in vitro pepsin digestibility and amino acid composition ... › 33408 › 1 › pey175.pdfsion...
Post on 30-Jan-2021
1 Views
Preview:
TRANSCRIPT
-
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 contactjournals.permissions@oup.com.
Received December 19, 2017.Accepted April 19, 2018.1Corresponding author: steffen.adler@nibio.no
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
1
Downloaded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pey175/5001673by Norsk institutt for bioøkonomi useron 27 June 2018
mailto:steffen.adler@nibio.no
-
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
Downloaded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pey175/5001673by Norsk institutt for bioøkonomi useron 27 June 2018
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
Downloaded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pey175/5001673by Norsk institutt for bioøkonomi useron 27 June 2018
-
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,
Downloaded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pey175/5001673by Norsk institutt for bioøkonomi useron 27 June 2018
-
FRACTIONS OF HYDROLYZED CHICKEN FEATHERS 5
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,
Downloaded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pey175/5001673by Norsk institutt for bioøkonomi useron 27 June 2018
-
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
-
FRACTIONS OF HYDROLYZED CHICKEN FEATHERS 7
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
le5.
Invi
tro
peps
indi
gest
ibili
tyan
dch
emic
alco
mpo
siti
onin
feat
hers
,co
mm
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
Downloaded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pey175/5001673by Norsk institutt for bioøkonomi useron 27 June 2018
-
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
-
FRACTIONS OF HYDROLYZED CHICKEN FEATHERS 11
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.
Downloaded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pey175/5001673by Norsk institutt for bioøkonomi useron 27 June 2018
-
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
-
FRACTIONS OF HYDROLYZED CHICKEN FEATHERS 13
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
0.84
0.00
90.
002
Phe
+T
yr12
913
012
912
112
513
413
614
214
014
012
714
213
412
94.
90.
080.
340.
32H
is45
d,e
59a,
b,c,
d51
b,c,
d,e
49c,
d,e
44d,
e43
d,e
67a,
b47
c,d,
e71
a49
b,c,
d,e
64a,
b,c
44d,
e57
a,b,
c,d
36e
3.1
0.00
2<
0.00
1<
0.00
1A
rg87
g95
g11
9f13
0e,f,
d12
3f14
9b,c
,d14
9b,c
,d15
2b,c
143b
,c,d
,e16
0a,b
134c
,d,e
,f17
3a12
9e,f
145b
,c,d
,e3.
4<
0.00
1<
0.00
10.
003
a-g
Mea
nsw
ithi
na
row
wit
hdi
ffer
ent
supe
rscr
ipts
differ
(Tuk
ey-K
ram
erte
st,P
<0.
05).
1 H=
effe
ctof
hydr
olys
istr
eatm
ent,
F=
effe
ctof
frac
tion
atio
n,H
×F
=in
tera
ctio
n.2 A
utoc
lavi
ngat
2.4
bar,
133◦
C.
3 Che
mic
alsc
ores
=A
Ain
test
feed
[g/1
6g
N]/
AA
inid
ealpr
otei
n[g
/16
gN
]×
100.
Ava
lue
of10
0in
dica
tes
that
the
leve
lof
apa
rtic
ular
AA
wit
hin
the
feed
prot
ein
isid
enti
calto
the
diet
ary
AA
requ
irem
ent
leve
lfo
rA
tlan
tic
salm
on(1
.4to
2.8
kgbo
dyw
eigh
t;R
ollin
etal
.,20
03).
The
low
est
valu
ein
each
colu
mn
indi
cate
sth
efir
stlim
itin
gA
A.
4 Ana
lyze
das
dipe
ptid
ecy
stin
e.A
A,am
ino
acid
.
Downloaded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pey175/5001673by Norsk institutt for bioøkonomi useron 27 June 2018
-
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.
REFERENCES
AOAC International. 2012. Official methods of analysis of AOACInternational. Gaithersburg, MD, USA.
Bandegan, A., E. Kiarie, R. L. Payne, G. H. Crow, W. Guenter, andC. M. Nyachoti. 2010. Standardized ileal amino acid digestibilityin dry-extruded expelled soybean meal, extruded canola seed-pea,feather meal, and poultry by-product meal for broiler chickens.Poult. Sci. 89:2626–2633.
Chojnacka, K ., H. Górecka, I. Michalak, and H. Górecki. 2011. Areview: valorization of keratinous materials. Waste Biomass Valor2:317–321.
Coward-Kelly, G., V. S. Chang, F. K. Agbogbo, and M. T. Holtzap-ple. 2006. Lime treatment of keratinous materials for the genera-tion of highly digestible animal feed: 1. Chicken feathers. Biore-sour. Technol. 97:1337–1343.
EC. 2013. Commission Regulation (EC) 56/2013 of 16 January 2013amending Annexes I and IV to Regulation (EC) 999/2001 of theEuropean Parliament and of the Council laying down rules forthe prevention, control and eradication of certain transmissiblespongiform enc. EU.
Elmayergi, H. H., and R. E. Smith. 1971.Influence of growth of Strep-tomyces fradiae on pepsin-HCl digestibility and methionine con-tent of feather meal. Can. J. Microbiol. 17:1067–1072.
Eriksson, L., E. Johansson, N. Kettaneh-Wold, C. Wikström, and S.Wold. 2008. Design of Experiments: Principles and Applications.3rd ed. Umetrics AB, Ume̊a, Sweden.
FAOSTAT. 2017. No Title. Food Agric. Organ. UnitedNations, Rome, Italy. Accessed May 1, 2017.http://faostat3.fao.org/faostat-gateway/go/to/home/E
Ferrer, A., F. M. Byers, B. S. de Ferrer, B. E. Dale, and S.C. Ricke. 1999. Increasing nutrient availability of feather mealfor ruminants and non-ruminants using an ammonia pressuri-sation/depressurisation process. J. Sci. Food Agric. 79:828–832.
Florence, T. M. 1980. Degradation of protein disulphide bonds indilute alkali. Biochem. J. 189:507–520.
Glem-Hansen, N. 1980. The requirements for sulphur containingamino acids of mink during the growth period. Acta Agric. Scand.30:349–356.
Grazziotin, A., F. A. Pimentel, E. V. de Jong, and A. Brandelli.2006. Nutritional improvement of feather protein by treatmentwith microbial keratinase. Anim. Feed Sci. Technol. 126:135–144.
Gupta, R., R. Rajput, R. Sharma, and N. Gupta. 2013. Biotechnolog-ical applications and prospective market of microbial keratinases.Appl. Microbiol. Biotechnol. 97:9931–9940.
Gupta, R., and P. Ramnani. 2006. Microbial keratinases and theirprospective applications: an overview. Appl. Microbiol. Biotech-nol. 70:21–33.
Kim, W. K. K., E. S. S. Lorenz, and P. H. H. Patterson. 2002. Effectof enzymatic and chemical treatments on feather solubility anddigestibility. Poult. Sci. 81:95–98.
�Laba, W., and K. B. Szczeka�la. 2013. Keratinolytic proteases inbiodegradation of pretreated feathers. Polish J. Environ. Stud.22:1101–1109.
Lange, L., Y. Huang, and P. K. Busk. 2016. Microbial decompositionof keratin in nature—a new hypothesis of industrial relevance.Appl. Microbiol. Biotechnol. 100:2083–2096.
Lasekan, A., F. Abu Bakar, and D. Hashim. 2013. Potential ofchicken by-products as sources of useful biological resources.Waste Manage. 33:552–565.
MEROPS. 2017. Families of proteolytic enzymes. May 1,2017. http://merops.sanger.ac.uk/cgi-bin/family index?type=P.Accessed May 1, 2017.
Mokrejs, P., P. Svoboda, J. Hrncirik, D. Janacova, and V.Vasek. 2011. Processing poultry feathers into keratin hy-drolysate through alkaline-enzymatic hydrolysis. Waste Manag.Res. 29:260–267.
Moritz, J. S., and J. D. Latshaw. 2001. Indicators of nutritional valueof hydrolyzed feather meal. Poult. Sci. 80:79–86.
Morris, J. G., and Q. R. Rogers. 1978. Arginine: an essential aminoacid for the cat. J. Nutr. 108:1944–1953.
Mukesh Kumar, D. J., S. Lavanya, P. Priya, A. Immaculate NancyRebecca, M. D. Balakumaran, and P. T. Kalaichelvan. 2012.
Downloaded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pey175/5001673by Norsk institutt for bioøkonomi useron 27 June 2018
https://academic.oup.com/ps/article-lookup/doi/10.3382/ps/pey175#supplementary-datahttp://cycleweb.no/http://faostat3.fao.org/faostat-gateway/go/to/home/Ehttp://merops.sanger.ac.uk/cgi-bin/family_index?type=P
-
FRACTIONS OF HYDROLYZED CHICKEN FEATHERS 15
Production of feather protein concentrate from feathers by invitro enzymatic treatment, its biochemical characterization andantioxidant nature. Middle-East J. Sci. Res. 11:881–886.
NOVUS International. 2013. CIBENZA IND900, datasheet.:2 https://na38.salesforce.com/sfc/p/#E0000000Xz5h/a/E0000000LAYN/XLWNUGhoPmIjn5dqb1bmeECfiSWdvRTaPOICpeL5KUQ. Accessed May 1, 2017.
Owens, C. M., C. Alvarado, and A. R. Sams. 2001. Poultry MeatProcessing. AR Sams, ed. CRC Press, Boca Raton, FL.
Papadopoulos, M. C. 1986. The effect of enzymatic treatment onamino acid content and nitrogen characteristics of feather meal.Anim. Feed Sci. Technol. 16:151–156.
Papadopoulos, M. C., A. R. El Boushy, A. E. Roodbeen, and E. H.Ketelaars. 1986. Effects of processing time and moisture contenton amino acid composition and nitrogen characteristics of feathermeal. Anim. Feed Sci. Technol. 14:279–290.
Pedersen, M. B., S. Yu, P. Plumstead, and S. Dalsgaard. 2012.Comparison of four feed proteases for improvement of nu-tritive value of poultry feather meal. J. Anim. Sci. 90:350–352.
Rawlings, N. D., A. J. Barrett, and F. Robert. 2016. Twenty years ofthe MEROPS database of proteolytic enzymes, their substratesand inhibitors. Nucleic Acids Res. 44:D343–D350.
Rollin, X., M. Mambrini, T. Abboudi, Y. Larondelle, and S. J.Kaushik. 2003. The optimum dietary indispensable amino acid
pattern for growing Atlantic salmon (Salmo salar L.) fry. Br. J.Nutr. 90:865.
SAS Institute Inc. 2016. https://www.sas.com/en us/home.html.Accessed May 1, 2017.
Steiner, R. J. R., R. O. R. Kellems, and D. C. Church. 1983. Featherand hair meals for ruminants. IV. Effects of chemical treatmentsof feathers and processing time on digestibility. J. Anim. Sci.57:495–502.
Swisher, K. 2012. Market Report: Industry savors record prices andgrowing global demand. Placerville, CA.
Thannhauser, T. W., Y. Konishi, and H. A. Scheraga. 1984. Sensi-tive quantitative analysis of disulfide bonds in polypeptides andproteins. Anal. Biochem. 138:181–188.
Tiwary, E., and R. Gupta. 2012. Rapid conversion of chicken featherto feather meal using dimeric keratinase from Bacillus licheni-formis ER-15. J. Bioproces. Biotechniq. 2:1–5.
Wang, X., and C. M. Parsons. 1997. Effect of processing systems onprotein quality of feather meals and hog hair meals. Poult. Sci.76:491–496.
Yokote, Y., Y. Kubo, R. Takahashi, T. Ikeda, K. Akahane, and M.Tsuboi. 2007. Structural details of a fowl feather elucidated byusing polarized raman microspectroscopy. BCSJ 80:1148–1156.
Zhang, Y., R. Yang, and W. Zhao. 2014. Improving digestibilityof feather meal by steam flash explosion. J. Agric. Food Chem.62:2745–2751.
Downloaded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pey175/5001673by Norsk institutt for bioøkonomi useron 27 June 2018
https://na38.salesforce.com/sfc/p/#E0000000Xz5h/a/E0000000LAYN/XLWNUGhoPmIjn5dqb1bmeECfiSWdvRTaPOICpeL5KUQhttps://na38.salesforce.com/sfc/p/#E0000000Xz5h/a/E0000000LAYN/XLWNUGhoPmIjn5dqb1bmeECfiSWdvRTaPOICpeL5KUQhttps://na38.salesforce.com/sfc/p/#E0000000Xz5h/a/E0000000LAYN/XLWNUGhoPmIjn5dqb1bmeECfiSWdvRTaPOICpeL5KUQhttps://www.sas.com/en_us/home.html
top related