Characteristics of Protein-Based Biopolymer and Its
Application
Pratima Gupta, Kush Kumar Nayak
Department of Biotechnology, National Institute of Technology Raipur, Chhattisgarh 492010, India
This article aims to review the present scenario of protein
based natural polymer development, which has the ability
to stand against synthetic polymer. Demand of natural
polymers would increase in future considering their environmental safety aspect. Protein characteristics and their
suitability for polymer development are discussed here,
along with the polymer reinforcement techniques such as
development of blends, chemical block copolymerization,
and modification of existing protein material, which are
used for the development of biopolymer from protein. The
application of protein based polymer product range varies
from food and nonfood packaging stuffs to healthcare
C 2014 Society of
sectors. POLYM. ENG. SCI., 55:485–498, 2015. V
Plastics Engineers
INTRODUCTION
The primary goal of natural polymer based research is the
development of a system that can mimic the structure and function of native nondegradable synthetic polymer at some extend,
so that they can be replaced from extensive use thus making our
environment safe. A synthetic polymer, which is made-up of
petroleum products like polyethylene, polyvinyl chloride, polystyrene, are nondegradable and cause environmental damage
because they do not break down for tens of hundreds of years
[1] and persistence in the environment for a long time. The natural polymer over synthetic polymer offers a number of advantages such as complete degradation, increased soil fertility, low
accumulation of bulky plastic materials in the environment, and
reduction in the cost of waste management. Natural polymers
can obtained from three kinds of renewable resources: (a) plants
originated polymer such as starch [2], soy protein [3], and cellulose [4]; (b) from animals such as chitosan [5], keratin [6], silk
[7]; and (c) by microbial fermentation such as polyhydroxyalkanoates (PHA) and polyhydroxybutyrate (PHB) [8]. Technological advancement in polymer engineering gives new polymer
composites with novel characteristics for their desired application [9–11]. Cellulose [4], chitosan [12], starch [13], and PHB
[4, 13] etc., and their blends are explored considerably by various group of scientists because of its intrinsic ability to perform
very specific biochemical [14], mechanical, and structural roles
[3, 15].
Among natural polymer proteins are one of the strong candidates, which can be used for the development of new blend and/
or composite material. In natural state proteins are present as
either globular or fibrous structure form. The globular proteins
fold into complicated sphere-shaped structures held mutually by
Correspondence to: Pratima Gupta; e-mail: [email protected] or
Kush Kumar Nayak; e-mail: [email protected]
DOI 10.1002/pen.23928
Published online in Wiley Online Library (wileyonlinelibrary.com).
C 2014 Society of Plastics Engineers
V
POLYMER ENGINEERING AND SCIENCE—2015
an arrangement of hydrogen, ionic, hydrophobic, and covalent
(disulphide) bonds. The fibrous proteins are entirely extended
and coupled strongly together in parallel constructions, commonly through hydrogen bond to form fibers. The chemical and
physical characteristics of these proteins depend on the comparative amount of amino acid residues and its placement along the
polypeptide chain. The use of such amino acid sequence with
other natural and synthetic polymer can enrich polymer chemistry and science, which can be further exploited through chemical and protein engineering modifications to get novel polymer
design from proteins. Blends of protein with nonprotein, natural,
and synthetic molecules such as keratin-chitosan [16], glutenmethyl-cellulose [17], keratin-polypropylene, keratin-cellulosepolypropylene [18–20], and keratin-polyethylene [21] etc., are
explored by several scientists and they have reported that the
properties of the native protein film improved to some extend
(i.e., film strength, flexibility, and water vapor permeability,
etc.). Gluten [22], milk protein [23, 24], and soy protein [25] is
used in development of edible film, while keratin is used to
develop nanofiber [26], film [27], and composites [28] for material industries. The future application of protein based natural
polymer seems to be intense in the field of biomaterials [14,
29], packaging material and in coatings industries [30, 31]. The
present article provides a characterization of protein in its native
state for the formation of bio-based polymer along with the
polymer reinforcement technique for improving the characteristic/properties of peptide polymer to develop novel polymer
designs. The applications of such protein blends and composites
ranging to micro to macro structural level are comprehensively
discussed.
Protein Characteristics and Its Suitability for Polymer Development
Proteins are prepared from the basic unit called amino acids.
The protein’s structure is broadly categories into four structural
forms, which are called primary, secondary, tertiary, and quaternary structure. The primary structure of a protein is a linear
polymer with a string of amino acids coupled by peptide bonds.
Secondary structures of proteins are usually very regular in their
conformation and in point of fact, they are the spatial arrangements of primary structures. “Alpha Helices” and “Beta Pleated
Sheets” are two types of secondary structures and they are
majorly stabilized by hydrogen bonds. The tertiary structure of a
protein is the three-dimensional structure and is stabilized by
the series of hydrophobic amino acid residues and by disulfide
bonds formed among two cysteine amino acid. The tertiary peptide structure with less disulfide bonds form weak, rigid structures that are bendable, but still tough and can oppose rupture
such as hair and wool. While the structures that contain more
disulfide bonds lead to stronger, stiffer, and harder structures.
Quaternary structure of protein is the arrangement of more than
two chains of peptide, to form an entire unit. The interactions
between the chains are not like from those in the tertiary structure, but are differentiated solely by being an intermountain
range rather than an intrastring interaction. The quaternary structure occupies the bunch of abundant individual peptide chains
into an ultimate shape. A range of bonding interactions, including salt bridges, hydrogen bonding, and disulfide bonds hold the
variety of chains into a particular geometry. There are two foremost categories of proteins with quaternary structure, i.e.,
fibrous and globular protein. Fibrous proteins such as the keratins in hair and wool are composed of coiled alpha helical protein chains with other various coils analogs. Alternatively,
globular proteins may have an arrangement of the above types
of structures and are predominantly clumped into a shape of a
globe. Major examples include insulin, hemoglobin, and most
enzymes.
Keratin is a protein that contains disulfide bonds and has an
array of characteristics that ranges from a structurally robust,
impact-resistant material (horn) to a simple waterproof layer
(turtle shell). Keratin is together mechanically efficient in tension (wool) and compression (hooves) [32]. Keratins are found
in hair, wool, claws, nails, skin, fur, hooves, beaks, feathers,
horns, scales, actin, and myosin protein found in muscle tissues.
The main differences in various keratins arise from their sulfur
content. If there are many cysteine disulfide crosslinks, then
there is very little flexibility as in claws, hooves, horns, and
nails. In wool, skin, and muscle proteins, there are fewer disulfide crosslinks, which allow some stretching, but returns to normal upon relaxation of tension.
Silk is another outstanding biological polymer but has much
more complex structure. Actually, the final beta-pleated sheet
structure of silk is the result of the interaction of many individual protein chains. Specifically, hydrogen bonding on amide
groups on different chains is the basis of beta-pleated sheet in
silk proteins. Silks are produced by some insects such as from
spider and silkworm (Bombyx mori), but generally only the larvae silks have been used for textile manufacturing. Most silks
have extraordinary mechanical properties and demonstrate a
matchless combination of high tensile strength and extensibility.
The arrangement of strength and extensibility gives silks a very
high toughness, which equals that of commercial aromatic nylon
filaments [33], which themselves are benchmarks of current
polymer fiber technology. Spider silk has long been documented
as the marvel fiber for its unique combination of high strength
and break elongation. An earlier learning indicated spider silk
has strength as high as 1.75 GPa at a breaking elongation of
over 26% [34].
The quaternary structure of collagen consists of three lefthanded helices twisted into a right-handed coil. Collagen is a
cluster of naturally occurring proteins found in animals, because
of its unusual characteristics, such as biodegradability and weak
antigenicity [35], it is an important biomaterials for tissue engineering applications. Collagen fibers are commonly white, opaque, and viscoelastic matter acquire high tensile strength and
less extensibility. This collagen has been used as biomaterials in
drug delivery systems [36] and in tissue engineering [37]. A scientist reported that the Young’s modulus of the rat tail collagen
Type I vary between 3.7 and 11.5 GPa [38]. A series of studies
has focused on the structural and tensile properties of collagen
scaffolds for the purpose of designing functional biomaterials
for clinical application [39–42].
486 POLYMER ENGINEERING AND SCIENCE—2015
The investigations in a range of proteins such as gluten [43],
corn zein [44], soya [45], and milk [46] revealed that the proteins acquire the capability to form films, which can be used for
packaging. Such proteins have nutritional value as well, so it
can be also used for the development of edible film too.
Recently the progress of degradable films from protein has
drawn attention to a large extent due to protein’s skill to form
films and for its large quantity and renewable nature.
Polymer Enforcement Technique for Protein Material
Proteins/peptides are made up by several to thousands repeating unit of amino acid. The main chain of the peptide remains
constant throughout the length. The side chain of peptide may
consist of one of the 20 different functional groups acting on
their reactive side group “R”. These side chains verify the
nature and properties of the protein, and are accountable for the
infinite variety of protein shapes, functions, sequences, and its
nature [47]. Interaction phenomenon between natural and/or synthetic polymer with side chain groups of peptide allows to
develop natural polymer based film with enhanced properties
such as elasticity and toughness, etc. [48–50]. The different reinforcement approaches that are used for the development of protein based polymer can categorize in several ways that are
chemical agents and radiation treatment [51], chemical block
copolymerization [52–54], and preparation of blends [21, 55–
57]. Using these approaches it is possible to create a biodegradable and high performance polymer. For protein reinforcement
just four chemical targets account for the majority of crosslinking and chemical interaction, these targets are as follows:
1. Primary amines (ANH2): This group exists at the N-terminus
of each polypeptide chain and in the side chain of lysine
(Lys) residues.
2. Carboxyl (ACOOH): This group exists in the C-terminus of
each polypeptide chain and in the side chains of aspartic acid
(Asp) and glutamic acid (Glu).
3. Sulfhydryl (ASH): This group exists in the side chain of cysteine (Cys). Often, as part of a protein’s secondary or tertiary
structure, cysteine is joined together between their side chains
via disulfide bonds (ASASA), and
4. Carbonyls (RCHO): These aldehyde groups can be created by
oxidizing carbohydrate groups in glycoprotein.
Chemical and Physical Treatment
Interaction between natural and synthetic polymer and inbetween natural polymer is created by the use of chemical agents
and it is generally known as crosslinking agents. The aldehyde
such as formaldehyde and glutaraldehyde has widely used as
crosslinking agents to get better film characteristics. Formaldehyde is the common crosslinking agents and it interact with the
amino acids of peptide chain such as tryptophan, tyrosine, histidine, arginine, and cysteine, amino acids. Glutaraldehyde is more
specific than formaldehyde and it can interact with histidine, cysteine, tyrosine, and lysine. The effect of aldehyde as a crosslinker
on the properties of glutenin rich films have been studied by
Hernandez-Munoz et al. [58]. They mentioned that the value of
water vapor permeability of gluten rich films declines by around
30% when crosslinking agents such as glutaraldehyde, glyoxal,
DOI 10.1002/pen
caseinate film, suggested favorable interactions between whey
protein and calcium caseinate [61]. The soy protein isolates
film’s puncture strength have 37% higher than the nonirradiated
film [61]. The effect of c-irradiation on gluten film [62] and has
been investigated and the result indicates that irradiation treatment increases its tensile strength from 2.68 to 3.99 MPa and
decreases its water vapor permeability by 29% as compared to
nonirradiated sample. Irradiation treatment may be a useful as a
crosslinking agent to improve protein film properties.
Chemical Block Copolymerization
FIG. 1. The NHS Diaxirine ester is a hetero-bifunctional crosslinker, which
bind with primary amines of protein A and make diazirine-protein complex.
This complex again interacts with protein B when UV light exposed to
them.
Source:
http://www.piercenet.com/browse.cfm?fldID5F3324640A85B-7AB2-CBB8-CFD7065F70C6.
and formaldehyde has incorporated. Their results also describe
that the formaldehyde gives higher tensile strength values followed by glutaraldehyde and glyoxal. However, aldehydes also
have a major disadvantage that is their toxicity. This must be
taken into explanation when synthesizing biodegradable materials.
Because of the toxicity of aldehyde many researchers have been
trying to use the natural crosslinking agents to improve the film
properties. The study on the effects of natural crosslinking agents
(tannins and gallic acid) on the characteristics of thermo molded
films generated from sunflower protein isolate, elucidate the
incorporation of tannins and gallic acid in films gives higher tensile strength than for control films [59]. The Chestnut and Tara
tannin gave the largest gain in tensile strength from 2.8 to 4.2
MPa and 4.4 MPa for 3.5% and 6% of tannin respectively, but
inferior than those films obtained with an aldehyde. This may
possibly because they act through weak connections rather than
covalent bonds in the case of aldehyde. The addition of 1.5% glutaraldehyde increased the tensile strength of the film from 2.8 to
5.2 MPa with no loss of elongation [59].
Irradiation is a physical treatment, which is used to induce
modification in protein molecule [60]. It has found to be an efficient method for the enhancement of barrier (water and gas) and
mechanical (strength) properties of protein based edible films.
Proteins are affected by irradiation treatment (Fig. 1) either by
causing oxidation of amino acids, changes in amino acid conformation, split, or development of covalent bonds, and by the formation of protein free radicals [36]. In film forming solutions of
protein, super-oxide and hydroxyl anion radicals are generated
during the radiation. These anion radicals have the ability to
modify the molecular properties of proteins. Lacroix et al.
reported that c-irradiation was efficient in inducing crosslinks in
whey, casein, and soya protein edible films [61]. It is observed
that the puncture strength of c-irradiated whey protein film has
increased by 20% to 50% as compared with pure calcium
DOI 10.1002/pen
Over the years, block copolymers have attracted a great deal
of interest because they offer a unique platform to develop
material for diverse applications such as for drug delivery, tissue
engineering, and for food packaging. Block structures are placed
into various formats such as homo-block polymers, hetero-block
polymers, and hybrid block copolymers, which can made-up of
both natural and synthetic materials (Fig. 2). Combining polypeptide blocks with synthetic materials create hybrid block
copolymers with striking functional attributes that is the solubility, rubber elasticity, melt process ability of the synthetic block,
and the structure formation, mutual recognition, and biodegradability from the peptide block [53, 63]. Furthermore, advances
have been made in both the control of the assembly and function of homo-di-block copolypeptides. Hybrid block copolymers
exhibited both photo-activity and electro-activity, suggesting
applications in the field of biosensors, tissue engineering, and
nanoelectronic [64, 65]. In addition, hybrid block copolymers
containing pegylated peptides that respond to specific cellular
signals, such as the adhesion and migration of endothelial cells,
have been developed. The preparation of intelligent polymeric
micelles of functional polyethylene glycol-poly amino acid
(PEG-PAA) block [66] appear to be superior for both controlled
drug release and targeted delivery features with reduced toxicity
and improved efficacy significantly [66]. A few of examples of
block copolymer with polypeptide are shown in Table 1.
Preparation of Blends
A polymer blend is a mixture of materials in which at least
two different polymers are blended together to create a new
FIG. 2. Block copolymerization of peptide and synthetic polymer [53].
Synthetic polymer N-Carboxyanhydrides (NCA) bind with protein molecule
by interaction of primary amines (ANH2). By repeating of the above interaction long polymer chain formed.
POLYMER ENGINEERING AND SCIENCE—2015 487
TABLE 1. Instance applications of protein based block copolymers.
S. no.
1.
Block copolymer
2.
Peptide-synthetic hybrid blocks copolymers.
(polyisoprene-b-poly(epsilon-benzyloxycarbonyl-L-lysine)
PI-b-PZLys and polyisoprene-b-poly(L-lysine)
PI-b-PLys block copolymers)
Poly (ethylene glycol)-poly (amino acid) block copolymers
3.
4.
PEG/Peptide block copolymers
Poly (ethylene oxide) -block-peptide block copolymers
material with different physical properties. Some of the proteins
such as keratin [6], gluten [70], milk protein [71], zein [72], soy
[50], silk [73], and jatropha protein [74], etc., are well described
by the researchers and conclusions drawn from them are clearly
depicted that the composites made up of such proteins have a
promising future for natural polymer development. Protein such
as keratin and silk fibroin show high hydrophobic activity than
cellulose agriculture fibers and they have been used for development of composite with synthetic polymer like polyethylene [6,
48, 75]. This kind of composites is chemically compatible with
the hydrophobic polyethylene. Barone and Schmidt [21] have
been reported glass transition temperature (Tg) and crystalline
melting temperatures (Tm) of thermoplastic like polyethylene
and polypropylene become improved when they are blended
with keratin [21, 75]. The scanning electron microscope (SEM)
image (Fig. 3) of keratin and low-density polyethylene (LDPE)
blend demonstrate the fiber/polymer interface. A quantity of
matrix deformation occurs together with the fibers as the fibers
are pulled. A few of the fibers are fractured in the identical fracture plane as the polymer, which would point towards strong
fiber/polymer interactions [21].
Blends of keratin and chitosan have been developed, which
is used as a biomaterial since these proteins/carbohydrate composite possess biocompatibility and various biological functions such as wound healing and antibacterial activity [16].
Chitosan gave strength and flexibility to the keratin film.
Keratin-chitosan composite film had a softness judging from
Young’s modulus and composite film showed remarkably
improver water insoluble characteristics. However, the film
prepared from keratin mixed with 10 wt% chitosan have fairly
flexible and strong judged from ultimate strength (27 6 8
MPa), ultimate elongation (47 6 2%), and Young’s modulus
(152 6 76 MPa) [16]. Yoo and Krochta [46] studied the barrier, tensile, thermal, and transparency properties of whey protein and polysaccharide blend. They found that the blended
films are intermediate to properties of the pure polysaccharide
and whey protein film depends on the particular polysaccharide. In the case of methyl-cellulose or hydroxypropyl-methylcellulose with whey protein, the blended films reflect tensile
strength, and lower oxygen permeability [46]. The blending
technology gives an opportunity to develop blends of protein
polymer, which have different physical and mechanical properties, such as soy protein-agar blend film [50], of keratin-PEO
blend nanofibers [26], polylacticacid-keratin fibrous [76],
keratin-chitosan composite film [16] etc. Some of the examples
of protein blends are shown in Table 2.
488 POLYMER ENGINEERING AND SCIENCE—2015
Feature
References
Self assembled rod-coil copolymer nanostructures
[67]
The polymeric micelles feature a spherical 100 nm
core shell structure in which anticancer drugs are
loaded avoiding undesirable interactions in vivo
Its self assembly process
As for drug delivery, nontoxic, nonimmunogenic,
controlled-release systems for hydrophobic drugs
[66]
[68]
[69]
The SEM images of soy protein-agar blend film (Fig. 4) is a
sign of that the casting film possessed homogeneous interfaces
[50] and its Fourier transform infrared spectroscopy (FTIR)
study (Fig. 5) reveal that the interactions existed between soy
protein and agar by hydrogen bonding, and the active sites on
the soy protein molecular chains may be oxygen atoms of carbonyl groups [50]. The interactions between soy protein and
agar become stronger because of the enhanced blue-shift of
these bands with the increase of agar. The X-ray diffraction
(XRD) patterns of native agar and soy protein-agar blend films
(Fig. 6) gives a peak at 18.38 and a slight shoulder at around
14 , indicating a slightly crystalline [50]. After the use of
FIG. 3. SEM images of (a) 10 wt% and (b) 40 wt% 0.1 cm keratin feather
fiber in LD133A LDPE. The scale bar is 30 lm (Reproduced from Ref. 21,
with permission from Elsevier).
DOI 10.1002/pen
TABLE 2.
S. no.
Blend of proteins with natural and synthetic polymer with their improved properties.
Blend name
Improved property of the film of nanofiber
References
1.
2.
Carboxymethyl-cellulose/Soy protein
Keratin/Chitosan composite film
[3]
[16]
3.
Keratin/PEO blend nanofibers
4.
Whey protein/cellulose blend
5.
Soy protein/Agar blend
Enhanced tensile strength from 42.0 to 59.2 Mpa
Strong and flexible film, judged from ultimate strength 27–34
MPa, ultimate elongation 4–9% in the presence of 10–30 wt%
of chitosan.
Keratin/PEO blend at 30:70 ratio give relatively high tensile
strength, judged from young modulus 31 MPa, stress at break
6 MPa and strain at break 46.3 MPa.
Films show the better water insoluble property. Over 98% of the
recovery of blend film have been observed when treated at
100 C/30 min and over 99% recovery observed when treated
at 37 C/24 h
The tensile strength of the film raised from 4.1 to 24.6 MPa
glycerol as plasticizer, the shoulder peak at around 14 weaken
and a new weak peak at about 11.5 have been observed, which
indicate that the structure of agar have changed and it lead to
strong three-dimensional structure formation [50]. Figure 7
showed the SEM images of nanofibers produced electrospinning
the keratin/PEO blend solutions. The solution containing varying
wt% (90–10 wt%) of keratin, produced droplets, and bead-like
defect structure, solutions with a lesser content of keratin could
be electrospun without defects. Furthermore, SEM analysis
exposed that the diameter distribution of keratin-rich nanofibers
is narrow and the common diameter of the filaments reduced
with the raise of the keratin content.
Application of Protein Based Material in Packaging
Protein based edible films are attractive for food application
because of their high nutritional quality, excellent sensory properties, and good potential to adequately protect food products
from their surrounding environment. Such films act as a carrier
of antioxidant, flavor, and bacteriostats and can improve the
quality of food products. Protein based films have received considerable attention in recent years because of its uses in edible
and nonedible packaging materials. The proteins such as soybean [45], corn [44], wheat [44], peanut [78], and sunflower
seed [79] etc., as well as gelatin from collagen [80] and milk
proteins (casein and whey proteins) [81] are appropriate for the
production of edible film because of its nutritional properties as
aforementioned. Several globular proteins, including gluten [82],
[26]
[77]
[50]
corn protein [44], soy protein [50], and whey protein [83] etc.,
have been investigated for their film properties. Protein based
edible films have impressive gas barrier properties as compared
with those prepared from lipids and polysaccharides [84]. When
they are not moist, the O2 permeability of the soy protein film
was 500, 260, 540, and 670 times lower than that of low density
polyethylene, methyl-cellulose, starch, and pectin, respectively
[84]. Several researchers studied the application of protein based
edible films in food use [85, 86] and they reviewed the applications of protein films, such as soy protein film, casein emulsion
film, whey protein film, and corn-zein films on nut and fruit
products. The polymeric characteristics of the protein film have
been used for edible food packaging application [45, 72, 81, 87,
88] but for nonfood packaging application the major problems
are an advance of mechanical properties (such as toughness,
strength, and elasticity, flexural, shear strength, tensile modulus,
etc.). The step head blends of protein and nonprotein molecule
have been prepared with improved mechanical properties [26,
89–92]. Massive chances still exist to create a new kind of
blends with new characteristics, which could be used for both
food and nonfood packaging.
Gluten Films
The cohesiveness and elasticity of gluten facilitate the film
formation [43]. The gluten films are stronger and are also a
good barrier to O2 and CO2 [22] but are highly permeable to
water vapor and need to be made it impermeable for
FIG. 4. SEM micrographs of the cross-sections of the molding soy protein/agar blend films (a) SA15M and (b)
SA65M (Reproduced from Ref. 50, with permission from Elsevier).
DOI 10.1002/pen
POLYMER ENGINEERING AND SCIENCE—2015 489
face of sliced apples and potatoes. Whey protein fractions (blactoglobulin and b-lactalbumin) and pure whey protein isolates
has used successfully for film development [46] while caseinate
films have used for coating in apricot, papaya, chicken eggs,
apples, oranges, and for enzyme immobilization [94]. Whey protein concentrate have reported to be less permeable to water
vapor as compared to caseinate based film and whey protein
(fraction) based films. Also, the puncture strength of whey protein concentrates films was lowest and provided a good barrier
to O2, aroma and oil at low to intermediate relative humidity
[95]. Casein based films and biomaterials obtained from caseinate can find many applications in packaging [96–98], in edible
films and coatings for fruits and vegetables [98–100], or in
mulching films [101]. Some of the milk films formed with plasticizer are described in Table 3 with their mechanical strength.
FIG. 5. FTIR spectra of native agar, glycerol plasticized soy protein and
agar, and the soy protein/agar blend films (Reproduced from Ref. 50, with
permission from Elsevier). [Color figure can be viewed in the online issue,
which is available at wileyonlinelibrary.com.]
commercialization. Gluten has been used for coating dry roasted
peanuts and fried chicken pieces [87]. Experimental results
reveal that the casting films of gluten/methyl-cellulose blend
containing (25 wt %) glycerol plasticizer are superior in
mechanical properties of the molded composites. The wheat gluten/methyl-cellulose binary blend films show tailored mechanical and moisture barrier properties (17.3 to 33.4 wt % at 87%
RH), tensile strength (1.7 to 44.0 MPa for different blend fraction), and water vapor permeability (17.86 to 30.38 10211
gm21 s21 Pa21 at 87% RH) [17]. Cationic waterborne polyurethanes (CWPU) have been prepared and blended with gluten in
aqueous dispersion, this blend powders were thermally compressed molded into sheets with improved water resistance property [82]. The SEM image of gluten and the CWPU (Fig. 8)
indicates that homogeneous morphology and good interfacial
adhesion are beneficial and increase the impact strength of the
materials. While the FTIR study (Fig. 9) demonstrate the compatibility is exist in between gluten and CWPU [82]. Parnas and
coworkers [93] mentioned that the silica particles in the gluten
matrix (gluten/organo-silica composites) led to mechanical
strength i.e., tensile modulus 3.52 GPa, tensile strength 35.48
MPa, and elongation at break 1.03%, and similarly, coating the
alumina particles with saline coupling agents (ICEOS) in the
gluten matrix (gluten/Al2O3–ICEOS composite) gives the tensile
modulus 3.13 GPa, tensile strength 40.49 MPa, and elongation
at break 1.17% [93]. The fracture surface of composite as shows
on the SEM image (Fig. 10) describe that the composites made
from the two-step blending, provided better results for strength
and elongation [93].
Soy Protein Films
Soy proteins are inexpensive, abundant, and biodegradable
and have nutritional value as well. They have the potential to be
developed as biodegradable and edible films. Protein based edible films can form bonds at diverse positions and offer high
potential for forming numerous linkages. However, soy protein
films still have low moisture barrier properties because of their
hydrophilic property and the considerable amount of hydrophilic
plasticizer used in film preparation. One broadly used method to
enhance the water vapor barrier of films has been the integration
of hydrophobic compounds such as lipids into the film forming
solution. In addition, another way to get better the properties of
soy protein film is to alter the protein network arrangement
through crosslinking of the protein chains. The occurrence of
reactive functional groups in the amino acid side chain of protein makes this crosslinking process achievable through enzymatic, chemical, or physical treatments. The exceptionally low
O2 permeability values of soy protein isolates (SPI) films provide an opportunity for preserving foods from oxidative deterioration [45]. SPI film is used for delaying the oxidation and
hydrolysis reaction of packaged lard [45]. Therefore, SPI films
have potential as a packaging material, which will preserve the
qualities of stored food ingredients. A succession of SPI and
Milk Protein Films
Milk proteins (casein and whey) have excellent nutritional
value and possess numerous functional properties (its solubility
in water and ability to act as an emulsifier), which are important
for the formation of edible films [49]. The report of Tien et al.
[81] explain the effect of milk protein based edible coatings on
the browning reaction of sliced apples and potatoes [81]. The
results confirm that the film formulations become effective in
delaying browning reactions by acting as O2 barriers on the sur-
490 POLYMER ENGINEERING AND SCIENCE—2015
FIG. 6. XRD patterns of soy protein-agar blend films (Reproduced from
Ref. 50, with permission from Elsevier). [Color figure can be viewed in the
online issue, which is available at wileyonlinelibrary.com.]
DOI 10.1002/pen
FIG. 7. Scanning electron micrographs of keratin/PEO blend nanofibers: (a) 90/10, (b) 70/30, (c) 50/50, (d) 30/70,
and (e) 10/90 (Reproduced from Ref. 26, with permission from Elsevier).
agar blend films containing 33% glycerol as plasticizer have
fabricated and its tentative results exposed that hydrogen bonding interactions existed among soy protein and agar [50]. The
tensile strength of the blend films enhanced with the incorporation of agar at different concentration. With the increase of
agar, the tensile strength of the blend films raised from 4.1 to
FIG. 8. SEM images of fractured gluten-CWPU blend sheets with different types and contents of CWPU (a) Pristine gluten, (b) WP-20-1, (c) WP-50-1, (d) WP-20-2 (Reproduced from Ref. 82, with permission from Wiley).
DOI 10.1002/pen
POLYMER ENGINEERING AND SCIENCE—2015 491
FIG. 9. FTIR spectra of powders of CWPU and two molded gluten/CWPU
blends with PCL-1 diol and MDI (hard segment: 46%). (Reproduced from
Ref. 82, with permission from Wiley).
24.6 MPa [50]. In another study, food grade carboxy-methylcellulose (CMC) and SPI blend fruitfully employed to fabricate
novel edible films [3]. The XRD (Fig. 11) investigated reveals
that SPI and CMC are extremely compatible, and the addition
of glycerol reduces the crystallinity of CMC/SPI blends [3]. The
FTIR study (Fig. 12) explains that complex Maillard reactions
should happen between SPI and CMC. Additionally, in the SPI/
CMC blends the free amino groups of SPI/CMC films decreased
rapidly with the increasing CMC addition. The images of CMC/
SPI film (Fig. 13), which made-up by a continuous casting
method, demonstrate that it can be manufactured easily with no
cracks and puncture. Overall the films have a good durability,
and flexible as much as necessary to be rolled into forms for
sensible applications [3].
Researchers have tried to advance the properties of soy protein films that have prospective uses in the food packaging
industry [105–107]. In the United States it is estimated that,
about 25,000–50,000 metric tons of soy proteins are used in
paper coating industries [108]. It is found that SPI coated paper
FIG. 10. Fracture surfaces of composite prepared by the in situ blending at 1/1 silane/Al2O3 by mole (a) WG/Al2O3
in acetone, (b) WG/Al2O–TEOS in acetone catalyzed by TEA, (c) WG/Al2O3–MTMOS in acetone catalyzed by
TEA, (d) WG/Al2O3–GTMOS in acetone catalyzed by TEA, (e) WG/Al2O3–ICEOS in acetone catalyzed by TEA (f)
WG/Al–ICEOS in toluene catalyzed by Sn(Oct)2 (g) WG/Al2O3–TESBA in acetone catalyzed by TEA (Reproduced
from Ref. 93, with permission from Elsevier).
492 POLYMER ENGINEERING AND SCIENCE—2015
DOI 10.1002/pen
TABLE 3. Comparison of tensile strength and elongation at break of the
milk protein film formed in the presence of plasticizers.
Film
Elongation
at break
(%)
Tensile
strength
(MPa)
Sodium caseinate/Glycerol (4:1)
Sodium caseinate/Glycerol (2:1)
Sodium caseinate/PEG (4.54:1)
Sodium caseinate/PEG (1.9:1)
Whey protein/Glycerol (5.7/1)
Whey protein/Glycerol (2.3/1)
Whey protein/Sorbitol (2.3/1)
Whey protein/Sorbitol (1/1)
10.5
73.7–84.2
5.3
25.4
4.1
30.8
1.6
8.7
17.4–26.7
10.9–11.7
10.9–16.35
10.9–13.9
29.1
13.9
14.0
14.7
References
[102]
[103, 104]
act as gas and oil barrier as well as having enough mechanical
properties (strength, elasticity, etc.), for pull out the shelf life
time of food commodities [109]. Rhim et al. [110] give a statement that the SPI coated paper boards grant higher water resistance than that of alginate coated paper boards. Brother and
McKinney [111] reported plastics making by using soy protein
and various crosslinker agents at melt state. Paetau et al. [112]
reported the groundwork and processing environment for making biodegradable plastics from soy concentrate and soy isolate.
Soy concentrate and isolate, as well as acid treated soy concentrate and soy isolate, were compression molded at various molding temperatures and moisture levels. Sulfuric acid, acetic acid,
hydrochloric acid, and propionic acid have examined for their
appropriateness for treating soy protein with regard to ending
properties. The molded specimens have also tested for their percentage elongation, Young’s modulus, tensile, yield strength,
and water absorption. The plastics obtained by molded technique are rigid and brittle, with tensile values from 10 to 40
MPa, yield strength values from 30% to 167% weight [112].
Plastic specimens made from soy concentrate displayed similar
tensile value, but greater water absorption as compared with
plastics made from soy isolate.
Corn Zein Films
Zein, a component of corn, is a unique and complex material
and has long been investigated for its uses for a variety of purposes other than food and feed such as coatings, inks, adhesives,
and fibers, etc. [113]. Zein proteins have importance due to its
ability to solubilize in binary solvent system containing water
and a lower aliphatic alcohol, such as aqueous isopropanol and
aqueous ethanol. Corn zein films and its coatings are used as O2
and moisture barrier for nuts, candies, and other foods [72].
They have relatively insoluble in water and forms strong glossy
films resistant to grease and O2. Zein has natural resistance to
bacterial attack [114], it forms tasteless coating and has stability
in conditions of high humidity and high heat as well as corn
zein coated paper proved more effective films for wrapping O2
sensitive foods and in regards to O2 barrier properties [72]. Performance of zein films as barrier packaging for tomatoes,
cooked turkey, popcorn, and shell eggs has been evaluated by
Ersus and coworkers [115] and they found that the zein film
coating provided barrier effect and beneficial internal O2 composition for inhibiting microbial growth. It is valuable to conduct advanced researches on coating of intermediate moisture
DOI 10.1002/pen
fruits with different coating materials or different combinations
of edible films. Usage of different additives or antimicrobial
agents, determination of film thickness, could be alternative
research areas for improving film coating effect on intermediate
moisture fruits.
Lim and Jane [116] reported that the injection molded corn
starch-zein plastics at 6.6% water content and 11.5% glycerol
exhibited good 4.5–5.3% elongation at break and 22 to 25 MPa
tensile properties, in a molding temperature range of 150 to
160 C.
Rakotonirainy and Padua [117] used compression molding
technique to obtain ply laminated zein sheets. The individual
film components have been obtained by solution-casting and the
pressing carried out in at 120 C. The lamination procedure
induced melting and flow of the oleic acid-zein films, decreasing voids, and defects. As a result, mechanical and oxygen permeability properties improved. Pol et al. [118] took advantage
of the thermoplastic properties and differences in molecular
weight of soy protein and corn zein to produce a single and
double coat laminates by compression molding. Compression
molding can result in the development of a film based on protein with a range of barrier and mechanical properties that are
dependent on the formulation and processing conditions used.
Compression molding is an appropriate technology to inspect
the thermoplastic properties of plasticized proteins as well as
the properties of the resulting films. It can also serve as a step
toward the use of a more continuous, high speed technology for
film manufacture.
Gelatin Films
Gelatin is a transparent, flavorless solid colorless material,
derived from collagen obtained from diverse animal byproducts. It is commonly used in the pharmaceutical and food
industries, and produced on a large scale at comparatively low
price. On account of its functional properties it has been used
for the production of biodegradable films [119]. Although like
the mainstream of films based on protein, it has a partial barrier
to water vapor [119]. The gelation, thermal, mechanical, and
FIG. 11. X-ray patterns of (a) pure SPI powder, (b–g) CS-5, CS-10, CS-15,
CS-20, CS-25, and CS-30 CMC/SPI films, (h–l) CS-20-1, CS-20-2, CS-20-3,
CS-20-4, and CS-20-5 CMC/SPI glycerol films (Reproduced from Ref. 3,
with permission from Elsevier). [Color figure can be viewed in the online
issue, which is available at wileyonlinelibrary.com.]
POLYMER ENGINEERING AND SCIENCE—2015 493
FIG. 12. Maillard reactions tested by FTIR (a) FTIR spectra of CMC, (b) molecular structure of CMC with a
degree of substitution x, (c) FTIR spectra of SPI, (d and e) a and b molecular structure of SPI, (f) FTIR spectra of
CS-5, CS-10, CS-15, CS-20, CS-25, CS-30, CS-35, and CS-40 CMC/SPI blends, (g) a typical Maillard reaction
between CMC and an amino acid (glutamine, Gln) (Reproduced from Ref. 3, with permission from Elsevier). [Color
figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
oxygen permeability properties of gelatin films have studied by
Avena Bustillos et al. [120]. They described that the tensile
strength, percent elongation, and puncture deformation were
highest in mammalian gelatin films, followed by warm-water
fish gelatin film and then by cold-water fish gelatin films [120].
Further the property of gelatin film can improve by the chemical
treatment, for example films reticulated with formaldehyde and
glyoxal have higher tensile strengths, opacity, lower water vapor
permeability, and good color differences than the untreated films
[121].
Silk Protein Films
Silk is a fibrous protein and have a range of properties like
resistance towards oxidation, anti-bacterial, UV resistant, capability to absorb, and releases moisture. It can be copolymerized,
blended, and crosslinked with other natural/synthetic materials
with ease. The silk composites are useful to develop biomedical
materials, functional membranes, fabrics, and fibers. Jiang et al.
[122] developed robust, ultra-thin silk fibroin films, and characterized it by a high elastic modulus of 6–8 GPa (after treatment
with methanol) and by the ultimate tensile strength up to 100
MPa. They also suggested that these films have probable applications in biocompatible implants, micro-scale bio-devices, synthetic coatings for artificial skin [122], and coating material for
natural and artificial fibers, fabrics [123]. As well as such kind
494 POLYMER ENGINEERING AND SCIENCE—2015
of silk protein materials are useful preparation of degradable
shopping bags, wrapping film, and composting bags, etc. [124].
Application of Protein Based Material in Health Care
Biocompatible materials from proteins have been used to
develop biological matrix or scaffolds for various biomedical
applications including, tissue engineering, wound dressings,
membrane filters, and drug delivery. Protein materials give a
chance to develop new generation biomaterials because protein
is capable of carrying out a variety of functions such as carriers
for drug delivery, scaffold in tissue engineering. Protein such as
silk [125], gelatin [89], and elastin [126] etc., shows good compatibility within the human body and use as a scaffold in tissue
engineering with great success. Hu et al. [127] developed biomaterials from blends of silk fibroin and tropoelastin system.
These blends film offers a new material system for cell support
and tailored biomaterial properties to match mechanical needs
such as modification, of mechanical features, with resilience
from 68% to 97%, and elastic modulus between 2 to 9 Mpa,
depending on the ratio of the two polymers [127]. Researchers
developed homo-polymers, diblock copolymers, and triblock
copolymers of the protein [128], and such block copolymers
provide an extra level of control for drug delivery, and serve as
novel scaffolds for tissue engineers because they provide good
physical and biochemical support for both differentiated and
progenitor cells [129].
DOI 10.1002/pen
tion and use of these responsive protein polymers with specifically designed macroscopic and microscopic structural and
chemical features. The application of the protein polymers for
drug delivery shows great promise when protein is immobilized
in smart hydrogels [132]. It would then be possible to translate
the chemical signal into the environmental signal and then into
the mechanical signal, namely shrinking or swelling of smart
gel. The shrinking or swelling of smart hydrogels globule in
reaction by minute changes in temperature or pH can be utilized
effectively to organize drug release [134]. By using such drug
formulations incorporated into hydrogels, pharmaceutical companies will be able to increase the efficiency, cost effectiveness,
and range of applications for existing therapeutics.
Almany and Seliktar [135] described a biosynthetic hybrid
hydrogels scaffold [135] composed of a fibrinogen backbone
and crosslinked with bifunctional polyethylene glycol side
chains, which provides a distinct advantage over other hydrogel
scaffold materials because its mechanical properties are highly
malleable while the biological functionality is maintained by the
backbone of the polymeric network. Koutsopoulos et al. [136]
have demonstrated a gel known as a “nanofiber hydrogel
scaffold,” which is composed of small protein fragments, can
successfully carry and release drugs of different size, potentially
enabling delivery of drugs such as insulin and herceptin [134,
137]. And they can control the rate of release by changing the
density of the gel, allowing for continuous drug delivery over a
specific period of time [136]. These hydrogels enables, over
hours, days or even months, a gradual release of the drug from
the gel, and the gel itself is eventually broken down into harmless amino acids the building blocks of proteins. Peptide hydrogels are ideally suited for drug delivery as they are pure, easy to
design and use, nontoxic, nonimmunogenic, bio-absorbable, and
can be locally applied to a particular tissue.
Challenges for the Development of Protein Based Biopolymer
FIG. 13. Photographs of CMC/SPI film in expanded and rolled states fabricated by a continuous casting method (Reproduced from Ref. 3, with permission from Elsevier). [Color figure can be viewed in the online issue,
which is available at wileyonlinelibrary.com.]
The continuing developments in the protein based material
have been carried out by researchers, which offer the viewpoint
of substantial impact on clinical practice in surgery and regenerative medicine. Controlled drug delivery systems have become
increasingly important mainly because of the awareness of the
difficulties associated with a variety of old and new drugs. Biodegradable protein polymers can be used as drug delivery systems because of their biocompatibility and biodegradability
[130]. The protein based natural polymers can be used in the
form of micro particles, from which the incorporated drug is
released to the environment in a controlled manner [131].
Advances in protein polymer science have led to the development of several novel drug delivery systems [132]. A proper
consideration of surface and bulk properties can aid in the
designing of protein based polymers for drug delivery applications [133]. Much of the development of novel protein based
materials in controlled drug delivery is focused on the prepara-
DOI 10.1002/pen
In natural states, proteins generally exist in two form fibrous
proteins and globular proteins [138]. The former form is water
insoluble [139] and the later form is water soluble or soluble in
aqueous solutions of acids or bases [140]. The chemical/physical
properties of these proteins depend on the relative amounts of
amino acid residues and their position along the protein polymer
chain [83]. Films from peptide polymer are usually produced
from dispersions of the protein as the solvent evaporates, and
the solvent is generally limited to ethanol water mixtures, ethanol, or water. The challenge is to make possible to use some
more solvents have a good quality and that can help to make a
better crosslink in between protein molecules. Another challenge
is to control the degree of bond formation during the chain
interaction. Generally, acid, bases, heat, and/or solvents are usually used to denature the protein in order form film. Once the
extended film, chain to chain interaction of peptide can attach
throughout by ionic, hydrophobic, hydrogen, and by covalent
bonding, and such interaction of peptide chain could be controlled by the deviation in degree of bond formation. Protein
molecules are expected to have all kinds of feasibility to prepare
natural polymer and it is predicted that the major challenges are
to improve the physical and mechanical properties of protein
polymers, so that they can mimic the function of native synthetic polymer at some extend. The polymer characteristics of
POLYMER ENGINEERING AND SCIENCE—2015 495
the proteins have been successfully used for the formation of
edible food packaging by various researchers [45, 72, 81, 87,
88]. But in nonfood packaging the major problems in the development of protein based polymer are an enhancement of protein
film properties such as toughness, strength, and elasticity, flexural, shear strength, tensile modulus, etc. The step head blends
of protein and nonprotein molecule have been prepared successfully with improved characteristics [26, 89–92]. Use of suitable
binder/plasticizers or crosslinking agent is also a part of future
challenge in respect to the enhancement of the adhesive or cohesive property of the protein based biopolymer.
CONCLUSION
Protein molecules are one of the biological materials among
other natural polymers such as PHA, PLA, starch, and cellulose,
etc., and it has been used for the development of natural polymers. Many of the proteins like silk, gelatin, keratin, soy protein, and casein, etc., have executed very interesting features of
polymers such as flexural, shear strength, tensile modulus, as
well as exceptional material properties including toughness,
strength, and elasticity, for the creation of novel bio-polymer.
However, in native form protein polymers are weak and not
suitable for the product development, but this problem can be
overcome by the polymer reinforcement technology. Polymer
reinforcement technology offers an opportunity to change the
physical and mechanical properties of protein polymer as per
desired product. Such modification will be helpful to use them
for various applications from micro (as biomaterial for drug
delivery application) to macro scale (as material for packaging,
for tissue engineering and for bio-composite). Massive chances
still exist to create a new kind of blends of protein polymer
with new characteristics, which could be used for both food and
nonfood packaging.
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DOI 10.1002/pen