Carbohydrate Polymers 195 (2018) 631–641

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Effect of pH and TPP concentration on chemico-physical properties, release T

kinetics and antifungal activity of Chitosan-TPP-Ungeremine microbeads
Arash Moeinic, Alessio Cimminoc, Giovanni Dal Poggettoa, Mariaelena Di Biaseb,
⁎
Antonio Evidentec, Marco Masic, Paola Lavermicoccab, , Francesca Valeriob, Antonella Leoned,
⁎
Gabriella Santagataa, , Mario Malinconicoa
a
Institute for Polymers, Composites and Biomaterials, National Research Council, Via Campi Flegrei 34, 80078 Pozzuoli, Napoli, Italy
b
Institute of Sciences of Food Production, National Research Council, via Amendola 122/0, 70126 Bari, Italy
c
Department of Chemical Sciences, University of Naples “Federico II”, Complesso Universitario Monte S. Angelo, Via Cintia 4, 80126 Napoli, Italy
d
Institute of Science of Food Production, National Research Council, Via Provinciale Lecce Monteroni, 73100 Lecce, Italy

A R T I C LE I N FO A B S T R A C T

Keywords: In this study, chitosan based microbeads containing Ungeremine, an antimicrobial alkaloid particularly active
Chitosan against Penicillium roqueforti, a filamentous fungus responsible of the bakery products deterioration, were pre-
Ionic crosslinking pared by external gelation by using sodium tripolyphosphate (TPP) as crosslinking agent. The stability of the
Microbeads beads, as well as the loading efficiency of the bioactive molecule, were assessed at different pH and TPP con-
pH dependence
centrations resulting particularly enhanced at low pH. All the microbeads evidenced antimicrobial activity
Antifungal property
against Penicillium roqueforti. The release kinetics of Ungeremine was tailored by opportunely modulating pH and
Controlled release
TPP concentrations. Morphological analysis evidenced the improvement of the structural crosslinking density of
microbeads including Ungeremine and spectroscopic analysis emphasized the active participation of
Ungeremine to the crosslinking process occurring between chitosan and TPP. Finally, thermogravimetric ana-
lysis confirmed the increasing of free volume in three-dimensional networks and their liability to thermal de-
gradation.

1. Introduction
Foodstuffs are subjected to a post processing microbial spoilage
risks, mostly involving the mold growth, representing a serious and
economic drawback for both manufacturers and consumers (Saranraj &
Geetha, 2012). One of the most common filamentous fungus associated
to the food deterioration is the Penicillium roqueforti (PR), responsible,
inter alia, of the unsafe mycotoxins production (Cakmakci et al., 2015;
Ravimannan, Sevvel, & Saarutharshan, 2016). This fungal species,
especially resistant to weak acid preservatives, can contaminate several
foodstuffs but in particular cereals, bread and pastries, meat, eggs and
cheese (Dantigny, Guilmart, & Bensoussan, 2005). In particular, bakery
products are important staple food in many countries. They represent
valuable source of nutrients, even if, during shelf-life, many different
fungi are able to simultaneously colonize the substrate, by determining
its spoilage (Banwart, 1989; Filtenborg, Frisvad, & Thrane, 1996).
Therefore, due to the increasing global consumers demand, the devel-
opment of novel, friendly and ecologically sustainable preservation
techniques, aimed to extend the shelf life of bakery products, by
hampering the mold growth, represents a worthy output for the bakery
industry, from the safety and economic point of views. The current
research is focusing the attention on the use of biopreservatives, in-
cluding antifungal plant extracts, such as Ungeremine.
Ungeremine (identification code: Un) (Fig. S1) is a betaine type
alkaloids isolated from Pancratiummaritimim L., a well know species of
Amaryllidaceae (Abou-Donia et al., 1992). In particular, this alkaloid is
an antifungal agent, mainly active against PR, responsible of the food
spoilage previously discussed (Valerio et al., 2017). The applicative
potentialities of Ungeremine are strictly correlated to its stereo-
structure, easy achievement and source renewability (Cimmino, Masi,
Evidente, Superchi, & Evidente, 2017). Detailed information related to
Ungeremine are reported in Supplementary data (SD), introduction
section.
Nevertheless, free Ungeremine is not suitable to be handled; so, it is
necessary to select a carrier system able to vehicle the bioactive mo-
lecule, by modulating its releasing kinetics. Actually, as far as we know,
no attempts have been performed in this regard. The encapsulation in a
biodegradable polysaccharide matrix, such as chitosan (identification

⁎
Corresponding authors.
E-mail addresses: paola.lavermicocca@ispa.cnr.it (P. Lavermicocca), santagata@ipcb.cnr.it (G. Santagata).

https://doi.org/10.1016/j.carbpol.2018.05.005
Received 16 February 2018; Received in revised form 13 April 2018; Accepted 2 May 2018
Available online 04 May 2018
0144-8617/ © 2018 Elsevier Ltd. All rights reserved.
A. Moeini et al.
code: Ch) could represent a suitable way by which Un, from an aqueous
solutions could be converted to solid forms, more easily to handle and
to tailor, depending on the peculiar application (Trifković et al., 2014).
Chitin and chitosan are biopolymers supporting the structural compo-
nent of numerous living organisms, such as crustacean, insect exoske-
leton and the cell walls of fungi and some algae. In particular, chitosan
is obtained from the partial N-deacetylation of chitin; so chitosan is a
linear copolymer based on linked β-1,4-glucosamine and N-acetyl glu-
cosamine residues at different extents, depending on the deacetylation
degree (Ravi Kumar, 2000). Differently from chitin which is insoluble
in common solvent for less than chemical modifications (Kumar,
Muzzarelli, Sashiwa, & Domb, 2004), the presence of a prevailing
number of free 2-amino-2-deoxyglucose units in chitosan, showing a pK
of 6.3, allows its solubilization in aqueous acidic solutions, where the
amine groups NH2 are easily protonated in NH3+. The wide and ver-
satile range of chitosan functions and applications are therefore related
to its cationic polyelectrolyte behavior, which is unique among poly-
saccharides and natural polymers (Tolaimate et al., 2000). These pe-
culiar and worthy properties, together with the biodegradability, bio-
compatibility, non-toxicity and filmogenicity features, are responsible
of the wide chitosan applications in the food industry as antimicrobial
and antioxidant agent (Ferreira, Nunes, Castro, Ferreira, & Coimbra,
2014; Harris, Lecumberri, Mateos-Aparicio, Mengíbar, & Heras, 2011;
Valdés, Ramos, Beltrán, Jiménez, & Garrigós, 2017), in biomedical and
pharmaceutical fields as polymeric drug carrier (Catanzano et al., 2015;
Muzzarelli, 2010; Straccia, Romano, Oliva, Santagata, & Laurienzo,
2014), in agricultural and environmental areas as protective film and
metal ion based complexes (Deshpande, Dapkekar, Oak, Paknikar, &
Rajwade, 2017; Malinconico, Santagata, & Schettini, 2006). Under the
acidic conditions, the resultant positive charge allows to formulate
microspheres by ionic crosslinking of the polymeric matrix with specific
polyanions, such as multivalent salts. In particular, sodium tripoly-
phosphate (identification code: TPP) is widely used because of its non-
toxic property and quick gelling ability (Fwu-Long, Shyu, Lee, & Wong,
1999; Gan, Wang, Cochrane, & McCarron, 2005). It can form gel by
means of ionic interaction between positively charged amino groups of
chitosan NH3+, and its negatively charged counterions, OH−,
HP3O104− and P3O105−. The physical crosslinking of chitosan with TPP
represents a valuable method for obtaining micro-nanoparticles with
controlled size and favorable biomolecule encapsulation efficiency
(Cho, Chun, Kim, & Park, 2014). In addition, reversible physical cross-
linking by electrostatic interaction, instead of chemical cross-linking,
avoids the toxicity of reagents and other undesirable effects (Ghanem &
Ghaly, 2004; Shu & Zhu, 2000). Ungeremine structure is typical of a
zwitterion, with free azomethine cation and negatively charged phenate
group (see Fig. S1). Hence, it could be hypothesized the development of
antimicrobial microbeads based on ionic crosslinking occurring be-
tween positive and negative charged groups of Ch, TPP and Un.
The purpose of this work was the preparation of novel functional
chitosan tripolyphosphate based microbeads (Ch_TPP) (control sam-
ples), including the same amount of Ungeremine as antifungal com-
pound (Ch_TPP_Un), and their investigation by means of chemico-
physical properties. In particular, the structural and morphological
Table 1
Sample identification codes, Un concentration (mmol), Ungeremine loading efficiency (%), Beads porosity (%), Weight Loss percentage at 100 °C (WL%100 °C) and at
150 °C (WL%150 °C), Temperature of degradation onset (T onset °C), Temperature of maximum degradation rate (Tpeak °C).
Data/Sample Un.conc. (mmol) LE(%) Por (%) ± 10%
Ch – – – 8.2
Ch_TPP4 – – 0.17%
Ch_TPP5 – – 0.25%
Ch_TPP6 – – 0.29%
Ch_TPP_Un4 11.39 94.67% 0.85%
Ch_TPP_Un5 11.52 95.75% 1.1%
Ch_TPP_Un6 11.62 96.55% 1.3%
Carbohydrate Polymers 195 (2018) 631–641
properties of the microbeads were investigated by means of Fourier
transform-infrared spectroscopy (FTIR), scanning electron microscopy
(SEM) and porosity test, whereas the thermal stability was evaluated by
thermogravimetric analyses (TGA). The antifungal activity against PR
was assessed and confirmed in all the prepared Ch_TPP_Un composi-
tions, whereas the beads stability, encapsulation efficiency and re-
leasing profile of Ungeremine were monitored at different pH and
concentration of TPP, in order to tailor the antifungal performances of
Ch_TPP_Un microbeads. This paper should be considered as a pre-
liminary step to further deep investigations (object of upcoming work),
related to the inclusion of the best promising Ch_TPP_Un formulations
inside a biodegradable polymer, in order to have a bioactive food
packaging material.
2. Materials and methods
2.1. Materials
Chitosan (molecular weight 310,000–375,000 Da and deacetylation
degree 75%) and sodium tripolyphosphate (TPP) were purchased from
Sigma Aldrich (Milan, Italy). Acetic acid and sodium hydroxide were
purchased from Sigma Aldrich and used as received. Potato dextrose
agar (PDA) was purchased from Biolifeitaliana, Milan, Italy.
Ungeremine was obtained by following themethod reported in SD,
Section 2.1.
2.2. Preparation of Ch_TPP_Un and Ch_TPP based microbeads
Chitosan solution was prepared by dissolving chitosan (1 g) in
125 mL of dilute acetic acid (1% v/v), kept under stirring for 24 h.
Different concentration of TPP solutions were prepared by dissolving
4%, 5% and 6% w/v of TPP in 25 mL deionized water. The choice of the
above TPP concentrations was performed on the base of previous at-
tempts in which lower percentages of TPP led to unstable and irregu-
larly shaped beads, as showed in Fig. S2 of SD file Section S2.2.
Hence, 2.5 mL of the prepared chitosan solutions were dropped into
tripolyphosphate aqueous solutions and kept under slight stirring for
about 30 min, up to obtain spherical water stable beads, following used
as control (for more details, see SD Section, 2.2). The microbeads were
isolated by vacuum filtration on a paper filter and washed with 100 mL
of deionized water to remove the excess of unreacted TPP from the
beads surface. Finally, the microparticles were freeze-dried for 24 h.
As far as concerning the Ungeremine inclusion inside Ch_TPP mi-
crobeads, Chitosan-Ungeremine solution was prepared by dissolving
chitosan (1 g) and Ungeremine (0.320 g) in 125 mL of dilute acetic acid
(1% v/v), kept under stirring for 24 h. The amount of Ungeremine used
was based on data reported by Valerio et al. (2017); the authors cal-
culated the minimum amount of Ungeremine responsible of its anti-
fungal activity against Penicillium roqueforti (Valerio et al., 2017).
Hence, the same amount of Ungeremine was used for the preparation of
the beads, i.e. 12.03 mmol.
The experimental procedure for the obtainment of Ch_TPP_Un beads
was the same one previously reported for the control samples.
W.Loss (%) at 100 °C ± 5% W.Loss% at 150 °C ± 5% Tonset (°C) Tpeak (°C)
8.9 – 295
12.4 14.8 165 252
13.4 14.9 155 269
15 18 156 268
10 12.7 – 256
11.5 12.4 – 250
12.2 13.6 – 250
632
A. Moeini et al.
Nevertheless, in this case, 2.5 mL of the chitosan-Ungeremine solutions
were used. The concentration of Ungeremine (mmol), reported in
Table 1, was evaluated by means of an indirect method, exploiting
UV–vis methodology. Briefly, the Ungeremine content was calculated as
the drug released from the whole beads prepared in the different TPP
solutions (mother liquors). The method used was deeply described in
SD file, Section S2.3.
The microbeads thus obtained were stored in a desiccator for the
following analyses.
In Table 1, the identification codes of chitosan based beads and the
Ungeremine concentration (mmol) were reported.
2.3. Loading efficiency test of Ungeremine in Ch_TPP_Un beads
The loading efficiency (LE) tests were performed following the in-
direct method, by which the amount of Ungeremine found in mother
liquid was evaluated exploiting UV–vis spectroscopy. The following
equation (Eq. (1)) was used to calculate the percentage of the Un-
geremine loading in the beads prepared at different TPP concentrations.
The amount of Ungeremine found in mother liquid (see SD file Section
S2.3) was divided for the starting amount of Ungeremine used, (Starting
Ungeremine content 12.03 mmol), and the percentage value obtained
was the non-absorbed Ungeremine amount. Thus, the complement to
unity represented the percentage of loading efficiency (Eq. (1)).
Anyway, in order to not weigh down the paper, a detailed description of
the methodology used here was reported in SD file, Section S2.3.
Ungeremine content in mother liquor(mmol)
%LE = 100 − × 100
Starting Ungeremine content(12.03mmol)
(1)
2.4. Releasing kinetics
In order to assess the releasing profile of Ungeremine from the
beads, buffer solutions based on sodium hydroxide (pH = 5.7) and
potassium phosphate (pH = 6.2), were prepared. Equivalent volumes of
the buffer solutions were casted in six vials. Three of them were up-
loaded with one Ch_TPP_Un microbead at different concentrations of
TPP, whereas the other three vials were added with the corresponding
neat Ch_TPP microbeads at different concentrations of TPP, and used as
the control. The concentration of the Ungeremine released from the
single microbeads was regularly checked during the time, by per-
forming UV–vis spectroscopic analysis of buffer solutions. The values of
Ungeremine releasing were averaged on the three specimens.
2.5. Biological assays
2.5.1. Viability of cancer and normal cell lines in the presence of
Ungeremine
Human breast adenocarcinoma cell line MCF-7, containing the es-
trogenic receptor, was obtained from the European Collection of Cell
Cultures (ECACC, London, UK). Cell line was routinely grown in RPMI-
1640 medium without phenol red, supplemented with 10% FBS, 2 mM
glutamine, 50 U/mL penicillin G, 50 μg/mL streptomycin (all from
GIBCO) in 75 cm2 plastic flasks (Corning, NY, USA) at 37 °C in a 5% CO2
humidified atmosphere.
Normal Human Epidermal Keratinocytes, adult (HEKa) (from
Invitrogen), were cultured in Epilife Medium supplemented with HKGS
(Human Keratinocyte Growth Supplement), 50 U/mL of penicillin G
and 50 μg/mL of streptomycin (all from GIBCO) in 75 cm2 plastic flasks
and incubated at 37 °C in a 5% CO2 humidified atmosphere. Cells were
seeded at a starting density of 2.5 × 105 cells/mL and trypsinized twice
a week when reached 80%–90% confluences.
Both cell types were seeded in flat bottomed 96-wellplates (Corning,
NY, USA) at 104 cells/well in 200 μL of the respective complete medium
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Carbohydrate Polymers 195 (2018) 631–641
and allowed to attach for 24 h at 37 °C in a humidified,5% CO2 atmo-
sphere. After 24 h, the culture medium was replaced with complete
medium containing Ungeremine at the following final concentrations:
100, 50, 25, 12.5, 6.3, 3.1, 1.6 and 0.8 μg/mL for MCF-7 and 100, 50,
25, 12.5, 6.3, 3.1, 1.6, 0.8, 0.4 and 0.2 μg/mL for HEKa. For negative
controls, the test compound was replaced with solvent only (1.5% v/v
methanol) or medium only, controls were included on each plate. Cells
treated with all the Ungeremine concentrations were incubated for
24 h, 48 h or 72 h at 37 °C in a humidified, 5% CO2 atmosphere. Six
replicates in two independent experiments, for each treatment and for
each incubation time were performed.
Cell viability was assessed by MTS assay using CellTiter 96®AQueous
One Solution Assay (Promega Co. USA) according to the manufacturer’s
instructions. Briefly, after the treatment periods, 20 μL of MTS reagent
was added to each well and the multiplates were incubated at 37 °C in a
humidified 5% CO2 atmosphere for 3 h. Cell viability was determined as
assessment of metabolic activity by measuring the absorbance at
490 nm (A490nm) using Infinite® 200 PRO plate reader (Tecan Austria
GmbH). The possible interaction of MTS reagents and the test com-
pound (absorbance at 490 nm) has been excluded by incubation of extra
plates with the different concentrations of Ungeremine in each of the
complete medium in absence of cells. Cell viability was expressed as
percentages of the control and calculated by the following equation:
cell viability (%) = (ACn-treated/Acontrol) × 100, in which ACn-treated and
Acontrol represented the absorbance at 490 nm of cells treated with the
different Ungeremine concentrations and the absorbance at 490 nm of
the respective controls. The IC50 were obtained from nonlinear re-
gression analysis of concentration-effect curves by the GraphPad Prism
5 Demo program.
2.5.2. Antifungal assay
Penicillium roqueforti IBT18687 (PR) was obtained from the Culture
Collection of the Technical University of Denmark, Lyngby, Denmark.
Fungal conidia were collected from 7-day-old cultures on PDA, washed
twice with sterile water, and a 50 μL aliquot of conidial suspension was
spread on PDA plates, and incubated at 25 °C for 72 h. Conidia were
collected using Triton X 100 0.05% (v/v), counted in the Thoma
chamber and used in antifungal activity tests.
Antifungal assays were performed as previously described
(Milovanovic, Stamenic, Markovic, Ivanovic, & Zizovic, 2015; Seo,
Nguyen, Park, & Jung, 2014), with some modifications. Briefly, chit-
osan beads (Ch_TPP and Ch_TPP_Un) were sterilized by UV light for
30 min. Afterwards, 20–25 chitosan beads were placed onto PDA plates
which were then inoculated with 20 μL of a spore suspension
(1 × 105spores/mL) of the test microorganism PR. Mycelial growth was
observed after 1,2, and 3 days of incubation at 25 °C.
2.6. Scanning electron microscopy (SEM) and porosity test
Morphological analysis of films was performed by means of a FEI
Quanta 200 FEG Scanning Electron Microscope (SEM) on microbeads
surface. SEM observations were performed in low vacuum mode
(PH2O: 0.7 Torr), using a large field detector (LFD) and an acceleration
voltage of 5–20 kV. Prior to the observation, the sample surfaces were
coated with a homogeneous layer (18 ± 0.2 nm) of Au–Pd alloy by
means of a sputtering device (MED 020, Bal-Tec AG). In order to better
map each sample, three different beads for each compositions were
observed. In particular, for each sample, more than five different areas
were scrutinized and for each one of them several magnifications were
performed. In order to evaluate the porosity of the microbeads, porosity
tests were evaluated by following the method reported by Schettini
et al. (2013) and Bundela and Bajpai (2008), via a liquid displacement
process (Bundela & Bajpai, 2008; Schettini et al., 2013). As non-solvent,
n-hexan was used, since its ability to easily penetrate into the pores of
beads without inducing shrinkage, swelling or solubilization of the
polymeric matrix (Zhang & Ma, 1999). The porosity of the beads was
A. Moeini et al.
Fig. 1. Ch_TPP bead formation at basic (a), and acidic (b) pH.
evaluated by following Eq. (2): a known weight of beads (W0) was
immersed in a graduated cylinder containing a known volume (V0) of n-
hexane. The beads were kept in n-hexane for 24 h. Then the beads were
extracted and weighted (W1). By knowing the density (ρ) of n-hexan,
the porosity (P) of the beads was obtained. The measurements were
performed in triplicate.
W1 − W0
P= × 100
ρ × V0 (2)
2.7. Attenuated total reflection Fourier Transform Infrared (FTIR-ATR)
spectroscopy
Attenuated Total Reflection Fourier Transform Infrared (FTIR-ATR)
spectroscopy was carried out by means of a Perkin-Elmer Spectrum 100
spectrometer (Waltham, USA), equipped with a Universal ATR diamond
crystal-sampling accessory. All the samples were analyzed at room
temperature. Spectra were recorded as an average of 64 scans in the
range 4000–480 cm−1, with a resolution of 4 cm−1. No mathematical
correction (e.g.smoothing) was done, while spectroscopic manipula-
tion, such as baseline adjustment and normalization were performed
using the Spectracalc software package OMNIC 9 (Thermo Fisher
Scientific, Inc., MA, USA). Before testing, all samples were dried in
desiccator for 48 h.
2.8. Thermogravimetric analysis (TGA)
Thermogravimetric analyses (TGA) were carried out with a Mettler
Thermogravimetric Analyzer Mod. TG50. Measurements were per-
formed on samples of about 3–5 mg, placed in open ceramic crucibles
and heated from room temperature to 600 °C at 20 °C/min in air at-
mosphere, with a nominal gas flow rate of 30 mL/min. Before the tests,
a blank curve was measured and subtracted from the single thermo-
grams, to correct from instrumental drift (Vyazovkin et al., 2014). The
measurements were performed in triplicate.
3. Results and discussion
3.1. Chitosan based beads stability, loading efficiency test and controlled
releasing kinetics
Ch_TPP micro-beads have been widely investigated for improving
the drug delivery of both small and large molecular weight drugs (Dash,
Chiellini, Ottenbrite, & Chiellini, 2011) and may therefore be con-
sidered a promising encapsulating system of Ungeremine, since the
bioactive molecule, like chitosanis soluble in acetic acid water solution
and can be easily dissolved inside the polymeric solution before the
gelation process. Moreover, as previously detailed, Un is a zwitterion
(Carey & Sundberg, 2008; Von & Silversmith, 1987). Hence, it could be
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Carbohydrate Polymers 195 (2018) 631–641
hypothesized that during the crosslinking process between chitosan and
TPP, Ungeremine could keenly take part to the ionic interaction, thus
inducing the formation of a strong three-dimensional network by means
of ionic physical entanglements. The nature and extent of ionic inter-
actions between chitosan and TPP are reliant to parameters as the
charge density of both electrolytes, strongly dependent on the solution
pH. Indeed, Shu and Zhu (2000, 2001) reported that the chitosan beads
prepared with TPP could both increase the loading efficiency of active
compounds and prolong their release period, by opportunely mod-
ulating the pH (Shu & Zhu, 2000, 2001). As concerning the investigated
system, the pristine TPP water solution (pH 8.6) contained both OH−
groups and P3O105−. Therefore, the ionic interaction with the available
eNH3+ binding sites could occur by deprotonation (interaction be-
tween OH− and NH3+ groups) of charged amine groups or by ionic
crosslinking (interaction between P3O105− and NH3+ groups) pro-
cesses. Anyway, when chitosan solutions were dropped inside TPP basic
solution, gelled microbeads, by ionotropic gelation, were suddenly
obtained. Anyway, they showed irregular shape and their easy
smashing in TPP solutions, supported their instability (see Fig. 1a). It is
worthy to highlight that, in the pristine TPP solution, the coexistence of
both counterions triggered a sort of their competition in reacting with
the protonated amine groups; it was likely that, when the chitosan
droplets were in contact with TPP solution, the gelling mechanism of
the chitosan beads was mainly ruled by the phase inversion process
(coacervation of eNH3+ and OH- groups), at the expense of only few
crosslinking junction points between eNH3+ and P3O105−. Thus, at
high pH, the coacervation process was characterized by a weak ionic
crosslinking and the randomly coiled chitosan repeating units provided
a slackened loop conformation. This effect, sharpened by the increasing
of TPP concentration and hydroxyl groups, induced further deproto-
nation of chitosan amine groups with following weakening of ionic
complex formation (Fig. 2a). By progressively adding acetic acid to TPP
solution, the pH was reduced, passing by pH = 7 up to pH = 6.1. The
concentration of hydroxyl ions was drastically reduced and an in-liquid
curing mechanism occurred, involving the chitosan amine groups pro-
tonation, the strong positive charge repulsion, and the ionic cross-
linking process between P3O105− and NH3+ groups. Therefore, the
beads formed were more spherical, homogeneous and stable over the
time. In addition, they were smaller with respect to the ones obtained at
basic pH (see Fig. 1b). These outcomes were consistent with a tight
crosslinked Ch-TPP structure, responsible of both higher stability and
lesser swelling ability of the beads. Hence, at low pH, crosslinked
chitosan resulted in a more extended loop and ladder shaped three-
dimensional network (Fwu-Long et al., 1999), as evidenced in Fig. 2b
and c.
As concerning Ch_TPP_Un beads preparation, chitosan and
Ungeremine were previously dissolved providing a yellow-orange co-
lored solution, since the chromophores groups present in the alkaloid.
Anyway, the presence of the color played a key role in the
A. Moeini et al.
Fig. 2. In liquid curing mechanism of Chitosan in TPP solutions at basic (a), neutral (b) and acidic pH (c).
Fig. 3. In-liquid curing mechanism of Ch_TPP_Un beads in TPP solution: (a)
basic, (b) neutral and (c) acidic pH.
enlightenment of the crosslinking process occurring during the external
gelation method (see Fig. 3). Actually, when Ch_Un solution was
dropped into TPP basic solutions, two macroscopic opposite phe-
nomena could be observed: at the beginning, the beads formed and
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Carbohydrate Polymers 195 (2018) 631–641
were yellow, thus indicating that the crosslinking process physically
involved Ungeremine. Anyway, after few seconds, Ungeremine was
fastly squeezed out from the beads, as evidenced by the drastically
changing of beads color from yellow to white. Actually, the fast gelation
process between chitosan and TPP basic solutions allowed Ungeremine
charged molecules to be physically entrapped in the weak ionic cross-
linked layer formed on the bead surface, that for this reason, became
yellow; anyway, as previously described, at high pH, the beads coa-
cervation is controlled by phase-inversion gelation process, since both
the higher concentration of hydroxyl groups and their smaller size with
respect to TPP anions. Moreover, it should be stressed that the Un steric
hindrances due to the presence of aromatic and heterocycles residues,
induced its squeezing out from the surface gelation layer at basic con-
dition, thus allowing beads becoming newly white (Fig. 3a). By in-
creasing Ch_Un concentration inside TPP solution, pH decreased and
the active molecule started to diffuse into the beads, even if some
yellowness could be still observed in TPP solution, indicating that Un-
geremine was not completely absorbed in Ch_TPP microbeads.
(Fig. 3b). With the following lowering of pH at about 6.1, the gelation
process, as previously detailed, was controlled by the ionic crosslinking
between P3O105− and NH3+. This experimental condition matched
with the higher loading efficiency of the antifungal molecules inside the
crosslinked network, resulting in the development of yellow and stable
A. Moeini et al.
Ch_TPP_Un beads (Fig. 3c). It could be assumed that, being a zwitterion,
Ungeremine could take actively part to the ionic crosslinking process
with both TPP and NH3+ groups, by means of ionic physical en-
tanglements (Carey & Sundberg, 2008; Ko, Park, Hwang, Park, & Lee,
2002; Von & Silversmith, 1987).
In Table 1, the loading efficiency of Ungeremine in Ch_Ung_TPP
beads at different concentration of TPP has been detailed. The analysis
of data highlighted that for all the samples, the loading efficiency was
very high; in particular, it is possible to underscore that by increasing
TPP concentration, the loading efficiency of the antifungal metabolite
was slightly enhanced. This outcome could be explained by considering
the increase of the crosslinking junction points occurring between Un-
geremine positive charged nitrogen and TPP polyanion. At the same
time, this worthy result evidenced that, besides being physically en-
trapped in the Ch_TPP network, Ungeremine played an active role
during the crosslinking process, as following confirmed by FTIR data.
In order to follow the kinetics of Ungeremine releasing from chit-
osan based beads, UV–vis spectroscopic analysis was performed by
using two different pH buffer solutions 5.7 and 6.2 as releasing media.
These two pH were selected as they are typical of bakery products. An
intense UV absorption at 370 nm was observed (spectrum not reported)
according to the presence of Ungeremine conjugated chromophore and
the percentage of Ungeremine released from the chitosan microbeads
formulations vs time, at pH = 5.7 and pH = 6.2, was plotted in Fig. 4a
and b respectively. The diffusion properties and the release kinetics
were functions of Ungeremine concentration, changing of beads stabi-
lity at different pH, water absorbed, dissolution of Ungeremine and
desorption of the same via swelling controlled mechanism (Dini,
Alexandridou, & Kiparissides, 2003). Each curve was built by evalu-
ating the Ungeremine content inside the buffer solutions at different
times. Specifically, three beads of each compositions were subsequently
dipped inside buffer solutions and periodically collected at the same
time intervals. The average value of the Ungeremine amount detected
from the beads was reported as a function of the time. From the analysis
of the curves, worthy difference in releasing behavior could be marked
at the two pH. Specifically, at pH = 5.7, Ch_TPP_Un beads highlighted a
similar trend independently from TPP concentration, with a high Un
releasing in the first three hours and a slower one up to 48 h, where
about 20% of Un was squeezed out (Fig. 4a). At pH = 6.1, the kinetics
pattern followed a substantial different profile, depending on TPP
concentration. During the first 24 h, a burst and faster Ungeremine
releasing was observed (Fig. 4b), particularly accentuated in
CH_TPP_Un4 composition. In the following 24 h, a sort of a plateau was
detected for all formulations, even if Ch_TPP_Un4 sample experienced
the highest releasing, reaching up to 35% of Ungeremine weight de-
livery. It could be hypothesized that the starting fast release was mainly
Fig. 4. Kinetics release profile of Ungeremine (%w) in buffer solution at pH = 5.7 (a) and pH = 6.2 (b) vs time (hours).
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Carbohydrate Polymers 195 (2018) 631–641
caused by desorption of the Ungeremine from the outer layer of
crosslinked systems. Anyway, at both pH, from 24 up to 48 h of treat-
ment, the releasing process followed a tailoring-off behavior, likely due
to a more convoluted pathway for Ungeremine dissolution from the
inside core of Ch_TPP crosslinked network (Forni, Vandelli, &
Cameroni, 1992) towards the solution. Moreover, it deserves of note
that, generally, by increasing TPP concentration, the Ungeremine re-
leasing from the beads decreased since the stronger interaction occur-
ring between Ungeremine and rising concentration of TPP counterions,
as previously shown by the loading efficiency percentage and following
detailed by FT-IR spectra evaluations. Probably, since the degree of
swelling and following desorption of Ungeremine is mostly ruled by the
crosslinking density of the polymeric network and by the strength of the
network formed, as previously described and reported in literature
(Harris et al., 2011; Higuera-Ciapara, Felix-Valenzuela, Goycoolea, &
Argüelles-Monal, 2004), a decrease in the amount of crosslinking agent
and increasing of pH as in the case of Ch_TPP4%_Un system, induced as
expected effect, the enhancement of Ungeremine releasing. Thus, at
higher pH (Fig. 4b), as expected, the curves followed a faster releasing
kinetics, since the increasing of OH− residues and following decreasing
of ionic complex. Hence, depending on the specific bakery substrate, it
could be possible to modulate Ungeremine release.
3.2. Biological activities
3.2.1. Effect of Ungeremine on cancer and normal cell line viability
Since Ungeremine is released from the Ch_TPP microbeads, the
absence of toxicity of the neat substance was demonstrated both on
breast cancer cell line (MCF-7), and on normal human epidermal ker-
atinocytes-adult (HEKa) in a range of concentrations from 0.2 to
100 μg/mL (equivalent to about 0.8–376 μM, respectively). In parti-
cular Ungeremine, at all the tested concentrations was not cytotoxic for
both the considered cell types, treated for 24 h. At longer incubation
time, 48 h and 72 h, only the two higher concentrations of Ungeremine
slightly decreased cell viability in MCF-7 cells, but none relevant cy-
totoxicity was detectable, as demonstrated by the IC50 values
(106.6 μg/mL, 124.4 μg/mL and 30.8 μg/mL, respectively, equivalent to
396, 459 and 115 μM), confirming previous data on a panel of cancer
cells (Van Goietsenoven et al., 2010). Significantly, none cytotoxicity
was detectable for normal epidermal keratinocytes at the tested con-
centrations and at all treatment times as the IC50 values for HEKa were
very high (IC50 > 111 μg/mL) (Fig. S4). At our best knowledge, there
is no literature data about exposure by the skin or epithelial mucosae
(short or long-term exposure) to Ungeremine.
A. Moeini et al. Carbohydrate Polymers 195 (2018) 631–641

Fig. 5. Antifungal assay of Ungeremine released from the Ch_TPP beads after 72 h, at different TTP concentration.

Fig. 6. SEM micrographs of Ch_TPP4 (a) and Ch_Un_TPP4 (b) surface.

637
A. Moeini et al.
Fig. 7. FT-IR spectra of Ch, Ch_TPP6% and Ch_TPP6_Un in the range of 3600–2200 cm−1 (a) and in the range of 1800–1100 cm−1 (b).
3.2.2. Antifungal activity of Ungeremine contained in Ch_TPP beads against
Penicillium roqueforti (PR)
The antifungal properties of neat Ungeremine against PR was pre-
viously shown and detailed by Valerio et al. (2017). The antifungal test
was performed using the Ch_TPP_Un based beads and Ch_TPP, used as
control, to assess the release of Ungeremine from the Ch_TPP beads into
the agar plates previously inoculated with the fungal strain. The assay
confirmed the antifungal activity of Ungeremine released from the
beads at different concentrations of TPP, as evidenced in Fig. 5.
Antifungal activity was determined by evaluating the inhibition
zones formed around the beads. All the tested samples were strongly
active in inhibiting the fungal growth; indeed, after 72 h, fungal my-
celia was observed only in all control beads (Ch_TPP), with a con-
tamination mostly starting from the beads core, maybe because the UV
sterilization was not completely effective.
This outcome suggested that already at the lowest concentration of
TPP, Ungeremine could exploit its antifungal activity. For this reason,
aimed to use the above formulated microparticles as additives in
bioactive food packaging material, a first challenge could be re-
presented by testing Ch_TPP_UN4 composition (work in progress).
3.3. Scanning electron microscopy (SEM) analysis and porosity test
SEM analyses were performed on three samples for each composi-
tion in order to obtain more detailed information on morphology of
chitosan microbead surfaces. In Fig. 6a and b, as an example, the mi-
crographs of Ch_TPP4 and Ch_TPP4_Un are respectively reported. The
neat beads evidenced a microporous and wrinkled surface with regular
distribution of holes, whose diameter was in the range of 200 μm,
The Ungeremine loading induced a strong effect on the architectural
morphology of the microbeads; indeed, it provided a deeper regularity
of the surface associated with more closely packed holes, showing
thicker walls and smaller size diameters of about 100 μm. This peculiar
morphology suggested a substantial increment of the structural cross-
linking density, since the presence of a tighter network, as previously
detailed. Similar results were found by Srinatha, Pandit, and Singh
(2008) and by Chung and Park (2007) (Chung & Park, 2007; Srinatha
et al., 2008).
The evaluation of void spaces of chitosan based beads, by means of
porosity tests, played a key role in providing information related to the
polymeric network structure, following to the crosslinking process oc-
curring with different concentration of TPP and Un. The porosity data,
reported in Table 1, evidenced a peculiar behavior of Ch_TPP and
Ch_TPP_Un samples. By the increasing of crosslinking junction points,
638
Carbohydrate Polymers 195 (2018) 631–641
following to the high concentration of TPP, the porosity of samples
increased and the effect was particularly marked in samples containing
Ungeremine. This peculiar result could be explained considering that
the crosslinking process of chitosan beads, responsible of the junction
points development, occurred during the samples soaking in water so-
lutions of TPP; so chitosan crosslinked while swelling, i.e., it adsorbed
some water during the formation of three-dimensional stable network,
as also shown by thermal analysis. Hence, the conformation of cross-
linked polymeric backbone attained in wet condition did not change
after freeze-drying process (Russo, Malinconico, & Santagata, 2007;
Russo, Abbate, Malinconico, & Santagata, 2010). This outcome induced
the increasing of free volume and porosity of the samples, particularly
heightened in the beads containing Ungeremine where more cross-
linking junction sites were formed and preserved after drying process.
3.4. Attenuated total reflection Fourier Transform Infrared (FTIR-ATR)
spectroscopy
All chitosan based beads cured in TPP solution at pH ≈ 6.1, were
analyzed by FT-IR spectroscopy, in order to follow the structural
changes of chitosan after crosslinking process with trypolyphosphate
and Ungeremine. In order to compare the spectroscopic behavior of the
crosslinked based beads with the ones of the neat polymer, a film of
plain chitosan was prepared by casting from a water acidic solution
(pH ≈ 6.1). In Fig. 7a and b, as an example, FT-IR spectra of Ch,
Ch_TPP6% and Ch_TPP6_Un are reported. In particular, in the region of
high frequency absorption peaks (Fig. 7a), FT-IR spectrum of neat
chitosan evidenced a band with two submaxima at 3362 cm−1 and
3292 cm−1 attributed to −OH and −NH groups stretching vibrations.
In Ch_TPP6 and Ch_TPP6_Un systems, this bands became wider, flat-
tened and underwent a shift towards lower frequency (at about
3166 cm−1), thus suggesting the physical interactions between the
functional polar groups of chitosan, sodium polyphosphate and Un-
geremine molecules, mainly occurring via hydrogen bonding (Jia-hui,
Yu-min, & Hua, 1999). Carbonyl and finger print regions of the spectra
are reported in Fig. 7b. Neat chitosan displayed two strong vibration
peaks at 1653 cm−1 and 1564 cm−1. These peaks were respectively
assigned to C]O stretching vibration (amide I) and to NeH bending
vibration (amide II) of N-acetylglucosamine residues (Smitha, Sridhar,
& Khan, 2005). However, since the chitosan used showed a DA of 75%,
only 25% of the nitrogen atoms occurred as amides, whereas the re-
maining atoms were neutral and protonated aminesas expected, since
both films and beads were prepared in acid conditions. Considering that
protonated amines display asymmetric deformation in
A. Moeini et al.
1650–1570 cm−1 range and symmetric vibrational mode in
1550–1505 cm−1 range, it is plausible that besides the amide I, the
band at 1653 cm−1 also contains the asymmetric eNH3+ deformation,
whereas the peak at 1564 cm−1 resulted from the overlapping of both
amide II, primary deacetylated amines NeH stretching vibrations and
the symmetric eNH3+ vibrational mode. Actually, the band at
1564 cm−1 showed a larger intensity than the one at 1653 cm−1, thus
indicating the prevalence of amine groups (Lawrie et al., 2007). FT-IR
technique is a valid tool aimed to assess the physical or chemical in-
teractions between components in polymeric based systems. By com-
paring FT-IR spectra of plain chitosan and Ch_TPP, it is worthy to
highlight several differences correlated to the evidence of the ionic
crosslinking process occurring. Indeed, following to the development of
chitosan three-dimensional networks, the peak of 1653 cm−1, assigned
to the amide ν(C]O) mode shifted towards 1635 cm−1 and the peak at
1564 cm−1 moved to 1543 cm−1. These results could be ascribed to the
formation of physical entanglements occurring among the functional
groups of Ch_TPP beads during the crosslinking process by means of
both ionic interactions and hydrogen bonding. The development of
tighter linkages was responsible of the local energy stretching vibra-
tions inhibition with following decreasing of corresponding absorption
frequency (Bhumkar & Pokharkar, 2006; Xu & Du, 2003). Moreover, at
1545 cm−1, a new shoulder weak peak could be evidenced, likely as-
signed to the δ vibrational mode of protonated amine groups in chit-
osan, as confirmed by Souza, Salles, Brandão, Edwards, and De Oliveira
(2015). In Ungeremine loaded beads, the previous bands were fur-
thermore shifted towards lower frequencies; in particular, the amide I
peak moved to 1622 cm−1 whereas the peak at 1564 cm−1 shifted to
1526 cm−1. These results evidenced that Ungeremine molecules were
not only physically entrapped inside the crosslinked network, but
keenly took part to the new linkages occurring in the three-dimensional
network, thanks to the their positive nitrogen and negative oxygen
reactive residues. Finally, at 1218 cm−1, in Ch_TPP and Ch_TPP_Un
spectra, it was possible to detect the P]O stretching peak, related to
the presence of tripolyphosphate groups (spectrum not reported) (Fwu-
Long et al., 1999).
3.5. Thermogravimetric analysis (TGA)
In Fig. 8, the mass loss (TGA) of all chitosan based beads (Fig. 8a)
and their derivative curves (DTG) (Fig. 8b) are reported. In Table 1, the
weight loss percentage at 100 °C and 150 °C (WLoss%100 °C and
WL%150 °C), the temperature of main degradation step (Tonset °C), and
the temperature of maximum degradation rate (Tpeak °C) are reported.
All data were opportunely normalized with respect to the sample sizes.
Thermogravimetric analysis is a valid tool to determine the different
Fig. 8. Chitosan based beads thermograms: Weight loss (%) (TGA) (a) and thermal degradation rate (DTG) (b).
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Carbohydrate Polymers 195 (2018) 631–641
water adsorption of films and to investigate the different pattern of
polymeric degradation; moreover, information related to the physical
interaction between the components of a polymeric system, are de-
tectable too (El-hefian, Nasef, & Hamid, 2012). From the analysis of the
curves, three different stages of mass loss may be recognized: the first
from room temperature to about 100 °C, the second between 100 °C and
about 200 °C, and the third at temperatures above 200 °C. Higuchi and
Iijima proposed that water exists in three states in hydrophilic polymer
matrices: (i) freewater, (ii) freezable bound water, (iii) non-freezable
bound water (Higuchi & Iijima, 1985). On the base of the above claims,
analyzing the thermograms in Fig. 8a, it is possible to claim that the
first two steps of weight loss up to about 200 °C concerned the elim-
ination of water (Russo et al., 2010). Lastly, around 200 °C, the de-
composition process of polysaccharides starts, involving a random split
of the glycosidic bonds, vaporization and elimination of volatile pro-
ducts (Nieto, Peniche-Covas, & Padrón, 1991). Nevertheless, it is
worthy to highlight a severe different thermal behavior passing by neat
chitosan to Ch_TPP and Ch_TPP_Un samples. In fact, the water loss of Ch
was of about 9% at 150 °C, whereas, at the same temperature, the
crosslinked samples without and with Ungeremine released about 18%
(Ch_TPP6) and 14% (Ch_TPP6_Un) of their water contents. This out-
come suggests that the three-dimensional networks induced the ab-
sorption of high water amount. Moreover, this trend is enhanced by
increasing TPP concentration. This outcome is cleared up by taking in
account that crosslinking occurred while swelling as previously de-
tailed. Since in the swollen samples some macromolecular chain seg-
ments, before interacting with each other, could interact with water
molecules via hydrogen bonds, the free volume of the polymeric based
system increased (Russo et al., 2007).
The degradation profile of the films (DTG curves) and the relative
temperature pattern are reported in Fig. 8b and Table 1, respectively.
The first worthy observation is related to the hastening of thermal de-
composition process of all cross-linked samples, particularly accen-
tuated in Ch_TPP_Un samples, where the lowest Tonset.degr and Tpeak
could be found. Similar results were also found by Kim and Lee (1993),
Neto et al. (2005) and Russo et al. (2007), when working with cross-
linked samples of chitosan (Kim & Lee, 1993; Neto et al., 2005; Russo
et al., 2007). As a matter of fact, usually cross-linked samples experi-
ence a delay in the onset of degradation. The lowering of chitosan
thermal stability after the crosslinking process could be associated to
changes in the macromolecular backbone structure of the polymer. In
particular, the heterogeneous cross-linking reactions between poly-
saccharides chains and crosslinking agent induced the weakening of
attractive intra-inter-molecular hydrogen bonds. Thus, according to
literature data, the less packed chitosan macromolecular chains, re-
sulting more exposed to the random splitting of the glycosidic bonds,
A. Moeini et al.
fastly underwent to the thermo-degradation process. This phenomenon
is particularly stressed in Ungeremine-loaded samples (see Table 1),
where the main degradation process was severely anticipated with re-
spect to both pristine polymer and unloaded beads. It was likely that,
even at higher concentration of TPP, the excess of protonated amine
groups on chitosan backbone could not be stabilized by negative
counterions. Hence, some domains in which the electrostatic repulsion
prevailed could trigger a less packed structure more prone to the
thermal degradation (Pieróg, Ostrowska-Czubenko, & Gierszewska-
Druzyńska, 2012).
4. Conclusion
In this paper, the preparation and chemico-physical characteriza-
tion of chitosan based microbeads containing Ungeremine, were re-
ported. The beads stability and the highest loading efficiency were
reached at low pH. Both morphological and spectroscopic analysis
evidenced that Ungeremine took actively part to the threedimensional
network involving chitosan and TPP. The Un releasing kinetics profile
could be tailored by modulating pH on the base of the food products.
The results emphasized that the highest Ungeremine enfolding was
reached by Ch_TPP_Un4 formulation at pH = 6.2. Moreover, all the
compositions were active against Penicillium roqueforti, the fungus
mainly associated with bakery products spoilage. Finally, thermo-
gravimetric analysis confirmed the hypothesis of “crosslinking while
swelling” mechanism, occurring during the development of the three-
dimensional network; indeed, the increasing of microbeads free vo-
lume, particularly enhanced in Ungeremine-loaded samples, was re-
sponsible for a faster thermal degradation.
Based on the whole screening of the sample properties, Ch_TPP_Un4
formulation matches the best performances allowing it to be pre-
liminarily exploited as bioactive additive for the development of novel
biodegradable packaging materials for food preservation (work in
progress).
Acknowledgements
The authors thank Cristina Del Barone, SEM skilled worker, for her
support in performing morphological analysis at LAMeST Laboratory of
Scanning and Trasmission Electron Microscopy (Pozzuoli IPCB-CNR).
The authors thank Dr. Salvatore Mallardo, researcher, for the key
role in the technical support of the experimental data elaboration.
The authors acknowledge the project “SOSTENIBILITA’ DELLA
FILIERA AGROALIMENTARE (SO.FI.A)”, in the frame of National
Technological Cluster of Ministry of University and Research (MIUR),
for the financial support.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at https://doi.org/10.1016/j.carbpol.2018.05.005.
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