International Journal of Food Microbiology 164 (2013) 135140

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International Journal of Food Microbiology

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

Effects of nisin-incorporated lms on the microbiological and

physicochemical quality of minimally processed mangoes
Ana Andra Teixeira Barbosa a,, Hyrla Grazielle Silva de Arajo b, Patrcia Nogueira Matos b,
Marcelo Augusto Guitierrez Carnelossi b, Alessandra Almeida de Castro b
a
Departamento de Morfologia, Universidade Federal de Sergipe, So Cristvo, Sergipe, Brazil
b
Departamento de Tecnologia de Alimentos, Universidade Federal de Sergipe, So Cristvo, Sergipe, Brazil

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

Article history: The aim of this study is to examine the effects of nisin-incorporated cellulose lms on the physicochemical and
Received 12 November 2012 microbiological qualities of minimally processed mangoes. The use of antimicrobial lms did not affect the phys-
Received in revised form 3 April 2013 icochemical characteristics of mangoes and showed antimicrobial activity against Staphylococcus aureus, Listeria
Accepted 6 April 2013
monocytogenes, Alicyclobacillus acidoterrestris and Bacillus cereus. The mango slices were inoculated with S. aureus
Available online 11 April 2013
and L. monocytogenes (107 CFU/g), and the viable cell numbers remained at 105 and 106 CFU/g, respectively, after
Keywords:
12 days. In samples packed with antimicrobial lms, the viable number of L. monocytogenes cells was reduced
Bacteriocin below the detection level after 4 days. After 6 days, a reduction of six log units was observed for S. aureus. In con-
Minimally processed fruits clusion, nisin showed antimicrobial activity in mangoes without interfering with the organoleptic characteristics
Food preservation of the fruit. This result suggests that nisin could potentially be used in active packing to improve the safety of
Active packing minimally processed mangoes.
2013 Elsevier B.V. All rights reserved.

1. Introduction
The increasing demand for fresh-tasting, healthy, nutritious and
ready-to-eat foods has stimulated the expansion of minimally processed
fruit and vegetable markets worldwide (Abadias et al., 2008; Oms-Oliu et
al., 2010; Xiao et al., 2011). These products have a limited shelf life due to
processing, which accelerates biochemical reactions and increases the
susceptibility of these products to microbial contamination and growth
(Abadias et al., 2008; Botelho et al., 2010; Mukherjee et al., 2004).
The increased consumption of these products has been associ-
ated with foodborne illness (Beuchat, 2002; Mukherjee et al., 2006;
Olaimat and Holley, 2012; Rosa and Carvalho, 2000). Studies have indi-
cated that these foods can be contaminated with various bacterial path-
ogens, including Salmonella, Shigella, Escherichia coli O157:H7, Listeria
monocytogenes and Staphylococcus aureus (Beuchat, 1996; De Roever,
1998; Nguyen-the and Carlin, 1994). Recently, Alegre et al. (2010) dem-
onstrated the growth of E. coli O157:H7, Salmonella and Listeria innocua
in minimally processed apples. Pinheiro et al. (2005) also isolated coli-
forms, Salmonella sp. and molds from several minimally processed
fruits.
Thus, to further expand the minimally processed food market, it
is necessary to develop preservation techniques that maintain the
nutritional and sensory quality of the product and ensure microbio-
logical safety. Several studies have proposed alternative strategies in
the preservation of these products (Almenar et al., 2007; Considine
et al., 2008). Among these, the use of bacteriocins from lactic acid
bacteria has been widely considered, as these substances are natural
preservatives, avoiding the addition of synthetic compounds to food
(Carvalho et al., 2007a,b, 2008; Cotter et al., 2005).
Bacteriocins have been used for food preservation in active packing
(Appendini and Hotchkiss, 2002; Jin et al., 2010; Santiago-Silva et al.,
2009) and represents a potential alternative for the preservation of min-
imally processed fruits. Nisin is a bacteriocin that has been approved for
use in some foods and is a great candidate for fruit preservation, as this
compound exhibits high antibacterial activity, stability at an acidic pH,
an inability to affect changes in the food product and a relatively low tox-
icity to humans (Cleveland et al., 2001). To our knowledge this bacterio-
cin has not yet been tested in the preservation of minimally processed
fruits.
In this study, we examined the effects of nisin in cellulose lms on
the physicochemical and microbiological quality of minimally processed
mangoes.
2. Materials and methods

2.1. Microorganisms and growth conditions

Corresponding author at: Departamento de Morfologia, Universidade Federal de
Sergipe, So Cristvo, SE 49100-000, Brazil. Tel.: + 55 79 2105 6618; fax: + 55 79
2105 6620. S. aureus ATCC 8095, Bacillus cereus ATCC 4504 and L. monocytogenes
E-mail address: ana.barbosaufs@yahoo.com.br (A.A.T. Barbosa). ATCC 7644 were grown in BHI media (HIMDEDIA, Mumbai, India) at

0168-1605/$ see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ijfoodmicro.2013.04.004
136 A.A.T. Barbosa et al. / International Journal of Food Microbiology 164 (2013) 135140
37 C. Alicyclobacillus acidoterrestris DSMZ 2498 was grown at 40 C in
AAM (A. acidoterrestris media) according to the methods of Yamazaki
et al. (2000). The indicator strain Lactococcus lactis ATCC 19435 was cul-
tivated in MRS media (HIMDEDIA, Mumbai, India) at 30 C.
2.2. Film production
The lms were produced using cellulose acetate (Rhodia, Freiburg,
Germany) dissolved in acetone P.A. (Synth, SP, Brazil) using the cast-
ing method according to Santiago-Silva et al. (2009). The increasing
concentration of cellulose acetate was tested (3 to 12% v/w). Nisin
(Chrisin C; CHL Hansen) was incorporated into the emulsion at 25%
and 50% (w/w) of the cellulose weight, with a nal concentration of
7,500 and 15,000 IU (International Units) per mL of acetone, respec-
tively, if 3% cellulose acetate was used. The antimicrobial lms were
UV sterilized for 2 min on each side for use in these experiments.
The lm thickness was measured using a micrometer (precision:
025 mm, Pantec, Taubat, SP). The values were calculated as an av-
erage of ve measurements of three randomly selected lms. The
upper surface of the lms was analyzed using intermittent contact
atomic force microscopy (IT-AFM; NT-MDT Co., Ntegra Prima, Russia)
in air at room temperature. Scan areas of 40 m 2 were obtained.
2.3. The antimicrobial activity and stability of the lms
The antimicrobial activity of the lms was tested against S. aureus
ATCC 8095, B. cereus ATCC 4504, A. acidoterrestris DSMZ 2498 and
L. monocytogenes ATCC 7644 using the diffusion method. Approxi-
mately, 10 6 CFU/mL of the microorganisms was inoculated in solid
media and dispensed into Petri dishes. Sterile 2-cm diameter antimi-
crobial lms were placed in the dish and incubated at the optimal
growth temperature for each microorganism. After 24 h, the inhibi-
tion zones formed on the lms were analyzed. A control treatment
without nisin was also included.
The stability of nisin on the lms was tested using L. lactis ATCC
19435 as an indicator strain according to the diffusion method de-
scribed above. The antimicrobial lms were produced and sterilized,
and the samples were incubated at room temperature and 4 C. After
15, 30 and 70 days, the samples were collected, and the antimicrobial
activity was tested.
2.4. Minimal processing of mangoes
Tommy Atkins mangoes were purchased from a local market at the
mature-green stage, showing a purple-green color, which is considered
perfect for eating. The fruits were initially immersed in a solution of so-
dium hypochlorite (200 mg/L) for 10 min and rinsed with chlorinated
water (50 mg/L) for supercial disinfection. Following good manufactur-
ing practices, the fruits were manually peeled and sliced.
2.5. The antimicrobial activity of the lms on minimally processed
mangoes
Approximately 25 g of mango slices was processed as described in
2.4 and sterilized under UV light for 3 min on each side. The slices
were immersed for 5 min in a 0.1% peptone solution containing ap-
proximately 10 8 cells/mL of S. aureus or L. monocytogenes. The inocu-
lated mango slices were covered with cellulose lms containing 25%
nisin and then packed in polystyrene trays covered with PVC lm.
Control treatments without nisin were also conducted.
The microbial analyses were performed after 0, 2, 4, 6 and 12 days at
refrigeration temperature (5 1 C). The mango slices (25 g) were
manually homogenized in 225 mL of saline solution (0.85% NaCl)
using a sterile bag (540 mL, TWIRL'EM, QC Canada). The samples
were collected, serially diluted and plated onto BHI agar to deter-
mine the viable cell number.
2.6. Effect of lms on the physicochemical characteristics of minimally
processed mangoes
The mango slices (60 g) were processed as described in Section 2.4
and covered with cellulose lms containing 25% nisin and then packed
in polystyrene trays covered with PVC lm. The trays were stored at
5 C for 12 days. Samples were collected every 3 days, and the soluble
solids (expressed in Brix), total titratable acidity, pH level, vitamin C
content and browning index were evaluated (Carnelossi, 2000).
The experimental design was completely randomized in a 1 5 fac-
torial (1 storage temperature) with 5 periods of storage and three
replicates. The control treatments without nisin and without cellu-
lose lms were also performed.
2.7. Statistical analysis
All treatments were repeated at least three times and each repetition
was conducted in triplicate. The reported data represents arithmetic
mean values, and the error bars in the gures refer to the standard devi-
ation of the mean.
3. Results
3.1. Properties, antimicrobial activity and stability of the lms
As the acetate concentration doubled, an increase in the lm
thickness of approximately 25% was observed (Table 1). However,
the cellulose acetate concentration in the lms with sufcient exi-
bility and malleability for fruit packing was 3% and 6%. The AFM im-
ages of the lms showed no morphological difference at these acetate
concentrations (results not shown). When a nisin concentration of 25
and 50% was added to the cellulose emulsion, the lm thickness in-
creased above 50% (Table 1). No signicant difference was observed in
lms containing 25 or 50% of nisin in the same concentration of acetate
(Table 1). However, when 6% acetate was used, the lms with bacterio-
cin were more rigid and difcult to handle. The AFM images indicated
that lms produced using 3% cellulose acetate resulted in a large quan-
tity of pores when compared to the 6% cellulose acetate (Fig. 1). The an-
timicrobial tests also indicated that when 6% acetate was added, lower
inhibition zones were observed (Fig. 2).
After the addition of nisin, the lms produced with 3% acetate
were as thick as the lms produced with 9% acetate without bacteri-
ocin (Table 1). However, the lms generated from 3% acetate did not
display the same stiffness as observed with the lms generated from
9% acetate. Therefore, based on these results, 3% cellulose acetate was
sufcient to generate antimicrobial lms suitable for packaging min-
imally processed fruit. Considering that at this acetate concentration,
25 or 50% nisin showed the same inhibition zones (Fig. 2B and C), 25%
nisin was added to the lms used in the subsequent experiments.
Antimicrobial tests using the diffusion method showed that the
antimicrobial lm inhibited S. aureus, L. monocytogenes, B. cereus
and A. acidoterrestris strains in vitro (Fig. 3). The antimicrobial activity
Table 1
Thickness of cellulose lms without nisin (control) and with 25% and 50% of nisin.
Films (% cellulose acetate) Thickness (m)
Controld Nisind
25% 50%
c bc
3 34.00 0.13 63.00 0.20 66.00 0.16bc
6 45.00 0.01bc 118.00 0.21a 123.00 0.01a
9 59.00 0.07bc
12 76.60 0.02b
Mean of 5 repetitions () not tested.
d
Mean followed by the same letter don't differ at 1% of signicance.
 A.A.T. Barbosa et al. / International Journal of Food Microbiology 164 (2013) 135140 137

Fig. 1. AFM images of cellulose lms obtained using the casting method. Nisin was incorporated into the lms at 0 (A) and 25% (B; C). Acetate cellulose was used at 3% (A; B) and 6%
(C). Three-dimensional images are also shown (D; E; F, respectively). Scan areas of 40 m2 were analyzed.

of nisin incorporated lms remained stable, even after 70 days of storage
at refrigeration (4 C 2) or room (30 C 2) temperatures (Fig. 4).
3.2. Antimicrobial activity of lm incorporated with nisin in minimally
processed mango
After two days of storage a decrease in two log units in viable cell
number was observed in mango slices inoculated with approximately
10 7 CFU/g of S. aureus and treated with antimicrobial lm containing
nisin (Fig. 5A). The number of viable cells in the control treatment
(without nisin) exhibited a reduction of only one log unit after six
days of storage (Fig. 5A). However, after the sixth day, a 100,000-fold re-
duction in the viable cell number was observed for nisin compared with
the control treatment (Fig. 5A). After 12 days, the number of viable cells
in the control treatment was 105 CFU/g, and no viable cells were detected
in mangoes packed with nisin (Fig. 5A).
For L. monocytogenes, the number of viable cells in the control treat-
ment was maintained at 107 CFU/g for six days, and a reduction of only
one log unit was observed on the 12th day (Fig. 5B). The samples
packed with antimicrobial lms showed a 1000-fold reduction in the
number of viable cells after two days of storage, and no viable cells
were detected after the 4th day (Fig. 5B).

3.3. Effect of nisin on the physicochemical characteristics of minimally

processed mangoes

Mango slices were stored at 5 C and no signicant difference (at 5%

Fig. 2. Antimicrobial activity of lms containing nisin against the indicator strain L. lactis
ATCC 19435. Nisin was used at 0 (A), 25% (B and D) and 50% (C and E) of the acetate
weight. The acetate concentrations tested were 3% (A, B and C) and 6% (D and E) of the
acetone volume (v/w). The antimicrobial activity of the lms was assessed using the dif-
fusion method in solid media previously inoculated with L. lactis at 106 CFU/mL. The plates
were incubated for at least 24 h.
by Tukey's test) in the pH, total titratable acidity, vitamin C content,
browning index and soluble solids (SS) was observed for samples
packed with or without nisin (Table 2). The physicochemical character-
istics of mangoes were maintained for 9 days of storage under both
treatments (Table 2). The same result was observed for samples packed
with PVC lms alone (results not shown).
In all treatments, the pH was reduced during the rst 3 days,
remaining constant until the end of the storage period. Consequently,
the total acid content increased during the rst 3 days and was subse-
quently maintained between 0.2 and 0.3% citric acid until the 12th
day of storage (Table 2). The vitamin C content in all treatments was re-
duced during the rst 3 days and remained constant until the 9th day.
The soluble solids were increased during the rst 6 days of storage,
and the browning index generally remained unchanged throughout
the storage period (Table 2).
4. Discussion
The use of minimally processed fresh-cut fruits have raised food
safety concerns due to the increased availability of nutrients on cut
138 A.A.T. Barbosa et al. / International Journal of Food Microbiology 164 (2013) 135140

Fig. 3. Antimicrobial activity of lms containing nisin (N) against S. aureus ATCC 8095 (A), A. acidoterrestris DSMZ 2498 (B), B. cereus ATCC 4504 (C) and L. monocytogenes ATCC 7644
(D). The antimicrobial activity of the lms was assessed using the diffusion method in solid media previously inoculated with microorganisms at 106 CFU/mL. The plates were in-
cubated for at least 24 h, and the inhibition zones were analyzed and are indicated with the arrows in the gure (N). Control treatments are also shown (C).

surfaces and the greater potential for contamination from the increased
amount of handling (Leverentz et al., 2003). Generally, the safety of
these products primarily reects adequate chill chain and hygiene
Fig. 4. Antimicrobial activity of nisin-incorporated lms after 70 days of incubation at
4 C (A) or room (30 C 2) temperature (B). The antimicrobial activity of the lms
was assessed using the diffusion method in solid media previously inoculated with
the indicator strain L. lactis ATCC 19435 at 106 CFU/mL. The plates were incubated
for at least 24 h and the inhibition zones were analyzed.
practices, which are difcult to implement and control (Belletti et al.,
2008). Thus, the development of strategies to increase the safety of
these products is needed, and bacteriocins are great candidates for use
in hurdle technology.
In this study, we examined the effects of nisin-incorporated lms
against pathogen and spoilage microorganisms associated with fruit
products. The advantage of using bacteriocin in lms instead of apply-
ing it directly onto food surfaces is associated with the increased sta-
bility of the compound and the control of its release in foods, which
ensures food safety during storage (Guiga et al., 2010).
Atomic force microscopy (AFM) was used for lm surface charac-
terization. La Storia et al. (2008) exploited AFM analysis to correlate
the surface topography of antimicrobial lms with antimicrobial ac-
tivities. These authors observed that a homogeneous distribution of
the bacteriocin in lms was directly related to a homogeneous anti-
microbial activity. Our analyses also showed a homogeneous nisin
distribution in acetate lms as well a homogeneous antimicrobial ac-
tivity in solid media.
The AFM images showed a more homogeneous and compact struc-
ture in antimicrobial lms produced with 6% acetate. However, lms
produced with this acetate concentration showed smaller inhibition
zones than lms produced with 3% of acetate. Our ndings indicate
that cellulose acetate may interact with the bacteriocin, interfering in
its activity or release to food surface. We suggest that, at an increased
concentration of acetate (6%), the bacteriocin is released more slowly
from the lm leading to smaller inhibition zones on the solid medium
during the incubation time. Further studies are needed to clarify this
issue.
 A.A.T. Barbosa et al. / International Journal of Food Microbiology 164 (2013) 135140 139

life of minimally processed fruits is typically 5 to 7 days (Watada, 1997).

Fig. 5. Effects of nisin on the viability of S. aureus (A) and L. monocytogenes (B) on minimally
processed mangoes. A total of 25 g of mango slices were inoculated with 107 CFU/g of each
microorganism and packed with (white bars) and without (black bars) nisin. The trays were
incubated at 5 C for 12 days, and samples were collected every 3 days, serially diluted and
plated onto BHI agar to determine the number of viable cells.
The antimicrobial lms inhibited strains of L. monocytogenes, S. aureus,
B. cereus and A. acidoterrestrisin vitro. These microorganisms have been
isolated as pathogens or spoilage microorganisms from many fruit
products (Carvalho et al., 2007a,b; Durak et al., 2010; Frder et al.,
2007; Vantarakis et al., 2011). The antimicrobial activity of lms remained
stable, even after 70 days at refrigerator or room temperatures. The shelf
Therefore, the antimicrobial activities of lms remain stable to ensure the
microbiological safety of the product.
Our results showed that physicochemical characteristics of min-
imally processed mangoes were maintained during 9 days and that
nisin did not change the physicochemical characteristics of the
product. Interestingly, nisin exhibited antimicrobial activity against
L. monocytogenes and S. aureus without interfering in the organolep-
tic characteristics of mangoes. Thus, the use of bacteriocins as a nat-
ural food preservative seems advantageous (Cleveland et al., 2001).
Further testing of the effect of nisin-incorporated lms on the total
viable counts of processed mango is needed to fully justify the use
of nisin-incorporated lms to extend shelf-life of packed mangoes.
Cellulose lms incorporated with nisin were efcient in eliminating
S. aureus and L. monocytogenes contamination from minimally processed
mango slices. We selected these microorganisms to do the in situ assay
because among the microorganisms tested in vitro, these gram-positive
pathogens were the most commonly isolated from minimally processed
vegetables (Abadias et al., 2008; El-Hadedy and El-Nour, 2012; Eni et al.,
2010; Frder et al., 2007; Nguz et al., 2005; Viswanathan and Kaur,
2001).
S. aureus produces an enterotoxin that can cause nausea, stomach
cramps, vomiting, and diarrhea (Hait, 2012). A dose of less than 1.0 g
in contaminated food will produce these symptoms, and this level is
obtained when S. aureus populations exceed 10 5 cells per gram (Hait,
2012). The antimicrobial lms used in this study inhibited S. aureus
cells inoculated in mango slices even when the initial contamination
was as high as 107 CFU/g. After 4 days of storage, the viable cell number
was reduced below infectious doses of this microorganism.
Outbreaks of listeriosis typically occur at pathogen populations great-
er than 103 CFU/g or CFU/mL (Tompkin, 2002). In this study, Listeria
remained viable in mango slices at populations above the infectious
dose, even after 12 days of storage. However, in the presence of nisin,
no viable cells were detected in mangoes after 4 days, even after an ini-
tial contamination of 107 CFU/mL.
The results obtained in this study suggest that the lms containing
nisin can potentially be used as a hurdle strategy to improve the safety
of minimally processed fruits. Indeed, nisin inhibited the growth of
S. aureus and L. monocytogenes in mangoes without interfering with
the appearance, texture and nutritional value of the fruit.
Acknowledgments
The authors would like to thank the Conselho de Desenvolvimento
Cientco e Tecnolgico (CNPq), Braslia, Brazil and the Fundao de
Apoio Pesquisa e inovao Tecnolgica do Estado de Sergipe
(FAPITEC), Sergipe, Brazil, for providing fellowships and nancial
support for this research. We thank Dr. Sukarno Olavo Ferreira, from
the Physics Department at the Universidade Federal de Viosa for tech-
nical assistance with the AFM analyses.

Table 2
Effect of nisin-incorporate lms on physicochemical characteristics of minimally processed mango. Control treatments without nisin are also shown.

Treatment Storage day

0 3 6 9 12

pH Control 4.30Aa 3.80Ab 4.03Aa 4.33Aa 4.29Aa

Nisin 4.30Aa 3.95Ab 4.32Aa 4.25Aa 4.20Aa
Acidity (% citric acid) Control 0.24Aa 0.47Ab 0.29Aa 0.24Aa 0.28Aa
Nisin 0.24Aa 0.41Ab 0.32Aa 0.23Aa 0.24Aa
Vitamin C (% ascorbic acid) Control 44.44Aa 29.07Ab 29.41Ab 26.04Ab 18.18Ac
Nisin 46.51Aa 23.25Ab 27.94Ab 31.25Ab 21.59Ac
Browning Index Control 188.94Aa 213.87Aa 194.45Aa 188.26Aa 206.83Aa
Nisin 188.94Aa 183.33Aa 199.89Aa 198.38Aa 201.73Aa
Solids soluble (Brix) Control 11.25Ab 11.69Ab 12.56Aa 12.12Aa 11.12Ab
Nisin 11.25Ab 10.81Ab 11.50Aa 11.75Aa 10.81Ab
Means for the same parameter followed by the same letter, uppercase in column and lowercase in line, did not differ signicantly by Tukey test at 5% probability.
140 A.A.T. Barbosa et al. / International Journal of Food Microbiology 164 (2013) 135140
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