Sustainable and practical utilization of feather keratin by an innovative physicochemical pretreatment: High density steam flash-explosion

Abstract

Currently, great attention is being paid to the utilization of biomass, such as feather keratins. It is imperative to extract and dissolve keratins from animal keratinous materials for exploitation of innovative biopolymers. However, most of the current processes are based on strong acid and alkali hydrolysis, chemical cleavage and other violent reactions, which are not eco-friendly and/or result in severe degradation and destruction of feather keratins. In this study, high density steam flash-explosion (HDSF) as an innovative pretreatment of biomass was firstly employed to treat feather waste. In HDSF treatment, steam with a powerful seepage force first penetrates into fibrous tissues and cells of feathers, and then quickly expands and breaks free of the structure upon an explosive decompression at supersonic speed (within 0.0875 s). HDSF effectively destabilized β-sheet crystals and intermolecular disulfide bonds without causing substantial damage to the keratin protein chain, dramatically increasing the extraction and dissolubility of feather keratins in polar solvents like water, salt solution and weak bases, as well as enzymatic accessibility. HDSF treatment could be a sustainable and practical pretreatment for extraction of feather keratin for exploitation of biomaterials and conversion of feathers to nutrient animal feed instead of the current chemical hydrolysis and hydrothermal treatment.

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Sustainable and practical utilization of feather keratin by an innovative
physicochemical pretreatment: high density steam flash-explosion
Wei Zhao,* Ruijin Yang,* Yiqi Zhang and Li Wu
Received 5th August 2012, Accepted 27th September 2012
DOI: 10.1039/c2gc36243k
Currently, great attention is being paid to the utilization of biomass, such as feather keratins. It is
imperative to extract and dissolve keratins from animal keratinous materials for exploitation of innovative
biopolymers. However, most of the current processes are based on strong acid and alkali hydrolysis,
chemical cleavage and other violent reactions, which are not eco-friendly and/or result in severe
degradation and destruction of feather keratins. In this study, high density steam flash-explosion (HDSF)
as an innovative pretreatment of biomass was firstly employed to treat feather waste. In HDSF treatment,
steam with a powerful seepage force first penetrates into fibrous tissues and cells of feathers, and then
quickly expands and breaks free of the structure upon an explosive decompression at supersonic speed
(within 0.0875 s). HDSF effectively destabilized β-sheet crystals and intermolecular disulfide bonds
without causing substantial damage to the keratin protein chain, dramatically increasing the extraction and
dissolubility of feather keratins in polar solvents like water, salt solution and weak bases, as well as
enzymatic accessibility. HDSF treatment could be a sustainable and practical pretreatment for extraction
of feather keratin for exploitation of biomaterials and conversion of feathers to nutrient animal feed
instead of the current chemical hydrolysis and hydrothermal treatment.
1. Introduction
Poultry feathers are waste products generated in large quantities
from commercial poultry processing and down product manufac-
ture, which have become a crucial industrial waste over the
world. An estimated 1.5–2 million tonnes are produced annually
as a waste from the poultry industry in the United States and
over 65 million tones are produced worldwide.
1,2
Poultry feath-
ers contain about 90% protein (keratin) and are a cheap and
renewable source for protein fibers. However, feather keratin is
tightly packed in β-sheets into a supercoiled polypeptide chain
with a high degree of disulfide cross-linkages, hydrophobic inter-
actions, and hydrogen bonds. The peculiar structure of keratin
confers indissolubility, mechanical stability and resistance to
common proteolytic enzymes and chemicals to feathers.
3
Thus,
most feathers are currently disposed of as waste by incineration
or landfilling.
4
Currently, greater attention has been paid to the utilization of
biomass.
5,6
In order to effectively exploit biomass, a pretreat-
ment is generally required to destroy the structure preventing
degradation of biomass or to release the constitutive com-
ponents, thereby increasing enzyme and solvent accessibility.
6
If preferred processing techniques can be found, the insoluble
and hard-to-degrade animal keratin could be converted into
biomaterials with innovative properties, such as highly efficient
absorbent materials,
7
bioplastics and biomedical materials for
tissue engineering and regenerative medicine.
8
However, keratins
are stabilized by a large amount of intra- and intermolecular
disulfide cross-links plus other protein structural features, like
crystallinity and hydrogen-bonding. It gives keratin high strength
and stiffness, and insolubility in polar solvents, like water, weak
acids and bases, as well as in apolar solvents. Therefore, it has
become imperative to extract and dissolve keratins from animal
keratinous materials for the exploitation of innovative biopoly-
mers. Advances in the extraction and dissolution of keratins
(Table 1), led to the exponential growth of keratin materials and
their derivatives.
The chemical methods (Table 1) mostly employ strong acid
and alkali hydrolysis and chemical cleavage of the disulfide
bonds to extract soluble keratins. However, these chemicals used
in chemical methods, such as sulfites, thiols, DTT, or peroxides,
are harmful, often toxic, and difficult to handle. The physico-
chemical methods, such as hydrothermal, superheated water
treatments, are mostly based on violent reaction conditions.
Although conventional steam explosion and extrusion treatments
are feasible for pretreatment of lignocellulosic biomass, reduc-
tants such as sodium sulfite are required to synergistically
combine with these physicochemical methods for pretreatment
of keratinous materials because keratins are more robust than
carbohydrates.
21
These treatments always result in severe degra-
dation of keratin with reduction of molecular weight and loss of
mechanical properties.
21
In addition, enzymatic hydrolysis and
microbial fermentation are recently receiving considerable
State Key Laboratory of Food Science & Technology and School of
Food Science and Technology, Jiangnan University, No. 1800 Lihu
Road, Wuxi 214122, China. E-mail: zhaow@jiangnan.edu.cn,
yrj@jiangnan.edu.cn; Fax: +86 510 85919150
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attention.
23
However, the higher cost of enzymes themselves,
with a long production cycle, has so far limited the development
of industrial processes. Moreover, most keratins are converted
into much shorter oligopeptides and amino acids. Thus, research-
ers have focused on finding simple and eco-friendly processing
methods to extract and dissolve feather keratins.
High density steam flash-explosion (HDSF) is an innovative
method for pretreatment of biomass, which could be performed
on a large scale. This method is based on pressurization and
forcing steam into fibrous tissues and cells of biomass, followed
by rapidly releasing the pressure in an explosive decompression
event. This process functions as an adiabatic expansion process
and a conversion process of thermal energy into mechanical
energy.
24
HDSF is developed from conventional steam
explosion, extrusion or steam spout or swollen technologies, but
it is dramatically different from these physicochemical methods.
The comparison in detail between HDSF and other conventional
explosion technologies has been clearly illustrated in a recent
study.
24
HDSF adopts a structure in catapult explosion mode that
is principally composed of a cylinder and piston, which could
complete the explosion within 0.0875 s. The completion of
release of high density energy in an extremely short time could
provide enough force to disrupt and unfold the compact structure
of fibrous proteins and avoid a long time of violent treatment
under high temperature and pressure. Conventional steam
explosion technologies adopt the classical structure in valve
blow mode, which need at least several tens of seconds or
minutes to finish the release of high pressure steam.
24
Steam
with a powerful seepage force will penetrate the entirety of an
organic tissue structure. Given a rapid enough decompression
(HDSF), most of the steam in the biomass will quickly expand
and break free of the structure. Thus, the internal structure of the
biomass is disrupted by a mechanical shearing force. In a slow
decompression (conventional steam explosion technologies), the
pressure will have time to equalize across the structure, resulting
in a much smaller shearing force.
24
Moreover, HDSF could reduce the time of violent treatment of
biomass under high temperature and pressure. The temperature
of biomass treated by HDSF could immediately decrease to
50 °C or lower, which is also very beneficial for the pretreatment
of biomass. Yu et al.
24
employed HDSF and conventional steam
explosion with the same thermochemical effect to treat maize
stalk, demonstrating that the most prominent factor in the action
of HDSF is the kinetic energy produced by steam explosion
rather than thermochemical reaction. This is also confirmed in
our unpublished study, as HDSF treatment could effectively
improve the extraction of heat-sensitive procyanidins with high
antioxidant activity from plants. The aim of this study is to
develop a novel, simple and eco-friendly pretreatment method to
extract and dissolve feather keratin, which could reduce or
eliminates the use of hazardous substances in the utilization of
keratins.
2. Materials and methods
2.1. Materials
Dry and clean feathers from chickens were supplied by
Hangzhou Venus Biological Nutrition Co., Ltd., China. The
feathers (6.0% moisture content (w/w, wet basis), 90.2% crude
protein and 1.6% crude fat (w/w, dry basis)) were defatted by
Soxhlet extraction with petroleum ether, washed with distilled
Table 1 Methods to extract and dissolve keratins from different biomaterials
Method Biomaterial Treatment conditions Aim Result (dissolubility)
Chemical methods Wool
8
7 M urea, SDS (2%, w/v), and 2-mercaptoethanol
(5%, v/v) at 50 °C for 12 h
Biodegradational film
preparation
48%
Wool, chicken
feathers
9
2.4 M thiourea, 15 M urea, in 25 mM Tris-HCl, pH
8.5 containing 5% (v/v) DTT at 50 °C for 1–3d
Extraction 67–70%
Wool
10
8 M urea, 0.14 M DTT, 5 mM Tris, pH 8.6 at 65 °C
for 2 h
Extraction 50%
Feather
11
50 g L
−1
Na
2
S solution at 30 °C for 25 h Bio-polymer About 60%
Feather
12
A two-stage alkaline-enzymatic hydrolysis Keratin hydrolysate
Stage 1 : 0.1–0.3% KOH solution at 70 °C, for 24 h 24.08% (0.1% KOH)
Stage 2: 1–5% proteinase at 50–70 °C for 4–8 h 90.83% (0.3% KOH)
Wool fibres
13
8 M urea, 0.5 M sodium bisulphate,
SDS g/wool g = 0.6, pH = 6.5 at 65 °C for 5 h
Film preparation 33%
Feathers
14
Hydrophobic ionic liquid at 80 °C for 4 h Extraction About 21%
Silk fibroin
15
Ionic liquids at 30 °C after cooling from 100 °C Dissolution 12.24%
Wool keratin
5
Ionic liquids at 100–130 °C for over 10 h Dissolution 11%
Physicochemical
methods
Silk fibroin
16
Hydrothermal treatment at 220 °C for 60 min Protein and amino acid
preparation
23.21% of protein;
75.39% of amino acid
Silk fibroin
fibers
17
Radiation with 1 MeV electron beam, 1000 kGy
irradiation
Extraction 20% at 121 °C
Wool fiber
18
Steam explosion at 0–0.8 MPa Modification The dissolving time in
2.5% NaOH increased.
Wool
19
Steam explosion at 220 °C for 10 min Conversion 18.66%, amino acids were
seriously damaged.
Feather
20
Extrusion with sodium sulfite Bioplastic preparation 30%
Feather
21
Extrusion at 120 °C for 30 min, using a
combination of glycerol, water, and sodium sulfite
as processing aids
Extrusion Much shorter
oligopeptides
Feather
22
Superheated water at 220 °C for 120 min Hydrolysis Oligopeptides
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water and dried at 40 °C for 48 h (the degreasing procedure in
this study was for the elimination of interference in the analysis
of treated samples. It was not found to influence the treatment
efficiency and could be omitted in the practical application of
HDSF in pretreatment of feathers.). All chemicals were of
analytical grade.
2.2 High density steam flash-explosion (HDSF) treatment
The experiments were carried out in the QBS-200B HDSF
device with a 5 L chamber from Gentle Science & Technology
Co. Ltd., China. The diagram of the HDSF process is shown in
Fig. 1. The structure, working principle and calculation of para-
meters can be obtained from a recent study.
24
The difference
between the catapult explosion mode of HDSF and the valve
blow mode of conventional steam explosion has been clearly
elaborated.
24
The whole structure (Fig. 1A and B) in catapult
explosion mode is composed of a cylinder and piston. The
piston is driven by three tailored pneumatic linear actuators. The
cylinder and piston set is installed in the shadowed area of
Fig. 1A. In the steam pressurization phase, the two parts are
tightly coupled (Fig. 1C). During the explosion, the piston,
driven by three pneumatic linear actuators, bursts out of the
cylinder, a process that is equivalent to a vessel suddenly frac-
turing into two halves (Fig. 1D). In the process of opening the
piston, the piston rapidly accelerates due to the kinetic energy of
the steam and material, as well as the force from the devices
driving the process. When the stroke of the piston reaches one
quarter of the cylinder diameter, the effective gas deflation
passage –the exposed area between the piston and cylinder –
reaches the same area as the cylinder cross section.
24
The steam
was quickly forced into the chamber through the inflation inlets.
When the pressure in the chamber reached expected the value
(3–5 s), inflation inlets were closed and then the explosion was
completed within 0.0875 s. We also prolonged the time of
pressurization to 160 s (keeping the sample in the chamber
under expected pressure) to investigate the effects of the thermo-
chemical reaction. All experiments were carried out on the same
amount (1 kg) of air-dried feathers. The stream pressures were
set at 0–2.0 MPa. After HDSF treatment, samples were received
and frozen at −20 °C until the analyses.
2.3 Morphological characterization
Scanning electron microscopy (SEM) analysis was performed
with a Quanta 200 microscope (PHILIPS, Netherlands).
2.4 Dissolubility in different solvents and enzymatic
accessibility (pepsin digestibility) of feather keratins
The samples were dried in vacuum and ground to 40 mesh.
Protein solubility in several solvents: deionized water (pH 5.9),
sodium phosphate buffer (0.01 M, pH 7.5), sodium phosphate
buffer (0.01 M, pH 7.5) with 2% urea, sodium phosphate buffer
(0.01 M, pH 9.0) and 0.2% potassium hydroxide solution were
determined after a 20 min incubation at room temperature as
described previously by Araba and Dale (1990).
25
The solubility
was determined by measuring protein in the filtrate by the
Kjeldahl method. Pepsin-digestible N of each treatment was
determined by the procedure of the AOAC Official Method
971.09 (AOAC, 2000)
26
using 0.2% pepsin (activity 1 : 10 000)
in 0.075 mol L
−1
hydrochloric acid. In this assay, 1.000 g
(±0.010 g) of sample was incubated in 150 mL of pepsin solu-
tion at 45 °C for 16 h.
2.5 Amino acid analysis
The samples were hydrolyzed with hydrochloric acid
(6 mol L
−1
) at 110 °C for 24 h in sealed tubes. An Alliance
(Agilent 1100) high performance liquid chromatograph (HPLC)
was used, and the eluate was detected at 338 nm. Tryptophan
was determined separately, as described by Molnar-Perl and
Pinter-Szakacs (1989).
27
2.6 Disulfide-sulfhydryl analysis
Free thiol groups and disulfide bonds were determined by a
solid-phase assay according to Chan and Wasserman (1993).
28
Colorimetric reaction with 2-nitro-5-thiosulfobenzoate (NTSB)
was conducted under the conditions described by Thannhauser
et al. (1987)
29
with the aim of disulfide bond content determi-
nation. An extinction coefficient of 13 600 M
−1
cm
−1
was used
to calculate the number of sulfur containing groups.
2.7 Molecular weight distribution determination
The molecular weight distribution of feather keratins dissolved
in phosphate buffer (pH 7.5) with 2% urea and phosphate buffer
(pH 9.0) was assessed by gel permeation chromatography (GPC)
with a Sephadex G-50 column (1 × 100 cm). Feather keratins
Fig. 1 The diagram of the HDSF process. The whole structure (A and
B) in catapult explosion mode is composed of a cylinder and piston. The
cylinder and piston set is installed in the shadowed area of (A). In the
steam pressurization phase, the two parts are tightly coupled (C). During
the explosion, the piston, driven by three pneumatic linear actuators and
the kinetic energy of the steam and material, bursts out of the cylinder,
a process that is equivalent to a vessel suddenly fracturing into two
halves (D).
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(30 mg) extracted using phosphate buffer ( pH 7.5) with 2% urea
and phosphate buffer (pH 9.0) were loaded onto a Sephadex
G-50 gel filtration column, pre-equilibrated and eluted with the
corresponding dissolved solutions. Separation was obtained at a
flow rate of 20 mL h
−1
and eluted fractions (5.0 mL) were
pooled after spectrophotometric measurements at 280 nm. A
molecular weight calibration curve was prepared from the
average retention time of the following standards: bovine serum
albumin (66.2 kDa), ovalbumin (45.0 kDa), lactate dehydrogen-
ase (35.0 kDa), restriction endonuclease Bsp981 (25.0 kDa),
lysozyme (14.4 kDa), cytochrome C (12.5 kDa), aprotinin
(6.5 kDa), bacitracin (1450 Da).
2.8 X-ray diffraction analysis
The samples were dispersed onto a stub and placed within the
chamber of the analytical X-ray powder diffractometer (Bruker
AXS, Rheinfelden, Germany). Generator intensity was 40 kV,
generator current was 50 mA. The sample was then scanned
from 2θ=5–70°, in steps of 0.02°. The resultant graphs were
printed out on the Origin graph plotting package.
2.9 Fourier transform infrared (FTIR) spectroscopy
Infrared spectroscopy was performed using a FTIR 510 Nicolet
spectrophotometer using the diffuse reflectance technique, spec-
tral resolution 4 cm
−1
and 32 scans. The KBr pellet sampling
method was used to prepare the thin film for testing. Spectra
were baseline corrected, smoothed with a nine-point Savitzky–
Golay function. All spectra were shown after band-narrowing by
Fourier self-deconvolution (FSD) to resolve overlapping IR
bands using a half-bandwidth of 30.0 cm
−1
and enhancement
factor k= 3.0. Second derivative spectra were obtained to
support the initial identification of band positions by deconvolu-
tion. These were carried out using the OMNIC software (Nicolet
Instrument Co., Madison, WI). The band positions obtained
from the above steps were then used as the initial guess for
curve-fitting of the original spectra with Gaussian bands. For the
final fits, the positions, heights, and widths of all bands were
varied simultaneously. The curving fitting procedure was calcu-
lated on Peakfit v4.12 software (SeaSolve Software Inc.).
2.10 Statistical analyses
Each experiment was carried out at least in triplicate. All statis-
tical analyses were conducted with SAS software (Version 8.0),
and P-0.05 was used to determine statistical significance in
all tests.
3. Results and discussion
Extraction, dissolubility and enzymatic accessibility of feather
keratins after HDSF treatment
As is known, extraction and dissolubility of keratins from feath-
ers are the prerequisites for further utilization of feather keratins.
Many methods (Table 1) have been employed to directly extract
and dissolve keratins (chemical methods) or as pretreatments
(physicochemical methods) to improve the extraction and dis-
solubility of keratins. In this study, HDSF treatments were
employed to treat feathers. The exciting results could be seen
from Fig. 2. The dissolubility of feather keratins in all the sol-
vents tested in this study dramatically increased with the increase
of HDSF pressure. It was worth noting that the insoluble and
hard-to-degrade feather keratins could not dissolve in deionized
water (pH 5.9) and phosphate buffer (0.01 M, pH 7.5) at all, but
had a dissolubility of 12.9% and 22.0% after HDSF treatment at
2.0 MPa in the two solvents, respectively. This result is very
important and promising for the utilization of feather keratins,
because feathers are probably the most abundant and cheapest
keratinous materials in nature.
30
Although the dissolubility is
lower when compared with the current chemical methods in
Table 1, this makes possible the recycling of feathers using treat-
ments performed free of chemicals, only with water. As illus-
trated in Fig. 2, the dissolubility of feather keratins after 2.0 MPa
HDSF treatment dramatically increased to 65.0% from 2.0%
before HDSF treatment upon addition of 2.0% urea in phosphate
buffer (0.01 M, pH 7.5). Feather keratin primary chains are
10.2–10.4 kDa linear molecules containing a high number of
hydrophobic residues. The hydrophobic interaction is one of the
dominant stabilizers, which could be destroyed by urea. The
phosphate buffer (0.01 M, pH 9.0) also showed excellent dis-
solubility (68.0% at 2.0 MPa) for feather keratins treated by
HDSF. Compared with the severe chemical reactions in Table 1,
the phosphate buffer (0.01 M, pH 7.5) with 2% urea and phos-
phate buffer (0.01 M, pH 9.0) used in this study are milder, more
eco-friendly and efficient, indicating HDSF pretreatment could
efficiently reduce or even eliminate the use of hazardous sub-
stances to improve the extraction and dissolubility of keratins.
The protein solubility in 0.2% potassium hydroxide solution is
always the common procedure to evaluate the pretreatment of
keratins. After HDSF pretreatment, the dissolubility of feather
keratins could reach 72.8% at 2.0 MPa from only 3.0% in the
original sample. It is much higher than those of the chemical
methods in Table 1 using harsh chemicals. From the point of
Fig. 2 The extraction, dissolubility and enzymatic accessibility of
feather keratins treated by HDSF treatment.
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view of eco-friendly conversion and utilization of extracted
keratins, it is not necessary to use 0.2% potassium hydroxide to
extract feather keratins.
Feathers are rich in crude protein (90%). However, they are
poorly susceptible to common proteolytic enzymes, such as
trypsin, pepsin, and papain, due to their intact and compact
structures.
3
Without pretreatment, only 10.5% of feather keratins
could be hydrolyzed by pepsin (Fig. 2). The pepsin digestibility
dramatically increased to 93.2% after HDSF treatment at
2.0 MPa, indicating that HDSF treatment could destroy the
tightly packed structures and increase the enzymatic accessibility.
Several researchers have employed chemical treatments to
increase the enzymatic accessibility of feather keratins. A recent
study
12
performed a two-stage alkaline-enzymatic hydrolysis
procedure using 0.1–0.3% potassium hydroxide solution at
70 °C for 24 h and 1–5% proteinase hydrolysis at 50–70 °C for
4–8 h to obtain 24.08% and 90.83% of degradation in 0.1% and
0.3% potassium hydroxide, respectively. Moreover, many
researchers screened microorganisms that could secrete kerati-
nase for the degradation of feathers. This represents an environ-
mentally friendly approach, but requires a long production cycle
and tedious preparation. In a recent study,
23
twenty thermophilic
actinomycetes were selected that were able to degrade 87–91%
of feather wastes after 72 h of cultivation. Obviously, the present
study could obtain higher enzymatic accessibility (93.2% pepsin
digestibility) after HDSF treatment without any harsh chemicals
for a shorter time. Nowadays, the growing demand for food,
materials, energy and reducing greenhouse gases has motivated
research on development of technologies to utilize renewable,
bio-based resources. Recycling of feathers is also a subject of
interest among animal nutritionists, because of their potential as
a cheap and alternative protein source. The present study demon-
strates that HDSF could convert more than 90% of feather
keratin to the pepsin-digestible form, which has extraordinary
appeal to the feed industry.
Morphological changes of feathers treated by HDSF
The effect of HDSF treatment (2.0 MPa) on the morphology of
feathers is illustrated in Fig. 3. SEM photos of untreated
(Fig. 3A and B) and treated feathers (Fig. 3C–F) showed
obvious morphological changes induced by HDSF treatment. As
shown in Fig. 3A and B, feathers have a hierarchical structure
beginning with the level of the central barbs, which grow
directly from the quill. These central barbs are tiny ‘quills’
which also grow barbs. Quill fractions are composed of both the
inner and outer quill; the outer quill is more densely structured
than the inner quill, which is porous.
31
The hollow keratin fibers
could increase the steam–solid contact area, facilitating the
penetration of steam with a powerful seepage force.
After HDSF treatment, most of the feathers were completely
destroyed and the structure changed from fibers into an amor-
phous structure (Fig. 3C). The central axis of the feather dis-
appeared and the original structure of the feather could not be
identified. HDSF treatment destructured the fibers and increased
the reactive area. It was beneficial to enhance the extraction, dis-
solubility and enzymatic accessibility of feather keratins. In
addition, only a small proportion of feather fibers still retained
their structure after HDSF treatment (Fig. 3D–F). The untreated
feather has a smooth surface covered with a clutter of fibrils,
playing an important role in protecting the feather from damage.
The kinetic energy of the steam explosion fractured the feather
fiber, along with defibrillation of the fiber, due to the removal of
the cementing materials (Fig. 3D–F). After HDSF treatment,
the surface of the feather became damaged and rough (Fig. 3D
and F). It is interesting to note from Fig. 3D–F that there are
many holes in the rough surface of feather fibers, which indicates
the action of HDSF on the feather. During the HDSF process,
steam with a powerful seepage force will penetrate the interior
cells of the feather. Upon an explosive decompression (within
0.0875 s), most of the steam in the feather will quickly expand
and disrupt the internal structure of the feather, finally forming
the holes in the surface of feather when the steam rushes out.
This process is dramatically different from the conventional
steam explosion, steam spout or swollen or extrusion, which
mainly employ boiling at high temperature and pressure. In the
present study, we prolonged the time of the feather in the high
pressure to 160 s and then carried out steam explosion, but no
Fig. 3 SEM photos of untreated (A × 160 and B × 500) and treated
feathers (C × 600, D × 50, E × 500 and F × 1000) by HDSF pretreat-
ment (2.0 MPa).
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additional effect was found on the treatment efficiency, indicat-
ing that the kinetic energy produced in HDSF plays a major role
in the action of HDSF on feather keratins.
Effects of HDSF treatment on the molecular weight profile and
amino acids of feather keratins
The molecular weight profile of dissolved feather keratins is
related to the degree of degradation of keratin under HDSF treat-
ment. Most of the physicochemical methods in Table 1 caused
substantial damage to the protein chain due to the harsh con-
ditions, resulting in severe degradation of keratin and loss of
mechanical properties for application in eco-composites and
bioplastics. Whereas the chemical methods, such as the 2-mer-
captoethanol method, are a benchmark for good yield and un-
damaged keratin, but environmental harmfulness and high cost
mean they are not industrially viable. Based on the results of
extraction and dissolubility of feather keratins treated by HDSF
in this study, it is not necessary to use 0.2% potassium hydroxide
to dissolve feather keratins from the point of view of eco-friendly
processing and application of extracted keratins. The molecular
weight profiles of dissolved feather keratins in phosphate buffer
(pH 7.5) with 2% urea and phosphate buffer (pH 9.0) were
assessed (Fig. 4). As shown in Fig. 4, the dissolved feather kera-
tins in phosphate buffer (pH 7.5) with 2% urea exhibited two
peaks of molecular weight distribution. One is around 10 kDa,
which represents the natural monomer of keratin with primary
chains of 10.2–10.4 kDa linear molecules.
32
Another is above
30 kDa, which represents soluble keratin of high molecular
weight. With the increase of pressure of HDSF treatment, the
peak of molecular weight of above 30 kDa increased. The dis-
solved feather keratins in phosphate buffer ( pH 9.0) displayed a
Table 2 Amino acids composition (g per 100 g crude protein) of the
samples from the steam explosion compared to reference feathers
a
Amino acids Control 1.4 MPa 1.6 MPa 1.8 MPa 2.0 MPa
ASP 5.42
a
5.37
a
5.64
a
5.32
a
4.88
a
GLU 8.41
a
8.59
a
8.72
a
8.41
a
8.25
a
SER 9.33
a
9.53
a
9.41
a
8.99
a
8.69
a
HIS 0.50
a
0.55
a
0.60
a
0.53
a
0.48
a
GLY 7.16
a
8.14
a
7.97
a
7.88
a
7.97
a
THR 4.04
a
3.99
a
4.01
a
3.86
a
3.75
a
ARG 4.96
a
5.15
a
5.11
a
5.00
a
4.94
a
ALA 3.31
a
3.90
a
3.80
a
3.73
a
3.84
a
TYR 4.15
a
4.11
a
4.10
a
3.99
a
3.93
a
CYS-S 5.07
a
2.16
b
2.21
b
1.66
c
1.16
d
VAL 6.06
a
6.49
a
6.48
a
6.41
a
6.32
a
MET 0.41
a
0.46
a
0.50
a
0.49
a
0.47
a
PHE 3.18
a
3.81
a
3.66
a
3.56
a
3.62
a
ILE 3.51
a
3.83
a
3.85
a
3.78
a
3.77
a
LEU 6.16
a
7.04
a
6.92
a
6.74
a
6.68
a
LYS 1.12
a
1.11
a
1.18
a
1.10
a
1.05
a
PRO 10.03
a
12.13
a
10.13
a
10.68
a
11.52
a
a
The value is the average of three replicates. The data marked with
different letters in the same row were statistically different at P= 0.05.
Fig. 4 The molecular weight distribution of feather keratins dissolved
in phosphate buffer (pH 7.5) with 2% urea and phosphate buffer (pH
9.0) assessed by gel permeation chromatography (GPC) with a Sephadex
G-50 column (1 × 100 cm).
Fig. 5 The changes of disulfide bonds and free thiol groups of feather
keratins in HDSF treatment.
Fig. 6 The X-ray diffraction pattern of feather keratins before and after
HDSF treatment.
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similar molecular weight distribution (data not shown), indicat-
ing HDSF treatment did not cause substantial damage to the
protein chain of feather keratin.
The effects of HDSF treatment on the amino acids of feather
keratins were further evaluated in this study (Table 2). A signifi-
cant reduction (p< 0.05) in cystine as a function of HDSF
pressure was found. Other amino acids in feathers were
unchanged during HDSF treatment ( p> 0.05). Feather keratin
contains a large number of disulfide bonds, associated with the
amino acid cystine, which cross-link the protein chains.
Disulfide bonds are mainly responsible for the high stability,
hardness and insolubility of feather keratin. Thus almost all the
chemical and physicochemical methods (Table 1) exert their
action by first breaking the intermolecular disulfide bonds. Fig. 5
illustrated the quantitative change of disulfide bonds and free
sulfhydryl in feather keratins treated by HDSF. With the increase
of pressure of HDSF, the disulfide bonds significantly decreased
from 330 to 60 nmol mg
−1
, whereas free sulfhydryls increased
from 5 to 27 nmol mg
−1
, indicating that disulfide bonds of
feather keratins were destroyed by HDSF. From the results of
Fig. 5 and Table 2, it could be deduced that breaking of disulfide
bonds as a result of cystine destruction caused a considerable
amount of keratins to be soluble and susceptible to enzyme.
Generally, HDSF treatment is not a severe process, which could
cleave the intermolecular disulfide bonds without causing sub-
stantial damage to the protein chain. Different from the HDSF
treatment, serious losses of lysine, cystine, serine and threonine
were found in other treatments of feathers, such as heat treat-
ment,
33,34
steam extrusion
35
and high temperature and pressure
treatment in combination with lime treatment.
36
Fig. 7 FTIR spectra (A) and resolved amide III of feather keratins before (B) and after HDSF treatment at different pressures (C: 1.4 MPa; D:
1.6 MPa; E: 1.8 MPa; F: 2.0 MPa).
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Effects of HDSF treatment on the structure of feather keratins
Besides disulfide bonds, the crystallinity also takes an important
role in the high strength and stiffness of feather keratins. The
original and HDSF treated feather samples were taken to
examine the crystal structure by wide angle X-ray diffraction
(Fig. 6). As shown in Fig. 6, the original feather sample dis-
played a typical diffraction pattern of β-keratins (feather keratins)
with a prominent 2θpeak at 9.2° corresponding to the α-helix
configuration of the feather keratin, and a more intense band at
19.6° indexed as its beta strand secondary structure.
7
After
HDSF treatment, the samples at different pressures of HDSF
presented the identified X-ray diffraction patterns, characterized
by decrease in the intensity of the peaks, indicating that crystals
were significantly destroyed under HDSF treatment. Moreover, it
was observed that the peak at 9.2° became more intense and
dominant than the band at 19.6°, which was ascribed to the
decomposition of the β-sheet of feather keratins. As is known,
feathers’mechanical stability, insolubility and resistance to pro-
teolytic digestion are consequences of the tight packing of the
protein chain in β-sheets (β-keratin) into a supercoiled polypep-
tide chain.
37
The decrease of crystallinity and decomposition of
the β-sheet structure could definitely contribute to the improve-
ment of extraction, dissolubility and enzymatic accessibility of
feather keratins. Interestingly, no significant changes even
increase of the crystallinity of feather keratins were found in
chemical treatments, such as alkali treatment (0.75 mol L
−1
sodium hydroxide)
38
and physicochemical treatments, such as
conventional steam explosion
8
or extrusion,
21
demonstrating the
different mechanisms and actions of these means on the keratins.
The FTIR spectra in the region of 4000–400 cm
−1
of untreated
and HDSF treated feather are shown in Fig. 7A. It can be seen
from Fig. 7A that the samples before and after HDSF treatments
at 1.4–2.0 MPa exhibited similar absorption bands at 3300 cm
−1
(amide A: N–H and O–H), 2850 cm
−1
(–CH
2
), 1627 cm
−1
(amide I: CvO), 1520 cm
−1
(amide II: N–H and C–H) and
1233 cm
−1
(amide III: C–H, N–H, C–C and CvO). This indi-
cates that neither production of new functional groups and
chemical bonds nor substantial damage to the protein chain of
feather keratin was induced by HDSF treatment. Methods cur-
rently being used to extract information on protein secondary
structure from infrared spectra are based on empirical correlation
between the frequencies of certain vibrational modes and
types of secondary structure of polypeptide chains, such as
α-helix, β-sheet, β-turn, and random coil.
39
As is known,
the amide I region (1700–1600 cm
−1
) and amide II region
(1580–1480 cm
−1
) as the candidates for secondary structure
characterizations suffer from several limitations, such as strong
interference from water vibrational bands and overlap of
revolved bands corresponding to secondary structures.
39
In this
study, the easily resolved and better defined amide III region
(1350–1200 cm
−1
) was employed for quantitative analysis of
protein secondary structure, due to the in-phase combination of
N–H in-plane bending and C–N stretching vibrations.
39
The
amide III mode was further resolved in Gaussian bands
(Fig. 7B–F) corresponding to α-helix (1330–1295 cm
−1
), β-turn
(1295–1270 cm
−1
), random coil (1270–1250 cm
−1
) and β-sheet
(1250–1220 cm
−1
). The bands not related to the secondary struc-
ture are defined as “other”. From the resolved amide III of
feather keratin (Fig. 8), the β-sheet decreased and the unordered
structure (random coil) increased after HDSF treatment, which
was consistent with the observation of X-ray diffraction. The dis-
sociation of secondary structures, especially the decrease of
β-sheet is essential for the destabilization and rearrangement of
β-keratin.
Conclusions
Based on the present study, a diagram could be proposed to
describe the action of HDSF treatment on feather keratins
Fig. 8 The changes of secondary structures of feather keratins after
HDSF treatment.
Fig. 9 A diagram to describe the action of HDSF treatment on feather
keratins.
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(Fig. 9). In HDSF treatment, steam with a powerful seepage
force first penetrates into the fibrous tissues and cells of feather,
and then quickly expands and breaks free of the structure
upon an explosive decompression at supersonic speed (within
0.0875 s).
This process effectively destroyed or destabilized β-sheet crys-
tals and intermolecular disulfide bonds without causing substan-
tial damage to the keratin protein chain, dramatically increasing
the enzymatic accessibility, extraction, and dissolubility of
feather keratins in polar solvents like water, salt solution and
weak bases. HDSF is a physicochemical process, which includes
two phases: the pressurization phase (3–5 s) and the explosion
phase (within 0.0875 s). Definitely, the thermal factor plays a
role in the action of HDSF treatment of feathers. However, the
time of materials in high temperatures is very short compared
with the conventional physicochemical methods, including
steam explosion, steam spout or swollen or extrusion and the
temperature of materials could decrease to 50 °C or lower
immediately after HDSF treatment, indicating that HDSF treat-
ment is not a severe process.
HDSF treatment could be a sustainable and practical pretreat-
ment for the extraction of feather keratin for exploitation of bio-
materials and conversion of feathers to nutrient animal feed,
instead of the current chemical (strong acid, alkali or catalytic)
hydrolysis and hydrothermal treatment (high pressure and temp-
erature). Moreover, HDSF treatment could be performed on a
large scale now that HDSF equipment with a 5 m
3
chamber has
been developed.
24
In the catapult explosion mode of HDSF, the
valve opening speed transforms into the piston movement speed
in the cylinder. Thus the large diameter piston can get an equal
proportion of driving force to the small diameter piston, and
achieve the same speed breaking away from cylinder. Therefore,
the explosion speed and treatment efficiency in scale-up appli-
cations of catapult explosion can remain consistent with the
small equipment.
Acknowledgements
The authors gratefully acknowledge the supported by Project of
the National Natural Science Foundation of PR China
(31271977). This study was also supported by 111 project-
B07029, Program for Changjiang Scholars and Innovative
Research Team in University and Project Funded by the Priority
Academic Program Development of Jiangsu Higher Education
Institutions.
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