In vitro pepsin digestibility and amino acid composition ... › 33408 › 1 › pey175.pdfsion (Zhang et al., 2014) or other processes where the pressure decreases suddenly (Ferrer

In vitro pepsin digestibility and amino acid composition in soluble andresidual fractions of hydrolyzed chicken feathers

S. A. Adler,∗,1 R. Slizyte,† K. Honkapää,‡ and A-K. Løes§

∗NIBIO-Norwegian Institute of Bioeconomy Research, P.O. Box 115, 1431 Ås, Norway; †SINTEF Ocean, 7465Trondheim, Norway; ‡VTT Technical Research Centre of Finland Ltd, P.O. Box 1000, 02044 VTT, Finland; and

§Norwegian Centre for Organic Agriculture, 6630 Tingvoll, Norway

ABSTRACT Beta-keratin in poultry feathers is astructural protein that is resistant to degradation dueto disulfide and hydrogen bonds. Feather meal can bea valuable feed compound if the digestibility can be in-creased. The objective of the present study was to an-alyze the effects of chemical, enzymatic, and pressure-thermic treatments for chicken feathers on solubility,in vitro protein digestibility (IVPD), and amino acidcomposition of solubilized and residual fractions. Twoexperiments were conducted. In experiment 1, mod-els for solubility and IVPD were developed includingthe above factors applying a central composite face-centered design. Addition of sodium hydroxide (NaOH)and sodium sulfite (Na2SO3), and autoclaving time af-fected solubility and IVPD of the feather hydrolysates,but not addition of keratinolytic enzyme. In experi-ment 2, 7 combinations of the hydrolysis factors NaOH,Na2SO3, and autoclaving time with a predicted IVPDof 900 g/kg of DM, calculated for the sum of solu-bilized and residual feather fractions, were includedto measure effects on IVPD and amino acid composi-

tion in each fraction. The IVPD values were higher forsolubilized than residual fractions when treated withNaOH and autoclaving, but no differences were foundwhen treated with Na2SO3 and autoclaving. Losses ofcystine were substantial for all treatments, but lowerfor Na2SO3 than for NaOH. Furthermore, use of lowerNa2SO3 concentration and longer autoclaving time re-duced losses of cystine. Compared with NaOH treat-ments, Na2SO3 gave lower losses of threonine, arginine,serine, and tyrosine. With reference to the ideal proteinprofile for Atlantic salmon (Salmo salar L.), the treat-ments with 60 or 90 min autoclaving and 0.36 or 0.21%Na2SO3 had the highest chemical scores. The scoreswere generally higher for amino acids in residual thansolubilized fractions, but with 90 min autoclaving and0.21% Na2SO3 differences were small. In conclusion, hy-drolysis of chicken feathers with low concentrations ofNa2SO3 combined with autoclaving results in feathermeal with high nutritional value for Atlantic salmon;separation of solubilized and residual fractions is notnecessary.

Key words: beta-keratin, keratinolytic protease, sodium hydroxide, sodium sulfite, Atlantic salmon2018 Poultry Science 0:1–15

http://dx.doi.org/10.3382/ps/pey175

INTRODUCTION

Poultry feathers are animal by-products with a highprotein content. Approximately 900 g/kg of the featherdry matter (DM) consists of beta-keratin, a structureprotein rich in the essential amino acids (AA) leucine,valine, arginine, isoleucine, phenylalanine, and threo-nine, but with smaller proportions of lysine, methio-nine, histidine, and tryptophan (Yokote et al., 2007;

C© The Author(s) 2018. Published by Oxford University Press onbehalf of Poultry Science Association. This is an Open Access articledistributed under the terms of the Creative Commons AttributionNon-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribu-tion, and reproduction in any medium, provided the originalwork is properly cited. For commercial re-use, please [email protected].

Received December 19, 2017.Accepted April 19, 2018.1Corresponding author: [email protected]

Bandegan et al., 2010). The sulfur-containing cysteineand methionine (Glem-Hansen, 1980), together withthreonine, tyrosine, and phenylalanine, are importantfor the synthesis of hair and feather keratin; arginineplays an important role in the urea cycle of cats (Mor-ris and Rogers, 1978). However, a surplus of AA suchas valine and isoleucine in feed can be toxic for someanimals. In some countries, feather meal is used as afeed component for animals such as pigs, pets, fish,poultry, and ruminants, but due to the unbalanced AAcomposition, feather meal can only be a complemen-tary feedstuff in diets for monogastrics (Papadopouloset al., 1986) and supplementation with lysine, histi-dine, and other AA may be required. Because of disul-fide bonds, hydrogen bonds, and hydrophobic interac-tions between AA, feather keratin is insoluble in wa-ter and has a low digestibility with enzymes such aspepsin (Papadopoulos et al., 1986). The digestibilityof feathers can be improved and AA made biologically

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2 ADLER ET AL.

available through cleaving the bonds by pressure-thermic treatments, chemical hydrolysis, and steam ex-plosion (Zhang et al., 2014), and by action of ker-atinolytic microorganisms and keratinolytic enzymes(Gupta et al., 2013; Lasekan et al., 2013). Degradationof keratin, as a result of pressure-thermic treatment,which is typically applied in commercial production offeather meal, is often accompanied by a decrease in cys-tine content and processing may also decrease the di-gestibility or availability of AA in general (Moritz andLatshaw, 2001). A growing volume of studies suggestsimproved digestibility, lower AA losses, and decreasedenergy requirements for cooking if enzymatic hydrolysisis included in the process (Gupta and Ramnani, 2006;Pedersen et al., 2012). Applying more gentle treatmentsto feathers has a potential to decrease losses of valuableAA and increase the digestibility of feather meal.

According to EU regulations, hydrolyzed proteins de-rived from parts of non-ruminants such as feather mealmay be used as feed for non-ruminant farmed animalsand aquaculture animals (EC, 2013). In 2013, 109 mil-lion tonnes of poultry meat were produced worldwide(FAOSTAT, 2017). Assuming a meat yield of 460 g/kgbody weight and a feather proportion 75 g/kg of bodyweight (Owens et al., 2001) in the live birds, the esti-mated potential yield of poultry feathers accounted for17.8 million tonnes worldwide in 2013. However, onlya small proportion of poultry feather are processed tofeather meal in Europe and the USA, about 175,000and 600,000 tonnes of feather meal are producedannually, respectively (Swisher, 2012). Feather mealproduced by pressure-thermic treatment has a typicalprotein content of over 800 g/kg of DM with a largevariation in true AA digestibility depending on the pro-cessing method (Wang and Parsons, 1997). In the studyof Moritz and Latshaw (2001), increased pressure from2.1 to 5.2 bar for 36 min at 149◦C increased in vitropepsin (0.2%) digestible protein from 704 to 938 g/kg,but true available AA content, determined in WhiteLeghorn cockerels, was reduced for most AA. In thesame study, the content of cystine decreased with in-creased pressure during cooking and was converted tolanthionine, whereas other AA were less affected.

Efforts have been made in testing alternative treat-ments to increase digestibility without diminishing ef-fects on content and availability of essential AA such ascystine (Moritz and Latshaw, 2001). Chemical hydrol-ysis has been studied with sodium hydroxide (NaOH),potassium hydroxide (KOH) (Mokrejs et al., 2011),or calcium hydroxide (Ca(OH)2) (Coward-Kelly et al.,2006), sodium sulfite (Na2SO3), and phosphoric acid(H3PO4) (Steiner et al., 1983). Furthermore, enzymatichydrolysis with commercial proteases or supernatantsof keratinolytic microorganisms (Tiwary and Gupta,2012), microbial fermentation (Elmayergi and Smith,1971), and physical extractions, by steam-flash explo-sion (Zhang et al., 2014) or other processes where thepressure decreases suddenly (Ferrer et al., 1999) havebeen tested. In most research aiming to increase the

digestibility of chicken feathers, the focus has been onutilizing hydrolyzed feathers without separating solu-bilized and residual fractions (Steiner et al., 1983; Pa-padopoulos et al., 1986; Moritz and Latshaw, 2001;Grazziotin et al., 2006; Mukesh Kumar et al., 2012;Zhang et al., 2014). Others have analyzed either solu-bilized (Coward-Kelly et al., 2006) or residual feathers(Kim et al., 2002; �Laba and Szczeka�la, 2013). To ourknowledge, little attention has been paid to the differ-ent characteristics of solubilized and residual fractionsof feather hydrolysates.

The objective of the present study was to mea-sure and analyze the effects of combinations of chem-ical, enzymatic, and pressure-thermic treatments forchicken feathers on solubility, in vitro pepsin digestibil-ity (IVPD), and AA composition of solubilized andresidual fractions, and to evaluate the usability as feedfor Atlantic salmon (Salmo salar L.).

MATERIALS AND METHODS

Two experiments were conducted to study the effectsof hydrolysis treatments on IVPD and AA composi-tion of solubilized and residual feather fractions. Exper-iment 1 was designed to model the effects of chemical,enzymatic, and pressure-thermic hydrolysis on solubil-ity and IVPD. Sodium hydroxide and Na2SO3 were se-lected as chemical agents, and 2 commercial enzymeswere tested. Experiment 2 was designed to verify themodel, which was established from data achieved inexperiment 1, and to study effects on IVPD and AAcomposition in a series of treatments predicted to pro-duce constant IVPD values for the sum of solubilizedand residual fractions of hydrolyzed feathers (hereafterreferred to as total IVPD).

Data Availability: All relevant raw data arewithin the paper and its supporting information files(http://dx.doi.org/10.17632/p62xptkt4j.1).

Materials

Feathers from white broiler chicken (Gallus gallus do-mesticus, breed Ross 308) were collected at a slaughter-house in Eidsberg municipality in Norway (Nortura SA,Oslo, Norway). Chicken slaughtered at Nortura are typ-ically 50 d old at slaughtering and feathers are removedmechanically, transported in a water bath, and collectedafter the water is removed mechanically. Feathers werestored frozen (–20◦C) until experimental use. In exper-iment 1, the feathers were washed (by hand in tap wa-ter), sterilized in an autoclave (2 bar, 121◦C, 15 min),dried (at 45◦C for 48 h), and kept frozen (–20◦C). Inexperiment 2, the feathers were washed, dried, and keptfrozen. Prior to experiment 1, the feathers were milledin an ultrafine friction grinder (MKCA6–2, MasukoSangyo Co. Ltd, Japan). The grinder was equipped withMKC type stainless steel fillings. During grinding thefeathers were fed into the hopper and forced through a


http://dx.doi.org/10.17632/p62xptkt4j.1

FRACTIONS OF HYDROLYZED CHICKEN FEATHERS 3

Table 1. Coded, actual, and observed levels in the central composite design matrix of experiment 1 including 4 factors and 3 levelsof each factor applied to study second-order response surfaces on pH, DM solubility, and IVPD.

Coded level Actual level Observed level

IVPD

Run χ1 χ2 χ3 χ4NaOH, %(v/v)

Na2SO3,%(v/v)

Enzyme, %(v/v)1

AC time,min2 pH

DMsolubility,g/kg of DM

Solubilizedfraction,g/kg of DM

Residualfraction,g/kg of DM

Total,g/kg ofDM

1 –1 –1 –1 –1 0 0 0 0 6.3 30 735 158 1692 1 –1 –1 –1 0.5 0 0 0 12.4 577 850 395 6543 –1 1 –1 –1 0 0.25 0 0 7.4 62 846 294 3284 1 1 –1 –1 0.5 0.25 0 0 12.5 588 877 384 6795 –1 –1 1 –1 0 0 1 0 7.0 40 808 215 2326 1 –1 1 –1 0.5 0 1 0 12.4 650 860 446 7107 –1 1 1 –1 0 0.25 1 0 7.4 121 853 258 3318 1 1 1 –1 0.5 0.25 1 0 12.5 597 887 389 6939 –1 –1 –1 1 0 0 0 120 6.8 136 963 658 69210 1 –1 –1 1 0.5 0 0 120 9.9 953 920 632 88911 –1 1 –1 1 0 0.25 0 120 7.0 230 939 890 89912 1 1 –1 1 0.5 0.25 0 120 9.9 993 922 682 92513 –1 –1 1 1 0 0 1 120 6.8 138 970 751 75814 1 –1 1 1 0.5 0 1 120 9.9 960 919 678 89715 –1 1 1 1 0 0.25 1 120 7.2 271 949 875 89416 1 1 1 1 0.5 0.25 1 120 9.9 1024 915 630 94717 –1 0 0 0 0 0.125 0.5 60 6.9 142 919 841 83418 1 0 0 0 0.5 0.125 0.5 60 10.2 971 899 618 88619 0 –1 0 0 0.25 0 0.5 60 9.7 755 961 695 87720 0 1 0 0 0.25 0.25 0.5 60 9.8 860 947 724 91621 0 0 –1 0 0.25 0.125 0 60 9.7 800 945 705 88922 0 0 1 0 0.25 0.125 1 60 9.8 820 947 686 89723 0 0 0 –1 0.25 0.125 0.5 0 12.0 208 820 286 39424 0 0 0 1 0.25 0.125 0.5 120 9.5 872 941 735 90125 0 0 0 0 0.25 0.125 0.5 60 9.8 819 940 693 89026 0 0 0 0 0.25 0.125 0.5 60 9.7 809 952 699 89427 0 0 0 0 0.25 0.125 0.5 60 9.8 821 927 729 878N-5 0 0 1 0 6.1 56 842 156 189N-13 0 0 1 120 6.6 121 993 639 658N-16 0.5 0.25 1 120 10.0 992 913 642 915N-25 0.25 0.125 0.5 60 9.7 785 940 709 869

1Cibenza IDN900 in runs 1 to 27; NovoProD in runs N-5 to N-25; runs N-5 to N-25 were not used in modeling.2AC = autoclaving, 2.4 bar, 133◦C.IVPD, in vitro protein digestibility.

gap between rotary and stator grinding plates. Featherswere ground successively with gap widths of 7, 2, and1 mm. Dry matter content of the milled feathers was947 g/kg. In experiment 2, whole feathers with a DMcontent of 957 g/kg and a fat content of 22.6 g/kg ofDM were used. These feathers were not autoclaved orground in order to have a more realistic approach. Com-mercial feather meal, GoldMehl FM (GePro, Diepholz,Germany) was used as a reference in the digestibil-ity studies. Two commercial enzymes, both describedby the producers to be efficient in hydrolyzing feath-ers, were compared in experiment 1. Cibenza IDN900was kindly donated by Novus International, Inc. (St.Charles, MO). The product contains sodium sulfate,dried Bacillus licheniformis fermentation solubles, min-eral oil, and natural flavor. The producer stated a min-imum enzyme activity of 1.1 mkat/g. NovoProD waskindly donated by Novozymes, Bagsværd, Denmark.This product is a non-specific protease, and containssubtilisin initially obtained from B. subtilis. We foundan activity of 12.6 μkat/g at pH 7.5 with casein. Na2SO3was obtained from BDH Prolabo (VWR International,Pty Ltd., Tingalpa, Australia) and porcine pepsin (ac-

tivity 167 μkat/g with casein) was obtained from Sigma(Sigma-Aldrich Co. LLC., St. Louis, MO).

Design of Experiment 1

Response surface methodology was used to map theeffects of a set of hydrolysis factor levels (NaOH,Na2SO3, enzymes, pressure-thermic treatment time) onsolubility and IVPD of the products. A central compos-ite face-centered design with 4 factors and 3 levels ofeach factor was applied to study second-order responsesurfaces (Table 1). A total of 27 runs were conductedin 2 replicates. The measured effects included solubil-ity, IVPD in solubilized and residual fractions, and totalIVPD was calculated. The treatments were performedon 3 subsequent days, where day 1 comprised the runswith boiling for 30 min, day 2 autoclaving (2.4 bar,133◦C) for 60 min, and day 3 autoclaving (2.4 bar,133◦C) for 120 min.

Chemical Hydrolysis Twenty grams of milledfeathers were placed in 1,000 mL plastic bottles andmixed with pre-heated (80◦C) stock solutions of NaOH,Na2SO3, and de-ionized water adding up to 400 mL


4 ADLER ET AL.

according to the experimental plan. The bottles wereplaced in a water bath heated to 80◦C for 60 min. Dur-ing the treatment, the temperature in the bottles var-ied between 71 and 75◦C. Thereafter, the bottles werechilled in an ice bath to 55 to 58◦C to prepare for theenzymatic treatment step.

Enzymatic Hydrolysis Cibenza IDN900 or Novo-ProD were dissolved in water with continuous steer-ing overnight to a stock solution of 2%. The enzymesolution and de-ionized water were added to the bot-tles, resulting in enzyme concentrations of 0, 0.5, and1.0% (w enzyme/w feather). The bottles were continu-ously agitated in a shaker (INFORS HT Ecotron, InforsAG, Bottmingen, Switzerland) at 55◦C with 100 rpmfor 60 min, before heating or autoclaving.

Pressure-thermic Hydrolysis In the treatmentswithout autoclaving, enzyme activity was stopped byheating the bottles in boiling water for 30 min. After10 min the temperature in the bottles was 70◦C. Auto-claving was conducted in a steam sterilizer (GE 6610,Getinge Sterilization AB, Gothenburg, Sweden) for 60or 120 min at 133◦C and 2.4 bar.

After heating or autoclaving, the bottles were chilledin an ice bath prior to separation of solubilized andresidual fractions by centrifugation. Four of the runswith Cibenza IDN900 (runs 5, 13, 16, 25) were alsoconducted with NovoProD (runs N-5, N-13, N-16, N-25) to compare the effects of these enzymes.

Design of Experiment 2

The model for total IVPD achieved in experiment1 was used to design treatments with predicted totalIVPD of 900 g/kg of DM (Table 2). We aimed to de-velop feather meals with high digestibility comparedto commercial feather meals. However, maximizing thetotal IVPD implies a risk of degrading AA. Solubility,IVPD, and concentrations of N, C, Na, S, and ash weremeasured in solubilized and residual material with theaim to detect the fraction with highest feed value.

Adding enzyme had no effect on solubility or di-gestibility in experiment 1 and was therefore not in-cluded in experiment 2. Thirty grams of unmilled andunsterilized feathers were placed in 1,000 mL plasticbottles and mixed with pre-heated (80◦C) stock solu-tions of NaOH, Na2SO3, and de-ionized water addingup to 400 mL. The bottles were placed in a water bathheated to 85◦C for 60 min. After the chemical treat-ment, the bottles were autoclaved for 30, 60, or 90 minat 133◦C and 2.4 bar and thereafter chilled in an icebath to 40 to 45◦C before centrifugation and separa-tion.

Separation of Solubilized and ResidualFractions

In both experiments, solubilized and residual frac-tions were separated by centrifugation (Sorvall RC

Table 2. Hydrolysis treatments in experiment 2 with a predictedtotal IVPD of 900 g/kg of DM (n = 2).

Treatment NaOH, % Na2SO3, % AC time, min1

1 0.528 0.000 302 0.343 0.000 603 0.274 0.000 904 0.000 0.693 305 0.000 0.357 606 0.000 0.210 907 0.210 0.219 60

1Autoclaving at 2.4 bar, 133◦C.IVPD, in vitro protein digestibility.

12BP, Thermo Fisher Scientific Inc., Waltham, MA;3,963 × g for 15 min, ambient temperature) and the liq-uid phase was poured into separate containers. The pHin the liquid fraction was measured (Knick pH-Meter766, Calimatic, Knick Elektronische Messgeräte GmbH& Co. KG, Berlin, Germany) and both fractions werefreeze dried (Christ Epsilon 2–25 DS, Martin ChristGefriertrocknungsanlagen GmbH, Osterode am Harz,Germany) and weighed. The solubilized fraction wascrushed with a spoon and stored in tight plastic bags at–20◦C until analysis. The residual fraction was groundthrough a 0.5-mm screen (Fritsch Pulverisette 14, Idar-Oberstein, Germany) and stored in tight plastic bagsat –20◦C until analysis. Separation of soluble and non-soluble fractions is denoted fractionation in the follow-ing.

Analytical Measurements

In vitro pepsin digestibility was analyzed accordingto the AOAC method 971.09 (AOAC International,2012) with some modifications. Briefly, 2 parallels of0.5 g were incubated in a 2 mg pepsin/mL solution for16 h at 40◦C in 2 M hydrochloric acid (HCl). After di-gestion, samples were centrifuged (3,230 × g for 20 min)and separated. Both fractions (solubilized and resid-ual) were then dried at 55◦C overnight. The remainingmoisture content in the samples was determined gravi-metrically after drying at 105◦C until constant weightof samples was achieved (typically 24 h). Ash contentwas determined after heating dry samples at 590◦C for12 h. Total N and C were determined by CHNS-O el-emental combustion system (ECS 4010, Costech Ana-lytical Technologies Inc., Valencia, CA) in 4 parallels(experiment 1 only). Concentrations of Na were ana-lyzed by inductively coupled plasma atomic emissionspectrophotometry after dry ashing according to theAOAC method 999.11, and S by inductively coupledplasma atomic emission spectrophotometry after mi-crowave oven digestion under pressure according to theAOAC method 991.10 (AOAC International, 2012). Fatcontent was analyzed by acid hydrolysis (Soxtec Sys-tem, Foss Analytical, Denmark). The AA concentra-tions in freeze-dried ground samples were analyzed bya high-performance liquid chromatography system (Ag-ilent Infinity 1260, Agilent Technologies, Santa Clara,



CA) coupled to an online post-column derivatizationmodule (Pinnacle PCX, Pickering laboratories, Moun-tain View, CA), using nynhydrin (Trione) as a deriva-tizing reagent and a Na+ exchange column (4.6 ×110 mm, 5 μm). Amounts of AA, taurine, and am-monium (NH4+) were quantified from standard curves.Prior to the analysis, the samples were hydrolyzed in6 M hydrochloric acid containing 0.4% merkaptoethanolfor 24 h at 110◦C. Glutamine and asparagine wereconverted to glutamic and aspartic acid. Cysteine wasquantified in its dipeptide form, cystine, but cysteineand its oxidation products could not be detected. Thesamples were filtered by microfilter, the pH was ad-justed to 2.2, and the samples were further dilutedwith a citrate buffer (pH 2.2) for the high-performanceliquid chromatography analysis. Tryptophan was ana-lyzed, but the method was not optimized for that AA.

Calculations

Dry matter solubility was calculated as DM yield ofsolubilized feather hydrolysates divided by the initialsample weight (DM). For calculation of N and C solu-bility, the weights were multiplied with the concentra-tions of N or C. The IVPD was calculated as weight ofthe initial sample subtracted the weight of the resid-ual fraction after dissolution in pepsin and HCl, di-vided by weight of the initial feather sample. TotalIVPD was calculated as sum of IVPD for the solubi-lized and the residual fractions from feather hydrolysis,weighted by the proportions of solubilized and resid-ual fractions measured in the hydrolysis step. Recoveryof a specific AA was calculated as: Yield of solubilizedfeather hydrolysates multiplied with concentration ofthe AA in the solubilized fraction added the yield ofresidual feather hydrolysates multiplied with the con-centration of the AA in the residual fraction, dividedby initial feather weight multiplied with the concentra-tion of total AA. Dietary protein quality was assessedby determining its chemical score, i.e., the ratio of thelimiting AA in the tested feed (g/16 g of N) divided bythe AA in ideal protein for the specific animal (g/16 gof N), multiplied with 100.

Statistical Analysis

In experiment 1, the results were modeled using mul-tiple linear regression procedures of the MODDE statis-tical software (version 11.0.1, Umetrics AB, Ume̊a, Swe-den). The replicated center-point experiments (runs 25,26, and 27) were used to estimate the replicative error.For each measured characteristic (solubility, pH, IVPDof solubilized and residual fraction), and calculated to-tal IVPD, a complete model with all linear, interaction,and quadratic terms was first developed. Then, statisti-cally insignificant terms were removed to maximize thelevel of prediction (Q2) and the goodness of fit (R2).The model was considered good if Q2 > 0.5 and the

difference between R2 and Q2 < 0.2 to 0.3 (Eriksson etal., 2008). The quadratic model of the system is pre-sented in Eq. 1:

y = α0 + α1χ1 + α2χ2 + α3χ3 + α4χ4 + α12χ1χ2

+α13χ1χ3 + α14χ1χ4 + α23χ2χ3 + α24χ2χ4

+α34χ3χ4 + α11χ12 + α22χ22 + α33χ32 + α44χ42

(1)

where y is the predicted response; α0 is a constant co-efficient (intercept); α1, α2, α3, and α4 are linear effects;α12, α13, α14, α23, α24, and α34 are interaction effects;and α11, α22, α33, and α44 are quadratic effects, whereasχ1, χ2, χ3, and χ4 are the independent variables NaOHconcentration (%), Na2SO3 concentration (%), enzymeconcentration (%), and autoclaving duration (min).

In experiment 2, solubility, total IVPD, and recov-ery of AA (Eq. 2), and chemical composition and pro-portions of AA (Eq. 3) were analyzed using the mixedmodel procedure in SAS (SAS Institute Inc., 2016).

yij = μ + αi + εij (2)

where y was the individual dependent variable(n = 14); μ was the average of all observations; α wasthe fixed effect of treatment (i = 1 to 7); and εij wasthe random residual error, assumed to be independentand N(0, σ2).

yijk = μ + αi + βj + (αβ)ij + εijk (3)

where y was the individual dependent variable(n = 28); μ was the average of all observations; α wasthe fixed effect of treatment (i = 1 to 7); β was thefixed effect of fraction separation (i = 1, 2; where 1= solubilized fraction, 2 = residual fraction); (αβ) wasthe interaction of the fixed effects; and εij was the ran-dom residual error, assumed to be independent and N(0, σ2).

Statistical significance of differences between meanswas tested with the Tukey–Kramer test (P < 0.05).

RESULTS

Experiment 1

Dry matter solubility and pH of feathers were af-fected by NaOH and autoclaving time (Table 3). En-zyme or Na2SO3 had no effect. The enzyme activity ofCibenza IDN900 measured with casein in a 10-min in-cubation at 30◦C gave 170 nkat/g at pH 7.5, 310 nkat/gat pH 9.0, and 220 nkat/g at pH 12.8, which was lowcompared to a minimum of 1.1 mkat/g stated by theproducer.

The IVPD of the solubilized and residual fractions,and the total IVPD were affected by NaOH, Na2SO3,


6 ADLER ET AL.

Table 3. Effects of the hydrolysis factors NaOH and Na2SO3 concentrations, proteolytic enzyme (E)1 concentration, and autoclaving(AC) time on chicken feather solubility, pH, and IVPD expressed as corresponding unscaled coefficients in the models for the selectedresponses in experiment 1.

IVPD

Factor Dry matter solubility pH Solubilized fraction Residual fraction Total

Constant 2.234 7.169 78.60 17.58 19.54E2 NS3 NS NS NS NSNaOH 240.9 22.26 28.61 44.15 91.76Na2SO3 NS NS –17.61 52.11 57.91AC 0.7393 –0.02668 0.3179 1.107 1.194NaOH × NaOH –258.7 –23.24 –35.45 NS NSNa2SO3 × Na2SO3 NS NS 148.1 NS NSAC × AC –0.004942 0.0002076 –0.001404 –0.005286 –0.006066NaOH × Na2SO3 NS NS NS –120.1 –101.2NaOH × AC 0.4158 –0.04117 –0.1568 –0.5173 –0.5253Na2SO3 × AC NS NS –0.2139 NS NSR2 0.956 0.979 0.924 0.962 0.974Q2 0.907 0.958 0.810 0.932 0.957

1Cibenza IDN900.2Effects of E × E, E × NaOH, E × Na2SO3 and E × AC had effect on any of the variables.3Not significant effects, P > 0.05.IVPD, in vitro protein digestibility.

Figure 1. Contour plot for pH (a) and dry matter solubility (%) (b) in chicken feather hydrolysates as affected by NaOH concentration andautoclaving time.

and autoclaving time. The contour plots illustrate theeffects on pH, DM solubility, and IVPD (Figures 1 to3). Application of the enzyme Cibenza IDN900 had noeffect on DM solubility, pH, or IVPD of solubilized,residual, or combined fractions. NovoProD resulted inlow values for DM solubility and IVPD similar to thoseof Cibenza IDN900. Consequently, the enzymatic treat-ment step was excluded from the models.

Total IVPD increased with increasing concentrationsof both NaOH and Na2SO3. For autoclaving time themodel indicated increase in total IVPD between 80 and100 min, but a rapid reduction of IVPD for longer au-toclaving times. None of the studied factors alone wasenough to achieve a total IVPD of 900 g/kg of DM,but the model indicates that this could be achieved bycombining the factors NaOH, Na2SO3, and autoclav-

ing time. The predicted solubility varied significantlyamong the treatments with a predicted total IVPD of900 g/kg of DM. The model for total IVPD was usedto design experiment 2.

Experiment 2

The average total IVPD across all treatments was863 g/kg of DM (SEM 16.8, P = 0.18), which wasslightly lower than predicted, and considerably higherthan the IVPD of the untreated feathers and the com-mercial feather meal (Tables 4 and 5). The solubilityof DM was considerably higher (P


Figure 2. Contour plot for in vitro pepsin digestibility (%) in solubilized (a) and residual (b) chicken feather fractions as affected byconcentrations of NaOH, Na2SO3 and autoclaving time.

N (P

8 ADLER ET AL.

Figure 3. Contour plot for total in vitro pepsin digestibility (solubilized and residual fractions combined) (%) of chicken feather hydrolysatesas affected by concentrations of NaOH, Na2SO3 and autoclaving time.

Table 4. Chemical composition, solubility of N, C, and DM for hydrolysis of chicken feathers, and sum of IVPD in solubilized andresidual feather fractions in experiment 2 (n = 2).

Treatment 1 2 3 4 5 6 7 SEM P-value

AC time, min1 30 60 90 30 60 90 60NaOH, % 0.53 0.34 0.27 0.00 0.00 0.00 0.21Na2SO3, % 0.00 0.00 0.00 0.69 0.36 0.21 0.22

Solubility, g/kg of DMDM 903a 815b 735c 238e 194f 162f 620d 5.6

FRACTIONS OF HYDROLYZED CHICKEN FEATHERS 9Tab

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erci

alfe

athe

rm

eal,

and

solu

biliz

edan

dre

sidu

alfe

athe

rfr

acti

ons

inex

peri

men

t2

(n=

2).

Tre

atm

ent

Feat

hers

1Fe

athe

rm

eal2

12

34

56

7SE

MP

-val

ue3

AC

tim

e,m

in4

––

3060

9030

6090

60H

2F

3H

×F

NaO

H,%

––

0.53

0.34

0.27

0.00

0.00

0.00

0.21

Na 2

SO3,

%–

–0.

000.

000.

000.

690.

360.

210.

22

Frac

tion

–S

RS

RS

RS

RS

RS

RS

R

Item

,g/

kgof

DM

IVP

D15

654

986

5a,b

673e

903a

,b70

4e93

8a74

6c,d

,e85

8a,b

868a

,b82

9b,c

,d88

0a,b

838a

,b,c

878a

,b86

0a,b

732e

,d18

.1<

0.00

1<

0.00

1<

0.00

1N

151

128

131c

,d95

h13

5b,c

,d11

9e,f

139a

,b,c

,d13

2c,d

83i

142a

,b,c

104h

,g14

6a,b

112f

,g14

8a13

0d,e

140a

,b,c

,d2.

1<

0.00

1<

0.00

1<

0.00

1C

495

461

470c

,d51

4a,b

456d

529a

481b

,c,d

511a

,b30

5f50

3a,b

,c36

5e51

9a37

5e50

7a,b

449d

499a

,b,c

6.3

<0.

001

<0.

001

<0.

001

Na

<0.

250

0.74

651

.2c

27.1

f35

.1e

14.2

g30

.2e,

f9.

6h,g

,i11

6.1a

12.6

h,g

69.1

b7.

6h,i

52.3

c3.

9i42

.2d

13.8

h,g

1.12

<0.

001

<0.

001

<0.

001

S18

.914

.920

.0e

12.8

e20

.2e

15.2

e21

.0e

15.8

e11

0.8a

21.8

e83

.4b

20.2

e72

.1c

17.7

e34

.8d

18.4

e1.

79<

0.00

1<

0.00

1<

0.00

1A

sh5.

813

.528

5a,b

103d

,e21

0b,c

,d57

e20

8b,c

,d35

e36

4a43

e21

3b,c

,d23

e17

1c,d

13e

248b

,c40

e16

.8<

0.00

1<

0.00

10.

005

a-iM

eans

wit

hin

aro

ww

ith

differ

ent

supe

rscr

ipts

differ

(Tuk

ey-K

ram

erte

st,P

<0.

05).

1 >U

ntre

ated

cut

feat

hers

.2 C

omm

erci

alfe

athe

rm

eal,

Gol

dMeh

lFM

.,G

ePro

,D

ieph

olz,

Ger

man

y.3 H

=ef

fect

ofhy

drol

ysis

trea

tmen

t,F

=ef

fect

offr

acti

onat

ion,

H×

F=

inte

ract

ion.

4 Aut

ocla

ving

at2.

4ba

r,13

3◦C

.IV

PD

,in

vitr

opr

otei

ndi

gest

ibili

ty.

The chemical scores showed that lysine and histidinewere the first and second limiting essential AA in com-bined soluble and residual fractions, assessed as feed forAtlantic salmon in the growth interval from 1.4 to 2.8 kgbody weight (Table 8). For treatments 5 and 6 only ly-sine and histidine had scores below 100, which was alsothe case for the commercial feather meal. For the treat-ments 3 to 5 AA had a chemical score below 100. Thechemical scores of the separated fractions showed thatthe residual fractions of treatment 5 and 6 had only 2scores below 100; however, the solubilized fractions oftreatments 6 and 7 had chemical scores close to 100for tryptophan and are therefore similar to the residualfractions of treatments 5 and 6 (Table 9).

DISCUSSION

Enzyme Treatment

Novus International claims that Cibenza IND900 im-proves nutritional value of feather meal, lowers heatrequirements of the rendering process, and supportsfeather meal profitability and environmental sustain-ability (NOVUS International, 2013). However, in thepresent study the enzyme did not affect DM solubilityor IVPD when used alone or in combination with othertreatments. A possible explanation may be the low ac-tivity of the enzyme measured in casein. It is not clearwhy the activity was low.

Positive effects of proteolytic enzymes have beenclaimed in several studies. Papadopoulos (1986) re-ported positive effect on IVPD of proteolytic en-zyme treatment (Maxatase, Gist-Brodcades NV, Delft)of feathers hydrolyzed by pressure-thermic treatment.However, adding NH4+ to maintain pH at 8.5 may haveconfounded the effects of the enzyme in that study.Mokrejs et al. (2011) found that increasing enzyme(Savinase, EC 3.4.21. 62, Ultra 16 L, Novozymes A/SBagsvaerd, Denmark) concentration from 1 to 5% ina 2-stage hydrolysis of degreased feathers, using 0.3%potassium hydroxide and enzyme treatment (62◦C for4 h), increased feather solubilization from about 705 to785 g/kg. Kim et al. (2002) found that INSTA-PROenzyme (INSTA-PRO International, Des Moines, IA50,322) treatment for 24 h after NaOH treatment (1.0 Nfor 2 h at 37◦C) increased N solubility, but not IVPD.This enzyme product includes B. subtilis fermentationextract and Na2SO3 that may both have contributedto the observed solubilization. In other studies, super-natants from keratinolytic bacteria or bacteria cultureshave been used, which may contain mixture of severalenzymes. Grazziotin et al. (2006) used supernatants orwhole culture of the keratinolytic bacterium Vibrio sp.strain kr2 to hydrolyze autoclaved and hammer milledfeathers. After cultivation, the whole culture and thesupernatants were autoclaved. Supernatant culture hy-drolysate (985 g/kg) had higher IVPD than whole cul-ture hydrolysate (834 g/kg) and a commercial feathermeal (578 g/kg). It is not clear if the autoclaving after


10 ADLER ET AL.

Table 6. Amino acid (AA) composition and NH4+ concentration in chicken feathers, commercial feather meal, and chicken featherhydrolysates (solubilized and residual fractions combined) in experiment 2 (n = 2).

Treatment Feathers1 Feather meal2 1 2 3 4 5 6 7 SEM P-value

AC time, min3 – – 30 60 90 30 60 90 60NaOH, % – – 0.53 0.34 0.27 0.00 0.00 0.00 0.21Na2SO3, % – – 0.00 0.00 0.00 0.69 0.36 0.21 0.22

AA, g/kg of total AAEssential AAHis 7.5 8.1 8.5 9.1 8.0 8.6 8.9 7.6 9.2 0.58 0.54Lys 23.4 20.2 22.4 21.0 17.8 20.6 19.8 18.2 18.0 1.30 0.24Leu 87.5 86.4 97.0a 92.3b 91.6b 84.1c 84.3c 83.4c 93.5a,b 0.74


Tab

le7.

Am

ino

acid

(AA

)co

mpo

siti

onan

dN

H4+

conc

entr

atio

nin

solu

biliz

edan

dre

sidu

alfe

athe

rfr

acti

ons

inex

peri

men

t2

(n=

2).

Tre

atm

ent

12

34

56

7SE

MP

-val

ue1

AC

tim

e,m

in2

3060

9030

6090

60H

2F

3H

×F

NaO

H,%

0.53

0.34

0.27

0.00

0.00

0.00

0.21

Na 2

SO3,

%0.

000.

000.

000.

690.

360.

210.

22

Frac

tion

SR

SR

SR

SR

SR

SR

SR

AA

,g/

kgof

tota

lA

AE

ssen

tial

AA

His

8.4c

,d,e

11.3

abc

9.0b

,c,d

,e9.

4a,b

,c,d

,e8.

1c,d

,e7.

8d,e

11.9

a,b

8.0c

,d,e

12.5

a8.

3c,d

,e11

.8a,

b7.

1e10

.9a,

b,c,

d6.

5e0.

600.

02<

0.00

1<

0.00

1Lys

21.7

c,d,

e38

.9a

19.5

c,d,

e30

.7b

16.0

e,f

23.8

c8.

2g22

.6c,

d11

.0f,g

21.3

c,d,

e12

.0f,g

19.0

c,d,

e16

.6d,

e,f

20.2

c,d,

e1.

08<

0.00

1<

0.00

1<

0.00

1Leu

97.5

a86

.1b

93.4

a85

.3b

93.3

a85

.9b

85.0

b84

.0b

84.9

b84

.2b

87.7

b82

.8b

97.7

b86

.8b

0.89

<0.

001

<0.

001

<0.

001

Ile

56.4

a,b,

c,d

48.3

e54

.6a,

b,c,

d51

.3d,

e54

.4a,

b,c,

d51

.1d,

e57

.1a,

b51

.6c,

d,e

56.9

a,b,

c51

.9b,

c,d,

e58

.3a

51.7

b,c,

d,e

57.6

a53

.7a,

b,c,

d0.

940.

03<

0.00

10.

18M

et7.

1c,d

10.3

a,b,

c7.

8b,c

,d7.

5b,c

,d6.

4c,d

5.6d

12.7

a8.

0b,c

,d12

.4a

6.0d

11.2

a,b

5.6d

8.2b

,c,d

5.8d

0.73

0.00

1<

0.00

1<

0.00

1P

he57

.0a

49.8

c,d

54.5

a,b

48.6

c,d

54.1

a,b

50.5

b,c,

d50

.4b,

c,d

48.7

c,d

52.5

b,c

48.5

c,d

51.3

b,c,

d47

.9d

57.8

a50

.6b,

c,d

0.79

<0.

001

<0.

001

0.02

Trp

8.6a

,b10

.0a,

b8.

5a,b

5.9a

,b6.

2a,b

7.1a

,b12

.3a

8.7a

,b7.

7a,b

8.2a

,b7.

2a,b

8.1a

,b7.

0a,b

5.5b

1.18

0.03

0.37

0.24

Val

92.1

a84

.8a,

b85

.8a,

b83

.2b,

c84

.4a,

b82

.8b,

c71

.0e

79.8

b,c,

d71

.8e,

d78

.7b,

c,d,

e75

.6c,

d,e

80.5

b,c

86.4

a,b

86.9

a,b

1.43

<0.

001

0.09

70.

001

Thr

19.9

d23

.0c,

d23

.4b,

c,d

25.8

a,b,

c,d

23.3

b,c,

d31

.4a,

b,c

27.0

a,b,

c,d

31.6

a,b,

c26

.3a,

b,c,

d31

.1a,

b,c

31.8

a,b

32.2

a26

.0a,

b,c,

d32

.0a,

b1.

53<

0.00

1<

0.00

10.

3N

on-e

ssen

tial

Asx

(Asn

+A

sp)

74.2

a,b

70.3

a,b,

c,d,

e71

.6a,

b,c,

d61

.4d,

e,f

75.4

a63

.3c,

d,e,

f73

.2a,

b,c

63.3

c,d,

e,f

80.8

a60

.4e,

f75

.6a

59.6

f64

.8b,

c,d,

e,f62

.1d,

e,f

1.86

0.00

8<

0.00

10.

004

Glx

(Glu

+G

ln)

127.

5b,c

148.

2a12

0.4b

,c,d

,e12

9.7b

120.

7b,c

,d,e

124.

0b,c

,d11

6.3c

,d,e

120.

3b,c

,d,e

127.

4b,c

115.

1d,e

,f11

9.1b

,c,d

,e11

1.8e

,f10

3.5f

125.

4b,c

,d2.

07<

0.00

1<

0.00

1<

0.00

1A

rg42

.8e

48.5

d,e

56.5

c,d

66.7

a,b

59.7

b,c

70.9

a70

.5a

69.0

a,b

67.8

a,b

71.4

a65

.5a,

b,c

74.0

a65

.0a,

b,c

69.3

a,b

1.71

<0.

001

<0.

001

0.03

Ala

64.3

a,b

57.8

b63

.1b

58.1

b62

.5b

53.5

b57

.1b

52.5

b55

.0b

55.1

b63

.7a,

b52

.3b

77.0

a52

.3b

2.43

0.01

<0.

001

0.00

6C

ys3

7.3d

8.5c

,d10

.0b,

c,d

14.1

b,c,

d9.

7b,c

,d7.

8c,d

10.4

b,c,

d10

.7b,

c,d

7.3d

21.3

a,b,

c17

.1b,

c,d

31.3

a22

.7a,

b8.

6c,d

2.42

<0.

001

0.07

<0.

001

Gly

98.3

a82

.7a,

b92

.2a,

b78

.4a,

b92

.1a,

b80

.7a,

b81

.3a,

b74

.8b

81.2

a,b

80.6

a,b

72.6

b84

.3a,

b76

.2b

79.2

a,b

3.83

0.03

0.04

0.03

Hyl

3.7a

3.9a

2.3a

,b1.

3a,b

1.7a

,b2.

3a,b

0.0b

0.0b

0.0b

0.0b

0.0b

0.0b

1.7a

,b1.

6a,b

0.54

0.00

10.

910.

84P

ro10

.6a,

b11

.0a

10.2

a,b

11.0

a10

.4a,

b10

.2a,

b10

.2a,

b9.

8a,b

10.2

a,b

9.6a

,b10

.5a,

b9.

2b10

.8a

10.3

a,b

0.26

0.01

0.08

0.02

Ser

71.5

f64

.2f

92.0

d,e

88.6

e10

1.7c

,d10

4.6c

133.

6a12

4.8a

,b12

4.3a

,b12

2.2b

120.

0b12

0.6b

93.5

d,e

107.

8c1.

94<

0.00

10.

55<

0.00

1Tau

0.0b

0.0b

0.0b

0.0b

0.0b

0.0b

7.8a

0.0b

2.3b

0.0b

0.0b

0.0b

0.0b

0.0b

0.59

<0.

001

<0.

001

<0.

001

Tyr

22.6

c,d

33.5

a21

.8d

29.1

a,b,

c21

.9d

29.1

a,b,

c30

.2a,

b31

.9a,

b30

.3a,

b29

.8a,

b26

.3b,

c,d

28.3

a,b,

c,d

26.3

b,c,

d26

.5a,

b,c,

d1.

250.

003

<0.

001

0.00

4Tot

alA

A,g/

kgof

DM

776e

,d54

1h,i

828c

,d67

6e,f,

g83

1c,d

810c

,d51

3i91

4a,b

,c61

4g,h

950a

,b66

5f,g

1,00

4a75

3d,e

,f85

1b,c

,d19

.7<

0.00

1<

0.00

1<

0.00

1N

H4+

,g/

kgof

DM

17.0

b11

.4f,g

16.0

b11

.4f,g

15.5

b,c

12.5

e,f

9.9g

12.5

e,f

14.3

c,d

13.2

d,e

19.4

a13

.6d,

e14

.2c,

d13

.3e,

d0.

29<

0.00

1<

0.00

1<

0.00

1

a-iM

eans

wit

hin

aro

ww

ith

differ

ent

supe

rscr

ipts

differ

(Tuk

ey-K

ram

erte

st,P

<0.

05).

1H

=ef

fect

ofhy

drol

ysis

trea

tmen

t,F

=ef

fect

offr

acti

onat

ion,

H×

F=

inte

ract

ion.

2 Aut

ocla

ving

at2.

4ba

r,13

3◦C

.3 A

naly

zed

asdi

pept

ide

cyst

ine.


12 ADLER ET AL.

Table 8. Chemical score of essential AA in chicken feathers, commercial feather meal, and chicken feather hydrolysates (solubilizedand residual fractions combined) in ideal proteins for Atlantic salmon (Salmo salar L.) in experiment 2 (n = 2).

Treatment Feather1 Feather meal2 1 2 3 4 5 6 7 SEM P-valueAC time, min3 - - 30 60 90 30 60 90 60NaOH, % - - 0.53 0.34 0.27 0.00 0.00 0.00 0.21Na2SO3, % - - 0.00 0.00 0.00 0.69 0.36 0.21 0.22

Chemical score4Lys 45 41 40 38 32 40 39 37 32 1.9 0.09Met+Cys5 287 198 52c 67b,c 56c 75b,c 102b 145a 87b,c 6.6


Tab

le9.

Che

mic

alsc

ore

ofes

sent

ialA

Ain

chic

ken

feat

her

hydr

olys

ates

inso

lubi

lized

(S)

and

resi

dual

(R)

frac

tion

sin

idea

lpro

tein

sfo

rA

tlan

tic

salm

on(S

alm

osa

lar

L.)

inex

peri

men

t2

(n=

2).

Tre

atm

ent

12

34

56

7SE

MP

-val

ue1

AC

tim

e,m

in2

3060

9030

6090

60H

FH

×F

NaO

H,%

0.53

0.34

0.27

0.00

0.00

0.00

0.21

Na 2

SO3,

%0.

000.

000.

000.

690.

360.

210.

22

Frac

tion

SR

SR

SR

SR

SR

SR

SR

Che

mic

alsc

ore3

Lys

39c

67a

36c,

d53

b29

d,e

44b,

c15

f44

b,c

20e,

f42

c22

e,f

39c

29d,

e37

c,d

1.7

<0.

001

<0.

001

<0.

001

Met

+C

ys4

52c

64b,

c66

b,c

74b,

c59

b,c

50c

86b,

c73

b,c

73b,

c10

7a,b

102a

,b,c

151a

108a

,b53

c9.

2<

0.00

10.

430.

002

Thr

85e

94e

104c

,d,e

105b

,c,d

,e10

0d,e

138a

,b,c

,d11

9a,b

,c,d

,e14

5a,b

116a

,b,c

,d,e

145a

,b,c

136a

,b,c

,d15

7a10

8b,c

,d,e

140a

,b,c

,d7.

3<

0.00

1<

0.00

10.

24Trp

118a

,b13

1a,b

122a

,b78

b86

a,b

102a

,b17

4a13

0a,b

111a

,b12

3a,b

99a,

b12

7a,b

93a,

b77

a,b

16.9

0.02

0.56

0.18

Val

358a

316a

,b34

6a,b

310a

,b33

1a,b

332a

,b28

7b33

6a,b

290b

337a

,b29

5a,b

359a

328a

,b34

7a,b

11.5

0.20

0.03

0.00

2Ile

240a

,b19

7c24

1a,b

209b

,c23

4a,b

,c22

5a,b

,c25

2a23

8a,b

252a

243a

,b24

9a25

2a23

9a,b

235a

,b,c

6.9

0.00

30.

001

0.06

Leu

232a

196b

231a

195b

225a

,b21

2a,b

211a

,b21

7a,b

210a

,b22

1a,b

210a

,b22

6a,b

228a

,b21

3a,b

5.8

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14 ADLER ET AL.

However, in vivo measurements are necessary for a morereliable evaluation of the digestibility. Na and S fromthe added salts contributed heavily to the ash contentin the hydrolysates. With regard to Na+ and ash con-centrations the residual fractions are preferred as a feedcomponent. Higher Na concentration can be easily ac-cepted in salmon feed compared with feed for terres-trial animals, but increased salt content will in any caselower the feed value due to dilution of the feed.

CONCLUSIONS

The present study confirmed that NaOH, Na2SO3,and autoclaving time affect solubility, IVPD, and AAcomposition in chicken feather hydrolysates. Under theconditions in experiment 1, the tested proteolytic en-zymes alone or in combination with the above treat-ments did not have any effects on these response vari-ables. A total IVPD of 900 g/kg of DM can be achievedby different combinations of NaOH, Na2SO3, and au-toclaving. Adding low concentrations of Na2SO3 com-bined with an autoclaving time of 60 to 90 min results inhydrolysates with high IVPD and low losses of AA. Forthis hydrolysis treatment, lysine and histidine are theonly limiting essential AA compared to ideal protein forAtlantic salmon. Separation of solubilized and residualfractions is most likely not feasible due to small differ-ences between the fractions in chemical scores for themost relevant treatments (treatments 5 and 6) studiedhere. A limiting factor for the use of hydrolyzed chickenfeathers as a feed component is the high concentrationof Na+ and ash. Fractionation can be used in treat-ments with low solubility to keep ash concentration ofthe residual fraction low. If fractionation is not an op-tion, treatments resulting in slightly lower IVPD maybe considered to further improve AA composition andlower salt content.

SUPPLEMENTARY DATA

Supplementary data are available at Poultry Scienceonline.

Supplementary Table S1. Apparent recovery of AAand NH4+ in chicken feather hydrolysates in the sumof solubilized and residual fractions in experiment 2(n = 2)

ACKNOWLEDGMENTS

This work was funded by The Research Coun-cil of Norway as part of the research project “To-tal utilization of raw materials in the supply chainfor food with a bio-economical perspective (CYCLE)”(http://cycleweb.no/) [225349/E40, 2013]. The au-thors thank Nortura SA for supplying raw materials.The authors thank Riitta Alander (VTT) and PanuLahtinen (VTT) for skillful technical assistance andfeather millings.

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