Total solubilisation of the chicken feathers by fermentation with a keratinolytic bacterium, Bacillus pumilus A1, and the production of protein hydrolysate with high antioxidative activity

Process Biochemistry 46 (2011) 1731–1737 Contents lists available at ScienceDirect Process Biochemistry journa l homepage: www.e lsev ier .com Total so fer bacteri tio high an Nahed Fa am Ines Dah Laboratoire de s de Sf a r t i c l Article history: Received 14 Ja Received in re Accepted 27 M Keywords: Feather protei Antioxidant ac Animal feed Bacillus pumilu ctive grad rs fo pepti com eval engin an IC ein an 1. Introduction Feathers at poultry- throughout acterized b protein sou produce fea high-energy methionine Nutritional with feath nous waste represented bioconversi tial applica reported by isms were k [3–5], actin ens fed by and supplem by soybean ∗ Correspon E-mail add In recent years, hydrolysed proteins from many animal and plant sources have been found to possess antioxidant activity, 1359-5113/$ – doi:10.1016/j. are produced in large amounts as a waste by-product processing plants, reaching millions of tons annually the world. Feathers, consisted mainly of keratin char- y its high recalcitrant nature, could be an important rce in animal feedstuff. Currently, some industries ther meal by steam pressure cooking, which require input. The resulted feather meal is deficient in and histidine which limit its use in animal feed [1]. enhancement of feathers can be achieved by hydrolysis er-degrading microorganisms. Hydrolysis of kerati- s by microorganisms possessing keratinolytic activity an attractive alternative method for their efficient on and improving their nutritional value. The poten- tions of such microbial keratinases have been recently Gupta and Ramnani [2]. In fact, several microorgan- nown to degrade keratinous substrates such as fungi omycetes [6,7] and some Bacillus species [8–13]. Chick- feather hydrolysate produced by Bacillus licheniformis ented with amino acids grow as well as chickens fed meal [14]. ding author. Tel.: +216 74274088. ress: nahedfakh [email protected] (N. Fakhfakh). such as milk casein [15], soybean [16], rice bran [17], quinoa seed protein [18], canola [19], egg yolk protein [20] and porcine myofibrillar proteins [21]. However, no antioxidant activity was reported in the poultry feather protein hydrolysate. To the best of our knowledge, this is the first report highlighting the antioxidant potential of such keratinous waste hydrolysate which could be an important protein source in feed formulations for fish or domestic animals. Fish feeds containing high levels of polyunsaturated fatty acids are susceptible to lipid oxidation. Presently, fish feeds are protected with synthetic antioxidants, mainly ethoxyquin (EQ) and butylated hydroxytoluene (BHT) [22]. Recently, there is a substantial carry- over of these antioxidants to the fish fillet and the mandatory 2 weeks starvation period before slaughter of farmed fish is not suf- ficient for clearance of these antioxidants from the fillet [22,23]. Therefore, a switch to natural antioxidants in fish feed ingredi- ents would be an advantage both for the aquaculture industry and with regard to consumer health and well-being. Furthermore, the relationship between nutrition and animal health has long been recognized. Animal’s morbidity and mortality can be explained partly by impaired immune responsiveness. Therefore, the role of natural antioxidants in immunity and health in food animals is of prime importance [24]. This study reports the production of feather protein hydrolysate (FPH) by Bacillus pumilus A1, isolated from a local slaughter house see front matter © 2011 Published by Elsevier Ltd. procbio.2011.05.023 lubilisation of the chicken feathers by um, Bacillus pumilus A1, and the produc tioxidative activity khfakh ∗, Naourez Ktari, Anissa Haddar, Ibtissem H men, Moncef Nasri Génie Enzymatique et de Microbiologie, Université de Sfax, Ecole Nationale d’Ingénieur e i n f o nuary 2011 vised form 26 May 2011 ay 2011 n hydrolysate tivity s a b s t r a c t A feather protein hydrolysatewas effe strain A1. In fact, complete feather de feathers. Cultivation of 50g/l of feathe mum production of amino acids and a very high in vitro digestibility (98%) the antioxidant activities of FPH were 2-picrylhydrazyl (DPPH) radical-scav important antioxidant potential with may be useful as supplementary prot / locate /procbio mentation with a keratinolytic n of protein hydrolysate with za Mnif, ax, B.P. 1173-3038, Sfax, Tunisia ly produced using the keratinolytic bacterium Bacillus pumilus ation was achieved in medium containing up to 50g/l of raw r two days, at 45 ◦C and at initial pH of 10.0, resulted in maxi- des (42.4 g/l). The feather protein hydrolysate (FPH) presents pared with that of the untreated feathers (2%). Furthermore, uated using in vitro antioxidant assays, such as 1,1-diphenyl- g activity and reducing power. Interestingly, FPH presents an 50 value of 0.3±0.01mg/ml. These results indicate that FPH d antioxidants in animal feed formulations. © 2011 Published by Elsevier Ltd. 1732 N. Fakhfakh et al. / Process Biochemistry 46 (2011) 1731–1737 polluted water [25]. The in vitro digestibility and the antioxidant potential of feather protein hydrolysate were also investigated. 2. Materials and methods 2.1. Feathers Chicken fe were washed feathers were microbial trea 2.2. Bacterial B. pumilus house polluted rRNA gene seq 2.3. Culture m The strain composed of mediumused 0.5 g/l KH2PO4 [27]. The med in a 1 l Erlenm at 30 ◦C and 25 the effect of fe individually ev tion. Aliquots o and the supern uble protein a protein concen (mg/l/h). All ex 2.4. Caseinoly Protease a bhavi et al. [2 Tris–HCl buffe crude enzyme stopped by th 15minat room absorbance of tometer, PG. in curve was gen activity was d per minute un the means of a 2.5. Analytica Dry matte Protein conten [30]. Soluble p of Biuret [31] peptideswasd by Soxhlet ext content was d 2.6. Antioxida 2.6.1. DPPH ra DPPH rad described by ent concentra DPPH in 99.5% dark for 60mi a UV–Visible s China). The p calculated acc Scavenging ac where Ac is th sample extract from the grap (FPH) concent DPPH radical-scavenging activity. Butylated hydroxyanisole (BHA) was used as a standard. The test was carried out in triplicate. 2.6.2. Reducing power assay The ability of the hydrolysate to reduce iron (III) was determined according to hod o ent co 2.5m follow then wasm er 10m Increa he va itro d tro pr as des g/ml ubati al 16 n, sam tants ent of l prote ults oduc side , the tion as be ects o duct gate se in Effect micr ing n so ed af nt. T /ml) ). high pro dium /l of s pr gh fe sion her d es pe ). Ye /l/h) ed in Effect effe prod ysate ing se pr valu by ph to pr athers (CF), supplied by a local poultry industry (Chahia, Tunisia), threefold with tap water and finally with distilled water. The washed dried at 90 ◦C for 22h and then stored at room temperature prior to tment. strain A1 producing alkaline keratinases was isolated from the slaughter water in Sfax city (Tunisia). It was identified on the basis of the 16S uencing (EU719191) [25]. edia and growth conditions B. pumilus A1 was routinely grown in Luria–Bertani broth medium 10g/l peptone, 5 g/l yeast extract, and 5g/l NaCl [26]. The initial for feather protein hydrolysate productionwas composed of 30g/l CF, , 0.5 g/l K2HPO4, 2.0 g/l NaCl, 0.1 g/l KCl, 0.1 g/l MgSO4·7 H2O, pH 6.0 ia were autoclaved at 121 ◦C for 20min. Cultivations were conducted eyer flask containing 100ml culture medium maintained for 5 days 0 rpm. To optimize the composition and the condition of the culture, ather concentration (10–70g/l), initial pH and the temperature were aluated for their performance in feather protein hydrolysate produc- f 1ml were periodically removed, centrifuged for 5min at 15,000× g atant was stored at 4 ◦C for determination of proteolytic activity, sol- nd amino acid concentration. Productivity was determined as soluble tration per time to reach 5-day-value or 2-day-value concentration periments were performed in duplicate for the mean calculation. tic activity ctivity of the crude enzyme was measured by the method of Kem- 8] using casein as a substrate (Sigma–Aldrich). A 0.5ml of 100mM r (pH 8.5) containing 1% (w/v) casein was mixed with 0.5ml of the suitably diluted, and incubated for 15min at 60 ◦C. The reaction was e addition of 0.5ml 20% (w/v) trichloroacetic acid allowed to rest for temperature and then itwas centrifuged for 15minat 17,000× g. The the supernatant was measured at 280nm (T70, UV/VIS Spectropho- struments Ltd. Wiftaft, UK) using the appropriate blank. A standard erated using solutions of 0–50mg/l of tyrosine. One unit of protease efined as the amount of enzyme required to liberate 1�g of tyrosine der the experimental conditions used. Protease activity represents t least two determinations carried out in duplicate. l methods r was determined by oven-drying at 105 ◦C to constant mass [29]. t (dry weight basis) was analyzed according to the Kjeldahl method rotein and peptide concentrations were determined by the method using ovalbumin as the standard. Concentration of amino acids and eterminedby theninhydrinmethod [32]. Fat contentwasdetermined raction with hexane for 8h at boiling point of the solvent. The ash etermined by combustion of the sample at 550 ◦C for 8h. nt activity dical-scavenging assay ical-scavenging activity of the hydrolysates was determined as Bersuder et al. [33]. A volume of 500�l of each sample at differ- tions was mixed with 500�l of 99.5% ethanol and 125�l of 0.02% ethanol. The mixture was then kept at room temperature in the n, and the reduction of DPPH radical was measured at 517nm using pectrophotometer (T70, UV/VIS spectrometer, PG Instruments Ltd., ercentage inhibition of the DPPH radical (scavenging activity) was ording to the formula: tivity (%) = [ Ac − As Ac ] × 100 e absorbance of the control reaction and As is the absorbance of the . Sample concentrationproviding 50% inhibition (IC50)was calculated h plotting inhibition percentage against feather protein hydrolysate ration. A lower absorbance of the reaction mixture indicated a higher the met at differ 6.6) and 30min, ture was solution ride. Aft 700nm. power. T 2.7. In v In vi creatin, with 2m After inc addition digestio superna D= cont the tota 3. Res 3.1. Pr Con keratin Produc teria h the eff the pro investi protea 3.1.1. The contain nitroge obtain consta (560U (Fig. 1a The soluble the me at 70g protein that hi repres of feat peptid (Fig. 1d (0.25g obtain 3.1.2. The on the hydrol contain protea this pH uated strain f Yildirim et al. [34]. An aliquot of 1ml sample of each hydrolysate ncentrations was mixed with 2.5ml of 0.2M phosphate buffer (pH l of 1% potassium ferricyanide. The mixture was incubated at 50 ◦C for ed by addition of 2.5ml of 10% (w/v) trichloroacetic acid. The mix- centrifuged at 15,000× g for 10min. Finally, 2.5ml of the supernatant ixedwith 2.5ml of distilledwater and 0.5ml of 0.1% (w/v) ferric chlo- in reaction, the absorbance of the resulting solutionwasmeasured at sed absorbance of the reaction mixture indicated increased reducing lues are presented as the means of triplicate analyses. igestibility oteolytic digestion of samples was performed with pepsin and pan- cribed by Ikeda et al. [35]. One gram of each sample was incubated of pepsin (from porcine stomach, Sigma) for 2h at 37 ◦C in 2M HCl. on, the pH was adjusted to 8.0 with 2M NaHCO3, and incubated for h with 2mg/ml of pancreatin (from porcine pancreas, Sigma). After ples were centrifuged and protein concentration was determined in by the Kjeldahl method [30]. Protein digestibility was calculated as: protein released upon the digestion of 1 g of sample/the content of in of 1 g of sample before digestion. tion of feather protein hydrolysates ring that feathers are composed by at least 90% of use of this protein source can be of great interest. of feather protein hydrolysates using keratinolytic bac- en considered as an interesting alternative [2]. Thus, f feather concentrations, initial pH and temperature on ion of amino acids, peptides and soluble proteins were d by B. pumilus A1. The activity of the crude alkaline the culture broth was also determined. of feather concentration oorganismB. pumilusA1was grown inmineralmedium different amounts of raw feathers as the sole carbon and urce. The highest levels of proteolytic activities were ter cultivation for 3 days and then activities remained he strain exhibited the highest enzyme production in culture medium containing 50g/l of chicken feathers est levels of amino acids and peptides (30±2.5 g/l) and teins and peptides (7.3±2g/l) were also obtained in containing 50g/l of feathers (Fig. 1b and c). However, feathers, proteases, amino acids, peptides and soluble oduction were reduced. In fact, it was demonstrated ather concentration may cause substrate inhibition or of keratinase production, resulting in a low percentage egradation. The highest ratio of soluble proteins and r gram of feathers was reached with 10g/l of feathers t, maximum productivity of amino acids and peptides and soluble proteins and peptides (0.06 g/l/h) were culture medium containing 50g/l of feathers (Fig. 1d). of initial medium pH ct of the initial medium pH values from 6.0 to 11.0 uction of proteolytic enzymes and on feather protein was studied by cultivating the strain A1 in themedium 50g/l of intact feathers. As shown in Fig. 2a, the highest oduction level was obtained at pH 6.0. Nevertheless, at e no complete feather degradation was achieved (eval- ysical appearance). The optimum initial pH of the A1 oduce FPH was observed to be 9.0–11.0, and complete N. Fakhfakh et al. / Process Biochemistry 46 (2011) 1731–1737 1733 Fig. 1. Effects hydrolysate pr solubilisatio (data not sh and soluble observed at 3.1.3. Effect Several a significan optimize FP of feather concentration on caseinolytic activity (a), amino acids and peptides (b), so oductivity (d). Cultivations were performed in media with initial pH of 6.0 at 30 ◦C. n of feathers was obtained after 4 days of fermentation own). The highest amino acids and peptides (40±2g/l) proteins and peptides (10.6±0.7 g/l) productions were pH 10.0 (Fig. 2b and c). of cultivation temperature works showed that the fermentation temperature has t effect on the protein production [8,36]. In order to H production, different temperatures values (between 25and60 ◦C containing As show mumactivi protease pr peratures ( production 3 days of fe The kine ble protein luble proteins and peptides (c) productions and on feather protein )were testedby cultivating the strainA1 in themedium 50g/l of feathers at initial pH of 10.0. n in Fig. 3a, at 40, 45 and 50 ◦C, protease reached amaxi- ty after 24hof cultivationand thendecreased.However, oduction curve was completely different at lower tem- 25, 30 and 35 ◦C). In fact, at these temperatures, the of proteolytic enzymes by A1 strain was maximal after rmentation (Fig. 3a). tics of production of amino acids, peptides and solu- s are shown in Fig. 3b and c. The maximum levels of 1734 N. Fakhfakh et al. / Process Biochemistry 46 (2011) 1731–1737 1 2 3 4 500 600 a C as ei no ly tic a ct iv ity (U /m l) So lu bl e pr ot ei ns a nd p ep tid es (g /l) A m in o ac id es a nd p ep tid es (g /l) Fig. 2. Effects (b), soluble pr media contain amino acid peptides (13 mentation ( and peptid increased w and0.88g/l (Fig. 3d). 3.2. Biochem The A1 s cating its s peptides an degradation of raw fea natantwas shown in Fi appears ind in peptides order to ch the culture estingly, af constant m Table 1 Physico-chemical composition of FPH and raw feathers. Composition (%) FPH Raw feathers n re o dige ete s vern in dby epsi high he p so de t (84% hydr y of tioxi resu idant ng po 0 00 00 00 00 b c 6543210 Cultivation time (days) pH 6 pH 7 pH 8 pH 9 pH 10 pH 11 10 12 0 5 10 15 20 25 30 35 40 45 6543210 Cutivation time (days) pH 6 pH 7 pH 8 pH 9 pH 10 pH 11 Protei Fat Moistu Ash In vitr compl dried o The directe using p a very (2%). T was al ponen of this stabilit 3.3. An The antiox reduci 0 2 4 6 8 6543210 Cultivation time (days) pH 6 pH 7 pH 8 pH 9 pH 10 pH 11 of medium pH on caseinolytic activity (a), amino acids and peptides oteins and peptides (c) productions. Cultivations were performed in ing 50g/l of feathers at 30 ◦C. s and peptides (42.4±1.5 g/l) and soluble proteins and .7±0.5 g/l)wereobtainedat 45 ◦Cafter twodaysof fer- Fig. 3b and c). It is worthy to note that soluble proteins es as well as amino acids and peptides productivities ith temperature to reach maximum levels of 0.29 g/l/h /h, respectively at 45 ◦C and after twodays of incubation ical and physical characterization of FPH train was able to complete feather degradation, indi- trong keratinolytic activity. Maximum amino acids, d proteins production as well as more efficient feather were observed under the following conditions (50g/l thers; 45 ◦C; pH 10.0 and 48h). The culture super- analyzedbySDS–polyacrylamidegel electrophoresis.As g. 3e, no band with molecular mass higher than 14kDa icating the efficacy of A1 strain in hydrolysing feather and proteins with low molecular masses. Besides, in eck that chicken feathers were completely solubilised, was filtrated throughWhatmanNo. 3 filter paper. Inter- ter washing and drying the filter paper at 105 ◦C to ass, no residual feathers were obtained showing their 3.3.1. DPPH DPPH is at 517nm. substrate s enged and in absorban The DPPH r concentrati Fig. 4a clea scavenging 3.3.2. Redu The redu of an antio reports hav antioxidant pounds. In wasdeterm hydrolysate to the ferro bymeasurin shows the r trations (0. The results with the FP thehighest interest. 4. Discussi Microbia tial approac to cost-effe present wo used to op Different ch duction of 5 days of c 84 ± 0.5 85.31 ± 0.43 0.057 ± 0.01 2 ± 0,7 3.72 ± 0.4 6.07 ± 0.87 10.68 ± 0.5 0.69 ± 0.08 stibility (%) 98 ± 0.7 2 ± 0.2 olubilisation. Finally, the filtrate was autoclaved and ight at 90 ◦C to obtain FPH. vitro digestibility of the feather protein hydrolysate B. pumilusA1 in theoptimizedconditionswasmeasured n and pancreatin. As shown in Table 1, the FPH presents digestibility (98%) compared to that of raw feathers hysico-chemical characterization of the produced FPH termined (Table 1). Proteins represent the major com- ) of the FPH followedbyash (10.68%). The lowhumidity olysate (3.72%) may contribute to the microbiological the product during its storage. dant activity of feather protein hydrolysate lting feather protein hydrolysate was assayed for its activity using DPPH radical-scavenging activity and wer. free radical-scavenging activity a stable free radical that shows maximum absorbance When DPPH radicals encounter a proton-donating uch as an antioxidant, the radicals would be scav- the absorbance would be reduced [37]. The decrease ce is taken as a measure for radical-scavenging activity. adical-scavenging activity was investigated at different ons (0.1–3mg/ml) of the FPH. The results presented in rly show that the FPH exhibited an interesting radical- activity with an IC50 value of 0.3mg/ml. cing power cing power assay is often used to evaluate the ability xidant to donate an electron or hydrogen [38]. Many e revealed that there is a direct correlation between activities and reducing power of certain bioactive com- this assay, the ability of the FPH to reduce Fe3+ to Fe2+ ined. Thepresenceof antioxidants in the featherprotein results in reduction of the Fe3+/ferric cyanide complex us form. Therefore, the Fe2+ complex can be monitored g the formationof Perl’s Prussianblueat700nm.Fig. 4b educing power activities of the FPH at different concen- 1–3mg/ml) compared with BHA as positive standard. showed that the reducing power has increased linearly H concentration. Although the synthetic BHA presents antioxidant activity, natural antioxidants areof growing on l conversion of keratinous wastes constitutes a poten- h for their biodegradation and valorisationwith respect ctiveness and environment protection [39,40]. In the rk, a feather-degrading bacterium B. pumilus A1 was timize the production of feather protein hydrolysate. icken feather concentrations were tested for the pro- FPH. Complete feather degradation was achieved after ultivation in the presence of 50g/l of chicken feather. N. Fakhfakh et al. / Process Biochemistry 46 (2011) 1731–1737 1735 Fig. 3. Effects hydrolysatepr Lane 1: molec However, in after incub those repor Kr2. pH is on of the bact degradation ing only ch degradation amino acids nolytic activ for proteol degradation This result enzymes pr ditions (pH similar to th shows that FK 46 was r [42] report level of solu 8.0.The ferm of cultivation temperature on caseinolytic activity (a), free amino acids and peptides (b), oductivity (d). Cultivationswereperformed inmedia containing50g/l of featherswith initi ular mass markers, lanes 2, 3 and 4 contained 25, 40 and 55�g of protein, respectively (e the presence of 70g/l, residual feather rachis remained ation (data not shown). Our results are in line with ted by Grazziotin et al. [41] using the strain Vibrio sp. e of the most important factors affecting the growth eria, keratinase production and percentage of feather . Since cultures were conducted in media contain- icken feather as nitrogen and carbon source, complete of chicken feather to produce an hydrolysate rich in and peptides could only be achievedwhen high kerati- ity was attained. Thus, different pH values were tested ytic enzyme and FPH production. Complete feather by theA1 strainwas observed at initial pHsof 9.0–11.0. could be explained by the fact that the proteolytic oduced by A1strain are highly active at alkaline con- 8.5 on casein and pH 9.0 on keratin) [27]. This finding is at published by Suntornsuk and Suntornsuk [8] which complete feather degradation by the strain Bacillus sp. eached with initial pH of 9.0. However, Grazziotin et al. that the strain Vibrio sp. Kr2 produced the maximum ble proteins with initial pH comprised between 6.0 and entation temperature is also an interesting parame- ter that ca fermentatio ers (42.6± acids andpe amino acid tein concen determined whole chick was obtain of fermenta feather solu obtained w with the st acids and p containing The in v found to be [43]. Howe fermentatio It is wo exhibited h parable to t soluble proteins and peptides (c) productions and on feather protein alpHof10.0. FPHanalysisbySDS–polyacrylamidegel electrophoresis. ). n influence the production of enzymes. After 48h of n at 45 ◦C, the keratin present into the 50g of feath- 0.43g proteins) was completely solubilised to amino ptides, detectedby theninhydrinmethod (42.4±0.52g s and peptides). This result was confirmed by the pro- tration in the culture supernatant (41.8±0.5 gproteins) by the Kjeldahl method. However, the degradation of en feathers by B. licheniformis PWD-1 and Vibrio sp. Kr2 ed only after 6 days of cultivation at 50 ◦C and 5 days tion at 30 ◦C, respectively [41,42]. Because of the total bilisation, the concentration of amino acids (42.4 g/l) ith the strain A1 is very high compared to that obtained rain Vibrio sp. Kr2 which released only 0.2 g/l of amino eptides after 5 days of fermentation in the medium 60g/l of feathers [41]. itro digestibility of the FPH produced by A1 strain was very close to that obtainedwith the strain Vibrio sp. Kr2 ver, the digestibility of feather hydrolysate obtained by n of the strain Kocuria rosea was only 88% [44]. rthy to note that the FPH, produced in this study, igh DPPH free radical-scavenging activitywhich is com- hat obtained from the shrimp waste [45]. The IC50 value 1736 N. Fakhfakh et al. / Process Biochemistry 46 (2011) 1731–1737 25 50 75 100 en gi ng a ct iv ity (% ) BHA Fig. 4. Antiox activity (a) and of FPH prod protein hyd assay [46]. ferent conc value increa by severalw FPH, with h free radical potential of time. In fact antioxidant thermore, w be able to p natural ant tate or asco were studie important t they functio the immun efficiency o 5. Conclus In the pr ried out by obtained un pH 10.0 and activity usi power. Inte potential. T high amoun a very high feathers. Th tein and na aquaculture being. Furth food of rats tion. The ox FPH should Acknowled This stud istry of High References [1] Papadopoulos MC, El Boushy AR, Roodbeen AE, Ketelaars EH. Effects of processing time and moisture content on amino acid composition and nitro- gen characteristics of feather meal. Animal Feed Science and Technology 1986;14: pta R, overvi tos R ergillu drich Dorato 3;21: u P, atinol hophy logy 2 atova erizati inomy logy 1 dDG, ion of 9;100 torns rged c nani ductio olving y 200 ai P, A atinol robio led Ha atin b –94. hfakh lecula ther-d 9;55: ar R ial pa TCC91 nt. Ap lliams enta 1;70: nca H estion ional bs BF bioact earch rado J ion of d Che ko RE rolys by N acity anaka k prot 4;86: 0 a b 3,532,521,510,50 Concentration (mg/ml) Sc av FPH 0 0,5 1 1,5 2 2,5 3,532,521,510,50 Concentration (mg/ml) A bs or ba nc e at 7 00 n idant activity of feather protein hydrolysate (FPH). DPPH-scavenging reducing power (b). uced by A1 strain (0.3mg/ml) was lower than that of rolysate from smooth hound (0.6mg/ml) using DPPH The reducing power of the FPH was investigated at dif- entrations and was to be concentration dependent. Its sedwith thehigher FPHconcentrationsaswas reported orks [45–47]. The reducing power results revealed that igh amino acid and peptide contents, could react with s to form stable products. This interesting antioxidant feather protein hydrolysate was reported for the first , the FPHcouldbeusedas anatural sourceofprotein and s for animal nutrition (fish or domestic animals). Fur- hen FPH was used in animal feed formulations, it can rotect feed against oxidation. The effects of different ioxidants, such as rosemary extract, ascorbyl palmi- rbic acid, in fish feed made from marine raw materials d [22].Moreover, natural antioxidantswere found to be o animal health by enhancing its immunity [24]. Indeed, n to remove harmful free radicals and to strengthened e system. Consequently, lower mortality and increased f livestock production could be reached. ion esent study, feather degradation was successfully car- fermentation of the strain B. pumilus A1. The FPH der optimum conditions (50g/l chicken feathers, 45 ◦C, 2 days of fermentation) was assessed for antioxidant [2] Gu an [3] San Asp [4] Frie of 200 [5] Anb ker Tric bio [6] Ign act act bio [7] Sye cat 200 [8] Sun me [9] Ram pro inv istr [10] Pill ker Mic [11] Ou ker 687 [12] Fak mo fea 200 [13] Kum ent (M me [14] Wi ferm 199 [15] Bla dig nat [16] Gib of Res [17] Par rat Foo [18] Alu hyd [19] Cum cap [20] Sak yol 200 ng DPPH free radical-scavenging activity and reducing restingly, FPH was found to possess good antioxidant he characterization of the FPH showed that it contained ts of peptides and amino acids. Moreover, it presents in vitro digestibility compared to that of the untreated e use of FPH in fish feed formulations as source of pro- tural antioxidants would be an advantage both for the industry andwith regard to consumer health andwell- er works should be done to incorporate the FPH in the to study its effect on growth and in vivo lipid peroxida- idative stability of an animal diet formulated using the also be investigated. gement y was carried out with financial support from the Min- er Education and Scientific Research – Tunisia. [21] Saiga A, T porcinem Food Che [22] Hamre K, raw mate [23] Bohne VJ thetic ant Food and [24] Chew BP mals. Ani [25] Fakhfakh metallok feather m istry and [26] Miller JH Cold spri [27] Fakhfakh tical expe pumilusA Biochem [28] Kembhav protease nology 19 279–90. Ramnani P. Microbial keratinases and their prospective applications: ew. Applied Microbiology and Biotechnology 2006;70:21–33. MDB, Firmino AA, de Sá CM, Felix CR. 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Total solubilisation of the chicken feathers by fermentation with a keratinolytic bacterium, Bacillus pumilus A1, and the production of protein hydrolysate with high antioxidative activity 1 Introduction 2 Materials and methods 2.1 Feathers 2.2 Bacterial strain 2.3 Culture media and growth conditions 2.4 Caseinolytic activity 2.5 Analytical methods 2.6 Antioxidant activity 2.6.1 DPPH radical-scavenging assay 2.6.2 Reducing power assay 2.7 Inprotect protect unhbox voidb@x penalty @M {}vitro digestibility 3 Results 3.1 Production of feather protein hydrolysates 3.1.1 Effect of feather concentration 3.1.2 Effect of initial medium pH 3.1.3 Effect of cultivation temperature 3.2 Biochemical and physical characterization of FPH 3.3 Antioxidant activity of feather protein hydrolysate 3.3.1 DPPH free radical-scavenging activity 3.3.2 Reducing power 4 Discussion 5 Conclusion Acknowledgement References