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Critical Reviews in Food Science and Nutrition
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Recent progress in using zein nanoparticles-loaded
nanocomposites for food packaging applications
Farhad Garavand, Diako Khodaei, Niaz Mahmud, Joinul Islam, Injeela Khan,
Shima Jafarzadeh, Reza Tahergorabi & Ilaria Cacciotti
To cite this article: Farhad Garavand, Diako Khodaei, Niaz Mahmud, Joinul Islam, Injeela Khan,
Shima Jafarzadeh, Reza Tahergorabi & Ilaria Cacciotti (2022): Recent progress in using zein
nanoparticles-loaded nanocomposites for food packaging applications, Critical Reviews in Food
Science and Nutrition, DOI: 10.1080/10408398.2022.2133080
To link to this article: https://doi.org/10.1080/10408398.2022.2133080
Published online: 12 Oct 2022.
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REVIEW
CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION
Recent progress in using zein nanoparticles-loaded nanocomposites for food
packaging applications
Farhad Garavanda, Diako Khodaeib, Niaz Mahmudc, Joinul Islamc, Injeela Khanc, Shima Jafarzadehd, Reza
Tahergorabic and Ilaria Cacciottie
aDepartment of Food Chemistry and Technology, Teagasc Moorepark Food Research Centre, Co. Cork, Ireland; bDepartment of Sport,
Exercise, and Nutrition, Atlantic Technological University, Galway, Ireland; cFood and Nutritional Sciences Program, North Carolina
Agricultural and Technical State University, Greensboro, North Carolina, USA; dSchool of Engineering, Edith Cowan University, Joondalup,
Western Australia, Australia; eDepartment of Engineering, INSTM RU, University of Rome ‘Niccolò Cusano’, Rome, Italy
ABSTRACT
Biopolymers are important due to their exceptional functional and barrier properties and also
their non-toxicity and eco-friendly nature for various food, biomedical, and pharmaceutical
applications. However, biopolymers usually need reinforcement strategies to address their poor
mechanical, thermal, and physical properties as well as processability aspects. Several natural
nanoparticles have been proposed as reinforcing agents for biopolymeric food packaging materials.
Among them, zein nanoparticles (ZNPs) have attracted a lot of interest, being an environmentally
friendly material. The purpose of the present review paper is to provide a comprehensive overview
of the ZNPs-loaded nanocomposites for food packaging applications, starting from the synthesis,
characteristics and properties of ZNPs, to the physicochemical properties of the ZNPs-loaded
nanocomposites, in terms of morphology, permeability, solubility, optical features, hydrophobic/
hydrophilic behavior, structural characteristics, thermal features, and mechanical attributes. Finally,
at the end of this review, some considerations about the safety issues and gastrointestinal fate of
ZNPs, as well as the use of ZNPs-based nanocomposites as food packaging, are reported, taking
into account that, despite the enormous benefits, nanotechnology also presents some risks
associated to the use of nanometric materials.
Introduction
Nanotechnology is gaining a lot of interest in several sectors
of science, engineering and technology, such as the phar-
maceutical, biomedical and food ones (Garavand, Nooshkam,
et al., 2022). Recently, the use of nanotechnology in food
packaging is constantly increasing in order to improve the
package performance and overcome some limits in terms
of thermal, mechanical and barrier properties (Ashfaq,
Khursheed, Fatima, Anjum, & Younis, 2022). Nanotechnology
includes the synthesis, processing, characterization, and/or
manipulation of structures and materials characterized, at
least, by one dimension less than 100 nm (Cerqueira, Vicente,
& Pastrana, 2018). The employment of nanosized structures
allows designing packaging materials with improved and
unique features, able to provide higher shelf life and better
quality preservation, with a remarkable impact on food pro-
cessing, packaging, and storage (Garavand et al. 2022). In
particular, the interest in nanocomposites for food packaging
applications is remarkably incrementing during the last two
decades, as testified by the relevant growth in the number
of publications from 2010, with a more than the five-fold
increase in the number per year during the period 2010–
2019 (Sarfraz, Gulin-Sarfraz, Nilsen-Nygaard, & Pettersen,
2020). The nanocomposites are composed of a matrix and
nanofillers (e.g., nanoparticles (NPs), nanofibers, nanorods.),
in order to provide specific functionalities, such as improved
barrier, mechanical, and thermal properties, as well as anti-
microbial and antioxidant actions (Khazaei, Nateghi, Zand,
Oromiehie, & Garavand, 2021).
Among the used nanofillers, NPs can be employed for
several aims: i) to improve the barrier properties; ii) to act
as reinforcing agents; iii) to act as a carrier for specific
biomolecules, antimicrobial/antioxidant agents, due to its
ability to encapsulate and efficiently deliver active sub-
stances, iv) to directly play as antimicrobial agents and as
nanosensors for the detection of food-relevant analytes (gas-
ses, small organic molecules and food-borne pathogens), for
the active and smart packaging sectors (Azizi-Lalabadi,
Garavand, & Jafari, 2021; Garavand, Cacciotti, et al., 2022;
Jafarzadeh et al., 2022). Organic and inorganic NPs are
commonly used as nanofillers within a polymeric matrix
(He, Deng, & Hwang, 2019).
Among the inorganic ones, nanoclays, carbon nanofibers/
nanotubes, metallic NPs (e.g., silver), and oxides (e.g., zinc
oxide, titanium dioxide, silica, diatomite, etc.) have been
explored (Benucci, Lombardelli, Cacciotti, & Esti, 2020;
© 2022 Taylor & Francis Group, LLC
CONTACT Farhad Garavand farhad.garavand@teagasc.ie; farhad.garavand@gmail.com
https://doi.org/10.1080/10408398.2022.2133080
KEYWORDS
Zein nanoparticles; food
packaging; nanocomposites;
nanoparticle synthesis; safety
2 F. GARAVAND ETAL.
Cacciotti, Fortunati, Puglia, Kenny, & Nanni, 2014; Cacciotti,
Lombardelli, Benucci, & Esti, 2019), whereas among the
organic ones, natural and synthetic polymeric NPs have
been tested, including proteins (e.g. zein), carbohydrates
(e.g. chitosan, starch), fats, nanocellulose, etc. (Babaee,
Garavand, Rehman, Jafarazadeh, Amini, & Cacciotti, 2022;
Y. Garavand, Taheri-Garavand, Garavand, Shahbazi, Khodaei,
& Cacciotti, 2022; Vahedikia et al., 2019).
Concerning the natural polymeric NPs, zein NPs (ZNPs)
have recently attracted a lot of interest. Zein is an environ-
mentally friendly material and is one of the most important
industrial biopolymers for the 21
St century (Soltani &
Madadlou, 2015). This protein represents around 50% of
the total protein content in maize, belongs to the prolamin
class, is made of lipophilic amino acid residues, is commonly
obtained as a co-product from processing corn in food,
feed, agriculture products, or as a by-product of fuel pro-
duction (Kasaai, 2018), and was approved as a Generally
Recognized As Safe (GRAS) excipient in 1985 by the United
States Food and Drug Administration (US-FDA)
(Weissmueller, Lu, Hurley, & Prud’homme, 2016). It has
been reported that the global corn fiber market size exceeded
USD 790 million in 2021 and is expected to grow at a
compound annual growth rate (CAGR) of 13% from 2022
to 2028 (Liang et al., 2022).
On the basis of the solubility and sequence homology,
four zein kinds, which are different in peptide chains,
molecular sizes, and solubility (Parris, Dickey, Tomasula,
Coffin, & Vergano, 2001), can be identified: α-zein (19 and
22 kDa, 70–85% of the total fraction of zein mass, commer-
cially available), β-zein (14 kDa), γ-zein (16 and 27 kDa),
and δ-zein (10 kDa) (Kim, Woo, Clore, Burnett, Carneiro,
& Larkins, 2002). Zein differs from other proteins for its
amino acid composition, since it almost completely lacks
lysine and tryptophan, leading to its unique solubility, mostly
limited to acetic acid, acetone, water alkaline solutions, and
ethanolic solutions (Lawton, 2002). Indeed, it is
water-insoluble and can be only solubilized in mixtures of
water with alcoholic solvents, anionic detergents, urea or
base (Giteru, Ali, & Oey, 2021). Alpha zein dissolves in
70-95% ethanol solution, while beta fraction is insoluble in
95% ethanol and only dissolves in 60% ethanol. Using reduc-
ing agents is proposed to make gamma fraction soluble in
ethanol (Reddy & Yang, 2011b; Shukla & Cheryan, 2001).
Additionally, zein presents high coating and film forming
capability, biodegradability, biocompatibility, amphiphilic
behavior (both hydrophobic and hydrophilic character), anti-
oxidant properties, high resistance to humidity, heat, abra-
sion, and water, the ability to enhance the shelf-life of
biomolecules (Vahedikia et al., 2019). It is suggested as an
appropriate encapsulating agent, nutrient and drug constit-
uent (Kasaai, 2018) and it is widely used as a biodegradable
food packaging materials.
Due to its positive charge, it is appropriate for the deliv-
ery of negatively-charged foods, nutrients, and drugs.
Moreover, it is suitable for the delivery of different nutrients
into the body, owing to the wide range of its isoelectric
point, as well as for the delivery of essential oils, drugs,
and enzymes (X. Wang et al., 2019). Owing to its
film-forming capability, zein is reported to form a tough
and brittle film during the casting of its ethanolic solution
(Cuq, Gontard, & Guilbert, 1998), with biodegradable prop-
erties, good mechanical and barrier features (Z.-M. Gao
et al., 2014; Y. Guo, Liu, An, Li, & Hu, 2005; Parris etal.,
2001; Shukla etal., 2001). For all these reasons, ZNPs have
been widely used for food and nutrition applications, par-
ticularly for the production of nanocomposite food packag-
ing materials (Reddy & Yang, 2011a). In this regard, ZNPs
can be used not only as reinforcing agents, but also as
environmentally friendly functional ingredients nanocarriers
to protect them against degradation, improving their stabil-
ity, and providing desirable release characteristics and deg-
radation (Oliveira et al., 2018).
To the best of our knowledge, no review paper about the
ZNPs-loaded nanocomposites for food packaging has been
published yet. This study aims to provide a comprehensive
overview about the synthesis, interactions, film forming
properties, and safety concerns of ZNPs-loaded nanocom-
posites for food packaging applications.
Synthesis of zein nanoparticles
Zein proteins can be easily converted to NPs, due to owning
a unique hydrophobic nature. Several methods have been
suggested for the preparation of ZNPs. The liquid-liquid
dispersion is one of the common methods for the prepara-
tion of these NPs as a food-grade nanosized delivery system.
In this method, zein molecules are solubilized in 80% eth-
anol solution and subsequently homogenized with water and
sheared into smaller droplets. The ZNP’s shape and dimen-
sion vary depending on the shearing speed, the zein quan-
tity, and the ethanol amount. By increasing the ethanol
content, the particle size of ZNPs reduces. At a higher con-
centration of ethanol, a homogenizer is employed to shear
the fluid into smaller droplets prior to dropping ethanol
concentration below the critical point of ZNPs participation
(Zhong & Jin, 2009). Penalva, Esparza, Larraneta,
González-Navarro, Gamazo, and Irache (2015) also prepared
ZNPs using a spray dryer after dissolving the zein protein
into a 65% ethanol/water solution. The size of ZNPs pre-
pared in this method was reported to be about 200 nm and
they showed a negative zeta potential.
Anti-solvent perception is another method suggested for
producing ZNPs (Figure 1A). In this method, after the zein
particles dissolution in alcohol, it is poured or sheared into
a water-based medium for phase separation, resulting in
ZNPs. The inter-diffusion between ethanol and water reduces
the solubility of zein and after the precipitation of the solu-
tion; the ZNPs are formed (Zhong et al., 2009). De Folter,
Van Ruijven, and Velikov (2012) added zein into an
ethanol-water mixture (80:20) using a syringe for a con-
trolled mixture. After the yellow milky color dispersions
preparation and the ethanol removal from the solution, a
more concentrated zein solution was achieved. After the
dispersion centrifugation and the large aggregates discarding,
ZNPs with an average size of 70 ± 13 nm and a mono-disperse
spherical morphology were collected. ZNPs prepared in this
CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION 3
method were stable at pH 4.0 and showed a positively
charged zeta potential (63 ± 8 mV). One of the drawbacks
of this method is that the NPs obtained are relatively unsta-
ble and do not easily disperse in water. Dispersing the zein
in the sodium caseinate and adjusting the pH or ethanol
concentration were other approaches suggested to overcome
this challenge (H. Chen & Zhong, 2014). The solvent evap-
oration technique is another strategy for the fabrication of
ZNPs-loaded films at neutral pH conditions. The solvent is
evaporated via cast drying in this technique (Song, Sun,
Gul, Mata, & Fang, 2021). Using supercritical fluids along
with methanol as the solvent is another method proposed
for the preparation of ZNPs, allowing for obtaining ZNPs
with an average size between 50 and 350 nm. These ZNPs
were reported to be in either sphere or the filament network
which was highly affected by the nozzle and flow rate of
CO2. However, this technique is not highly advised, due to
the possible toxicity of organic solvent residues and a high
production cost (Sining & Zhao, 2017). Rapid mixing of
solvent and antisolvents is another proposed method for
preparing ZNPs, called flash nanoprecipitation (K.-K. Li
etal., 2014). In this method, the sodium caseinate is applied
as the processing stabilizer and ZNPs with a size below
350 nm were achieved. The particle size of ZNPs was similar
to different ethanol solutions that can be attractive for indus-
trial and continuous production of ZNPs.
Due to the flammability risk of ethanol as the solvent
and possible encapsulation of lipophilic particles that exist
as the co-dissolve in ethanol, dissolving zein in propylene
glycol which is non-flammable is suggested. H. Chen and
Zhong (2015) used propylene glycol for dissolving zein
particles (diameter of 250 nm and height of 51.3 nm, high
stability in the pH range of 3-8), followed by stabilizing
them in gum Arabic. Electrohydrodynamic processing
(EHDP) is another user-friendly and cost-effective method
for the synthesis of micro and nanostructures (Figure 1B).
High efficiency, low cost, working at room/ambient condi-
tions, as well as controllable temperature and humidity, are
some of the advantages of this method (Marques, Azevedo,
Teixeira, Pastrana, Gonçalves, & Cerqueira, 2019; Silva
et al., 2021). Electrospinning and electrospraying are two
basic EHDP methods for preparing micro/nanofibers and
micro/NPs, respectively. In both methods, a similar basic
setup, composed of a syringe pump (to monitor the flow
rate), a typical metal needle with various diameters, a high
voltage power supply (applying the high fields to the tip
of the needle), and a grounded metal collected for depos-
iting samples, is needed. Although ethanol is the main
solvent used for this technique, other solvents such as meth-
anol, isopropanol, acetone, acetic acid, and dimethyl for-
mamide (DMF) are also used in EHDP. The concentration
of polymer has a significant influence on the size and
morphology of the resultant particles. Yao, Li, and Song
(2007) reported that increasing the zein concentration from
20 to 50% (w/w) significantly increased the diameter of
the fibers from 500 nm to 6 µm. Another research by Karim,
Fathi, and Soleimanian-Zad (2020) showed that the increase
in zein concentration from 5 to 12% (w/w) resulted in
nanobeads with a size range of 100-220 nm. While at higher
concentrations (over 25% w/w) the fibers were formed and
additional concentration increase up to 40% (w/w) resulted
in fibers with an average diameter of 200 nm. By further
increment in zein concentration, the size of fibers signifi-
cantly increased to values above 1 µm. Miri, Movaffagh,
Najafi, Najafi, Ghorani, and Koocheki (2016) also prepared
edible sub-micron zein fibers using the electrospinning
process and the acetic acid solution. Similarly, a higher
average diameter was reported for higher solution concen-
trations. Moreover, an increase in the electrospinning volt-
age also led to an increment in the average size of fibers,
Figure 1. A) Anti-solvent perception method (liquid-liquid dispersion/phase separation method and B) Electro spraying methods for preparation of zein
nanoparticles.
4 F. GARAVAND ETAL.
while no influence of the flow rate or distance between
needle tip and collector on the morphology of fibers was
reported.
Characterization of ZNPs
Several microscopic methods have been applied to study
the morphology and dimension of ZNPs, such as transmis-
sion electron microscopy (TEM), scanning electron micros-
copy (SEM), and atomic force microscopy (AFM). By
FE-SEM (field emission-SEM) observation, it has been
demonstrated that spherical or roughly spherical ZNPs with
a smooth surface have been usually achieved with a variable
size: for example, according to Penalva etal. (2015), ZNPs,
used for the delivery of resveratrol, are characterized by a
size close to 200 nm, whereas for other authors by an average
size of 64 nm and a bimodal size distribution (75–104 nm)
(Kasaai, (2018).), for Soltani etal. (2015) by a mean diam-
eter of 69 nm in a range of 60–263 nm, for Zhong et al.
(2009) an average dimension of 100–200 nm, with the for-
mation of smaller particles at higher shear rates, and at a
higher ethanol concentration or at a lower zein concentra-
tion, for ZNPs synthesized by liquid–liquid dispersion.
Similarly, TEM investigation evidenced the obtainment of
mono-disperse spherical NPs, synthesized by means of
anti-solvent precipitation, with an average diameter of
(70 ± 13 nm) (De Folter et al., 2012), as well as roughly
spherical and uniform particles with diameters ranging from
50 to 100 nm (Hu & McClements, 2015), and of homoge-
neous spherical NPs with smooth surfaces (De Melo etal.,
2019; de Souza Tavares, Pena, Martin-Pastor, & de Sousa,
2021). Xu, Jiang, Reddy, and Yang (2011) reported the
obtainment of hollow ZNPs with an average diameter of
65 nm. In particular, in the case of a single hollow zein
particle (100 nm in diameter), they were able to evidence a
wall thickness in the range of 3-10 nm, with cavity size
around 20 to 50 nm in diameter.
The same dimensional and morphological results have
been demonstrated by means of AFM characterization. AFM
has been proposed as a nondestructive characterization tech-
nique to investigate the food material’s three dimensional
structure (Y. Guo et al., 2005). AFM characterization allows
to achieve more details about the single protein particle,
related to the dimension, roughness and resolution, through
a precise surface, topographic map, with respect to other
microstructural techniques, such as TEM, SEM, and scan-
ning probe microscopies (SPMs) (Allison, Mortensen,
Sullivan, & Doktycz, 2010). From the acquired 2 D phase
and 3 D topographical AFM images, it was evidenced that
the produced ZNPs were homogeneously spherical and char-
acterized by a smooth surface, with an average dimension
of 119 ± 25 nm (Podaralla & Perumal, 2012). Similarly, Zhao
et al. (2020) and Chen, Ye, and Liu (2013) demonstrated
the spherical ZNPs by AFM.
Xu etal. (2011) compared the morphological features of
hollow and solid ZNPs by AFM. From AFM investigation,
hollow ZNPs presented an average diameter size of around
60 nm, whereas solid ZNPs were 120 nm. Based on the color
scale bars, the 2 D images evidenced that the heights were
about 6 nm and 100 nm for hollow and solid ZNPs, respec-
tively. Moreover, whereas the hollow ZNPs height was much
smaller than their average diameter (60 nm), for the solid
ones it was close to their average diameter (120 nm). This
experimental evidence suggested that the hollow ZNPs
tended to collapse after drying.
Furthermore, dynamic light scattering (DLS) measure-
ments were performed in order to investigate the diameter
size distribution and the zeta potential of the produced NPs.
For example, De Folter et al. (2012) demonstrated that the
ZNPs are positively charged and zeta potential of 63 ± 8 mV
was detected at pH 4.0. In another work an average size of
69 nm for ZNPs prepared by evaporation of a zein ethanolic
solution (80% v/v), after adjusting its pH to 4.0, was revealed
by DLS (Soltani etal., 2015). A particle size distribution of
suspensions containing relatively small particles (d ≤ 400 nm)
was measured by the DLS, whereas in the case of relatively
large particles (d > 400 nm) by a laser diffraction particle
size analyzer (static light scattering, SLS) (Hu et al., 2015).
Zhao et al. (2020) reported a size range of 30–200 nm for
the produced ZNPs (averaged particle diameter of 74 nm),
as well as a ξ-potential of +35.6 mV and the polydispersity
index (PDI) of 0.167, as DLS results.
On the basis of the collected papers, it is evident that the
ZNPs size distribution, charge, wetting properties, and emul-
sion stability are influenced by the preparation methods, their
degree of aggregations (G. Liu, Wei, Wang, Hu, & Jiang, 2016),
concentration, temperature, pH, and ionic strength (De Folter
et al., 2012). For example, ZNPs tend to strongly aggregate if
prepared in the absence of nonionic surfactant (e.g., Tween
80), as a consequence of the hydrophobic attraction between
zein surfaces non-polar groups (Hu et al., 2015). Similarly,
different degrees of aggregation promote the formation of the
non-uniform size of the globules: for example, for a zein con-
centration of 1 μg/mL, a narrow PDI (diameter 60–120 nm,
and height 15–50 nm) was revealed (De Folter et al., 2012;
Y. Guo et al., 2005). Regarding the temperature, ZNPs usu-
ally exhibit higher stability at fridge or room temperature.
Concerning the pH influence, Hu et al. (2015) reported that
the ZNPs tended to aggregate at pH 2, whereas they were stable
at high ionic strengths after heating at 90 °C. On the other
hand, at pH 7 and high ionic strengths (>100 mM NaCl), the
ZNPs tended to aggregate, but they were stable after heating
at 90 °C for 2 hours.
As a final consideration, in the considered cases, the
particles size strongly influences their proprieties and effi-
cacy. Indeed, most of the described ZNPs were proposed
for potential use as carriers for drugs and biomolecules in
food and pharmaceutical formulations (Hu & McClements,
2015; De Melo et al., 2019; Podaralla & Perumal, 2012;
Chen, Ye, and Liu, 2013), particularly for the potential devel-
opment of food-grade nanoscalar delivery systems (Zhong
etal., 2009; Soltani etal., 2015). Thus, the nanosized struc-
ture and the consequent ability to encapsulate high drug
amounts and to penetrate into cells allowed improving the
bioavailability of the encapsulated biomolecules, ensuring
their controlled release. For example, the oral bioavailability
CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION 5
of the resveratrol encapsulated within ZNPs increased in
mouse 19-fold with respect to free resveratrol (Penalva etal.,
2015). de Souza Tavares etal. (2021) demonstrated that the
ellagic acid encapsulation within ZNPs enhanced its anti-
oxidant and antibacterial activities, making its active phe-
nolic groups more accessible. Thus, the obtained system can
be proposed as a potential solution against free radicals and
a promising treatment for Gram-positive and Gram-negative
bacteria based infections.
Interaction between ZNPs and the encapsulated
biomolecules
The interaction between the produced ZNPs and the encap-
sulated biomolecules is pivotal. Taking into account that
ZNPs are usually employed as carriers of negatively charged
food compounds, due to their positive charge, the conse-
quent electrostatic interaction plays a significant impact on
the release and subsequent absorption of the encapsulated
compounds in the GI tract. Indeed, it has been demon-
strated that in the case of electrostatic interactions between
the nanocarriers and the considered molecules, a delayed
release was revealed, as expected (Pakulska et al., 2016).
Thus, this peculiar behavior can influence the absorption
of the encapsulated compounds in the GI tract, determining
the biological responsiveness of the developed NPs system.
Different characterization techniques have been employed
to study the interaction between the produced ZNPs and
the encapsulated biomolecules. FTIR spectroscopy has been
used in order to study the zein functional groups, as well
as their interaction with the encapsulated biomolecules.
ZNPs FTIR spectrum is commonly similar to that of the
used zein powder, as expected: the detected peaks/bands at
3307 cm−1, 2959 cm−1, 1653 cm−1, 1537 cm−1 and 1241 cm−1
can be ascribed to the OH stretching, C-H stretching, C=O
axial deformation of the primary amide, CN angular defor-
mation and N-H axial deformation, respectively (C.-E. Park,
Park, & Kim, 2015).
In the case of ellagic acid (EA) loaded ZNPs, the bands
associated with EA at 3562 cm−1 and 3468 cm−1 were not
visible, suggesting a homogeneous EA distribution within
the ZNPs (de Souza Tavares et al., 2021). Moreover, the
detection of an axial OH stretching superimposed with the
NH stretching at 3306 cm−1indicated an interaction of the
EA OH group with the zein NH groups through hydrogen
bonds, leading to the development of a strong network
structure (Tavares, Tavares-Júnior, Otero-Espinar,
Martín-Pastor, & Sousa, 2020). Additionally, the C=O
stretching band of amide I (zein) was shifted at 1659 cm−1,
as a consequence of the formation of hydrogen bonds
between EA and the amide groups of zein (amide conju-
gate). The angular deformation of the amide II N-H bond
and the EA phenyl ring peak in the zein spectrum was
shifted to 1539 cm−1 and to 823 cm−1, in the EA-loaded
NPs spectrum, respectively. These results confirm the pres-
ence of aromatic condensation with the aromatic aminoac-
ids present in the zein hydrophobic portions (Tavares
et al., 2020).
The ZNPs interaction with the encapsulated biomole-
cules can be also studied by fluorescence quenching, which
is considered an ideal characterization to investigate the
interactions of small molecules with proteins (Y. Liu, Li,
Zhang, Li, & Hou, 2019). Indeed, zein molecules are char-
acterized by an intrinsic fluorescence associated with the
tyrosine tryptophan, and phenylalanine residues (X. Wang
etal., 2019): in particular, ZNPs excited at 278 nm present
a characteristic fluorescence at 309 nm, mostly ascribed to
the intrinsic fluorescence from tyrosine residues present in
the zein hydrophobic cavities. Thus, the alterations in the
intrinsic fluorescence of zein (fluorophore) can be ascribed
to the interaction with a specific molecule (quencher). It
has been reported that zein owns a great affinity for the
molecule’s hydrophobic parts, particularly the aromatic rings
(Sousa, Luzardo-Álvarez, Blanco-Méndez, & Martín-Pastor,
2012). For example, the ellagic acid encapsulation within
ZNPs led to a significant quenching of ZNPs fluorescence
(de Souza Tavares etal., 2021), due to the EA binding with
the zein hydrophobic groups.
Similarly, NMR studies have been performed in order to
study the interaction between the ZNPs and the encapsulated
biomolecules. In this manner, it was possible to identify the
signals related to the formation of transient complexes
between the zein and the considered molecules, such as EA
(de Souza Tavares et al., 2021), tetracycline (Sousa et al.,
2012), with a phenolic structure, and anacardic acid (de
Araujo, Martin-Pastor, Pérez, Pinazo, & de Sousa, 2021)
which led to two forms involving its aromatic and aliphatic
moieties. It has been reported that according to the com-
plexation of the aromatic rings with the protein, the phar-
macophore moieties (–OH groups) were more available for
the biological interactions, leading to a remarkable improve-
ment in the biological effectiveness of the produced NPs.
Impacts of ZNPs on composite attributes
Composite films and coatings, or multicomponent systems,
have become a key focus of food packaging research. The
preparation of composite with the complexation of a hydro-
philic layer with a hydrophobic layer gives these films their
exceptional barrier characteristics (Mahmud, Islam, &
Tahergorabi, 2021; Tavassoli-Kafrani, Shekarchizadeh, &
Masoudpour-Behabadi, 2016). In addition, several carbohy-
drate–carbohydrate, carbohydrate-protein, and protein-protein
combinations are feasible in these systems (Podshivalov,
Zakharova, Glazacheva, & Uspenskaya, 2017; Suppakul,
Boonlert, Buaphet, Sonkaew, & Luckanatinvong, 2016;
Thakur etal., 2016). The notion of a NPs-based composite
has recently attracted a lot of interest because of its effec-
tiveness in preserving food and increasing film’s physical,
mechanical, and structural properties. NPs-based composites
are composed of a mix of active and matrix molecules joined
by covalent or non-covalent bonds that sometimes share
electron pairs between atoms, resulting in a more robust
and stable system for food packaging (Bacskay, Reimers, &
Nordholm, 1997; S. Li et al., 2021). ZNPs are undergoing
substantial study and development in this area, emphasizing
6 F. GARAVAND ETAL.
mixing with other agents to create composite films with
improved characteristics. The typical feature of ZNP-based
composite films is meshwork made of doughnut structures
generated by asymmetric rods linked to each other (Y. Guo
etal., 2005). The zein rods are held together by hydrophobic
contacts and intermolecular solid disulfide bonds, which
modulate the film integrity (Argos, Pedersen, Marks, &
Larkins, 1982; Y. Guo et al., 2005). As a result, zein films
are highly brittle and lack flexibility. Furthermore, the sol-
vents, plasticizers, and drying processes used in the film’s
production have a considerable impact on the composite
film’s final characteristics (Ghanbarzadeh, Musavi, Oromiehie,
Rezayi, Rad, & Milani, 2007; Shi, Kokini, & Huang, 2009).
There is significant promise for edible coatings and bio-
degradable packaging materials made of zein-based com-
posites. The composite-forming properties of ZNPs have
long been recognized, and they serve as the foundation for
the majority of commercial applications of zein in food
packaging. Despite the fact that it is a protein, zein possesses
an exceptionally high water resistance. Moreover, it can act
as an effective plasticizer, and more research should be
conducted in this area in the future. Because of its hydro-
phobic and solvent-friendly properties, zein is particularly
well-suited for blending, as its mechanical and moisture
barrier properties may be greatly improved when compared
to other biopolymers (Bayer, 2021). Zein can be used as a
matrix and combined with additional functional substances
that have antioxidant or flavoring properties to create an
active packaging solution for food. It is important to men-
tion that the Food and Drug Administration (FDA) has
approved it for oral use as well. According to early literature,
as compared to other polysaccharides and plant proteins,
zein food packaging increases the shelf life of goods while
simultaneously improving their quality.
Recent research evaluated the impact of adding ZNPs to
whey protein isolate (WPI) films. The inclusion of ZNPs
considerably enhanced the mechanical characteristics and
water vapor barrier with no negative impact on the films’
elongation. The hydrophilicity and fractional free volume
of the WPI films were reduced after the addition of NPs.
To achieve a consistent distribution of ZNPs throughout the
film, sodium caseinate was also added. With both hydro-
philic and hydrophobic groups present in sodium caseinate,
an effective interface was formed between the hydrophobic
ZNP and the hydrophilic WPI matrix, allowing for homog-
enous NPs dispersion, even at different NPs amounts. The
authors concluded that the WPI/ZNP nanocomposite films
have the potential to be suitable for food packaging (Oymaci
& Altinkaya, 2016). Another study created a film based on
corn starch (CS)-zein/rutin nanocomposite (RNs) and
assessed the impact of RNs at various concentrations. With
increasing NPs loading, the tensile strength (TS) and elon-
gation at break (EAB) rose from 1.19 to 2.42 MPa and 42.10
to 78.84%, respectively. The addition of RNs resulted in the
creation of a net-like structure, which reduced the RN–CS
film’s water vapor permeability and water solubility (S.
Zhang & Zhao, 2017). Farajpour, Djomeh, Moeini,
Tavakolipour, & Safayan (2020) employed ZNPs as fillers in
a bio-composite film constructed on potato
starch-glycerol-olive oil. Olive oil and different concentra-
tions of ZNPs were combined and the effects of this com-
bination on the structural and physicomechanical features
of the plasticized potato starch films were investigated. With
the addition of oil, the water vapor permeability was
reduced. Higher ZNPs contents increased barrier character-
istics even further. The TS of the material rose as the ZNPs
concentration increased. The application of ZNPs and olive
oil improved the apparent color and UV transmission of
potato starch-glycerol films. Furthermore, the storage mod-
ulus dropped with the addition of olive oil but was increased
by adding NPs, as expected.
It was possible to develop an active-packaging biopolymer
film utilizing a composite made of hydroxypropyl methylcel-
lulose and ZNPs with reduced vapor permeability. ZNPs film
raised the water contact angles to values > 70° and lowered
water vapor permeability of films by 10–30% after incorpora-
tion. Mechanical tests revealed decreased elongation capacity
and increased TS from 27 kPa to 49 kPa. With rising the
ZNPs fraction, an initial increase was followed by a progres-
sive drop in Young’s modulus (YM). At larger ZNPs volume
percentages, decreased elasticity was seen within microscale
portions of the films, and the trends were highly associated
with the bulk YM values of the composite films. The initial
rise in YM followed by a steady drop in YM might be due
to a shift in ZNP dispersion/clustering mixed with a collapse
of the interfacial zone around ZNP (Gilbert, Cheng, & Jones,
2018). In order to develop sustainable high-performance
nanocomposite materials, it is necessary to exploit nanoscale
building blocks based on naturally renewable resources and
efficient manufacturing techniques. As a response, a physi-
cally solid and multifunctional nanocomposite was created
employing natural building blocks such as protein ZNPs and
bacterial cellulose nanofibrils (BCNs). A well-established,
scalable papermaking technique successfully incorporated the
ZNPs into the BCNs networks, resulting in a homogenous
nano-paper composite. The resultant BCN-ZNPs nanocom-
posites had significantly better tensile mechanical characteris-
tics and thermal stability, owing to high interfacial adhesion
and interactions caused by hydrogen bonds between ZNPs
and the BCNs matrix. Furthermore, the biocompatibility of
the nanocomposites is improved following the addition of
ZNPs, owing to the creation of a rougher surface structure
and the edible zein’s high biocompatibility. The authors con-
cluded that the green and straightforward preparation process
for these mechanically robust and multifunctional BCN-ZNPs
nanocomposites would encourage their sustainable application
in food packaging, with both environmental and economic
benefits (Q. Li et al., 2020).
Physical characteristics
Permeability
Water vapor permeability (WVP) is a pivotal criterion for
determining the viability of biodegradable packaging mate-
rials manufactured from agricultural polymers (Garavand et
al. 2017). It’s vital to keep the film from expanding during
permeation studies for an accurate assessment (Oymaci
et al., 2016). The water vapor mass transfer of composite
CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION 7
films from proteins has been intensively investigated, not
only because controlling moisture content is critical for
maintaining food quality, but also because the findings can
aid in understanding probable mass transfer processes. When
compared to wax films, protein films have higher WVP
values, which are two to four times higher than low-density
polyethylene films (Gennadios, Weller, Hanna, & Froning,
1996). Because of the high cohesive energy density of the
polymer, protein-based composite films, such as ZNPs-based
films, are particularly brittle without the addition of plas-
ticizers. Plasticizers like glycerol are needed to make films
more flexible and processable. Plasticizers, on the other
hand, often alter the system’s ability to draw water, resulting
in an increase in the film permeability (Gennadios et al.,
1996). Furthermore, the temperature and relative humidity
(RH) of a medium have a significant impact on the WVP
of ZNPs-based composite films. Since permeability is a prod-
uct of the penetrant’s diffusivity (kinetic factor) and solu-
bility (thermodynamic factor), a change in the permeability
can be induced by a kinetic or thermodynamic component
or a combination of both of them. The reduction in the
penetrant permeability through a nanocomposite film has
been previously explained by a prolonged diffusive trajectory
since penetrant molecules can only go around the particles.
While this is a good argument, other factors can also affect
the structure of the films and, as a result, their water-vapor
barrier efficacy (Oymaci et al., 2016). Several studies have
been conducted to determine the effect of ZNPs on the
WVP of composite films. The hydrophobic character of the
zein molecule had a role in improving the water barrier
qualities of composite films with the ZNPs inclusion. The
inclusion of ZNPs promoted the hydrogen bonding between
the zein molecules and the film matrix, resulting in the
creation of film networks with a smaller free volume, result-
ing in a more compact structure with better water tolerance
and moisture resistance (C. Li, Wu, Su, Riffat, Ni, & Jiang,
2019; Wu, Tong, Wu, & Pang, 2022). The ZNPs loading
increase was observed to decrease the film’s WVP in a ZNP/
WPI composite film. When the ZNP:WPI ratio was adjusted
at 1.2, they discovered that WVP decreased from 0.322 to
0.052 g mm/m2 h kPa (Oymaci et al., 2016). The WVP of
films containing ZNPs was lower than that of pure films
that did not include ZNPs. Similarly, ZNPs-konjac gluco-
mannan (KGM)-chitosan (CS) composite films presented a
lower WVP than neat KGM film (Wu etal., 2022).
The presence of oxygen, which is commonly related to
the phenomena of oxidation in foods, makes oxygen per-
meability (OP) one of the most essential criteria in food
packaging. The temperature has also been demonstrated to
have a significant impact on the oxygen (O2) permeability
of protein composites. Corn zein, wheat gluten, and wheat
gluten/soy protein isolate films have greater O2 permeability
as the temperature rises. Zein films have greater O2 perme-
ability than wheat gluten and wheat gluten/soy protein com-
posite films evaluated under the same circumstances (X.
Guo, Lu, Cui, Jia, Bai, & Ma, 2012). The O2 permeability
values found for their zein films were lower than those
published in the literature for edible cellulosic films and
their composites with lipids, as well as for other plastic
films (Arcan, Boyacı, & Yemenicioğlu, 2017). Therefore,
depositing ZNPs on the surface of various composite films
has been proposed as a novel way of producing plastic
packaging materials with better oxygen and water vapor
barrier qualities. For example, it was observed that the OP
of corn distarch phosphate-zein bilayer composite films was
0.8786 × 10−2 g·m−2 ·d−1 ·Pa−1, whereas the control film had
1.1600 × 10−2 g·m−2 ·d−1 ·Pa−1, with a 25% reduction (Sun,
Shao, Jiang, Shen, & Ma, 2018).
The gas barrier characteristics of ZNP composites are
challenging to quantify since they are highly dependent on
the film formulation, production process, and thickness, as
well as the temperature and relative humidity (RH) of the
test circumstances (Arcan etal., 2017). Carbon dioxide per-
meability (CDP) is especially important in the field of food
packing since carbon dioxide was the key component of
enhanced environment packaging systems where fresh fruit
and vegetables were interchanged during the postharvest
period (Kashiri, Cerisuelo, Domínguez, López-Carballo,
Hernández-Muñoz, & Gavara, 2016). L. Zhang, Liu, Wang,
Dong, Sun, and Zhao (2019) created a ZNPs—chitosan com-
posite film and discovered that adding ZNPs significantly
reduced the OP and CDP values of the films. Due to the
existence of polar interactions in their structure, blend films
had a superior oxygen barrier and produced a perpendicular
layer to prevent gas transport (Valencia-Sullca, Vargas,
Atarés, & Chiralt, 2018). WVP, OP, and CDP are all key
components of food packaging since they all have a part in
the film’s and product’s quality. The final film’s gas barrier
characteristics are affected by the whole composite manu-
facturing process. In general, the use of ZNPs in films
results in lower WVP, OP, and CDP values. More study is
needed, however, to better understand the important ele-
ments that may influence the barrier qualities during the
composite manufacturing. A short summary of recent find-
ings related to the physical characteristics of ZNPs-based
composites is presented in Table 1.
Solubility
Water solubility represents the composite’s water resistance
capability, indicating its affinity for water. The solubility of
composites in water can provide information about their
behavior in an aquatic environment and is a measure of
their water resistance. This is also a key aspect in deter-
mining the use of biodegradable composites as packaging
materials (Gnanasambandam, Hettiarachchy, & Coleman,
1997). When it comes to maintaining intermediate or high
moisture foods, low solubility coatings are critical for
long-term storage preservation. The dissolution of antimi-
crobial compounds in low-water-resistance films causes the
film to lose its antimicrobial action; this increases the dif-
fusion of the considered substances from the surface to the
bulk of the food, resulting in a low concentration at the
food surface, where they should be at their highest concen-
tration (Ozdemir & Floros, 2008). The zein, plasticizers and
other active agents added to ZNP-based composites play a
key role in controlling the final composite’s solubility. For
example, adding oleic and linoleic acids to a zein-based film
8 F. GARAVAND ETAL.
reduced the water absorption; the linoleic acid was shown
to be more efficient than the oleic acid in reducing the
water absorption, due to the linoleic acid’s stronger polym-
erization inside the film matrix (Budi Santosa & Padua,
1999). Another study demonstrated that adding ZNPs to a
zein-oleic acid composite film reduced its solubility, probably
owing to the zein hydrophobic property, as well as to the
high oleic acid volume percentage. A lower solubility was
observed in the case of zein-oleic acid composite films (≈10)
with respect to another film based on xanthan gum (≈13)
(Pena Serna & Lopes Filho, 2015). Similarly, according to
(Wu etal., 2022), the solubility of ZNPs included films was
lower than that of other films including chitosan and KGM.
When submerged in distilled water, the neat KGM films
swiftly vanished during the solubility experiments. The sol-
ubility index for KGM and ZNPs-KGM-CS composite film
were found ≈100 and ≈13, respectively. According to the
researchers, the hydrophilicity of carbohydrate-based mate-
rials (KGM and CS) encourages the film matrix to absorb
more water, resulting in comparatively poor moisture barrier
properties of neat films, whereas the hydrophobic nature of
ZNPs prevents the water absorption by the polysaccharide
matrix, lowering the solubility (Vahedikia et al., 2019). It
has been recommended in recent years that techniques such
as extrusion be used for protein-based materials since they
can induce their crosslinking, which results in increased
water resistance, but also enhanced toughness and brittleness
of the packing materials (Pommet, Redl, Guilbert, & Morel,
2005). One method of overcoming the poor mechanical
qualities of the protein-based material is to include a chem-
ical that will improve the water-resistance while still allowing
the material to be processed.
Color
Color is an important characteristic in food packaging since
it may have a direct impact on the acceptance of the product
by the consumers. In the L* a* b* color system, L* indicates
the lightness, and a* and b* are color coordinates, where + a*
is in the red direction, -a* is in the green direction, +b* is
in the yellow direction, -b* is in the blue direction, low L*
is dark, and high L* is light (Vicente, Cerqueira, Hilliou, &
Rocha, 2011). An investigation of the color characteristics
of corn distarch phosphate-zein bilayer films (C-Z) was
carried out. The addition of ZNPs resulted in a decrease in
the L, which indicated a darker film. This effect was mostly
attributable to the fact that zein has the ability to scatter
and refractive light in various ways. When it comes to pack-
aging light-sensitive goods, dark-colored films provide a
distinct advantage. Because of the yellow color of zein, it
considerably lowered the a values of C-Z bilayer films and
raised the b values, indicating that the films became yellow
with the integration of ZNPs (Sun et al., 2018). A similar
trend was observed in another study by L. Zhang et al.
Table 1. Summary of recent ndings on physical characteristics of ZNPs-based composites.
Composite
Water Vapor
Permeability
(WVP) Solubility
Color
Opacity
Contact
Angle ReferenceL* a* b*
ZNPs-Whey Protein Isolate 0.05A— — — — — — (Oymaci et al.,
2016)
ZNPs-Corn Distarch
Phosphate
0.42B—88.50 −2.74 17.32 — — (Sun et al., 2018)
ZNPs-Chitosan 1.57C—90.52 −1.75 11.59 — — (L. Zhang et al.,
2019)
ZNPs-chitosan
NPs-Cinnamon Essential
Oil
2.05D39.13 27.05 −14.22 19.00 1.87◊—(Vahedikia et al.,
2019)
ZNPs/Natamycin-loaded
casein-Gelatin
— — 85.35 −0.52 5.44 —≈122 (Mo et al., 2021)
ZNPs-Konjac
Glucomannan-Chitosan
2.78E13.18 — — — — 96.68° (Wu et al., 2022)
ZNPs-Chitosan 2.2A— — — — — — (Escamilla-García
et al., 2013)
ZNPs-Whey Protein Isolate —≈85 90.24 −2.18 18.49 — — (Tsai & Weng,
2019)
ZNPs-Hydroxypropyl
Methylcelluose
60.01F— — — — — 71.34° (Gilbert et al.,
2018)
ZNPs-Potato Starch-glycerol-
Olive oil
1.28G—92.59 −10.09 16.75 — — (Farajpour etal.,
2020)
ZNPs/Rutin NPs-Corn Starch 1.47H32.79 — — — — — (S. Zhang et al.,
2017)
ZNPs-Clove Essential
Oil-Potato Starch
5.57I— — — — — 91.10° (Alinaqi, Khezri, &
Rezaeinia, 2021)
ZNPs-Poly (Ethylene Oxide) — — 91.70 −3.85 26.48 20.62106.7° (da Rosa et al.,
2020)
ZNPs-Silicone
Molds-Propylene Glycol
—≈10 65.48 −1.91 32.00 ≈0.011□—(Spasojević et al.,
2019)
ZNPs-Oleic acid-Xanthan
Gum
—13.1 ———8.49√—(Pena Serna etal.,
2015)
ZNPs-Laponite — — — — — — ≈62° (Rouf, Schmidt, &
Kokini, 2018)
A g mm/m2 h kPa, B WVP*(10 − 12 g·cm−1 ·s−1 ·Pa−1), C WVP×1013 (kg·m·m−2 ·s−1 ·Pa−1), D g Pa−1 h−1 m−1, E WVP × 1011(g/m⋅s⋅Pa), F g mm m−2 h−1 kPa−1, G WVP(×10 − 7 g/m·h·Pa), H WVP/g mm−1 s−1 KPa−1, I WVP (g/
(m·s·Pa) × 10 − 10), ◊ AU nm/m, A mm−1, □ 1/m, √ mm–1
CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION 9
(2019). Without ZNPs, the film was clear, but when ZNPs
were added, the film took on a yellowish color. After mixing
with ZNPs in chitosan, the a and L values reduced, while
the b values increased. With the addition of ZNPs, the
brightness of the film progressively darkened and the yel-
lowness of the film grew (L. Zhang et al., 2019). When
compared to the control sample, the ZNPs-chitosan NPs
(CNPs)-cinnamon essential oil (CEO) films, including CNPs,
CEO, and a combination of CNPs and CEO, showed sub-
stantial changes in all color metrics. The original hue of
CNPs suspension, which altered the pale-yellow color of
zein films, reduced the redness and yellowness of zein films
(Vahedikia et al., 2019). Similarly, another composite film
based on natamycin-loaded casein/ZNPs-gelatin showed a
drop in L values and increment in both a* and b* values,
with increasing the NPs loading. The b* value increased
from 0.88 to 5.44, showing that the film went from colorless
to yellow over time. This might be related to the yellowish
nature of zein and natamycin. This suggests that adding
natural additives to ZNPs-based composite films can have
an impact (Mo etal., 2021). Apart from the natural yellow-
ish hue of the zein, several variables can impact the final
color metrics of ZNPs-based composites. The drying tem-
perature, pH, and other additives, for example, have been
shown to influence the color of composites. As a result,
further study is needed to better identify and comprehend
the color affecting elements during the composites manu-
facturing in order to evaluate how to make composites more
appealing to consumers.
Opacity
The opacity of a film indicates how much light travels
through it; this is significant in circumstances when light
incidence on food products must be controlled. In other
words, opacity is a measure of a film’s light barrier quality,
or its ability to shield a product from light-induced degra-
dation (Pena Serna etal., 2015; Vicente et al., 2011). Higher
opacity values indicate less transparency, hence the higher
the opacity values, the less transparent the film will be. The
opacity of composites is highly linked to the origin of the
components as well as the surface topography of the films
created during the drying process (Vahedikia etal., 2019).
The type of compounds used in ZNP films also has a sig-
nificant impact on the film’s ultimate opacity. For example,
in the same ZNP composite, it was found that adding CNPs
enhanced opacity while adding clove essential oil lowered
opacity. The addition of 4% CNPs to the zein films resulted
in a lit appearance with ≈10% increased opacity, but the
addition of CEO reduced the opacity of the neat zein film
from 1.56 to 1.23. The decline in opacity of zein films in
the presence of CEO was most likely attributable to the
uneven surface of zein films as a result of CEO droplets
migrating from the matrix to the surface during the drying
procedure (Vahedikia et al., 2019). Another research revealed
that combining ZNPs, oleic acid, and xanthan gum resulted
in a 60% increase in opacity. The film with only ZNPs and
oleic acid had an opacity of 10.8, whereas the opacity of
ZNPs/oleic acid/xanthan gum film was 13.1. The addition
of xanthan gum to a zein-oleic acid mix film reduced the
light transmission through the film, probably as a result of
the polysaccharide’s packed structure (Pena Serna et al.,
2015). The polymer matrices could absorb light in the ultra-
violet spectrum and, to a lesser extent, in the visible spec-
trum. Because many foods are unstable in the presence of
light, opaque materials are of great value in the food pack-
aging industry, as they provide a natural barrier to light
without the use of additives (Gennadios et al., 1996). For
instance, da Rosa and colleagues developed a film containing
ZNPs and poly (ethylene oxide) (PEO) and tested it for
opacity. When compared to the neat PEO control films, the
NPs enhanced the degree of opacity of the studied composite
films (da Rosa et al., 2020). As a result of the deeper hue
of zeins when added to the matrix, ZNPs composite can be
used to pack light-sensitive food goods. Additionally, further
study should be conducted to better understand the full
production process, from ingredient to finished product, in
order to assess the opacity index in its entirety.
Contact angle
Surface wetting property or water contact angle (WCA) is a
useful indicator of a film’s susceptibility to absorb moisture
or water, and so may be used to assess the film’s water barrier
capabilities. In general, the hydrophobicity of a surface can
be determined by measuring the contact angle of a water
droplet on the film surface. A WCA of less than 90° usually
implies a hydrophilic surface, whereas a WCA of more than
90° usually indicates a hydrophobic surface (Nephomnyshy,
Rosen-Kligvasser, & Davidovich-Pinhas, 2020). The contact
angle, on the other hand, is not time-independent and can
change in seconds or minutes owing to film expansion, liquid
composition changes, or liquid evaporation (Karbowiak,
Debeaufort, & Voilley, 2006; Kokoszka, Debeaufort, Lenart,
& Voilley, 2010). Initial contact angle measurements in this
scenario do not adequately reflect the surface’s hydrophilic/
hydrophobic characteristics. Estimating the rate of water
absorption into the film by evaluating the change in drop
volume may be important to better analyze the wetting prop-
erties of the films (Karbowiak et al., 2006; Kokoszka et al.,
2010). The impact of ZNPs addition on the WCA of the
composite film was described in a recent study on ZNPs/
KGM/chitosan composite. To compare with the composite
KGM/CS/ZNP, neat KGM, KGM/ZNP (KZ), and CS films
were produced. The pristine KGM film had the lowest WCA
of 33.45°, but the KZ and KGM/CS/ZNP films had a less
hydrophilic surface of 52.22° and a hydrophobic surface of
96.68°, respectively, which can be attributed to ZNPs’ very
hydrophobic nature, as well as to a NPs lotus effect. When
ZNPs are integrated into CS film, the WCA is reduced from
90.53° to 88.53°; this is likely owing to the increased rough-
ness generated by the etching action, validated by SEM with
solvent strength testing. As in prior work employing CS and
proteins, it was suggested that protein combinations in the
polysaccharide matrix controlled the surface hydrophilicity/
hydrophobicity of the final composite films (Wu etal., 2022;
Yu etal., 2017). A WCA of HPMC/ZNP composite film was
also explored and compared to the related neat films in
10 F. GARAVAND ETAL.
another study. Pristine HPMC films were found to be hydro-
philic, with an average water contact angle of 58.16°, which
was consistent with previous research (Kraisit, Luangtana-Anan,
& Sarisuta, 2014). The films got more hydrophobic as the
volume percent of ZNPs rose, resulting in higher contact
angles, albeit none of the composite films had a contact angle
more than 90° (Gilbert et al., 2018; Kraisit et al., 2014). As
the amount of ZNPs/CEO added to a starch-based composite
increased, the WCA climbed from 51.6° to 91.10°. Because
the zein and CEO are hydrophobic, it was hypothesized that
using these compounds in the matrix of starch-based
bio-nanocomposite films would boost their hydrophobicity
(Alinaqi et al., 2021). Another study on ZNPs-bacterial cel-
lulose nanofibrils (BCNs) composite reported interesting
findings on WCA: the ZNPs/BCN nanocomposites had
greater WCA values than neat bacterial cellulose, and the
trends in the WCA results were in good agreement with the
surface roughness (SR) data. Surface roughness is recognized
to be a crucial element in regulating the hydrophobicity of
material surfaces, and increasing surface roughness can
improve the surface hydrophobicity in general (Wan, Wang,
Ma, Sun, & Yang, 2017; Yoshimitsu, Nakajima, Watanabe, &
Hashimoto, 2002). As evident in Figure 2, the ZNPs addition
was associated with an increment in the film surface rough-
ness. The interspaces between the integrated ZNPs and the
particle aggregates might trap air, resulting in a solid-and-air
interface, potentially increasing the surface hydrophobicity of
the ZNPs/BCNs nanocomposites (Q. Li et al., 2020). The
surface hydrophobicity of nanocomposite films is linked not
only to interactions between the biopolymer and ZNPs, but
also to the nanomaterial’s own hydrophobicity (Sahraee,
Milani, Ghanbarzadeh, & Hamishehkar, 2017). Finally, the
surface of the biopolymer matrix is replaced in the structure
by NPs containing hydrophobic groups, and, thus, the increase
in the water contact angle is due to these hydrophobic groups.
Structural characteristics
FTIR
The development of new peaks or the shift of the old peaks
in Fourier transform infrared spectroscopy (FTIR) might
reveal information on the physicochemical environment of
functional groups inside a composite. The alterations might
indicate particular interactions between the polymer matrix
and the NPs, which could help researchers better understand
and create composite materials (Rouf et al., 2018). Recent
research compared corn distarch phosphate-zein bilayer films
(C-Z) to corn distarch phosphate-based films (C) and
zein-based films (Z) using FTIR analysis. The prominent
absorption band at 1715 cm−1 in the spectra of C and C-Z
films was due to C=O stretching vibration, while the absorp-
tion peak of the carbonyl group at 1715 cm−1 vanished in Z
films. The absorption band at 1448 cm−1 was attributed to
C—H stretching, the characteristics peak at 1647 cm−1 to the
stretching vibration of C=O (amide I), and the stretching and
bending vibration of C—N and N—H at 1538 cm−1 to the
stretching and bending vibration of CN and NH (amide II)
(Gu et al., 2013). The C and C-Z films had distinct peaks
between 1000 and 1200 cm−1, which were attributable to
starch’s C—O bond stretching. The new distinctive signal at
1589 cm−1 for C-Z bilayer films was attributed to N—H defor-
mation vibration, showing a strong connection between both
polymers and an improved intermolecular hydrogen bond,
comparable to previous results (Moreno, Cárdenas, Atarés, &
Chiralt, 2017). Furthermore, many unique peaks were iden-
tified at low wavenumbers, ranging from 1000–550 cm−1, which
were attributed to the skeleton’s C—CH3 and C—COO stretch-
ing vibrations (Lopez, Garcia, Villar, Gentili, Rodriguez, &
Albertengo, 2014). FTIR studies also indicated alterations in
C-Z bilayer films, which were mostly ascribed to the chemical
interactions between the starch and zein molecules via hydro-
gen bonding formation, with C-Z bilayer films retaining the
fundamental structure of C films (Sun etal., 2018). FTIR of
ZNPs/KGM/chitosan composite (ZNPs/KGM/CS) film was
reported by Wu et al. (2022). When compared to the spectrum
of pristine components, the ZNPs/KGM/CS composite films
showed the following changes: the absorption band around
3440 cm−1 shifted to a lower wavenumber, indicating intermo-
lecular hydrogen bonds between KGM and CS; the stretching
of carbonyl at 1730 cm−1 in KGM and the intensive band at
1080 cm−1 for neat CS disappeared; and the stretching of inter-
molecular hydrogen bonds at 1633 cm−1 in KGM shifted to
Figure 2. The trending link between WCA (b) and composite lm surface
roughness (SR) (a). The SR increases as the loading of ZNPs increases. The SR
and WCA have a positive association, meaning that as the SR rises, so does
the WCA. Adopted from Q. Li et al. (2020) with permission.
CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION 11
approximately 1550 cm−1, further suggesting that new hydrogen
bonds occurred between KGM and CS molecules in the ZNPs/
KGM/CS composite film (L. Wang, Mu, Li, Lin, Lin, & Pang,
2019; Wu et al., 2022; L. Zhang et al., 2019). When ZNPs
were added to a potato starch (PS) and olive oil emulsion
film, the —OH absorption band migrated to a lower wave-
number (3439 cm−1) with respect to neat PS. The infrared
absorption peak at 1542 cm−1 in the ZNPs FTIR spectrum,
ascribed to the amid II of zein, vanished in the PS/ZNPs
spectrum, indicating that ZNPs and film components may
interact with the film component (Farajpour etal., 2020). The
FTIR spectra of the bacterial cellulose (BC) nanofibrils
(BCNs)/ZNPs nanocomposites show all the distinctive peaks
of neat BC, as well as the adsorption peaks at 1654 and
1544 cm−1, which correspond to the characteristic bands of
amide I (C—O stretching) and amide II (N—H bending and
C—N stretching) in the zein protein, respectively. This shows
that ZNPs have been successfully integrated into the BCNs
network. With increased ZNPs concentration in the nano-
composites, the centered band at 3435 cm−1, attributed to the
stretching vibrations of the BCNs hydroxyl groups, moved to
lower wavenumbers. This was due to the creation of inter-
molecular hydrogen bonds between BCNs and ZNPs, resulting
in a reduction in the number of free hydroxyl groups on the
BCNs surface and, as a result, a drop in the intensity of the
centered band (Wan et al., 2017). The high compatibility and
uniformity were due to the interactions between ZNPs and
additives. Thus, the functional groups inside the composite
generate a unique environment that influences the composite’s
ultimate physicochemical characteristics, making FTIR analysis
a crucial aspect of the composite production process.
XRD
X-ray diffractometry (XRD) is a technique for studying the
phase composition of materials, as well as revealing the struc-
tural information of crystals. Further clarification of informa-
tion from the crystalline state, employing XRD spectra of the
films, is necessary to better understand the compatibility
between NPs and the film matrix (Wu etal., 2022). In a ZNP/
KGM/CS composite, the XRD analysis showed interesting
findings. Pristine KGM exhibited a nanocrystalline state with
a broad peak around 2θ=20°, neat CS with a diffraction peak
around 2θ=19.89°, which is usually due to strong intermolec-
ular interactions between CS chains via intermolecular hydro-
gen bonding; and neat zein two diffraction peaks around
2θ=8.89° and 19.5° (Gang et al., 2007; Wu et al., 2022). A
prominent distinctive peak at about 22° was detected, similar
to prior studies in which ZNPs were included inside a con-
tinuous CS matrix, which became flatter and broader with
both position and intensity adjustments, demonstrating high
compatibility between these two components. In addition,
when ZNPs were introduced into KGM matrices, the diffrac-
tion patterns of the ZNP/KGM/CS composite films showed
two peaks that were identical to those of the zein, although
with lesser intensity. These findings also pointed to the exis-
tence of strong hydrogen bonding interactions between the
hydroxyl groups of ZNPs and film matrices, implying that
numerous film components were compatible (Sun etal., 2018).
XRD patterns of neat zein and zein films containing CEO
and CNPs were also compared. Two strong peaks were
detected at 9° and 20° in all instances. These peaks had inter-
molecular intervals of 4.6 Å and 8.96 Å, respectively, which
were linked to the average skeleton distance in the α-helix
structure of protein and the distance of -inter helix peaks in
zein. Adding CEO alone had no effect on the location or
intensity of zein’s characteristic XRD peaks, suggesting that
CEO had no effect on the intermolecular or spectramulecular
structure of zein. In the case of films containing CNPs, how-
ever, an increase in the XRD peak intensity was noticeable,
particularly for the first peak, which was also displaced to
the lower 2θ°. Based on these findings, it is possible to spec-
ulate that the presence of CNPs had no effect on the helical
structure (α-helix), but that the molecular accumulation of
the matrix (interhelix peaks) was weakened, most likely due
to the interaction of CNPs with zein macromolecular chains
(Vahedikia et al., 2019). L. Wang et al. (2019) used KGM,
curcumin, and zein nanofibrils to develop a composite film.
They found that neat KGM film had a significant characteristic
broad peak centered at 2θ=20.3° range, indicating that KGM
is in an amorphous form. The backdrop of the zein nanofibrils
was diffuse, with two diffraction haloes at 2θ=8.87° and 17.3°.
Because the KGM and zein molecules fully intertwined, the
peak at 2θ=20.3° vanished. As the concentration of zein rose,
a peak at roughly 2θ=8.87° gradually emerged. The XRD
patterns of all the KGM/Cur/zein nanofibrils films were very
similar to the KGM film, demonstrating that KGM and zein
nanofibrils are quite compatible.
Mechanical characteristics
Mechanical properties are the primary requirements for food
packaging films. One of the main drawbacks of biodegradable
food packaging films is their low mechanical strength com-
pared to those made with a traditional petrochemical-derived
polymer. This weak strength of biodegradable packaging makes
it vulnerable to mechanical stress during the storage, process-
ing, handling, and transportation (Ebrahimi, Fathi, & Kadivar,
2019; Pająk, Przetaczek-Rożnowska, & Juszczak, 2019).
Therefore, researchers are working to reinforce the packaging
films, increasing their mechanical strength by the addition of
proper nanofillers. Recent studies reported that the incorpo-
ration of ZNPs within the polymer matrix of a packaging
film can improve its mechanical properties (S. Zhang et al.,
2017). TS, EAB, and YM are the three important factors that
describe the mechanical properties of the food packaging
films, as described in detail in the following paragraphs.
Tensile strength
TS refers to the maximum stress or stretch that a film can
withstand before breaking. In food packaging, different fac-
tors, especially the composition of biopolymer films, influ-
ence the TS (F. Garavand, Rouhi, Razavi, Cacciotti, &
Mohammadi, 2017). The incorporation of ZNPs into pack-
aging films has become a well-known strategy for enhancing
their mechanical properties, due to the creation of hydrogen
bonds. Farajpour etal. (2020) reported that the addition of
12 F. GARAVAND ETAL.
ZNPs into the potato starch-olive oil-based emulsion films
increased the density of these films, and, thus, their TS.
This characteristic is known as the Pickering effect, which
was also seen in other ZNPs-containing emulsion films.
ZNPs can create oil-ZNPs or glycerol-ZNPs interactions,
which help to impede the starch chain movement. Besides,
ZNPs formed hydrogen bonds with the starch matrix and
developed electrostatic interactions between its amino group
and the hydroxyl group of the starch matrix. Oymaci etal.
(2016) incorporated the ZNPs into WPI-based food pack-
aging films and evaluated the improvement of mechanical
and barrier properties (Figure 3). The study reported that
the incorporation of ZNPs at a ratio of 1.2 (w/w of WPI)
into the film significantly increased the TS of WPI films
from 2.5 MPa to 10.2 MPa. The incorporation of ZNPs in
WPI film allowed good stress transfer by attributing an
efficient interfacial interaction between the ZNPs and the
matrix. However, since zein has an isoelectric point at
around 6.2, it is very difficult to disperse the ZNPs in the
WPI solution, which makes the NPs unstable at pH 7 in
the film-forming solution and leads to aggregation. Another
study reinforced starch-based bio-nano-composite films with
electro-sprayed ZNPs (EZNs) and reported that mechanical
properties such as TS were greatly affected by the EZNs-CEO
levels in the starch matrix (Alinaqi etal., 2021). The TS of
the starch-based films increased up to a certain EZN-CEO
concentration (10%), whereas higher levels of incorporation
(10%-15%) significantly decreased the TS. Gilbert et al. (Q.
Li et al., 2020) examined the barrier and mechanical prop-
erties of hydroxypropyl methylcellulose and ZNPs composite
films and reported that the ZNPs incorporation increased
their TS. Similar influences on composite TS have been
previously observed in ZNPs-loaded whey protein isolate
films. Enhanced TS of composite films with increased vol-
ume fraction of filler particles indicates a significant influ-
ence on particle-matrix adhesion and nanometer-scale size
of ZNPs. While low adhesion between matrix and NPs typ-
ically reduces the TS of composite films, higher
particle-matrix adhesion can increase the composite TS
(Dekkers & Heikens, 1983). Hua et al. (2021) reported that
the incorporation of clove essential oil-loaded zein-sodium
caseinate NPs (C/ZN) increased the TS of chitosan-based
composite films. Usually, the TS is related to the microscopic
network structure and film’s intermolecular force. Since the
NPs were cross-linked with chitosan, reducing the mobility
of the film structure, a reinforcement of the film network
structure was revealed (Nisar, Wang, Yang, Tian, Iqbal, &
Guo, 2018). Besides, the hydroxyl groups presented in both
chitosan and C/ZN NPs were responsible for making strong
interaction between them which led to a higher TS of the
composite films.
Elongation at break (EAB)
EAB, also known as tensile EAB, refers to the ratio between
the increased length and initial length after breakage. It
generally indicates the flexibility of films and has an inverse
relationship to the TS (Jafarzadeh, Rhim, Alias, Ariffin, &
Mahmud, 2019). Recent studies reported that the incorpo-
ration of ZNPs in food packaging film improves the flexi-
bility of the packaging films. Gilbert etal. (2018) evidenced
a decrease in the EAB as a result of incorporating ZNPs in
hydroxypropyl methylcellulose films. However, the incorpo-
ration of low-level loading (1 and 3 wt%) of ZNPs into the
potato starch-olive oil-based emulsion film increased the
EAB values (Farajpour et al., 2020). In this case, a plasti-
cizing effect led to an increase in spacing and mobility
between the chains (Oymaci etal., 2016). Nonetheless, when
the ZNPs content increased to (9 wt%), the higher amount
of NPs restricted the chains, and the EAB value was reduced
compared to the elongation of neat emulsion film. Moreover,
Hua etal. (Hua etal., 2021) reported that the incorporation
of CEO-loaded zein-sodium caseinate NPs (C/ZN) increased
the EAB value of the composite films compared to the neat
chitosan films enriched with different concentrations of
orange peel essential oils. Q. Li et al. (2020) also showed
that the ZNPs incorporation in bacterial cellulose nanofibrils
increased the EAB value of the nanocomposites with respect
to the neat bacterial cellulose nanofibrils films.
Young modulus
YM demonstrates the stiffness of a film, and it is unswerv-
ingly connected to the TS (Jafarzadeh, Alias, Ariffin, &
Mahmud, 2017). Generally, YM illustrates how easily a film
can stretch and deform and is determined by the slope of
stress-strain at the elastic limit. The strength of nanocom-
posites is affected by various factors, including the nature
of the polymer, type and concentration of the plasticizer,
chemical cross-linking, and macrostructure. Recent studies
revealed that the incorporation of ZNPs in food packaging
films could improve the YM value of the packaging com-
posites. Alinaqi etal. (2021) reported that the incorporation
of EZN-CEO up to 10% in the starch-based bio-nano-com-
posite films improved the YM and TS of starch-based food
packaging film, but higher levels of incorporation signifi-
cantly reduced them. Gilbert et al. (2018) stated that the
relative stiffness of HPMC films was greatly increased by
the addition of ZNPs. Wu et al. (2022) also demonstrated
that the ZNPs incorporation into a different
polysaccharide-based matrix, such as KGM and chitosan,
film increased the YM values compared to the neat films.
Figure 3. Tensile strength (TS) and elongation at break (EAB) value of WPI
lms as a function of the ZNPs concentration. Adapted from Oymaci et al.
(2016) with permission.
CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION 13
Morphological characteristics
The microstructural properties of the food packaging films
greatly affect their mechanical and barrier properties,
which, thus, can be easily modified by changing the micro-
structure. Recent studies revealed that the ZNPs incorpo-
ration in the food packaging films significantly improved
their morphological characteristics, depending on the ZNPs
performance in the packaging films on the NPs size, shape,
surface area, and inter-particle interactions. SEM, AFM,
and TEM are generally used to demonstrate the morpho-
logical characteristics of the food packaging films or
nanocomposites.
Scanning electron microscopy (SEM)
Generally, SEM uses a high-frequency electron beam on the
surface and reveals information about the film’s morphology,
crystalline structure, and materials orientation, and is applied
for studying the surface and cross-sections of the packaging
films. The researchers commonly use the SEM investigation
to understand the improved properties and uniformity of
the composite film components distribution. A smooth and
uniform microstructure testifies the homogeneity of the
obtained composite film. The polymer nanocomposites are
characterized by significant interfacial properties between
the polymer matrix and the nanofillers such as interactions
or adhesion at the interface (Zare, Rhee, & Park, 2017). A
high amount of interfacial properties develops another phase
around the NPs, demonstrating the benefits of the nano-
composites compared to conventional micro-composites
(Issa, Schimmel, Worku, Shahbazi, Ibrahim, & Tahergorabi,
2018). However, the high surface area of NPs and the robust
interaction between the particles promote their aggregation,
which has a negative impact on the properties of the nano-
composites and reduces the potential improvement of the
mechanical behavior. Farajpour et al. (2020) evaluated the
morphological properties of potato starch-olive oil-based
nanocomposites incorporated with ZNPs by using SEM
(Figure 4). The SEM results showed that the addition of
olive oil in the starch films increased their roughness and
induced discontinuities in the film matrix (Figure 4b).
However, the incorporation of ZNPs made the film’s surface
smoother (Figure 4c). The reason behind the smoothness
of the ZNPs incorporated films was that the ZNPs worked
as an emulsifier (Pickering effect) and helped the dispersion
and stabilization of oil droplets in the matrix (Farajpour
etal., 2020). Besides, the cross-section structure of the neat
control film (Figure 4d) showed a continuous, compact, and
homogeneous phase, with no pores or air bubbles. In con-
trast, the samples containing olive oil without ZNPs (Figure
4e) showed pores of different sizes. However, these pores
were diminished after adding ZNPs (Figure 4f), obtaining
a homogenous and dense structure. Oymaci et al. (2016)
also reported a smooth, non-porous, and homogenous struc-
ture in the ZNPs-loaded WPI films compared to the control
film. SEM micrographs reported in Wu et al. (2022) showed
that polysaccharide-based films with or without ZNPs incor-
poration had smooth surfaces without having any holes or
cracks. In addition, the cross-sectional morphology of
ZNPs-loaded polysaccharide-based films evidenced a com-
pact matrix, characterized by a good ZNPs distribution, due
to the slower dehydration rate. In another study, neat chi-
tosan film was found tight in the cross-section structure
without any visible cracks or apparent particles, but after
the addition of C/ZN NPs, micro-voids were seen in the
chitosan composite films with rough surface (Hua et al.,
2021). Moreover, a smooth, non-porous, and homogeneous
structure was found in the kappa-carrageenan-loaded gelatin
film. In contrast, SEM micrographs of gelatin films incor-
porated with thymol-loaded ZNPs showed a reduction in
smoothness and an irregular surface (Yavari Maroufi,
Ghorbani, & Tabibiazar, 2020).
Atomic force microscopy (AFM)
AFM is used to investigate the film’s surface topography
and roughness and visualize the dispersion of the NPs in
the composite films. Farajpour et al. (2020) evaluated the
surface roughness of potato starch-olive oil-based nano-
composites incorporated with ZNPs by using AFM and
reported that the surface roughness of the nanocomposites
was minimized with the addition of ZNPs in the emulsion
film and this AFM derived results was also supported by
the SEM microstructural evaluation outcome. In addition,
the study also found a relationship between contact angle/
water vapor permeability and surface roughness and
reported that a reduction in surface roughness in the com-
posite films reduced the contact angle/water permeability.
A similar result was also reported by Ghanbarzadeh and
Oromiehi (2008) for the whey protein and edible zein
films. Moreover, AFM analysis in Oymaci et al. (2016)
evidenced a globular microstructure of neat WPI films.
Still, a homogeneous distribution of ZNPs was observed
in the ZNPs-loaded WPI composite films. The NPs in the
composites were individually scattered, and the phase dif-
ference between the WPI matrix and ZNPs was evidently
identifiable. In addition, Gilbert et al. (2018) performed
the AFM topographical analysis of HPMC films and
reported that the ZNPs incorporation within the HPMC
films was correlated to an increased number of observable
quite dispersed particles in the composite films. Besides,
AFM images did not show any significant differences in
the surface roughness except for some composites contain-
ing a higher amount of added ZNPs.
Transmission electron microscopy (TEM)
TEM provides detailed information about the structure, micro-
structure, material characterization, and chemical composition.
Hu etal. (2015) evaluated the morphology of surfactant-coated
ZNPs with fairly uniform dimensions and a diameter ranging
from 50 to 100 nm by using TEM. The study also found similar
morphological results for surfactant-coated ZNPs when they
were coated with alginate. Moreover, the TEM micrograph of
the nanocomposite prepared by bacterial cellulose nanofibrils
and ZNPs (Q. Li et al., 2020) showed that the ZNPs were
uniformly adhered to the BCNs aqueous suspensions by inter-
molecular hydrogen bonds.
14 F. GARAVAND ETAL.
Thermal characteristics
Thermal analysis methods identify suitable processing con-
ditions, application, and polymer chain structure. The ther-
mal characteristics of nanocomposites are determined by
differential scanning calorimetry (DSC) and thermogravi-
metric analysis (TGA).
Dierential scanning calorimetry
DSC is widely used to confirm the entrapment of an active
compound in a polymer matrix and analyze the possible
interactions that occur between the active compound and
the polymer with thermal transitions. DSC is used to obtain
information about materials, including polymers and
organic-inorganic composites. The energy changes during
continuous heating and cooling can be obtained from the
DSC analysis, which enables the determination of the
transition temperatures such as crystallization temperature
(Tc), melting temperature (Tm), and glass transition tem-
perature (Tg). da Rosa et al. (2020) evaluated the thermal
characteristics of PEO bioactive nanocomposite films func-
tionalized with ZNPs by DSC and reported a direct rela-
tionship between the melting enthalpy and the crystallinity.
The increase of NPs in the polymeric matrix promoted a
reduction of melting enthalpy and films crystallinity.
According to Sánchez-Soto, Ginés, Arias, Novák, and
Ruiz-Conde (2002), many factors, such as molecular mass,
DSC scan speed, and polymer kind, can affect the thermal
properties of PEO. M. S. Park and Kim (2002) attributed
this behavior to the variability in crystallization kinetics.
This was explained by the fact that rapid crystallization of
PEO requires little energy for crystal formation. However,
with the incorporation of NPs in films, the PEO crystalli-
zation becomes slower, increasing the crystallization enthalpy.
Figure 4. a) SEM micrograph of the control lm. b) SEM micrograph of the potato starch lm incorporated with 10% olive oil. c) SEM micrograph of the potato
starch/10% olive oil emulsion lms loaded with 9% ZNP. d) Cross-section structure of the neat lm. e) Cross-section structure of the potato-starch lm con-
taining olive oil without ZNPs. f) Cross-section structure of the potato-starch lm incorporated with ZNPs. Adopted from Farajpour et al. (2020) with
permission.
CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION 15
Xiang, Xia, Wang, Wang, Wu, and Ni (2021) evaluated
the thermal properties of KGM-based composite film using
the DSC curve and reported that the incorporation of the
NPs brings a significant change in the composite films
(Figure 5). In Figure 5, KGM film presented a noticeable
endothermic peak at 79.8 °C that is linked to its
glass-transition temperature (Tg), in agreement with a pre-
vious study (K. Wang et al., 2017). KZN, KNC, KNT, and
KNS showed Tg at 120.9, 111.7, 110.3, and 109.2 °C, respec-
tively. The higher the glass transition temperature, the
higher the temperature needed to break the molecular
chains, suggesting enhanced molecular interaction (Ahmad,
Hani, Nirmal, Fazial, Mohtar, & Romli, 2015). The inter-
molecular interactions between KGM and the NPs may
change the original crystalline structure, and molecular
network, which is the reason behind the lower Tg value of
KGM compare to the others. Zuo, Chen, Li, He, Li, and
Wu (2020) reported that the incorporation of nano-SiO2
in the polylactic acid-grafted-bamboo fibers composites
improved the crystallinity of the film, and the increased
crystallinity made the composites better heat resistance.
Moreover, all the blend films exhibited a single Tg, which
indicated good miscibility between the film components.
In addition, the DSC curves allowed to identify the thermal
decomposition temperature (Td), which was 318.9, 334.5,
323.1, 322.8, and 319.0 °C for KGM, KZN, KNC, KNT, and
KNS films, respectively. Therefore, a change in Tg and Td
was observed, which indicates that the incorporation of the
NPs enhanced the heat resistance of the film, where KZN
had the highest thermal stability because of its strong
molecular interaction.
Thermo-gravimetric analysis (TGA)
The thermo-gravimetric analysis describes the relationship
between the weight change and the temperature. In a con-
trolled atmosphere, the amount of mass decreases and the
time/temperature relationship can provide information about
the thermal and oxidative stability of materials. In addition,
the composition of materials can also be identified by using
the TGA thermogram. Yavari Maroufi etal. (2020) evaluated
the thermal characteristics of gelatin-based films prepared
using covalent interaction through dialdehyde
kappa-carrageenan (DAK-car) and thymol-loaded ZNPs con-
tent. The obtained result was related to the generation of
intermolecular chemical cross-linking with covalent bonds
amongst the amino groups of GEL and the dialdehyde groups
of DAK-car (Akrami-Hasan-Kohal, Ghorbani, Mahmoodzadeh,
& Nikzad, 2020; Yavari Maroufi etal., 2020; Zuo etal., 2020).
It indicated that by creating aldehyde groups and forming
Schiff-base bonds, thermal stability was achieved for GEL.
Wu et al. (2022) evaluated the production and character-
ization of composite films incorporated with ZNPs based on
the complexity of the continuous film matrix. Results indi-
cated that ZNPs incorporation enhanced the thermal stability
of all matrices, either with a single component or in combi-
nation. In addition, when evaluated with the TGA results,
the KCZ composite film with ZNPs incorporated into the
KC composite matrix exhibited greater remaining mass con-
tent when compared to the composite films, where the com-
bination of ZNPs was introduced into a single-component
film matrix, e.g. KZ and CZ composite films. Generally,
improved thermal stability corresponded to the higher deg-
radation temperature and lower mass loss, indicating a more
significant interaction between ZNPs and the film network
matrix and a higher crystallinity, which could also be sup-
ported by the FTIR and XRD analyses (Y. Liu et al., 2019).
Q. Li etal. (2020) studied the thermal properties of the
nanocomposites prepared with BCNs and ZNPs by using
TGA. TGA curves of pure BC and BCNs-ZNPs nanocom-
posites showed that all the samples slightly lost weight at
100 °C because of the moisture loss from the membrane.
Under elevated temperatures, the neat BC showed two
decomposition steps: the first rapid decomposition step,
which occurred at 280-365 °C, was attributed to the degra-
dation of cellulose; when the temperature went beyond
400 °C, the full decomposition of BC was achieved and
formed various pyrolysis products (W.-H. Gao, Chen, Yang,
Yang, & Han, 2010). The BCNs-ZNPs nanocomposites
showed a similar thermal decomposition. However, the
nanocomposite residues also increased with the increase of
ZNPs content. The residues were increased from 14.8% to
20.3% for neat BC and 40% for BCN-ZNP nanocomposites.
The study also reported a lower weight loss rate of
BCNs-ZNPs nanocomposites compared to pure BC.
Therefore, the study concluded that the BCNs-ZNPs nano-
composites showed better thermal stability than the neat
BC film and which can be attributed to the successful incor-
poration of ZNPs into the BCNs network.
Safety issues and gastrointestinal fate of zein
nanoparticles
Along with the increased use of nanomaterials in different
industries, the concern regarding their safety, biocompati-
bility, and toxicity in food materials has grown. Nanosized
materials generally possess exclusive characteristics do not
present in their natural form. Several physical, chemical,
Figure 5. DSC curves of KGM, KNZ, KNC, KNT and KNS lms. Adopted from
Xiang et al. (2021) with permission.
16 F. GARAVAND ETAL.
and biotechnological parameters can be involved during the
transition of organic materials into NPs with sizes ranging
from 1 to 100 nm (Garavand et al. 2022). Therefore, it is
necessary to understand the behavior of these nanomaterials
during their development and application (Sandoval, 2009).
Due to the high surface volume ratio of nanosized materials,
they may exhibit inimitable physicochemical properties such
as high solubility, toxicity, high strength, magnetic proper-
ties, diffusivity, optical properties, color, and thermodynamic
properties (Singh, Shukla, Kumar, Wahla, Bajpai, & Rather,
2017; J. Zhang, Hu, Du, Cao, Wang, & Yuan, 2020).
Some parameters of nanomaterials, such as the particle
size, zeta potential, surface groups, and aggregation state,
influence the toxicity of these materials (Jain, Kumar Mehra,
& K Jain, 2015). As mentioned earlier, due to their small
size and high surface area, NPs can easily pass through
tissues or cell membranes, and, thus, they can destruct the
biological systems. Moreover, the behavior of NPs in the
gastrointestinal (GI) tract is reported to be different from
that of the conventional particles and can induce some
allergic and toxic reactions (Pathakoti, Manubolu, & Hwang,
2017). Due to the possible accumulation of NPs in human
tissue or organs and also their possible toxic reactions with
other compounds, it is necessary to evaluate their recom-
mended daily allowance (RDA) or tolerable upper intake
levels (J. Chen & Hu, 2020).
Protein-based NPs such as ZNPs are fabricated using
different processes of denaturation, injection, molding, and
spray drying (Matalanis, Jones, & McClements, 2011). The
stability of these NPs during processing or storage of food
is affected by different intrinsic factors such as pH, ionic
strength, water activity, temperature, and the presence of
proteases (Gonçalves, Martins, Duarte, Vicente, & Pinheiro,
2018). Moreover, processing and storage conditions such as
thermal processing, chilling, freezing, drying, and homog-
enization are the other parameters affecting the stability and
physiological properties of NPs (X. Liu, Zhang, Sohal, Bello,
& Chen, 2019). Due to the expectation of similar transpor-
tation behavior for proteins and protein-based NPs in the
GI tract, there are few studies that investigate in vitro and
in vivo transportation of NPs in food throughout the GI
tract (X. Liu etal., 2019). In general, these NPs will undergo
the degradation process throughout the GI tract, due to the
presence of proteolytic enzymes and pH variations from the
oral cavity to the intestine (Alger, Momcilovic, Carlander,
& Duncan, 2014). The gastric emptying, degradation, and
aggregation of protein-based NPs in the GI tract by altering
the size of particles and surface properties initiate the deg-
radation of these particles (Marciani etal., 2008).
Different chemical and biological methods are used to
study the carcinogenicity, teratogenicity, reproductive tox-
icity, developmental toxicity, and neurotoxicity of nanoma-
terials. In vitro models of the GI tract, such as monocultures,
co-cultures, or in vivo models, including oxidative/nitro-
sative stress assessments of NPs, simulating the GI tract
passage, are some of the methods for the pathophysiolog-
ical study of nanomaterials (X. Liu et al., 2019). Although
the metal and metal oxide NPs can be traced in food
systems using different methods such as TEM, SEM, or
splCPMS (Single particle-inductively coupled plasma-mass
spectrometry) methods, the organic-based NPs such as
protein-based are more difficult to monitor and more
sophisticated analytical methods are required to study them
in the food system (Öztürk Er, Dalgıç Bozyiğit, Büyükpınar,
& Bakırdere, 2022).
Several studies have evaluated the food allergy aspects
of diets based on maize and also the related celiac diseases
(Cabrera-Chávez etal., 2009; Johnson, Labrooy, & Skerritt,
2008; Krishnan, Jang, Kim, Kerley, Oliver, & Trick, 2011;
Lee, Benmoussa, Sathe, Roux, Teuber, & Hamaker, 2005;
Pasini et al., 2002). The immunogenicity of ZNPs in mice
after two intramuscular injections within 12 weeks intervals
was reported for the first time by Hurtado-López and
Murdan (2006). F. Li et al. (2019) reported that the immu-
nogenicity of ZNPs in subcutaneously injected mice samples
was influenced by the NP’s size. However, some studies have
investigated the in vivo antigenicity of the zein which might
contribute to provoking an immune response in presence
of zein and form the anti-zein antibodies which hinder it’s
in vivo application (Abdelsalam et al., 2021). The in vivo
immunogenicity of ZNPs in female BALB/c mice was studied
by F. Li et al. (2019). These authors studied the effect of
particle size, dose, and inoculation routes on the in vivo
antigenicity of NPs. The results showed that the size of NPs
had no influence on the zein-specific Immunoglobulin-G
antibody titers. A higher IgG antibody titers and inflamma-
tory cell infiltration were observed for samples from intra-
muscular ZNPs injected than those from subcutaneous
injected samples with the same dose of NPs. The predom-
inant Th2-type immune response was observed for both
administration routes during IgG subtype assays. A system-
atic recall immune response was shown for mice samples
after week 50 by a significant increase in specific IgG titer
at all dose levels. Samples of those subjected to subcutane-
ously injection showed a delayed decrease in IgG antibody
level compared to the intramuscular injection groups in all
dose levels.
Luo, Wang, Teng, Chen, Sun, and Wang (2013) developed
ZNPs stabilized in casein and studied their stability in cell
culture medium and HBSS buffers. These researchers
reported that ZNPs had no cytotoxic activity for Caco-2
cells during 27 h of the study. The stabilization of NPs into
caseinate significantly improved the cell uptake of NPs in
concentration and time-dependent approaches and enhanced
the epithelial transport through Caco-2 cells monolayer. The
cell uptake of zein-casein NPs under blocking conditions of
4 °C, sodium azide, and colchicine showed an
energy-dependent endocytosis procedure (Luo, Teng, Wang,
& Wang, 2013). While these studies help to understand the
potential toxicity of zein NPs, the intramuscular route of
administration or cell culture cytotoxicity tests used in these
studies limits direct comparison to oral ingestion.
Reboredo, González-Navarro, Martínez-Oharriz,
Martínez-López, and Irache (2021) evaluated the oral drug
delivery possibility of polyethylene glycol (PEG)- ZNPs.
These authors reported the mean size of around 200 nm for
nanocarriers with the negative zeta potential. Coating ZNPs
with PEG reduced the hydrophobicity of nanocarriers. The
CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION 17
mobility of nanocarriers in pig intestinal mucus is signifi-
cantly increased by the hydrophobicity of PEG-coated NPs.
In animal models, PEG-coated NPs were able of crossing
from the mucus mesh and reach the epithelium, while
uncoated NPs retained in the protective mucus layer. These
authors suggested that PEG-coated ZNPs can be used to
increase the oral bioavailability and biological activity of
compounds with low permeability properties. Mucosal per-
meability of ZNPs and ZNPs-coated with a Gantrez®
AN-thiamine conjugate (GT) was evaluated by Inchaurraga
etal. (2019). The results showed that the diffusivity of ZNPs
in pig intestinal mucus increased by 28 times after coating
the NPs. In vivo analysis also confirmed that uncoated NPs
were restricted to the mucus layer while coating them
increased their permeation to the intestinal epithelium.
Mucoadhesive studies also revealed that ZNPs accumulated
in the stomach of animals while the mucus-penetrating
ZNPs showed to leave the stomach quicker to access the
small intestine of animals.
Conclusions
Synthetic petrol-based packaging composites are still dom-
inating the food packaging industry due to the economic
issues and their excellent performance. On the other hand,
finding appropriate alternatives instead of synthetic poly-
meric composites is investigated by various researchers and
manufacturers because of increasing pollution concerns,
waste disposal, and human health. The expansion in aware-
ness about environmental issues has encouraged the food
and nutraceutical manufacturers to develop biopolymer-based
edible films and coatings as promising green alternatives to
petroleum-based packaging materials. Also, alongside edible
packaging materials; these films serve different capacities
such as delivery vehicle for antimicrobials, antioxidants,
nutraceuticals, and other functional ingredients. These films
don’t thoroughly bargain the desirable physical and mechan-
ical attributes as packaging materials for extensive commer-
cial uses. Reinforcement of these biopolymers by ZNPs could
enhance their further applications in industrial scales by
modifying their structural pattern to obtain biopolymers
with improved mechanical, physical, barrier, and thermal
characteristics. However, the further use of ZNPs-based films
and coatings would need a comprehensive investigation on
their composition, interactions, the effect of various addi-
tives, processing parameters, safety aspects, etc. Concerning
the safety aspects, indeed, it could lead to potential health
hazards, ascribed to the higher ZNPs reactivity, due to the
reduced size and incremented specific surface area, the
migration of nanomaterials into the food from a package,
and consumer exposure to NPs. It should be noted that the
advanced techniques for using more environmentally friendly
solvents, novel extrusion techniques, and new developments
in compounding ZNPs with other natural products, in addi-
tion to the unique characteristics and properties of zein,
have opened a vast new area of research for the development
of zein and ZNP-based materials and their potential appli-
cations in the food and nutrition industries.
Disclosure statement
No potential conict of interest was reported by the authors.
Funding
e author(s) reported there is no funding associated with the work
featured in this article.
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