International Journal of Biological Macromolecules 104 (2017) 687–707

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Review

Improving the integrity of natural biopolymer films used in food

packaging by crosslinking approach: A review
Farhad Garavand a , Milad Rouhi b , Seyed Hadi Razavi a,∗ , Ilaria Cacciotti c ,
Reza Mohammadi b
a
Bioprocess Engineering Laboratory (BPEL), Department of Food Science and Engineering, Faculty of Agricultural Engineering and Technology, University of
Tehran, Karaj, Iran
b
Department of Food Science and Technology, School of Nutrition Sciences and Food Technology, Kermanshah University of Medical Sciences, Kermanshah,
Iran
c
Department of Engineering, University of Rome “Niccolo Cusano”, INSTM RU, Via Don Carlo Gnocchi, 3, 00166 Rome, Italy

a r t i c l e i n f o a b s t r a c t

Article history: Currently used approaches for biopolymer modification are either expensive, poisonous or do not lead
Received 14 March 2017 into the well-desired characteristics to the final film materials. Development of crosslinking procedure is
Received in revised form 17 June 2017 an innovative strategy to improve mechanical, physical and thermal properties of biopolymer films. This
Accepted 21 June 2017
review provides a brief description of film-forming biopolymers (e.g. chitosan, whey protein, alginate
Available online 23 June 2017
and starch) followed by a detailed introduction to definition and classification of various crosslinkers,
the effect of crosslinking on emerging attributes of biopolymer films including physical, mechanical and
Keywords:
thermal properties, identification of crosslinking occurrence, and cytotoxicity status of commonly used
Biopolymer
Crosslinking
crosslinkers in the field of food and food-related packaging materials.
Film © 2017 Elsevier B.V. All rights reserved.
Food packaging
Mechanical properties

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 688
2. Biopolymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 688
3. Crosslinking agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 689
3.1. Crosslinking at ambient temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 689
3.2. Crosslinking at intermediate temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 689
4. Crosslinking agents in biopolymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 690
5. Influence of crosslinking on biopolymers characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 691
5.1. Physical properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 691
5.1.1. Water absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 691
5.1.2. Water vapor permeability (WVP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 691
5.1.3. Solubility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 691
5.1.4. Film thickness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 698
5.1.5. Light barrier properties and transparency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 698
5.1.6. Color changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 698
5.2. Mechanical properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 698
5.3. Thermal properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 699
5.3.1. Differential scanning calorimetry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 700
5.3.2. Thermogravimetric analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 701

∗ Corresponding author.
E-mail address: srazavi@ut.ac.ir (S.H. Razavi).

http://dx.doi.org/10.1016/j.ijbiomac.2017.06.093
0141-8130/© 2017 Elsevier B.V. All rights reserved.
688 F. Garavand et al. / International Journal of Biological Macromolecules 104 (2017) 687–707

6. Complementary techniques for identification of crosslinking occurrence in biopolymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 701

6.1. X-ray diffraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 703
6.2. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 703
6.3. Fourier transform infrared spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 703
6.4. Nuclear magnetic resonance (NMR) spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 704
7. Cytotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 704
8. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 704
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 705

1. Introduction Crosslinking agents are suitable for biopolymer materials partic-

ularly those originated from carbohydrates or proteins, providing
The main mission of food packaging is to maintain the quality a reduced water vapor and gases permeability in food packaging
and safety of food products during storage and transportation, and materials. Crosslinkers can overcome the inherent deficiencies in
to broaden their shelf-life by avoiding unlikeable circumstances, mechanical and barrier properties of biopolymers, making them
such as hazardous microorganisms and their corresponding toxins, more applicable compared to petroleum-based counterparts for
external physical forces, chemicals compounds, sunlight, perme- food packaging purposes. A heat-light resistance biopolymer along
able volatile compounds, oxygen and moisture [1]. In order to with a reduced water solubility and swelling can be expected by
accomplish such a mission, packaging materials should arrange introducing crosslinking agents. Chemical crosslinking is the most
for physical protection and establish appropriate physiochemical accredited procedure than the others. Protein-containing biopoly-
and microbiological situations to products that are indispensable mers are inclined to participate more in the reactions induced by
to achieve an acceptable endurance, as well as uphold the quality- crosslinkers, because of their various reactive and functional groups
safety issues of food items [2,3]. [13]. The typical crosslinking process involves the use of a wide
Currently, a large number of petroleum based polymers are range of crosslinking agents (e.g. glutaraldehyde, epichlorohydrin,
used in food, beverage and foodstuff packaging industries [4], with Ca2+ ions, sodium trimetaphosphate, sodium benzoate, phosphoryl
consequent accumulation of non-degradable and non-recyclable chloride, citric acid, boric acid and Maillard reaction) [11,14–17].
waste materials and concerns over environmental issues. Thus, This review will present recent developments of crosslinked
there is an increasing demand for bio-based raw materials in order natural biopolymeric films for food packaging applications. It will
to unravel the waste disposal problems to an assured magni- start with an introduction to definition and classification of biopoly-
tude [5]. In this framework, biopolymers, particularly those from mers and crosslinkers, followed by a comprehensive report about
renewable organic resources, have been regarded as promising film-forming natural biopolymers. Then the effect of crosslink-
green substitutes of non-biodegradable plastic materials [6,7]. ing on emerging properties of film-forming biopolymers such
Numerous subjects must be addressed before commercial use of as chitosan, gelatin, whey protein and starch, will be discussed.
bio-based packaging raw materials. These subjects comprise the Finally, the different characterization methods to demonstrate the
rates of degradation in different situations, possible alterations of crosslinking occurrence and the environmental concerns regarding
some physical, mechanical and thermal properties during stor- crosslinkers will be presented and discussed.
age, their resistance against the growth of microorganisms, and
the risk of harmful compounds release into wrapped foodstuffs
[8]. Among biopolymer materials that can be used for construc- 2. Biopolymers
tion of biocomposite films, a lot of attention has been dedicated to
polysaccharides, proteins, lipids and polyesters alone or in combi- The use of plastics over glass and metals for packaging means
nation as materials for the film production [9]. Most of the protein has continued to increase due to their good properties, accessi-
and polysaccharide-based films commonly present suitable barrier bility, easy processability, low weight, and low cost. Classically,
properties against oxygen at low and intermediate relative humid- plastic materials are prepared by condensation polymerization
ity, and have relatively acceptable mechanical strength, alongside process or polymerization of hydrocarbon or hydrocarbon-like
poor moisture and water vapor barrier features, due to their high monomers from different raw materials. Therefore, they find
hydrophilic nature [9]. Although many studies were focused on their origin in the petrochemical industry, making them non-
improving the film-forming aspects of biopolymers and designated biodegradable and non-renewable [18]. With particular measures
a noteworthy progress in this field, some physical, structural, ther- taken to discover substitutes to petroleum-based materials and
mal, and mechanical attributes are not pleasing so far and there with regard to environmental issues, several studies have been
are a number of complications for their industrial scale-up and pushed to propose new biodegradable packaging materials and
employment. In recent years various strategies have been followed polymers achieved starting from renewable resources. Employing
to overcome such drawbacks, including chemical modifications, biopolymer-based materials can overcome waste disposal prob-
plasticizer addition, blending with other biodegradable polymers, lems to a considerable magnitude. Nevertheless, in some cases,
inclusion of nanoparticles as reinforcing fillers, and addition of weakened mechanical and physical attributes, as well as inappro-
compatibilizers to improve miscibility of some incompatible poly- priate gas and water vapor barrier properties, in comparison with
mers [10,11]. frequently used petroleum derived plastic materials, restrict their
In details, chemical processes, such as etherification, esterifica- commercial use. Consequently, several researches have been done
tion, grafting and crosslinking, are able to prevent the excessive to boost new procedures aimed to improve the mechanical and
moisture uptake and macromolecular reformations, in addition barrier traits of biopolymer-based packaging raw materials, along
to the accessibility and affordable costs compared to the novel with incorporation of bioactive compounds to provide antioxidant
approaches like the addition of nanoparticles as fillers [12]. and antimicrobial properties which are indispensible in the food
A polymer with an integrated network can be obtained by packaging sector [10,19].
crosslinking process, in which the polymer chains are connected The biodegradable biopolymers can be classified in three main
intra- or intermolecularly by covalent or non-covalent links. categories on the basis of their origin (Fig. 1) [10,20–23] as follows:
 F. Garavand et al. / International Journal of Biological Macromolecules 104 (2017) 687–707
Fig. 1. Classification of different film-forming biopolymers.
1. Biopolymers originated from agricultural resources, including
polysaccharides (e.g. starch), ligno-cellulosic products (e.g. cel-
lulose and its derivatives), proteins (e.g. whey and collagen),
lipids (e.g. bee wax) and free fatty acids;
2. Biopolymers achieved by means of microbial fermentation, such
as pullulan and polyhydroxyalkanoates;
3. Chemically synthesized biopolymers using monomers attained
by natural raw materials such as poly(lactic acid).
Recent advances in case of natural-based or natural-occurred
biopolymer films are donating considerable progress in food
packaging, pharmaceutical and surgery means. In particular, the
present review will be mainly focused on the modification of agro-
resources derived biopolymers and on the influence of the added
crosslinkers on their properties.
3. Crosslinking agents
The crosslinking method involves the formation of chemical
bonds between different molecular chains to generate a stronger
three-dimensional network (Fig. 2). There are various classifica-
tions for the employed crosslinking agents. They can be classified
on the basis of the occurred bond kind, leading to well-stabilized
covalent crosslinking, formation of ionic bonds, and physical
crosslinking created by hydrogen and Van der Waals bonds. In other
classifications, crosslinkers are divided into three main types: phys-
ical, chemical, and enzymatical [24]. In the case of polymers, it
is possible to follow two different approaches: crosslinking and
post-crosslinking. In the former case, the crosslinker is directly
added during the polymer preparation in order to obtain the
ultimate material at one stage, whereas in the latter case the
crosslinking occurs after polymer formation. Moreover, if the
desired efficiency was not achieved, a complementary stage (post-
crosslinking) should be considered, although pre-crosslinking may
occur as a result of the first-stage crosslinking. Moreover, crosslink-
689
ing and post-crosslinking reactions can occur either at ambient
temperature or at intermediate temperature (<150 ◦ C) [25].
3.1. Crosslinking at ambient temperature
Development of integrated networks in heat-sensitive sys-
tems and improving energy efficiency are interesting subjects in
crosslinking reactions of polymers at ambient temperature. Sev-
eral examples of crosslinkers commonly used at room temperature
are reported in Table 1, evidencing drawbacks, advantages and
applications [25]. Various fundamental functional groups includ-
ing carboxylic acid reactions, reactions which activated amines
with carbonyl functions, acetoacetyl groups, acetals, acrylamide
occupations, crosslinkers which capable to react with water
and derivatives containing isocyanate functions, enzymatically
occurred reactions and physical crosslinking can be used in room
(ambient) temperature crosslinking. It is well understood that the
reactivity is a crucial limitation in these reactions and it should
be considered to avoid processes with over-reactive components
prior to introducing crosslinkers. This type of crosslinking is exten-
sively used in biology, coatings and paints, particularly to develop
chemical resistance and improved mechanical properties.
3.2. Crosslinking at intermediate temperature
Table 2 shows main crosslinking agents involved in inter-
mediate temperature approach [25]. Intermediate temperatures
are referred to temperatures in the range of 40–150 ◦ C, while
high temperatures are 150–300 ◦ C. According to Table 2, car-
boxylic acid reacts with oxazoline-epoxide functions, azetidines,
alcohols reacting with sheltered urethanes, azlactones and methy-
lol amide, reversible Diels–Alder reaction, and Huisgen reactions
between azido and triple bonds (click chemistry) are key functions
involved in intermediate crosslinking. The above-mentioned reac-
tions require particular conditions and in certain circumstances
(e.g. bisfuran/bismaleimide) they may be reversible as the temper-
690 F. Garavand et al. / International Journal of Biological Macromolecules 104 (2017) 687–707

Fig. 2. A schematic of the crosslinker’s mode of action in biopolymer chains.

Table 1
Main crosslinking agents applied at ambient (room) temperature.

Crosslinking agent Examples Drawbacks Benefits Applications

Carboxylic acid
Acetoacetyl groups
Amines
Acetal function
Acrylamide derivative
Enzymes
Physical crosslinkers
Other crosslinking moieties
Aziridine and carbodiimide
Acetylacetonate,
acetoacetoxyethyl
methacrylate
Hydrazine derivatives such
as piperazine and tertiary
amines
N-(2,2-dimethoxyethyl)
methacrylamide
N-methylolacrylamide and
isobutoxymethylacry-
lamide
Transglutaminase
Ultraviolet beams, ␥-rays,
electron beams, ozone,
X-rays etc.
Water (isocyanates and
sol–gel reactions)
Oxygen (reaction
generating peroxide and
acetylenic coupling)
Poor water stability, and some
irritant, toxic and mutagen
properties
pH- dependency
Time consuming of some
reactions
Self- reaction under acidic
conditions
Formation of toxic
formaldehyde
by-products due to undesirable
side reactions
Heat sensitivity, specificity
Inherent dangers due to
working with radiation
Shrinkage and fracture
during the curing process
Possibility of some unwanted
plasticizing effect due to long
side chains
Capability to use in in vivo Emulsions preparation and
conditions fabrication of composite films
and coatings
Reaction with various active Polymer synthesis, acrylic
groups, reduction of the latexes production
viscosity and the glass
transition temperature of
polymers, less sensitivity to
hydrolysis
Occurring several reactions at Use in waterborne acrylic
room temperature because of dispersions
high nucleophily
pH responsive Production of acrylic coatings
crosslinking agent and chemically stable latex for
paints
Copolymerization with Self-crosslinkable latexes
monomers containing
hydroxyl groups and
self-crosslinkability
Bio-based environment friend Blend film especially
crosslinking agent protein-polysaccharide ones
Successful crosslinking Genetic aims, hydrogel
between proteins and nucleic formation and etc.
acids
Production of homogenous Generation of silane
networks with high purity at latex and emulsions
low temperature, coexisting of
organic and inorganic phases
to form polymers with hybrid
materials, green process
Induction of the inter-chain Alkyde resins, mummification
crosslinking though various and dental printings
side reactions

ature reaches to about 150 ◦ C. Well-known uses of such reactions
are strengthening some properties in coatings, composite films,
self-healing polymers and textile finishing.
4. Crosslinking agents in biopolymers
The possibility to introduce crosslinking agents into the
structure of biopolymers extremely depends on the chemical
arrangement, presence of active groups and molecular weight of
biopolymers as well as compatibility of crosslinker and polymer
to form appropriate interactions. Accordingly, biopolymers can be
divided into two main classes depending on their molecular weight,
namely high molecular weight and low molecular weight with
molecular weights more than 105 g/mol and lower than 104 g/mol
(frequently in the range of 2–3 × 103 g/mol), respectively. Defi-
nitely, low molecular-weight biopolymers often need a consecutive
crosslinking approach to achieve acceptable mechanical, thermal
and structural properties [25]. Some examples of the biopolymers
modified by such strategy are summarized in Table 3. As it is
observed carboxylic acids and calcium ions improved the physio-
chemical, thermal and mechanical properties of most biopolymers
such as alginate, pectin, whey proteins, chitosan, starch and gelatin.
 F. Garavand et al. / International Journal of Biological Macromolecules 104 (2017) 687–707 691

Table 2
Main crosslinking agents applied at intermediate temperatures.

Crosslinking agent Examples Drawbacks Benefits Applications

Carboxylic acid
Alcohol function
Azetidine functions
Diels–Alder reactions
1,3-Dipolar cycloaddition
and “click chemistry”
reaction
Reaction with epoxides and
reaction involving oxazolines
Protected urethanes, azlactone
function and methylol amide
function
Acrylate and methacrylate
monomers
Furane/bismaleimide reaction
Huisgen reactions between
azido and triple bond
Not reported Production of durable coatings, Powder coatings and
easily introduction into the crosslinking of polyesters
polymers and
multi-functionality
Evolution of blocking agents Exhibiting UV-proofing Use in glass or glossy
properties coatings and
thermo-crosslinkable gels
Not reported Possessing double reactivity, Copolymers
self- crosslinkability, and avoid
wool-felting during washing
Sometimes reversibility is not Providing wide variety of Polyacrylic-type coatings
quantitative functional groups for both
polymerization and
crosslinking, obtaining
thermally reversible
crosslinked polymers
Explosiveness and toxicity of azides Reactions occur at low Synthesize some
temperatures (<100 ◦ C), high organogels
yields and tolerant to the
media condition

5. Influence of crosslinking on biopolymers characteristics
5.1. Physical properties
5.1.1. Water absorption
Kinetic diagrams of water absorption are imperative means to
anticipate the stability and quality attributes of packaging materi-
als during loading, transport and storage of various foodstuffs [54].
In particular, crosslinking of polysaccharide-based films strength-
ens the intermolecular interactions by generating covalent bonds
that enhance natural intermolecular hydrogen bonds, resulting
in improved water resistibility [43]. For example, crosslinking of
O-acetyl galactoglucomannan, a softwood-derived polysaccharide
from wood and pulping industry, by glyoxal caused a significant
decrease in moisture uptake of sorbitol containing films. Simi-
larly, the moisture resistance of hemicellulose-based films can be
strongly reinforced by crosslinkers such as glyoxal or citric acid
[52]. In a research performed by Goetz et al. [44] about crosslinking
of cellulose whiskers with poly(ethylene glycol) and poly(methyl
vinyl ether-co-maleic acid) and the resulting films, it was found
that the crosslinked films containing 25% whiskers absorb signif-
icantly less amount of water compared to the both 50% and 75%
non-crosslinked whisker films.
5.1.2. Water vapor permeability (WVP)
The solubility and diffusivity of water molecules are important
factors to control permeability in polymeric matrix [55]. Biopoly-
mers based films generally present relatively high inclination to
WVP. Therefore, crosslinking approach allows to deliver novel
arrangements of tightly linked three dimensional networks which
can be established to obstruct migration of water within the food
packaging materials. As stated by Reddy et al., crosslinking of
peanut protein films using citric acid could slightly reduce the WVP
(about 12% reduction for 1% (w/w) citric acid) and no significant
difference was revealed between the films crosslinked with dif-
ferent amounts of citric acid (1–3% w/w) [37]. In another work,
Ghanbarzadeh et al. demonstrated that the addition of different
concentrations of citric acid (0–20%, w/w) as crosslinker to the plas-
ticized starch film caused a significant reduction in the WVP with
respect that of the untreated film (i.e. 4.64 × 10−7 g Pa−1 h−1 m−1 )
[43]. As an example, the starch films containing 10% (w/w) of citric
acid presented the lowest WVP value (2.62 × 10−7 g Pa−1 h−1 m−1 ).
The reason of such occurrence can be related to the substitution of
hydrophilic OH groups with hydrophobic ester groups inside the
film structure. On the other hand, crosslinker addition may intro-
duce a tortuous pathway for water molecules to transfer through
the matrix. It is also observed that higher concentrations of citric
acid, from 10% to 20% (w/w), cause considerable enhancement in
WVP, probably due to the plasticizing role of extra-added citric acid.
Free excessive citric acid molecules may be included between the
polymer chains, increasing the mobility of the inter-chain and chain
spaces and, thus, encouraging the film diffusivity and accelerating
the rate of water vapor transmission. In opposition with the results
reported the remarkable reduction in WVP as affected by incor-
poration of crosslinkers, Cao et al. [28] pointed out that different
concentrations of crosslinking agents (i.e. ferulic acid and tannin
acid) played negligible effects on WVP of gelatin biofilms. It was
also stated that addition of ferulic acid did not influence the WVP of
the films obtained by soya protein isolate [56]. The supposed reason
may be due to the presence of numerous hydroxyl groups in fer-
ulic acid and/or tannin acid which tend to interact with water. The
introduction of transglutaminase to the casein, gelatin and their
blends, caused WVP enhancement, mainly because of improved
chain mobility which resulted in an increase in the water diffusion
coefficient [31].
In order to reduce WVP, it is also possible to use physical
crosslinking by ␥-irradiation as an operative technique to crosslink
proteins and to develop barrier and mechanical characteristics
of protein-based films. Lacroix et al. [57] demonstrated that ␥-
irradiation induces the formation of crosslinks in casein, soy, and
whey protein-based biofilms and, hence, improves the WVP, while
Ouattara et al. [58] affirmed that ␥-irradiation crosslinking causes
a decline in WVP of milk protein-based films and expands their
microbial resistance and enzymatic biodegradation. Lee et al. [59]
also revealed a decreased WVP in ␥- irradiated soy protein films,
mainly because of the generation of crosslinking and aggregation
of the polypeptide chains. On the basis of all the collected data, it
can be concluded that incorporation of crosslinking agents in the
desired range can modify the WVP of biopolymer films, otherwise
the excess amount acts as a plasticizer agent.
5.1.3. Solubility
Solubility is one of the main features of packaging because of
its protective role surroundings the food products. Water insolu-
bility of biopolymers supports their integrity as well as guarantees
the quality of foods [60]. The film properties of wheat straw hemi-
cellulose, crosslinked by addition of citric acid, revealed that the
most intense influence of citric acid was on insoluble matter of
 692
Table 3
Common crosslinking agents used for some biofilms.

Biopolymer(s) Crosslinking agent Concentration Crosslinking method Improved properties Reference

Whey proteins Formaldehyde and calcium 5.5 and 9% (w/w) Mixing crosslinking agents Physical, thermal and [26]
chloride formaldehyde and 0.05% (w/w) with whey protein solution mechanical properties
CaCl2 on the basis of film (10% (w/w)) at pH 7, drying at
solution dry matter 25 ◦ C for 18–24 h
Corriedale sheep wool keratin Ethylene glycol diglycidyl 75–300 mg/g of protein Mixing with keratin solution Physical and mechanical [27]
ether (EGDE) or glycerol (6.5% w/v protein), drying at properties
diglycidyl ether (GDE) 50 ◦ C overnight
Bovine bone gelatin Ferulic acid 5–40 mg/g of gelatin Mixing with gelatin solution Physical and mechanical [28]
(12% w/w) with pH 7 at 50 ◦ C properties

F. Garavand et al. / International Journal of Biological Macromolecules 104 (2017) 687–707

for 20 min, drying at ambient
temperature
Bovine bone gelatin Tannin acid 5–40 mg/g of gelatin Mixing with gelatin solution Physical and mechanical [28]
(12% w/w) with pH 9 at 50 ◦ C properties
for 20 min, drying at ambient
temperature
Bovine hide gelatin Dialdehyde carboxymethyl 1–10% (w/w) of gelatin Dissolving in distilled water Optical, physical, thermal and [29]
cellulose (DCMC) under stirring for 12 h at room mechanical properties
temperature to produce a 1%
(w/v) solution, mixing with
gelatin solution (10% w/v) at
60 ◦ C for 1 h, drying at 40 ◦ C for
24 h
Bovine hide gelatin Microbial transglutaminase 10 U/g of protein Mixing with gelatin solution Physical properties [30]
(10% w/w) at 50 ◦ C for 15 min
and subsequently enzyme
inactivation at 85 ◦ C for 10 min,
drying at room temperature for
24–48 h
Bovine hide gelatin Formaldehyde 8.8 mmol/100 ml of film Mixing with gelatin solution Physical, thermal and [30]
solution (10% w/w) at 50 ◦ C for 15 min, mechanical properties
drying at room temperature for
24–48 h
Bovine hide gelatin Glyoxal 26.5 mmol/100 ml of film Mixing with gelatin solution Physical and thermal [30]
solution (10% w/w) at 50 ◦ C for 15 min, properties
drying at room temperature for
24–48 h
Bovine hide gelatin/casein Microbial transglutaminase 10 U/g of protein Mixing with gelatin/casein Mechanical properties [31]
solution (7% w/w) at 50 ◦ C and
pH 7.0 for 15 min and
subsequently enzyme
inactivation at 85 ◦ C for 10 min,
drying at room temperature for
24 h
Pig skin gelatin Glutaraldehyde Glutaraldehyde solution Immersing films into solution Physical, thermal and [32]
0.125–2.5% (w/w) in phosphate at pH 7.4 for 24 h at room mechanical properties
buffer temperature, washing and
drying at room temperature
Pig skin gelatin Genipin Genipin solution 0.07–2.0% Immersing films into solution Physical, thermal and [33]
(w/w) in phosphate buffer at room temperature and pH mechanical properties
7.4 for 24 h, washing and
drying at room temperature
Fish gelatin Ultraviolet (UV)-crosslinking
Fish gelatin Maillard reaction using ribose
Fish gelatin UV-crosslinking and Maillard
reaction using ribose
Fish gelatin Lactose
Fish gelatin UV-crosslinking and lactose
Fish gelatin Microbial transglutaminase
Fish gelatin/nanoclay Microbial transglutaminase
␭ = 253.7 nm, 30 W
1–2% w/w of gelatin granule
(with 10.93% moisture)
1–2% ribose (w/w of gelatin
granule (with 10.93%
moisture))
UV (␭= 253.7 nm, 30 W)
1–2% w/w of gelatin granule
(with 10.93% moisture)
1–2% lactose (w/w of gelatin
granule (with 10.93%
moisture))
UV (␭= 253.7 nm, 30 W)
20–80 mg/ml enzyme solution
800 mg transglutaminase
powder (1% purity and 100
units of enzyme activity per
gram of powder)/5 ml enzyme
solution
Irradiating gelatin solution (5% Physical and mechanical [34]
w/v) by UV at a distance of properties
30 cm from the surface for
60 min (the film solution
received a UV radiation dose of
2.158 J/m2 ), drying at 40 ◦ C for
24 h
Mixing with gelatin solution Physical, optical and [34]
(5% w/v) at room temperature mechanical properties
F. Garavand et al. / International Journal of Biological Macromolecules 104 (2017) 687–707
(25 ◦ C), drying at 40 ◦ C for 24 h
Mixing with gelatin solution Physical, optical and [34]
(5% w/v) at room temperature mechanical properties
(25 ◦ C), irradiating gelatin
solution (5% w/v) by UV at a
distance of 30 cm from the
surface for 60 min (the film
solution received a UV
radiation dose of 2.158 J/m2 ),
drying at 40 ◦ C for 24 h
Mixing with gelatin solution Physical properties [34]
(5% w/v) at room temperature
(25 ◦ C), drying at 40 ◦ C for 24 h
Mixing with gelatin solution Physical and mechanical [34]
(5% w/v) at room temperature properties
(25 ◦ C), irradiating gelatin
solution (5% w/v) by UV at a
distance of 30 cm from the
surface for 60 min (the film
solution received a UV
radiation dose of 2.158 J/m2 ),
drying at 40 ◦ C for 24 h
Mixing 5 ml enzyme solution Thermal and mechanical [35]
with 200 ml fish gelatin properties
solution 10% (w/v) at 50 ◦ C and
pH 7 for 50 min and
subsequently enzyme
inactivation at 100 ◦ C for
15 min, degassing by filter
layers, drying at 25 ◦ C for 2 days
Mixing 5 ml enzyme solution None [36]
with 100 ml fish gelatin
solution 40% (w/v) at 50 ◦ C and
subsequently enzyme
inactivation at 100 ◦ C for
15 min, mixing with nanoclay
solution (containing 5%
nanoclay and 20% sorbitol
(w/w of gelatin) at 35 ◦ C for
24 h, drying
693
Table 3 (Continued)

694
Biopolymer(s) Crosslinking agent Concentration Crosslinking method Improved properties Reference

Peanut protein Citric acid 1–3% w/w of film solution Mixing with sodium Physical, thermal and [37]
hypophosphite (as the catalyst, mechanical properties
50% w/w of citric acid), adding
to the peanut protein alkali
solution (containing 1% (w/w)
sodium hydroxide), heating to
90 ◦ C during 20 min with
vigorous stirring, holding at
90 ◦ C for 5 min, drying under
ambient conditions, curing
films at 150 ◦ C for 30 min
Soy protein Maillard reaction – Mixing at pH 10.0 at 80 ◦ C, Physical, thermal and [38]

F. Garavand et al. / International Journal of Biological Macromolecules 104 (2017) 687–707

isolate/carboxymethyl vacuum defoaming, drying at mechanical properties
cellulose (CMC) 50 ◦ C for 6 h
Soy protein isolate/CMC Maillard reaction – Mixing at pH 10.0 at 80 ◦ C, Optical, thermal and [17]
vacuum defoaming, drying at mechanical properties
50 ◦ C for 6 h

Sodium alginate Calcium chloride
Sodium alginate Calcium carbonate
Sodium alginate Calcium chloride
Sodium alginate Calcium chloride and barium
chloride
1st stage: 0.03% calcium
chloride (w/v) in film solution
2nd stage: 2–7% (w/v) of
calcium chloride solution
0.01–0.03 g/g of alginate
1 st stage: 0.27% w/v of film
solution
2nd stage: 1.2% (w/v) of
calcium chloride solution
1 st stage: 0.27% calcium
chloride (w/v of film solution)
2nd stage: 0.6% (w/v) of
barium chloride solution
1 st stage: mixing with sodium Physical and mechanical [39]
alginate solution (1.5% w/v) at properties
70 ◦ C for 30 min, drying at 40 ◦ C
for 18 h
2nd stage: spraying aqueous
solution of calcium chloride
over both sides of the film,
maintaining for 3 h at room
temperature (25 ◦ C), removing
the surface excess solution by
flushing water, drying at 30 ◦ C
for 15 h
Mixing with glucono-␦-lactone Physical and mechanical [40]
(5.4 g/g CaCO3 ), mixing with properties
alginate solution (to provide
1.5% w/v alginate) at
13500 rpm and room
temperature for 3 min, gently
stirring overnight, drying at
30 ◦ C for 24 h
1 st stage: adding to the Not compared with blank [41]
alginate solution (1.5% w/v), treatments
drying at 40 ◦ C for 20 h
2nd stage: immersing films in
50 ml of calcium chloride
solution (containing 3% v/v
glycerol) for 10 min, removing
the surface excess solution,
drying at room temperature
(25 ◦ C) for 5 h
1 st stage: adding to the Not compared with blank [41]
alginate solution (1.5% w/v), treatments
drying at 40 ◦ C for 20 h
2nd stage: immersing films in
50 ml of calcium chloride
solution (containing 3% v/v
glycerol) for 10 min, removing
the surface excess solution,
drying at room temperature
(25 ◦ C) for 5 h
Sodium alginate Barium chloride and calcium 1 st stage: 0.12% barium
chloride chloride (w/v of film solution)
2nd stage: 1.2% (w/v) of
calcium chloride solution
Alginate/pectin Calcium chloride 1 st stage: 0.02–0.04 g calcium
chloride dihydrate/g
biopolymers 2nd stage: 3%
(w/v) of calcium chloride
solution
Starch Citric acid 5% (w/w) of starch
Corn starch Citric acid 5–20% (w/w) of starch
Corn starch/CMC Citric acid 10% (w/w) of starch
Maize starch/microcrystalline UV-crosslinking using sodium Sodium benzoate (0.15% (w/w)
cellulose benzoate (as a of dry starch)
photo-sensitizer) UV (␭ > 290 nm) irradiated by
medium-pressure mercury
vapor lamps (400 W)
1 st stage: adding to the Not compared with blank [41]
alginate solution (1.5% w/v), treatments
drying at 40 ◦ C for 20 h
2nd stage: immersing films in
50 ml of calcium chloride
solution (containing 3% v/v
glycerol) for 10 min, removing
the surface excess solution,
drying at room temperature
(25 ◦ C) for 5 h
F. Garavand et al. / International Journal of Biological Macromolecules 104 (2017) 687–707
1 st stage: adding a dilute Not compared with blank [14]
aqueous calcium chloride treatments
solution slowly (1 ml/min) to
the sodium alginate/pectin
solution (1.5% w/v) at 70 ◦ C for
30 min, strong mixing, drying
at 40 ◦ C for 20 h
2nd stage: immersing films in
50 ml of calcium chloride
solution for 30 min, removing
the surface excess solution,
drying at room temperature
and RH > 60% for 6 h
Mixing with sodium Physical, thermal, optical and [42]
hypophosphite (as a catalyst, mechanical properties
50% w/w of citric acid) and
starch solution 3% (w/w) at
90 ◦ C for 20 min, cooling
solution to 65 ◦ C, drying for
48 h, treating films at 165 ◦ C for
5 min
Mixing with starch solution Physical and mechanical [43]
(5% w/v) at room temperature properties
(25 ◦ C) for 5 min, agitating at
500 rpm and 90 ◦ C for 30 min,
drying at 60 ◦ C
Mixing with starch solution Not compared with blank [43]
(5% w/v) at room temperature treatments
(25 ◦ C) for 5 min, mixing with
CMC solution at 75 ◦ C for
10 min, degassing with stirring
at 40 ◦ C for 30 min, drying at
60 ◦ C
Mixing sodium benzoate with Physical and mechanical [12]
starch/cellulose solution (6.66% properties
w/v) at 100–105 ◦ C, cooling to
85–90 ◦ C and degassing with
stirring by vacuum, drying at
50–60 ◦ C, irradiating films by
UV at 60 ◦ C for 2–60 min
695

Table 3 (Continued)
Biopolymer(s)
Cellulose nanowhiskers
Methyl cellulose
Hydroxy propyl methyl
cellulose (HPMC)
Bacterial cellulose nanocrystals
(BCNC)/Poly(vinyl alcohol)
(PVA)
Wheat straw hemicellulose
Chitosan
Crosslinking agent Concentration
Poly(methyl vinyl 8.375% (w/v) of crosslinking
ether-co-maleic acid) solution (containing 1.25%
(w/v) poly(ethylene glycol))
Glutaraldehyde 0.5–12.0% (w/w) of film
solution
Citric acid 5–15% w/w of HPMC
Boric acid 3–15% w/w of PVA
Citric acid 5–30% w/w of hemicellulose
H2 SO4 H2 SO4 solution 2% (w/w)
696
Crosslinking method Improved properties Reference
Mixing 40 ml crosslinking Physical properties [44]
solution with cellulose
nanowhiskers, drying
overnight, curing at 135 ◦ C for
6.5 min
Mixing with methyl cellulose Physical, thermal, mechanical [45]
F. Garavand et al. / International Journal of Biological Macromolecules 104 (2017) 687–707
solution (2% w/w of film and biodegradation properties
solution) and 2 drops of
hydrochloric acid (HCl, as a
catalyst to yield a solution of
pH 3) at room temperature and
700 rpm for 1 h, washing and
neutralizing dried films by
distilled water for 3 h
Mixing with Sodium Physical properties [46]
dihydrogenophosphate
(NaH2 PO4 , as a catalyst, 0–50%
w/w of citric acid) and HPMC
solution (3% w/w) for 15 min
(pH was reached to 2.65),
drying at 60 ◦ C for 60 min,
Treating films at 190 for
15 ◦ C min
Mixing with BCNC/PVA Mechanical properties [11]
solution at 80 ◦ C and 1600 rpm
for 30 min (to achieve film
solution with 6% (w/v) dry
matter), synchronically
ultrasonicating (35 kHz) and
agitating (13000 rpm) at 60 ◦ C
for 5 min, degassing at 60 ◦ C
and 500 mmHg for 15 min,
drying at 50 ◦ C for 12 h
Mixing with hemicellulose Physical properties [47]
solution (5% w/v), degassing
under vacuum, drying at room
temperature for 24 h,
conditioning (50% RH, 24 ◦ C) in
an environmental chamber for
24 h to reach 11.91–12.32%
w/w moisture contents, curing
dried films at 150 ◦ C for 10 min
Immersing films into solution Not compared with blank [48]
for 1 h, washing and drying treatments
Crab shell chitosan
Shrimp chitosan
Tannic acid
Main-chain-type benzoxazine
polymer, (MCBP)
20–80 mg/g of chitosan
25–400% (w/w) of chitosan
Mixing with chitosan solution Physical and mechanical [49]
(1.5% w/w), drying at 37 ◦ C for properties
24 h
Dissolving in acetic acid Thermal and Mechanical [50]
solution (1% w/w acetic acid) of Properties
at ambient conditions, adding
slowly to the chitosan solution

F. Garavand et al. / International Journal of Biological Macromolecules 104 (2017) 687–707

(containing 1% w/w acetic acid)
under vigorous agitation to
obtain a total 2% (w/w) solid
content, degassing under
vacuum at room temperature,
drying at room temperature for
2 days, drying under vacuum at
50 ◦ C for 24 h, heating in
sequence at 75, 100, 125, 150,
and 175 ◦ C for 2 h
Chitosan/PVA Glutaraldehyde Glutaraldehyde solution 0.01% Immersing films into Not compared with blank [48]
(w/w) glutaraldehyde solution treatments
containing 0.5% (w/w) sulfuric
acid (as a catalyst) for 1 h,
washing and drying
Chitosan/corn cob Glutaraldehyde Glutaraldehyde solution 1% Mixing with chitosan solution Thermal and mechanical [51]
(v/v) (1.5% w/v) and corn cob properties
powder, drying at room
temperature for 48 h
Galactomannan/bovine serosa Glutaraldehyde Glutaraldehyde solution Immersing films into solution Physical and thermal [16]
collagen 0.001–1.5% in phosphate buffer at room temperature and pH properties
7.4 for 24 h, treating in
glycine solution (0.025 M
glycine:0.05 M borate, pH 9.2)
for 10 min, washing with water
and drying
Spruce Glyoxal 5% w/w of GGM Mixing with GGM solution (1% Physical, thermal and [52]
O-acetyl-galactoglucomannan w/v) at 60 ◦ C for 4 min, mechanical properties
(GGM) degassing by ultrasonication
under vacuum for 5 min,
drying at 50% RH and 23 ◦ C
Spruce GGM Ammonium zirconium 10% (w/w) of GGM Mixing with GGM solution (1% Physical and mechanical [53]
carbonate w/v), treating at pH 8.5 and properties
80 ◦ C for 5 h, degassing by
ultrasonication under vacuum
for 5 min, drying at 50% RH and
23 ◦ C

697
698 F. Garavand et al. / International Journal of Biological Macromolecules 104 (2017) 687–707
hemicellulose, whose insolubility was amplified 5-fold by incor-
poration of 20% citric acid [47]. Former studies corroborated the
above-mentioned changes in solubility [46,61], ascribed to the
generated ester bonds between citric acid and the polysaccharide
[62], resulting in a denser structure and corresponding improved
water insolubility. The reduced and/or unavailable hydroxyl groups
and their replacement by hydrophobic ester groups could be also
related to the effects of citric acid concentration on water solubility
[47,63]. Consequently, crosslinking agents can overcome the poor
moisture resistance of polysaccharide films, originated from their
hydrophilic nature, reducing the water absorption by formation of
some interconnects between polysaccharide molecules.
5.1.4. Film thickness
Thickness of films and coatings which usually range from 80 ␮m
to 200 ␮m plays a pivotal role in some barrier properties, including
WVP and gases transport. Since most of the studied biopolymer-
based films are originally hydrophilic, their thickness presumably
influences WVP because of differences between the water vapor
pressure and the accumulation of moisture at the film/air inter-
face. As well, occurrence of crosslinking outwardly toughens the
generated internal bonds of the biopolymer films [64] and vari-
ous denser composites will be achieved, depending on the amount
of crosslinking agent incorporated into film structure. Denser
structural biopolymeric patterns owing to crosslinking addition,
along with the improvement in some physical characteristics, were
reported in numerous documents [40,46,65–67].
5.1.5. Light barrier properties and transparency
Because of consumer’s preference to see the food items, higher
transparency is greatly recommended for food packaging films and
coatings [68]. Film transparency is extensively related to the func-
tionality of film ingredients because of their great influence on
the appearance of the food products. As well, light barrier prop-
erties are considered another fundamental requirement for food
packaging materials. Since the emitted UV light ranging from 200
to 280 nm is considered as one of the oxidation initiators in food
products, how to promote the barrier properties of packaging mate-
rials against UV light has received a great deal of attention [69]. In
particular, lipid oxidation represents a great concern for the food
manufacturers due to the development of undesirable off-flavors
and generated hazardous products, especially occurring free radical
reactions. As a result, food manufacturers must improve procedures
to avoid or slow down lipid oxidation in food products. Leera-
hawong et al. [69] reported that protein-based biofilms are good
UV protectors, mainly because of the presence of aromatic amino
acids which can captivate the UV light. In this regard Mu et al. [29]
investigated the light barrier and transparency features of edible
gelatin films crosslinked with dialdehyde carboxymethyl cellulose
(DCMC). Crosslinked gelatin films prohibited lipid oxidation by
constructing proper barrier against UV light in the food system.
Furthermore, incorporating more DCMC content led to an increase
in transparency of gelatin–DCMC films at 280 nm, although it was
insignificant at 600 nm. It was hypothesized that DCMC improves
the UV barrier characters of gelatin films by generating abundant
C N groups through formation of Schiff’s base between ␧-amino
groups of lysine or hydroxylysine side groups of gelatin and the
aldehyde groups in DCMC.
In agreement with the aforementioned declarations, Guo et al.
expressed that the transmission of UV light was so slight at 200 nm
and 280 nm for all gelatin-oxidized xanthan gum films, even though
transparency is high at 280 nm [70]. Therefore, the all films exhib-
ited appropriate barrier properties against UV light, associated
again to their high content of aromatic amino acids which can
absorb UV rays. Furthermore, the transparency of gelatin-oxidized
xanthan gum films at 280 nm is about 3 times as that of gelatin-
xanthan gum film. This result revealed that the transparency of
the films is less than 1 at 600 nm and slightly improved with
the increase in oxidization level of oxidized xanthan gum. The
slight increase in transparency at 600 nm could be a consequence
of darker color caused by crosslinking. The light transmission in
the UV and visible light range evidenced that the oxidization
of xanthan gum can significantly improve the UV light barrier
attributes and has little effect on the transmission of visible light.
The results obtained by Dou et al. on feather keratin film crosslinked
by dialdehyde starch validated the transparency enhancement by
introducing crosslinking agents [71].
5.1.6. Color changes
The color of biopolymeric films is regarded as an important fac-
tor to evaluate their quality. It is stated that the color is greatly
influenced by various factors, such as pH value, degree crosslinking,
plasticizer content, thermal process and fabrication procedure. In
addition, the color of protein-based biopolymers is more altered by
the type and concentration of amino acids and proteins, rather than
other treatments and processes [38]. Most of the polysaccharide-
based films usually appear colorless, even though there are some
exceptions such as chitosan which displayed a slightly yellow
color. Crosslinking of chitosan by tannic acid showed that addition
of tannic acid triggered the major color changes in the obtained
films due to its affinity to oxidize, in turn yielding brownish films
[49]. Similarly, the crosslinking of proteins by citric acid results
in yellow-brown films principally when exposed to high tempera-
tures and/or after extended periods of time [72]. Reddy and Yang
reported that yellowness index (YI) of the starch films was not
affected by different amounts of citric acid before curing treatment
[42]. Indeed, curing of starch films at 165 ◦ C for 5 min, followed
by addition of citric acid (1–20 wt%), intensified the yellowness
of the prepared films. In details, films crosslinked with 1–5 wt%
citric acid showed YI values of about 10% higher than their corre-
sponding YI before curing, whereas YI values of the films treated
with 10% citric acid sharply increased after curing treatment and
those of films treated with 20% citric acid were approximately 13
times higher compared to non-crosslinked films after curing. The
revealed remarkably higher yellowness of films can be ascribed
to the dehydration of citric acid owing to curing at high tempera-
tures, with consequent development of an unsaturated acid [42,73].
Comparable intensify in yellowness-pattern was also observed for
glutenin [74], soy protein isolate [75], and castor bean proteins [76]
crosslinked by different aldehydes. On the other hand, Benavides
et al. noted that incorporation of CaCO3 did not significantly alter
the total color difference of the alginate films so that calcium ions
at low concentration (0.01–0.03 g CaCO3 /g alginate) was not suffi-
cient to change the light scattering emitted by alginate background
[40].
5.2. Mechanical properties
Maintaining the integrity of packaging films is important during
processing, transport, and handling [5]. Generally, the incorpo-
ration of more amounts and/or dosages of crosslinking agents
improve the tensile strength (TS) of biopolymer films and decrease
their elongation at break (EAB) [43]. Mechanical properties of
some biofilms are compared in Table 4. All considered film making
biopolymers display almost identical trend in case of incorporat-
ing crosslinkers so that significant and progressive increment of
TS is detected once the amount/dosage of crosslinkers is amplified
(commonly less than 10% for enzymatic and chemical crosslinkers
and less than 20 kGy for irradiation). Gou et al. reported that aver-
age TS value of gelatin-xanthan gum film is about 24 MPa, which is
considerably higher than that of gelatin film (about 2 MPa) [70].
The significant increase of TS value by the addition of xanthan
 F. Garavand et al. / International Journal of Biological Macromolecules 104 (2017) 687–707 699

Table 4
Mechanical properties of various biopolymer films as affected by crosslinkers.

Biopolymer(s) Crosslinker Mechanical properties Reference

Type Dosage Elongation at break Tensile strength

Pectin/gelatin Gamma irradiation 10 kGy 15% ↓ 34% ↑ [77]

Soy protein isolate Gamma irradiation 16 kGy 21% ↓ 25% ↑ [59]
Gelatin Ferulic acid 4% 35% ↓ 11% ↑ [28]
Gelatin Tannin acid 4% 4% ↓ 4% ↑ [28]

Starch Citric acid 5% – 150% ↑ [42]

Un-plasticized chitosan Tannic acid 4% 40% ↑ 15% ↑ [49]
Plasticized chitosan Tannic acid 4% 53% ↓ 25% ↑ [49]

Soy protein isolate Genipin 1% 103% ↑ 29% ↑ [78]

Fish gelatin/oxidized Sodium periodate 2.4% 50% ↓ 25% ↑ [70]
xanthan gum
Bovine gelatin/calcium Transglutaminase 8% 33% ↑ 58% ↑ [79]
carbonate
Soy protein isolate/fish Transglutaminase 3% 69% ↓ 27% ↑ [80]
gelatin
Fish gelatin Transglutaminase 3% 42% ↑ 14% ↑ [80]
Bacterial cellulose Boric acid 1.96% 13% ↓ 10% ↓ [11]
nanocrystals/Poly(vinyl
alcohol)

gum could be related to the created interactions between both
biopolymers and the entanglement of gelatin chains within the
film matrix. After introducing oxidized xanthan gum into gelatin
as crosslinker, the obtained films presented substantially higher TS
(average around 33 MPa) and reduced EAB values (average 11.3%),
suggesting the occurrence of crosslinking between gelatin and oxi-
dized xanthan gum by addition of sodium periodate. Increase in
TS value of protein films due to crosslinking often goes along with
a reduction in EAB (less extensible films), because the crosslinks
allow to obtain a tougher structure.
It is important to highlight that some crosslinking agents may
act both as a crosslinking agent and a plasticizer in the biopolymer
films. For example, Ghanbarzadeh et al. [43] showed such bilat-
eral effect by addition of wide range of citric acid in the starch
films. By increasing citric acid content up to 10 wt%, the TS of the
films increased and EAB decreased. Indeed, for low amounts, cit-
ric acid probably induces the formation of highly linear structure
by hydrolyzing branched chains of starch molecules, in turn gen-
erating more hydrogen connections between the starch chains and
increasing the TS of the obtained films. For high contents, the extra
(not-reacted) portion of citric acid probably acts as a plasticizer and
reduces the interactions between the macromolecules, with con-
sequent TS decrement and EAB increase. Comparable results were
reported by Ning et al. about the effect of citric acid on the mechan-
ical behavior of the blend films based on thermoplastic starch
and linear low-density polyethylene [81]. Contrariwise, Shi et al.
remarked that increasing citric acid content (from 5 wt% to 30 wt%)
in polyvinyl alcohol-starch films did not significantly reduce the TS;
however, the EAB remarkably increased [82]. On the other hand, Ma
et al. [83] showed an extraordinary crosslinking effect of citric acid
at 9 wt% concentration on pea starch biocomposite film, in such a
manner that TS increased and EAB reduced.
Interestingly, decrease in EAB is typically attended by increase
in TS, which apparently conflicts with some results reported in
Table 4. Controversial results regard the effect of introducing
crosslinkers on EAB values can be interpreted and justified with dif-
ferent reasons. It seems that the crosslinker amount and the nature
of film-forming constitutes are important items which strongly
influence EAB. For example, Gonzalez et al. [78] observed two dif-
ferent behaviors of soy protein crosslinked by genipin, in terms of
EAB. When the genipin amount was fewer than or equal to 1%, more
expansible networks (high EAB) were established because of some
intrinsic interruptions in protein interactions as a result of genipin
addition. On the other hand, an increase in genipin concentration
(more than 1%) led to a reduction in EAB due to the formation of
new crosslinks, obtaining a stiffer structure in which the movement
between the chains is limited. In another study performed by Wang
et al. [79] the EAB percent of gelatin-calcium carbonate compos-
ite films was considerably enhanced with increase in the amount
of transglutaminase enzyme. These experimental evidences were
in agreement with those related to food proteins derived films
prepared using gelatin, soy protein isolates, sodium caseinate and
whey protein concentrate which crosslinked by transglutaminase
[84]. Different kinds and means of the interactions between plasti-
cizers and protein chains and combination of plasticizers into some
particular areas in the polymeric matrix result in boosting their
distance, and thus, affect their mobility. Protein chain composi-
tions, molecular weight, shape, and amino acid content of proteins
derived from various origins could responsible for the aforesaid
results. The mechanical properties of the most of the crosslinked
film-forming biopolymers were close to that of traditional syn-
thetic films including polyvinylidene chloride (PVDC), polylactic
acid (PLA), polyethylene and so on [85].
Rouhi et al. reported that increases in the concentrations of boric
acid improve the TS of the poly(vinyl alcohol) (PVA)/bacterial cel-
lulose nanocrystals (BCNC) films due to the crosslinking reactions.
However, the TS of films decreased with increasing the content
of boric acid at high concentrations (5% (w/w)) of bacterial cellu-
lose nanocrystals. The reason was BCNC coalescence and decrease
in their aspect ratio due to intra and inter-molecular crosslinking
[11].
5.3. Thermal properties
Thermodynamic characteristics, established by thermal anal-
ysis, are important to comprehend the quality of substances
under cooling/heating rates, inert/reduction/oxidation atmosphere
or under gas pressures. Recently, several techniques were used to
determine the thermo-physical properties of materials, including
differential scanning calorimetry (DSC), thermogravimetric analy-
sis (TGA), differential thermal analysis (DTA), dynamic mechanical
analysis (DMA), evolved gas analysis (EGA), dilatometry (DIL),
dielectric analysis (DEA). The principal application of the TGA and
DSC is to study phase transitions under different atmospheric con-
ditions, cooling/heating rates and temperatures [86]. Table 5 shows
700 F. Garavand et al. / International Journal of Biological Macromolecules 104 (2017) 687–707

Table 5
Thermal properties of various crosslinked biopolymer films.

Biopolymer(s) Crosslinking agent DSC TGA Reference

Gelatin Immersing films into Increased thermal stability- – [32]

glutaraldehyde solutions endothermic peak at 72 ◦ C, and
(0.125–2.5% (w/w)) the denaturation enthalpy
amounts to about 6 J/g
Gelatin Immersing films into genipin Endothermic peak at 86–98 ◦ C – [33]
solutions (0.07–2.0% (w/w))
Galactomannan/collagen Immersing films into First transition at 48 ◦ C – [16]
glutaraldehyde solutions second transition at
(0.001–1.5%) 58.7–66.7 ◦ C
Gelatin Transglutaminase (10 U/g of Melting temperature – [30]
protein) decreased to 62.95 ◦ C
Gelatin Formaldehyde Melting temperature increased – [30]
(8.8 mmol/100 ml of filmogenic to 77.43 ◦ C
solution)
Gelatin Glyoxal (26.5 mmol/100 ml of Melting temperature increased – [30]
filmogenic solution) to 87.09 ◦ C

Fish gelatin Microbial transglutaminase The melting temperature – [35]

(5–20 mg/g of biopolymer) increased from 124 ◦ C to 158 ◦ C
Chitosan/poly(vinyl Immersing films into Broad exothermic peak at Weight losses at 40–120 ◦ C [48]
alcohol) glutaraldehyde solution (0.01% 310 ◦ C and 170–300 ◦ C
(w/w)) containing sulfuric acid
(0.5% (w/w)) as the catalyst
Chitosan Immersing films into sulfuric Sharp exothermic peaks at Weight losses at 40–150◦C [48]
acid solution (2% (w/w)) 290 ◦ C, and 200–300 ◦ C
endothermic peak at 100 ◦ C

Starch Citric acid 5% (w/w) of starch Increase the temperature of Lower weight loss between [42]
water evaporation 120 and 220 ◦ C, higher
endothermic peak weight loss between 220
and 320 ◦ C, 20% lower
weight loss at 600 ◦ C
Soy protein Maillard reaction A single Tg in the range A single and higher [38]
isolate/Carboxymethyl 75–100 ◦ C and melting decomposition
cellulose between 140 and 160 ◦ C temperature in the range
250–350 ◦ C
Chitosan/corn cob Glutaraldehyde – Weight losses at 50 ◦ C, [51]
300 ◦ C and 310–350 ◦ C

selected publications focused on the thermal properties, including ing comparable values. In details, both genipin and glutaraldehyde
TGA and DSC, of crosslinked biopolymeric films. generally induce a decrease in the denaturation enthalpy [32,33].
Furthermore, the thermal stability was increased by 2% (w/w)
genipin in the crosslinked treatment, due to the slight but sig-
5.3.1. Differential scanning calorimetry
nificant increase of the denaturation temperature from 91 ◦ C to
DSC is a thermal analysis instrument which is widely applied to
98 ◦ C. However, the denaturation temperature of gelatin films
determine the difference in the amount of heat required to increase
showed non-significant changes with 2.5% (w/w) glutaraldehyde,
the temperature of a sample and reference as a function of time and
exhibiting a lower denaturation temperature and a less thermal
temperature [87]. Both the sample and reference are maintained at
stability than films crosslinked by genipin. In consistent with
equal temperature throughout the analysis. The program of a DSC
these results, de Carvallho and Grosso [30] observed that the
analysis is usually designed such that the temperature of sample
Tm increased from 65.06 ◦ C in native gelatin films to 87.09 ◦ C
and reference holders linearly increases as a function of time.
and 77.43 ◦ C in glyoxal- and formaldehyde-treated gelatin films,
For example, in a paper focused on chitosan and poly(vinyl alco-
respectively. They reported a decrease in ␧-amino groups in the
hol) (PVA) blend films crosslinked by glutaraldehyde [48], DSC was
transglutaminase-modified film, because of an increase in molecu-
used to examine their thermal properties. An endothermic peak at
lar weight. However, no change was observed in the melting point
about 60 ◦ C was related to water loss, whereas a broad exothermic
of the resulting film compared to the native film, and the values of
peak at 310 ◦ C was ascribed to the chitosan thermal degradation
melting enthalpy (Hm ) showed a decrease as compared to those
that occurred at 290 ◦ C in neat chitosan films, suggesting that the
of the native film. Increase in the number of crosslinking covalent
thermal stability of crosslinked chitosan–PVA film was significantly
bonds, which exothermically break, resulted in a decrease of the
improved.
hydrogen bonds number, which endothermically break, demon-
Yi et al. [35] investigated the thermal properties of micro-
strating the decrease in the Hm , despite the increase in Tm [32,88].
bial transglutaminase crosslinked fish gelatin films. Their results
Considering collagenous materials, an endothermic peak in the
showed an increase in melting temperature (Tm ) caused by the
DSC thermogram is usually recorded and ascribed to the helix-coil
incorporation of crosslinker. The Tm measured by DSC increased
transition of collagen. The amount of the denaturation enthalpy
from 124.78 ◦ C to 158.49 ◦ C as the enzyme reaction time increased
related to this peak is depended on the relative amount of triple
from 0 to 50 min. There was no significant difference of Tm and
helical structure in the samples, and is significantly higher for col-
crosslinking degree between 10 and 30 min reactions. Bigi et al.
lagen compared to gelatin [88,89]. Rault et al. [90] studied collagen
investigated the influence of both genipin and glutaraldehyde
films crosslinked by different chemical crosslinking agents, observ-
crosslinkers on the thermal properties of gelatin films, evidenc-
 F. Garavand et al. / International Journal of Biological Macromolecules 104 (2017) 687–707
ing an increase in the Tm of the film modified with glutaraldehyde.
Usha and Ramasami [91] also reported an increase in the Tm of col-
lagen films processed with formaldehyde due to the formation of
crosslinks. Kopp et al. [92] observed a decrease in the Hm caused
by crosslinking of collagen fibers. As well, Figueiró et al. [16] stud-
ied the effects of glutaraldehyde crosslinking agent on the thermal
properties of galactomannan-collagen films, using DSC. The pres-
ence of two transitions in modified galactomannan–collagen films
indicated two structures with different thermal stability because of
the heterogeneous reaction of glutaraldehyde in a mixture of galac-
tomannan and collagen. The first transition at low temperature
was due probably to the not-occurring the crosslinking in collagen
by glutaraldehyde. Therefore, they concluded that the presence of
galactomannan could avoid the glutaraldehyde access to collagen
molecules in the internal of film matrix [93]. The second transition
at higher temperature, such as 60.7 and 66.7 ◦ C (samples treated
with 0.05 and 1% glutaraldehyde, respectively), showed that the
fixation of collagen molecules on the film surface was a function of
glutaraldehyde concentration. The galactomannan blocked by the
network of collagen fibers stabilized by glutaraldehyde. The tran-
sition at 58.7 ◦ C for the galactomannan–collagen film treated with
1.5% glutaraldehyde could present the occurrence of polymeriza-
tion reactions by higher concentrations of glutaraldehyde, resulting
glutaraldehyde-induced polymers which could also block the pen-
etration of glutaraldehyde into the internal regions of the collagen
matrix, correspondingly decreasing the denaturation temperature.
Glutaraldehyde stabilized the collagen molecules and resulted in
an increase of the denaturation temperature [94].
It has been reported that citric acid could act as a crosslinker
by forming strong hydrogen bond interactions with starch, which
improve its thermal stability, and prevent retrodegradation [42,95].
According to DSC data reported by Reddy et al. [42], the DSC plot of
the non-crosslinked film during the first heating cycle showed an
endothermic peak at about 100 ◦ C, whereas that of the crosslinked
film with 5% citric acid had an endothermic peak at about 150 ◦ C.
This peak in the both non-crosslinked and crosslinked dried films
disappeared after the second heating cycle, suggesting that it can
be ascribed to the moisture in the films. Moreover, the shift of the
identified endothermic peak at higher temperatures in the case of
crosslinked films confirmed that the crosslinking is able to act as
obstruction to water evaporation in the film.
The Maillard reaction can take place when protein is mixed
with carbohydrates at high temperatures [96]. Lii et al. [97] formed
covalent bonds between carboxymethyl cellulose (CMC) and casein
by electro-synthesis. These CMC-casein complexes exhibited good
emulsifying properties and thermal stability. Moreover, edible
films based on CMC and soy protein isolate (SPI), plasticized by
glycerol and crosslinked by Maillard reactions were studied [38].
Investigation of films by DSC showed that pure SPI films had glass
transition temperature (Tg ) at about 60 ◦ C, in agreement with their
previously observations [98]. CMC-SPI blends exhibited a Tm value
in the range 140–160 ◦ C and a single Tg between 75 and 100 ◦ C,
indicating that SPI and CMC are highly compatible and miscible.
All crosslinked films showed an endothermic Tg curve peaked at
a higher temperature than that of SPI and CMC. Consequently,
increase in Tg with increasing proportion of CMC was due to an
increasing extent of crosslinking.
5.3.2. Thermogravimetric analysis
TGA is a technique in which changes in weight of materials
are determined as a function of temperature with constant heat-
ing rate, or isothermally as a function of time [99]. TGA allows
to study physical phenomena, such as second-order phase tran-
sitions, including desorption, absorption, adsorption, vaporization
and sublimation. Moreover, TGA can supply information about
chemical phenomena, including decomposition, chemisorption,
701
desolation and solid-gas reactions (such as oxidation or reduction)
[99]. TGA is commonly used to measure characteristics of sub-
stances that show either mass loss or gain because of oxidation,
decomposition or loss of volatiles (such as moisture), and it is also
applicable to study the polymers [100].
For example, Tripathi et al. [48] investigated the thermal decom-
position of chitosan-PVA films crosslinked with glutaraldehyde.
Thermographs of chitosan films showed that the weight loss
between 40 and 150 ◦ C was as a result of the moisture vaporiza-
tion. The weight loss between 200 and 300 ◦ C was associated to
the chitosan thermal degradation. Furthermore, two considerable
weight losses were exhibited in the TGA plot of chitosan–PVA film
crosslinked by glutaraldehyde: one at 40–120 ◦ C was ascribed to
the moisture vaporization and the other at 170–300 ◦ C was due to
the degradation of crosslinked chitosan–PVA film. They concluded
that introducing crosslinker in the network of the composite causes
a remarkable improvement in the thermal stability. Furthermore,
Yeng et al. [51] prepared the unmodified and modified chitosan-
corn cob composite films with glutaraldehyde as a crosslinking
agent and performed TGA measurements, identifying three main
weight losses: the first one started at about 50 ◦ C and was related
to the evaporation of absorbed water; the second one at 300 ◦ C
was ascribed to the thermal decomposition of hemicellulose, cel-
lulose and lignin; the third one took place in the temperature of
310–350 ◦ C due to polymer decomposition. The thermal stability
of chitosan-corn cob films reduced with increasing corn cob con-
tent. However, they showed that glutaraldehyde as a crosslinker
increased the thermal stability of films due to the presence of imine
linkages.
Su et al. [38] studied the effects of the Maillard reaction on
the thermal stability of edible films based on CMC-SPI blends.
CMC-SPI blend films exhibited about 15% weight loss in the tem-
perature range between 0 and 150 ◦ C as a result of the water
evaporation. However, these samples showed different decompo-
sition ratios in the range of 30–80% residual mass. The slope of
the weight–temperature line was reduced with increasing CMC
content of the CMC-SPI blends, indicating greater thermal sta-
bility compared to neat SPI that decomposed at a relatively low
temperature (about 110 ◦ C), as demonstrated by the related TGA
curve. In details, the CMC-SPI blends decomposed at temperatures
250–350 ◦ C, temperatures higher than those of both neat SPI and
CMC films. The increase in thermal stability of the blend films
could be assigned to crosslinking stimulated by Maillard reaction
between CMC and SPI. Finally, TGA curves of all blends showed a
single decomposition temperature, indicating good compatibility
and miscibility between CMC and SPI.
6. Complementary techniques for identification of
crosslinking occurrence in biopolymers
Identification of biopolymeric microstructures is essential since
it determines their mechanical and barrier properties. Moreover,
structural studies of polymers would improve our concep-
tion about crosslinking reactions. The investigation of physical,
mechanical and/or thermal properties of polymers exposed to
crosslinking agents is considered an acceptable way to deter-
mine whether reticulated network in polymers has formed
or not. However, as shown in Table 6, some complementary
methods, such as X-ray diffraction (XRD), SDS-PAGE (sodium
dodecyl sulfate–polyacrylamide gel electrophoresis), FTIR (Fourier
transform infrared) and Nuclear magnetic resonance (NMR) spec-
troscopy, can approve the crosslinking reaction occurrence in
polymers.
 702
Table 6
Complementary techniques for identification of crosslinking occurrence in some biopolymers.

Biopolymer(s) Crosslinking agent Crosslinling bond XRD SDS-PAGE FTIR NMR Reference

Hydroxyl propyl methyl
cellulose
Casein/gelatin
Methyl Cellulose
Citric acid Ester bond – – Increase in absorption peak – [46]
intensity of the ester band
vibration (1735 cm−1 )
Transglutaminase Isopeptide bond – Formation of new high – – [31]
molecular mass
polymers (≥212 kDa)
Glutaraldehyde Hydrogen bond – – Decrease in the peak of – [45]
C O stretching from
aldehyde group of free
Glutaraldehyde

F. Garavand et al. / International Journal of Biological Macromolecules 104 (2017) 687–707

(1710 cm−1 )
Starch/poly(vinyl alcohol) Citric acid Ester bond Decrease in the crystal – Increase in the peak of the – [82]
Hydrogen bond peak at 19.3◦ C O stretching vibration
(1729 cm−1 )
Increase in the peak of the
stretching vibration of the
hydroxyl groups
(3340 cm−1 )
Fish gelatin/nanoclay Transglutaminase Isopeptide bond – Decrease in band – – [36]
intensity <75 kDa
Increase in band
intensity >250 kDa
Carboxymethyl Maillard reaction C N – – Decrease in the intensity of – [38]
cellulose/soy protein C N the bands at 3100–2900,
isolate 1600–1400 and 890 cm−1
(amide I, П and Ш)
The absorption band at
1241–1472 cm−1 (C N)
and the new absorption
band appeared at
1647 cm−1 (C N)
Chitosan Tannic acid Hydrogen bond Shift the peaks toward – Decrease in the intensity of – [49]
lower 2␪ values absorption peaks
Starch Citric acid Hydrogen bond Increase in the diffraction – The additional peak in the – [42]
intensity of peaks at 17◦ crosslinked film of the
and 22◦ , decrease in the% carboxyl and ester carbonyl
crystallinity bands (1724 cm−1 )
Soy protein Maillard reaction C N – – Decrease in the intensity of Decrease in the [17]
isolate/carboxymethyl C N the bands at 3100–2900, intensity of 13 C peak at
cellulose 1600–1400 and 822 cm−1 173 ppm (COO
(amide I, П and Ш) backbone carbonyl
The n band at group)
1278–1296 cm−1 (C N) No signal at 50 ppm
and the new n band at (NH2 group)
1645 cm−1 (C N)
Bacterial cellulose Boric acid Hydrogen bond – – Increase in the wave – [11]
nanocrystals/Poly(vinyl B O C ether bond number of OH stretching
alcohol) peak (3000–3550 cm−1 )
A new and weak
absorption peak at about
1035 cm−1 attributed to
the stretching vibration of
B OC
 F. Garavand et al. / International Journal of Biological Macromolecules 104 (2017) 687–707
6.1. X-ray diffraction
X-ray crystallography allows identifying the atomic and molec-
ular structure of crystals and, thus, can be used to investigate the
influence of the crosslinking agent on the polymer crystallization.
For example, in the case of chitosan, according to Wang et al.,
the two peaks at 2␪ 11.6◦ and 8.4◦ correspond to the hydrated crys-
talline structure, while the broad peak at around 2␪ 23◦ indicates
the presence of an amorphous structure [101]. XRD of tannic acid
crosslinked chitosan films was investigated by Rivero et al. [49].
They reported that neat chitosan films exhibited a slight trend to
higher 2␪ values during the storage, which indicates that acetic
acid, used in order to completely solubilize chitosan, along with
water molecules, was spontaneously removed from the chitosan
matrix. Ogawa et al. ascribed this shift to a gradual conformational
evolution toward the anhydrous polymorph of chitosan, related
to the equilibrium moisture [102]. Brown et al. reported a simi-
lar shift [103]. Films containing plasticizer, tannic acid or both of
them, showed similar XRD patterns, such that a shift of their char-
acteristic peaks was observed during the storage. The magnitude
of these shifts was different depending on the used additives. The
incorporation of tannic acid shifted the peaks toward lower 2␪ val-
ues, while the effect was more considerable with the addition of
plasticizer. However, a lower percentage of displacement of the
aforementioned peaks was observed with the addition of both tan-
nic acid and glycerol as a plasticizer, than in the case of plasticized
chitosan films, attributing to a synergic effect of tannic acid and
glycerol.
XRD was also performed to investigate the physical structure of
citric acid crosslinked starch films by Reddy and Yang [42]. They
reported that XRD patterns of the crosslinked and non-crosslinked
starch films showed similarly two distinguished peaks at about 2␪
17◦ and 22◦ . However, both films did not present the typical starch
peaks at about 2␪ 5◦ and 14◦ , suggesting changes in crystal struc-
ture and/or the crystallinity. The non-crosslinked and crosslinked
starch films had comparable crystallinity of about 17% and 14%,
respectively. Also the crosslinked starch films with epichlorohy-
drin compared to native starch, did not show any change in peak
positions or crystallinity [104,105]. Some of the amorphous regions
were oriented well after crosslinking that was indicated by the
increase in the diffraction intensity followed by a decrease in the
crystallinity of the crosslinked starch films.
The effect of citric acid on crystalline structure of the crosslinked
starch-polyvinyl films was studied by Shi et al. [82]. XRD patterns
showed a high percentage of crystallinity (␹c = 54%) in PVA which
presents the typical diffraction peak at 2␪ 19.3◦ [106], while corn
starch presents peaks at 2␪ 15◦ , 17◦ , 18◦ , 23◦ and 26.5◦ [107].
Most of the diffraction peaks in the PVA-starch-glycerol blend films
were more similar to PVA ones than those of the neat corn starch.
This indicated that the crystalline structure of starch granules was
decomposed due to gelatinization during the blending with PVA
at temperatures >100 ◦ C. XRD patterns exhibited no retrogradation
of starch, indicating that the PVA and glycerol molecules strongly
prevented the crystalline structure of starch and PVA diffraction
dominated in the blends. The intensity of the peak related to the
PVA at 2␪ 19.3◦ decreased with the addition of glycerol and cit-
ric acid. This could be due to plasticizing and crosslinking effects
of citric acid that reduced the crystallinity of the PVA. Moreover,
XRD pattern of PVA-starch-citric acid blend showed a new peak
at around 2␪ 31.5◦ , ascribable to the residual unreacted citric acid.
On the other hand, this peak was not detected in the XRD curve
of PVA-starch-citric acid with glycerol, probably because glycerol
kept the citric acid from assembles, and the citric acid attended in
the blends in the molecular level.
703
6.2. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis
Protein based polymerization can be evaluated by means of SDS-
PAGE method, a very common technique for separating proteins by
electrophoresis, as described by Laemmli [108].
In a study by Chambi and Grosso [31], the molecular weight
profile of the films treated with transglutaminase was evaluated.
In the absence of transglutaminase, the casein films showed intense
bands around 30 kDa, related to ␣s1-, ␤- and ␬-casein [109], and the
prominent band of the gelatin film had a molecular mass of 212 kDa,
ascribed to the ␤-gelatin subunit [110]. In the casein/gelatin blend
films, the intensities of the bands of the individual proteins differed
as a function of the gelatin and casein contents. The occurrence of
crosslinking in the proteins resulted in the formation of new high
molecular weight polymers (≥212 kDa) and a partial disappearance
of the bands related to the non-crosslinked casein. Oh et al. achieved
complete crosslinking of the casein fractions using casting method
soon after the addition of transglutaminase, which could allow the
polymerization during drying at 23 ◦ C for 12 h, despite of the high
moisture content [111].
Gomez-Guillen et al. [112] stated that crosslinking with micro-
bial transglutaminase (MTGase) could lead to an increase in the
molecular weight of fish gelatin. Kolodzlejska et al. [113] reported
that reaction time and concentration of MTGase-treated Baltic cod
skin gelatin considerably increased, leading to an increment in the
speed of gel formation and of the gelatin viscosity during MTGase
treatment. Bae et al. [36] investigated the SDS–PAGE of neat fish
gelatin and MTGase-treated fish gelatin. In MTGase-treated fish
gelatin, the band intensity with molecular masses below 75 kDa
decreased with increasing the treatment time, whereas the band
intensity above 250 kDa increased. They indicated that crosslinking
reaction in the fish gelatin matrix was induced by MTGase, lead-
ing to increased molecular mass. Indeed, the molecular weight of
MTGase is 38 kDa with 331 amino acid residues [114,115] and, the
probability of band overlap is very low; thus the revealed bands
between 37 kDa and 75 kDa have to be associated to fish gelatin
molecules. Similarly, other authors observed the decrease of band
intensity at about 100 kDa and the increase of band intensity in the
upper position when treated with MTGase [112,116].
6.3. Fourier transform infrared spectroscopy
FTIR can be considered as a practical and supplementary method
to characterize the microstructures of crosslinked biopolymer
films. In fact, it allows identifying the possible functional chemical
groups and molecular interactions [11].
Coma et al. [46] assessed the degree of reticulation of hydrox-
ypropyl methyl cellulose (HPMC) with citric acid, comparing the
results of FTIR analysis at ester band (1735 cm−1 ) with acid–base
titration method, to determine the rate of crosslinking and ester
bond in crosslinked cellulosic derivatives. The comparison between
the reticulation rates calculated from both methods showed a pos-
itive linear relationship. They concluded that FTIR spectroscopy,
compared to titration method, is an easy, rapid and acceptable
method for the approximate determination of the ester bond
formation rate directly on cellulosic films crosslinked by polycar-
boxylic acid. In another work, Reddy and Yang investigated the
crosslinking of starch films by citric acid through FTIR [42]. The
comparison between the FTIR spectra of the non-crosslinked films
and films crosslinked with 5% citric acid revealed an extra peak
in the crosslinked film at around 1724 cm−1 that is related to the
C O stretching vibrational mode [117]. It was improbably that all
the carboxyl groups are esterified and this peak could be a coales-
cent one due to both the ester bond and carboxyl carbonyl groups
in citric acid. However, all the samples were washed before the
test, to remove thoroughly the effects of unbound citric acid in the
704 F. Garavand et al. / International Journal of Biological Macromolecules 104 (2017) 687–707
blend. Therefore, the presence of this peak confirmed the chemical
linkages between starch and citric acid. Similarly, Shi et al. [82]
investigated the effects of citric acid as a crosslinker on chemi-
cal structure of starch-PVA films. The IR spectra of PVA-citric acid
and starch-citric acid exhibited the peak at 1729 cm−1 , demonstrat-
ing the occurrence of esterification between citric acid and either
starch or PVA. However, the peak at 1729 cm−1 of PVA-citric acid
was very weaker with respect to that revealed in the case of starch-
citric acid, suggesting more esterification between the citric acid
and starch than PVA. In addition, as expected, the carbonyl peak
intensity increased with increasing the citric acid concentration.
On the other hand, the carbonyl peak of PVA-starch-citric acid with
glycerol was lower than the blend without glycerol. The reason was
probably that some citric acid molecules reacted with glycerol to
obtain glycerol citrates which were solubilized into the water dur-
ing immersion. As a result, the addition of glycerol prevented the
crosslinking reaction between citric acid and starch or PVA. Fur-
thermore, it is worthy to underline that the intensity of the peak
around 3340 cm−1 (stretching vibration of the hydroxyl groups) of
PVA-starch-glycerol was lower than that of PVA-starch-citric acid,
probably due to the fact that citric acid can form stronger hydro-
gen bonds with PVA or starch compared to glycerol. As expected,
higher citric acid concentrations caused an increase in the number
of the hydroxyl and carboxyl groups and, thus, of the hydroxyl peak
height.
The rate of Maillard reactions in CMC-SPI films plasticized by
glycerol was studied by Su et al. using FTIR spectroscopy [38].
The FTIR spectra of various weight ratios of CMC-SPI blend films
showed that the intensity of the bands at 3100–2900, 1600–1400,
and 890 cm−1 gradually decreased as CMC was added to SPI. These
changes revealed modifications of the amide I, II and III vibra-
tions. These results showed that the hydroxyl group in CMC and
amino groups in SPI were consumed during the blending process
at high temperature. Furthermore, a new band at 1647 cm−1 indi-
cated a C N stretching vibration assigned to the Maillard reaction
[118–120]. Therefore, complex Maillard reactions are expected to
occur between CMC and SPI.
Rimdusit et al. [45] characterized the structure of methyl cellu-
lose (MC)-glutaraldehyde crosslinked films by FTIR spectroscopy.
For crosslinked MC, the peaks at 1710 cm−1 (carbonyl stretch-
ing band from aldehyde group of glutaraldehyde) indicated the
crosslinkage between MC and glutaraldehyde. With an increase
in the glutaraldehyde above 6.0 wt%, the peak at 1710 cm−1 sub-
stantially increased, due to an increment in unbound aldehyde.
Therefore, the optimal glutaraldehyde content was below 6.0 wt%
and this peak was not found at the concentration of 4.5 wt%. More-
over, the appearance of this absorption spectrum suggested the
formation of inter-molecular hydrogen bonding, similar to the
results reported for chitosan crosslinked with glutaraldehyde [121].
The potential crosslinking activity of tannic acid was studied in
the chitosan films by FTIR [49]. Rivero et al. [49] applied the sec-
ond derivative method in FTIR spectra to enhance the resolution
and verify the peak positions because the exact wavenumber posi-
tion of biological samples is sometimes difficult to be identified.
The IR spectrum of crosslinked chitosan films was similar to that
of non-crosslinked ones. Nevertheless, the intensity of absorption
peaks decreased as a result of crosslinking reaction; similarly, the
second derivative method proved this result. According to Aelenei
et al. [122] typical bands of the tannic acid were either weakened
or covered, as shoulders under other bands, which confirmed that
chitosan matrix consumed the free tannic acid. Furthermore, a syn-
ergistic effect of components glycerol and tannic acid was shown in
the spectra of crosslinked chitosan films plasticized with glycerol.
Effect of boric acid (as a crosslinker) on FTIR spectrum of
poly(vinyl alcohol) (PVA)/bacterial cellulose nanocrystals (BCNC)
films was studied by Rouhi et al. [11]. They observed a new and
weak absorption peak related to the stretching vibration of B OC
at wave number of about 1035 cm−1 . Moreover, the peak of OH
stretching (3000–3550 cm−1 ) was shifted to a higher wave number.
These results indicated the occurrence of the crosslinking reaction
between boric acid with PVA and/or BCNC.
6.4. Nuclear magnetic resonance (NMR) spectroscopy
NMR spectroscopy uses the nuclear magnetic resonance of spe-
cific atoms to provide information about the chemical and physical
properties of atoms or their related molecules, as well as reaction
state, dynamics, structure and chemical environment. This tech-
nique can be used to verify the occurred crosslinking of polymers
and to investigate the Maillard reaction. Su et al. used solid-state
13 C NMR analysis to prove crosslinking by Maillard reaction in SPI-
CMC blend films [17]. 13 C NMR spectra of pure SPI showed that a
characteristic sharp peak of C1 (the carboxylic carbon of SPI, back-
bone carbonyl group) could be clearly identified at approximately
173 ppm [123]. The C2 carbon spectrum contained mainly bands in
the alkyl region, with a prominent peak at about 50 ppm from non-
transformed NCH2 moieties [123], implying that Maillard reaction
occurred by consuming −NH2 . The intensity of the characteristic
13 C peak of pure SPI at 173 ppm gradually decreased as the propor-
tion of CMC in the blends increased. There was no signal at 50 ppm,
approving occurrence of Maillard reactions between SPI and CMC
blends.
7. Cytotoxicity
Several studies have shown that the synthetic crosslinking
agents, including carbodiimide and hexamethylenediisocyanate,
are all cytotoxic and dangerous for human body and nature. There-
fore, it is delightful to provide a safe crosslinking agent to be used
in packaging materials that has low cytotoxicity and to construct a
stable and biocompatible film or coating [124]. Natural crosslinking
agents, such as those originated from microbial fermentation and
those from plant materials, show appropriate potential both in case
of constructing powerful bonds between biopolymer chains and
safe or less-cytotoxic effects. Genipin, transglutaminase, carboxylic
acids, and crosslinking via Maillard reaction are examples of safe
and naturally-occurred crosslinking agents applied in biopolymers
till now.
For example poly(carboxylic acids), including boric acid and
citric acid, are cost-effective and non-toxic components which
improve the functionality of a wide range of biopolymer materials,
from proteins to polysaccharides [42]. Some of the crosslinkers, like
borate, not only are accessible and cost-effective but also introduce
some advantages to the soil and environment [125]. As well, some
of the complications, such as toxicity and calcification as a result of
crosslinking by aldehyde groups, especially glutaraldehyde, have
been effectively lightened by applying glycine or glutamic acid
as post-treatment. Glutamic acid treatment is also supposed to
quench reactive aldehyde groups by connecting amino groups from
the acid end [126].
8. Concluding remarks
Biopolymer films, based on various carbohydrates and proteins
and, in a fewer extent, on some lipids, possess favorable barrier
characteristics, but, in some cases, exhibit weak mechanical, struc-
tural and thermal attributes, along with their moisture-sensitivity,
compared to well-established commercial polymers. Several
endeavors were recently devoted to investigate the possibility
to improve permeability and/or thermo-mechanical attributes of
film-forming biopolymers by introducing crosslinks into the film
 F. Garavand et al. / International Journal of Biological Macromolecules 104 (2017) 687–707
network. Crosslinking reactions enable the preparation of materials
with enhanced mechanical, thermal and physicochemical prop-
erties. Particularly, naturally-occurred crosslinking agents have
attracted more attention in order to take into account environmen-
tal and health concerns, along with the economic issues. It can be
concluded that crosslinking approach is a cost-effective and effi-
cient method compared to other reinforcement strategies, e.g. the
addition of nanomaterials in order to obtain biopolymeric matrix
composites which present inherent drawbacks.
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