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In: Food Processing: Methods, Techniques and Trends ISBN 978-1-60692-414-3

Editor: Valerie C. Bellinghouse. © 2009 Nova Science Publishers, Inc.

Chapter 19

NISIN ADSORPTION ON
TWO FOOD CONTACT SURFACES

Nelson Pérez-Guerra*, Clara Fuciños-González†, Paula Fajardo-


Bernárdez‡, Isabel Rodríguez-Amado, Raquel Rial-Otero,
Carmen González-Barreiro, Lorenzo Pastrana-Castro
Nutrition and Bromatology Group, Department of Analytical and Food Chemistry, Food
Science and Technology Faculty, Ourense Campus, University of Vigo,
E-32004 Ourense, Spain

ABSTRACT
Some antimicrobial substances including organic acids (sorbate, propionate and
benzoate) and fungicides have been commonly used to control microbial growth in food
products and extend their shelf-life. However, the application of these antimicrobial
substances as preservatives in the food industry has some drawbacks. In some cases, their
use could involve a safety risk to consumers if their release is not tightly controlled by
some mechanisms within the packaging material. In other cases, the levels of
antimicrobial substances required for inhibition may introduce a strong flavour to the
food. For these reasons and taking into account the increasing consumer demands for
minimally processed foods, the interest has been focused on finding natural and effective
preservatives such as the bacteriocins. These antimicrobial substances have been
commonly incorporated directly into the foods, in the formulation of the packaging
material or adhered on the food packaging surfaces. This last technology is studied in the
present work by determining the effects of Nisaplin concentration (0-12 g/L) and
temperature (8, 25, 40 and 60ºC) on the adsorption of nisin activity on two surfaces,

*
Author whom correspondence should be addressed. Tel. +34-988-387000. Fax +34-988-387001. E-mail:
nelsonpg@uvigo.es

cfucinos@alumnos.uvigo.es

pfajardo@uvigo.es
494 N. Pérez-Guerra, Clara Fuciños-González, Paula Fajardo-Bernárdez et al.

polyethyleneterephthalate (PET) and cellophane, which are commonly used as packaging


materials in the food industry.
For both surfaces, the increase in temperature led to a decrease in the affinity
between nisin and the surface. The PET adsorbed a higher amount of nisin activity (1.78
BU/cm2) in comparison with cellophane (1.67 BU/cm2) at 8ºC (P < 0.05). Adsorption of
nisin activity to cellophane surface described L-2 type curves for 40 and 60ºC and L-3
type curves for 8 and 25ºC. However, for PET surface, isotherms were L-2 type (at
60ºC), L-3 type (at 25 and 40ºC) and L-4 type (at 8ºC) curves. Nisin retained its
antibacterial activity once adsorbed to the food contact surfaces and was able to inhibit
the growth of Enterococcus hirae CECT 279 on Rothe agar medium. The effect of PET
or cellophane-based bioactive packaging in food was very encouraging. Their
effectiveness in inhibiting the microbial growth was indicated by reduced specific growth
rates and low total bacteria counts in milk and chopped veal meat.

Keywords: adsorption, antibacterial activity, food contact surfaces, isotherms, nisin

INTRODUCTION
The direct application of antimicrobials in food systems is a preservation method that has
been commonly used to extend shelf-life of food products and improve their quality and
safety by controlling the growth of undesirable organisms. However, effectiveness of these
antimicrobials in foods could be reduced because of leaching of the antimicrobial products
into the food matrix and the cross-reactions between the antimicrobials and some food
components (Dawson et al., 2005; Mauriello et al., 2005). Therefore, the interest has been
focused on the use of packaging films containing antimicrobial products to prevent the
growth of spoilage organisms and many food-borne pathogenic bacteria and their spores
(Dawson et al., 2005; Mauriello et al., 2005; Guerra et al., 2005a; 2005b). These
antimicrobial-containing packaging materials can provide a microbiological hurdle
downstream by a controlled release of the preservative into the food (Bower et al., 1998; Han,
2000; Cutter, 2002; Quintavalla and Vicini, 2002; Dawson et al., 2005).
However, the use of some antimicrobial compounds could have some major drawbacks.
Then, the antimicrobial could have a safety risk to consumers if its migration into the food is
not tightly controlled by some mechanisms within the packaging material (Ronner and Wong,
1993; Vermeiren et al., 1999; Scannell et al., 2000). For example, excessive migration of SO2
from pads of sodium metabisulphite-incorporated microporous material to the foods could
cause toxicological problems (Ozdemir and Sadikoglu, 1998; Vermeiren et al., 1999). In the
same way, the use of butylated hydroxytoluene in contact with foods (Vermeiren et al., 1999;
Suppakul et al., 2003) has been questioned due to its tendency to accumulate in human
adipose tissue (Vermeiren et al., 1999). On the other hand, the level of antimicrobial required
for inhibition may introduce a strong flavour to the food that leads to a loss in its sensory
properties, thus reducing its quality (Guerra et al., 2005b).
For these reasons, the production of antimicrobial films using natural preservatives such
as bacteriocins (mainly nisin produced by Lactococcus lactis) has received a great attention.
Nisin is active against a variety of Gram-positive bacteria, including spoilage organisms and
food-borne pathogens such as L. monocytogenes, Clostridium botulinum and Staphylococcus
Antibacterial Activity of Nisin-absorbed Surfaces 495

aureus (Guerra and Pastrana, 2002). On the other hand, when combined with other inhibitory
factors, nisin can inhibit growth of some Gram-negative bacteria, thereby reducing the
amount of chemicals added to the food and decreasing the intensity of the processing
conditions (Daeschel et al., 1992). In addition, nisin is heat stable, non-toxic and sensitive to
the action of digestive proteases (Guerra and Pastrana, 2002).
In the design of an antimicrobial food contact surface coated with bacteriocins for active
packaging applications, particular adsorption of the antimicrobial agent should be increased
as much as possible to deliver a high antimicrobial activity to food systems. However, the
amount of bacteriocin adsorbed is greatly influenced by the properties of the food contact
surface (absorbent), the bacteriocin (absorbate) concentration in the solution and the
temperature (Daeschel et al., 1992; Kim and Hong, 2000; Guerra et al., 2005a; 2005b). In this
way, the adsorption behaviour of bacteriocins on a surface and their effect in preventing
bacterial adhesion have been studied with increased interest in food processing (Table 1) and
in other fields such as biotechnology and medicine (Fontana et al., 2006; Fontana et al.,
2007).
In this investigation, the adsorption kinetic of nisin activity from two Nisaplin solutions
(containing 1.2 and 12 mg of nisin per liter) to PET and cellophane surfaces was firstly
studied. After determining the time to reach adsorption equilibrium, the effect of temperature
on the adsorption of nisin activity on both surfaces was investigated. Then, the stability of the
Nisaplin-adsorbed surfaces was ascertained using an activity retention test with Enterococcus
hirae CECT 279 as a target bacterium. Finally, the functionality of adsorbed nisin activity on
PET and cellophane surfaces was determined by checking its effectiveness in reducing
populations of natural microbiota of pasteurized skim milk and fresh veal meat.

MATERIALS AND METHODS

Bacterial Strains

Enterococcus hirae CECT 279, the target organism used in the bacteriocin photometric
activity assay, was obtained from the Spanish Type Culture Collection (CECT). This
bacterium was maintained at 4ºC on Rothe agar (Cultimed, Madrid, Spain) slants.

Bacteriocin Preparation and Quantitation

Nisaplin® was obtained from Aplin & Barrett, Ltd (Danisco Cultor, England) with the
following composition (in %): nisin, 2.5, sodium chloride, 77.5, milk protein, 12.0,
carbohydrates, 6.0 and moisture, 2.0. Sodium phosphate buffer solutions were prepared using
chemicals of analytical grade and distilled, deionised water. Nisaplin was added to 0.01 M
monobasic sodium monophosphate (pH 4.5) to assure complete solubilisation of nisin. This
suspension was then centrifuged (5000 × g for 10 min) to remove insoluble impurities.
Subsequently, 0.01 M dibasic sodium monophosphate (pH 9.0) was added until reaching a pH
of 6.0 and a final concentration of 12.0 g/L of Nisaplin (0.3 mg of nisin/mL). Different
496 N. Pérez-Guerra, Clara Fuciños-González, Paula Fajardo-Bernárdez et al.

concentrations of Nisaplin solutions were prepared by dilution of stock solution with sterile
0.01 M sodium phosphate buffer (pH 6.0).

Table 1. Some examples on the use of bacteriocins in food packaging materials

Bacteriocin Material Food Mode of Reference


inclusion
or
adherence
Bacteriocin Polythene Frankfurters Adsorption Ercolini et
32Y al., 2006
BLIS Polythene Pork steak Adsorption Mauriello et
and ground al., 2004
beef
Enterocins A Biodegradable films (alginate, Cooked ham Inclusion Marcos et al.,
and B zein and polyvinyl alcohol) 2007
Enterocin Low density polyethylene Frankfurters Adsorption Iseppi et al.,
416K1 (LDPE) and fresh 2008
cheeses
Lacticin Cellulose Sliced cheese Adsorption Scannell et
3147 Polyethylene/polyamide and ham al., 2000
Nisaplin Cellophane Chopped meat Adsorption Guerra et al.,
2005a
PET Pasteurized Adsorption Guerra et al.,
skim milk 2005b
Cellulose Sliced cheese Adsorption Scannell et
Polyethylene/polyamide and ham al., 2000

Nisin Palmitoylated alginate-based Beef Inclusion Millette et


films and activated alginate al., 2007
beads
Methylcellulose/hydroxypropyl- Vacuum- Adsorption Franklin et
methylcellulose packaged hot al., 2004
dogs
Soy-based films Turkey Adsorption Dawson et
bologna al., 2002
Low-density polyethylene Milk Adsorption Mauriello et
al., 2005
Calcium alginate, 0.75% Smoked Inclusion Datta et al.,
agar edible coating, and zein salmon 2008
coatings
Protein- and polysaccharide- Poultry skin Adsorption Natrajan and
based films Sheldon,
2000
Antibacterial Activity of Nisin-absorbed Surfaces 497

Paperboard coated with vinyl Milk and Adsorption Lee et al.,


acetate-ethylene copolymer orange juice 2004
incorporating nisin and/or
chitosan
Pediocin PD- Stainless steel Wine Adsorption Nel et al.,
1, nisin, and 2002
plantaricin
423

The antimicrobial activity of each solution of Nisaplin was determined against


Enterococcus hirae CECT 279 using a turbidimetric bioassay (Cabo et al., 1999). Briefly, the
method is based on the determination of growth inhibition (at 700 nm) of the target bacterium
(E. hirae CECT 279) caused by serial dilutions of bacteriocin samples. Firstly, Nisaplin
samples were diluted as needed in distilled sterile water. Secondly, equal volumes (2.5 mL) of
diluted bacteriocin samples and a culture of E. hirae CECT 279 (diluted to an absorbance of
0.2 at 700 nm with sterile buffered Rothe broth (pH 6.3)) were mixed in sterile culture tubes.
Finally, the tubes were incubated for 6 h at 30ºC. Controls consisted in three culture tubes in
which the diluted bacteriocin samples were substituted by distilled sterile water. Growth
inhibition was measured spectrophotometrically at 700 nm. Dose/response curves were
obtained from these data. Nisin activity was calculated as Bacteriocin Units (BU) per mL (1
BU/mL = amount of bacteriocin needed to obtain 50% growth inhibition compared to control
tubes) as indicated by Murado et al., (2002).

Preparation of Solid Surfaces

PET was provided as rectangular plates (18.4 cm by 3.0 cm by 0.02 cm) by Catalana de
Polímeros (El Prat de Llobregat, Barcelona). The cellophane “P” type surfaces were provided
by La cellophane Española, S. A. (Burgos, Spain). These materials were cut into pieces of
convenient dimensions for each experiment. The pieces were washed with 1% (w/v) sodium
dodecyl sulphate (SDS, Sigma, Madrid, Spain), rinsed with distilled, deionised water and
washed again with ethanol (Panreac, Barcelona, Spain) during 10 min under shaking
conditions each time. Finally, materials were rinsed with distilled, deionised water and air-
dried.

Kinetics of Nisin Adsorption

The times needed to reach adsorption equilibrium of nisin activity to the two surfaces
were determined using two Nisaplin solutions (1.2 and 12.0 g/L) prepared as described above.
Rectangular pieces of PET and cellophane “P” type with surface areas of 110.4 and 53.8 cm2,
respectively, were cleaned as described before. Each surface was placed into individual sterile
glass bottles (50 mL) and covered with 20 mL (in case of cellophane) or 25 mL (in case of
PET) of the corresponding Nisaplin solution. Then, they were incubated at four different
temperatures (8, 25, 40 and 60ºC) under static conditions to allow adsorption. Triplicate
498 N. Pérez-Guerra, Clara Fuciños-González, Paula Fajardo-Bernárdez et al.

samples were run simultaneously. Control samples (20 and 25 mL) of each Nisaplin solution
were placed into sterile glass bottles (50 mL) and subjected to the above adsorption
conditions, without the presence of any surface. At pre-established times (6, 9, 12, 24 and 36
h), the surfaces were removed and both the remaining Nisaplin solutions and the control
solutions of Nisaplin were analyzed for antibacterial activity using the above mentioned
photometric assay (Cabo et al., 1999). This procedure eliminated the need to quantify the
amounts of nisin adsorbed to the walls of the glass bottles.
The amounts of antibacterial activity (BU) adsorbed to each material were determined
from the difference between the total amounts of nisin in the controls and in the remaining
solutions (Norde and Zoungrama, 1998; Scannell et al., 2000). To be consistent, the results
were divided by the area of each surface and expressed as adsorbed BU/cm2 of surface.

Nisin Adsorption Isotherms

To obtain the adsorption isotherms, the PET and cellophane surfaces were placed into
individual sterile glass bottles (50 mL) and exposed to the different Nisaplin solutions (with
concentrations ranged from 0.6 to 12.0 g/L) under static conditions as described above. The
samples were then incubated for 12 h at four different temperatures: 8, 25, 40 and 60ºC.
Triplicate samples of each material were run simultaneously. For each surface, control
samples of each Nisaplin solution were placed into sterile glass bottles (50 mL) and subjected
to the above adsorption conditions, without the presence of surface. The nisin activities
adsorbed on each surface (expressed as BU/cm2) were calculated as described in the previous
section.

Antibacterial Activity of Nisin-adsorbed Surfaces

Bacteriocin activity of surfaces with adsorbed nisin was determined using the agar
diffusion assay (Guerra and Pastrana, 2002). Molten Rothe agar was cooled to 45ºC and
subsequently seeded with 0.1 mL of an overnight culture of the indicator strain, Enterococcus
hirae CECT 279. Inoculated medium was dispensed in sterile Petri dishes and allowed to
solidify in a laminar flow hood.
To prepare a material with adsorbed nisin activity, cleaned rectangular plates (2.5 cm by
1.8 cm) of PET (0.05 cm in thickness) and cellophane (0.04 cm in thickness) were placed into
individual sterile glass bottles (50 mL) and covered with 25 mL of a 10.0-g/L Nisaplin
buffered solution (0.01M NaH2PO4, 0.01M Na2HPO4, pH=6.0) in case of PET, or with 20 mL
of a 12.0-g/L Nisaplin buffered solution in case of cellophane. The surfaces immersed in the
Nisaplin solutions were stored at 8ºC during 12 h under static conditions. Subsequently, the
surfaces with adsorbed nisin activity were rinsed twice with distilled and deionised water and
placed on the agar surface. Cleaned surfaces contacted with phosphate buffer (0.01M
NaH2PO4, 0.01M Na2HPO4, pH=6.0) without Nisaplin were used as controls. All agar plates
were held at 4ºC for 4 h to allow nisin diffusion prior to incubation at 30ºC for 48 h (Guerra
et al., 2005a; 2005b).
Antibacterial Activity of Nisin-absorbed Surfaces 499

Antibacterial Efficacy of the Nisaplin-adsorbed Surfaces (PET and


Cellophane) in Food Systems

Five-hundred-mL PET bottles (cleaned as described above for pieces of materials) of the
type used for mineral water (Cabreiroá, S.A., Ourense, Spain) were filled with 500 mL of a
10.0 g/L Nisaplin buffered solution and maintained at 25ºC for 12 h. After adsorption, the
bacteriocin solution was removed under sterile conditions and PET bottles were rinsed and
dried as described above. Subsequently, the bottles were filled with 500 mL of pasteurized
skim milk, which was obtained from a local supermarket 24 h after production. Controls
consisted of clean PET bottles without adsorbed Nisaplin. These samples were stored at 4ºC
and analyzed daily over a 24-day period. At selected intervals, two samples (1 mL) of skim
milk were withdrawn for bacterial enumeration. Each sample was serially diluted with sterile
buffered peptone water. Total aerobic plate counts (TAPC) were determined by the spread
plate technique on Plate Count Agar (Merck, Darmstadt, Germany). Plates were incubated at
30ºC for 48 h. Colonies were counted and results expressed as CFU per mL of milk. All
counts were carried out in triplicate using two samples of milk in each trial. Data obtained
were presented as mean values and standard deviations (Guerra et al., 2005a).
Disks of cellophane “P” type (9 cm in diameter) were used for the preparation of
bioactive packaging. These surfaces were previously prepared as described above for pieces
of materials and then contacted with a 12 g/L Nisaplin buffered solution (0.01M NaH2PO4,
0.01M Na2HPO4, pH=6.0) for 12 h at 8ºC.
The fresh veal meat, which was obtained from a local supermarket 24 h after slaughter,
was ground in a sterilized household blender (Moulinette Coupe-Coupe, Ecully, France).
Aliquots of 50 g of chopped meat were extended with a sterile spatula on the dried cellophane
surfaces treated with Nisaplin, resulting in thick layers of about 5 mm. Then, they were
covered with other treated cellophane surface such that the entire area was covered. The
controls consisted on 50 g samples of chopped meat extended on both cellophane surfaces
without Nisaplin and on cellophane surfaces previously treated with 0.01 M sodium
phosphate buffer (pH=6.0). All samples were incubated at 4ºC.
At selected intervals (1 day) up to 12 days, samples of chopped meat were withdrawn for
bacterial enumeration. From each sample of 50 g of chopped meat, a 10-g sample was mixed
with 90 mL of buffered peptone water and pummelled in a Stomacher (model 400, Seward,
London, UK) for 1 min. The homogenate was then serially diluted with sterile buffered
peptone water. An aliquot (0.1 mL of each serial dilution) was inoculated on prepoured plates
containing Plate Count agar (Merck, Darmstadt, Germany). Agar plates were incubated
aerobically at 30ºC for 48 h. Colonies were counted and results expressed as CFU per g of
meat. All calculations were based on six repetitions: all counts were performed three times
using two samples in each trial. Data were then reported as means and standard deviations
(Guerra et al., 2005b).

Statistical Methods

Individual experiments and analytical replications were performed in duplicate and


triplicate, respectively. All data points are represented by the mean with the standard error.
500 N. Pérez-Guerra, Clara Fuciños-González, Paula Fajardo-Bernárdez et al.

Data sets were analyzed by analysis of variance (ANOVA) on SPSS 8.0 for Windows.
Statistical significance occurs for a standard level of significance (P< 0.05).

RESULTS AND DISCUSSION

Nisin Adsorption Isotherms

To determine the potential protective activity of PET and cellophane surfaces with
adsorbed antibacterial activity against the attachment of pathogen bacteria, the nisin
adsorption capacity of these materials was determined throughout the construction of their
adsorption isotherms.

Figure 1. Kinetics of antimicrobial activity adsorption on rectangular pieces of PET (with a surface of
110.4 cm2) and cellophane (with a surface of 53.8 cm2) after their contact at 8ºC (Ο), 25ºC (∆), 40ºC ( )
and 60ºC (∇), with two solutions of Nisaplin (1.2 and 12.0 g/L) containing 30 (A) and 300 (B) mg of
nisin per liter, respectively. Experimental data reported are means ± standard deviations of three
replicates.
Antibacterial Activity of Nisin-absorbed Surfaces 501

The results of nisin activity adsorption kinetics from 1.2 and 12 g/L Nisaplin solutions to
the PET and cellophane surfaces at 8, 25, 40 and 60ºC are shown in Figure 1. The maximum
of adsorption was detected after 12 h of contact and remained constant thereafter, thus
showing that this was the time needed to reach equilibrium in all cases. Therefore, the nisin
adsorption isotherms at 8, 25, 40 and 60ºC were obtained after contacting the two surfaces
with the different Nisaplin solutions for 12 h.
The adsorption isotherms of nisin activity for PET and cellophane surfaces are presented
as plots of amounts of adsorbed nisin activity (BU/cm2) versus Nisaplin concentration (g/L)
in Figure 2. As it can be observed, the temperature influenced both the shape of the isotherms
obtained and the amounts of nisin activity adsorbed on each surface.
At 8ºC, adsorption of nisin activity on PET surface increased with the increase in
Nisaplin concentration, displaying three phases (left part of Figure 2). In the first phase, the
adsorbed amounts of nisin activity increased rapidly in the range of 0-1.2 g of Nisaplin/L. In
the second phase, the concentration of nisin activity increased slowly with the increase in
Nisaplin concentration from 1.2 to 8.0 g/L. However, when the Nisaplin concentration in
solution increased from 8.0 to 12.0 g/L, a new rise in the concentration of nisin activity
adsorbed was observed. At 25 and 40ºC, adsorption of nisin activity on this surface also
displayed three phases. The first exhibited a steep slope in the Nisaplin concentration range of
0-1.2 g/L (in case of isotherm at 25ºC) or 0-0.6 g/L (in case of isotherm at 40ºC). In the
second phase, the slope was less pronounced as Nisaplin concentration in solution raised from
1.2 to 4.0 g/L (isotherm at 25ºC) or from 1.2 to 10.0 g/L (isotherm at 40ºC). In the third
phase, the adsorption of nisin activity on the surface increased as concentration of Nisaplin
raised from 4.0 (at 25ºC) or 10.0 (at 40ºC) to 12.0 g/L. However, nisin activity adsorption at
60ºC exhibited only two phases, a steep initial slope in the Nisaplin concentration range of 0-
1.6 g/L, followed by a steady increase in the Nisaplin range of 1.6 to 12.0 g/L (left part of
Figure 2). With regard to cellophane surface (right part of Figure 2), it can be noted that
adsorption of nisin activity increased with the increase in Nisaplin concentration displaying
three phases at 8 and 25ºC or two phases at 40 and 60ºC.

Figure 2. Experimental adsorption isotherms of antibacterial activity on PET and cellophane surfaces at
8ºC (Ο), 25ºC (∆), 40ºC ( ) and 60ºC (∇). Adsorbed activity data were obtained after 12 h of
incubation of the surfaces with solutions containing different Nisaplin concentrations. Experimental
data reported are means ± standard deviations of three replicates.
502 N. Pérez-Guerra, Clara Fuciños-González, Paula Fajardo-Bernárdez et al.

Thus, in the first phase, in which nisin showed a higher surface affinity, the rapid increase
in nisin activity adsorption on both surfaces could be related to the existence of sites capable
of adsorbing the nisin molecules. The decrease in adsorption observed in the second phase
probably occurred because vacant sites became more difficult to find with the progressive
covering of the surfaces. In the third phase, the increase in the adsorbed amounts of nisin
activity could be attributed to the development of a fresh surface in which, the nisin
molecules are attracted by the own nisin molecules forming the first layer (Giles and Smith,
1974; Guerra et al., 2005a; 2005b). In addition, it can be noted the slope in the first phase for
the two surfaces was higher than that of the second phase (Figure 2). This was probably
because the forces generating the second and subsequent adsorbed layers are weaker than
those generating the first (Giles and Smith, 1974; Guerra et al., 2005a; 2005b).
These observations suggest that the adsorption of nisin activity on PET and cellophane
surfaces follows the monolayer (for PET at 60ºC and cellophane at 40 and 60ºC) and then
multilayer mechanism (for PET at 8, 25 and 40ºC and cellophane at 8 and 25ºC).
The experimental equilibrium data of nisin activity adsorbed were fitted with the
Langmuir isotherm by using least-squared regression analysis. The Langmuir isotherm
(Chang et al., 2000), in the present study, could be given by:

AAm ⋅ K ⋅ [N ]
AA =
1 + K ⋅ [N ]
(1)

Where AA is the adsorbed activity (BU/cm2 of surface), AAm and K are the maximum
adsorbed activity (BU/cm2 of surface) and the adsorption equilibrium constant (L/g),
respectively. [N] is the Nisaplin concentration in the solution (g/L).
The Langmuir isotherm predicts that the adsorbate concentration in the solid surface rises
monotonically with increasing adsorbate concentration in the solution until a plateau
(constant adsorbate concentration in the surface) is reached. This plateau adsorbate
concentration represents the monolayer concentration (Young et al., 1988). Therefore, this
isotherm is based on the assumption that the adsorbate molecules are adsorbed on a
energetically uniform surface forming only a monolayer. Thus, molecules of adsorbate are
adsorbed only on the free surface of the adsorbent (Young et al., 1988; Chang et al., 2000).
The Langmuir isotherms and the values of AA, K and correlation coefficient (r2) for both
the PET and cellophane surfaces at 8, 25, 40 and 60ºC are shown in Figure 3 and Table 2,
respectively.
Although the values of AAm and K decreased with the increase in temperature for both
surfaces (Table 2), the Langmuir model only provided a good fit of the experimental
isotherms obtained for PET at 60ºC (r2 = 0.941) and cellophane at 40 (r2 = 0.979) and 60ºC
(r2 = 0.995). These results confirm that, in these cases, the adsorption of nisin activity on both
surfaces might be limited to monolayer (Figures 2 and 3). According to the system of
isotherm classification (Giles and Smith, 1974), adsorption of nisin activity on PET surface
(left parts of Figures 2 and 3) described L-2 type (at 60ºC), L-3 type (at 25 and 40ºC) and L-4
type curves (at 8ºC). In the same way, the isotherms obtained for cellophane surface were
similar to the L-2 type curves at 40 and 60ºC and to the L-3 type curves for 8 and 25ºC (right
parts of Figures 2 and 3).
Antibacterial Activity of Nisin-absorbed Surfaces 503

Table 2. Parameter values of the Langmuir model for the isotherms of nisin activity on
PET and cellophane surfaces at four different temperatures

PET Cellophane
8ºC 25ºC 40ºC 60ºC 8ºC 25ºC 40ºC 60ºC
K 0.824 0.813 0.809 0.787 0.937 0.926 0.895 0.862
AAm 1.698 1.504 0.945 0.796 1.565 1.131 0.927 0.769
r2 0.893 0.890 0.874 0.941 0.901 0.881 0.979 0.995

On the other hand, the increase in temperature led to a decrease in the amount of nisin
activity adsorbed on the two surfaces (Figures 2 and 3). This finding is in agreement with a
previous observation (Kim and Hong, 2000) in which the increase in temperature produced a
reduction in the adsorbed amounts of both cellobiohydrolases I and II on microcrystalline
cellulose. This phenomenon could be a result of an increase in the excitation state of protein
molecules at higher temperature that could decrease the attractive forces between them and
the solid surfaces. On the other hand, the increase in the amounts of nisin adsorbed at lower
temperatures may be related to a reduction in translational energy of the bacteriocin, which
could favour its positioning for adsorption or to a reduction in energy available for desorption
of nisin once adsorbed (Kim and Hong, 2000).
From the comparison between the adsorption isotherms obtained for both surfaces, it can
be noted that the adsorption capacity of PET was significantly higher (P < 0.05) than that of
the cellophane surface for the four temperatures assayed (Figures 2, 3 and 4). Thus, the
maximal amounts of nisin activity adsorbed on PET and cellophane surfaces were
respectively, 1.78 and 1.67 BU/cm2 at 8ºC, 1.68 and 1.36 BU/cm2 at 25ºC, 1.11 and 0.91
BU/cm2 at 40ºC, and 0.77 and 0.69 BU/cm2 at 60ºC (Figures 2, 3 and 4). These differences in
the adsorption capacity of both surfaces could be related with their different chemical and
physical characteristics, including the surface elemental composition, hydrophobicity,
roughness, the amounts of pores on the surface, as well as their charge and free energy
(Gugala and Gogolewski, 2004).

Figure 3. Comparison between Langmuir model isotherm (full lines) and experimental adsorption data
(symbols) of antibacterial activity on the PET and cellophane surfaces at 8ºC (Ο), 25ºC (∆), 40ºC ( )
and 60ºC (∇). Experimental data reported are means ± standard deviations of three replicates.
504 N. Pérez-Guerra, Clara Fuciños-González, Paula Fajardo-Bernárdez et al.

Figure 4. Antibacterial activity adsorbed on PET and cellophane surfaces as a function of temperature.
Data of antibacterial activity adsorbed were obtained after immersing each surface in 0.6 (Ο), 4.0 (∇),
8.0 ( ) and 12.0 (∆) g/L Nisaplin solutions for 12h. Data reported are means ± standard deviations of
three replicates.

Antibacterial Activity of the Nisin-adsorbed Surfaces

The antibacterial activity of the nisin-adsorbed surfaces against Enterococcus hirae


CECT 279, a nisin-sensitive strain (Guerra and Pastrana, 2002), was detectable on both the
PET and cellophane surfaces pre-treated with Nisaplin. Thus, the thicknesses of the clear
zones of inhibition observed around the periphery of the surfaces treated with 10.0 g/L (in
case of PET) and 12.0 g/L Nisaplin buffered solution (in case of cellophane) were 0.6 mm
(Guerra et al., 2005a; 2005b). These observations may result from the diffusion of nisin into
the medium (Daeschel et al., 1992) or due to the fact that nisin retained its antibacterial
activity in the adsorbed state (Bower et al., 1998; Siragusa et al., 1999). Other researchers
have observed that both nisin and pediocin retained their antibacterial activity when adsorbed
onto different surfaces (Bower et al., 1995a, 1995b; Daeschel and McGuire. 1995; Ming et
al., 1997). In addition, it has been observed that nisin incorporated in different surfaces
retained its antibacterial activity against Lactobacillus helveticus and Brochothrix
thermosphacta (Siragusa et al., 1999), Micrococcus luteus ATCC 10240 (Mauriello et al.,
2005), Listeria monocytogenes ATCC 15313 (Dawson et al., 2005).

Antibacterial Activity of Nisaplin-adsorbed PET in a Food System

Fresh pasteurized skim milk packaged in the control PET bottles (untreated with
Nisaplin) showed a continue increase in the TAPC until the 10th day of incubation at 4ºC
(Table 3). Subsequently, the TAPC increased slightly and by the end of the experiment (24
days) had increased by approximately 1.2 log units (P < 0.05) in comparison with the TAPC
of the first day. In contrast to this, in PET bottles to which Nisaplin had been adsorbed, there
was an initial drop of 0.6 log units (form day 0 to day 1 of experiment), which was followed
by a slight increase from day 6 until the end of the incubation (day 24). However, at this
Antibacterial Activity of Nisin-absorbed Surfaces 505

point, the final TAPC was lower than the initial level (P < 0.05). In addition, it can be noted
that PET bottles prepared with Nisaplin reduced levels of TAPC on skim milk by
approximately 1.5 log units (P < 0.05) compared with the controls (PET bottles untreated
with Nisaplin) after 24 days of incubation. This represents a mean reduction of 96.4%, when
the mean counts (CFU/mL) of the pasteurized skim milk in the untreated PET bottles at each
sampling time are taken as 100%. These results indicate that PET bottles impregnated with
nisin significantly (P < 0.05) improved the microbial stability of milk.
Thus, it is reasonable to suppose that the use of bioactive PET bottles would give a good
protection against the outgrowth of nisin-sensitive strains, including species of Listeria,
Clostridium, Micrococcus and Staphylococcus that result from postpasteurization
contamination. If so, the use of PET bottles treated with Nisaplin could contribute to the
extension of the shelf life of skim milk. Similar results were obtained when samples of
pasteurized milk and orange juice were stored in contact with paperboards coated with nisin
and/or chitosan in a blinder of vinyl acetate ethylene copolymer at 3 and 10ºC (Lee et al.,
2004). At 3ºC, the use of these antimicrobial paperboards significantly extended lag time,
reduced the specific growth rates and consequently, decreased the maximum cell
concentration of the total aerobic bacteria and yeasts in both milk and orange juice (Lee et al.,
2004).

Table 3. Effect of Nisaplin-coated PET and untreated PET on the total aerobic plate
counts of pasteurized skim milk stored at 4ºC

Total aerobic plate counts of pasteurized skim milka


Days of storage Untreated PET bottles Treated PET bottles Reduction (%)b
0 2.5 ± 0.13 2.5 ± 0.13 0.0
1 2.7 ± 0.14 1.9 ± 0.17 81.8
2 3.0 ± 0.12 1.9 ± 0.12 91.9
4 3.3 ± 0.09 1.9 ± 0.28 96.0
6 3.4 ± 0.12 1.9 ± 0.09 96.3
8 3.5 ± 0.09 2.0 ± 0.04 97.2
10 3.6 ± 0.10 2.1 ± 0.05 96.8
12 3.7 ± 0.09 2.2 ± 0.08 96.8
16 3.6 ± 0.04 2.2 ± 0.09 96.8
20 3.7 ± 0.06 2.2 ± 0.08 97.1
24 3.7 ± 0.04 2.2 ± 0.07 96.4
a
Mean values of results for two milk samples and three analytical replications in log(CFU/mL of milk)
± SD.
b
Calculated as ((No-N)×100)/No, where No is the mean count of the pasteurized skim milk in the
untreated PET bottles and N is the mean count of the pasteurized skim milk in the Nisaplin-coated
PET bottles (both expressed as CFU/mL of milk).
506 N. Pérez-Guerra, Clara Fuciños-González, Paula Fajardo-Bernárdez et al.

Antibacterial Activity of Nisaplin-adsorbed Cellophane in a Food System

The total bacteria counts in the control inserts (non nisin-containing cellophane) appeared
to be significantly higher (P < 0.05) than those in the samples treated with nisin after storage
at 4ºC for 1 day and longer (Table 4).
The initial total population of these bacteria after chopping was in the region of 4.0 log
units. Fresh veal meat packaged in the control inserts showed only a small increase in the
total bacteria count after the first 4 days of incubation. However, from this time, the rate of
proliferation of these bacteria increased and by the end of the experiment the initial total
population had increased by approximately 1.4 log unit (Table 4). Contrarily, in nisin-
impregnated cellophane surfaces, there was an initial drop of 0.7 log units. Then, the cells
surviving nisin treatment resumed growth from the second day of incubation. However, the
final total bacteria count level reached after 12 days of incubation (approximately 3.9 log
units) was significantly lower (P < 0.05) than the initial total bacteria count level. This
represents a mean reduction of 96.7%, when the mean counts (CFU/g) of the veal meat
covered with the untreated cellophane surfaces at each sampling time are taken as 100%.
These results showed that bioactive cellophane to which nisin was adsorbed, gave a good
protection against bacterial growth and the possibility for extending the shelf life of chopped
meat.
Other researchers observed similar reductions in the total populations of bacteria, when
different meat samples were packaged with food packaging materials containing bacteriocins
(Ming et al., 1997; Scannell et al., 2000; Dawson et al., 2002; Lee et al., 2003; Mauriello et
al., 2004).

Table 4. Effect of Nisaplin-coated cellophane and untreated cellophane on the total


endogenic bacteria plate counts of fresh veal meat stored at 4ºC

Total aerobic plate counts (CFU/mL) of fresh veal meata


Days of storage Untreated cellophane Treated cellophane Reduction (%)b
0 4.0 ± 0.10 4.0 ± 0.10 0.0
1 4.2 ± 0.13 3.3 ± 0.11 89.0
2 4.3 ± 0.15 3.3 ± 0.15 90.2
4 4.5 ± 0.18 3.5 ± 0.16 88.3
6 4.8 ± 0.19 3.7 ± 0.11 91.4
8 5.3 ± 0.12 3.8 ± 0.17 96.7
10 5.3 ± 0.13 3.9 ± 0.15 95.7
12 5.4 ± 0.17 3.9 ± 0.10 96.7
a
Mean values of results for two veal meat samples and three analytical replications in log (CFU/g of
meat) ± SD.
b
Calculated as ((No-N)×100)/No, where No is the mean count of the meat packaged with the untreated
cellophane surfaces and N is the mean count of the meat packaged with the Nisaplin-coated
cellophane surfaces (both expressed as CFU/g of meat).
Antibacterial Activity of Nisin-absorbed Surfaces 507

CONCLUSION
The effectiveness of PET bottles and the cellophane surfaces impregnated with nisin in
inhibiting the microbial growth in milk and chopped veal meat was indicated by reduced
specific growth rates of their total aerobic plate counts. Both antimicrobial surfaces
significantly (P < 0.05) improved the microbial stability of milk and fresh veal meat.
The use of antimicrobial packaging in combination with storage at low temperatures
would provide foods with enhanced microbial stability. In addition, the use of packaging
materials treated with Nisaplin could contribute to the reduction of the amounts of chemicals
added to the food and to a decrease in the intensity of the processing conditions.

ACKNOWLEDGEMENTS
The research presented in this paper was financially supported by the Vicerrectorado de
Investigación da Universidade de Vigo, Spain, the Instituto Nacional de Investigación y
Tecnología Agraria y Alimentaria (INIA), Spain (project CAL01-045-C2-2), The Xunta de
Galicia, Spain (project PGIDT00BIO1E), and the Ministerio de Educación y Ciencia (project
MAT2006-11662-C03-03).

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