Feather Broth Research Articles

Optimize and Purification of Keratinase Produced from Local Aspergillus Terreus A13 Isolate Using A Feather as Substrate

Keratin is a fibrous, insoluble structural protein that is highly cross-linked with hydrophobic, hydrogen, and disulfide bonds. Keratinases are enzymes that belong to the category of serine hydrolases that are capable of breaking down keratin. The results of the determination of the better fermentation system showed that the production of keratinase from local A.terreus A13 isolate by submerged fermentation (SmF) system was the best system to give the highest specific activity (113.4 U/mg) of keratinase compared with solid-state fermentation (SSF). The optimum conditions for keratinase production by SmF, were determined via cultivation conditions, including carbon source, nitrogen source, temperature, pH of the medium, and time of incubation were optimized to enhance the production of total keratinase production in a culture of A.terreus A13 with incubator shaker. The highest product of total keratinase was achieved in feather broth with 2 % sucrose, and 0.5 % soya bean, with a pH of 5.5 at 28 °C for 8 days. Separation and purification of keratinase from a local isolate of A.terreus A13 was done by precipitating with 0-75 % saturated ammonium sulfate, then by ion-exchange chromatography on DEAE-Cellulose column and sephadex G-150 gel. Partially purified keratinase gave an activity of 5.1 U/ml, protein concentration of 0.004 mg/ml, and specific activity of 1275 U/mg with purification fold of 4.96 and 49 % as yield. The aim of the present study was to optimize the production of keratinase from A. terreus A13, cultivated using optimum conditions, and its use for the biodegradation of feathers.

The Novelty of Amino Acid Production From Feathers Degradation Em-ploying Keratinase from Streptomyces Venezuelae AZ15

Keratinases are enzymes belonging to the serine hydrolases group which are capable of degradation of keratin, an insoluble and fibrous structural protein widely cross-linked with hydrogen, disulfide, and hydrophobic bonds. Attempts to find new sources of enzymes and amino acids for fundamental knowledge of enzyme evolution, structure‐function relationships, catalysis mechanisms of enzymes, and even for the identification of novel protein folds. In this study, seventy-nine samples were collected from different places in the University of Baghdad, and the best isolate for amino acid production by feathers degradation was by using Streptomyces venezuelae AZ15. The best fermentation system and the optimum culture conditions for amino acid production from feathers as substrate utilizing S. venezuelae AZ15 were elucidated. Using feathers as a substrate, the results showed that the submerged fermentation (SmF) system was the best to give the highest yield of amino acid. Furthermore, the highest yield of total amino acid was gained in feather broth supplemented with 2% glucose and 0.5 % ammonium nitrate, with a pH of 6.0 at 37 °C for 8 days.

Preparation, Purification, and Identification of Novel Feather Keratin-Derived Peptides with Antioxidative and Xanthine Oxidase Inhibitory Activities.

Feather keratin is an underappreciated protein resource of high quality, with limited bioavailability, and it urgently requires eco-friendly methods to enhance its value. Here, we report on the preparation, purification, and identification of novel peptides with antioxidant and xanthine oxidase (XOD) inhibitory activities from fermented feather broth, using Bacillus licheniformis 8-4. Two peptides, namely, DLCRPCGPTPLA (DA-12) and ANSCNEPCVR (AR-10), displayed remarkable 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical scavenging abilities with half-maximal inhibitory concentrations (IC50) values of 0.048, 0.034, and 0.95, 0.84 mg/mL, respectively. These values exceed those of the previously reported feather keratin-derived antioxidant peptides. Another peptide, GNQQVHLQSQDM (GM-12), demonstrated XOD activity inhibition, with an IC50 value of 12.15 mg/mL, and it quenched the fluorescence of XOD. Furthermore, after simulating gastrointestinal digestion, DA-12, AR-10, and GM-12 retained their biological activities. Meanwhile, DA-12 and GM-12 showed an unexpected synergistic inhibition on XOD activity accompanied by fluorescence quenching. This study provides new insights into the potential applications of feather keratin, including functionalized feed with antioxidative and antigout (anti-hyperuricemia) activities.

Bio-degradation of Feathers Waste by the Staphylococcus epidermidis P3A

Development of industrialization is directly proportional to the development of nations. Rising industrializations also increase the pollutions. One of them is poultry industry which discharges a huge amount of keratinous waste. The present study focused on the selection of feather degrading bacteria from Barshi region of Maharashtra. Bacterial strains were grown in whole feather broth medium. Out of 16 proteolytic bacterial isolates, 9 feather degrading isolates were selected from the Parande road side feather waste dumping area. Among these isolates, P3A was selected and classified on the basis of morphological and biochemical analysis. 16S rRNA studies confirmed that the isolated strain was Staphylococcus epidermidis. Staphylococcus epidermidis P3A shows 84% feather degradation and 72 U/ml maximum keratinase activity after 96 hrs. The isolate shows potential use in feather waste disposal methods which are safe and environment friendly.

Feathers as bioresource: Microbial conversion into bioactive protein hydrolysates

Abstract Feathers are recalcitrant protein-rich by-products generated by the poultry agro-industry, and valorization of residual biomasses from industrial processing is increasingly focused. Three feather-degrading Bacillus strains (CL18, CL33A, CL14) were used to convert feathers into feather hydrolysates (FH) during submerged cultivations, and the in vitro bioactivities of FHs were assessed. In feather broth (10 g/L feathers), Bacillus sp. CL18 degraded feathers more efficiently and the resulting FHs displayed enhanced antioxidant activities. With increasing feather concentrations, soluble protein was higher (9.02 mg/mL) after five days of cultivation with Bacillus sp. CL18 on feather broth containing 40 g/L feathers. This FH displayed antioxidant activities, as evaluated through the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) radical-scavenging assays (34.7% and 80.3%, respectively), Fe2+-chelating ability (80.6%), and reducing power (0.518 Abs700). In vitro antihypertensive and antidiabetic activities of FH were demonstrated through inhibition of angiotensin I-converting enzyme (ACE; 89.7%) and dipeptidyl peptidase-IV (DPP IV; 95.7%) activities, respectively. Half-maximal effective/inhibitory concentrations of FH were 5.39, 15.12, 10.50, 1.61 and 1.52 mg/mL in the ABTS, DPPH, Fe2+-chelating, ACE- and DPP IV-inhibitory assays, respectively. Antioxidant, antidiabetic and antihypertensive potentials of FHs indicate microbial conversion as a suitable eco-friendly technology for feathers reclamation and valorization within the biorefinery concept.

Protease production by the keratinolytic Bacillus sp. CL18 through feather bioprocessing

Bacillus sp. CL18 was investigated to propose a bioprocess for protease production using feathers as organic substrate. In feather broth (FB), containing feathers as sole organic substrate (1-100gl-1), maximal protease production was observed at 30gl-1 (FB30) after 6days of cultivation, whereas increased feather concentrations negatively affected protease production and feather degradation. Protease production peaks were always observed earlier during cultivations than maximal feather degradation. In FB30, 80% of initial feathers mass were degraded after 7days. Addition of glucose, sucrose, starch, yeast extract (2gl-1), CaCl2, or MgCl2 (10mmoll-1) to FB30 decreased protease production and feather degradation. FB30 supplementation with NH4Cl (1gl-1) resulted in less apparent negative effects on protease production, whereas peptone (2gl-1) increased protease yields earlier during cultivations (3days). Through a central composite design employed to investigate the effects of peptone and NH4Cl (0.5-4.5gl-1) on protease production and feather degradation, FB30 supplementation with peptone and NH4Cl (0.5-1.1gl-1) increased protease production within a shorter cultivation time (5days) and hastened complete feather degradation (6days). Feather bioconversion concurs with sustainable production of value-added products.

Proteolytic system of Bacillus sp. CL18 is capable of extensive feather degradation and hydrolysis of diverse protein substrates

ABSTRACT1. Feathers are recalcitrant protein-rich wastes produced in huge amounts by poultry processing for meat production. Hence, feather bioconversion and protease production by Bacillus sp. CL18 were investigated.2. Bacillus sp. CL18 demonstrated a remarkable feather-degrading potential. Through cultivations on feather broth (10 g l−1 feathers), 94.5% ± 3% of whole feathers were degraded after 4 d. Increases in soluble protein contents were observed and protease production was maximal also at d 4. This strain produced diverse proteolytic enzymes during growth.3. Crude protease displayed optimal activity at 55°C (50–62°C), pH 8.0 (7.0–9.0) and a low thermal stability. Proteolytic activity increased in the presence of Ca2+, Mg2+, Triton X-100, Tween 20 and dimethyl sulphoxide. Inhibition profile indicated that crude protease contains, mainly, serine proteases. Enzyme preparation hydrolysed mainly casein and soy protein isolate.4. The keratinolytic capacity of Bacillus sp. CL18 at moderate temperatures (30°C) might be appropriate for feather conversion, resulting in protein hydrolysates and proteolytic enzymes. Proteases are postulated to be added-value products that can be obtained from such a bioprocess.

Screening of Bacteria for Protease Production and Feather Degradation

Feathers are recalcitrant wastes produced by the poultry industry. Considering potential environmental hazards and the need for energy conservation/recycling, adequate approaches are demanded for feathers reclamation. Microbial conversion is an interesting alternative from both technological and economical perspectives. Therefore, 15 bacterial strains were isolated from a site containing waste feathers, and evaluated for proteolytic and keratinolytic potentials. From these bacterial isolates, seven produced extracellular proteases in milk agar plates, also demonstrating the ability to grow on feather meal agar plates, preferentially at pH values from 7 to 9, and at 30–37 °C. The isolate named CL33A displayed higher efficiency for feather degradation in qualitative assays, and selected for studies in feather broth (FB), a medium containing whole feathers (10 g/L) as the sole source of carbon, nitrogen and energy. CL33A degraded 29, 75, and 95 % of the feathers in FB, after 96, 144 and 216 h of growth, respectively. Feather degradation was corroborated by increases in soluble protein concentration and medium pH. Production of proteolytic enzymes reached maximal values after 216–240 h of growth on FB, and CL33A also produced proteases when cultivated in feather meal, peptone, and soy protein isolate. Through 16S rRNA gene sequencing, this feather-degrading isolate was identified as Bacillus sp. CL33A. Bioprocessing could be a suitable technology aiming the management and valorization of feathers/feather meal, due to the production of protein hydrolysates and also proteolytic enzymes that could be used as important biocatalysts.

English

  A unique standard starch casein medium has been implemented for the isolation of actinobacteria from the south Indian marine sediments. A combination of techniques, morphological, physiological and biochemical tests provided the evidence for the isolated actinobacteria. All the 56 isolates were inoculated on milk agar and soya meal agar plates for the primary proteolytic screening and the proportional study was made by ANOVA. Among the 56 isolates, nine showed proteolytic activity in terms of making clear zone around their colony on the plates. Then, nine isolates were subjected to the secondary screening on feather broth where three isolates (IS -1, 2 and 18) showed degradation of feather between seven and ten days. The keratinolytic characters of crude enzymes were scrutinized by feather keratin as substrate and the protein concentration was determined. Then, the isolates were identified at molecular level by 16S rRNA gene amplification technique.   Key words: Actinobacteria, keratinase, milk agar, soya meal agar, 16S rRNA gene amplification.

Keratinolytic potential of a novel Bacillus sp. P45 isolated from the Amazon basin fish Piaractus mesopotamicus

Bacillus sp. P45, isolated from the intestine of the Amazon basin fish Piaractus mesopotamicus, showed proteolytic activity when grown on skimmed milk and feather meal agar plates. The keratinolytic potential of this strain was evaluated on whole feather broth and human hair broth. Bacillus sp. P45 degraded almost 90% of chicken feathers after 72 h of submerged cultivation on whole feather broth, and the production of extracellular proteases was observed. The formation of thiol groups was also detected during growth, indicating the contribution of sulphitolysis to the efficient hydrolysis of feather keratin. Nevertheless, Bacillus sp. P45 was unable to degrade hair keratin, possibly due to the conformational diversity of this substrate in comparison to feather keratin. Additionally, preliminary results demonstrated that this strain might be utilized in the degradation of recalcitrant collagen-containing wastes. The keratinolytic character of Bacillus sp. P45 might be utilized in environmental-friendly processes such as bioconversion of waste feathers, representing an alternative way of waste management that could lead to the production of value-added products such as microbial biomass, protein hydrolysates and proteolytic enzymes.

Characterization of a protease of a feather-degrading Microbacterium species.

To characterize a new feather-degrading bacterium. The strain kr10 producing a high keratinolytic activity when cultured on native feather broth was identified as Microbacterium sp., based on phenotypical characteristics and 16S rDNA sequence. The bacterium presented optimum growth and feather-degrading activity at pH 7.0 and 30 degrees C. Complete feather degradation was achieved during cultivation. The keratinase was partially purified by gel filtration chromatography. It was optimally active at pH 7.0 and 55 degrees C. The enzyme was inhibited by 1,10-phenanthroline, EDTA, p-chloromercuribenzoic acid, 2-mercaptoethanol and metal ions like Hg(2+), Cu(2+) and Zn(2+). A new Microbacterium sp. strain was characterized presenting high feather-degrading activity, which appears to be associated to a metalloprotease-type keratinase. This micro-organism has enormous potential for use in biotechnological processes involving keratin hydrolysis.