Icef full paper - 11th International Congress on Engineering and Food

Release of natamycin from alginate and pectin films intended for food packaging
Andréa Cristiane Krause Bierhalz a , Mariana Altenhofen da Silva a , Theo Guenter Kieckbusch a
a School of Chemical Engineering, University of Campinas, Campinas, Brazil (theo@feq.unicamp.br)
ABSTRACT
Packages with antimicrobial activity provide a promising form of active packaging systems applicable in
food processing. Polysaccharides, especially alginate and pectin, are good candidates to be used in active
films since they show excellent functional properties and are biodegradable. In this study, single and
composite films based on alginate and pectin containing natamycin as active agent were prepared and the
release behavior in water and the diffusion coefficients were evaluated. The influence of natamycin on
opacity, soluble matter in water and mechanical properties of the films were also investigated. Diffusion
coefficients were determined using a solution to Fick’s Law for a plane sheet and the mechanism
involved in the diffusional process was investigated using the Power Law Model. Addition of natamycin
promoted an increase in soluble matter in water and opacity and decreased the tensile strength when
compared to films without the added anti-microbial agent. The natamycin mass released by immersion of
the film in water fitted well to Fick’s second law diffusional model, with effective diffusivity values
ranging from 9.53.10 -9 (single pectin films) to 9.22.10 -12 cm 2 /s (single alginate films). The values of the
diffusional exponents ranged between 0.5 and 1.0, suggesting that the transport process had non-Fickian
(anomalous) characteristics and a solvent diffusion rate of the same order of magnitude as the polymer
relaxation. The single alginate films exhibit more suitable attributes for use in packaging than the single
pectin and composite films, indicating a greater potential for use as systems for release of active
substances.
Keywords: active film; alginate; pectin; natamycin; diffusion coefficient
INTRODUCTION
The increasing demand for safer and minimally processed food products has intensified the research on
antimicrobial packaging. This innovative concept of packaging can prevent the product’s deterioration,
extending the shelf life and maintaining the sensorial attributes and safety of several food products [1].
Biodegradable films prepared from polysaccharide materials, such as algintate and pectin can be used to
incorporate antimicrobials agents, delivering them to the food surfaces, where deterioration by microbial
growth often begins.
Alginates are hydrophilic polysaccharides derived from brown algae known as Phaeophyceae Alginates
are composed of (1,4)-linked β-D-mannuronate (M) and α-L-guluronate (G) units, which are present in
the linear macromolecule as homopolymeric blocks (poly-M and poly-G), together with blocks in
alternating sequence (MG) [2]. Pectin is the main component of the citrus processing by-products and one
of the most widely studied ionic polysaccharides with useful and versatile properties in many
applications. The basic chemical structure of pectin is a linear polymer of D-galacturonic acid units with
their methyl esters connected through α-(1,4)-glycosidic bonds [3]. According to their degree of
methylation, pectins are divided into two categories: low-methoxyl pectins (LMP) and high methoxyl
pectins (HMP), with a degree of methylation respectively lower and higher than 50%. The degree of
methylation has a decisive effect on the mechanisms of gelation [4]. The extensive use of these two
polymers arises from their ability to form gels in the presence of divalent cations such as calcium ions [2].
The more commonly antimicrobials used to preserve food are organic acids such as benzoic and sorbic
acids, bacteriocins such as nisin and some plant extracts [5]. Natamycin is a natural antifungal agent
produced during fermentation by the bacterium Streptomyces natelensis and is widely used in the food
industry for the prevention of mold contamination of meats, cheese and fruits [6].
Several studies have been done on the release of antimicrobials incorporated into different packaging [7,
8, 9, 10]. Since the efficiency of antimicrobial films is based on the diffusion of active substances from
the polymeric matrix to the food, the knowledge of the diffusivity of these compounds is a determinant
factor in the development of an antimicrobial food packaging system [8]. A slower release and, therefore,
a lower diffusion coefficient is desirable in this application to maintain a critical surface concentration of
preservative [10].

The physical and chemical properties of the film are also important for packaging system efficiency.
Addition of antimicrobial agents may cause changes in the polymeric structure of the film, affecting its
mechanical properties and effectiveness as a barrier [11,12] Thus, possible interactions between
antimicrobial agents and biopolymers should be considered in the development of active packaging.
In this work, single and composite films based on alginate and pectin containing natamycin as active
agent were prepared and the release behavior in water and the diffusion coefficients were evaluated. The
influence of natamycin on thickness, opacity, soluble matter in water and mechanical properties of the
films were also investigated.
MATERIALS & METHODS
Materials
Medium viscosity sodium alginate, extracted from Macrocystis pyrifera seaweed, was purchased from
Sigma–Aldrich (St. Louis, USA) and amidated low methoxy pectin (LM 101AS) donated by of CPKelco
(Limeira, Brazil), were used as biopolymers matrices in the composite and single films. Calcium chloride
dihydrate (Merck, Darmstadt, Germany) was used as crosslinking agent and glycerol (Synth, Diadema,
Brazil) as plasticizer. Natamycin (Natamax ® ), kindly donated by Danisco (São Paulo, Brazil), was used as
antimicrobial agent.
Film preparation
Alginate, pectin and alginate/pectin (1:1) films were made by casting in a two-stage crosslinking
procedure. In the first stage, a film forming solution of biopolymer (1.5 g/100 mL) was prepared in 400
mL distilled water already containing 0.6 g glycerol/g biopolymer at room temperature. The solution was
mechanically stirred at 900 rpm (Tecnal, TE-139, Piracicaba, Brazil) for about 1 h to ensure homogeneity.
Afterwards, the temperature of the system was raised to 70ºC and a dilute aqueous calcium chloride
solution (30 mL) was slowly added to the biopolymer solution at a flow rate of 1 mL/min delivered by a
peristaltic pump (Masterflex C/L, model 77120-70, Vernon Hills, USA) until a total amount of 0.05 g
CaCl 2 .2H 2 O/g biopolymers was transferred. Aliquots of the solution (50 mL) were poured into square
plaxiglass frames (225 cm 2 ) and dried in a convection oven (Fanem, model 099EV, Guarulhos, Brazil) at
40ºC for about 20 h.
After detaching the resulting film from the support, the crosslinking was complemented in a second stage
contact, by total immersion of the films in 50 mL of an aqueous calcium chloride solution (5% w/v)
containing glycerol (3% v/v) for 20 min. The excess surface liquid was removed and the films were dried
in a ventilated ambient for about 5 h, at room temperature. All films were conditioned at room
temperature and 52% relative humidity inside desiccators for 3 days before submission to physical
characterization.
The active films were prepared as described previously using 4g natamycin/100g alginate added to the
polymeric solution containing calcium chloride (first stage). The system was further stirred for another 10
minutes before casting.
Film thickness (δ)
The film thickness was controlled by pouring a constant mass (50 g) of the film forming solution over the
support. Digital micrometer (Mitutoyo, MDC-25S, Naulcapan de Juárez, Mexico) was used to measure
the thickness of conditioned films. Ten measurements were taken at random positions.
Soluble matter in water (S w )
The soluble matter in water of the films was measured as proposed by Irissin-Mangata et al. [13]. The
moisture weight fraction, ω, of the film was gravimetrically determined in a vacuum oven (Lab-Line,
Squaroid, USA) at 105ºC for 24 h. Disks cut from the same film, were weighed and immersed in 50 mL
of distilled water using a 250 mL beaker maintained under mild agitation (150 rpm) at 25ºC for 24 h
(Shaker Bath Orbit, Lab-Line, USA). The final dry matter of the sample was determined in the same
vacuum oven (105ºC/24 h). The fractional solubilized matter (S w ) was calculated as a function of the
initial dry matter.
Mechanical properties
Tensile strength (TS) and elongation at break (TE) of the preconditioned films were determined at room
temperature using a TA.XT2 (Stable Microsystems SMD, Surrey, UK) according to ASTM standard
method D882 [14]. Films were cut into strips (10 x 2.54 cm) and mounted between the tensile grips of the
instrument. The initial grip spacing and cross-head speed were set at 5 cm and 0.1 cm/s, respectively. A

microcomputer was used to record the stress–strain curves. The tensile strength was expressed as the
maximum force at break per initial transversal area of the film and the elongation as a percentage of the
original length.
Opacity (O p )
The opacity of the films was determined in triplicate using a Colorquest II colorimeter (Hunterlab,
Virginia, USA) operating in the transmittance mode according to Hunterlab method [15]. Opacity was
calculated by the equipment software, as the relationship among the opacity of each sample over the black
standard and the opacity of each sample over the white standard.
Natamycin migration test
Square film samples (4 cm 2 ) were immersed in a beaker containing 25 mL of distilled water under gentle
agitation (150 rpm) in a shaker (Shaker Bath Orbit, Lab-Line, USA) at a temperature of 25°C. After a
predetermined period of time, the film was quickly transferred to a second water container. This
procedure was repeated from one beaker to another until no further release of natamycin could be
detected. The natamicyn concentration in the beakers was determined with UV/VIS spectrophotometer
(HP, model 8453, USA) over a wave length range of 290 to 350 nm. The natamycin concentration was
obtained by measuring the amplitude of the peak at 317 nm. The accumulated mass of natamycin
released, M t , was calculated and plotted as M t /M ∞ as a function of time, where M t /M ∞ is the fractional
natamycin release. The mean film thickness was determined before and after each experiment.
Diffusion coefficient determination
Diffusion coefficients were determined from the data obtained using a relationship derived from the
solution to Fick’s Law for a flat plate [16]. Under the conditions of the experiments, and assuming a
constant total film thickness, δ, the following equation (Eq. 1) can be adapted for the fractional natamycin
release.
⎛ π
( )
⎟ ⎞
= − ∑ ∞ 2
Mt 8 1
2 D
1
exp
⎜−
2n + 1 t
(1)
2
2
2
M∞
π n=
0 (2n + 1) ⎝ δ ⎠
where D is the average effective diffusivity of natamycin, assumed constant.
For short contact times, when less than 60% of the solute mass is liberated, a simplified solution of Fick’s
Second Law can be used (Eq. 2):
M t Dt
= 4
(2)
2
M δ π
∞
Polymeric matrices are prone to undergo structure relaxation under the influence of the penetrating
solvent or solute molecules and anomalous transport mechanisms can prevail for some situations. A
common way to investigate the mechanism involved in the diffusion process for a planar system is by
fitting the early portion of the release curve (M t /M ∞

opacity, mechanical properties and soluble matter in water of single and composites alginate/pectin films
are shown in Table 1.
Table 1. Thickness (δ), soluble matter in water (S w ), tensile strength (TS), elongation at break (TE) and opacity (O p )
of films without natamycin (control films) and with natamycin (active films).
Film δ (µm)** S w (%)* TS (MPa)** TE (%)** O p (%)*
Pectin
Control films 19 (0.9) c 24.53 (0.54) b 65.49 (5.52) d 4.66 (0.44) b 4.55 (0.06) f
Active films 20 (1.1) c 45.52 (1.79) a 48.46 (5.93) e 4.75 (0.37) b 51.98 (0.89) b
Pec/Alg
Control films 25 (3.1) b 16.92 (1.97) d 94.53 (3.43) b 4.68 (0.53) b 7.85 (0.48) e
Active films 25 (1.9) b 25.66 (4.76) b 80.87 (7.08) c 4.81 (0.45) b 57.20 (0.86) a
Alginate
Control films 30 (2.2) a 19.03 (1.22) c 122.51 (3.27) a 6.59 (0.72) a 13.12 (0.06) d
Active films 30 (2.1) a 17.74 (1.98) cd 106.73 (4.99) b 6.84 (1.09) a 48.57 (0.39) c
Average (standard deviation) of three (*) and ten (**) experimental determinations.
Average with the same letter, in the same column, indicate no significant difference (p

Diffusion coefficients of natamycin from single and composite films were estimated by adjusting the
experimental points to Equation 1 using 15 terms in the summation. Initial experimental data (M t /M ∞ <
0.6) were also adjusted to the diffusion model for short contact times (semi-infinite solid model) using
Equation 2. These values, the correlation coefficient as well the time of equilibrium found for each
situation studied are shown in Table 2.
Table 2. Time to equilibration (t), diffusion coefficients (D), diffusion exponent (n) and diffusional constant (k) of
pectin, alginate and composite films containing natamycin
Film t (h) D 1 (cm 2 /s) R 1 D 2 (cm 2 /s) R 2 n k (s -1 )
Pectin 30 3.22.10 -9 0.9963 3.92.10 -9 0.9991 0.5744 4.54.10 -3
Pec/Alg 70 2.80.10 -10 0.9834 3.92.10 -10 0.9989 0.7258 6.56.10 -4
Alginate 800 9.18.10 -12 0.9986 1.11.10 -11 0.9988 0.6028 4.32.10 -4
1 Diffusion coefficient and correlation coefficient found from Equation 1.
2 Diffusion coefficient and correlation coefficient found from Equation 2.
Comparing the times to equilibration results (t), it can be observed that natamycin release was slower in
single alginate films. Pectin films reached equilibrium in approximately 30 hours, while this period
increased to 70 hours for composite films and over 800 hours for alginate films. These results reinforce
the hypothesis that natamycin presents higher compatibility with alginate than with pectin. The rapid
release of natamycin from pectin films also explains the high soluble matter in water value observed in
Table 1.
As expected, the diffusivity coefficients decreased significantly when alginate was added to the
formulation. The largest difference in results between the two fitting approaches used (D 1 and D 2 ), was
observed with the pectin film, which indicates that the diffusion is more influenced by swelling. Bajpai et
al. [21] produced alginate and pectin beads crosslinked with calcium and studied the release of potassium
nitrate in water. The authors observed that the release rate increased with an increase in the pectin
concentration. This behavior was attributed to the higher hydrophilicity of pectin, which causes relaxation
of the chains, resulting in faster release of the active agent. The films with higher proportions of alginate
also had a higher degree of crosslinking and created difficulties for natamycin mobility. Zactiti &
Kieckbusch [8] observed that the diffusivity of potassium sorbate in water decreased when the
concentration of calcium used in the crosslinking of alginate films was increased.
Although the diffusivity was higher for pectin films, the observed values were low compared to other
results on release of antimicrobial agents. For example, values found in the literature for potassium
sorbate, lisosyme and sorbic acid diffusivities are in the order of 10 -8 cm 2 /s [9, 22, 23].
Diffusion exponents for the Power Law Model (Equation 3) were obtained by plotting ln (M t /M ∞ ) versus
ln (t). The diffusional exponent (n) was calculated from the angular coefficients and the diffusional
constant (k) was obtained from the linear coefficients of the fitting line. As shown in Table 2, all
formulations presented a diffusional exponent (n) between 0.5 and 1, which are characteristic of
anomalous diffusion, a mechanism in which the diffusion rate of the solvent and the relaxation of the
polymeric chains are of the same order of magnitude. The deviation from Fickian behavior indicates that
the polymer relaxation phenomenon is more prominent and may affect the release of natamycin during
the first moments of the process [17]. An anomalous diffusion mechanism was also observed by Zactiti &
Kiecbusch [8] for alginate films with potassium sorbate. Flores et al. [24] and Ozdemir and Floros [22]
also observed deviations from ideal Fickian behavior during release of potassium sorbate from starch and
protein films, respectively.
CONCLUSION
The incorporation of natamycin as an antimicrobial agent affected tensile strength and, particularly, the
opacity of active films. When pectin was used as a biopolymer, the results revealed that natamycin was
not homogenously incorporated into the structure, which caused its rapid release as well as high water
solubility values. Composite films tended to present intermediary properties when compared to both
simple films. The diffusion coefficient obtained by adjusting the experimental data to Fick’s second law
varied from 9.53.10 -9 to 9.22.10 -12 cm 2 /s. The good compatibility of natamycin with alginate, the low
diffusion coefficient and the excellent physical properties indicate that this film has great potential for use
in antimicrobial packaging.

Acknowledgments
The authors acknowledge The State of São Paulo Research Foundation – FAPESP for financial support
(Proc. 2008/52830-9).
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