ARTICLE IN PRESS

Lebensm.-Wiss. u.-Technol. 37 (2004) 915

Use of entrapped microorganisms as biological oxygen scavengers in

food packaging applications
C. Altieria, M. Sinigagliaa, M.R. Corboa, G.G. Buonocoreb,
P. Falconea, M.A. Del Nobilea,*
b

a
University of Foggia, Via Napoli, 25-71100 Foggia, Italy
Department of Materials Engineering and Production, University of Naples Federico II, P.le Tecchio, 80-80125 Naples, Italy

Received 12 December 2002; accepted 6 May 2003

Abstract
In this work a new method is proposed to produce oxygen-scavenger lms using aerobic microorganisms as the active
compound. The manufacturing cycle of the investigated oxygen-scavenger lm was optimized both to prolong the microorganisms
viability during storage and to improve the efciency of the lm to remove oxygen from the package headspace. It was found that it
is possible to store the desiccated lm over a period of 20 days without monitoring any appreciable decrease of microorganism
viability. It was also pointed out that the highest respiratory efciency of the proposed active lm is obtained by entrapping the
microorganisms into polyvinyl alcohol, and by using the active lm as a coating for a high humidity food.
r 2003 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.
Keywords: Aerobic microorganisms; Environment-friendly materials; Oxygen scavengers; Food preservation

1. Introduction
Active packaging is an emerging and exciting area of
food packaging technology that can confer many
preservation benets to a wide range of foods (Day,
1998). In fact, in many cases food deterioration is caused
by oxidation reactions or by the presence of spoilage
aerobic microorganisms. For instance, to prolong the
shelf-life of dairy and bakery products it is of primary
importance to slow down the growth rate of molds and
aerobic bacteria. Also, to prevent damages of a wide
range of food it is necessary to reduce the deterioration
rate of constituents, such as oils, fats, pigments and
vitamins (Gill & McGinnes, 1995; Schozen, Ohshima,
Ushio, Takigichi, & Koizumi, 1997; Berenzon & Saguy,
1998). In all these cases oxygen scavengers are successfully used to prolong the shelf-life of packed food, by
reducing the oxygen concentration in the package
headspace.

*Corresponding author. Tel.: +39-0881-589-233; fax: +39-08-881740211.

E-mail address: ma.delnobile@unifg.it (M.A. Del Nobile).

Different kinds of oxygen scavengers are commercially available, and most of them are based on the
oxidation reaction of iron (Nakamura & Hoshino, 1983;
Smith, Ramaswarmy, & Simpson, 1990). A real risk in
using this type of oxygen scavenger could be an
accidental ingestion of a large amount of iron (Labuza,
1987). A different type of oxygen scavenger is that using
the enzyme reaction surface. The main shortcoming with
this type of oxygen scavengers is their sensitivity to
physicalchemical factors (pH, aw, salt concentration,
temperature) and they cannot be effectively used for
low-water foodstuffs (Graff, 1994). Ascorbate oxidation, photo-sensitive dye oxidation, unsaturated fatty
acid, immobilized yeast on a solid surface (Floros,
Dock, & Han, 1997) are further concepts in this type of
packaging technology. Most of the oxygen scavengers
reported above are available in form of sachet to be
introduced into the package. An alternative to the
sachet is the incorporation of the active compound into
the packaging structure itself. An example is Oxyguard
(Tokyo Seikan Kaisha, Japan), incorporated into a
laminate.
Other researchers proposed an alternative approach:
the use of entrapped aerobic microorganisms, capable of

0023-6438/03/$30.00 r 2003 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/S0023-6438(03)00115-4

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C. Altieri et al. / Lebensm.-Wiss. u.-Technol. 37 (2004) 915

consuming oxygen (Tramper, Luyben, & van der Tweel,

1983; Doran & Bailey, 1986; Gosmann & Rehem, 1986,
1988). Natural and biological oxygen scavengers, based
on the use of microorganisms entrapped in a polymeric
matrix, effective in preserving foods, safe to use,
agreeable to consumer, inexpensive, environment
friendly, could be a very interesting concept to modern
food technology. In fact, the possibility to create a new
package, having many desirable characteristics, is very
promising, also taking into account the new consumers
demand for mildly preserved convenience foods, having
fresh-like qualities and being environmental friendly. In
the eld of biotechnology, immobilization of whole cells
is gaining increasing importance (Gosmann & Rehem,
1988). Alginate, agar, and gelatin (Tramper et al., 1983;
Doran & Bailey, 1986; Gosmann & Rehem, 1986, 1988)
have been successfully used. Unfortunately, the above
study cannot be used for the development of a biological
oxygen scavenger. In fact, the cycle life of a biological
oxygen-scavenger lm includes the entrapment of the
microorganisms in an appropriate polymeric matrix
(lm manufacturing), the maintenance of the desiccated
lm till its use (lm storage and distribution), and the rehydration (lm usage, obtained by putting the lm in
contact with the food).
The present work is aimed to develop an environmental friendly oxygen-scavenger lm using microorganisms as the active component. In particular,
hydroxyethyl cellulose (HEC) and polyvinyl alcohol
(PVOH) were used to entrap two different kinds of
microorganisms: Kocuria varians and Pichia subpelliculosa. The optimal microorganism entrapment conditions
were determined by evaluating the inuence of the
following on the microorganism viability during storage:
type of microorganism, nutrient and polymeric matrix
concentrations. The inuence of both re-hydration
conditions and macromolecular mobility of the entrapping matrix on the respiratory efciency of the active
lm were also evaluated.

2. Materials and methods

2.1. Organisms
K. varians DSM 20033. In the following it will be
referred to as microorganism A.
P. subpelliculosa, isolated from apples and stored in
the Agricultural Faculty, University of Foggia, Italy. In
the following it will be referred to as microorganism B.

was grown in Saboraud Agar (SA) (Oxoid) and

incubated at 30 C for 48 h.
The same media without agar were used to perform
the working broth cultures.
2.3. Polymeric matrices
PVOH and HEC were purchased from Aldrich
Chimica, and were used as received.
2.4. Entrapment procedure
2.4.1. Microorganism entrapment into HEC
HEC was dissolved in distilled water and autoclaved
at 121 C for 15 min. Three different polymeric solutions
were prepared: (i) 1.5 g/100 g, (ii) 6 g/100 g, and (iii)
7.5 g/100 g. Each of the above solutions were mixed with
revitalized broth culture of either microorganisms A or
B (4:1). The concentration of nutrients in the revitalized
broth culture for microorganism A and B is reported in
the following. In the case of microorganism A three
nutrient broths were prepared with the following
nutrient concentration: 4, 8 and 16 g/L. In the case of
microorganism B, three different Sabouraud broths
containing 10, 20 and 30 g/L nutrient concentrations
were used. Films of about 20 g were aseptically prepared
by pouring the above solutions (i.e. water, HEC,
microorganisms and nutrient) on a polycarbonate
plate, and waiting until the lm equilibrates at room
humidity (about 50%). All the steps described above to
prepare the lms were conducted inside a laminar ux
hood. The produced lms were aseptically stored at
room temperature.
2.4.2. Microorganism entrapment into PVOH
To dissolve PVOH in water (15 g/100 g), it was mixed
with distilled water and then autoclaved at 121 C for
15 min. A single polymeric solutions were prepared:
15 g/100 g. The above solution was mixed with revitalized broth culture of microorganisms A (4:1). The
concentration of nutrients in the revitalized broth
culture for microorganism A is 8 g/L. Films of about
2 g were aseptically prepared by pouring the above
solutions (i.e. water, PVOH, microorganisms and
nutrient) on a polycarbonate plate, and waiting until
the lm equilibrates at room humidity (about 50%). All
the steps described above to prepare the lms were
conducted inside a laminar ux hood. The produced
lms were used as soon as they equilibrate at room
humidity (about 50%).

2.2. Culture media

2.5. Evaluation of entrapped microorganisms respiratory

activity

The strain A was grown in nutrient agar (NA) (Oxoid,

England) and incubated at 37 C for 72 h. The strain B

Films of about 2 g, obtained according to the above

procedures, were aseptically stored into hermetically

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closed vials. The respiratory activity of the entrapped

cells were monitored for two different conditions: the
water activity inside the vial was set equal to 1; the lms
were layed down on agar slants, which was previously
introduced into the vial. The vials used for determining
the microorganism respiratory activity have a capacity
of 20 mL, and a headspace volume of 10 mL. All vials
were stored at 37 C. The evolution of O2 and CO2 in the
vial headspace was determined by means a gas
chromatograph. In the present experimentation a gas
chromatograph DANI mod. GC 86.10 was employed,
equipped with a prepacked column CTR 1Alltech
(Milan, Italy). The analysis was carried out in the
following operative conditions: start temperature (35 C)
risen to 90 C at a rate of 30 C/min and cooled to 35 C;
gas carrier (He) speed 90 mL/min; detector (TCD)
125 C; injector 70 C. All the analyses were performed
in triplicate.
2.6. Evaluation of entrapped microorganisms viability
Count of viable cells entrapped in lm of HEC,
prepared according to the above procedure, were carried
out by decimally diluting the lm into a sterile saline

11

solution and spread (0.1 mL of each dilution) on plates

containing the same media described above: NA and
SA, and incubated at 37 C and 25 C for K. varians and
P. subpelliculosa, respectively. All analyses were performed in triplicate.

3. Results and discussion

The manufacturing cycle of the investigated oxygenscavenger lms must be optimized both to prolong the
microorganisms viability during storage and distribution and to improve the efciency of the lm to remove
oxygen from the package headspace. These two distinct
aspects of the process will be presented separately in the
following. In particular, the determination of the
optimal desiccation conditions will be rst addressed
by evaluating the inuence of the following on the
microorganism viability during storage: microorganism
type, nutrient concentration, polymeric matrix concentration. Subsequently, the inuence of the following on
the efciency of the active lm in consuming oxygen will
be discussed: re-hydration conditions and macromole-

8.0

6.0

log(CFU)

log(CFU)

2.0

0
(a)

4.0

0.0

20

10
Time [day]

10

15

20

Time [day]

(b)

8.0

log(CFU)

6.0

4.0

2.0

0.0
(c)

10

20
Time [day]

Fig. 1. Log(cfu) plotted as a function of time for microorganism A entrapped in HEC (HEC concentration in the casting solution equal to 6 g/100 g).
(a) Carbon source concentration in the casting solution equal to 4 g/L; (b) carbon source concentration in the casting solution equal to 8 g/L; and
(c) carbon source concentration in the casting solution equal to 16 g/L. The curves showed in the gure dont represent any model, they only facilitate the reading of the data.

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12

cular mobility of the polymeric matrix used to entrap

the microorganisms.
3.1. Microorganism maintenance during storage
The determination of the optimal desiccation conditions was carried out using HEC as polymeric matrix to
entrap the microorganisms. In fact, a lm of HEC can
be easily dissolved in water at room temperature,
allowing the evaluation of viable cells entrapped in the
lm after desiccation. On the contrary, PVOH needs to
be heated at 121 C for 15 min to be dissolved in water,
making impossible the determination of viable cells
entrapped in the desiccated lm.
Fig. 1 shows the concentration of viable cells plotted
as a function of storage time for the microorganism A;
the three sets of data were obtained by varying the initial
nutrient concentration (S0 ). As showed in Fig. 1 for
values of S0 equal or higher than 8 g/L, the concentration of viable cells do not change appreciably over a
period of 20 days (Fig. 1b and c). On the contrary, for a
value of S0 equal to 4 g/L the concentration of viable
cells steadily decreases with the storage time (Fig. 1a).
Fig. 2 shows the concentration of viable cells plotted as

log(CFU)

6.0

4.0

2.0

0.0
(a)

10

15

20

Time [day]

log(CFU)

4
0
(b)

10

a function of storage time for the microorganism A. In

this case the three sets of data were obtained by varying
the HEC concentration (C0 ). From the data shown in
Figs. 1 and 2, it can be inferred that for values of C0
equal or lower than 6 g/100 g the concentration of viable
cells do not change appreciably over a period of 20 days
(Fig. 1b and 2a); while, for a value of C0 equal to 7.5 g/
100 g the concentration of viable cells steadily decreases
with the storage time (Fig. 2b).
Fig. 3 shows the number of the viable cells plotted as a
function of storage time for the microorganism B; the
three sets of data were obtained by varying the initial
nutrient concentration. For this microorganism the
concentration of viable cells steadily decreases with the
storage time (Fig. 3ac) regardless of the value of S0 :
Fig. 4 shows the concentration of viable cells plotted as
a function of storage time for the microorganism B; in
this case the three sets of data were obtained by varying
the HEC concentration. Analogously to what was
shown in Fig. 3, the number of viable cells steadily
decreases with the storage time (Figs. 4a and b)
regardless of the values of C0 :
From the data shown in Figs. 14, it can be inferred
that microorganism A seems to be more appropriate to
be used as the active agent for the investigated
oxygen-scavenger lms. There are several factors that
could account for the viability of the two different types
of entrapped cells. Among them the most important
could be: (a) the extent of the compressive pressure that
the macromolecular matrix exerts on the cells while the
lm is in the desiccated state, (b) the local nutrient
concentration, and (c) the structures of cell walls. The
determination of the relationships among cell viability
and the above factors goes beyond the aim of this work,
and it will not be addressed. The obtained results also
suggest that the optimal HEC concentration for the
investigated oxygen-scavenger lms is 6 g/100 g. In fact,
using 6 g/100 g HEC the polymeric solution viscosity is
sufciently high to be processed to a lm, and the
viability of the entrapped cells is sufciently high for 20
days. Concerning the inuence of the initial nutrient
concentration on the cell viability, Fig. 1 points out that
for values of the initial nutrient concentration equal or
higher than 8 g/L, the local nutrient concentration do
not represent a limiting factor to the viability during
storage of the entrapped cells, suggesting that the
optimal value for the initial nutrient concentration is
8 g/L.

20

Time [day]

Fig. 2. Log(cfu) plotted as a function of time for microorganism A

entrapped in HEC (carbon source concentration in the casting solution
equal to 8 g/L. (a) HEC concentration in the casting solution equal to
1.5 g/100 g and (b) HEC concentration in the casting solution equal to
7.5 g/100 g. The curves showed in the gure do not represent any
model, they only facilitate the reading of the data.

3.2. Film respiration efficiency

Figs. 5 and 6 show the ratio between carbon dioxide
and oxygen concentrations in the vial headspace plotted
as a function of time for lm containing microorganism
A. The data was obtained by entrapping microorganism
A in PVOH and HEC and re-hydrating the lm in two

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13

6
log(CFU)

log(CFU)

2
0
(a)

10

15

20

10
Time [day]

(b)

Time [day]

15

20

log(CFU)

0
(c)

10
Time [day]

15

20

Fig. 3. Log(cfu) plotted as a function of time for microorganism B entrapped in HEC (HEC concentration in the casting solution equal to 6 g/100 g).
(a) carbon source concentration in the casting solution equal to 10 g/L; (b) carbon source concentration in the casting solution equal to 20 g/L;
(c) carbon source concentration in the casting solution equal to 30 g/L. The curves showed in the gure do not represent any model, they only
facilitate the reading of the data.

different manners: (a) by conditioning the lm at aw

equal to 1 (to simulate the use of the lm as the internal
layer of a multi-layer structure used to package a high
humidity food) and (b) by laying down the lm on agar
slant (to simulate the use of the lm as a coating of a
high humidity food). As shown in the above gures, the
evolution on storage of the oxygen concentration in the
vial headspace depends on both re-hydration conditions
and macromolecular mobility of the entrapping matrix.
In particular, there is a faster decrease of the oxygen
concentration in the vial headspace level during storage
either entrapping the microorganisms in PVOH or
laying down the lm on agar slant.
Before analysing the data showed in Figs. 5 and 6, few
comments are in order. The evolution on storage of the
oxygen concentration in the vial headspace is affected by
two simultaneously occurring phenomena: the microorganism respiration rate, and the rate at which oxygen
diffuse through the entrapping polymeric matrix (i.e. the
oxygen diffusion coefcient of the hydrated entrapping
matrix). The former factor depends on the viability of
the immobilized microorganisms and on the local
nutrient concentration (nutrient availability). While,

the oxygen diffusion coefcient depends on the nature

of the entrapping polymeric matrix and on its hydration
status (i.e. the amount of sorbed water).
The data shown in Figs. 5 and 6 suggest that PVOH is
more efcient in immobilizing the investigated microorganism. In fact, regardless of the re-hydration
condition, the microorganisms entrapped in PVOH is
faster in reducing the oxygen concentration in the vial
headspace. Considering that the oxygen diffusion
coefcient of hydrated PVOH is similar to that of
hydrated HEC, and that the nutrient concentration is
the same in the two investigated lms, it is reasonable to
suppose that the observed differences should be
attributed to a different viability of the immobilized
microorganisms.
By laying down the lm on agar slant, between the
two re-hydration conditions investigated, it is the
most advantageous condition in removing oxygen
from the vial headspace. Most probably the obtained
results have to be ascribed to the different amount of
sorbed water. In fact, as the sorbed water increases
both the macromolecular mobility of the entrapping
matrix and its oxygen diffusion coefcient increase,

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14

25
6

[O2]/[CO2]

log(CFU)

20
4

15
10
5

10
Time [day]

(a)

15

20

8
12
Time [day]

16

20

Fig. 6. Ratio between O2 and CO2 concentration in the headspace of

hermetically closed vials plotted as a function of time for lm
containing microorganism A re-hydrated by laying down the lm on
agar slant and stored at 37 C. (&) entrapped in PVOH and ()
entrapped in HEC.

8
6
log(CFU)

4. Conclusions
2
0

(b)

10
Time [day]

15

20

Fig. 4. Log(cfu) plotted as a function of time for microorganism B

entrapped in HEC (carbon source concentration in the casting solution
equal to 20 g/L. (a) HEC concentration in the casting solution equal to
1.5 g/100 g and (b) HEC concentration in the casting solution equal to
7.5 g/100 g. The curves showed in the gure do not represent any
model, they only facilitate the reading of the data.

[O2]/[CO2]

10

15
Time [day]

20

25

Fig. 5. Ratio between O2 and CO2 concentration in the headspace of

hermetically closed vials plotted as a function of time for lm
containing microorganism A re-hydrated at aw equal to 1 and stored at
37 C. (&) entrapped in PVOH and () entrapped in HEC.

In this work a new method is proposed to produce

oxygen scavenger lms using aerobic microorganisms as
the active compound. The manufacturing cycle of the
investigated oxygen-scavenger lms was optimized both
to prolong the microorganisms viability during storage
and to improve the efciency of the lm to remove
oxygen from the package headspace. The former aspect
was addressed by xing the immobilizing polymeric
matrix and varying the nutrient concentration, the type
of immobilized microorganism and the polymer concentration.
Regarding the efciency of the active lm to remove
oxygen from the vial headspace, it was shown that
polymer matrices with a high macromolecular mobility,
such as PVOH, are more suited to entrap microorganisms. Moreover, it was shown that the best results, in
terms of oxygen consumption rate, can be obtained by
laying down the lm on agar slant (i.e. using the active
lm as a coating of a high humidity food).

Acknowledgements
The work was funded under the MURST Piani di
Potenziamento della Ricerca Scientica e Tecnologica.

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