Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 03168825 2022-07-20
WO 2021/150744
PCT/US2021/014401
CONTINUOUS PRODUCTION OF KERATIN FIBERS
FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
This invention was made with government support under Grant No. 2019-67021-
29940, awarded by the United States Department of Agriculture, National
Institute of Food
and Agriculture. The government has certain rights in the invention.
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Provisional Application Serial No.
62/963,968, filed on January 21, 2020, the contents of which are incorporated
by
reference in their entirety herein.
TECHNICAL FIELD
The present disclosure relates to continuous processes for preparing a keratin
fiber.
Such processes can be useful for preparing a keratin fiber with a high draw
ratio, for
example.
BACKGROUND
Research on the preparation of high-end materials through green synthesis
technology has received more and more attention [1-3]. At the same time, the
preparation
of common products for daily use via green and sustainable approaches is
gaining
popularity, such as the use of renewable monomers to prepare sustainable
polyester [4, 5].
But many of these sustainable polymers are hardly degradable [6]. Long-term
accumulation of such materials will inevitably affect the environment and
human health. It
is reported that synthetic polymers are circulating in the atmosphere and
ocean after turning
into nano- or microparticles [7]. For instance, precipitations have been found
to contain
polymer particles in many districts even in the Artic [8]. Recently people
found that plastic
particles fell out of the sky with snow in the Arctic. These plastic particles
will inevitably
enter the human body and accumulate [9]. Recent studies show that synthetic
polymer
particles can cause various hazards to human health such as cancer, chronical
illness, and
1
CA 03168825 2022-07-20
WO 2021/150744
PCT/US2021/014401
damage to the reproductive system [10]. Therefore, the utilization of wastes
to develop
quality products with good degradability should also receive equal attention
in the future
[11], especially in the textile field which can be plagued by environmental
pollution. In
terms of textile fibers, more than 80 million tons of synthetic fibers are
produced every
year globally [12]. Almost all synthetic fibers are petroleum-based and hardly
degradable
in the environment [13]. Therefore, it is desirable to find alternatives to
replace petroleum-
based fibers [14, 15]. Alternatives should be sustainable, environmentally
responsible and
affordable.
Although studies for the utilization of keratinous wastes began decades ago,
very
few regenerated products with high quality have been developed due to damages
to the
primary structures during extraction and recovery of the secondary structures
in the
regeneration of keratin materials. Among the regenerated keratin products,
fibers have high
quality specifications, including resistance to repeated launderings and high
toughness.
SUMMARY
Provided herein are processes for preparing a keratin fiber. In some
embodiments,
the methods comprise extruding a keratin solution into a first solution to
form a first fiber;
drawing the first fiber and oxidizing the first fiber to form a treated fiber;
drawing the
treated fiber and oxidizing the treated fiber one or more times; and setting
the treated fiber
to form the keratin fiber.
In some embodiments, the keratin fiber has a draw ratio of above about 500%.
In
some embodiments, the keratin fiber has a draw ratio of about 800% to about
1000%. In
some embodiments, the keratin fiber has a draw ratio of about 1500%.
In some embodiments, the diameter of the keratin fiber is about 5 micrometers
to
about 30 micrometers. In some embodiments, the diameter of the keratin fiber
is about 15
micrometers.
In some embodiments, the keratin fiber comprises at least about 70% keratin.
In
some embodiments, the keratin fiber comprises at least about 85% keratin.
In some embodiments, the keratin fiber has a tenacity greater than about 0.8
g/den.
In some embodiments, the keratin fiber has a tenacity of about 1 g/den to
about 2 g/den.
2
CA 03168825 2022-07-20
WO 2021/150744
PCT/US2021/014401
In some embodiments, the keratin fiber has a strain greater than about 5%. In
some
embodiments, the keratin fiber has a strain of about 10% to about 30%. In some
embodiments, the keratin fiber has a strain of about 15%. In some embodiments,
the keratin
fiber has been dried prior to measuring the strain.
In some embodiments, the keratin fiber has a toughness higher than about 15
Fern'.
In some embodiments, the keratin solution has a consistency coefficient (K) of
about 2 Pa=sn to about 6 Pa=sn, wherein the keratin solution is at 25 C and
comprises keratin
at about 18% w/w of the composition. In some embodiments, the keratin solution
has a
consistency coefficient (K) of about 4.2 Pa= sn, wherein the keratin solution
is at 25 C and
comprises keratin at about 18% w/w of the composition. In some embodiments,
the keratin
solution has a flow behavior index of about 0.9 to about 0.94, wherein the
keratin solution
is at 25 C and comprises keratin at about 18% w/w of the composition. In some
embodiments, the keratin solution has a flow behavior index of about 0.91,
wherein the
keratin solution is at 25 C and comprises keratin at about 18% w/w of the
composition.
In some embodiments, the keratin solution comprises a reducing agent. In some
embodiments, the keratin solution comprises an electrolyte. In some
embodiments, the
electrolyte is a sulfate, an acetate, a chloride, a citrate, a carbonate, a
phosphate, or a
combination thereof In some embodiments, the keratin solution comprises sodium
dodecyl
sulfate (SDS). In some embodiments, the keratin solution is prepared from a
keratinous
material.
In some embodiments, the process further comprises preparing the keratin
solution.
In some embodiments, preparing the keratin solution comprises: extracting
keratin from a
keratinous material to form extracted keratin; and dissolving the extracted
keratin in an
aqueous solution comprising a reducing agent to form the keratin solution. In
some
embodiments, the aqueous solution further comprises SDS.
In some embodiments, the keratinous material comprises one or more of: animal
hair, horn, and feather. In some embodiments, the hair is the hair is wool,
camel hair, alpaca
hair, rabbit hair, or a combination thereof In some embodiments, the feather
is a duck
feather, a goose feather, a chicken feather, or a combination thereof.
3
CA 03168825 2022-07-20
WO 2021/150744
PCT/US2021/014401
In some embodiments, the keratin fiber comprises at least 70% of the disulfide
crosslinkages compared to the amount of disulfide crosslinkages in the
keratinous material.
In some embodiments, the keratin fiber comprises at least 85% of the beta-
sheet
crystallinity compared to the amount of beta-sheet crystallinity in the
keratinous material.
In some embodiments, the reducing agent comprises a thiol group. In some
embodiments, the reducing agent comprises mercaptoethanol, cysteine,
dithiothreitol, 1,2-
ethanedithi ol,
1,3 -b enzene dithi ol, bi s(2-m ercaptoethyl) ether, ethylene glycol
bisthioglycolate, or a combination thereof.
In some embodiments, the step of extruding a keratin solution into a first
solution
comprises using a spinneret to extrude the keratin solution. In some
embodiments, the
spinneret comprises a hole, wherein the hole has a diameter of about 50
micrometers.
In some embodiments, the first solution comprises sodium sulfate, zinc
sulfate, and
acetate buffer. In some embodiments, the pH of the first solution is about 2.
In some
embodiments, the first solution comprises sodium sulfate in an amount of about
15% w/w
of the composition, zinc sulfate in an amount of about 5% w/w of the
composition, and
acetate buffer with a pH of 2.
In some embodiments, the step of oxidizing comprises exposing the fiber to an
oxidizing solution comprising an oxidant selected from the group consisting
of: a peroxide,
a halogen oxoacid or salt thereof, a high-valent metal salt, and a combination
thereof. In
some embodiments, the peroxide is an alkali metal peroxide, an alkaline earth
metal
peroxide, or a combination thereof. In some embodiments, the oxidant is sodium
periodate.
In some embodiments, the oxidizing solution further comprises a buffer. In
some
embodiments, the oxidizing solution further comprises acetate buffer. In some
embodiments, the pH of the oxidizing solution is about 2. In some embodiments,
the
temperature of the oxidizing solution is about 35 C.
In some embodiments, the step of drawing the treated fiber and oxidizing the
treated
fiber is repeated two times. In some embodiments, the process further
comprises drawing
the treated fiber prior to setting the treated fiber.
In some embodiments, setting the treated fiber comprises exposing the treated
fiber
to a wash solution comprising a surfactant. In some embodiments, the
surfactant is selected
4
CA 03168825 2022-07-20
WO 2021/150744
PCT/US2021/014401
from the group consisting of: ammonium lauryl sulfate, SDS, sodium laureth
sulfate,
sodium myreth sulfate, sodium stearate, sodium lauroyl sarcosinate,
perfluorononanoate,
perfluorooctanoate,
(3- [(3 -chol ami dopropyl)dimethyl amm oni o]-1-prop anesulfonate),
cocamidopropyl hydroxysultaine, cocamidopropyl betaine, phosphatidylserine,
phosphatidylethanolamine, phosphatidylcholine, a sphingomyelin, cetrimonium
bromide
(CTAB), cetylpyridinium chloride (CPC), benzalkonium chloride (BAC),
benzethonium
chloride (BZT), dimethyldioctadecylammonium chloride,
dioctadecyldimethylammonium
bromide (DODAB), and a combination thereof
In some embodiments, the wash solution further comprises an acetate buffer. In
some embodiments, the wash solution has a pH of about 2. In some embodiments,
the wash
solution is at a temperature of about 40 C.
In some embodiments, setting the treated fiber comprises winding the treated
fiber
and oxidizing the treated fiber.
In some embodiments, exposing the treated fiber to a wash solution comprising
a
surfactant is performed prior to winding the treated fiber and oxidizing the
treated fiber. In
some embodiments, winding the treated fiber is at a rate of about 15
meters/minute.
In some embodiments, the keratin fiber is dried at about 85 C for about 1
hour. In
some embodiments, the keratin fiber is annealed at about 125 C for about 1
hour. In some
embodiments, the keratin fiber is annealed after it is dried.
In some embodiments, the process is a continuous process.
In some embodiments, the process further comprises exposing the keratin fiber
to
a solution comprising an oxidized saccharide. In some embodiments, the keratin
fiber is
exposed to the solution comprising an oxidized saccharide for about 3 to about
25 hours.
In some embodiments, the oxidized saccharide is a sucrose polyaldehyde.
In some embodiments, the step of exposing the keratin fiber to a solution
comprising an oxidized saccharide is performed prior to exposing the treated
fiber to a
wash solution comprising a surfactant.
Also described herein are keratin fibers prepared by any of the processes
described
herein.
5
CA 03168825 2022-07-20
WO 2021/150744
PCT/US2021/014401
In some embodiments, the term "about" is used herein to mean approximately, in
the region of, roughly, or around. When the term "about" is used in
conjunction with a
numerical range, it modifies that range by extending the boundaries above and
below the
numerical values set forth. In general, the term "about" is used herein to
modify a numerical
value above and below the stated value by a variance of 10%.
Unless otherwise defined, all technical and scientific terms used herein have
the
same meaning as commonly understood by one of ordinary skill in the art to
which this
invention belongs. Methods and materials are described herein for use in the
present
invention; other, suitable methods and materials known in the art can also be
used. The
materials, methods, and examples are illustrative only and not intended to be
limiting. All
publications, patent applications, patents, database entries, and other
references mentioned
herein are incorporated by reference in their entirety. In case of conflict,
the present
specification, including definitions, will control.
DESCRIPTION OF DRAWINGS
FIG. 1 is a diagram of a wet spinning line employed with stepwise oxidation
and
drawing.
FIG. 2 is a reducing SDS-PAGE gel with Lane 1 containing standard protein
markers; Lane 2 containing regenerated keratin; and Lane 3 containing chicken
feathers.
FIG. 3A is a comparison of disulfide bonds in keratin between chicken feathers
and spun keratin fibers using Raman spectra.
FIG. 3B shows recovery of disulfide bonds in keratin fibers measured by HPLC
at different oxidation stages via controlled disulfide bond assembly.
FIG 3C shows the effect of controlled disulfide bond assembly on spinnability
of
keratin fibers. Fine control of disulfide bond assembly was achieved using
stepwise
oxidation and drawing on a continuous spinning line.
FIG. 4A shows the relationship between recovery ratio of disulfide bonds in
fibers and highest draw ratio.
FIG. 4B shows the morphologies of spun keratin fiber from highest draw ratio
(scale bar= 501.tm).
6
CA 03168825 2022-07-20
WO 2021/150744
PCT/US2021/014401
FIG. 5 shows a morphological change in keratin fibers on a continuous spinning
line with controlled disulfide bonds assembly.
FIG. 6A shows a continuous line for keratin production.
FIG. 6B shows spun keratin fibers.
FIG. 7A is a plot showing typical stress-strain curves for feather barbs and
continuously spun keratin fibers.
FIG. 7B is a keratin filament that endured a high degree of twisting.
FIG. 8A is an XRD spectra of chicken feathers and keratin fibers.
FIG. 8B shows deconvolutions of the 13C NMR spectra (around 170 ppm) of
chicken feathers and keratin fibers.
DETAILED DESCRIPTION
Keratinous wastes, especially poultry feathers, are abundant, safe, cost-
effective
and readily available materials that could be used to produce fibers [16].
Among all
poultries, chicken has the largest consumption worldwide [17]. With an annual
chicken
consumption of about 65 million tons and a subsequent generation of chicken
feathers of
5 million tons worldwide annually [18], the potential production of protein
fibers from
chicken feathers is already 2.5 times higher than the current output of both
wool and silk.
Chicken feathers are also rich in protein (keratin) content as high as 90-92%
[19]. Having
linear polymeric backbones and an average molecular weight higher than 10 kDa
[20],
feather keratin meets the molecular specifications for fiber spinning. With
around 7%
cysteine served as crosslinking sites, feather keratin is expected to possess
good tensile
properties and aqueous stabilities [21]. Fibers made of feather keratin are
very likely to
have smooth touch, moisture transmission and thermal insulation due to similar
chemical
structures to those of wool and silk [15].
Unsuccessful continuous fiber production can result from the difficulty in
keratin
extraction from feathers, incomplete re-dissolution of keratin, limited
alignment of
molecular chains of protein, and inefficient recovery of disulfide
crosslinkages. For a long
period of time, strong alkali solutions were used to dissolve and extract
keratin [22].
7
CA 03168825 2022-07-20
WO 2021/150744
PCT/US2021/014401
However, high pH not only hydrolyzes the backbone of the protein but it can
also reduce
amounts of sulfhydryl groups on the keratin [23, 24]. Damaged protein
backbones and a
decline in sulfur groups can make it challenging to produce high-quality
fibers. Ionic
liquids have been used to extract keratin from wastes [25, 26]. Recently,
ionic liquids were
used to dissolve keratin for spinning. However, the properties of obtained
fibers were not
satisfactory [27]. One reason for such low mechanical property is poor
dissolution of
keratin. Liquids could break ionic interactions and hydrogen bonds; they are
not able to
interrupt disulfide bonds and hydrophobic interactions among keratin
molecules. A non-
destructive extraction system was developed and regenerated chicken keratin
fibers on a
lab-scale [28]. However, such fiber spinnings lacked techniques to efficiently
recover the
disulfide bonds and secondary structures. As a result, the spinnability of
keratin fibers was
not good. The stretchability of the regenerated fiber was also remarkably
limited. The
linearity of the regenerated fiber was poor, leading to long-distance between
keratin
backbones and low chances for the formation of intermolecular disulfide
crosslinkages.
The resulting fibers had a large diameter, low strength, and poor flexibility.
The tenacity
of the regenerated fiber was only 50% of the original chicken feathers and
strain was only
4%. In addition, the regenerated keratin fiber did not inherit good wet
properties of chicken
feathers. Although the research regarding the fiber production from keratinous
waste began
in the 1940s [29], there are few efficacious processes developed to
continuously produce
regenerated pure keratin fibers with high quality. It is desirable for fibers
to have stress
higher than 100 Mpa and strain higher than 10%. To achieve this, most research
focuses
on either post crosslinking or addition of high-performance polymers such as
PVA and
cellulose into keratin fibers to improve the properties [27, 30-33].
Accordingly, the present application provides keratin fibers and processes for
preparing the keratin fibers. Such fibers can include keratin fibers produced
on a
continuous line via stepwise oxidation and drawing. Stepwise oxidation and
drawing of a
fiber can result in one or more of: controlled assembly of disulfide
crosslinkages, optimum
recovery of secondary structures, satisfactory mechanical properties, and
scalable
production of keratin fibers. For example, the properties of the regenerated
keratin fibers
can be close to that of chicken feathers. Furthermore, through the use and
recycling of safe
8
CA 03168825 2022-07-20
WO 2021/150744
PCT/US2021/014401
and inexpensive chemicals, the continuous keratin fiber production disclosed
herein is
sustainable, environmentally responsible, and affordable. Continuous keratin
fiber
production via efficient recovery of secondary structures and disulfide bonds
can minimize
the use and generation of hazardous substances in the manufacturing process
and can open
a new window for the utilization of keratinous wastes.
A "keratin fiber" as used herein is a fiber comprising at least about 70%
keratin. As
used herein, the term "fiber" or "textile fiber" means a unit of matter which
is capable of
being spun into a yarn or made into a fabric by bonding or by interlacing in a
variety of
processes including weaving, knitting, braiding, felting, twisting, or
webbing, and which
is the basic structural element of a textile product.
In some embodiments, a keratin fiber described herein has at least about 70%
keratin. For example, the keratin fiber can have about 70% to about 99%, about
70% to
about 95%, about 70% to about 90%, about 70% to about 85%, about 70% to about
80%,
about 70% to about 75%, or about 95% to about 99%, about 90% to about 99%,
about 85%
to about 99%, about 80% to about 99%, about 75% to about 99%, or about 75% to
about
95% keratin. For example, the keratin fiber can have about 70%, about 75%,
about 80%,
about 85%, about 90%, about 95%, or about 99% keratin.
In some embodiments, a process for preparing a keratin fiber includes
extruding a
keratin solution into a first solution to form a first fiber; drawing the
first fiber and oxidizing
the first fiber to form a treated fiber; drawing the treated fiber and
oxidizing the treated
fiber one or more times; and setting the treated fiber to form the keratin
fiber. In some
embodiments, the process is a continuous process.
A keratin solution as described herein includes keratin, e.g., extracted
keratin. In
some embodiments, the keratin solutions further includes sodium dodecyl
sulfate (SDS),
and/or a reducing agent. Reducing agents as described herein can include a
thiol-based
group, e.g., monothiols and dithiols. Non-limiting examples of such reducing
agents
include mercaptoethanol, cysteine, dithiothreitol, 1,2-ethanedithiol, 1,3-
benzenedithiol,
bis(2-mercaptoethyl) ether, and ethylene glycol bisthioglycolate. In some
embodiments,
the keratin solution includes a reducing agent at a concentration of about
0.5% to about 3%
w/w of the keratin. For example, about 0.5% to about 2.5%, about 0.5% to about
2%, about
9
CA 03168825 2022-07-20
WO 2021/150744
PCT/US2021/014401
0.5% to about 1.5%, about 0.5% to about 1%, about 2.5% to about 3%, about 2%
to about
3%, about 1.5% to about 3%, about 1% to about 3%, about 1.5% to about 2.5%, or
about
1.75% to about 2.25% w/w of the keratin. In some embodiments, the keratin
solution
includes a reducing agent at a concentration of about 1.4%, about 1.6%, about
1.8%, about
2%, about 2.2%, about 2.4%, about 2.6%, or about 2.8% w/w of the keratin.
In some embodiments, the keratin solution includes extracted keratin at about
20%
to about 35% w/w of the solution. For example, about 20% to about 25%, about
20% to
about 30%, about 30% to about 35%, or about 25% to about 35% w/w of the
solution. In
some embodiments, the keratin solution include extracted keratin at about 25%
to about
30% or about 26% to about 28% w/w of the solution. In some embodiments, the
keratin
solution includes extracted keratin at about 24%, about 25%, about 26%, about
27%, about
28%, about 29%, or about 30% w/w of the solution.
In some embodiments, the keratin solution includes SDS. In some embodiments,
the keratin solution includes SDS at about 5% to about 15% w/w of the
solution. For
example, about 5% to about 8%, about 5% to about 10%, about 5% to about 12%,
about
12% to about 15%, about 10% to about 15%, about 8% to about 15%, or about 8%
to about
12% w/w of the solution. In some embodiments, the keratin solution includes
SDS at about
5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%,
about
13%, about 14%, or about 15% w/w of the solution.
In some embodiments, the pH of the keratin solution is adjusted to be about 7
to
about 10. For example, about 7 to about 8, about 7.5 to about 8.5, about 8 to
about 9, about
8.5 to about 9.5, or about 9 to about 10. Any buffer suitable for maintaining
the desired pH
can be used. Non-limiting examples of such buffers include carbonate-
bicarbonate buffer,
glycine-sodium hydroxide buffer, sodium borate buffer, TRIZMA buffer (e.g., 2-
Amino-
2-(hydroxymethyl)-1,3-propanediol buffer), and diethanolamine buffer. In some
embodiments, the keratin solution includes a buffer at about 0.1 M to about
0.3 M. For
example, about 0.1 M to about 0.15 M, about 0.1 M to about 0.2 M, about 0.1 M
to about
0.25 M, about 0.25 M to about 0.3 M, about 0.2 M to about 0.3 M, about 0.15 M
to about
0.3 M, about 0.15 M to about 0.2 M, about 0.18 M to about 0.22 M, or about 0.2
M to about
0.25 M.
CA 03168825 2022-07-20
WO 2021/150744
PCT/US2021/014401
In some embodiments, the amount of reducing agent in a keratin solution
described
herein is optimized such that the keratin is fully dissolved and/or the
molecular
entanglement is low. In some embodiments, the degree of molecular entanglement
is
indicated by the flow behavior index (n), which can be determined by measuring
shear
stress using a rotational rheometer (see Equation 1). The consistency
coefficient (K) for
the keratin solution, which is directly proportional to polymer viscosity in
solution, can
also be determined by measuring shear stress using a rotational rheometer.
Equation 1: T=Kyn
where y. is the shear rate (s-') measured in the range of 0-1000 s-1
In some embodiments, the keratin solution has a flow behavior index of about
0.8
to about 0.95. For example, about 0.8 to about 0.82, about 0.8 to about 0.84,
about 0.8 to
about 0.86, about 0.8 to about 0.88, about 0.8 to about 0.9, about 0.8 to
about 0.92, about
0.8 to about 0.94, about 0.92 to about 0.95, about 0.9 to about 0.95, about
0.88 to about
0.95, about 0.86 to about 0.95, about 0.84 to about 0.95, about 0.82 to about
0.95, about
0.88 to about 0.92, or about 0.90 to about 0.94. In some embodiments, the
keratin solution
has a flow behavior index of about 0.88, about 0.89, about 0.9, about 0.91,
about 0.92,
about 0.93, about 0.94, or about 0.95. In some embodiments, the keratin
solution is at about
C and includes extracted keratin at about 18% w/w of the solution during the
20 measurement of the shear stress.
In some embodiments, the keratin solution has a consistency coefficient (K) of
about 2 Pa=sn to about 6 Pa=sn. For example, about 2 Pa=sn to about 6 Pa=sn,
about 3 Pa=sn
to about 6 Pa=sn, about 4 Pa=sn to about 6 Pa=sn, about 5 Pa=sn to about 6
Pa=sn, about 4
Pa=sn to about 6 Pa=sn, about 3 Pa=sn to about 6 Pa=sn, about 3 Pa=sn to about
5 Pa=sn, about
25 3.5 Pa=sn to about 5.5 Pa=sn, about 3.5 Pass" to about 4.5 Pa=sn, or
about 4 Pass' to about
4.5 Pa=sn. In some embodiments, the keratin solution is at about 25 C and
includes
extracted keratin at about 18% w/w of the solution during the measurement of
the shear
stress.
In some embodiments, a process described herein further includes preparing the
keratin solution, e.g,. any of the keratin solutions as described herein. In
some
11
CA 03168825 2022-07-20
WO 2021/150744
PCT/US2021/014401
embodiments, preparing the keratin solution includes extracting keratin from a
keratinous
material to form extracted keratin; and dissolving the extracted keratin in an
aqueous
solution comprising a reducing agent, e.g., any of the reducing agents
described herein to
form the keratin solution. In some embodiments,
Keratin can be extracted from any keratinous material, e.g., a material
comprising
keratin. Non-limiting examples of keratinous materials include: hair, horn,
and feather. In
some embodiments, the keratinous material includes wool, camel hair, alpaca
hair, rabbit
hair, duck feather, goose feather, chicken feather, or a combination thereof
In some
embodiments, the step of extracting keratin from a keratinous material to form
extracted
keratin includes exposing a keratinous material to an extraction solution. The
extraction
solution can include one or more of SDS, urea, and a reducing agent.
In some embodiments, the extraction solution includes SDS. In some
embodiments,
the extraction solution includes SDS at about 5% to about 15% w/w of the
solution. For
example, about 5% to about 8%, about 5% to about 10%, about 5% to about 12%,
about
12% to about 15%, about 10% to about 15%, about 8% to about 15%, or about 8%
to about
12% w/w of the solution. In some embodiments, the extraction solution includes
SDS at
about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about
12%,
about 13%, about 14%, or about 15% w/w of the solution.
In some embodiments, the extraction solution includes urea at about 1 M to
about
3 M. For example, about 1 M to about 1.5 M, about 1 M to about 2 M, about 1 M
to about
2.5 M, about 2.5 M to about 3 M, about 2 M to about 3 M, about 1.5 M to about
3 M, about
1.5 M to about 2 M, about 1.8 M to about 2.2 M, or about 2 M to about 2.5 M.
In some embodiments, the extraction solution includes a reducing agent, e.g.,
any
of the reducing agents described herein. In some embodiments, the extraction
solution
includes the reducing agent at about 5% to about 15% w/w of the solution. For
example,
about 5% to about 8%, about 5% to about 10%, about 5% to about 12%, about 12%
to
about 15%, about 10% to about 15%, about 8% to about 15%, or about 8% to about
12%
w/w of the solution. In some embodiments, the extraction solution includes the
reducing
agent at about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about
11%, about
12
CA 03168825 2022-07-20
WO 2021/150744
PCT/US2021/014401
12%, about 13%, about 14%, or about 15% w/w of the solution. In some
embodiments, the
reducing agent is cysteine.
In some embodiments, the pH of the extraction solution is adjusted to be about
9 to
about 11.5. For example, about 9 to about 11, about 9 to about 10.5, about 9
to about 10,
about 11 to about 11.5, about 10.5 to about 11.5, about 10 to about 11.5,
about 9.5 to about
11.5, or about 10 to about 11. In some embodiments, the pH of the extraction
solution is
adjusted to be about 9, about 9.5, about 10, about 10.5, about 11, or about
11.5.
In some embodiments, the step of extracting keratin from a keratinous material
to
form extracted keratin includes exposing a keratinous material to an
extraction solution for
a period of time. In some embodiments, the period of time is about 8 hours to
about 15
hours. For example, about 8 hours to about 9 hours, about 8 hours to about 10
hours, about
8 hours to about 11 hours, about 8 hours to about 12 hours, about 8 hours to
about 13 hours,
about 8 hours to about 14 hours, about 14 hours to about 15 hours, about 13
hours to about
hours, about 12 hours to about 15 hours, about 11 hours to about 15 hours,
about 10
15 hours
to about 15 hours, about 9 hours to about 15 hours, about 10 hours to about 14
hours,
or about 11 hours to about 13 hours. In some embodiments, the period of time
is about 8
hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13
hours, about
14 hours, or about 15 hours.
In some embodiments, the temperature of the extraction solution is held at
about 60
C to about 80 C. For example, about 60 C to about 65 C, about 60 C to
about 70 C,
about 60 C to about 75 C, about 75 C to about 80 C, about 70 C to about
80 C, about
65 C to about 80 C. In some embodiments, the temperature of the extraction
solution is
held at about 65 C, about 66 C, about 67 C, about 68 C, about 70 C, about
71 C,
about 72 C, about 73 C, about 74 C, or about 75 C.
In some embodiments, the step of extracting keratin from a keratinous material
to
form extracted keratin further includes centrifuging the extraction solution
comprising the
keratinous material to form a centrifuged extraction solution comprising the
keratinous
material. In some embodiments, the supernatant of the centrifuged extraction
solution
comprising the keratinous material is adjusted to the isoelectric point using
any suitable
acid (e.g., hydrochloric acid). In some embodiments, sodium sulfate is added
to the
13
CA 03168825 2022-07-20
WO 2021/150744
PCT/US2021/014401
supernatant of the centrifuged extraction solution comprising the keratinous
material to
precipitate the extracted keratin. In some embodiments, the extracted keratin
is washed to
remove impurities. In some embodiments, the extracted keratin is vacuumed
dried.
A keratin solution as described herein can be installed on a spinning line
prior to
the step of extruding the keratin solution into a first solution to form a
first fiber. The
spinning line can be any suitable spinning line, e.g., a wet spinning line. In
some
embodiments, the step of extruding a keratin solution into a first solution
comprises using
a spinneret to extrude the keratin solution. For example, the keratin solution
is extruded
into the first solution using a spinneret. In some embodiments, the spinneret
comprises one
or more holes, wherein the holes have a diameter of about 50 micrometers.
In some embodiments, the first solution includes an electrolyte. Non-limiting
examples of suitable electrolytes include sulfate, acetate, chloride, citrate,
carbonate, and
phosphate. The electrolytes can be paired with any suitable cation. Non-
limiting examples
of such cations include alkali metals and transition metals such as lithium,
sodium,
magnesium, and zinc. Accordingly, in some embodiments, the first solution
includes
lithium sulfate, sodium sulfate, sodium acetate, zinc sulfate, zinc acetate,
zinc chloride,
sodium carbonate, sodium phosphate, zinc carbonate, or a combination thereof
In some embodiments, the first solution includes an electrolyte in an amount
of
about 10% to about 30% w/w of the composition. For example, about 10% to about
15%,
about 10% to about 20%, about 10% to about 25%, about 25% to about 30%, about
20%
to about 30%, about 15% to about 30%, about 15% to about 20%, or about 18% to
about
22% w/w of the composition. In some embodiments, the first solution includes
an
electrolyte in an amount of about 10%, about 12%, about 14%, about 16%, about
18%,
about 20%, about 22%, about 24%, about 26%, about 28%, or about 30% w/w of the
composition. In some embodiments, the first solution includes sodium sulfate
at 15% w/w
of the composition, zinc sulfate at 5% w/w of the composition.
In some embodiments, the first solution further includes a buffer. Any buffer
suitable for maintaining the desired pH can be used. Non-limiting examples of
such buffers
include hydrochloric acid (HC1)-potassium chloride buffer, glycine-HC1buffer,
and acetate
buffer. In some embodiments, the first solution includes a buffer at a
concentration of about
14
CA 03168825 2022-07-20
WO 2021/150744
PCT/US2021/014401
0.1 M to about 0.3 M. For example, about 0.1 M to about 0.15 M, about 0.1 M to
about 0.2
M, about 0.1 M to about 0.25 M, about 0.25 M to about 0.3 M, about 0.2 M to
about 0.3
M, about 0.15 M to about 0.3 M, about 0.15 M to about 0.2 M, about 0.18 M to
about 0.22
M, or about 0.2 M to about 0.25 M.
In some embodiments, the pH of the first solution is adjusted to be about 1 to
about
4. For example, about 1 to about 1.5, about 1 to about 2, about 1 to about
2.5, about 1 to
about 3, about 1 to about 3.5, about 3.5 to about 4, about 3 to about 4, about
2.5 to about
4, about 2 to about 4, about 1.5 to about 4, or about 1.5 to about 2.5. In
some embodiments,
the pH of the first solution is adjusted to be about 1, about 1.4, about 1.6,
about 1.8, about
2, about 2.2, about 2.4, about 2.6, about 3, about 3.5, or about 4.
In some embodiments, the first solution includes sodium sulfate, zinc sulfate,
and
acetate buffer. In some embodiments, the first solution includes sodium
sulfate at 15% w/w
of the composition, zinc sulfate at 5% w/w of the composition, and acetate
buffer.
Stepwise drawing and oxidizing after extruding a keratin solution into a first
solution to form a first fiber can help establish disulfide bonds and ordered
structures in
the first fiber. In some embodiments, the step of oxidizing comprises exposing
the first
fiber to an oxidizing solution comprising an oxidant selected from the group
comprising a
peroxide, a halogen oxoacid or salt thereof, a high-valent metal salt, or a
combination
thereof. Non-limiting examples of a peroxide include alkali metal peroxides
and alkaline
earth metal peroxides such as sodium periodate, hydrogen peroxide, chlorite,
hypochlorite,
and sodium ferrate(VI). In some embodiments, the oxidant is present in an
amount of about
2 g/L to about 6 g/L. For example, about 2 g/L to about 2.5g/L, about 2 g/L to
about 3g/L,
about 2 g/L to about 3.5g/L, about 2 g/L to about 4g/L, about 2 g/L to about
4.5g/L, about
2 g/L to about 5g/L, about 2 g/L to about 5.5g/L, about 5.5 g/L to about 6g/L,
about 5 g/L
to about 6g/L, about 4.5 g/L to about 6g/L, about 4 g/L to about 6g/L, about
3.5 g/L to
about 6g/L, about 3 g/L to about 6g/L, about 2.5 g/L to about 6g/L, about 3
g/L to about
5g/L, or about 3.5 g/L to about 4.5 g/L. In some embodiments, the oxidant is
present in an
amount of about 2g/L, about 2.5g/L, about 3g/L, about 3.5g/L, about 4g/L,
about 4.5g/L,
about 5g/L, about 5.5g/L, about 6 g/L.
CA 03168825 2022-07-20
WO 2021/150744
PCT/US2021/014401
In some embodiments, the oxidizing solution further comprises a buffer. In
some
embodiments, the first solution further includes a buffer. Any buffer suitable
for
maintaining the desired pH can be used. Non-limiting examples of such buffers
include
HC1-potassium chloride buffer, glycine-HC1 buffer, citrate buffer, and acetate
buffer. In
some embodiments, the first solution includes a buffer at a concentration of
about 0.1 M to
about 0.3 M. For example, about 0.1 M to about 0.15 M, about 0.1 M to about
0.2 M, about
0.1 M to about 0.25 M, about 0.25 M to about 0.3 M, about 0.2 M to about 0.3
M, about
0.15 M to about 0.3 M, about 0.15 M to about 0.2 M, about 0.18 M to about 0.22
M, or
about 0.2 M to about 0.25 M.
In some embodiments, the pH of the first solution is adjusted to be about 1 to
about
4. For example, about 1 to about 1.5, about 1 to about 2, about 1 to about
2.5, about 1 to
about 3, about 1 to about 3.5, about 3.5 to about 4, about 3 to about 4, about
2.5 to about
4, about 2 to about 4, about 1.5 to about 4, or about 1.5 to about 2.5. In
some embodiments,
the pH of the first solution is adjusted to be about 1, about 1.4, about 1.6,
about 1.8, about
2, about 2.2, about 2.4, about 2.6, about 3, about 3.5, or about 4.
In some embodiments, the temperature of the oxidizing solution is about 30 to
about
40. For example, about 30 C to about 35 C, about 35 C to about 40 C, or
about 33 C to
about 38 C. In some embodiments, the temperature of the oxidizing solution is
about
30 C, about 31 C, about 32 C, about 33 C, about 34 C, about 35 C, about 35 C,
about
36 C, about 37 C, about 238 C, about 39 C, or about 40 C.
In some embodiments, the step of drawing the treated fiber and oxidizing the
treated
fiber is repeated two or more times. For example, the step of drawing the
treated fiber and
oxidizing the treated fiber is repeated two, three, four, or five times. In
some embodiments,
the step of drawing the treated fiber and oxidizing the treated fiber is
repeated two times.
In some embodiments, the process further comprises drawing the treated fiber
prior to
setting the treated fiber.
Setting and enhancing the fiber
In some embodiments, setting the treated fiber comprises exposing the treated
fiber
to a wash solution comprising a surfactant. Non-limiting examples of suitable
surfactants
16
CA 03168825 2022-07-20
WO 2021/150744
PCT/US2021/014401
include ammonium lauryl sulfate, SDS, sodium laureth sulfate, sodium myreth
sulfate,
sodium stearate, sodium lauroyl sarcosinate, perfluorononanoate,
perfluorooctanoate, (3-
[(3 -chol ami dopropyl)dim ethyl amm oni 0] -1-prop ane sul fonate),
cocamidopropyl
hydroxysultaine, cocamidopropyl betaine, phosphatidylserine,
phosphatidylethanolamine,
phosphatidylcholine, a sphingomyelin, cetrimonium bromide (CTAB),
cetylpyridinium
chloride (CPC), benzalkonium chloride (BAC), benzethonium chloride (BZT),
dimethyldioctadecylammonium chloride, and dioctadecyldimethylammonium bromide
(DODAB). In some embodiments, the surfactant is present in an amount of about
0.2 to
about 2 g/L. For example, about 0.2 g/L to about 0.6 g/L, about 0.2 g/L to
about lg/L, about
0.2 g/L to about 1.4 g/L, about 0.2 g/L to about 1.8 g/L, about 1.6 g/L to
about 2 g/L, about
1.2 g/L to about 2 g/L, about 0.8 g/L to about 2 g/L, about 0.4 g/L to about 2
g/L, about 0.5
g/L to about 1.5 g/L, or about 0.8 g/L to about 1.2 g/L.
In some embodiments, the wash solution further comprises a buffer. Any buffer
suitable for maintaining the desired pH can be used. Non-limiting examples of
such buffers
include HC1-potassium chloride buffer, glycine-HC1 buffer, citrate buffer, and
acetate
buffer. In some embodiments, the wash solution includes a buffer at a
concentration of
about 0.05 M to about 0.3 M. For example, about 0.05 M to about 0.1 M, about
0.05 M to
about 0.15 M, about 0.05 M to about 0.2 M, about 0.05 M to about 0.25 M, about
0.25 M
to about 0.3 M, about 0.2 M to about 0.3 M, about 0.15 M to about 0.3 M, about
0.1 M to
about 0.3 M, about 0.15 M to about 0.2 M, about 0.18 M to about 0.22 M, or
about 0.2 M
to about 0.25 M.
In some embodiments, the pH of the wash solution is adjusted to be about 1 to
about
4. For example, about 1 to about 1.5, about 1 to about 2, about 1 to about
2.5, about 1 to
about 3, about 1 to about 3.5, about 3.5 to about 4, about 3 to about 4, about
2.5 to about
4, about 2 to about 4, about 1.5 to about 4, or about 1.5 to about 2.5. In
some embodiments,
the pH of the wash solution is adjusted to be about 1, about 1.4, about 1.6,
about 1.8, about
2, about 2.2, about 2.4, about 2.6, about 3, about 3.5, or about 4.
In some embodiments, the wash solution is at a temperature of about 35 C to
about
45 C. For example, about 35 C to about 40 C, about 40 C to about 45 C, or
about 38
C to about 42 C. In some embodiments, the wash solution is at a temperature
of about 35
17
CA 03168825 2022-07-20
WO 2021/150744
PCT/US2021/014401
C, about 36 C, about 37 C, about 38 C, about 39 C, about 40 C, about 41
C, about
42 C, about 43 C, about 44 C, or about 45 C.
In some embodiments, setting the treated fiber comprises winding the treated
fiber
and oxidizing the treated fiber.
In some embodiments, exposing the treated fiber to a wash solution comprising
a
surfactant is performed prior to winding the treated fiber and oxidizing the
treated fiber. In
some embodiments, winding the treated fiber is at a rate of about 15
meters/minute.
In some embodiments, the keratin fiber is dried at about 85 C for about 1
hour. In
some embodiments, the keratin fiber is annealed at about 125 C for about 1
hour. In some
embodiments, the keratin fiber is annealed after it is dried.
In some embodiments, the process further comprises exposing the keratin fiber
to
a solution comprising an oxidized saccharide. Non-limiting examples of
suitable
saccharides include glucose, sucrose, raffinose, cellobiose, dextran, and
alginate. A
saccharide can be oxidized using any of the oxidants described herein. In some
embodiments, the keratin fiber is exposed to the solution comprising an
oxidized
saccharide for about 3 to about 25 hours. For about 3 to about 5, about 3 to
about 10, about
3 to about 15, about 3 to about 25, about 20 to about 25, about 15 to about
25, about 10 to
about 25, about 5 to about 25. In some embodiments, the keratin fiber is
exposed to the
solution comprising an oxidized saccharide for about 1 hour, about 2 hours,
about 3 hours,
about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours,
about 9 hours,
about 10 hours, about 15 hours, or about 20 hours. In some embodiments, the
oxidized
saccharide is a sucrose polyaldehyde.
In some embodiments, the step of exposing the keratin fiber to a solution
comprising an oxidized saccharide is performed prior to exposing the treated
fiber to a
wash solution comprising a surfactant.
A keratin fiber produced using the processes described herein can have a high
draw
ratio. The draw ratio is the ratio of the drawing rate and extrusion rate. In
some
embodiments, the keratin fiber can have a draw ratio of at least about 500%,
e.g., at least
about 600%, about 700%, about 800%, about 900%, about 1000%, about 1100%,
about
1200%, about 1300%, about 1400%, about 1500%, about 1600%, about 1700%, about
18
CA 03168825 2022-07-20
WO 2021/150744
PCT/US2021/014401
1800%, about 1900%, or about 2000%. In some embodiments, a keratin fiber
produced
using the processes described herein can have a draw ratio of about 500% to
about 2500%.
For example, about 500% to about 2300%, about 500% to about 2100%, about 500%
to
about 1900%, about 500% to about 1700%, about 500% to about 1500%, about 500%
to
about 1300%, about 500% to about 1100%, about 500% to about 900%, about 500%
to
about 700%, about 2300% to about 2500%, about 2100% to about 2500%, about
1900%
to about 2500%, about 1700% to about 2500%, about 1500% to about 2500%, about
1300% to about 2500%, about 1100% to about 2500%, about 900% to about 2500%,
or
about 700% to about 2500%. In some embodiments, a keratin fiber produced using
the
processes described herein can have a draw ratio of about 500%, about 600%,
about 700%,
about 800%, about 900%, about 1000%, about 1100%, about 1200%, about 1300%,
about
1500%, about 1600%, about 17000%, about 1800%, about 1900%, about 2000%, about
2100%, about 2200%, about 2300%, about 2400%, or about 2500%.
In some embodiments, a keratin fiber produced using the processes described
herein can have a diameter of about 5 micrometers to about 30 micrometers. For
example,
about 5 micrometers to about 25 micrometers, about 5 micrometers to about 20
micrometers, about 5 micrometers to about 15 micrometers, about 5 micrometers
to about
10 micrometers, about 25 micrometers to about 30 micrometers, about 20
micrometers to
about 30 micrometers, about 15 micrometers to about 30 micrometers, or about
10
micrometers to about 30 micrometers.
Several processes can be used to assess the tenacity and strain of a fiber as
described
herein. Non-limiting examples of such processes include the ASTM standard D-
3822 and
the ISO 5079:1995. In some embodiments, the keratin fiber is equilibrated at
21 C and
65% relative humidity for 24 h prior to the test. In some embodiments, the
gauge length
and extension speed are 1 inch and 18 mm/min, respectively.
In some embodiments, a keratin fiber produced using the processes described
herein can have a tenacity at least about 0.8 g/den. For example, about at
least about 1
g/den, about 1.2 g/den, or about 1.4 g/den. In some embodiments, a keratin
fiber produced
as described herein can have a tenacity of about 0.8 g/den to about 2.5 g/den.
For example,
about 0.8 g/den to about 2.4 g/den, about 0.8 g/den to about 2.2 g/den, about
0.8 g/den to
19
CA 03168825 2022-07-20
WO 2021/150744
PCT/US2021/014401
about 2.0 g/den, about 0.8 g/den to about 1.8 g/den, about 0.8 g/den to about
1.6 g/den,
about 0.8 g/den to about 1.4 g/den, about 0.8 g/den to about 1.2 g/den, about
0.8 g/den to
about 1 g/den, about 2.4 g/den to about 2.5 g/den, about 2.2 g/den to about
2.5 g/den, about
2 g/den to about 2.5 g/den, about 1.8 g/den to about 2.5 g/den, about 1.6
g/den to about 2.5
g/den, about 1.4 g/den to about 2.5 g/den, about 1.2 g/den to about 2.5 g/den,
about or about
1 g/den to about 2.5 g/den.
In some embodiments, the keratin fiber has a strain of at least about 5%. For
example, at least about 5%, at least about 6%, at least about 7%, or at least
about 8%. In
some embodiments, the keratin fiber has a strain of about 10% to about 30%.
For example,
about 10% to about 15%, about 10% to about 20%, about 10% to about 25%, about
25%
to about 30%, about 20% to about 30%, or about 15% to about 30%. In some
embodiments,
the keratin fiber has a strain of 10%, about 11%, about 12%, about 13%, about
14%, about
15%, about 16%, about 17%, about 18%, about 19%, or about 20%. In some
embodiments,
the keratin fiber has been dried prior to measuring the strain.
Toughness of a keratin fiber described herein can be determined by measuring
total
area under the stress-strain curve of keratin fibers. Any method that can
obtain a stress-
strain curve can be used. In some embodiments, the keratin fiber is
equilibrated at 21 C
and 65% relative humidity for 24 h prior to the test. In some embodiments, the
gauge length
and extension speed are 1 inch and 18 mm/min, respectively. In some
embodiments, the
keratin fiber has a toughness of at least about 15 J/cm3. For example, at
least about 20
J/cm3, or at least about 25 J/cm3. In some embodiments, the keratin fiber has
a toughness
of about 15 J/cm3 to about 30 J/cm3.
Beta-sheet crystallinity of a keratin fiber and/or keratinous material can be
determined using, for example, X-ray diffraction. In some embodiments, the
keratin fiber
comprises at least 85% of the beta-sheet crystallinity compared to the amount
of beta-sheet
crystallinity in the keratinous material.
Disulfide bonds in a keratin fiber and/or keratinous material can be
determined
using, for example, using Raman spectroscopy. In some embodiments, the keratin
fiber
comprises at least 70% of the disulfide crosslinkages compared to the amount
of disulfide
crosslinkages in the keratinous material.
CA 03168825 2022-07-20
WO 2021/150744
PCT/US2021/014401
Also provided herein are keratin fibers prepared by any of the processes
described
herein. For example, provided herein is a keratin fiber prepared by extruding
a keratin
solution into a first solution to form a first fiber; drawing the first fiber
and oxidizing the
first fiber to form a treated fiber; drawing the treated fiber and oxidizing
the treated fiber
one or more times; and setting the treated fiber to form the keratin fiber. In
some
embodiments, the process is a continuous process. In some embodiments,
provided herein
is a keratin fiber prepared by extracting keratin from a keratinous material
to form extracted
keratin; dissolving the extracted keratin in an aqueous solution comprising a
reducing agent
to form the keratin solution; extruding a keratin solution into a first
solution to form a first
fiber; drawing the first fiber and oxidizing the first fiber to form a treated
fiber; drawing
the treated fiber and oxidizing the treated fiber one or more times; and
setting the treated
fiber to form the keratin fiber.
Additional details of the embodiments provided herein can be found, for
example,
in references 39 and 40, both of which are incorporated by reference in their
entireties
herein.
EXAMPLES
Example 1. Continuous production of keratin fiber from chicken feathers
Materials
Feather fiber corporation, Nixa, CO provided the chicken feather barbs. Other
chemicals, with ACS reagent grade, such as sodium dodecyl sulfate (SDS),
cysteine,
mercaptoethnaol and urea, were purchased from VWR International (Radnor, PA).
Chemical reagents used in SDS-PAGE analysis, including LDS sample buffer (4x),
Nupage 20x MES running buffer and NuPAGE 4-12% Bis-Tris gel, were purchased
from
Invitrogen, Inc., Grand Island, NY.
Keratin extraction from chicken feathers
The extraction of keratin from chicken feathers was conducted using an aqueous
solution containing different amounts of urea and SDS. To determine the
optimal recipe,
21
CA 03168825 2022-07-20
WO 2021/150744
PCT/US2021/014401
viscosity of supernatant of extraction and keratin yield were measured.
Detailed results are
in Table Si. 2 M urea and 10% SDS based on weight of feathers were chosen. 10%
of
cysteine was used to fully cleave the disulfide bonds in feathers for optimal
keratin
dissolution. According to the previous work, the pH of the extraction solution
was adjusted
to 10.5 using 15 wt% NaOH solution. The extraction was held for 12 hours at 70
C. After
the extraction, the dispersion was centrifuged at 9800 rcf for 20 minutes to
get the
supernatant, which was adjusted to the isoelectric point using hydrochloric
acid
accompanied by sodium sulfate to precipitate the keratin inside. The
precipitated keratin
was further washed to remove other impurities before being vacuumed dried.
Table Si. Viscosity and keratin yield from various extraction systems
Specific viscosity of
Extraction systems Yield
supernatant
8M urea 2.52 68.64 2.52%
4M urea 2.14 58.40 3.42%
10% SDS 1.46 31.20 2.51%
2M urea 5%SDS based on feather 1.72 48.50 2.43%
2M urea 8%SDS based on feather 2.01 61.80 3.56%
2M urea 10%SDS based on feather 2.52 66.90 3.14%
2M urea 15%SDS based on feather 2.73 67.14 2.97%
2M urea 20%SDS based on feather 3.04 67.51 3.25%
Preparation of keratin spinning dope
Spinning dope for continuous pilot-scale spinning was prepared by dissolving
27%
extracted keratin and 10% SDS based on weight of keratin in 0.2 M carbonate-
bicarbonate
22
CA 03168825 2022-07-20
WO 2021/150744
PCT/US2021/014401
buffer at pH of 8. To fully dissolve keratin and obtain the optimal molecular
entanglement
in solution, different amounts of a reducing agent (mercaptoethanol), were
added for
controlled cleavage of disulfide bonds in keratin molecules.
Stepwise oxidation and drawing were applied to continuous wet spinning lines
(ALEX JAMES AND ASSOC, US) for controlled assembly of disulfide bonds in
keratin
fibers. Stepwise oxidation and drawing can help improve the spinnability,
keratin
molecular alignment, degree of crosslinkages and fiber properties. Detailed
design is
demonstrated in FIG. 1. Prepared keratin spinning dope was centrifuged before
being
installed onto the spinning line. Keratin solution was extruded via a
spinneret with multiple
50 pm-diameter holes to the coagulation bath containing 15 wt% sodium sulfate,
5 wt%
zinc sulfate and acetate buffer with pH 2. Fibers from the coagulation bath
were drawn for
the first time before entering the first oxidation bath. The oxidation bath
contained 4 g/L
sodium periodate as the oxidation agents and acetate buffer with pH 2.
Oxidation
temperature was 35 C to ensure the fast disulfide bond assembly and fine fiber
stretchability. Fibers then went through multiple drawings and oxidations
before going to
the washing bath. Multiple oxidation and drawing steps can help the
establishment of
disulfide bonds and ordered structures in keratin fibers. The washing bath
contained
surfactants with concentrations from 1 g/L and acetate buffer with a pH of 2.
The
temperature was 40 C to ensure high washing efficiency. Reaching the final
winding
roller, the fibers went through the oxidation bath once again for the
immobilization of
ordered molecular structures in keratin fibers. The final speed of fiber
collecting was 15
meters/min. The dry fibers obtained were dried in an oven at 85 C for 1 h and
later
annealed at 125 C for about 1 h. Annealing was done to improve the mechanical
properties
of the fibers.
Characterizations
Rheological properties of spinning dope
Shear stress (T, Pa) of keratin spinning dopes with various concentrations of
reducing agents was measured using a rotational rheometer, R/S plus
(Brookfield, U.S.A.)
for determination of consistency coefficient (K, Pass") and flow behavior
index (n) based
23
CA 03168825 2022-07-20
WO 2021/150744
PCT/US2021/014401
on Equation 1. K is directly proportional to polymer viscosity in solution and
n indicates
the degree of molecular entanglement in solution. The smaller the value of n,
the better the
molecular entanglement. To precisely measure the rheological properties, 18
wt% of
keratin solution was used.
T=Kyn Equation 1
where y. is the shear rate (s-1) measured in the range of 0-1000 s-1.
Molecular weight of keratin backbones
About 1 mg of feather and keratin fiber was dissolved in 100 [EL of NuPAGE LDS
sample buffer (1 x ) with excess mercaptoethanol, heated at 70 C for 5 h. The
solution was
centrifuged prior to loading. Each sample of 10 [EL was loaded into an
individual slot of
the gel. A molecular marker from Spectra Multicolor Low Range Protein Ladder
was used.
The molecular weights of the protein standard mixture ranged from 4.6 to 42
kDa.
Mechanical properties
Keratin fibers were conditioned at 21 C and 65% relative humidity for 24 h
prior
to tests. Tensile properties of keratin fibers were obtained according to ASTM
standard D-
3822 using an Instron tensile testing machine (Norwood, MA). Gauge length set
for testing
was 1 inch and crosshead speed was 18 mm/min. Denier of fibers was used to
describe the
fineness of keratin fibers. For each test, at least 20 specimens were used.
Qualitative measurement of disulfide bonds in keratin using Raman Spectroscopy
Feathers and keratin fibers were characterized on a Raman spectrometer (The
DXR
Raman microscope, Thermo, USA). The laser wavelength was set at 532 nm with a
power
of 10 mW. The sample collect exposure time was 15 seconds with 15 cycles of
exposures
per sample. To compare the disulfide bonds in keratin, the ratio of peak areas
around 500
cm-1 (S-S) and 1450 cm-1 (C-H) was used.
Quantification of cystine in fibers
24
CA 03168825 2022-07-20
WO 2021/150744
PCT/US2021/014401
Fibers collected from each step of continuous spinning line were frozen
immediately after washing in distilled water and then freeze-dried. The
cystine content in
fibers was determined based on the process developed by Campanella et al [34].
In detail,
dried fibers were hydrolyzed using 6 N HC1 under 110 C for 24 hours to obtain
amino
acids. Phenylisothiocyanate was used for pre-column quantitative
derivatization of amino
acids by a HPLC, UltiMate 3000 series, USA equipped with a C-18 column
(Acclaim 120,
120 A, 4.6 x 250 mm, 5 [tm) and UV detector with wavelength set at 254 nm. The
flow
rate was 1 mL/min and a ternary gradient was employed using 0.7 M sodium
acetate with
pH 6.4 (phase A), water (phase B) and acetonitrile/water with volume ratio of
8:2 (phase
C). The gradient is shown in Table 1. Total retention time was 30 min with
additional 10
min for column re-equilibration.
Table 1. Gradient for HPLC analysis
Time Phase A Phase B Phase C
0 20 75 5
25 20 30 50
26 10 10 80
We used mercaptoethanol as the reducing agent in the spinning dope, therefore,
newly formed cystine on regenerated fibers could be considered as intra or
inter
molecular crosslinkings. In addition, cystine was formed during the fiber
drawing.
Drawing could increase the degree of linearity of molecular chains. Therefore,
majority
of cystine came from intermolecular crosslinking.
Analysis of secondary structures
X-ray diffraction and solid 13C NMR studies were carried out for secondary
structure analysis of feathers and keratin fibers. X-ray diffraction was
obtained using a
Rigaku D/Max-B X-ray diffractometer with Bragg¨Brentano parafocusing geometry,
diffracted beam monochromator, and conventional copper target X-ray tube set
(X, = 1.54
A) to 40 kV and 30 mA at 26 C. Diffraction intensities were recorded with 20
ranging
CA 03168825 2022-07-20
WO 2021/150744
PCT/US2021/014401
from 3 to 40 at a scan speed of 0.05 per second. The degree of
crystallinity was
calculated using Jade 6.0 software (Materials Data Incorporated: Livermore,
CA, USA)
with Gaussian peak fittings. The '3C solid-state NMR spectra were obtained
using a triple-
resonance (1H/13C/15N) magic angle spinning probe (3.2 mm) was equipped on the
NMR
spectrometer (Bruker, Avance 600, USA).
Statistical analysis
One-way analysis of variance using Scheffe test with a confidence interval of
95%
was used for all obtained data. The statistical analysis was conducted on SAS
9.4 software
(Cary, North Carolina) and survey procedures of PROC GLIMMIX.
Results and discussion
FIG. 2 compares molecular weights of protein backbones from regenerated
keratin
and the chicken feather. The results show that the damages to backbone of
regenerated
keratin is minimized. Compared to the chicken feather, contents of proteins
with 21 kDa
of regenerated keratin were slightly lower than that of the chicken feather
while contents
of protein with molecular weight from 8 kDa and12 kDa were higher than those
of chicken
feathers. The results in FIG. 2 also indicate that the regenerated keratin
contained a high
amount of y-keratin, which has a molecular weight of 11 kDa [35] and contains
high sulfur
content. y-keratin would facilitate formation of disulfide bonds in the
process of fiber
regeneration. The above results indicate that molecular structures in keratin
fibers could be
optimized via controlled cleavages and assembly of disulfide bonds on
continuous
production line. As a result, the final properties of keratin fibers were
improved.
Table 2. Rheological properties of 18% w/w keratin solution at 25 C
Concentration of reducing K value (Pass") n value
agent (wt% based on
keratin)
0.5 18.90 0.97
26
CA 03168825 2022-07-20
WO 2021/150744
PCT/US2021/014401
1 14.23 0.97
2 4.19 0.91
3 1.50 0.95
4 1.31 0.96
K is the consistency coefficient, directly proportional to polymer viscosity.
n is the flow
behavior index. The smaller value of n indicates the better molecular
entanglement.
Table 2 shows the effect of different concentrations of reducing agents on the
rheological properties of keratin spinning dope. The results show that
cleavage degrees of
disulfide linkages in the spinning solution directly affect the viscosity and
entanglement of
the keratin molecule in the solution. Specifically, as the concentration of
the reducing agent
in dope increased, the viscosity of the solution gradually decreased, but the
degree of
molecular entanglement increased first and then decreased. Increase in
concentrations of
reducing agent led to cleavage of disulfide bonds. As a result, as the
molecular weight of
keratin gradually decreased, so did the viscosity. In terms of degrees of
molecular
entanglement, when the amount of the reducing agent increased from 0.5% to 2%,
most
ordered structures became uncoiled as the cleavage of disulfide bonds, leading
to the better
solubility of keratin molecules. Therefore, the entanglement of molecules
increased. When
the concentration of the reducing agent continued to rise, the molecular
weight was further
lowered while the solubility of keratin remained unchanged. As a result,
degrees of
molecular entanglement decreased.
In order to ensure better spinnability of keratin fibers on a continuous
spinning line,
the disulfide bonds in keratin should be partially retained because of the
following three
benefits: 1) ensuring that the protein still has good solubility; 2) ensuring
that the protein
molecules are most entangled; and 3) keeping the molecular weight of the
protein high
enough to allow the spinning dope to be solidified rapidly in the coagulation
bath.
Therefore, 2% reducing agent was selected to partially cleave the disulfide
linkages in
spinning dope.
FIG. 3 shows the efficient recovery of disulfide bonds in keratin fibers via
controlled disulfide bond assembly on a continuous spinning line and resultant
spinnability
27
CA 03168825 2022-07-20
WO 2021/150744
PCT/US2021/014401
of keratin fibers. The results show that the rapid establishment of disulfide
crosslinkages
in continuous spinning is an important factor ensuring the good spinnability
of keratin
fiber. FIG. 3A qualitatively compares disulfide bonds in keratin fibers and
chicken
feathers. Results show that keratin fibers recovered high degree of disulfide
crosslinkages.
The peaks at about 500 and 1450 cm' were considered to be S¨S bonds and C¨H
bonds,
respectively. The Raman spectra were normalized according to the C¨H band,
whose peak
area was relatively large and not affected by the chemical treatments. Keratin
fibers had an
intensity of S¨S band slightly lower than chicken feathers, demonstrating high
recovery of
disulfide crosslinkages. Disulfide linkages were quantified using HPLC. FIG.
3B shows
that less than 10% of the crosslinkage bonds in fibers were recovered by
stretching in the
coagulation bath. After initial drawing and introduction of the first
oxidation bath, nearly
20% of the disulfide bonds in the fiber were recovered. As further
introductions of
oxidation baths accompanied by constant fiber drawing, the degree of disulfide
linkages
recovery could further increase. When fibers reached the final collection
roller on a
spinning line, nearly 70% of the disulfide bonds have been recovered, with a
degree of
crosslinking of about 5%. The rapid and high recovery in degrees of disulfide
linkages in
the continuous spinning process ensured good fiber spinnability. As shown in
FIG. 3C, the
spinnability of the fibers was substantially higher via fine control of
disulfide bond
assembly than that via non-control and simple control of disulfide bond
assembly. The only
oxidation process for non-control of disulfide bond assembly is the air
oxidation. Simple
control only contained single-step oxidation. Via controlled disulfide bond
assembly, the
final collection speed of the fiber can reach 15 m / min, 160% of spinning
using simple
control of disulfide bond assembly, 300% of spinning with the air oxidation.
FIG. 4A shows that recovery of degree of crosslinkages in the keratin fibers
determined the maximum draw ratio on the continuous spinning line. The results
show that
the high disulfide linkages recovery was the key to high drawing ratios of
fiber. A high
drawing ratio is useful for producing high-quality fibers. As shown in FIG.
4A, when the
degree of the disulfide bond recovery was less than 10%, the drawing ratio of
the fiber was
only 2 times. As recovery degree of disulfide bonds increased, the maximum
draw ratio of
the fiber can be increased to 10 times. FIG. 4A also shows that draw ratio of
fibers on
28
CA 03168825 2022-07-20
WO 2021/150744
PCT/US2021/014401
continuous spinning line had a linear relationship with the degree of recovery
of the
disulfide bond. Specifically Maximum drawing ratio = 0.2* Disulfide bond
recovery ratio
+ 0.5 with R2 0.98. The increase in draw ratio substantially reduced the
diameter of spun
fibers. As shown in FIG. 4B, fibers after 10 times of drawing had diameter of
only 15
which is lower than most natural wool fibers (30) and slightly larger than
silk fibers (10).
The fine fibers ensure a good hand, breathability and dyeability. The results
further indicate
that continuous production of quality keratin fibers was heavily dependent on
the rapid
formation of crosslinkages via controlled disulfide bond assembly. Without
controlled
disulfide bond assembly, the recovery degree of the disulfide bond was not
more than 20%,
resulting in a limited fiber draw ratio and subsequently poor fiber
properties.
FIG. 5 is the description of how high degree of ordered protein structures was
formed in keratin fibers via controlled disulfide bond assembly under external
stretch force.
The distance between protein backbones in newly solidified fiber can be
reduced to some
extent because of the existence of limited disulfide linkages inside. The
reduced distance
between protein backbones can facilitate the formation of intermolecular
disulfide bond
linkages during the first oxidation process. Then the formed crosslinkages in
oxidation bath
can help to improve the fiber stretchability and drawing ratios. High draw
ratio can
contribute to the linearity of molecular chains in keratin fibers and the
decrease in distance
between protein backbones. In turn, formation of intermolecular disulfide
bonds was
further facilitated. Via the controlled cleavage and assembly of disulfide
bonds,
intermolecular disulfide crosslinkages were gradually increased and secondary
structures
were gradually recovered in keratin fibers. At the last step of continuous
spinning,
collection step, fibers were once again oxidized to immobilize ordered
structures of keratin.
Recovered secondary structure in keratin fibers
Table 3. Comparison of secondary structures between chicken feather and
keratin fibers
Materials Degree of crystallinity
Total crystallinity Portion of a-helix Portion of 13-
sheet
Chicken feathers 31% 11% 20%
29
CA 03168825 2022-07-20
WO 2021/150744
PCT/US2021/014401
Keratin fibers 24% 5% 19%
The data obtained from XRD and '3C solid NMR. These spectra are shown in FIG.
8A and 8B, respectively. In XRD, two peaks, a minor peak at around 9 and a
major peak
at around 19 are used to analyze the secondary structure. In NMR, the
crystalline
structures of chicken feathers and keratin fibers were analyzed using the
chemical shift of
carbonyl groups. The deconvolution of carbonyl groups usually results in two
peaks at 176
ppm, attributed to a-helix and 172 ppm, attributed to both random coil and 13-
sheet
conformations.
Table 3 compares the secondary structure of keratin fibers with chicken
feathers.
The results showed the beta-sheet secondary structure and total crystallinity
of keratin
fibers recovered 95% and 80%, respectively. The high crystallinity in keratin
fiber is due
to the high degree of ordered structures resulting from fast controlled
disulfide bonds
cleavages and assembly. The reason for high degree of beta-sheet recovery is
as below.
Controlled disulfide assembly contributed to the high stretchability of fibers
and increase
in the fiber drawing ratios. As a result, a portion of the alpha-helix
structures in the fiber
transformed into beta-sheet structures. The degree of crystallinity in keratin
fiber was lower
than that of the original chicken feathers mainly because the disulfide bond
in the chicken
feathers cannot be completely recovered, and the ordered structures in keratin
fibers were
less than those in chicken feathers. In addition, slight damages on keratin
backbone also
contributed to the lower degree of crystallinity in keratin fibers.
FIG. 6A shows the continuous production of keratin fiber was achieved via
controlled cleavage and assembly of disulfide bonds. FIG. 6B shows a specimen
of
continuously spun keratin fibers. FIG. 7A compares stress-strain curves of the
original
feathers and keratin fibers from continuous spinning. The results show that
although the
feather barbs had a strain of about 10%, the feather barbs demonstrated a
curve of brittle
pattern. However, keratin fibers underwent a "strain hardening" stage before
break. The
reason is that chicken feathers had a high degree of cross-linkages and
ordered molecular
structure, providing a strong interaction between intermolecular chains.
Therefore, under
external forces, the molecular segment was unlikely to have dislocation. As a
result, fibers
CA 03168825 2022-07-20
WO 2021/150744
PCT/US2021/014401
broken before yielding point. In keratin fibers, interaction between molecular
chains was
relatively weak but most of the protein backbones were connected via
controlled recovery
of disulfide crosslinkages. Weak interaction can help the movement of protein
chains while
long molecular chains could help increase the slip distance between two
molecular chains.
Under the external force, dislocation was likely to happen between molecular
segments,
resulting in the strain hardening. In addition, the keratin fibers were
through high drawing
ratios, leading to the molecular chains in stretched state. Therefore, the
degree of molecular
entanglement was high. Under the external force, the overall orientation of
the molecular
segment in fibers would increase, contributing to the strain hardening.
Keratin fibers possessed high ductility due to the strain hardening process.
FIG. 7B
shows that keratin fibers can endure a high degree of twisting. The other two
Supporting
videos further demonstrate the high ductility of keratin fiber. The fiber
ductility was high
because of the high toughness of the fiber, stemming from substantial
restoration of the
secondary protein structures via controlled disulfide bond cleavage and
assembly in the
continuous spinning process.
Table 4. Mechanical properties of keratin fibers compared to other common
fibers.
Fiber Dry state Wet state
source Strength Strain Toughness Strength Strain
Toughness
(Mpa) (J/cm3) (Mp a) (J.cm3)
Feather 161 27.5 9.7 3.3% 21 2 127 24.5 18 3% 31.5 3
barbs
Keratin 138 30.5 11 2.8% 18.5 2 81 23 25 3% 28.7 4
fibers
Wool 173 23 36 5% 32 5 140 22 46 5% 36 4
Cotton13 420 46 6.2 1.5% 10.5 5 472 34 9 1% 24.6 6
Linen 700 45 3.1 0.4% 5 1 800 40 5 1% 7 1
Viscose13 276 20 21 5.2% 24 6 120 20 25 4% 21.1 4.3
Our work is shown in bold font.
31
CA 03168825 2022-07-20
WO 2021/150744
PCT/US2021/014401
Table 4 shows the mechanical properties of the fibers with restored secondary
protein structures via controlled cleavage and assembly of disulfide bonds in
the
continuous production process and compares properties with other commonly
fibers. The
results show that keratin fibers recovered 86% of stress properties at dry
state, 64% of wet
stress, 89% of dry toughness and 91.55 of wet toughness of original chicken
feathers. The
good properties of the feathers were preserved. Keratin fibers with restored
secondary
structures had a slightly lower strength than feather barbs because of damages
of the protein
backbone and a decrease in the degree of disulfide crosslinkages. The strain
of keratin
fibers was slightly higher than the original feathers because of easier
dislocation of the
protein segments. Compared with other commonly used fibers, keratin fibers had
merits.
For example, keratin strain and toughness were substantially higher than
cotton and linen.
The toughness was close to viscose fibers. The above results show that keratin
fibers with
restoration of secondary structures from continuous production meet the
specificaitons for
actual uses.
Table 5. Comparison of properties of regenerated keratin fibers developed from
various
approaches.
Continuous
Properties
Approach to regeneration
production on pilot Reference
recovery
scale
Controlled disulfide cleavage 86% of tenacity This
Yes
and assembly 113% of strain work
48% of tenacity
Control of disulfide cleavages No [28]
41% of strain
14% of tenacity
Regeneration from ionic liquids Strain not No [36]
reported
4% of tenacity
Applied glycerol as plasticizer No [37]
280% of strain
32
CA 03168825 2022-07-20
WO 2021/150744
PCT/US2021/014401
Blend poly(ethylene oxide) 3% of tenacity
No [38]
(containing 10% keratin) 1100% of strain
Property recovery based on the original keratin materials, such as feathers
and wool.
Table 5 compares the properties of keratin fibers from various regeneration
approaches. All keratin fibers except this work were regenerated on a lab-
scale. Results
show that due to the low recovery of secondary structures, tenacity recovery
was low, with
the highest was less than 50%. With poor recovery of secondary structure, the
strain of
regenerated fibers was even poor. To increase the strain, incorporation of
plasticizer or
other polymers into keratin was developed. As a result, the strain increases
at the cost of
sacrifice of fiber tenacity. For example, after incorporation of glycerol, the
tenacity of
regenerated keratin fibers was only 4% of raw fibers.
Cost-effective production with minimal environmental impact
Continuous fiber production via controlled cleavage and assembly of disulfide
bonds used non-toxic chemicals. Furthermore, baths such as coagulation and
oxidation on
spinning lines could be reused. Therefore, the discharges of continuous
spinning have
minimal discharges. Keratin content in continuous spun fibers was higher than
98%,
indicating the full degradability of fibers. Our process to produce keratin
fibers from
chicken feathers does not contain toxic chemicals that impose negative impacts
on the
environment. In order to estimate market potential of the regenerated keratin
fibers, the
material consumption and costs in our process are assessed, as shown in Table
S2.
Table S2. Material cost to produce lkg of pure keratin fibers
Unit Consumption to Total
Materials &
price produce 1 kg of Cost ($)
material
Chemicals
($/kg) t keratin fibers (kg) cost ($)
Extraction Chicken feathers 0 11 1.33 0
0.83
Cysteine 5.5 0.09 0.495
33
CA 03168825 2022-07-20
WO 2021/150744
PCT/US2021/014401
Urea & SDS 0.39 0.28 0.11
Alkali 0.2 0.20 0.040
Hydrochloric acid
0.10 0.07 0.007
(31%)
Sodium sulfate 0.06 0.08 0.005
Sodium carbonate 0.13 0.016 0.002
Sodium dodecyl
0.9 0.05 0.045
sulfate
Mercaptoethanol 4 0.02 0.08
Spinning Acetic acid 0.2 0.02 0.004
Sodium sulfate 0.17 0.15 0.003
Zinc sulfate 0.62 0.05 0.03
Oxidants 0.8 0.004 0.003
Surfactant 0.3 0.001 0.003
Calculation is based on our previous urea-cysteine based extraction method
t All the prices of chemicals were obtained from Alibaba.com or 1688.com
(accessed on 9/30/2019).
11 Chicken feathers are deemed as wastes and thus could be obtained at no
cost.
As shown in Table S2, the total material cost to produce 1 kg of pure keratin
fibers
is about $0.83. Since it is unable for us to obtain the material cost of
commercial protein
fibers and compare with that of the regenerated keratin fibers, the retail
prices of
commercial proteins such as wools and silk are used. Comparing to the bulk
price (metric
ton scale) of wool at about $7-30/kg and silk at about $45-80/kg, keratin
fibers from
chicken feathers have its cost at least 91% and 99% lower than sale prices.
Considering
34
CA 03168825 2022-07-20
WO 2021/150744
PCT/US2021/014401
other costs in large-scale production, the final price of keratin fibers from
chicken feathers
will be competitive. If keratin fibers from poultry feathers are sold at about
$4 per kg,
which is close to some natural cellulose fibers like linen, a ton of poultry
feathers will
produce fibers worth at least $3,000. If the 5 million tons of poultry
feathers worldwide
can be fully exploited, the market value of regenerated keratin fibers will
exceed $15
billion.
Example 2. Chemical crosslinking using oxidized saccharides
Five percent of sucrose in water reacted with sodium periodate at room
temperature
for 5 h. The molar ratio of sucrose to periodate was 1:3. The pH of reaction
medium was
kept at 5.5 0.1. After the reaction, slightly excessive barium dichloride was
added to
completely precipitate the oxidation agents. The mixture was filtrated to
obtain the
polyaldehyde derivatives of sucrose. Spun fibers from chicken feathers were
dipped in
solutions containing sucrose polyaldehydes for 5 h at room temperature before
the washing
process. After washing, fibers were dried in an oven at 85 C for 1 h and
later annealed at
125 C for about 1 h. The stress and strain of obtained fibers were 1.5 0.2
g/den and
16 2.1%.
REFERENCES
1. Y. Zhang, N. Du, Y. Chen, Y. Lin, J. Jiang, Y. He, Y. Lei and D. Yang,
Nanoscale, 2018, 10, 5626-5633.
2. A. G. Nandgaonkar, Q. Wang, K. Fu, W. E. Krause, Q. Wei, R. Gorga and L.
A.
Lucia, Green Chemistry, 2014, 16, 3195-3201.
3. Y. Li, H. Liu, J. Song, 0. J. Rojas and J. P. Hinestroza, ACS applied
materials &
interfaces, 2011, 3, 2349-2357.
4. M. Rabnawaz, I. Wyman, R. Auras and S. Cheng, Green Chemistry, 2017, 19,
4737-4753.
CA 03168825 2022-07-20
WO 2021/150744
PCT/US2021/014401
5. L. Lin, J. Liang, Y. Xu, S. Wang, M. Xiao, L. Sun and Y. Meng, Green
Chemistry, 2019, 21, 2469-2477.
6. S. Raj endran, R. Raghunathan, I. Hevus, R. Krishnan, A. Ugrinov, M. P.
Sibi, D.
C. Webster and J. Sivaguru, Angewandte Chemie International Edition, 2015, 54,
1159-1163.
7. J. Christensen, 'It is raining plastic': Scientists find colorful
microplastic in rain,
cnn.com/2019/08/14/health/plastic-rain-colorado-trnd/index.html).
8. M. Bergmann, S. Miltzel, S. Primpke, M. B. Tekman, J. Trachsel and G.
Gerdts,
Science Advances, 2019,5, eaax1157.
9. L. Parker, Nat. Geogr. Mag. Online, 2018.
10. Z. Singh and S. Bhalla, Adv Res Text Eng, 2017,2, 1012.
11. Z. Yang, A. Kumar, A. W. Apblett and A. M. Moneeb, Green Chemistry,
2017,
19, 757-768.
12. Industrievereinigung-Chemiefaser, Worldwide production volume of
chemical
and textile fibers from 1975 to 2016 (in 1,000 metric tons), statista-
com.libproxy .unl . edu/stati stics/263154/worldwide-production-volume-of-
textile-
fibers-since-1975/ (accessed 7/7, 2018).
13. N. Reddy and Y. Yang, Green Chemistry, 2005, 7, 190-195.
14. G. Gao, M. A. Karaaslan, J. F. Kadla and F. Ko, Green Chemistry, 2014,
16,
3890-3898.
15. B. Mu, H. Xu, W. Li, L. Xu and Y. Yang, Food Hydrocolloids, 2019, 90,
443-
451.
16. B. Mu, W. Li, H. Xu, L. Xu and Y. Yang, Journal of Materials Chemistry
A,
2018, 6, 10320-10330.
17. F. Allievi, M. Vinnari and J. Luukkanen, Journal of Cleaner Production,
2015,
92, 142-151.
18. L. Gao, H. Hu, X. Sui, C. Chen and Q. Chen, Environmental science &
technology, 2014, 48, 6500-6507.
19. N. Reddy, L. Chen, Y. Zhang and Y. Yang, Journal of Cleaner Production,
2014,
65, 561-567.
36
CA 03168825 2022-07-20
WO 2021/150744
PCT/US2021/014401
20. A. J. Poole, J. S. Church and M. G. Huson, Biomacromolecules, 2008, 10,
1-8.
21. K. M. ARAI, R. TAKAHASHI, Y. YOKOTE and K. AKAHANE, European
journal of biochemistry, 1983, 132, 501-507.
22. C. Tonin, A. Aluigi, C. Vineis, A. Varesano, A. Montarsolo and F.
Ferrero,
Journal of thermal analysis and calorimetry, 2007, 89, 601-608.
23. K. Ziegler, Journal of Biological Chemistry, 1964, 239, PC2713-PC2714.
24. W. Crewther, L. Dowling, A. Inglis and J. Maclaren, Textile research
journal,
1967, 37, 736-745.
25. N. Singh and K. Prasad, Green Chemistry, 2019.
26. S. Zheng, Y. Nie, S. Zhang, X. Zhang and L. Wang, ACS Sustainable
Chemistry
& Engineering, 2015, 3, 2925-2932.
27. K. Kammiovirta, A.-S. Jaaskelainen, L. Kuutti, U. Holopainen-Mantila,
A.
Paananen, A. Suurnakki and H. Orelma, RSC Advances, 2016, 6, 88797-88806.
28. H. Xu and Y. Yang, ACS Sustainable Chemistry & Engineering, 2014, 2,
1404-
1410.
29. R. Wormell and F. Happey, Nature, 1949, 163, 18.
30. J. G. Rouse and M. E. Van Dyke, Materials, 2010, 3, 999-1014.
31. Y. Li, X. Zhi, J. Lin, X. You and J. Yuan, Materials Science and
Engineering: C,
2017, 73, 189-197.
32. N. V. Patil and A. N. Netravali, ACS Omega, 2019, 4, 5392-5401.
33. J. Li, Y. Li, L. Li, A. F. Mak, F. Ko and L. Qin, Polymer Degradation
and
Stability, 2009, 94, 1800-1807.
34. L. Campanella, G. Crescentini and P. Avino, Journal of Chromatography
A,
1999, 833, 137-145.
35. T. Posati, D. Giuri, M. Nocchetti, A. Sagnella, M. Gariboldi, C.
Ferroni, G.
Sotgiu, G. Varchi, R. Zamboni and A. Aluigi, European Polymer Journal, 2018,
105, 177-185.
36. X. Fan, 2008.
37. B. Ma, X. Qiao, X. Hou and Y. Yang, International journal of biological
macromolecules, 2016, 89, 614-621.
37
CA 03168825 2022-07-20
WO 2021/150744
PCT/US2021/014401
38. A. Aluigi, C. Vineis, A. Varesano, G. Mazzuchetti, F. Ferrero and C.
Tonin,
European Polymer Journal, 2008, 44, 2465-2475.
39. Mu, B.N., Hassan, F., and Yang, Y.Q. Controlled assembly of secondary
keratin
structures for continuous and scalable production of tough fibers from chicken
feathers. Green Chemistry. 22(5), 1726-1734 (2020).
40. Mi, X., Mu, B.N., Li, W., Xu, H.L. and Yang*, Y.Q. From poultry wastes
to
quality protein products via restoration of secondary structure with extended
disulfide linkages. ACS Sustainable Chemistry & Engineering. 8(3). 1396-1405
(2020).
OTHER EMBODIMENTS
It is to be understood that while the invention has been described in
conjunction
with the detailed description thereof, the foregoing description is intended
to illustrate and
not limit the scope of the invention which is defined by the scope of the
appended claims.
Other aspects, advantages, and modification are within the scope of the
following claims.
38
