U.S. patent application number 12/252243 was filed with the patent office on 2009-03-05 for production of bypolymer film, fibre, foam and adhesive materials from soluble s-sulfonated keratin derivative.
This patent application is currently assigned to Keratec Limited. Invention is credited to Warren Glenn Bryson, Robert James Kelly, Douglas Alexander Rankin, Alisa Dawn Roddick-Lanzilotta.
Application Number | 20090062513 12/252243 |
Document ID | / |
Family ID | 19928462 |
Filed Date | 2009-03-05 |
United States Patent
Application |
20090062513 |
Kind Code |
A1 |
Kelly; Robert James ; et
al. |
March 5, 2009 |
PRODUCTION OF BYPOLYMER FILM, FIBRE, FOAM AND ADHESIVE MATERIALS
FROM SOLUBLE S-SULFONATED KERATIN DERIVATIVE
Abstract
Film, fibre, foam and adhesive materials are produced from
soluble S-sulfonated keratins. Once formed, the films, fibres,
foams or adhesives are treated to modify the properties of the
materials, in particular to improve the wet strength of the
materials. Treatments used include removal of the S-sulfonate group
by treatment with a reducing agent, treatment with an acid or
treatment with a common protein crosslinking agent or treatment
with a reduced form of keratin or keratin protein. The films are
made by solvent casting a solution of S-sulfonated keratin
proteins, the foam made by freeze-drying a solution of S-sulfonated
keratin proteins and the fibres made by extruding a solution of a
S-sulfonated keratin protein.
Inventors: |
Kelly; Robert James;
(Christchurch, NZ) ; Roddick-Lanzilotta; Alisa Dawn;
(Canterbury, NZ) ; Rankin; Douglas Alexander;
(Christchurch, NZ) ; Bryson; Warren Glenn;
(Christchurch, NZ) |
Correspondence
Address: |
OFFICE OF NAVAL RESEARCH
OFFICE OF COUNSEL, 875 NORTH RANDOLPH STREET
ARLINGTON
VA
22203-1995
US
|
Assignee: |
Keratec Limited
Canterbury
NZ
|
Family ID: |
19928462 |
Appl. No.: |
12/252243 |
Filed: |
October 15, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10488188 |
Mar 1, 2004 |
7465321 |
|
|
12252243 |
|
|
|
|
Current U.S.
Class: |
530/350 ;
264/202; 264/204 |
Current CPC
Class: |
Y10T 428/14 20150115;
Y10T 428/2852 20150115; C08H 1/06 20130101; Y10T 428/28 20150115;
D01F 4/00 20130101; C08J 9/28 20130101; C08J 2201/048 20130101;
C08J 2389/00 20130101; C08J 5/18 20130101 |
Class at
Publication: |
530/350 ;
264/202; 264/204 |
International
Class: |
C07K 14/00 20060101
C07K014/00; D01F 4/00 20060101 D01F004/00 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 31, 2001 |
NZ |
511492 |
Claims
1-12. (canceled)
13. A method for making protein fibres, the method comprising the
step of extruding a solution of S-sulfonated keratins into an
aqueous solution containing salts and a reductant that causes the
S-sulfonated keratins in solution to become insoluble.
14. The method as claimed in claim 13 wherein the reductant is a
thoil or thioglycollate salt.
15. A method for making protein fibres, the method comprising the
step of extruding a solution of S-sulfonated keratins into an
aqueous solution containing salts, a reductant and a crosslinking
agent that causes the S-sulfonated keratin in solution to become
insoluble.
16. The method as claimed in claim 15 wherein the reductant is
thiol or thioglycollate salt.
17. The method as claimed in claim 15 wherein the crosslinking
agent is formaldehyde.
18. A method for making protein fibres, the method comprising the
step of extruding a solution of S-sulfonated keratins into an
aqueous solution containing salts and an acid that causes the
protein S-sulfonated keratins in solution to become insoluble.
19. The method as claimed in claim 18 wherein the acid is sulfuric
acid.
20. A method for making protein fibres, the method comprising the
step of extruding a solution of S-sulfonated keratins in a solvent
into a hot environment and evaporating away the solvent to leave a
fibrous material behind.
21-41. (canceled)
42. The method as claimed in claim 16 wherein the crosslinking
agent is formaldehyde.
43. The method as claimed in claim 20 further comprising the step
of treating the fibrous material with a reductant.
44. The method as claimed in claim 43 wherein the reductant is a
thiol or a phosphine.
45. The method as claimed in claim 20 further comprising the step
of treating the fibrous material with a reduced form of keratin or
a reduced form of keratin peptide.
46. The method as claimed in claim 20 further comprising the step
of treating the fibrous material with an acid to protonate
S-sulfonate groups within the S-sulfonated keratin protein and any
other polar groups.
47. The method as claimed in claim 20 further comprising the step
of treating the fibrous material with formaldehyde or
glutaraldehyde.
48. A fibre produced by a method comprising the step of extruding a
solution of S-sulfonated keratins into an aqueous solution
containing salts, a reductant and a crosslinking agent that causes
the S-sulfonated keratin in solution to become insoluble.
49. The fibre as claimed in claim 48 wherein the reductant is thiol
or thioglycollate salt.
50. The fibre as claimed in claim 48 wherein the crosslinking agent
is formaldehyde.
51. A fibre produced by a method comprising the step of extruding a
solution of S-sulfonated keratins into an aqueous solution
containing salts and an acid that causes the S-sulfonated keratins
in solution to become insoluble.
52. The fibre as claimed in claim 51 wherein the acid is sulfuric
acid.
53. A fibre produced by a method comprising the step of extruding a
solution of S-sulfonated keratins in a solvent into a hot
environment and evaporating away the solvent to leave a fibrous
material behind.
Description
FIELD OF THE INVENTION
[0001] This invention relates to the preparation and use of soluble
keratin derivatives in the production of a range of biopolymer
materials such as films, fibres, foams and adhesives, and the
improvement of those materials using further chemical
treatments.
BACKGROUND TO THE INVENTION
[0002] Keratins are a class of structural proteins widely
represented in biological structures, especially in epithelial
tissues of higher vertebrates. Keratins may be divided into two
major classes, the soft keratins (occurring in skin and a few other
tissues) and hard keratins, forming the material of nails, claws,
hair, horn and (in birds and reptiles) is feathers and scales.
[0003] The hard keratins may in turn be further subdivided into
structural types described as .alpha.-keratin, .beta.-keratin, or
feather keratin. Keratins of the .alpha. and .beta. types have
different predominant structural motifs in their proteins, in the
former case supramolecular structures based on the .alpha.-helix
secondary structure of protein chains, and in the latter case on
the .beta.-pleated sheet motif.
[0004] All keratins are characterised by a high level of the
sulphur-containing diamino-acid cystine, which acts as a
cross-linking point between protein chains. This feature of a
high-level of interchain crosslinking through cystine gives the
keratins, especially the hard keratins, their characteristics of
toughness, durability, resistance to degradation, and desirable
mechanical properties. Cystine contents vary widely in the
keratins, which is reflected in their variation in mechanical
properties.
[0005] Wool and hair are examples of hard .alpha.-keratin. However,
even in a given .alpha.-keratin, there are many classes of
structural protein present, and the mechanical properties arise
from a sophisticated supramolecular organisation of proteins of
many different types to create a complex morphology with a
correspondingly complex mechanical behaviour.
[0006] An object of the invention is to provide biopolymer
materials derived from soluble keratin derivatives and production
methods for producing the biopolymer materials.
SUMMARY OF THE INVENTION
[0007] According to a broadest aspect of the invention there are
provided materials derived from S-sulfonated keratin proteins, as
herein defined, in the form of films, fibres, foams or adhesives.
The S-sulfonated keratin proteins can be derived from wool keratin
and be enriched in intermediate filament protein(s).
[0008] According to another aspect of the invention there is
provided a process method for the formation of films from
S-sulfonated keratin proteins in which a solution of the proteins
is cast and the solution solvents evaporated to leave a protein
film.
[0009] The solution(s) used can be aqueous based, including some
proportion of organic solvents.
[0010] The films produced by this process method are inherently
soluble in water or the solvent mix used for casting the film.
[0011] Another aspect of the invention describes a method for
improving the wet strength of films, produced by the process
method, by using chemical agents, such as thiols and phosphines,
that remove the sulfonate group and allow the formation of
disulfide bonds within the protein film. The difsulfide bonds
provide the film with wet strength.
[0012] Another method of improving the wet strength of a film,
produced by the process method, is described in which acidic
solutions are used to treat the protein film, and through a process
of protonation of the sulfonate groups and any other suitable polar
groups within the protein, the film becomes insoluble in water and
has significant wet strength.
[0013] Another aspect of the invention describes introduction of
crosslinks into a film, produced by the process method, through the
use of crosslinking agents such as those commonly used in protein
modifications, that target a range of functional groups present
within the protein.
[0014] A further aspect of the invention is a method for the
production of protein films using a solution comprising a
combination S-sulfonated keratin proteins and reduced keratin
proteins or peptides containing reactive cysteine residues. The two
species combine to form a crosslinked keratin network and
subsequently a protein film with good wet strength properties. This
approach of combining S-sulfonated and reduced keratins can also be
applied to the production of keratin fibres, foams and
adhesives.
[0015] A further aspect of the invention is a method for the
production of keratin fibres through the extrusion of a solution
comprising of S-sulfonated keratin proteins through a spinnerette
into a coagulation bath that causes the protein to become
insoluble. In particular the coagulation bath may contain
reductants, such as thiols or phosphines, that cause the removal of
the sulfonate group from the protein and lead to disulfide groups
forming. In addition the coagulation bath can contain crosslinking
agents, such as formaldehyde or glutaraldehyde, which cause the
protein(s) to become insoluble on contact with the coagulation
bath. In addition the coagulation bath can be at acidic pH, which
also causes the protein solution to become insoluble.
[0016] A further aspect of the invention is a method for the
production of keratin fibres through the extrusion of a solution
comprising of S-sulfonated keratin proteins through a spinnerette
into a hot environment through which the solvent is rapidly removed
and a fibrous keratin material remains. Fibres produced in this way
can be further processed through wet chemical treatments to improve
the wet strength of the fibres through the formation of crosslinks,
or by protonation of the protein in manners similar to those
described above for keratin films.
[0017] A further aspect of the invention is a method for the
production of keratin foams through the freeze drying of a solution
of S-sulfonated keratin proteins. Foams produced in this way can be
modified using similar methods to those described for keratin
films, that is through the use of a reductant such as a thiol or
phosphine to remove the S-sulfonate group, through the use of
reduced keratin proteins or peptides to remove the S-sulfonate
group, through the use of an acidic solution to protonate the
S-sulfonate group and the protein, or through the use of
crosslinking agents such as formaldehyde and glutaraldehyde to
modify the protein.
[0018] A further aspect of the Invention is a range of keratin
based adhesives, comprising at least in part a solution of
S-sulfonated keratin proteins. These adhesives can be made to have
superior wet strength properties through the use of reducing
agents, such as thiols or phosphines. Alternatively wet strength
can be imparted through the use of a reduced keratin protein or
reduced keratin peptide, to create a crosslinked keratin network.
These two sets of reagents can form a `two pot` adhesive.
[0019] The flexibility of the films, fibres, foams and adhesives
produced by the methods described can be modified through the use
of plasticizers such as those from the glycerol or polyethylene
glycol families.
[0020] According to further aspect of the invention there is
provided a film, fibre, foam or adhesive material derived from
keratin derivates of high molecular weight as described and claimed
in PCT/NZ02/00125 whereby the process includes a first stage
digestion is step of sulfonating a keratin source by oxidative
sulfitolysis followed by a second stage repetitive aqueous
extraction involving separation of soluble and insoluble keratin
and subsequent re-extraction of the insoluble keratin to thereby
produce a highly S-sulfonated keratin derivative. The protein
keratin source can be a naturally occurring protein source.
[0021] According to yet a further aspect of the invention there is
provided a film, fibre, foam or adhesive material derived from
either highly S-sulfonated keratin intermediate filament proteins,
soluble keratin peptides or a purified protein with little or no
damage to the structural integrity of the protein as produced from
an impure protein source as described above.
[0022] According to yet a further aspect of the invention there is
provided a combination of engineering solutions to produce a film,
fibre, foam or adhesive material derived from S-sulfonated keratin
proteins.
[0023] According to yet another aspect of the invention there is
provided a film, fibre, foam or adhesive material obtained from a
protein produced from a large scale recovery method as described
and claimed in PCT/NZ02/00125.
DESCRIPTION OF PREFERRED EXAMPLES
[0024] The features of this invention specifically cite some
methods and applications based on hard .alpha.-keratins from wool.
However, the principle can equally well apply to alternative
.alpha.-keratins, or any source of keratin which is able to yield
proteins of the intermediate filament (IF) type.
[0025] Similar preparative methods have been applied by the
applicants to other keratin sources such as feathers, to produce
materials equally well suited for some of the applications
described below. The features of this invention are intended to
cover the utilisation of such keratins as well, in applications
which are not dependent on the presence of proteins of the
.alpha.-type (IF proteins). This includes applications where
preparations based on .beta. or feather keratin may be combined
with IF proteins.
[0026] The characteristics of toughness and insolubility typical of
hard keratins are desirable properties in many industrial
materials. In addition, keratin materials are biodegradable and
produced from a sustainable resource and as such they have
significant potential for use as a substitute for oil-based
polymers in many applications, such as films, fibres and adhesives.
Their use in cosmetics and personal care applications is already
well established and an extension to medical materials is proposed
using materials such as those outlined in this specification.
[0027] Wool represents a convenient source of hard
.alpha.-keratins, although any other animal fibre, or horns, or
hooves, would serve equally well as a source of the desired
proteins. Wool is composed of approximately 95% keratin, which can
be broadly divided into three protein classes. The intermediate
filament proteins are typically of high molecular weight (45-60
kD), with a partly fibrillar tertiary structure and a cysteine
content of the order of 6%. They account for approximately 58% of
the wool fibre by mass although only part of this mass is actually
helix-forming in structure. The high- and ultra-high-sulphur
proteins, approximately 26% of the wool fibre, are globular in
structure, have a molecular weight range of 10-40 kD and can
contain cysteine levels up to 30 mol %. The high-glycine-tyrosine
proteins are a minor class comprising 6% of the wool fibre, have
molecular weights of the order of 10 kD and are characterised by
their high content of glycine and tyrosine amino acid residues.
[0028] Proteins from the different classes of wool keratins possess
characteristics that will give them unique advantages in specific
applications.
[0029] This invention pertains largely to the use of intermediate
filament proteins, and the use of them to produce films, fibres,
foams and adhesives.
[0030] Nonetheless the other non-fibrillar proteins have
applications in their own right in more restricted fields.
[0031] Likewise feather keratins, derived by extractive procedures
similar to those applied to wool, have specific valuable
applications in certain areas as defined below, but do not contain
the IF proteins deemed to be desirable in some end-uses.
[0032] The soluble keratin derivatives used In the method and
subsequent chemical treatments described in this specification were
obtained from wool or feathers either by reduction using sodium
sulphide or by oxidative sulphitolysis. An example of process for
the production of soluble keratin derivates is described in the
applicant's PCT/NZ02/00125 patent specification, the description of
which is incorporated herein by way of reference and outlined
above. The reduction of wool or feather keratin using sodium
sulphide involves dissolution in a dilute sodium sulphide solution
(or other sulphide solution). The combination of high solution pH
and sulphide ion concentration results in the keratin being
degraded to some extent, with possible hydrolysis of some of the
peptide bonds occuring, as well as the disulphide bonds being
reduced to yield protein rich in thiol and polysulphide
functionality. The rich thiol function of the isolated protein can
be confirmed using reagents such as nitroprusside. Oxidative
sulfitolysis involves the conversion of the cysteine in keratin to
S-sulfocysteine by the action of sodium sulphite and an oxidant, No
peptide hydrolysis occurs and the solubilised keratin has a
molecular weight distribution very similar to that in the
unkeratinised state. Proteins derivatised in this way are referred
to herein as S-sulfonated keratin proteins throughout the process
methods, and are isolated from an oxidative sulfitolysis solution
in the acid form, that is as kerateine S-sulfonic acid.
[0033] S-sulfonated keratin protein is soluble only as the salt,
which can be prepared by the addition of base to the S-sulfonated
keratin protein. For the preparation of Films from S-sulfonated
wool keratin Intermediate filament protein it is convenient to
prepare a 5% protein solution by suspending S-sulfonated keratin
protein in water and adding base such as sodium hydroxide or
ammonia to give a final composition of 1 ml 1 M NaOH, or equivalent
base, per gram of protein to a give a solution with a final pH In
the range 9-10. Casting this solution onto a flat surface, such as
a glass plate, and allowing the water and/or ammonia to evaporate
at room temperature results in the formation of a keratin film.
These keratin films have a high degree of clarity and have the
physical properties detailed in Table 1 below. In untreated films
there is likely to be little or no covalent bonding occurring
between keratin proteins within the material as, the disulphide
bonds present in the original keratin have been converted to
S-sulfocysteine. The hydrogen bonding and other non-covalent
interactions occurring between the proteins are clearly
significant, as the tensile strength of the material in the dry
state is relatively high. The hydrogen bonding type interactions
are overcome in the presence of water, reflected by the large
decrease in tensile strength under wet conditions.
[0034] The physical properties of the materials derived from
S-sulfonated keratin proteins depend to a large extent on the
nature of the interactions between the proteins comprising the
material. These can be affected significantly by a range of
chemical treatments, with one of the most significant of these
treatments being the use of a reductant to remove the sulfonate
group from the protein to leave a thiol function. Under atmospheric
conditions, or in the presence of an oxidant such as dilute
hydrogen peroxide, these thiol functions recombine to form
disulfide bonds and return the chemical nature of the keratin
material to one much closer to the original form, that is proteins
containing a high proportion of cystine disulfide links.
[0035] Treatment with a reducing agent, such as ammonium
thioglycollate at pH 7 for 30 minutes, or tributylphosphine for 24
hours, is an effective way to remove the sulfonate function from
S-sulfonated keratin. This can be confirmed using infra-red studies
as the S-sulfonate group gives rise to a strong, sharp absorbance
at 1022 cm.sup.-1 which is observed to disappear on exposure of the
S-sulfonated to the reagents described.
[0036] In one aspect of the invention the reductant used to remove
the sulfonate function and introduce cystine disulfides is itself a
keratin protein. Reduced keratin proteins, or keratin peptides,
containing the thiol function can be readily produced by the
process of sulphide dissolution described above. Keratin proteins
prepared in this way contain the cysteine reducing group which may
covalently attach directly to the S-sulfonate group to form a
cystine disulfide. In this way a crosslinked keratin network is
formed without the use of other agents.
[0037] In the case of S-sulfonated wool keratin intermediate
filament protein films reductive treatment significantly improves
the wet strength properties of the material, as indicated by Table
1. The material retains a good degree of flexibility when wet.
Other chemical treatments also affect the film properties.
Treatment with an acid, such as 1M hydrochloric acid, protonates
the basic groups within the protein and converts the
S-sulfocysteine, present as the sodium or ammonium salt, to
S-sulfonic acid. This can improve the hydrogen bonding
interactions, as the wet strength of the film clearly improves and
no covalent bonds have been introduced. The S-sulfonate
functionality, as determined by infra-red absorption, remains
intact. Standard protein crosslinking treatments, such as the use
of formaldehyde or glutaraldehyde, also improve the wet strength of
the film, and introduce rigidity in both the wet and dry states.
This is achieved through crosslinking the proteins in a way that
does not specifically target the sulfonate functionality and many
of the amino acid residues containing nucleophilic side groups such
as lysine, tyrosine and cystine may be involved in
crosslinking.
TABLE-US-00001 TABLE 1 Strength, extension and swelling data for
protein films. Dry strength Wet strength % extension % extension
Film and .times.10.sup.-7 Nm.sup.-2 .times.10.sup.-7 Nm.sup.-2 at
break dry at break wet treatment (cv) (cv) (cv) (cv) Untreated 1.3
(11) 0.06 (15) 151 (24) 227 (20) Reductant 5.9 (7) 2.2 (21) 6 (16)
208 (15) Acid 6.1 (3) 1.6 (14) 6 (31) 387 (6) Glutaraldehyde 5.0
(8) 1.9 (14) 4 (11) 4 (8) Formaldehyde 2.8 (16) 0.96 (8) 7 (41) 13
(25) cv = coefficient of variation, %, n = 5
[0038] Solutions of S-sulfonated keratin proteins can be used to
produce reconstituted keratin fibres by a variety of extrusion
methods. Using a wet spinning approach, similar in concept to the
spinning of viscose rayon in which a solution of a material is
extruded into a coagulation bath in which the material is
insoluble, solutions of S-sulfonated keratin proteins can be
extruded into solutions containing chemicals that make the protein
become insoluble. Any of the three approaches described for
chemically treating S-sulfonated keratin films can be employed in
the coagulation bath used to generate keratin fibres. By employing
reductants, such as ammonium thioglycollate, in the coagulation
bath, the S-sulfonated keratin proteins are converted back to
keratins containing cystine disulfides through a wet spinning
process, thereby producing reconstituted keratin fibres that have a
multitude of disulfide links and good physical properties. By using
acidic conditions the S-sulfonated keratin proteins become
protonated and subsequently insoluble. By using crosslinking
agents, such as formaldehyde or glutaraldehyde, the protein also
becomes insoluble. The coagulation baths can also contain high
concentrations of salt or solvent to assist the process of fibre
formation. In each case precipitation of the extruded protein
occurs, possibly only in an outer skin of the extruded filament,
and a fibre is formed with sufficient mechanical integrity to allow
it to be collected from the coagulation bath and subjected to
further treatments such as drawing or other chemical processes.
[0039] A dry spinning approach can also be employed for the
production of reconstituted keratin fibres. The method is similar
in concept to the formation of S-sulfonated keratin films described
above, in which solvent is removed from an S-sulfonated keratin
protein solution and a keratin material remains. In the formation
of fibres this approach is employed by extruding a solution of
S-sulfonated keratin protein that has a composition typically of
6-10% protein and up to 50% of a solvent such as acetone, ethanol
or isopropylalcohol, with the remaining portion of the solution
being water and a base such as sodium hydroxide to give a pH of
9-10. This solution is extruded downwards into a chamber containing
a continuous downward hot air stream which causes the solvent to
rapidly evaporate, and an S-sulfonated keratin fibre remains.
Subsequent chemical treatments, such as the reductive, acidic or
crosslinking treatments described for keratin films described
above, can be employed to impart wet strength properties to keratin
fibres produced by this method.
[0040] Solutions of S-sulfonated keratins can be used to prepare
highly porous protein foams. This is achieved by freeze drying a
solution, prepared as described for the casting of keratin films.
In order to produce foams the solution is cast onto an appropriate
dish or surface and frozen, prior to being freeze dried. The
resulting porous network is a foam of S-sulfonated keratin protein.
As with the film and fibre forms of this material, applying
chemical modifications to the protein has a significant effect on
the wet properties of the material. In particular, applying
reductants such as ammonium thioglycollate or tributylphosphine
under similar conditions to those applied to the protein film,
results in the removal of the S-sulfonate group and the formation
of a network of disulfide bonds, and subsequently decreases the
solubility and increases in the wet strength of the foam. A reduced
form of keratin can also be used to similar effect, again resulting
in the formation of foam comprising of a keratin protein
interconnected through a network of disulfide bonds. Treatment of
the foam with an acid, such as 1M hydrochloric acid, results in
protonation of any available groups within the material, such as
the S-sulfonate group, and a subsequent increase in the wet
strength of the material. Crosslinking agents, such as formaldehyde
or glutaraldehyde, can also be used to significantly modify the wet
properties of the foam.
[0041] All the above applications relate preferentially to the case
of IF-type proteins prepared from hard .alpha.-keratins such as
wool, but other applications such as the following one can use
keratins from other sources, such as feather keratin.
[0042] Solutions of keratins obtained from wool or feathers by
either reduction using sodium sulphide or by oxidative
sulphitolysis as described above show significant adhesive
properties in various applications. However, the wet strength of
both of these adhesives is limited. Keratin made soluble by
sulphide reduction is degraded to some extent and contains protein
chains of lower molecular weight than in the original wool.
S-sulfonate derived keratin polymers contain no covalent crosslinks
and hydrogen bonding interactions are weakened significantly in
water, as demonstrated by the keratin films described above.
However the wet strength and adhesive properties can be greatly
enhanced by reforming disulphide cross-links, by adding an oxidant
in the case of sulphide-derived proteins, or a reducing agent in
the case of the S-sulfonated keratin proteins. By such means very
effective adhesive bonding can be achieved, for example in
wood-particle composites bonded with oxidised sulphide-derived
proteins.
[0043] A particular feature of this invention relates to the
recognition that the sulphide-derived protein and the S-sulfonated
keratin proteins can be used in conjunction to create highly
cross-linked structures with very superior properties. As noted
above, the former class of protein can be crosslinked by oxidation,
and the latter by reduction. The two protein classes, one being in
a reduced state and the other in an oxidised state, will when mixed
form a self-crosslinking system. In effect, in such a system, an
addition of sulphide-derived protein is acting as a reductant and
crosslinking agent to convert the S-sulfonate groups in the other
component to disulfide bonds.
[0044] Such a two-pot self-crosslinking system is a particular
aspect of the invention which will have applications in many forms
of product, and has the advantage of eliminating volatile low
molecular weight materials and the necessity to use solvents in
some forms of product fabrication. Thus it is to be expected that
such composites can be formed from mixtures of solids or viscous
dispersions without shrinkage.
[0045] In such two-component systems, the respective
sulphide-derived and S-sulfonate keratin proteins can be produced
from the same or different keratin sources. For example, if the
mechanical property characteristics associated with IF proteins
were desirable, the S-sulfonated keratin protein could be derived
from a hard .alpha.-keratin such as wool, and the sulphide-derived
protein from another keratin source such as feathers.
[0046] An alternative two-component system is one which utilises a
reductant from the thiol or phosphine family in addition to
S-sulfonated keratin proteins. Combining solutions of these two
materials results in the removal the sulfonate group and formation
of cystine disulfides in the manner described above for keratin
films and fibres. This gives rise to an adhesive formulation with
good wet strength properties.
[0047] By such means, proteins from sources other than hard
keratins can be incorporated into many of the product classes
described above, and therefore the features in this invention
encompass keratin sources in general and are not restricted to hard
.alpha.-keratins.
[0048] Polar, soluble reagents of low molecular weight, such as
polyethylene glycol or glycerol, can be employed as plasticising
agents to give keratin materials flexibility. These agents are best
employed by inclusion in the keratin solutions used as the starting
point for the formation of films, fibres or adhesives.
EXAMPLES
Example 1a
Preparation of a Keratin Film
[0049] In order to prepare an S-sulfonated keratin film, a 5%
keratin protein solution was prepared by suspending 0.5 g
S-sulfonated wool keratin intermediate filament protein in water,
followed by the gradual addition of 0.5 ml of 1M sodium hydroxide
to the vigorously stirred solution over approximately 2 hours. The
pH of the solution was carefully monitored and observed to elevate
to .about.pH10 upon immediate addition of base, and gradually fall
as the base was absorbed by dissolution of the protein. A final pH
of 9.5 was obtained. The protein solution was centrifuged at 34,000
g to remove any insoluble material and the resulting solution was
cast onto a 100 mm square petri dish and allowed to dry under
ambient conditions. Following drying a clear protein film remained
which could be easily removed from the petri dish.
Example 1b
Disulfide Crosslinking of Protein Films
[0050] In order to improve the wet strength of S-sulfonated keratin
films, disulfide crosslinks were introduced to the film by
immersing the films produced in Example 1a in a solution containing
a reducing agent. One example is a solution comprising 0.25M
ammonium thioglycollate and 0.1 M potassium phosphate buffer
adjusted to pH 7.0. Another example is a solution comprising 1M
thioglycollic acid. Another example is a solution containing 85
microlitres of tributyl phosphine in 20 ml of 10% (v/v) 0.2M borate
buffer in dimethyl formamide buffered to pH 9.0. Following
immersion in the solution with gentle agitation for 30 minutes in
the case of the thiols and 24 hours in the case of the phosphine,
the keratin film was removed, rinsed briefly with water and allowed
to dry under ambient conditions.
Example 1c
Protonation of Protein Films
[0051] In order to improve the wet strength of S-sulfonated keratin
films, acid was used to protonate all available sites on the
proteins. This was achieved through immersion of the film produced
in Example 1a in 1M hydrochloric acid for 30 minutes. Following a
brief wash with water the film was allowed to dry under ambient
conditions.
Example 1d
Non-Disulfide Crosslinking of Protein Films
[0052] In order to improve the wet strength of S-sulfonated keratin
films crosslinking agents were used to chemically bond proteins
together. In one case this was achieved through the use of a
solution of 8% formaldehyde in 0.1M phosphate buffer at pH 7.0. The
film was immersed in this solution for 30 minutes, washed briefly
with water and allowed to dry under ambient conditions. In another
case, crosslinking was achieved through the use of a solution of 5%
glutaraldehyde in 0.1M phosphate buffer at pH7.0. The film was
immersed in this solution for 30 minutes, washed briefly with water
and allowed to dry under ambient conditions for 30 minutes.
Example 1e
Plasticising of Protein Films
[0053] In a variation of Example 1a, flexible protein films are
made by incorporating glycerol or polyethylene glycol into the
protein solution described in Example 1a at a level up to 0.2 g per
g of protein prior to casting the film. The resulting films have a
greater flexibility, as determined by extension at break
measurements, than the analogous films containing no
plasticiser.
Example 2a
Production of Keratin Fibres through Wet Spinning and Disulfide
Crosslinking
[0054] In order to prepare fibres derived from S-sulfonated keratin
proteins a spinning dope was prepared in a similar manner to that
prepared in Example 1a, with the difference being that for the
extrusion of fibres, the concentration of protein in the solution
was in the range 6-15%. A plasticiser, such as those described in
Example 1e, was added to the spinning dope. Following centrifuging
to remove solids and entrained air the dope was forced, using a
positive displacement pump such as a syringe or gear pump, or air
pressure, through a spinnerette into a coagulation bath. The
coagulation bath had a composition of 1M ammonium thioglycollate,
0.4M sodium phosphate, 0.25M sodium sulfate, 2% glycerol all set to
pH 7.0.
Example 2b
Production of Keratin Fibres Through Wet Spinning and Non-Disulfide
Crosslinking
[0055] In a variation to Example 2a, fibres were extruded into a
coagulation bath with a composition of 0.25M ammonium
thioglycollate, 0.1M sodium phosphate, 8% formaldehyde and 2%
glycerol. This served to form tough fibres without forming
disulfide bonds, as shown by infra red analysis which clearly
indicated the presence of the S-sulfonate group. Subsequent
treatment of the fibres with solutions containing reductants, such
as ammonium thioglycollate at a concentration of 0.25M and a pH of
7.0 with 0.1M potassium phosphate buffer, was sufficient to remove
the S-sulfonate group and reform disulfide bonds.
Example 2c
Production of Keratin Fibres Through Dry Spinning
[0056] In order to produce fibres through a dry spinning process,
first a spinning dope was prepared in a similar manner to that
described in Example 2a. In variation to the dope preparation a
solvent such as acetone or isopropylacohol was added to the dope to
give a final composition protein in the range 6-15%, solvent in the
range 20-50% and plasticiser in the range 1-3%. The dope was
extruded through a spinnerette, using similar technology to that
described in Example 2a, downwards into a chamber with a continuous
downwards hot air stream. This caused the solvent to rapidly
evaporate leaving a keratin fibre. Subsequent wet processing of the
fibre, through the use of acid, reductant and crosslinking agents,
of the type described in Examples 1, was used to improve the wet
strength properties of the fibre.
Example 3a
Production of a Keratin Foam
[0057] A solution of S-sulfonated keratin protein, prepared to a
protein concentration of 5% as described in Example 1a, was used to
create a keratin foam by freezing the solution in a 100 mm square
petri dish and freeze drying the resulting solid.
Example 3b
Chemical Modification of Keratin Foam
[0058] Chemical solutions containing reductants, acids or
crosslinking agents, of the described in Examples 1b, c, and d were
applied to the keratin foam, in a manner identical to that
described for the keratin film. A keratin foam with significantly
reduced solubility and improved wet strength resulted.
Example 4a
Application of a Keratin Adhesive to Bind Wood
[0059] A solution of S-sulfonated keratin protein, prepared to a
protein concentration of 5% as described in Example 1a, was used to
bind woodchips by mixing the keratin solution with woodchips in a
ratio of 1 ml solution per gram of woodchips. The mixture was then
pressed and heated in a similar manner to the production of
commercial urea formaldehyde bound particle board (3 MPa,
180.degree. C., 300 s), and a solid wood keratin composite
resulted.
Example 4b
Application of a Keratin Adhesive to Bind Textiles
[0060] A solution of S-sulfonated keratin protein, prepared to a
protein concentration of 5% as described in Example 1a, was used to
bind woollen textiles by coating one of the textile surfaces with
the keratin solution and pressing another textile onto the coated
textile with the use of a pinch roller system. Following the drying
of the composition at elevated temperature a bonded textile was
produced. In a small variation, plasticiser was included in the
protein solution, in a manner similar to that described in example
1d, to produce a flexible adhesive.
Example 4c
A Two Pot Adhesive System using a Reductant
[0061] An adhesive was made by combining a solution of S-sulfonated
keratin protein, prepared in the manner described in Example 1a,
with a solution of a reductant. The reductant solution contained
10% triscarboxyethylphosphine hydrochloride. When mixed in a ratio
of 10 parts keratin solution to 1 part reductant solution and
applied to two wood surfaces this two pot formulation dried over 12
hours to create a strong bond between the wooden surfaces that
remained strong in a moist environment. In a variation of this
application a reductant solution was used which contained 0.25M
ammonium thioglycollate buffered to pH 7.0 with 0.1M potassium
phosphate. When mixed in a ratio of 10 parts keratin solution to 1
part reductant solution and applied to two wood surfaces this two
pot formulation dried over 12 hours to create a strong bond between
the wooden surfaces that remained strong in a moist
environment.
Example 4d
A Two Pot Adhesive System using Two Forms of keratin
[0062] An adhesive was made by combining a solution of S-sulfonated
keratin protein, prepared in the manner described in Example 1a,
with a reduced keratin peptide solution which contained sulphur
amino acids primarily in the form of cysteine and had a composition
of 5% protein and 2% sodium sulphide. When mixed in equal parts and
applied to two wood surfaces this two pot formulation dried over 12
hours to create a strong bond between the wooden surfaces that
remained strong in a moist environment. In a variation of this
application, the reduced keratin peptide was used in the form of a
solid and mixed with the S-sulfonated keratin protein solution in a
ratio of 5 parts S-sulfonated keratin solution to 1 part reduced
keratin solid and applied to two wood surfaces this two pot
formulation dried over 12 hours to create a strong bond between the
wooden surfaces that remained strong in a moist environment.
[0063] Where in the description particular integers are mentioned
it is to be appreciated that their equivalents can be substituted
therefore as if they were set forth herein.
[0064] Thus by the invention there is provided a method for the
preparation and use of soluble keratin derivatives in the
production of a range of biopolymer materials such as films,
fibres, foams and adhesives, and the improvement of those materials
using further chemical treatment.
[0065] Particular examples of the invention have been described and
it is envisaged that improvements and modifications can take place
without departing from the scope of the attached claims.
* * * * *