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 RESEARCH PAPER New Biotechnology  Volume 31, Number 5  September 2014

Production of feather hydrolysates with

antioxidant, angiotensin-I converting
Research Paper

enzyme- and dipeptidyl peptidase-IV-

inhibitory activities
Roberta Fontoura1, Daniel J. Daroit2, Ana P.F. Correa1, Stela M.M. Meira1,
Mauricio Mosquera3 and Adriano Brandelli1
1
Laboratorio de Bioqumica e Microbiologia Aplicada, Instituto de Ciencia e Tecnologia de Alimentos (ICTA), Universidade Federal do Rio Grande do Sul (UFRGS),
Porto Alegre 91501-970, RS, Brazil
2
Universidade Federal da Fronteira Sul (UFFS), Campus Cerro Largo, Cerro Largo 97900-000, RS, Brazil
3
Instituto de Ciencia y Tecnologa de Alimentos y Nutricion (ICTAN, CSIC), C/Jose Antonio Novais, 10. Ciudad Universitaria, Madrid 28040, Spain

The antioxidant and antihypertensive activities of feather hydrolysates obtained with the bacterium
Chryseobacterium sp. kr6 were investigated. Keratin hydrolysates were produced with different
concentrations of thermally denatured feathers (1075 g l1) and initial pH values (6.09.0). Soluble
proteins accumulated in high amounts in media with 50 and 75 g l1 of feathers, reaching values of 18.5
and 22 mg ml1, respectively, after 48 hours of cultivation. In media with 50 g l1 of feathers, initial pH
had minimal effect after 48 hours. Maximal protease production was observed after 24 hours of
cultivation, and feather concentration and initial pH values showed no significant effect on enzyme
yields at this time. Feather hydrolysates displayed in vitro antioxidant properties, and optimal
antioxidant activities were observed in cultures with 50 g l1 feathers, at initial pH 8.0, after 48 hours
growth at 308C. Also, feather hydrolysates were demonstrated to inhibit the angiotesin I-converting
enzyme by 65% and dipeptidyl peptidase-IV by 44%. The bioconversion of an abundant agroindustrial
waste such as chicken feathers can be utilized as a strategy to obtain hydrolysates with antioxidant and
antihypertensive activities. Feather hydrolysates might be employed as supplements in animal feed, and
also as a potential source of bioactive molecules for feed, food and drug development.

Introduction mainly by keratins (90%, w w1). The recalcitrance of keratin-

Keratins are structural insoluble proteins found in the epidermis
and epidermal appendages of vertebrates, such as hair, wool and
feathers, presenting high stability towards common proteolytic
enzymes, chemical agents and mechanical stresses. However, the
same properties of keratin proteins that provide the essential
features for epidermis and its appendages to fulfill their biological
functions, also represents a challenge for waste management, since
enormous amounts of keratin-rich wastes are produced globally
from agroindustrial activities [1]. The increment in chicken meat
production results, concomitantly, in an increased generation of
wastes such as feathers, nails and beaks, which are composed
Corresponding author: Brandelli, A. (abrand@ufrgs.br)
rich wastes, and the energetic, economic and environmental
constraints associated with the existing management methods,
has led to an increased interest towards keratinolytic microorgan-
isms [2].
Diverse bacteria, isolated from various environments, are
shown to efficiently degrade feathers, and such a biological
process is considered as a suitable strategy to manage and add
value to keratin-rich materials. The conversion products include
keratinases, a class of proteolytic enzymes with application in
leather, feed, detergent, and biomedical industries. Also, micro-
bial biomass and feather protein hydrolysates might be
employed as nitrogen fertilizers and supplements to animal
feed [3].

www.elsevier.com/locate/nbt http://dx.doi.org/10.1016/j.nbt.2014.07.002
506 1871-6784/ 2014 Elsevier B.V. All rights reserved.
New Biotechnology  Volume 31, Number 5  September 2014
Research on natural antioxidants, particularly bioactive pep-
tides obtained through protein hydrolysis, is profusely reported.
Such peptides, encrypted in protein sequences, only demonstrate
biological activities after their release from the native protein.
Therefore, several proteins and proteolytic enzymes are investi-
gated, aiming to obtain bioactive peptides with potential utiliza-
tion in food science, technology and nutrition [4]. Also, bacterial
fermentations are reported to be beneficial for the bioactivity of
food products [5]. Recently, feather hydrolysates obtained through
microbial conversion were reported to present antioxidant activi-
ties [6].
In this context, Chryseobacterium sp. kr6 is a bacterium previ-
ously isolated from a poultry processing plant that present great
potential for feather keratin hydrolysis [7]. The aim of this work
was to investigate the production of proteolytic enzymes and
bioactive hydrolysates with antioxidant, antihypertensive and
antidiabetic potential during feather degradation by Chyseobacter-
ium sp. kr6.
Materials and methods
Feathers
Chicken feathers (CF), supplied by a local poultry industry (Avipal,
Porto Alegre, RS, Brazil), were washed threefold with tap water and
finally with distilled water. The washed feathers were dried at 458C
for 48 hours and then stored at room temperature before microbial
treatment.
Bacterial strain
Chryseobacterium sp. kr6, a feather-degrading bacterium which
produces alkaline keratinases, was isolated from feather waste
obtained at a poultry processing plant. Feathers were flooded in
5 g l1 peptone broth and after 24 hours at 308C this suspension
was used to streak feather-meal agar plates. Strain kr6 was isolated
and selected by its ability to produce clear zones (keratin hydroly-
sis) in feather-meal agar plates [7]. The strain was identified based
on morphological and biochemical tests, and 16S rDNA sequenc-
ing. Keratinolytic activity was estimated as described elsewhere
using azokeratin as substrate [7]. The strain was routinely main-
tained in Brain-Heart Infusion agar (BHA) plates.
Culture medium and growth conditions
The initial medium used for feather protein hydrolysate production
was composed of (g l1): chicken feather 50.0; KH2PO4 0.4; NaCl 0.5;
CaCl2 0.015; pH 8.0. The media were autoclaved at 1208C for
20 min. After inoculation, cultivations were conducted in 1 l Erlen-
meyer flasks, with a working volume of 100 ml, for up to 5 days at
308C in a rotary shaker (125 rev min1). To optimize the composi-
tion and conditions of the culture, the effect of feather concentra-
tion (1075 g l1) and initial pH (6.09.0) were individually
evaluated for their performance in feather protein hydrolysate
production. Aliquots of 1 ml were periodically removed, centrifuged
for 10 min at 10,000  g, and the supernatants were stored at 48C for
the determination of proteolytic activity, soluble protein and anti-
oxidant activity. All cultivations were performed in duplicate.
Proteolytic activity assay
Proteolytic activity was determined as described elsewhere [8]
using azocasein (Sigma Co., St. Louis, MO, USA) as substrate.
RESEARCH PAPER
Briefly, the reaction mixture contained 120 ml of suitably diluted
culture supernatant in 480 ml of 10 g l1 azocasein solution pre-
pared in 50 mmol l1 TrisHCl buffer (pH 8.0). The mixture was
incubated at 458C for 40 min, and the reaction was stopped by
adding 600 ml of 10% (w v1) trichloroacetic acid (TCA). After
centrifugation (10,000  g for 5 min), 800 ml of the supernatant
were mixed with 200 ml of 1.8 mol l1 NaOH, and the absorbance
was measured at 420 nm. One unit of enzyme activity was consid-
ered as the amount of enzyme that caused a change of 0.01
absorbance units under the above assay conditions.
Research Paper
Determination of soluble protein
After centrifugation (10,000  g for 15 min) culture supernatants
were utilized for determining the soluble protein concentration by
the Folin phenol reagent method [9].
Tricine SDS-PAGE of feather hydrolysates
Feather hydrolysates, obtained through cultivation of strain kr6
for 0, 1, and 2 days in feather medium (50 g l1 of feathers, initial
pH 8.0), were subjected to Tricine SDS-PAGE [10], using a 5%
stacking gel, 10% separating gel and a 16.5% resolving gel. Hydro-
lysate supernatants, with a 20 mg ml1 protein concentration,
were filtered through a 10 kDa cut-off membrane (Amicon Ultra
10k, Millipore, Ireland), and both permeates and retentates were
analyzed. Samples were mixed with loading buffer (50 mmol l1
TrisHCl, 40 g l1 SDS, 120 ml l1 glycerol, 20 ml l1 mercap-
toethanol, and 0.1 g l1 Coomassie blue G-250), heat-denatured
at 908C for 10 min, and electrophoresed in a Mini Protean II unit
(Loccus Biotecnologia). Protein bands were stained with Coomas-
sie Brilliant Blue G-250. The approximate molecular mass (MW) of
proteins in the hydrolysates was determined using a prestained
protein standard consisting of proteins between 2.5 and 250 kDa
(Benchmark Protein Ladder, Invitrogen).
Analysis of feather hydrolysates by reversed phase HPLC
For this analysis, the feather hydrolysates were initially centri-
fuged, and then filtered through a 10 kDa-cutoff membrane. The
filtrates were subjected to RP-HPLC (model E2995, Waters, Mil-
ford, USA) using a C18 column (Atlantis T3 5 mm 4.6  250 mm,
Waters, Milford, USA). The injection volume was 20 ml, and the
flow rate was 1 ml min1 using an acetonitrile gradient from 20 to
60% in 1 ml l1 trifluoroacetic acid (TFA) for 30 min.
DPPH (2,2-diphenyl-1-picrylhydrazyl) radical-scavenging assay
This method was performed as described by Brand-Williams et al.
[11], based on the capture of the DPPH radical by antioxidants,
producing a decrease in absorbance at 517 nm. The DPPH was used
at a concentration of 60 mmol l1, dissolved in methanol. The
solution was homogenized and transferred to a dark glass bottle.
The prepared solution was used only on the day of analysis. In the
dark, 1 ml of hydrolysate supernatants were transferred to test
tubes containing 3.9 ml of DPPH solution (60 mmol l1 DPPH) and
homogenized by shaking. After 45 min, the scavenging activity
was measured spectrophotometrically by the decrease in absor-
bance at 517 nm. Likewise, these sample proportions (1 ml dis-
tilled water and 3.9 ml DPPH radical solution) were used as a
control. Methanol was used as a blank. The standard curve was
performed using DPPH concentrations from 0 to 60 mmol l1.
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 RESEARCH PAPER
Results were expressed as scavenging rate (%) = [1  (A/A0)]  100,
where A is the absorbance of the test and A0 is the absorbance of
the blank.
ABTS (2,20 -azino-bis-(3-ethylbenzothiazoline)-6-sulfonic acid)
radical scavenging activity
Experiments were carried out using the ABTS assay [12], which
involves the generation of ABTS radical chromophore by the
oxidation of ABTS with potassium persulfate. This method is
applicable for both hydrophilic and lipophilic compounds. The
ABTS radical cation was produced by reacting 7 mmol l1 ABTS
Research Paper
stock solution with 140 mmol l1 potassium persulfate (final con-
centration), and allowing the mixture to stand in the dark for at
least 12 hours at room temperature before use. For the assay, the
ABTS*+ solution was diluted with 5 mmol l1 phosphate-buffered
saline (PBS, pH 7.0) to an absorbance of 0.7 (0.02) at 734 nm. A
10 ml of sample was mixed with 1 ml diluted ABTS*+ solution and
an absorbance (734 nm) reading was taken after 6 min.
Angiotensin I-converting enzyme (ACE)-inhibitory activity
Inhibition of ACE was assayed by the method of Cushman and
Cheung [13], with some modifications. Each sample aliquot
(20 ml) was mixed with 200 ml buffered substrate solution
(5 mmol l1 hippuryl-histidyl-leucine in 50 mmol l1 4-(2-hydro-
xyethyl)-1-piperazineethanesulfonic acid (HEPES)-HCl buffer con-
taining 300 mmol l1 NaCl, pH 8.3 at 378C). After adding 40 ml of
ACE, the reaction mixture was further incubated at 378C for
30 min, and finished with 150 ml of 1 mol l1 HCl. The hippuric
acid released was extracted with 1 ml ethyl acetate, and the organic
phase was transferred to a glass tube to be heat evaporated. The
residue was dissolved with 800 ml distilled water and measured
spectrophotometrically at 228 nm. The ACE-inhibitory activity
was expressed as percentage, according to the formula:
AB
% of inhibitory activity  100
AC
where A is the absorbance without sample (feather hydrolysate), B
is the absorbance without ACE, and C is absorbance in the presence
of both ACE and sample.
Dipeptidy peptidase-IV (DPP-IV) inhibitory activity
Inhibition of DPP-IV was measured in 96-well microplates using
Gly-Pro-p-nitroanylide as substrate [14]. Hydrolysate samples
(25 ml) were added of 25 ml of 1.5 mmol l1 substrate (in
100 mmol l1 Tris pH 8.0). The mixture was incubated for
10 min at 378C, followed by the addition of 50 ml of DPP-IV
(diluted in the same buffer to 0.01 U ml1). The reaction mixture
was incubated for 60 min at 378C, and the reaction was stopped by
adding 100 ml of 1 mol l1 acetate buffer pH 4.0. The absorbance of
the resulting solution was measured at 405 nm.
Results
Proteolytic activity and soluble protein release during feather
degradation
Extracellular protease production by Chryseobacterium sp. kr6 and
release of soluble proteins were investigated as a function of
cultivation time in media with different concentrations of ther-
mally denatured feathers and initial pH values. Regarding protease
activity, maximal levels were reached at 24 hours of cultivation,
508 www.elsevier.com/locate/nbt
New Biotechnology  Volume 31, Number 5  September 2014
independently of the feather content in the medium. At 24 hours,
slight differences on azocaseinolytic activities were detected; how-
ever, strain kr6 exhibited a tendency for higher enzyme produc-
tion in culture media containing 75 g l1 of chicken feathers
(Fig. 1a). The concentration of soluble proteins in the different
media is presented in Fig. 1c, and higher values
(27.2  0.6 mg ml1) were also observed in medium containing
75 g l1 of feathers, after four days of cultivation. A substantial
decline on proteolytic activity was observed as the cultivation time
increased in media containing 10 and 25 g l1 of feathers. Addi-
tionally, a decreased content of soluble protein was observed at 48
hours, gradually increasing afterwards (Fig. 1c). At 25 g l1 of
feathers, soluble protein concentration reached a plateau; and
at higher feather amounts (50 and 75 g l1 of feathers), soluble
proteins accumulated into the medium (Fig. 1c).
The effect of initial medium pH values (from 6.0 to 9.0) on the
production of proteolytic enzymes by Chryseobacterium sp. kr6 and
the content of soluble proteins were investigated in media con-
taining 50 g l1 of thermally denatured feathers. Initial pH caused
no effect on the protease yield at 24 hours of cultivation (Fig. 1b).
On the other hand, medium pH showed to affect the amount of
soluble proteins among experiments, particularly at 24 hours of
cultivation (Fig. 1d), where initial pH values of 8.0 and 9.0 showed
higher soluble protein contents (16.9 mg ml1) when compared to
cultivations at initial pH values of 6.0 and 7.0 (11.5 mg ml1).
After 48 hours, these differences were minimized and soluble
protein content at different initial pH values were around
18.5 mg ml1 (Fig. 1d).
The analysis of molecular mass distribution of feather hydro-
lysates, obtained with 50 g l1 of thermally denatured feathers and
initial pH 8.0, was performed. Each hydrolysate (obtained at 0, 1,
and 2 days of cultivation) was centrifuged, filtered through a
10 kDa-cutoff membrane, and both permeate and retentate were
submitted to Tricine SDS-PAGE (Fig. 2). No evident protein bands
were observed for feather hydrolysate permeates, suggesting that
peptide molecular masses were lower than 2.5 kDa. In the case of
retentates, six bands ranging from 21.5 to 36.5 kDa were detected
for feather hydrolysates obtained at 1 day of cultivation. Although
a similar profile was observed for retentate of hydrolysates
obtained at 2 days of cultivation, the protein bands showed a
higher intensity when compared to those at day 1 (Fig. 2), which
indicates a higher peptide content, and a higher degree of feather
hydrolysis.
Feather hydrolysates, obtained with 50 g l1 of thermally dena-
tured feathers, and initial pH 8.0, at different cultivation times,
were centrifuged and filtrated through a 10 kDa-cutoff membrane.
These permeates were then submitted to RP-HPLC, and the results
are presented in Fig. 3. Hydrolysates at time zero showed a minor
absorbance peak at around 8 min elution, which increased as the
cultivation time progressed. Also, feather hydrolysates of cultiva-
tions carried out for 48 hours displayed discrete curve shoulders at
11 and 14 min, and a distinct peak at 18 min (Fig. 3), indicating the
appearance of different peptides and also an increased intensity of
the peptide peaks as cultivation time increased.
Biological activities of feather hydrolysates
The antioxidant properties of feather hydrolysates produced by
Chryseobacterium sp. kr6 were investigated using the DPPH and the
New Biotechnology  Volume 31, Number 5  September 2014 RESEARCH PAPER

Research Paper
FIGURE 1
Protease production and release of soluble proteins during growth of Chryseobacterium sp. kr6 on feather media at 308C. (a) Azocaseinolytic activity of culture
supernatants obtained with different feather concentrations (* 10 g l1; 5 25 g l1; &50 g l1; ^ 75 g l1) and initial pH 8.0. (b) Azocaseinolytic activity on
culture supernatants obtained with 50 g l1 of feathers at different initial medium pH values (* pH 6.0; ! pH 7.0; & pH 8.0; ^ pH 9.0). (c) Soluble protein on
culture supernatants obtained with different feather concentrations and initial medium pH 8.0. (d) Soluble protein on culture supernatants obtained with 50 g l1
of feathers at different initial medium pH values.

ABTS assays. DPPH is a free radical that accepts an electron or a
hydrogen radical, becoming a stable molecule. The DPPH scav-
enging activity of feather hydrolysates peaked at 48 hours of
cultivation, reaching maximum values (83%) in cultivations with
25 g l1 and 50 g l1 of feathers. These values tended to remain
nearly unaltered (in the case of 50 and 75 g l1 of feathers) or to
decrease (in the case of 10 and 25 g l1 of feathers) at higher
cultivation periods (Fig. 4a). Particularly, the decrease in DPPH
scavenging by feather hydrolysates produced with 10 and 25 g l1
of feathers might be attributed to further hydrolysis of the peptides
responsible for the observed activity. Initial pH of feather medium
(with 50 g l1 of feathers) also affected the DPPH scavenging
activity of the feather hydrolysates. Specifically, lower scavenging
activities were observed for hydrolysates obtained at initial medi-
um pH values of 6.0 and 7.0, when compared to those obtained at
pH 8.0 and 9.0 (Fig. 4b).
In the presence of antioxidants with hydrogen-donating or
chain-breaking properties, the ABTS radical is reduced, with con-
comitant conversion to a colorless product. The ABTS scavenging
capability after 24 hours of cultivation was higher for feather
hydrolysates at initial feather concentration of 50 g l1. At 48
hours, ABTS scavenging was similar (around 90%) for hydrolysates
obtained at feather concentrations from 25 to 75 g l1 of feathers
(Fig. 4c). Conversely, antioxidant activities of feather hydrolysates
produced with 10 g l1 of feathers were lower throughout the
cultivation time. The effect of initial pH in a 50 g l1 feather
medium on the ABTS scavenging activity of feather hydrolysates
(Fig. 4d) was not as marked as that observed for the DPPH assay
(Fig. 4b). However, similarly to the latter method, higher ABTS
quenching was observed for 24-hour hydrolysates with an initial
medium pH of 8.0. A series of dilutions of supernatants from
feather hydrolysates (obtained with 50 g l1 of feathers, initial
pH 8.0) were performed to assess the protein concentration result-
ing in 50% of ABTS radical scavenging (IC50). For the 24 hour and
48 hour hydrolysates, IC50 values were 16 and 18.3 mg ml1,
respectively.
The ACE-inhibitory ability of hydrolysates obtained with
50 g l1 of feathers and initial pH 8.0 was then evaluated. At these
conditions, feather hydrolysates obtained at 24 hour and 48 hour
of cultivation displayed 53% and 65% of ACE inhibition, respec-
tively (Fig. 5). DPP-IV inhibitory activity was also measured. The
feather hydrolysates obtained at 24 and 48 hour of cultivation
caused a very similar dose-dependent inhibition of DDP-IV, reach-
ing maximum values around 4245% (Fig. 6).

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 RESEARCH PAPER
Research Paper
FIGURE 2
Molecular mass profile of hydrolysates produced by Chryseobacterium sp. kr6
during cultivation on feather medium (50 g l1 of feathers, initial pH 8.0, at
308C). Culture supernatants were filtered through a 10 kDa-cutoff membrane
before electrophoresis (Tricine SDS-PAGE). Lane 1, permeate of hydrolysate
supernatant obtained at t = 0 hour; lane 2, retentate of hydrolysate
supernatant obtained at t = 0 hour; lane 3, permeate of hydrolysate
supernatant obtained at t = 24 hours; lane 4, retentate of hydrolysate
supernatant obtained at t = 24 hours; lane 5, permeate of hydrolysate
supernatant obtained at t = 48 hours; lane 6, retentate of hydrolysate
supernatant obtained at t = 48 hours.
Discussion
The keratinolytic potential of Chryseobacterium sp. kr6 was
employed for the production of feather hydrolysates at different
substrate concentrations and initial pH. The effects of these
variables were first investigated on protease production and
soluble protein release. The soluble protein values obtained for
strain kr6 with 50 g l1 of feathers after 48 hour cultivation were
higher than those observed for several other microorganisms
grown on chicken feathers, including Bacillus species [15,16],
which are regarded as prominent feather-degrading strains and
keratinase producers [2]. These values were also superior when
compared to those obtained by Bacillus pumilus A1
(13.7 mg ml1) grown for the same period in a medium contain-
ing 50 g l1 feathers [6]. As can be observed from Fig. 1, the
kinetics of enzyme production and feather degradation do not
overlap, indicating that higher protease production occurs earlier
than soluble protein peaks.
Particularly, at 10 g l1 of feathers, the increased inaccessibility
of the insoluble growth substrate might have affected protease
production, and thus soluble protein release. It was observed that
the feather rachises remained until the 4th day of cultivation (data
not shown), corroborating a previous report in which Chryseobac-
terium sp. kr6 reached stationary growth phase after 15 hours of
growth on whole feathers medium, attacking the feather barbules
at first, and slowly degrading the feather rachises only after
36 hours of cultivation [17]. Therefore, the decrease on soluble
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New Biotechnology  Volume 31, Number 5  September 2014
FIGURE 3
RP-HPLC analyses of the <10 kDa fractions of feather hydrolysates obtained
at cultivation periods of 0 hour (), 24 hours (), and 48 hours ().
Hydrolysates were produced by Chryseobacterium sp. kr6 during growth on
feather medium (50 g l1 of feathers, initial pH 8.0, at 308C). Culture
supernatants were filtered through a 10 kDa-cutoff membrane, and the
permeates were applied into the RP-HPLC column. Elution was performed for
30 min.
proteins in cultivations with 10 g l1 of feathers could be related,
concomitantly, to the strain deficiency in exploring the feather
structures remaining in the medium, and to the consumption of
solubilized peptides for microbial biomass construction [15]. Bac-
terial strains might sequentially produce different proteolytic
enzymes targeted against increasingly inaccessible proteinaceous
growth substrates. This phenomenon, related to the decreased
nutritional quality of the medium [18], might partly explain
the increases in soluble protein in medium with 10 g l1 of feathers
at days 3 and 4. At higher feather concentrations, solubilized
proteins could have been sufficient to support microbial growth,
also suggesting that microbial activity was responsible for observed
protein surplus.
Alkaline pH values facilitate keratin degradation [3], and Chry-
seobacterium sp. kr6 was previously demonstrated to significantly
increase the medium pH during growth on raw feathers [7]. Also
considering that keratinolytic enzymes from strain kr6 are highly
active at pH values ranging from 7.4 to 9.2 [19], these factors could
explain the absence of important differences on the soluble pro-
tein content among cultivations starting with different pH values.
The molecular mass distribution of peptides resulting from
proteolysis is affected by enzyme specificity [20]. Strain kr6 was
reported to produce at four extracellular proteases during growth
on raw feather media; two of these enzymes were postulated to
possess broad substrate specificity, whereas the other two were
produced only in the presence of feathers [17]. Electrophoretic and
chromatographic profiles of feather hydrolysates indicate that the
type and abundance of peptides are affected as a function of
cultivation time. Specially, small peptides (<10 kDa) are not prop-
erly detected by Tricine SDS-PAGE [21]. Hence, feather hydroly-
sates permeates were also subjected to RP-HPLC, and higher
intensity of peaks related to peptides of increased hydrophobicity
were observed as cultivation periods increased to 48 hours. The
New Biotechnology  Volume 31, Number 5  September 2014 RESEARCH PAPER

Research Paper
FIGURE 4
Antioxidant activities in supernatants of feather hydrolysates produced by Chryseobacterium sp. kr6, as a function of cultivation time on feather media at 308C. (a)
DPPH radical scavenging activity of hydrolysates obtained with different feather concentrations (* 10 g l1; 5 25 g l1; &50 g l1; ^ 75 g l1) and initial pH 8.0.
(b) DPPH radical scavenging activity of hydrolysates obtained with 50 g l1 of feathers at different initial medium pH values (* pH 6.0; ! pH 7.0; & pH 8.0; ^ pH
9.0). (c) ABTS radical scavenging activity of hydrolysates obtained with different feather concentrations and initial medium pH 8.0. (d) ABTS radical scavenging
activity of hydrolysates obtained with 50 g l1 of feathers at different initial medium pH values.

50
DPP-IV inhibition (%)

40

30

20

10

0
0 5 10 15 20
Peptide concentration (mg/ml)

FIGURE 5
ACE-inhibitory activity of hydrolysates produced by Chryseobacterium sp. kr6
during cultivation on feather medium (50 g l1 of feathers, initial pH 8.0,
308C). Culture supernatants were employed in evaluations. Values are the
means  SEM of three independent determinations.
FIGURE 6
DPP-IV-inhibitory activity of hydrolysates produced by Chryseobacterium sp.
kr6 during cultivation on feather medium (50 g l1 of feathers, initial pH 8.0,
308C). Culture supernatants from day 0 (~), 1 (&) and 2 (*) were employed
in evaluations. Values are the means  SEM of three independent
determinations.

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 RESEARCH PAPER
presence of few predominant peaks would indicate that keratin
was hydrolyzed at specific sites, as previously suggested by Wang
et al. [22].
Feather hydrolysates might be potentially employed as supple-
ments to animal feed. These products are shown to possess higher
nutritional values in comparison to feather meal obtained
through hydrothermal processing [23], and could be employed
in feed formulations [24,25]. In this sense, the strain kr6 shows a
remarkable biotechnological potential, linking the effective pro-
cessing of feather wastes with the production of proteases and
hydrolysates with antioxidant, antihypertensive and antidiabetic
Research Paper
properties.
The antioxidant activity of feather hydrolysates was checked by
DPPH and ABTS assays, which are usual methods to evaluate the
antioxidant potential of peptides and protein hydrolysates [26].
Antioxidant properties have been associated with diverse protein
hydrolysates, and research on this field focuses on the production
of hydrolysates from food proteins (particularly caseins) with
commercial proteases [27]. However, few investigations deal with
the production of bioactive peptides from residual or waste pro-
teins [28]. Although keratinases have been employed to generate
casein hydrolysates possessing antioxidant properties [29,30], on-
ly one report describes the utilization of a microbial strain to
generate feather hydrolysates with antioxidant activities [6],
and until now, inhibition of ACE and DPP-IV was not associated
to keratin hydrolysates.
For Chryseobacterium sp. kr6, culture conditions resulting in
feather hydrolysates with optimal DPPH and ABTS scavenging
activities were set at 50 g l1 of thermally denatured feathers
and initial pH 8.0, after 48 hours of growth at 308C. At these
conditions, quenching of the ABTS radical by hydrolysates reached
approximately 90%. For comparison purposes, the ABTS scaveng-
ing activities of ovine caseinate hydrolysates, obtained after treat-
ment with enzyme preparation from Bacillus sp. P45, was 66% [29].
Fermentation with Bacillus subtilis B2 was observed to increase the
antioxidant properties (measured through ABTS and DPPH meth-
ods) of okara and soybean [5]. The DPPH scavenging by the
Chryseobacterium sp. kr6 feather hydrolysates reached approxi-
mately 83%. Ovine caseinate hydrolysates obtained with a prote-
ase preparation from Bacillus sp. P7, showed a 31% DPPH
reduction [31]; and 50% of DPPH quenching was achieved by
bovine caseinate hydrolysates [30] and feather hydrolysates
obtained with B. pumilus A1 [6].
As previously reported, distinct behavior of the ABTS and DPPH
radical scavenging assays could be expected by the different
stereoselectivity of the radicals, distinct peptides in the sample
are capable of reacting and quenching different radicals, different
stoichiometry of reactions between the antioxidant compounds in
hydrolysates and the ABTS and DPPH radicals, and also to the
solubility and diffusibility of the radicals [5,32]. Thus, a higher
activity with the ABTS method does not necessarily imply an
increased ability to scavenge DPPH radicals. In this perspective,
the ABTS method is one of the most effective and commonly used
protocols to monitor antioxidant activities [33].
The ability of feather hydrolysates to inhibit ACE and DPP-IV
activities was demonstrated for the first time in this work. ACE
performs the conversion of angiotensin I to angiotensin II (a strong
vasoconstrictor), and also degrades bradykinin (a vasodilator
512 www.elsevier.com/locate/nbt
New Biotechnology  Volume 31, Number 5  September 2014
peptide). Hence, ACE inhibitors act as potential agents for control-
ling blood pressure and lowering hypertension, with consequent
usefulness in preventing and/or treating cardiovascular diseases
[34]. In this sense, several investigations report the ACE-inhibitory
capability of milk protein hydrolysates and milk-derived peptides,
due to their potential positive impact on human health [35,36]. The
results obtained in this work (65% inhibition) are comparable to
those obtained for lactic acid bacteria during milk fermentation
[27], and also to soybean hydrolysates [37] obtained with commer-
cial enzymes. Otherwise, DPP-IV inhibitors have arise as a new class
of substances for treatment of type 2 diabetes, since this enzyme
rapidly degrade the glucagon-like peptide 1, an incretin hormone
that normalizes blood glucose concentration [38]. The DPP-IV-
inhibitory activity has been demonstrated for milk-derived protein
hydrolysates [39,40], but studies on other protein hydrolysates are
limited.
The biological activities of protein hydrolysates depend on
enzyme specificity, which determines the extent of protein hy-
drolysis and also the possible peptides to be generated from a
parent protein [41]. Usually, bioactivities of protein hydrolysates
are related to small peptides (<6 kDa) [4,36]. Protein hydrolysis
commonly results in the exposure of hydrophobic groups in the
surface of generated peptides [29] and, particularly, keratins
contain around 5060% of hydrophobic and aromatic amino acid
residues [42,43]. In this sense, aromatic and hydrophobic amino
acid residues appear to be involved in the antioxidant activities of
protein hydrolysates [4,41], and there are also indications that
hydrophobic amino acids at the three C-terminal positions influ-
ence the inhibitory action of peptides towards ACE [35]. Since in
RP-HPLC the solutes are eluted in order of increased hydropho-
bicity [44], the profiles of peptides with less than 10 kDa represent
the appearance and accumulation of peptides with higher hydro-
phobic properties (particularly at 11, 14 and 18 min; Fig. 5), that
might contribute to the observed bioactivities of feather hydro-
lysates.
In summary, Chryseobacterium sp. kr6 was successfully
employed to obtain feather hydrolysates demonstrating antioxi-
dant, DPP-IV and ACE-inhibitory activities. Considering the huge
amount of feathers generated as waste by the worldwide poultry
processing industry [1], such bioconversion strategy might be a
suitable alternative to expand the options for utilization of these
abundant and recalcitrant wastes. From a technological perspec-
tive, antioxidative properties of peptides and protein hydrolysates
might be employed in the elaboration of functional foods, and as
agents retarding the lipid peroxidation that leads to food deterio-
ration [4,26]. Particularly, as feather hydrolysates might be
employed as supplements in animal feed, their antioxidant prop-
erties could also protect feed against oxidation. These hydrolysates
and peptides might be potentially employed directly as a source of
biologically active molecules, or indirectly as a source of informa-
tion for the chemical synthesis of bioactive peptides, which might
lead to feed, food and drug development.
Acknowledgements
This work was supported by grants from the Coordenacao de
Aperfeicoamento de Pessoal de Nvel Superior (CAPES) of the
Ministry of Education from Brazil, and Conselho Nacional de
Desenvolvimento Cientfico e Tecnologico (CNPq), Brazil.
New Biotechnology  Volume 31, Number 5  September 2014
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