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693
Journal of Food Protection, Vol. 69, No. 3, 2006, Pages 693698
Copyright Q, International Association for Food Protection

Research Note

Extended Shelf Life of Soy Bread Using Modified

Atmosphere Packaging
URSULA FERNANDEZ, YAEL VODOVOTZ, POLLY COURTNEY,

AND

MELVIN A. PASCALL*

Department of Food Science and Technology, Ohio State University, 2015 Fyffe Road, Columbus, Ohio 43210, USA
MS 05-257: Received 31 May 2005/Accepted 28 October 2005

ABSTRACT
This study investigated the use of modified atmosphere packaging (MAP) to extend the shelf life of soy bread with and
without calcium propionate as a chemical preservative. The bread samples were packaged in pouches made from low-density
polyethylene (LDPE) as the control (film 1), high-barrier laminated linear low-density polyethylene (LLDPE)nylonethylene
vinyl alcoholnylonLLDPE (film 2), and medium-barrier laminated LLDPEnylonLLDPE (film 3). The headspace gases
used were atmosphere (air) as control, 50% CO250% N2, or 20% CO280% N2. The shelf life was determined by monitoring
mold and yeast (M1Y) and aerobic plate counts (APC) in soy bread samples stored at 218C 6 38C and 38% 6 2% relative
humidity. At 0, 2, 4, 6, 8, 10, and 12 days of storage, soy bread samples were removed, and the M1Y and APC were
determined. The preservative, the films, and the headspace gases had significant effects on both the M1Y counts and the
APC of soy bread samples. The combination of film 2 in the 50% CO250% N2 or 20% CO280% N2 headspace gases
without calcium propionate as the preservative inhibited the M1Y growth by 6 days and the APC by 4 days. It was thus
concluded that MAP using film 2 with either the 50% CO250% N2 or 20% CO280% N2 was the best combination for shelflife extension of the soy bread without the need for a chemical preservative. These MAP treatments extended the shelf life
by at least 200%.

Soy products are finding greater acceptance in the

United States partially due to a U.S. Food and Drug Administration (FDA) allowable claim linking the consumption of soy protein with a reduced risk of heart disease (1,
18). In response to this niche, we have developed a highly
accepted soy-enriched bread that contains sufficient soy
proteins per serving to meet the FDA-approved health
claim on coronary heart disease. When compared with
wheat bread of similar dimensions and ingredient content,
the soy bread has approximately three times the amount of
protein and stales at a significantly slower rate (19). One
factor that may affect the staling process is the difference
in water content and mobility between the two breads.
Zhang (20) reported that soy bread has a moisture content
of approximately 44.7% when compared to 39.9% in wheat
bread. This higher moisture content may significantly impact the microbial shelf life of the soy bread. Thus, any
technique that reduces the growth rate of microorganisms
in the soy bread will be advantageous to the baking industry and the consuming public.
Modified atmosphere packaging (MAP) is a technique
in which normal air within a sealed package is changed to
contain gases that are capable of increasing the shelf life
of the packaged product. MAP has been shown to extend
* Author for correspondence. Tel: 614-292-0287; Fax: 614-292-0218;
E-mail: pascall.1@osu.edu.
Present address: H-E-B Grocery Company, 5105 Rittiman Road, San
Antonio, TX 78218, USA.

the shelf life of bakery products in the past (2, 8, 12, 13).
This technique reduces or eliminates the need to use chemical preservatives while maintaining a desired shelf life for
the packaged product.
Most microorganisms that cause spoilage in bakery
products such as bread and cakes are aerobic, and a reduction or removal of oxygen from the environment should
thus result in a reduction of their growth rate and the spoilage rate of the product. Reduction of oxygen is one of the
primary uses of active packaging and MAP for nonsterile
foods (3). Although CO2 is not known to be lethal to microorganisms, it has shown both bacteriostatic and fungistatic properties and will hinder the growth of certain aerobic organisms (15). Daniels et al. (6) reported that CO2
penetrates the microbial cell and causes a rapid acidification
of its internal environment. This subsequently leads to an
alteration of the metabolic and enzymatic activities of the
microorganism. Thus, by removing the headspace oxygen
in the packaged soy bread samples, then flushing them with
varying levels of CO2, an environment unfavorable for the
growth of these organisms would be created. Soy bread was
selected as the substrate for the microbial growth because
this type of bread is relatively new to the United States
market and the development of techniques to maximize its
shelf life is essential for its marketing success.
Microorganisms differ greatly in their sensitivity to
CO2, and this relates to their oxygen requirements. For example, CO2 is known to be most effective against aerobic

694

FERNANDEZ ET AL.

J. Food Prot., Vol. 69, No. 3

TABLE 1. Ingredients and their amounts used to produce the soy

bread
Ingredients

Amount in soy dough

(%)

Water
Wheat flour
Soy flour
Soy milk powder
Sugar
Yeast
Gluten
Shortening
Salt
Dough conditionera
Calcium propionate (antimicrobial)

44.3
16.7
19.3
6.5
4.4
1.4
3.9
1.7
0.9
1
0.4b

Dough conditioner supplied by Caravan Products Co. Inc. (Totowa, N.J.).

b This level of calcium propionate is a percentage of wheat flour,
soy flour, and soy milk powder.

bacteria and molds when compared with other types of bacteria and yeast. Gram-negative bacteria are more sensitive
to CO2-enriched atmospheres than gram-positive types.
Notwithstanding this, the literature reports the successful
extension of shelf life in various bakery products using various mixtures of CO2 and nitrogen gases (4, 5, 8, 11, 15,
16). To determine the appropriate gas mixture for shelf-life
extension of a given bakery product, a study of the variables influencing the spoilage of that product must be done
(16).
The objective of our study was to evaluate the effect
of MAP and various packaging films on the growth of mold
and yeast and aerobic plate count in soy bread treated with
and without calcium propionate as a chemical preservative.
To meet this objective, we conducted shelf life studies during a 12-day storage period.
MATERIALS AND METHODS
Soy bread. The soy bread samples were prepared using a
patent-pending process and the ingredients shown in Table 1. The
resultant dough from these ingredients was divided into 1-kg portions, proofed at 438C for 40 min, and then baked for 70 min at
1708C. After cooling, the loaves were sliced and packaged in lowdensity polyethylene (LDPE) pouches. Each loaf had average
weight, volume, and density of 900 g, 1,744 cm3, and 0.52 g/cm3,
respectively. Forty loaves were baked in each batch. From these,
38 were used for microbial testing and 2 were used for volume
determination. The water activities (aw) of the crust and crumb of
the bread were 0.922 and 0.959, respectively. The bread had an
average pH of 6.12.
Packaging materials. The films used to package the soy
bread were (i) a 4-mil-thick (50.8 mm) LDPE (the control); (ii) a
laminated 5.5-mil-thick (139.7 mm) linear low density polyethylene (LLDPE)nylonethylene vinyl alcoholnylonLLDPE obtained from Cryovac Inc. (Duncan, S.C.); and (iii) a laminated
5.5-mil-thick (139.7 mm) LLDPEnylonLLDPE obtained from
Cryovac Inc. These films will be subsequently referred to as film
1, film 2, and film 3, respectively.

Headspace gases. For the MAP of the soy bread, the headspace gases used were 50% CO250% N2, 20% CO280% N2
obtained from Praxair (Columbus, Ohio), and air (as a control).
The mixed gases had a purity of 99.9%.
Pouch fabrication. Using films 2 and 3, pouches measuring
25 by 36 cm were fabricated on a Cryovac model 2070 vertical
form-fill-seal machine. The pouches made from film 1 measured
26 by 41 cm and were obtained in a preformed manner from
Central Ohio Bag & Burlap, Inc. (Columbus, Ohio). To minimize
and unify the headspace volume within each package, all pouches
were manually resized with a model 12SC/1 Sencorp impulse
sealer (Sencorp Systems, Hyannis, Mass.).
Packaging. Approximately 4 h after baking, all soy bread
samples with and without calcium propionate (the preservative)
were moved from the bakery to the packaging laboratory in their
original packages, then repackaged in pouches made from films
1, 2, and 3. All samples were repackaged with at least one of the
three headspace gases. For atmospheric packaged samples, the
pouches made from films 2 and 3 were sealed with the impulse
sealer set at 207 kPa (30 lb/in2), 14 V, and 1.2 s dwell time. For
film 1, the settings were the same except that the dwell time was
0.85 s. An Ultravac 2100 D vacuum-packaging machine (Koch
LLC, Kansas City, Mo.) was used to package the samples with
CO2 and N2 headspace gases. This sealer was set at 99% vacuum
for 10 s, 30% headspace gas, and a dwell time of 1.2 s for films
2 and 3. For film 1, the settings were the same except that the
dwell time was 1 s. All samples were stored at 218C 6 38C and
38% 6 2% relative humidity.
Microbial analyses. Microbial analyses were performed on
the soy bread samples after 0, 2, 4, 6, 8, 10, and 12 days of
storage.
For all samples prepared for the aerobic plate count (APC),
a half of a slice (30 g of sample) from the middle of each loaf
was homogenized in a model STO-400 Tekmar stomacher (Lorton, Va.) for 2 min with 270 ml sterile 0.1% peptone solution
(homogenate). A 0.1-ml aliquot of the homogenate was removed
from each sample, and a series of 10-fold dilutions were made
with the peptone solution. From the final diluted solution, a 0.1ml aliquot was removed and plated in duplicate on standard plate
count agar (Difco, Becton Dickinson, Sparks, Md.). All plates
were incubated at 378C 6 18C for 1 to 2 days, and then the CFU
were determined. All results were expressed as log CFU per gram.
For all samples prepared for the mold and yeast count
(M1Y), approximately 100 g of the bread crust was homogenized
in the stomacher for 2 min with 400 ml of sterile 0.1% peptone
solution. From this solution, a volume of 1 ml was removed, and
a series of 10-fold dilutions were prepared as needed. From the
final dilution, a 0.25-ml aliquot was removed and plated in duplicate on standard plate count agar containing tetracycline (5 mg/
ml) and chloramphenicol (5 mg/ml). All plates were incubated at
238C 6 18C for 4 days. CFU were counted manually and expressed as log CFU per gram.
Physical analysis. The average loaf volume (cubic centimeters) was determined by the rapeseed displacement method
(AACC 2000, Method 10-05). In addition, the weight (grams),
and density (grams per cubic centimeter) of the bread samples
from each lot were also measured. The average values for these
measurements were 1,744 cm3 (volume), 900 g (weight), and 0.52
g/cm3 (density).
Experimental design. A 2 3 3 3 3 3 6 (preservative 3
film 3 atmosphere 3 time point) full factorial design with six

J. Food Prot., Vol. 69, No. 3

EXTENDED SHELF LIFE OF SOY BREAD

695

blocks (batches) was used for the analysis of the microbial count.
The treatments were defined as preservative levels, film types, and
headspace gas combinations. A total of 18 treatments (2 preservative levels 3 3 films 3 3 headspace gases) were studied, and
each treatment replicated twice. Each batch consisted of one of
the two preservative levels (0.4 and 0% calcium propionate) assigned randomly. Two of the three films were randomly assigned
to each batch. All three headspace gases and all six times (2, 4,
6, 8, 10, and 12 days) were included in each batch.
Statistical analyses. The data collected from the microbial
analyses were statistically analyzed by analysis of variance with
significance determined at P , 0.05. Samples that were designated as nondetectable counts were those with numbers of #2 log
CFU/g (limits of detection for M1Y and APC). The statistical
analyses were conducted separately for the samples with and without preservative to determine (i) the effect of headspace gases on
the M1Y of the samples packaged with each of the three test
films and (ii) the effect of the three films on the M1Y of the
samples packaged in each headspace gas. The same effects were
analyzed for the APC response. If the analysis of variance found
a significant effect, Tukeys multiple comparison tests were used
to determine which treatment levels were significantly different
from each other as well as to rank the treatment levels. The statistical analyses were performed with SAS computer software
(SAS Institute, Inc., Cary, N.C.), version 9.1. Means and standard
deviations were calculated for the loaf volumes measurements
with Microsoft Excel (Ontario, Canada).

RESULTS AND DISCUSSION

M1Y counts: soy bread with preservative. Results
of the M1Y counts for samples with preservative and
packaged with films 1, 2, and 3 in the three headspace gases
(air, 50% CO250% N2, and 20% CO280% N2) are shown
in Figure 1A through 1C. These results show that there was
little difference between the M1Y for all the bread samples
packaged with each of the three films in the control atmosphere. However, for the soy bread samples with preservative and packaged in 50% CO250% N2 and the 20%
CO280% N2 headspace gases, there seems to be sharper
increases in the M1Y for bread samples packaged with
film 1 when compared with the other two films. The statistical analyses showed that the films had no significant effect
(P . 0.05) on the M1Y of soy bread packaged in the
control atmosphere, but the films had a significant effect on
the M1Y in the samples packaged in 50% CO250% N2
and the 20% CO280% N2 (P , 0.01) gases. For samples
packaged in 50% CO250% N2 and 20% CO280% N2, the
statistical analyses showed that the M1Y were significantly
higher (P , 0.01) for film 1 when compared with the
counts in soy bread with films 2 and 3, respectively. There
were no significant differences (P . 0.05) between the
counts in bread packaged with film 2 when compared with
film 3 for the 50% CO250% N2 and 20% CO280% N2
headspace gases as shown in Figure 1B and 1C.
M1Y counts: soy bread without preservative. Figure 1A through 1C also show the results for the M1Y in
samples without preservative. These figures show that there
was little difference between the M1Y for the soy bread
without preservative and packaged with each of the three
films in the control atmosphere. However, the films had a

FIGURE 1. Mold and yeast counts in soy bread with and without
calcium propionate packaged with films 1, 2, and 3 and containing air (control), 50% CO250% N2, or 20% CO280% N2. (A)
LDPE, (B) LLDPEnylonethylene vinyl alcoholnylonLLDPE,
and (C) LLDPEnylonLLDPE.

significant effect (P , 0.01) on the M1Y in the samples

packaged in the control atmosphere, the 50% CO250% N2
gas, and the 20% CO280% N2 gas. For samples packaged
in the control atmosphere, the M1Y were significantly
higher (P , 0.01) for film 1 when compared with the
counts in the soy bread with film 3. However, there were
no significant differences (P . 0.05) between the counts in
the bread packaged with films 1 and 2 as well as between
the counts in the bread with films 2 and 3 for the control.
For samples packaged in the 50% CO250% N2 and 20%
CO280% N2, the M1Y numbers were significantly higher
(P , 0.01) for film 1 when compared with the counts for
the bread with films 2 and 3 for both mixtures of headspace

696

FERNANDEZ ET AL.

J. Food Prot., Vol. 69, No. 3

gases. The M1Y numbers were significantly higher (P ,

0.01) for film 3 when compared with the counts in bread
with film 2 for the 50% CO250% N2 and the 20% CO2
80% N2 headspace gases.
The results for the soy bread with and without the preservative also show that the high and medium barrier films
(films 2 and 3, respectively) had a significant effect on the
M1Y in these samples. This can be seen by comparing
Figure 1A through 1C. For the samples packaged in film
1, the low barrier film (Fig. 1A), there was little difference
between the M1Y counts irrespective of the type of headspace gas used to package the soy bread. In films 2 and 3,
the M1Y in the control atmosphere were significantly
higher than those in the mixed gases.
APC: soy bread with preservative. When viewed together, the results in Figure 2A through 2C show that there
was little difference between the APC for each time point
for all the bread samples with preservatives and packaged
with the three films in the control atmosphere. For the results of the bread with preservative in 50% CO250% N2,
there seems to be little difference in the counts for the samples packaged with the three films. However, the results
show a slight increase from day 6 to day 8 for the samples
packaged with film 1 when compared with the other two
films. After day 8, the APC reached its maximum and remained fairly constant for samples packaged with film 1
but continued increasing slightly for samples in the other
two films. For samples packaged in 20% CO280% N2, the
APC seemed slightly higher up to day 8 for samples packaged with film 1 when compared with films 2 and 3. However, the statistical analyses showed that the films had no
significant effect (P . 0.05) on the APC in soy bread packaged in the control, the 50% CO250% N2 headspace gas,
and the 20% CO280% N2 headspace gas.
APC: soy bread without preservative. The results in
Figure 2A through 2C show that the APC in the bread
samples without preservative and packaged in the three
headspace gases was greater in samples packaged with film
1 when compared with the other two films. The statistical
analyses show that the packaging films significantly affected (P , 0.01) the APC in the samples for all three headspace gases. The APC were significantly higher (P , 0.01)
in the samples packaged with film 1 when compared with
films 2 and 3. However, there was no significant difference
(P . 0.05) between the results for the samples packaged
with film 2 when compared with film 3 for the three headspace gases.
When the results for the APC in the soy bread with
and without the preservative are compared, they show that
the medium- and high-barrier films had less of an effect on
their growth when compared with the M1Y count. Although in most cases there were significant differences (P
, 0.05) between the APC for the samples with air as the
headspace gas when compared with those having the mixed
gases, there were instances when there were no such differences. For example, there was no significant difference
(P . 0.05) between the counts for the controls and the 20%
CO280% N2 samples with preservative in film 2.

FIGURE 2. Aerobic plate counts in soy bread with and without

calcium propionate packaged with films 1, 2, and 3 and containing air (control), 50% CO250% N2, or 20% CO280% N2. (A)
LDPE, (B) LLDPEnylonethylene vinyl alcoholnylonLLDPE,
and (C) LLDPEnylonLLDPE.

DISCUSSION
MAP of bakery products with chemical preservatives
has been investigated in the past and has been shown to
increase the shelf life of these products when compared
with similar MAP products not treated with chemical preservatives (10, 17). Because there was little reference to
MAP of soy bread in the literature, we compared our results
with those of previously published research to see if baked
soy bread would produce similar finds.
The antimicrobial activity of CO2 in a culture medium
or food system depends on many factors. These include the
partial pressure of CO2, O2 concentration, volume of headspace gas, temperature, acidity, and aw (9). For this soy
bread MAP study, the effect of temperature and the volume
of the headspace gas were kept constant for all samples

J. Food Prot., Vol. 69, No. 3

during the storage period. The CO2 and O2 concentrations

in the headspace of the samples were not reported because
the focus of this paper was on the effect of the initial headspace gas and the barrier property of the packaging materials and on how these affected the M1Y and APC in the
soy bread samples. As a result, the ingredients and their
concentrations in the dough were also kept constant. This
helped to reduce large variability in the pH and aw of the
bread samples.
To successfully increase the shelf life of the soy bread
in our study, it was necessary to maintain the concentration
of the CO2N2 headspace environment inside of the package as long as was reasonably possible. Thus, great care
was taken in ensuring that there were no leaks in the pouches used to package the samples. Any leaks in the seals of
the packages would allow rapid changes to the headspace
gases and result in erroneous results. Although plastic films
are the packaging type of choice in most bread packaging
operations, plastic films are more permeable to gases and
vapors when compared with glass or metal. Thus, the use
of plastic to package the soy bread in our experiment presented a challenge that needed to be addressed. Monolayer
LDPE is widely used to package various bakery products.
However, LDPE films laminated to ethylene vinyl alcohol,
aluminum foil, or polyvinylidene chloride provide a better
barrier to the ingression of oxygen and other gases. These
high-barrier laminates are also capable of maintaining the
beneficial characteristics of MAP. This was proven when
we compared the oxygen transmission rates for films 1, 2,
and 3 and found that they had an OTR of 2,061.40, 1.20
and 36.50 cm3/m2/day, respectively.
The normal microbiological load for baked goods on
routine quality-control tests in the baking industry is
,103/g for molds, yeast, and yeast-like fungi and APC (7).
Consumers can reject bread either on the basis of a change
in its taste and/or texture and/or by visible signs of molds.
From visual observation on all samples throughout storage,
microbial growth was visible on the bread when the M1Y
was at least 3 log CFU/g. Even though this critical threshold for M1Y or APC (3 log CFU/g) did not necessarily
occur at the same time, it only requires one of the critical
values to be reached for the end of shelf life to be declared.
When all the results are viewed together, they show the
M1Y was more sensitive to the MAP when compared with
the APC. This can be seen in the reduced M1Y count
shown in Figure 1B and 1C. Although most of the APC
counts were significantly lower as a result of the modified
atmosphere, Figure 2B and 2C shows that its impact was
much less when compared with the M1Y. Indeed, other
researchers have obtained similar findings and reported that
MAP is more effective in limiting the growth of M1Y
when compared with bacteria (10, 14).
Table 2 shows the minimum time it took each treatment
to reach #103 CFU/g. This table shows that film 2 and the
20% CO280% N2 or the 50% CO250% N2 mixed gas
without the calcium propionate was able to delay the M1Y
from reaching this minimum by 6 days. In the case of the
APC, the combination of film 2 and 20% CO280% N2 or
the 50% CO250% N2 and no calcium propionate was able

697

EXTENDED SHELF LIFE OF SOY BREAD

TABLE 2. Average time (days) for microbial growth to reach #3

log CFU/g per treatment
No. of days
Film types

Air

20% CO280% N2

50% CO250% N2

1
2
3

M1Y, calcium propionate

2
2
2
8
2
8

2
6
6

1
2
3

M1Y, no calcium propionate

2
2
2
8
2
4

2
8
4

1
2
3

APC, calcium propionate

2
4
4
6
4
4

4
4
6

1
2
3

APC, no calcium propionate

2
2
2
6
2
2

2
6
2

to delay its growth by 4 days. Thus, this treatment extended

the shelf life of the soy bread by 200%. This treatment
combination can be seen as the best MAP system for extending the shelf life of the soy bread samples tested in this
study.
From the data reported, it can be concluded that (i) the
microbial shelf life of the soy bread was extended by at
least 4 days (200%) as a result of MAP; (ii) the best MAP
conditions for extension of the microbial shelf life of the
soy bread was obtained by the use of film 2 and 20% CO2
80% N2 or 50% CO250% N2; (iii) this extension of microbial shelf life could be obtained without the addition of
calcium propionate to the dough used to make the soy
bread; and (iv) the effectiveness of film 2 in retarding the
growth of both M1Y and APC was attributed to its high
oxygen barrier properties (1.2 cm3/m2/day) when compared
with the other two films that had higher oxygen transmission rates.
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