review on fabrication and applications of ultrafine particles from animal protein fibres

Fibers and Polymers 2014, Vol.15, No.2, 187-194

187

Review on Fabrication and Applications of Ultrafine

Particles from Animal Protein Fibres

Kiran Patil, Rangam Rajkhowa, Xungai Wang, and Tong Lin*

Australian Future Fibres Research and Innovation Centre, Institute for Frontier Materials, Deakin University,

Geelong, VIC 3220, Australia

(Received April 12, 2013; Revised June 3, 2013; Accepted June 8, 2013)

Abstract: Protein fibre wastes from animal hairs, feathers and insect secreted filaments can be aptly utilized by convertingthem into ultra-fine particles. Particles from animal protein fibres present large surface-to-weight ratio and significantlyenhanced surface reactivity, that have opened up novel applications in both textile and non-textile fields. This review articlesummarizes the state-of-the-art routes to fabricate ultrafine particles from animal protein fibres, including direct route ofmechanical milling of fibres and indirect route from fibre proteins. Ongoing research trends in novel applications of proteinfibre particles in various fields, such as biomedical science, environmental protection and composite structures are presented.

Keywords: Animal protein fibre, Particle, Fabrication, Biomedical applications, Environmental protection, Composite structures

Introduction

Nature exhibits diverse and abundant structural materials

in the form of protein fibres, in addition to those present in

intracellular, extracellular matrices, cytoplasma and blood

plasma of living organisms. These protein fibres are synthesized

on exoskeletal portion of most animals and certain insects

for various purposes. Keratin hairs and feathers function to

protect animals from extreme weather conditions, whereas

fibroin silk filaments of spiders and insects form nests, traps

or cocoons. The majority of animal protein fibres serve as

industrial raw material and contribute 8.4 % (2,643,000 MT)

to the total world natural fibre production, comprising 7.1 %

of fine animal hairs and 1.3 % insect secreted silk filaments

[1]. Nevertheless, a significant quantity of them is segregated

as a waste during processing, which is suitable only for low

value applications. Similarly, coarse and lengthy guard hairs

of certain domesticated and harvested animals such as

Cashmere goats, angora rabbits and llama, segregated from

their valuable fine fibres [2-4], are also treated as a waste or

low value side-products and they mainly get used for making

brushes and interlinings. Additionally, pachydermatous animal

hairs, human hairs, bird feathers are considered as a mere

waste on shearing or death of the respective animal and do

not get utilized for any valuable applications.

Considering the tedious nature of proteinaceous waste

handling due to the release of harmful gases such as CO,

CO2, H2S and HCN on its incineration [5], considerably long

decomposition time on land filling [6] and high energy

requirements for other physical/chemical methods of pro-

teinaceous waste processing [7], the animal protein fibre

wastes have been mainly converted to protein hydrolysate

feedstuffs with proteolytic micro-organisms i.e. enzymatic

chemical hydrolysis [8,9]. However feedstuffs from processed

animal by-products have not been allowed in Europe since

July 1994 [10]. The ban aims to create an efficient system of

protecting human health within the European Union from

infections caused by pathogenic micro-organisms of animal

origin.

Particalization of protein fibers has recently emerged as a

new strategy to improve the high value-added applications

of protein fibers. The presence of functional groups, such as

amino (-NH2), hydroxy (-OH) or thiol (-SH) enables protein

fiber particles to bind certain functional molecules, targeting

specific applications [11-13]. Compared to those made of

synthetic polymers, particles from protein fibers are bio-

degradable and biocompatible. They are easier to be degraded

through land filling after use, benefiting the environment,

apart from the lower cost. Such a new protein fibre form has

attracted much attention in both science and engineering

fields.

The present review addresses a specific form of protein

fibres i.e. ultrafine protein particles, their different fabrication

techniques and novel applications under exploration, along

with the further possible scope in the field at certain places.

Routes of Particle Fabrication from Animal Protein Fibres

Animal protein fibres can be converted into ultrafine

particles either directly by mechanical milling - a physical

route with top-down approach or indirectly from solubilised

protein macromolecules - a chemical route with bottom- up

approach. The two distinct routes of protein fibre particle

fabrication are herewith graphically summarised in Figure 1,

with their respective pros and cons.

The direct fabrication of animal protein fibre particles

relies on the mechanical milling or crushing of parent fibres

into the fine particles, while following the mechanism of

fibre fracture and progressive separation of the micro and*Corresponding author: [email protected]

DOI: 10.1007/s12221-014-0187-y

188 Fibers and Polymers 2014, Vol.15, No.2 Kiran Patil et al.

nano-fibrillar structures [14]. On the contrary, the indirect

route of protein fibre particle fabrication relies on the

solubilisation of animal protein fibres into soluble proteins

or polypeptides, from which the protein particles are generated

via different techniques.

Indirect Routes

Indirect route of protein fibre particle fabrication is a two-

step process. The first step involves solubilising the natural

protein fibre. The solvent and the process used for the

protein fibre solubilisation restrict the fibre disintegration to

the polypeptide level without further proceeding it to the

basic building blocks i.e. amino acids. In the second step, the

solubilized polypeptide chains are solidified back into spherical

solid state particles, a bottom-up approach of reorganising

polypeptide chains.

Although the animal protein fibres are insoluble in water

due to the complicated conformation and crosslinking of

polypeptide chains, their solubility can be improved by the

presence of specific ions, organic solvents, polymers or

surfactants [15]. In a freshly prepared protein solution of an

animal protein fibre, hydrophobic and hydrophilic amino

acid residues of the polypeptide chains get randomly dispersed

and undergo reorganisation by driving non-polar hydrophobic

regions of each polypeptide molecule away from water and

towards each other [16]. As shown in Figure 2, the protein

molecule reorganisation gets impetus from physical or

chemical stimuli such as vibration, agitation, freezing and

addition of organic solvents to the denatured protein solution

and forms a colloidal solution of protein microcrystals with

the formation of induced β sheet structure. These microcrystals

further act as a seed for protein aggregation while experiencing

a shear force generated from different processing techniques,

viz. phase separation, freeze drying, spray drying and electro-

spraying.

In general, animal protein fibre particles prepared by a

bottom-up approach are spherical in shape, quite homogenous

in size (Figure 3) with a rebuilt molecular architect. These

protein fibre particles are often known as “microspheres”,

the morphological features, surface and bulk properties of

which can be controlled by the preparation conditions.

Different bottom-up approaches adopted to fabricate ultrafine

particles from natural protein fibres are summarised herewith.

Phase Separation

The presence of strongly hydrated kosmotropic ions near

the surface of proteins lead to the precipitation or salting out

of the proteins in the form of β sheet rich phase –

‘microspheres’, from the protein poor supernatant through

the liquid-liquid phase separation [17]. Lammel et al. [18]

fabricated silk fibroin microspheres by inducing phase

separation of the silk fibroin protein from its aqueous solution

by the addition of potassium phosphate. They prepared the

starting aqueous silk fibroin solution by dissolving degummed

silk in 9.3 M LiBr which was further purified by dialysing

and centrifuging. The lower threshold of potassium phosphate

ionic strength for silk fibroin microsphere formation was

determined to be approximately 0.75 M. The size of the silk

fibroin microspheres was reported to be dependent upon the

silk fibroin solution concentration. The silk fibroin microsphere

size increased linearly from 486 nm to 1.2 µm with increase

in the silk fibroin solution concentration from 0.25 mg/ml to

2 mg/ml. The secondary structure as well as the zeta potential

of the microspheres was reported to be controllable by

varying the pH value of the aqueous silk fibroin solution.

Slotta et al. [19] also followed a salting out technique to

fabricate protein microspheres from eADF4, an engineered

and recombinantly produced spider dragline silk protein by

the addition of potassium phosphate. They reported the

formation of nanofibrils below 300 mM of potassium

phosphate and microspheres above 400 mM of potassium

phosphate. The size of the microspheres was reported to be

Figure 1. Bottom-up and top-down approaches to fabricate protein

fibre particles.

Figure 2. Scheme of protein particle (microsphere) generation

from its solution.

Figure 3. Spherical and homogeneous eADF4 microspheres [19].

Ultrafine Animal Protein Fiber Particles Fibers and Polymers 2014, Vol.15, No.2 189

controllable by mixing intensity and protein concentration,

which was also reported by Lammel et al. [18]. High protein

concentration resulted in larger particle size, whereas

increasing mixing intensity resulted in smaller microspheres.

In a distinct attempt, Sun et al. [20] dissolved chicken

feathers in an ionic liquid of 1-butyl-3-methylimidazolium

chloride ([BMIM]Cl) under inert atmosphere of N2 at

100oC. Solid keratin particles were phase separated from the

chicken feather solution by the addition of water. The β-

sheet content of the raw chicken feather was decreased from

47.19 % to 31.71 % and this was accompanied by decrease

in the water contact angle from 138 o to 76 o. To the best of

our knowledge, this is the only report on animal feather

(keratin) particle fabrication by an indirect route. However,

the chicken feather particles prepared by this technique were

non-spherical in shape.

Freeze Drying

Freeze drying technique of silk fibroin microsphere fabrication

relies on the repeated refrigeration and thawing of a purified

concentrated silk fibroin solution to obtain a milky suspension,

which is further freeze dried or lyophilized to obtain the silk

fibroin microspheres.

Cao et al. [16] solubilised degummed silk in 9.5 M aqueous

LiBr solution, that was followed by the freeze drying

technique to obtain spherically shaped homogeneous silk

fibroin microspheres of around 200 nm in diameter with

polydispersity index of ~0.1. They also reported that the size

of the microsphere can be varied by controlled addition of

ethanol in the dialysed silk fibroin solution, which also helped

in inducing more β sheet structures in the microspheres and

thus achieving improved crystallinity.

Nam et al. [21] used CaCl2/ethanol/H2O solvent system

for dissolving silk filaments and adopted the freeze drying

technique to form fine microspheres. They also found that

the addition of methanol or ethanol to the silk fibroin solution

formed fine silk microspheres with enhanced thermal stability

and crystallinity, whereas the addition of hydrophobic alcohols

such as 1-butanol and 1-octanol to the silk fibroin solution

formed lump-like shape of morphology with no conformational

changes.

The freeze drying technique has also been used to load

drug/growth factor molecules to the silk fibroin microspheres.

This was achieved by encapsulating silk fibroin and drug

molecules with lipid vesicles and repeated freeze-thawing

assisted in the formation of homogeneous silk fibroin

microspheres with improved drug loading. The final drug

loaded silk fibroin microspheres were obtained by washing

lyophilized silk fibroin-drug lipid vesicles with a solvent

[22-24].

Wang et al. [22] prepared horseradish peroxidase loaded

silk fibroin microspheres by hydrating 1,2-Dioleoyl-sn-

snglycero-3-phosphocholine lipid film with the peroxidase-

silk fibroin mixture solution. Peroxidase-silk fibroin-lipid

vesicles were generated from the freeze-thaw process on the

diluted peroxidase-silk fibroin-lipid mixture in water. Repeated

freeze thaw cycles created smaller vesicles with homogeneous

size distributions and led to higher loading of the peroxidase

due to improved mixing between the silk fibroin and the

drug. Without this process the drug and silk remained in

separate layers or domains in microspheres. The peroxidase

loaded silk fibroin microspheres, consisting of β-sheet structure

were finally generated by adding methanol or sodium chloride

solution to the lyophilized material to remove the lipid

templates. The surface texture of the particles was reported

to be dependent upon the chemicals used for washing off the

lipid template.

Spray Drying and Electro-spraying

More simplistically, silk fibroin microspheres can also be

prepared by spray drying of an aqueous silk fibroin solution.

However, the dry as-sprayed silk fibroin microspheres,

prepared by Hino et al. [25] were unstructured with larger

diameters (approximately 5 µm) compared to those prepared

by the previously described techniques. Hino et al. also

reported that the as-sprayed microspheres underwent crystalli-

zation on storing at 89 % humidity for 24 h by inducing β

sheet structure and acquired water insolubility.

Electrostatic spraying, also known as electro-hydrodynamic

spraying, involves generation of solution droplets under the

action of a high electric field. Figure 4 shows a typical

electrostatic spraying process. The liquid fluid flowing out

from the capillary nozzle is charged with a high voltage

before spraying [26]. Compared to the conventional spray

drying in which a gas flow is used to atomise the solution

mechanically, electro-spraying generates finer droplets and

hence finer particles, with less particle aggregation, accompanied

with narrower particle size distribution.

Gholami et al. [27] fabricated nano-sized (80 nm) silk

fibroin particles by electro-spraying of the sponge fibroin

solution in 98 % formic acid. The sponge fibroin was

initially prepared by freeze drying of the refrigerated silk

Figure 4. Schematics of electro-spraying [56].


fibroin solution. Solid silk fibroin sponge obtained was then

dissolved in 98 % formic acid. Finer silk fibroin particles

were formed from solutions of lower fibroin concentration.

Lowering the solution feeding rate or increasing the spraying

distance also resulted in reduced particle size. The particle

size was initially decreased with an increase in the applied

voltage until 20 kV, and then increased when further

increasing the voltage.

Although different indirect routes of protein fibre particle

generation have been mainly attempted for silk fibroin,

similar attempts are lacking for hair keratin particle generation.

This is due to the difficulties in hair keratin molecular

dissolution caused by the presence of strong covalent disulfide

(-S-S-) bonds among different keratin molecules or the α

helix loops within a single keratin molecule. However, with

the recent advent of ionic liquids as molecular solvents for

biological macromolecules [28], the doors for dissolution

and regeneration of hair keratin fibres appear to be opened

up. Hence, further experimental studies are needed to explore

the great scope in hair keratin particle generation by exploring

ionic liquids as solvents for hair keratins.

Direct Route: Mechanical Milling Technique

Ultrafine particle preparation from animal protein fibres

by mechanical milling technique is a straightforward and

comparatively quick process to produce particles in bulk

quantity without altering the native protein fibre molecular

architecture and thus preserving the native physicochemical

characteristics of the parent fibre in the particles.

In general, protein fibre particles, fabricated by the

mechanical milling technique are available in various non-

spherical shapes and sizes, often with a wide size distribution

(Figure 5) and well retained molecular structures. Although

a few articles in literature have specified decreased crystalline

content on protein fibre milling [29,30], it is still most likely

to be far better than the protein fibre microspheres prepared

from the indirect route, i.e. Protein Fibre > Mechanically

milled protein fibre particle > Indirectly synthesized protein

fibre microspheres. However, specific experimental studies

in this regard are warranted.

The mechanical milling of protein fibres has mainly adopted

purpose built apparatuses or commercially available equipment

in the ceramic processing and powder metallurgy industries.

Li et al. [31] invented an apparatus for preparing fine

particles and reported a method for pulverizing natural

organic substances (such as wool, silk and cotton) into nano-

scaled fibrous materials [32]. In this method, clean dry fibres

were initially crushed into small pieces of 500 µm in length

by using a rotary crusher. A pure water suspension of these

small fibre pieces was further processed on a professional

ultrasonic crusher that resulted in a particle suspension with

sizes smaller than 20 µm both in diameter and length. The

particle suspension was further fed into a nano-colloid

machine to prepare nanoparticles of less than 100 nm in

diameter and shorter than 800 nm in length. Finally dry

particles were achieved by spray drying of the particle

suspension. The particle size was said to be controllable with

an adjustment of colloid pressure and colloid time in the

nano colloid machine.

Xu et al. [29] pre-treated shortened wool fibres (around

3 mm in length, cut by rotary blade) with 0.5 % NaClO,

before grinding on a specially designed machine equipped

with two milling pans. To get differently sized wool particles,

different durations of grinding were adopted. Wool particles,

smaller than 2 µm in diameter and with a length between 5

and 10 µm, were obtained after 3 hours of grinding. The

ground wool particles exhibited no change in chemical

composition with slight decrease in crystalline content and

increase in thermal stability.

Cheng et al. [30] weakened wool fibres by treatment of

the fibres with hydrogen peroxide and further subjected

them to a three-step pulverisation process to produce wool

particles smaller than 100 nm in diameter. It was mentioned

that the preparation of nano-wool particles was facilitated by

the hydrogen peroxide treatment. The pulverized wool particles

showed decrease in crystalline content with no substantial

change in their chemistry.

Joko et al. [33] reported dry and wet milling of native and

chemically pretreated (sodium sulphite/sodium hydroxide)

wool fibres using different mechanical techniques such as

homogeniser, ball milling, jet milling and high speed rotor

milling. Fractured wool products with different shapes and

sizes (particles less than 10 µm in diameter and spindle

shaped particles of around 80 µm in length) were reported

and were said to be dependent on the milling technique

adopted.

In yet another attempt, Miyamoto et al. [34] adopted a

distinct explosive puffing treatment with saturated steam of

6 kg/cm2 at 164.2 oC to fracture unmodified and reduced

wool fibres into fine particles.

At our research centre (Australian Future Fibres Research

and Innovation Centre), we have developed ultrafine protein

particles from wool [35,36], Cashmere guard hair [37] and

various silk fibre varieties such as Bombyx mori, Muga andFigure 5. Globular Cashmere guard hair particles with wide

particle size distribution [37].


Eri [14,38] for exploring the unconventional applications of

protein fibres. Different commercial powder fabrication

facilities, such as rotary cutter, rotary mill, planetary ball

mill, attritor mill, spray dryer and air jet mill, have been

employed for producing protein fibre particles with different

size ranges.

Rotary milling of the chopped silk cocoon snippets was

ineffective and generated mere segmented fibrous structures

after many passes. On the contrary, planetary ball milling

produced silk particle aggregates as fine as 200 nm (volume

based mean particle size) on optimising powder to media

ratio, media size, water content and fibre degumming

treatment [14]. In various different studies, spray drying of

5-6 h attritor milled slurries of silk, wool and Cashmere

guard hair fibres produced mushroom like globular particle

aggregates with a volume based mean particle size (d(0.5))

of ~5 µm [36-38]. Interestingly, this particle size is very

much similar to that reported by Hino et al. [25] for silk

fibroin particles prepared by an indirect route of spray drying,

using a similar laboratory scale spray dryer. It is therefore

likely that the spray dried particles have consistent mean

particle sizes for the given atomizer diameter, independent

of the mean particle size on the inlet slurry, at a certain limit.

We also found that severe chemical pre-treatment on

protein fibre did not assist in achieving small particle size,

while following the adopted fabrication route i.e. chopping→

attritor milling→ spray drying [37]. However, further air jet

milling of the spray dried particles resulted in the particle

disintegration and the ultimate particle size was found to be

linearly related to the severity of the chemical pre-treatment.

Based on BET surface area studies, it was reported that the

attritor milling-spray drying route promoted particle aggregation

whereas air jet milling assisted in disaggregation of the

aggregate particles.

Applications of Animal Protein Fibre Particles

Animal protein fibre particles have unique application

trajectories depending on the adopted fabrication route, due

to their distinct morphology, feasibility for scaled up

production, size homogeneity and molecular architecture

(Figure 6). Several typical applications of animal protein

fibre particles have been summarized herewith.

Drug and Growth Factor Delivery

Animal protein fibre particles are promising carriers for

drug and growth factor delivery as they possess biocom-

patibility, biodegradability and many active amino groups

and tyrosine residues that favour bioconjugation with active

drug molecules. In addition, as seen in the previous section,

protein fibre particles can be fabricated in different controllable

structures and morphologies via different techniques to

favour the desired drug release kinetics.

Shi et al. [39] fabricated silk fibroin particles by phase

separation technique and reported model drugs (rhodamine

B, rhodamine B isothiocyanate-dextran and fluorescein iso-

thiocyanate labelled bovine serum albumin) loading efficiencies,

release kinetics and delivery to cells. The silk fibroin particles

showed good loading and release behaviour to hydrophobic

drugs as well as protein model drugs. Additionally, silk

fibroin particles showed very good ability to deliver drugs to

osteoblasts cells and most cells were found proliferating by

accepting the particles as well as the drugs inside the

particles.

Wang et al. [22] reported efficient loading and controlled

release of protein drug horseradish-peroxidase in its active

form and enzyme 3,3',5,5'-tetramethylbenzidine to silk fibroin

microsphere using a freeze drying technique with the assistance

of a lipid vesicle template. Repeated freeze-thaw cycles were

also used in silk fibroin microsphere fabrication to enhance

drug loading and reduce particle size. It was also found that

physical cross-linking of β-sheet structure, which was induced

by treatment with methanol or NaCl and the residual lipids

in the microspheres played important roles in controlling

enzyme release profiles.

Previously mentioned eADF4 microspheres [40], prepared

by the phase separation technique, have demonstrated efficient

loading of low molecular weight, positively charged hydro-

phobic drugs with constant drug release rates realised for a

period of two weeks at physiological conditions in vitro.

Hofer et al. [41] have demonstrated that macromolecular

drugs like lysozyme proteins can also be loaded on to

eADF4 microspheres. The release of lysozyme was reported

to be dependent on the ionic strength and pH value of the

release medium.

Temporally and spatially controlled delivery of growth

factors in 3D porous polymeric scaffolds enables understanding

of natural tissue growth process and in vitro engineering of

complex tissue constructs. Growth factors, bone morphogenetic

protein 2 (rhBMP-2) and insulin like growth factor I (rhIGF-

I), have been encapsulated into silk fibroin microspheres

through a freeze drying technique with the aid of lipid vesicles

Figure 6. Application trajectories of different types of animal

protein fibre particles and their suitability.


[23]. The silk fibroin microspheres were further incorporated

into alginate and silk scaffolds to form concentration gradient

impact on osteochondral differentiation of human bone

marrow-derived mesenchymal stem cells (hMSCs). The

regenerated silk fibroin microspheres were reported to be

more efficient than polylactic-co-glycolic acid (PLGA)

microspheres in delivering rhBMP-2, probably due to the

sustained release of the growth factor.

In short, animal protein fibre particles fabricated by

different indirect routes have shown potential in drug delivery

applications. These particles possess narrow particle size

distribution and therefore render long term stability to the

particle dispersions and in vivo distribution of the particles.

On the contrary, mechanically milled protein fibre particles

possess wide size distribution and have not yet found scope

in this trajectory of potential animal protein fibre particle

applications. Consequently, at our research centre different

experimental approaches are being explored to obtain

mechanically milled animal protein fibre particles with a

narrow size distribution, as they are likely to be favoured in

situations demanding drug release over a long duration, due

to their high crystallinity compared with their indirect route

counterparts. Additionally, mechanically milled animal protein

fibre particles, even with a wide particle size distribution,

may be used as drug/growth factor carriers in composite

scaffolds. However, experimental studies in this regard are

missing.

Environmental Protection

Due to the presence of nucleophilic groups such as amino

(-NH2), hydroxy (-OH) or thiol (-SH) groups and high

specific surface area (10-20 m2/g [37]), animal protein fibre

particles have inherent potential to effectively remove water

pollutants, both inorganic and organic species. Different

studies at laboratory level have demonstrated the higher

uptake and loading capacity of animal protein fibre particles

for toxic heavy metal ions and organic dyes compared to

their parent fibres and/or commercial resins.

Chicken feather particles, prepared by phase separation of

its solution in an ionic liquid, have been illustrated to be an

excellent candidate to remove Cr(VI) ions (efficiency 63.5-

87.7 %) from wastewater in the concentration range from 2

to 80 ppm [20]. The Freundlich constant (kF) for adsorption

of Cr(VI) ion by chicken feather particles was four times

larger than that of the raw chicken feather, possibly due to

the reorganization of amino and carboxyl groups towards the

surface of the regenerated chicken feather particles, while

electrostatically binding anionic Cr(VI) ions towards cationic

amino.

However, if commercialised, the heavy metal ion separation

from waste water streams is likely to require a huge quantity

of animal protein fibre particles. This would require large

scale production of such particles, using a mechanical milling

approach. Therefore, different studies involving variety of

heavy metal ions, organic dyes and mechanically milled

animal protein fibre particles have been undertaken at our

research centre [42-44].

During Cu2+ binding studies on the mechanically milled

wool particles, we found that the metal ion uptake rate of

wool particles was dramatically faster (~42 fold) than that of

the parent wool fibre. The wool particles also demonstrated two

to nine fold increase in the metal ion binding capacity in

comparison with commercial cation exchange resins [42]. In

a separate study, we have also reported ionic interactions of

transition metal ions with mechanically milled animal protein

fibre particles from Cashmere guard hair, Merino wool and

Eri silk. Their binding capacities for oppositely charged

metal species (Zn2+ and anionic species of Cr6+) [43] was

also examined. The maximum absorption yield of Zn2+ for

the studied particle samples was observed at pH 8, whereas

the anionic species of Cr6+ were efficiently absorbed from

aqueous solutions at pH 2, where protein fibre particles

demonstrated negative and positive zeta potential values

respectively. The absorbed metal ions were found to desorb

on exposing to a buffered aqueous solution at a pH value of

poor metal ion absorption. While exploring the removal of

water polluting organic dyes by using mechanically milled

wool particles, we have reported increased dye absorption

ability of wool with effective surface area [35]. The effect of

pH value on the sorption of hydrophilic dyes on wool

particles was more significant than that of the hydrophobic

dye uptake [44]. Comparison with activated charcoal and

other sorbents indicated that the fine wool particles had

excellent dye sorption capacity even at room temperature.

Composite Structures

Composite Films and Scaffolds

Animal protein fibre particles have been blended with

biocompatible polymer solutions to either efficiently load

bioactive drug/growth factor molecules or alter mechano-

chemisoprtion properties of the resultant casted film or

scaffold for biomedical applications. Mechanically milled

animal protein fibre particles have been aptly explored in

this field as the particles provide good mechanical strength

due to their retained native fibre molecular structure.

Synthetic polymer films such as polyurethane and poly-

propylene have been modified with milled keratin particles

[45-47]. The milled keratin particles have been either

blended with the polymer solution to cast it on a glass plate

or blended in an extruder with the polymer powder into

pallets which were further hot-pressed into films. The milled

keratin particle blended synthetic polymer films exhibited

increased water permeability, moisture regain and dynamic

storage modulus along with the decreased mechanical properties

with an increase in the particle/polymer ratio.

Mechanically milled silk particle - polyurethane blend

film has been prepared to realise a controlled heparin release

system for achieving antithrombogenic property in the film


[48]. Slow heparin release rate and low percentage cumulative

amount of the released heparin was realised with an increase

in the amount of the loaded heparin in the film, the silk

fibroin particles content in the film and the thickness of the

film.

As a scaffold needs to possess adequate mechanical

properties to match the intended implantation tissue such as

bone, Rockwood et al. [49] fabricated a composite scaffold

by using milled silk micro-particles to reinforce a silk

sponge for in vitro osteogenic tissue formation. The silk

micro-particle loading led to a substantial increase in the

scaffold compressive modulus from 0.3 MPa (non-reinforced)

to 1.9 MPa when the matrix : particle weight ratio was 1:2,

and this in 6 weeks dramatically improved the bone volume

fraction from 0.78 % for non-reinforced scaffold to 7.1 %

and 6.7 % respectively for 1:1 and 1:2 (sponge : particle)

loaded scaffolds. The improved mechanical properties in the

composite scaffold were reported to be resulted from the

high interfacial cohesion between the silk matrix and the

reinforcing silk particles [50] and increased material densifi-

cation [51].

Composite Textiles

Due to their high specific surface area and inherent

reactivity, the protein fibre particles have also been explored

to enhance the functional properties of textile substrates.

Xushan [52] has developed wool particle blended poly-

propylene fibres (2-3 % wool particles on weight basis) with

a fineness of 2.5-3.0 dtex/filament. Although the mechanical

properties of the blend fibre such as breaking strength, initial

modulus, breaking elongation and breaking work deteriorated,

the elastic recovery, moisture absorption and dye uptake

properties of the blend fibre were reported to be improved,

consequently conferring comfortable and linen like handle

to the ultimate blend fibre shirts.

Textile fabrics have also been functionalised by coating

with the milled animal protein fibre particles. A pure cotton

woven fabric has been coated with milled wool particles by

following the pad-dry-cure method [53]. During the treatment,

the cotton fabric specimen was padded after dipping into the

wool particle emulsion for 10 min, which was further dried

and cured in an oven at 130oC for 5 min. The warmth

retention property of the treated fabric showed improvement

as the thermal conductivity and Qmax values (represents

fabric cool sensation on touching) were reported to be

decreased from ~0.53 W/mk to ~0.48 W/mk and ~142 to

~129 respectively. However, the maximum moisture absorption

rate was found to be decreased from around 300 %/s for

untreated fabric to 20 %/s after the treatment.

Cheng et al. [54] studied the ultaviolet protection and

wrinkle recovery properties of the mechanically milled

nano-scale wool powder (mean particle size of 76.8 nm)

coated cotton fabrics. They also applied the wool particles

onto a cotton fabric using the pad-dry-cure method. The

wrinkle recovery angle of the treated cotton fabric increased

from 70-90 o to 120-130 o in both warp and weft directions,

whereas the Ultraviolet Protection Factor (UPF) enhanced

from ~7 to ~12.

By using an electrospraying technique to apply Cashmere

guard hair particles onto a fabric surface and subsequently

loading silver ions on the protein particle layer, we have

demonstrated that the silver loaded Cashmere guard hair

particles can considerably enhance the antibacterial properties

of the fabric treated [55]. The particle coating was stable

against rubbing and washing.

Summary and Outlook

High value of animal protein fibre wastes can be realised

by converting them into fine particles for many novel

technical applications. Different indirect chemical techniques

of protein fibre particle preparation rely on small scale

production in laboratory based processes and they can

produce uniform particles with good aqueous dispersability.

These fine and uniform particles may find applications in the

field of medical science. However, animal protein fibre

particles prepared by this technique suffers from the use of

harmful chemicals, scanty crystallinity compared to the

parent fibres, long processing time and limited viability

towards the scaled up production. On the contrary, the direct

mechanical milling to prepare the protein fibre particles can

largely retain the native fibre molecular architect and is easy

to scale up. Mechanically milled particles may find applications

in environmental protection and composite structures.

However, the mechanically milled particles are non-spherical

in shape and possess wide particle size distribution. Further

work is warranted to find ways of producing fine and uniform

protein fibre particles on a large scale, and to explore and

realise the full application potential of such ultrafine particles

from both protein and other natural fibre sources.

References

1. A. P. Brian Moir, Cotton Promo. Bull., 28, Spring (2011).

2. S. Herrmann, G. Wortmann, and F. Wortmann, World

Rabbit Sci., 4, 149 (1996).

3. S. Herrmann and F. Wortmann, Livest. Prod. Sci., 48, 1

(1997).

4. Z. Martinez, L. Iniguez, and T. Rodríguez, Small Ruminant

Res., 24, 203 (1997).

5. J. Hodgkin, M. Galbraith, and Y. Chong, Fire Mater., 7,

210 (1983).

6. B. Dent, S. Forbes, and B. Stuart, Environ. Geol., 45, 576

(2004).

7. T. Korni owicz-Kowalska and J. Bohacz, Waste Manag.,

31, 1689 (2011).

8. E. Vasileva-Tonkova, A. Gousterova, and G. Neshev, Int.

Biodeterior. Biodegrad., 63, 1008 (2009).

9. D. G. Syed, J. C. Lee, W. J. Li, C. J. Kim, and D. Agasar,

ll


Bioresour. Technol., 100, 1868 (2009).

10. EN SANCO/445/2004 Rev. 1, European Commission,

Brussels, 28 (2007).

11. T. Zhao and G. Sun, J. Appl. Polym. Sci., 103, 482 (2007).

12. M. Tsukada, T. Arai, G. Colonna, A. Boschi, and G. Freddi,

J. Appl. Polym. Sci., 89, 638 (2003).

13. Y. Kawahara and M. Shioya, J. Appl. Polym. Sci., 73, 363

(1999).

14. R. Rajkhowa, L. Wang, and X. Wang, Powder Technol.,

185, 87 (2008).

15. J. G. Hardy, L. M. Römer, and T. R. Scheibel, Polymer, 49,

4309 (2008).

16. Z. Cao, X. Chen, J. Yao, L. Huang, and Z. Shao, Soft

Matter, 3, 910 (2007).

17. K. D. Collins, Methods, 34, 300 (2004).

18. A. S. Lammel, X. Hu, S. H. Park, D. L. Kaplan, and T. R.

Scheibel, Biomaterials, 31, 4583 (2010).

19. U. K. Slotta, S. Rammensee, S. Gorb, and T. Scheibel,

Angew. Chem. Int. Ed., 47, 4592 (2008).

20. P. Sun, Z. T. Liu, and Z. W. Liu, J. Hazard. Mater., 170,

786 (2009).

21. J. Nam and Y. H. Park, J. Appl. Polym. Sci., 81, 3008

(2001).

22. X. Wang, E. Wenk, A. Matsumoto, L. Meinel, C. Li, and

D. L. Kaplan, J. Control. Release, 117, 360 (2007).

23. X. Wang, E. Wenk, X. Zhang, L. Meinel, G. Vunjak-

Novakovic, and D. L. Kaplan, J. Control. Release, 134, 81

(2009).

24. A. Wilz, E. M. Pritchard, T. Li, J. Q. Lan, D. L. Kaplan,

and D. Boison, Biomaterials, 29, 3609 (2008).

25. T. Hino, M. Tanimoto, and S. Shimabayashi, J. Colloid

Interf. Sci., 266, 68 (2003).

26. A. Jaworek and A. Krupa, Exp. Fluids, 27, 43 (1999).

27. A. Gholami, H. Tavanai, and A. Moradi, J. Nanopart. Res.,

13, 2089 (2011).

28. H. Xie, S. Li, and S. Zhang, Green Chem., 7, 606 (2005).

29. W. Xu, W. Cui, W. Li, and W. Guo, Powder Technol., 140,

136 (2004).

30. Y. Cheng, C. Yuen, Y. Li, S. Ku, C. Kan, and J. Hu, J. Appl.

Polym. Sci., 104, 803 (2007).

31. Y. Li and W. Xu, “Apparatus for Producing Fine Powder”,

The Hong Kong Polytechnic University, HK, US, 2006.

32. Y. Li and J. Hu, “Method for Pulverizing Natural Organic

Substance into Nano-scale Fibrous Material”, The Hong

Kong Polytechnic University, HK, 2004.

33. K. Joko, Sen'i Gakkaishi, 59, 30 (2003).

34. T. Miyamoto, T. Amiya, and H. Inagaki, Kobunshi

Ronbunshu, 39, 679 (1982).

35. G. Wen, J. Rippon, P. Brady, X. Wang, X. Liu, and P.

Cookson, Powder Technol., 193, 200 (2009).

36. R. Rajkhowa, Q. Zhou, T. Tsuzuki, D. A. V. Morton, and

X. Wang, Powder Technol., 224, 183 (2012).

37. K. Patil, R. Rajkhowa, X. J. Dai, T. Tsuzuki, T. Lin, and X.

Wang, Powder Technol., 219, 179 (2012).

38. R. Rajkhowa, L. Wang, J. Kanwar, and X. Wang, Powder

Technol., 191, 155 (2009).

39. P. Shi and J. C. H. Goh, Int. J. Pharm., 420, 282 (2011).

40. A. Lammel, M. Schwab, M. Hofer, G. Winter, and T.

Scheibel, Biomaterials, 32, 2233 (2011).

41. M. Hofer, G. Winter, and J. Myschik, Biomaterials, 33,

1554 (2012).

42. R. Naik, G. Wen, S. Hureau, A. Uedono, X. Wang, X. Liu,

P. G. Cookson, and S. V. Smith, J. Appl. Polym. Sci., 115,

1642.

43. K. Patil, S. V. Smith, R. Rajkhowa, T. Tsuzuki, X. Wang,

and T. Lin, Powder Technol., 218, 162 (2012).

44. G. Wen, P. G. Cookson, X. Liu, and X. G. Wang, J. Appl.

Polym. Sci., 116, 2216 (2010).

45. J. Huang, X. Liu, W. Li, and W. Xu, J. Thermoplast.

Compos. Mater., 25, 75 (2012).

46. W. Xu, J. Fang, W. Cui, and J. Huang, Polym. Eng. Sci.,

46, 617 (2006).

47. W. Xu, X. Wang, W. Li, X. Peng, X. Liu, and X. G. Wang,

Macromol. Mater. Eng., 292, 674 (2007).

48. X. Y. Liu, C. C. Zhang, W. L. Xu, and C. X. Ouyang,

Mater. Lett., 63, 263 (2009).

49. D. N. Rockwood, E. S. Gil, S. H. Park, J. A. Kluge, W.

Grayson, S. Bhumiratana, R. Rajkhowa, X. Wang, S. J.

Kim, G. Vunjak-Novakovic, and D. L. Kaplan, Acta

Biomater., 7, 144 (2011).

50. R. Rajkhowa, E. S. Gil, J. Kluge, K. Numata, L. Wang, X.

Wang, and D. L. Kaplan, Macromol. Biosci., 10, 599

(2010).

51. E. S. Gil, J. A. Kluge, D. N. Rockwood, R. Rajkhowa, L.

Wang, X. Wang, and D. L. Kaplan, J. Biomed. Mater. Res.

A, 99 A, 16 (2011).

52. G. Xushan, Melliand Int., 12, 267 (2006).

53. J. Hu, Y. Li, Y. Chang, K. Yeung, and C. Yuen, Colloids

Surf., A, 300, 136 (2007).

54. Y. Cheng, Y. Li, C. Yuen, and J. Hu, AATCC Review, 6, 41

(2006).

55. K. Patil, X. Wang, and T. Lin, Powder Technol., 245, 40

(2013).

56. A. Jaworek, Powder Technol., 176, 18 (2007).

review on fabrication and applications of ultrafine particles from animal protein fibres

Documents