather dissolution. Overall, for this part, the maximum feather dissolution percentage of 45.35% was obtained with sodium sulfite and urea concentrations of 0.05 M and 4 M, respectively, at 85°C in 4 hours. Figure 2 : Mean dissolution percentage as a function of sodium sulfite, urea, temperature, and time 3.2 Effect of SDS on feather dissolution The effects of SDS, urea, and incubation time on feather dissolution were investigated via a factorial design. The urea concentration and incubation time were lower than those examined above. The results are presented in Table 2 . These results indicate that SDS considerably affects feather dissolution. Figure 3 shows that the average feather dissolution percentage increased from 28.11% in the absence of SDS to 47.76% in the presence of 0.02 M SDS in the solution. The role of SDS as an anionic surfactant in the denaturation of proteins has been studied extensively. SDS can bind to protein molecules and help denature them [ 19 ]. The maximum feather dissolution percentage of 57.63% was obtained after 2 h of incubation at 85°C with urea and SDS concentrations of 2 M and 0.02 M, respectively. Under the same conditions but without SDS, the feather dissolution percentage was 49.66%. A comparison of the results in Tables 1 and 2 shows that the application of 2 M urea in solution gave almost the same results as the application of 4 M urea in the solution. In the absence of SDS, a feather dissolution percentage of 44.25% was obtained with 4 M urea in 2 h. This figure was 49.66% with 2 M urea in solution under the same conditions. This implies that increasing the urea concentration beyond 2 M has a marginal effect on the feather dissolution rate. Table 2 Factorial design for the effects of urea, SDS, and incubation time on feather dissolution. Temperature = 80°C, sodium sulfite conc. = 0.05 M urea(M) time(min) SDS (M) Feather dissolution percentage 1 0.5 120 0 30.30 2 0.5 60 0 29.25 3 0.5 30 0 14.67 4 1 120 0 30.20 5 1 60 0 27.07 6 1 30 0 22.39 7 2 120 0 49.66 8 2 60 0 27.30 9 2 30 0 22.15 10 0.5 120 0.02 45.27 11 0.5 60 0.02 42.21 12 0.5 30 0.02 39.76 13 1 120 0.02 54.85 14 1 60 0.02 51.38 15 1 30 0.02 40.66 16 2 120 0.02 57.63 17 2 60 0.02 54.19 18 2 30 0.02 43.89 Table 2 : Factorial design for the effects of urea, SDS, and incubation time on feather dissolution. Temperature = 80°C, sodium sulfite conc. = 0.05 M Figure 3 : Mean dissolution percentage as a function of the urea and SDS concentrations and incubation time 3.3 Analysis of precipitated keratin The protein resulting from feather dissolution was salted out with ammonium sulfate and analyzed via FTIR. The results are presented in Fig. 4 . The FTIR spectrum resembles the spectrum presented for keratin in the literature (Sharma et al. 2018; Welu et al. 2020; Khumalo et al. 2017). The broad peak in the range of 3300 cm − 1 is related to the hydrogen bonds of -N-H and -O-H and the external stretching mobility of amide functional groups and absorbed water. Low intensity peaks in the range of 3100–2900 cm − 1 indicate -N–H and -C–H stretching vibrations (Welu et al. 2020). The peak at 3200 cm − 1 is related to amide A, and the weak peak at 3000 cm − 1 is related to amide B (Khumalo et al. 2017). Absorption at 1624 cm − 1 occurs due to the stretching of the carbonyl group C = O of amide (I), and the peak at 1540.75 cm − 1 is related to amide (II), which is attributed to -N-H bending and -C-H stretching (Sharma et al. 2018). The absorption of amide(III) occurs at 1448.75 cm − 1 (Fagbemi et al. 2020), which is attributed to C–N stretching and -N–H bending, and signs of C = O bending and C–C stretching vibrations are observed. Amides I-III provide detailed information about protein conformation and changes in the main protein structure. Amide (I) is a combination of α- and β-sheets, and amide (III) can be attributed to the β-sheet structure. The range of amide (I) can provide more information about the present structure. The absorption between 600–750 cm − 1 is related to the mobility outside the simple bending of -N-H. The peak in the range of 990–1580 cm − 1 is related to the presence of S–S and C–S bonds in the extracted protein (Sharma et al. 2018). The peak in the range of 1100 − 1000 cm − 1 is related to thiosulfate ions, and the presence of thiosulfate ions is caused by the connection of sulfite ions to one of the sulfur atoms in a disulfide bond, which indicates the cleavage of this bond during the extraction process. The peak in the range of 1244.08 cm − 1 is related to carboxylic acid groups (Khumalo et al. 2017). The FTIR results confirmed the keratinous nature of the precipitated protein. 3.4 SDS‒PAGE test results To perform the SDS‒PAGE test, the proteins obtained from runs 7 (A) and 15 (B) in Table 1 were subjected to SDS‒PAGE. The resulting gel is shown in Fig. 5 . The figure shows that the molecular weight distribution has a peak at approximately 10 kDa, implying that the keratin molecules obtained here are mostly in the range of the 10 kDa molecular weight. This finding is consistent with previous studies reporting the molecular weight of keratin molecules from feathers (Kamarudin et al. 2017). 4. Conclusion The dissolution of feathers is a feasible way to reduce the amount of solid waste produced in chicken slaughterhouses. Sodium sulfite, as a reducing agent, initiates feather dissolution via the cleavage of disulfide bonds. Urea and SDS facilitate feather dissolution considerably. With a feather to solution ratio of 1:60 (kg:L), a urea concentration of 2 M, and an incubation time of 2 h were sufficient to reach the maximum dissolution of feathers. Under these conditions and in the presence of 0.02 M SDS, more than 57% of the feathers disintegrated. The protein molecules in the solution could be salted out by ammonium sulfate. FTIR confirmed that the precipitated molecules were keratin. Declarations Compliance with Ethical Standards The authors have no relevant financial or non-financial interests to disclose. All data generated or analyzed during this study are included in this published article. Author Contribution V. Zebhi-Ashkezari did the experiments. M.H. Fazaelipoor designed the experiments, and prepared the manuscript for publication. Data Availability All data generated or analyzed during this study are included in this published article. References C.W. Cheong, Y.S. Lee, S.A. Ahmad, P.T. Ooi, L.Y. Phang, Waste Manage. 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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-5105662","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":373997275,"identity":"c493060f-bdfd-4334-acea-5e84f01cd13d","order_by":0,"name":"Vahidesadat Zebhi-Ashkezari1","email":"","orcid":"","institution":"Yazd University","correspondingAuthor":false,"prefix":"","firstName":"Vahidesadat","middleName":"","lastName":"Zebhi-Ashkezari1","suffix":""},{"id":373997276,"identity":"b6c86474-4086-4315-9829-ec233860c0a2","order_by":1,"name":"Mohammad Hassan Fazaelipoor","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8klEQVRIiWNgGAWjYBAC9gYQWSABIhkfgAiwQAIeLTwHQKQBWAuzASlawGw2CbgWfICH/fjDDx8MLPL42XufVfPU1Mk2sB9+wPBwDx4tPAnJkjMMJIole46b3eY5xmbcwJNmwJDwDLcWe4aEA9I8BhKJG26ksd3mYeNJbGDIAfrlAB5b+B82//4D1VLM808isYH/DQEtEsls0gxQLcy8bQaJDRKEbJF4xmbZA/bLMWbJuX0Jxm0SzwwO4HdY+uMbPyrqgCHWxvjhzbc62X7+5IcPf+DRAgMJIIKJB0iwATERGqBaGH8Qo3QUjIJRMApGHAAANa9L/HrMpr8AAAAASUVORK5CYII=","orcid":"","institution":"Yazd University","correspondingAuthor":true,"prefix":"","firstName":"Mohammad","middleName":"Hassan","lastName":"Fazaelipoor","suffix":""}],"badges":[],"createdAt":"2024-09-17 21:32:46","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5105662/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5105662/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":72163424,"identity":"e57e854a-2787-45e2-a722-28be5ea0bb4f","added_by":"auto","created_at":"2024-12-23 10:01:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":37183,"visible":true,"origin":"","legend":"\u003cp\u003eNormal plot of the effects of sodium sulfite, urea, temperature, and time on feather dissolution\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5105662/v1/e15d3afefd553622862b9c64.png"},{"id":72163421,"identity":"f0786c41-6c81-4545-b8b2-3d989c7bc978","added_by":"auto","created_at":"2024-12-23 10:01:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":50097,"visible":true,"origin":"","legend":"\u003cp\u003eMean dissolution percentage as a function of the sodium sulfite and urea concentrations, temperature and incubation time\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5105662/v1/83d69ec9037ce7f2e7a949cd.png"},{"id":72163420,"identity":"c406cb20-3b7b-41b9-8e86-89eacacfca96","added_by":"auto","created_at":"2024-12-23 10:01:40","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":45533,"visible":true,"origin":"","legend":"\u003cp\u003eMean dissolution percentage as a function of the urea and SDS concentrations and incubation time\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5105662/v1/8c33448c6e7dce4768a1ef9f.png"},{"id":72163422,"identity":"80a04a7b-8015-42e5-a119-56922190f812","added_by":"auto","created_at":"2024-12-23 10:01:40","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":74372,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR analysis of the precipitated protein obtained by feather dissolution\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5105662/v1/36eee813318983bf2c8ebfd0.png"},{"id":72164937,"identity":"a858d957-9e25-41a6-a8fa-3b87b957ce2b","added_by":"auto","created_at":"2024-12-23 10:09:40","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":595310,"visible":true,"origin":"","legend":"\u003cp\u003eSDS‒PAGE results for extracted keratin\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5105662/v1/bb9d931f43994dbcaf70f839.png"},{"id":72165463,"identity":"82cddb99-100f-46e8-ac0a-7bbf4803929c","added_by":"auto","created_at":"2024-12-23 10:17:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1536224,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5105662/v1/b5287178-32af-4508-9e7d-c4a5e6c1a704.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Chicken feather dissolution to obtain keratin","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eChicken feathers are waste produced in poultry slaughterhouses in large amounts. It consists mainly of a protein named keratin (approximately 90%). Keratin is one of the most abundant structural proteins in animals and one of the many proteins found in the bodies of reptiles, birds and mammals. This protein is a structural component of nails, wool, feathers, hooves, etc. [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Keratin has high strength and hardness and is insoluble in polar and nonpolar solvents. Its stability is due to intra- and extramolecular disulfide cross-links, hydrogen bonds and crystallinity. These properties distinguish it from other fibrous proteins, such as myofibrillar and collagen [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Keratin is a protein rich in cysteine, which is connected to each other through intermediate strands with a diameter of 8\u0026ndash;10 nm and is available in two forms: α-keratin and β-keratin [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The presence of cysteine in high amounts makes keratin susceptible to hydrolytic reactions and oxidation [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. This polymer is composed of different amino acids that are able to increase intra- and extramolecular connections, and this feature allows the resulting materials to have diverse functional properties [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn recent years, attempts have been made to extract keratin via chemical and enzymatic methods. The methods of extracting keratin from biomass include acid hydrolysis, alkaline hydrolysis [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], enzymatic hydrolysis [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], ionic liquid hydrolysis [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], alkaline-enzymatic hydrolysis, and sulfitolysis [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Acid hydrolysis creates severe conditions that damage some useful amino acids during hydrolysis. In contrast, enzymatic hydrolysis results in fewer species changes, but the process is slow and expensive, which makes commercialization difficult [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Ionic liquids are also expensive, and their protein recovery is very low compared with that of other methods. In the sulfitolysis method, a reducing agent such as sodium sulfite is used to break disulfide bonds in the keratin structure. Urea is also used to denature protein structure, which leads to the extraction of keratin from keratin containing materials [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe extraction of keratin via sulfitolysis has been the subject of several studies. Isarankura et al. reported 87.6% keratin extraction from feathers in a solution containing 0.2 M sodium metabisulfite and 8 M urea [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Kamarudin et al. reached a maximum extraction yield of 80.2% in 6 h at 500 mM sodium sulfide and 50\u0026deg;C [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Khumalo et al. conducted an optimization study for the extraction of keratin from feathers. Sodium bisulfite at three concentrations of 0.2, 0.3, and 0.4 M; sodium dodecyl sulfate at three concentrations of 0, 0.035 and 0.07 M, temperature at two levels of 50, 70\u0026deg;C, and the reaction times at three levels 2, 3 and 4 h, were examined in a factorial experimental design. The concentration of urea was 1.5 M in all the runs. Maximum keratin extraction was reported at 0.3 M sodium bisulfite, 0.07 M SDS, 70\u0026deg;C, and 4 h [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Sinkiewicz et al. examined sulfitolysis to extract keratin in the presence of different reducing agents. 2-Mercaptoethanol and sodium bisulfite resulted in extraction yields ranging from 82\u0026ndash;84% in 2 hours. Further studies revealed that with sodium bisulfite as the reducing agent, the same range can be obtained in 1 hour [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Zeng and Qi performed a sulfitolysis reaction with sodium bisulfite and sodium dodecyl sulfate in the presence of lithium bromide to extract keratin from wool. Lithium bromide helps break hydrogen bonds in wool, which results in easier dissolution of wool. Zeng and Qi reported a keratin extraction yield of 50.2% from wool with sodium bisulfite, lithium bromide, and SDS concentrations of 0.5 M, 0.1 M, and 0.02 M, respectively, at 90\u0026deg;C for 4 h [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, sulfitolysis was studied for the extraction of keratin from chicken feathers, with an emphasis on minimizing the required chemicals, temperature, and time needed for feather dissolution. For this purpose, feather dissolution data were obtained in a series of factorial experimental designs with sodium sulfite, urea, SDS, incubation time, and temperature as effective factors.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials\u003c/h2\u003e \u003cp\u003eFresh chicken feathers were obtained from a chicken slaughterhouse in Yazd. Sodium sulfite and sodium dodecyl sulfate were purchased from Mojallali Chemical Company. Urea and ammonium sulfate were of industrial grade and were purchased from a local market. Standard proteins for SDS‒PAGE were obtained from Sigma Aldrich.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Factorial Experimental Design\u003c/h2\u003e \u003cp\u003eA full factorial experimental design was employed to determine the effects of sodium sulfite, urea, temperature, and incubation time on feather dissolution. Four factors, namely, sodium sulfite at two concentrations of 0.05 and 0.3 M; urea at two concentrations of 0 and 4 M; temperature at two levels of 50 and 85\u0026deg;C; and incubation time at two levels of 2 and 4 h, were used in the design. The feather to solution ratio was 1:60 (kg:L) for all the experiments.\u003c/p\u003e \u003cp\u003eTo investigate the effects of urea at concentrations less than 2 M, incubation times ranging from 0\u0026ndash;2 h, and the presence of SDS, a separate full factorial experimental design was employed. For urea, factor levels of 0.5, 1, and 2 M, for time, factor levels of 30, 60, and 120 min, and for SDS, factor levels of 0 and 0.02 M were selected. For all runs of this experiment, the concentration of sodium sulfite, temperature, and feather to solution ratio were 0.05 M, 85\u0026deg;C, and 1:60 (kg:L), respectively.\u003c/p\u003e \u003cp\u003eMinitab 18 was used for data analysis, and plotting the normal and mean effect plots.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Determination of feather dissolution\u003c/h2\u003e \u003cp\u003eAfter dissolution, the solid residues were separated from the solution using a filter cloth, washed with distilled water, and dried to a constant weight at ambient temperature. The dissolution percentage was calculated using the following relation:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\%Feather\\:dissolution=\\left(\\frac{initial\\:weight\\:of\\:feather-weight\\:of\\:dried\\:residues}{initial\\:weight\\:of\\:feather}\\right)100$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Precipitation of keratin from solutions\u003c/h2\u003e \u003cp\u003eSalting out with ammonium sulfate was used to precipitate keratin from the solutions. For this purpose, 500 g ammonium sulfate was added to each liter of the filtrate obtained with a filter cloth. The resulting mixture was centrifuged at 3000 rev/min for 5 min. The precipitated protein was washed with distilled water, dried at 50\u0026deg;C, and stored at 4\u0026deg;C for later use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.6 FTIR and SDS‒PAGE tests\u003c/h2\u003e \u003cp\u003eFTIR analysis was carried out to characterize the extracted keratin. This analysis was performed by Equinox manufactured by Bruker Company in attenuated reflectance mode in the range of 4000\u0026thinsp;\u0026minus;\u0026thinsp;650 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. KBr pellets were made from powdered keratin.\u003c/p\u003e \u003cp\u003ePolyacrylamide gel electrophoresis test (SDS‒PAGE) was used to obtain the molecular weight\u003c/p\u003e \u003cp\u003edistribution of the extracted keratin.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and Discussions","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Effects of urea, sodium sulfite, temperature, and incubation time on feather dissolution\u003c/h2\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the results of the factorial experimental design for the effects of sodium sulfite concentration (in the range of 0\u0026ndash;4 M), urea concentration (in the range of 0.05\u0026ndash;0.3 M), temperature (in the range of 50\u0026ndash;80\u0026deg;C), and incubation time (in the range of 2\u0026ndash;4 h) on feather dissolution. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the normal plot for the effects of the factors. In factorial analysis, on the basis of the experimental results, numerical values (called effects) are calculated for the factors and interactions among them. An effect is a quantity that shows how variations in a factor\u0026rsquo;s level affect the response. More effective factors produce larger effects, and a negative effect means that an increase in the factor level causes a decrease in the response. The normal probability plot is drawn for the effect values, and the significant factors are determined statistically [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The normal plot for the effects shows that sodium sulfite, urea, temperature, and time are all effective factors affecting feather dissolution in the investigated ranges, and among the interactions, only the interaction between urea concentration and temperature is statistically significant.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFactorial design for the effects of the sodium sulfite and urea concentrations, temperature, and incubation time on feather dissolution\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSodium sulfite (M)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eurea (M)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTemp (\u0026deg;C)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003etime (h)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eFeather dissolution percentage\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e14.13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e 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\u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e23.49\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e17.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e19.54\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e44.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e45.35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e10.53\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e12.73\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e16.55\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e19.08\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e13.53\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e15.86\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e43.35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e44.27\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e: Factorial design for the effects of the sodium sulfite and urea concentrations, temperature, and time on feather dissolution\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e: Normal plot of the effects of sodium sulfite, urea, temperature, and time on feather dissolution\u003c/p\u003e \u003cp\u003eAmong the investigated factors, temperature had the greatest effect on feather dissolution. The average feather dissolution in the experiment increased from 15\u0026ndash;32.12% when the temperature increased from 50\u0026deg;C to 80\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Higher temperatures facilitate the diffusion of urea and sodium sulfite into the rigid structure of the feather, increase the reaction rate of disulfide reduction, and promote protein denaturation. The addition of urea had a major effect on feather dissolution. The average degree of feather dissolution increased from 16.71% when there was no urea to 30.41% when 4 M urea was applied (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The use of urea as a protein denaturant disrupts the three dimensional structure of proteins formed by hydrogen bonding of the polar residues of the protein [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Sodium sulfite, as a reducing agent, plays an important role in feather dissolution. Protein fibers in keratin are cross-linked by disulfide bridges. Cleaving these bridges is essential for keratin extraction. Disulfide bonds in keratin are electrophile and react easily with sulfite anions as nucleophiles, resulting in the cleavage of disulfide bonds [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The average feather dissolution decreased from 25.13\u0026ndash;21.98% when the sodium sulfite concentration increased from 0.05 M to 0.3 M (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The reason for this reduction might be explained by the fact that higher concentrations of sodium sulfite result in higher pH and lower SO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e available for disulfide cleavage. The average feather dissolution percentage increased from 22.52% when the incubation time was 2 h to 24.61% when the incubation time was 4 h. This finding implies that extending the incubation time from 2 h to 4 h caused only a marginal increase in feather dissolution. Overall, for this part, the maximum feather dissolution percentage of 45.35% was obtained with sodium sulfite and urea concentrations of 0.05 M and 4 M, respectively, at 85\u0026deg;C in 4 hours.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e: Mean dissolution percentage as a function of sodium sulfite, urea, temperature, and time\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Effect of SDS on feather dissolution\u003c/h2\u003e \u003cp\u003eThe effects of SDS, urea, and incubation time on feather dissolution were investigated via a factorial design. The urea concentration and incubation time were lower than those examined above. The results are presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. These results indicate that SDS considerably affects feather dissolution. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows that the average feather dissolution percentage increased from 28.11% in the absence of SDS to 47.76% in the presence of 0.02 M SDS in the solution. The role of SDS as an anionic surfactant in the denaturation of proteins has been studied extensively. SDS can bind to protein molecules and help denature them [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The maximum feather dissolution percentage of 57.63% was obtained after 2 h of incubation at 85\u0026deg;C with urea and SDS concentrations of 2 M and 0.02 M, respectively. Under the same conditions but without SDS, the feather dissolution percentage was 49.66%. A comparison of the results in Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows that the application of 2 M urea in solution gave almost the same results as the application of 4 M urea in the solution. In the absence of SDS, a feather dissolution percentage of 44.25% was obtained with 4 M urea in 2 h. This figure was 49.66% with 2 M urea in solution under the same conditions. This implies that increasing the urea concentration beyond 2 M has a marginal effect on the feather dissolution rate.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFactorial design for the effects of urea, SDS, and incubation time on feather dissolution. Temperature\u0026thinsp;=\u0026thinsp;80\u0026deg;C, sodium sulfite conc. = 0.05 M\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eurea(M)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003etime(min)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSDS (M)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFeather dissolution percentage\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e120\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e30.30\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e29.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e14.67\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e120\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e30.20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e27.07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e22.39\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e120\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e49.66\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e27.30\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e22.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e120\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e45.27\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e42.21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e39.76\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e120\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e54.85\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e51.38\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e40.66\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e120\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e57.63\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e54.19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e43.89\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e: Factorial design for the effects of urea, SDS, and incubation time on feather dissolution. Temperature\u0026thinsp;=\u0026thinsp;80\u0026deg;C, sodium sulfite conc. = 0.05 M\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e: Mean dissolution percentage as a function of the urea and SDS concentrations and incubation time\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Analysis of precipitated keratin\u003c/h2\u003e \u003cp\u003eThe protein resulting from feather dissolution was salted out with ammonium sulfate and analyzed via FTIR. The results are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The FTIR spectrum resembles the spectrum presented for keratin in the literature (Sharma et al. 2018; Welu et al. 2020; Khumalo et al. 2017). The broad peak in the range of 3300 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is related to the hydrogen bonds of -N-H and -O-H and the external stretching mobility of amide functional groups and absorbed water. Low intensity peaks in the range of 3100\u0026ndash;2900 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e indicate -N\u0026ndash;H and -C\u0026ndash;H stretching vibrations (Welu et al. 2020). The peak at 3200 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is related to amide A, and the weak peak at 3000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is related to amide B (Khumalo et al. 2017). Absorption at 1624 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e occurs due to the stretching of the carbonyl group C\u0026thinsp;=\u0026thinsp;O of amide (I), and the peak at 1540.75 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is related to amide (II), which is attributed to -N-H bending and -C-H stretching (Sharma et al. 2018). The absorption of amide(III) occurs at 1448.75 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Fagbemi et al. 2020), which is attributed to C\u0026ndash;N stretching and -N\u0026ndash;H bending, and signs of C\u0026thinsp;=\u0026thinsp;O bending and C\u0026ndash;C stretching vibrations are observed. Amides I-III provide detailed information about protein conformation and changes in the main protein structure. Amide (I) is a combination of α- and β-sheets, and amide (III) can be attributed to the β-sheet structure. The range of amide (I) can provide more information about the present structure. The absorption between 600\u0026ndash;750 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is related to the mobility outside the simple bending of -N-H. The peak in the range of 990\u0026ndash;1580 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is related to the presence of S\u0026ndash;S and C\u0026ndash;S bonds in the extracted protein (Sharma et al. 2018). The peak in the range of 1100\u0026thinsp;\u0026minus;\u0026thinsp;1000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is related to thiosulfate ions, and the presence of thiosulfate ions is caused by the connection of sulfite ions to one of the sulfur atoms in a disulfide bond, which indicates the cleavage of this bond during the extraction process. The peak in the range of 1244.08 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is related to carboxylic acid groups (Khumalo et al. 2017). The FTIR results confirmed the keratinous nature of the precipitated protein.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.4 SDS‒PAGE test results\u003c/h2\u003e \u003cp\u003eTo perform the SDS‒PAGE test, the proteins obtained from runs 7 (A) and 15 (B) in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e were subjected to SDS‒PAGE. The resulting gel is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. The figure shows that the molecular weight distribution has a peak at approximately 10 kDa, implying that the keratin molecules obtained here are mostly in the range of the 10 kDa molecular weight. This finding is consistent with previous studies reporting the molecular weight of keratin molecules from feathers (Kamarudin et al. 2017).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThe dissolution of feathers is a feasible way to reduce the amount of solid waste produced in chicken slaughterhouses. Sodium sulfite, as a reducing agent, initiates feather dissolution via the cleavage of disulfide bonds. Urea and SDS facilitate feather dissolution considerably. With a feather to solution ratio of 1:60 (kg:L), a urea concentration of 2 M, and an incubation time of 2 h were sufficient to reach the maximum dissolution of feathers. Under these conditions and in the presence of 0.02 M SDS, more than 57% of the feathers disintegrated. The protein molecules in the solution could be salted out by ammonium sulfate. FTIR confirmed that the precipitated molecules were keratin.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompliance with Ethical Standards\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eV. Zebhi-Ashkezari did the experiments. M.H. Fazaelipoor designed the experiments, and prepared the manuscript for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eC.W. Cheong, Y.S. Lee, S.A. Ahmad, P.T. Ooi, L.Y. 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(2018) http://doi.org/10.1007/s10098-018-1498-2.\u003c/li\u003e\n \u003cli\u003eK.T. Welu, S.M. Beyan, S. Balakrishnan, H. Admassu, Curr. Appl. Sci. Technol. (2020) http://doi.org/10.14456/cast.2020.6\u003cspan dir=\"RTL\"\u003e\u003c/span\u003e\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":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"discover-chemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Discover Chemistry](https://link.springer.com/journal/44371)","snPcode":"44371","submissionUrl":"https://submission.nature.com/new-submission/44371/3","title":"Discover Chemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"chicken feather dissolution, keratin, urea, sodium dodecyl sulfate, sodium sulfite","lastPublishedDoi":"10.21203/rs.3.rs-5105662/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5105662/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe dissolution of chicken feathers was investigated for the purpose of reducing solid waste and obtaining keratin. For the first part, the effects of sodium sulfite (0.05–0.3 M), urea (0–4 M), temperature (50–85 °C), and incubation time (2–4 h) on feather dissolution were investigated via a factorial design. Another factorial design was applied to investigate the effects of sodium dodecyl sulfate (SDS) (0–0.02 M), and lower ranges of urea (0.5–2 M), and incubation time (0.5–2 h) on feather dissolution. Keratin was precipitated from the solutions by ammonium sulfate and characterized via FTIR and SDS‒PAGE methods. The results of the first part demonstrated the feasibility of feather dissolution in the presence of sodium sulfite, and the effectiveness of urea and temperature on feather dissolution. For this part, a maximum feather dissolution of 45.35% was obtained with sodium sulfite and urea concentrations of 0.05 M and 4 M, respectively, at 85 °C for 4 hours. The results of the second part revealed a positive effect of SDS on feather dissolution. A maximum feather dissolution of 57.63% was obtained with sodium sulfite, urea, and SDS concentrations of 0.05 M, 2 M, and 0.02 M, respectively, at 85 °C for 2 hours. FTIR analysis confirmed the keratinous nature of the precipitated protein, and SDS‒PAGE revealed that the mass of the protein molecules was less than 15 kDa. This work confirms the feasibility of chemical dissolution of chicken feathers to obtain keratin as a useful product.\u003c/p\u003e","manuscriptTitle":"Chicken feather dissolution to obtain keratin","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-23 10:01:35","doi":"10.21203/rs.3.rs-5105662/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-11-04T17:07:55+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-04T00:50:14+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-01T10:19:14+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-01T05:46:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"183525271805978964095532372014289823830","date":"2024-10-31T05:32:04+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-30T21:33:27+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-29T08:24:10+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-28T23:48:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"335060065558425089088712319233327599679","date":"2024-10-28T09:30:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"25860901309219177804763096611442360436","date":"2024-10-28T02:26:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"261472024527229334976055804486280604319","date":"2024-10-27T13:34:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"260779339598257914939104967339458664101","date":"2024-10-25T09:22:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"248072816785380687959899951079247898467","date":"2024-10-25T09:11:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"62814343767496433033699897700979019042","date":"2024-10-25T09:06:03+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-17T03:12:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"283259962737291633492781769096032021776","date":"2024-10-07T19:52:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"197881941950138608048729667409900362259","date":"2024-10-07T14:45:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"150929142066762110817754071355822453831","date":"2024-10-07T11:38:32+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-10-07T11:27:37+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-04T00:16:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-09-30T05:44:17+00:00","index":"","fulltext":""},{"type":"submitted","content":"Discover Chemistry","date":"2024-09-17T21:31:25+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"discover-chemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Discover Chemistry](https://link.springer.com/journal/44371)","snPcode":"44371","submissionUrl":"https://submission.nature.com/new-submission/44371/3","title":"Discover Chemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3f67a208-67e8-4680-a784-75b96c06bd16","owner":[],"postedDate":"December 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-04-08T14:23:36+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-23 10:01:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5105662","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5105662","identity":"rs-5105662","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}