Feather Hydrolysis Research Articles

Keratin-based pet food produced through proteolytic hydrolysis of chicken feather: Characterisation and palatability study

Valorisation of poultry co-products can fulfil the protein demand of the pet food sector thereby supporting a circular bioeconomy. In this investigation, keratin was produced through an optimised reduction (0.8 % sodium sulphite, for 90 min) and hydrolysis (using papain for 120 min) with a yield of 56.87 %. FTIR analysis, in vitro digestibility, SEM imaging, and amino acid profiling of the derived keratin were done and it was subsequently used for pet formulations. Results revealed significant alteration in secondary protein structure post-hydrolysis with in vitro digestibility of 90 %. Higher levels (>100 % increase) of histidine, arginine, lysine, and methionine were observed in the hydrolysate compared to conventional feather meal. Pet food was formulated with 20 % protein replacement that showed maximum palatability in single and two-bowl tests for 2–4 year old Labrador dogs. The developed pet food exhibited desirable colour, water activity (0.5), moisture (4.7 %), and relatively lower TBARS value (0.8 mg/kg) than the critical value.

Economic production of eco-friendly dehairing keratinase and antioxidant feather hydrolyzate using Bacillus halotolerans

AbstractFeather is a high recalcitrance keratin-based biomass, and keratin-degrading microorganisms have been seen as beneficial tools for its biodegradation and production of valuable products with biomedical and industrial applications. In the current study, the isolated keratinolytic bacteria was identified on the base of its 16 S rDNA sequencing data as Bacillus halotolerans, and its efficiency in the hydrolysis of feather with the preservation of most of its amino acid constituent was estimated by high-performance liquid chromatography Pico-Tag (HPLC PICO-TAG) method. The produced hydrolysate possessed antioxidant activity of 2,2-Diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity of 52.3 ± 5.6 mmole TE/g without possessing any cytotoxicity to human normal skin fibroplast cell line BJ-1 estimated by MTT assay. Moreover, the isolated strain was capable for producing keratinase under submerged fermentation of feather that was optimized by combining single-variable-at-a-time optimization with the application of statistical designs (Plackett-Burman and Box-Behnken). The optimum keratinase activity was 140.83 U/mL that was enhanced by 3.2-fold of the estimated initial activity, and the purified enzyme possessed optimum activity at pH 9.5 and 70 °C with a half-life of 69.3 min. Finally, the applicability of the purified enzyme in the dehairing of bovine hide was examined in comparison to sodium sulfide (Na2S) and lime (Ca(OH)2) conventional method. The results estimated the efficiency of the examined bio-treatment process, it achieved complete dehairing after 2 h with a good skin quality as manifested by scanning electron microscope (SEM). In addition, a significant reduction of the pollution load parameters was estimated.

A comprehensive comparative study of ultrasound-alkaline and thermal-alkaline hydrolysis of duck feather

Feathers, as a byproduct of the poultry industry, present a significant source of keratinous waste. Conventional methods have been widely used to extract keratin from feathers; however, they are associated with limitations such as high operational costs and environmental concerns. It is, therefore, crucial to develop cost-effective and time-efficient methods for extracting keratin on a large scale. In recent years, ultrasound-assisted alkaline hydrolysis has emerged as a promising and sustainable approach for efficient keratin recovery. This study compares the hydrolysis time, yield, and chemical properties of keratin extracted from feathers using ultrasound-assisted alkaline hydrolysis and thermal alkaline hydrolysis (hot plate method). The influence of factors such as particle size, alkali concentration, liquid-to-solid ratio, reactor geometry, temperature of keratin colloid upon precipitation, precipitation pH, and precipitating acid was investigated. Favorable conditions for ultrasound-assisted alkaline hydrolysis were found to be 3% NaOH, a 10:100 (w/v) solid-to-liquid ratio, using a cylindrical vessel, and an ultrasonic energy density of 360 kJ/L, with pH adjustment to 4.5 using citric acid after cooling to room temperature. This method outperformed the thermal approach, yielding 70% keratin in 25 min, compared to 23% in 90 min using a hot plate, due to the exothermic effect of cavitation. The results provide valuable insights into the potential of ultrasound-assisted alkaline hydrolysis as an eco-friendly and cost-effective approach to address the management of keratinous waste and enhance the overall recovery of keratin.

A synergistic catalytic system of keratinases and disulfide reductases for the bioconversion of feather wastes to feed alternative protein

Effective hydrolysis of feather waste by keratinase is an important way to develop high-quality alternative proteins for feed. It has been shown that the reduction of disulfide bonds is the key rate-limiting step in the hydrolysis of feathers by keratinases. However, the reductive activity of keratinase itself is low. In order to increase the reductive activity of the catalytic system, we screened endogenous disulfide reductase from Bacillus subtilis and constructed a synergistic catalytic system, KerZ1/yjbI-S2-P1, consisting of keratinase and disulfide reductase by co-expression optimization. The reductive activity and feather hydrolytic activity of the synergistic system were 5.67-fold and 5-fold higher than the original system. Feathers were hydrolyzed to a higher extent compared with the original system. The total amino acid and soluble protein concentrations in the products increased by 99% and 59%, respectively. This study provides a more environmentally friendly and efficient reduction capability for keratinase to hydrolysis waste feathers, and also demonstrates a new way of alternative protein feedstock mining.

Crop effect and mechanism of amino acid-modified biomass ash in remediation of cadmium-contaminated soil.

Lignocellulosic biomass ash (BA) has certain adsorption and passivation effects on heavy metals, but its function is generally weak. Amino acid salt can facilitate the leaching of heavy metals in soil. Therefore, modification of BA with amino acid salt may realize a higher leaching rate and better passivation of heavy metals in soil. In this study, BA was modified by amino acid hydrolysate obtained from the hydrolysis of chicken feathers by sulfuric acid. The physicochemical properties of BA and modified BA (MBA), their effects on Chinese cabbage (CC) yield and nutritional quality, and passivation effects on soil cadmium (Cd) were compared, and the related mechanisms were investigated. SEM-EDS, XRD, and FTIR demonstrated that BA was a CaCO3-type soil amendment, while MBA was a CaSO4-type soil amendment with the loading of amino acid. Compared with BA, MBA significantly increased the fresh weight, soluble sugar, vitamin C (Vc), and protein contents of CC in both non-Cd contaminated soil and Cd contaminated soil, and obviously decreased the nitrate content and Cd uptake of CC in Cd-contaminated soil. After the application of MBA, cadmium species in potted soil were transformed from higher plant availability, representing by exchangeable and carbonate-bound, into lower plant availability, representing by iron-manganese oxide bound, which was identified as the key reason for the significant reduction of Cd content in CC under MBA application.

Fusion of Substrate-Binding Domains Enhances the Catalytic Capacity of Keratinases and Promotes Enzymatic Conversion of Feather Waste.

The unique role of keratinases in keratin hydrolysis has garnered huge interest in the recovery of feather waste. However, owing to the high hydrophobicity of feather keratins, the catalytic capacity of keratinases for hydrolyzing feathers is typically low. In this study, we aimed to improve the keratinase feather hydrolysis efficiency by fusing a substrate-binding domain into the enzyme. We screened several carbohydrate-binding modules (CBMs) and linking peptides. We selected the most promising candidates to construct, clone, and express a fusion keratinase enzyme KerZ1/CBM-L8 with a feather hydrolysis efficiency of 7.8 × 10-8 g/U. Compared with those of KerZ1, KerZ1/CBM-L8 has a feather hydrolysis efficiency that is 2.71 times higher, a kcat value that is 179% higher, which translates to higher catalytic efficiency, and Km and binding constant (K) values that are lower, which indicate a higher KerZ1/CBM-L8-keratin binding affinity. Moreover, the number of binding sites to the substrate (N), determined using isothermal titration calorimetry, was 24.1 times higher than that of KerZ1. Thus, the fusion of the substrate-binding domain improved the binding ability of the keratinase enzyme to the hydrophobic substrate, which improved its feather hydrolysis efficiency. Therefore, using the fusion keratinase would significantly improve the recovery of feather waste.

New cold-adapted bacteria for efficient hydrolysis of feather waste at low temperature

A novel cold-adapted bacteria Arthrobacter oryzae BIM B-1663 isolated from Antarctic green snow showed keratinase activity and efficient poultry feather degradation. A. oryzae strain degraded more than 80 % of chicken feathers within 7 days of cultivation at 25 °C. The optimal keratinase activity for A. oryzae BIM B-1663 was observed at 50 °C, both for α-keratin (44.86 U/mL) and for β-keratin (94 mU/mL). The obtained results from sulfite and thiol groups tests and Fourier transform infrared spectroscopy (FTIR) showed that A. oryzae strain has a different keratin degradation mechanism than the reference strain Bacillus licheniformis CCM 2145T. FTIR fingerprinting can be used for monitoring of feather hydrolysis as it showed distinct chemical differences in feather meal hydrolysates, retentate and permeate from A. oryzae and B. licheniformis strains.

Modular Engineering to Enhance Keratinase Production for Biotransformation of Discarded Feathers.

Biotransformation of wasted feathers via feather-degrading enzyme has gained immense popularity, low conversion efficiency hinders its scale application, and the main purpose of this study is to improve feather-degrading enzyme production in Bacillus licheniformis. Firstly, keratinase from Bacillus amyloliquefaciens K11 was attained with the best performance for feather hydrolysis, via screening several extracellular proteases from Bacillus; also, feather powder was proven as the most suitable substrate for determination of feather-degrading enzyme activity. Then, expression elements, including signal peptides and promoters, were optimized, and the combination of signal peptide SPSacC with promoter Pdual3 owned the best performance, keratinase activity aggrandized by 6.21-fold. According to amino acid compositions of keratinase and feeding assays, Ala, Val, and Ser were proven as critical precursors, and strengthening these precursors' supplies via metabolic pathway optimization resulted in a 33.59% increase in the keratinase activity. Furthermore, keratinase activity reached 2210.66 U/mL, up to 56.74-fold from the original activity under the optimized fermentation condition in 3-L fermentor. Finally, the biotransformation process of discarded feathers by the fermented keratinase was optimized, and our results indicated that 90.94% of discarded feathers (16%, w/v) were decomposed in 12h. Our results suggested that strengthening precursor amino acids' supplies was an efficient strategy for enhanced production of keratinase, and this research provided an efficient strain as well as the biotransformation process for discarded feather re-utilization.

Fourier Transform Infrared Spectroscopy for Assessing Structural and Enzymatic Reactivity Changes Induced during Feather Hydrolysis.

Chicken feathers are major byproducts of the livestock processing industry with high potential in the feed sector. In this study, we present a new approach using Fourier transform infrared (FTIR) spectroscopy to detect the structural changes of feather keratin and its availability for enzymatic hydrolysis (AEH) induced by the thermal pressure hydrolysis (TPH) process. Compared to time-consuming in vitro measurement techniques, the proposed method provides rapid information about the structural changes during TPH which enables quick adaptation of TPH conditions as the quality of the incoming feather changes. By analyzing the FTIR spectra of raw and processed feathers, it was found that AEH negatively relates to the β-sheet content (represented by two IR peaks centered at 1635 and 1689 cm-1), while it positively relates to a new series of peaks centered around 1700 cm-1 appearing after the TPH process. The proposed FTIR technique provides a reliable and rapid approach to determine the digestibility indicated by AEH of the processed feather and may be used in process control and optimization.

Sequential Hydrolysis of Chicken Feathers Composed of Ultrasound and Enzymatic Steps: An Enhanced Protein Source with Bioactive Peptides

Chicken feather is a massive by-product. Its incorrect disposal can lead to serious environmental impacts. However, chicken feather is a promising low-cost keratin source. Keratin products have a wide application in the food and pharmaceutical industry. Mostly, chicken feathers are hydrolyzed by hydrothermal processes, and then applied into animal feed formulations. Despite the low cost, the hydrothermal hydrolysis leads to uncontrolled and low hydrolysis yield. Therefore, the aim of this work was to develop and optimize a sequential strategy of chicken feathers hydrolysis composed of ultrasound and enzymatic hydrolysis (savinase®) steps. In the first research step an experimental design was built and the optimum hydrolysis condition was obtained at 50 °C and 12.5% (enzyme/chicken feather), using three integrated rectors containing enzyme/substrate and sodium disulfite. Then, the ultrasound probe was added in the experimental apparatus in order to investigate the enzymatic hydrolysis assisted by ultrasound treatment. The enzymatic hydrolysis assisted by ultrasound treatment led to high concentrations of peptides, including a dipeptide (245.1868 m/z). Thus, the sequential hydrolysis strategy composed by two green technologies proposed in this study, enhanced the degree of hydrolysis of chicken feathers, producing bioactive peptides that can be used as ingredients in food products and other sectors.

Chicken Feather Waste Valorization Into Nutritive Protein Hydrolysate: Role of Novel Thermostable Keratinase From Bacillus pacificus RSA27

Microbial keratinases exhibit a momentous role in converting keratin biowastes into exceedingly valuable protein supplements. This study reports a novel, highly stable keratinase from Bacillus pacificus RSA27 for the production of pure peptides rich in essential amino acids from chicken feathers. Purified keratinase showed a specific activity of 38.73 U/mg, 2.58-fold purification, and molecular weight of 36 kDa. Kinetic studies using a chicken feather as substrate report Km and Vmax values of 5.69 mg/ml and 142.40 μg/ml/min, respectively, suggesting significant enzyme-substrate affinity/biocatalysis. Identification and in silico structural-functional analysis of keratinase discovered the presence of distinct amino acid residues and their positions. Besides, keratinase possesses a high-affinity calcium-binding site (Asp128, Leu162, Asn164, Ile166, and Val168) and a catalytic triad of Asp119, His151, and Ser308, known attributes of serine protease (subtilisin family). Furthermore, a scale-up to 5 L fermenter revealed complete feather hydrolysis (94.5%) within 24 h with high activity (789 U/ml) and total amino acid of 153.97 μmol/ml. Finally, cytotoxicity evaluation of protein hydrolysate resulted in negligible cytotoxic effects (1.02%) on the mammalian hepatoblastoma cell line, signifying its potential biotechnological applications.

ЭФФЕКТИВНОСТЬ КОРМЛЕНИЯ БРОЙЛЕРОВ ПЕРЬЕВОЙ МУКОЙ, ПОЛУЧЕННОЙ МЕТОДОМ КРАТКОВРЕМЕННОГО ГИДРОТЕРМИЧЕСКОГО ВОЗДЕЙСТВИЯ

In the poultry industry, animal flour is often used, which is prepared from a mixture consisting of the remains of bones, soft tissues and feathers. They are rarely processed separately, especially for feathers, which have a very low digestibility. The technology of short-term high-temperature hydrolysis of feathers in a thin layer leads to the destruction of the keratin structure and increases the availability of its amino acids. This processing method achieves 86–88% of protein digestibility. The efficiency of using this feather meal (PM) in feeding makes it possible to reduce the cost of rations and improve zootechnical indicators.

Subcritical water hydrolysis of poultry feathers for amino acids production

This study assessed the subcritical water hydrolysis (SWH) of poultry feathers to recover amino acids. Experiments were conducted in a semi-continuous flow-through subcritical reactor (110 mL), which was operated over a range of temperatures (210–250 °C) and water flow rates (5–15 mL min–1), combined through a 22 central composite design, at constant feed (10 g) and pressure (15 MPa). The results demonstrated that non-essential and essential amino acids were obtained from SWH of poultry feathers. The highest hydrolysis temperature resulted in the highest concentrations of valine, methionine, tryptophan, phenylalanine, isoleucine, leucine, and lysine. Otherwise, threonine, histidine, and arginine were obtained more effectively at lower temperatures. The response surface methodology was adopted to identify the best conditions for amino acid production, and it was possible to identify the ranges of temperatures and water flow rates to be used to recover specific amino acids. This study allowed concluding that SWH is a promising eco-friendly technology to recover amino acids from protein-rich wastes.

Chicken Feather Waste Hydrolysate as a Superior Biofertilizer in Agroindustry.

Billions of tons of keratinous waste in the form of feathers,antlers, bristles, claws,hair, hoofs, horns, and wool are generated by different industries and their demolition causes environmental deterioration.Chicken feathers have 92% keratin that can be a good source of peptides, amino acids, and minerals. Traditional methods of feather hydrolysis require large energy inputs, and also reduce the content of amino acids and net protein utilization values. Biological treatment of feathers with keratinolytic microbes is a feasible and environmental favorable preference for the formulation of hydrolysate that can be used asbioactive peptides, protein supplement, livestock feed, biofertilizer, etc. The presence of amino acids, soluble proteins, and peptides in hydrolysate facilitates the growth of microbes in rhizosphere that promotes the uptake and utilization of nutrients from soil. Application of hydrolysate enhances water holding capacity, C/N ratio, and mineral content of soil. The plant growth promoting activities of hydrolysate potentiates its possible use in organic farming, and improves soil ecosystem and microbiota. This paper reviews the current scenario on the methods available for management of keratinous waste, nutritional quality of hydrolysate generated using keratinolytic microbes, and its possible application as plant growth promoter in agroindustry.

Environmental aspects and economic evaluation of new green hydrolysis method for waste feather processing

In recent years, an increase in the chicken meat process industry has been growing quickly, which brings a large amount of difficult to process waste, chicken feathers. These billions of kilograms create a serious waste problem over the world, furthermore, poultry feather processing is not only particularly difficult but also relatively expensive. In fact, there is no technology suitable for processing such huge quantities of feathers to guarantee sustainable development of the chicken meat industry together with processing of waste feathers. This article introduces a newly designed and original, highly efficient and environmentally friendly method of physicochemical hydrolysis of waste feathers. The hydrolysis is carried out in the presence of a weak organic carboxylic acid; thus, the resulting hydrolysate does not contain any salts or ashes. Therefore, a resulting hydrolysate, which includes a mixture of amino acids, peptides, proteins, glycoproteins and free fatty acids, is suitable for a variety of applications; e.g. as a chondroprotective agent in the treatment of joint diseases, nutrients for crop plants or targeted biostimulants for agriculture. This paper is focused not only on the process scale-up but also on environmental aspects and economic evaluations to bring general view of the process.Graphic abstract

Keratin particles generated from rapid hydrolysis of waste feathers with green DES/KOH: Efficient adsorption of fluoroquinolone antibiotic and its reuse

Fluoroquinolone antibiotics are widely used in human and veterinary medicine. However, untreated fluoroquinolone seriously threatens the ecosystem and human health. In this study, deep eutectic solvents (DESs) were applied for the hydrolysis of waste feathers, and the keratin particles (KPs) in a low-cost teabag were utilized to adsorb fluoroquinolone norfloxacin. Results showed that choline chloride/ethylene glycol DES rapidly hydrolyzed feathers within 10 min, and the undissolved particles effectively adsorbed norfloxacin. Adding KOH markedly shortened the hydrolysis time (6 min) and increased the adsorption ability of KPs. The optimum hydrolysis conditions were DES ratio of 1 g: 4.67 g, KOH of 35.68 g L−1, and temperature of 90 °C. When KPDES+KOH of 2 g L−1, norfloxacin of 25 mg L−1, and pH0 7 were used, 94% of norfloxacin was removed in 60 min. A low-cost teabag effectively separated the KPs from the solution after adsorption and did not decrease the adsorption ability of the KPs. The Langmuir isotherm model well described the adsorption behavior of KPsDES+KOH (qmax = 79.36 mg g−1, R2 = 0.9972). In addition, acetone efficiently regenerated the exhausted KPsDES+KOH. The KPs maintained >80% of its adsorption ability after seven cycles of regeneration.

Chicken Feathers Waste Management by Microbial as a Sustainable and Tool Environmental Friendly

Valorizing chicken feather agro-wastes nowadays is imperative, if these wastes are disposed of without treatment, they could contribute to environmental problems. Conventional methods of processing chicken feathers such as landfilling, chemical treatment and burning are costly, non-time consumable and are toxic to the environment. Microbial hydrolysis, on the contrary, is now considered the main environmental-friendly recycling alternative. Hence, the aim of this study is to isolate keratinolytic bacteria with efficient feather hydrolysis, to optimize some of the physical parameters that could affect both bacterial growth and consequently its degrading ability. Results demonstrated an efficient feather degrading ability of newly identified Bacillus sp. D4 strain isolated from chicken feathers under optimal mesophilic temperature 37°C, pH 8.0 and 106 CFU/mL cell size inoculum, interpreted by highest keratin activity (55.0 ± 1.35 U/mL) and (54.3 ± 1.5 U/mL) respectively and higher total protein content in the cell free supernatant of 0.65 mg/mL. Beyond these parameters values, a moderate enzyme activity was observed at 40°C (35.1 ± 2 U/mL), 25°C (30.1 ± 2 U/mL), similarly at the initial pH 7.5 (52.3 ± 2 U/mL), pH 9.0 (49.0 ± 1.2 U/mL) and pH 10.0 (38.2 ± 1.35 U/mL). Bacillus sp. D4, on the other hand, was not able to tolerate high alkaline pH value 11.0 nor acidic pH 4.0 and 5.0 and high temperature of 55°C, correspondingly low enzyme activity was noted (19.0 ± 1 U/mL).

Effects of Ultrasound‐Assisted Alkaline Extraction on Antioxidant Activity and Functional Characteristics of Chicken Feather Keratin Peptides

AbstractIn this research, we focus on enzymatic hydrolysis of chicken feathers, after chemical or chemical and ultrasound pretreatment. Our result illustrated that ultrasound pretreatment preserved the main structure and amino acid composition of extracted keratin as confirmed by the Fourier‐transform infrared (FTIR) spectroscopy and gel‐electrophoresis analysis. In addition, investigation showed that ultrasound‐assisted chemical treatment was more effective on enzymatic hydrolysis and bioactivity as compared to only the chemical treatment. Thermo gravimetric analysis (TGA) characterization of hydrolyzed products, using Alcalase enzyme at different times, showed that decomposition temperature was decreased during enzymatic hydrolysis and the best antioxidant activity was achieved after 30 min of hydrolysis with the value of 90.43±4.03 % (5 mg/ml concentration). Also, it was found that hydrolyzed product indicated no cytotoxicity to SK‐N‐MC cells when the concentration is less than 0.312 mg/ml. In this paper, the results demonstrated that the ultrasonic‐assisted enzymolysis processing seems to be effective for producing keratin antioxidant hydrolysate.

Whole-genome Sequencing and Mining of Protease Coding Genes in Bacillus paralicheniformis MKU3, and its Degradomics in Feather Meal Medium.

Bacillus paralicheniformis MKU3 produces commercially important keratinolytic proteases by utilizing chicken feather. To unravel the genetics of these degrading keratinolytic proteases in B. paralicheniformis MKU3, we sequenced the genome of this bacterium and studied the protease distribution and their characteristics using bioinformatics tools. Also, a proteomic analysis was performed to identify the consortium of proteases involved in feather hydrolysis. A total of 2,531,755 quality reads were obtained in whole genome sequencing with an approximate coverage fold of 105. The draft genome consists of 4,370,039bp with 45 contigs. The draft genome codes for 4874 protein-coding genes. Furthermore, 109 genes coding for RNA, including 26 rRNA and 83 tRNA, were identified. Phylogenetic analysis of B. paralicheniformis MKU3 showed closest homolog to B. paralicheniformis F47. Genes coding for proteases belonging to five families were identified with the following proportions 37%, 36%, 9%, 14%, 2%, and 2% of serine-, metallo-, cysteine-, mixed-, and uncharacterized proteases, respectively. Metallo- and serine-protease represented more than 70% of the total proteases. Major protease families distributed in the genome were S8, S9, S33, M20, M50, C26, and C40. Most of the proteases showed significant similarity with the conserved domain database and also identified conserved catalytic sites and domains. SDS-PAGE and zymogram analysis of concentrated feather hydrolysis revealed the active proteases ranging from 10 to 250kD in size. Proteomic analysis on the feather hydrolysis of B. paralicheniformis MKU3 identified two proteases belonging to serine proteases (S8) and other two as metalloproteases.

Reaction Stages of Feather Hydrolysis: Factors That Influence Availability for Enzymatic Hydrolysis and Cystine Conservation during Thermal Pressure Hydrolysis

The vast amount of feathers generated (>1 Mtons/a in Europe) in the poultry industry is an opportunity of upcycling by-product materials and improving sustainable practices. Feathers are potentially interesting materials as feed protein ingredients due to their high protein (>85 wt%) and cystine content (>7 wt%). However, due to their challenging recalcitrant nature, they have to be processed to make feather protein suitably digestible. The objective was to investigate the effects of temperature (120oC–160oC) and time (10, 30, and 60 min) in thermal pressure hydrolysis of feathers on availability for enzymatic hydrolysis (AEH) and cystine conservation. AEH is defined as degree of degradation of processed feather protein by two digestive enzymes pepsin and pancreatin (Boisen). The present study identified and assessed four temperature stages that take place during feather processing. The four temperature stages are 120oC–135oC, 140oC–155oC, > 160oC, and the cooling-down phase. The second stage has the greatest influence on AEH. As well as temperature, hydrolysis time is also an essential parameter that had a major impact in the second stage (140oC–155oC). Both temperature and time influence negatively cystine content and stability. The present study demonstrates for the first time the importance of four reaction stages during feather hydrolysis and the impact of four stages on AEH of the obtained products.