JFS
M: Food Microbiology and Safety
Inhibition of Fungal Growth on Wheat
and Rye Bread by Modified Atmosphere
Packaging and Active Packaging Using
Volatile Mustard Essential Oil
K ARIN I. SUHR AND PER V. NIELSEN
Introduction
M
odified atmosphere packaging (MAP) of food has gained a
substantial success the past decades, facilitated by developments in the packaging industry of films and equipment and
the increasing unease among consumers toward chemical preservatives. Unpreserved bread and bakery products are prone to
mold spoilage within 2 to 3 d, which makes them obvious candidates for MAP and particularly for “brown-and-serve” products
(Brody 1993). The general recommendation for MAP of bakery
products has been a mixture of 60% CO2/40% N2, but specific gas
mixtures should be used for each type of product (Smith 1993),
and mixtures have varied from 0% to 100% CO2 balanced with N2
(Farber 1991; Zagory 1997). CO2 has an antimicrobial effect, but it
is soluble in water and fat, and excessive absorption can cause
package collapse. N2 on the other hand is an inert, tasteless gas
with low solubility. It is used for displacing O2 and prevents package collapse (Church 1994). A number of MAP studies have focused on the CO2:N2 balance with little or no attention to residual
O2 (Drulhe-Aleman 1996; El Halouat and others 1998; Rodriguez
and others 2000). However, experiments in our laboratory as well
as other studies have shown that the level of residual O2 plays a
significant role for mold germination (Smith and others 1986;
Abellana and others 2000), and the general rule of max 1% residMS 20040412 Submitted 6/21/04, Revised 7/26/04, Accepted 9/3/04. Authors
are with Biocentrum-DTU, Technical Univ. of Denmark, Building 221, 2800
Kgs. Lyngby, Denmark. Direct inquiries to author Nielsen (E-mail:
[email protected]).
© 2005 Institute of Food Technologists
Further reproduction without permission is prohibited
ual O2 in MAP of bakery products (Piergiovannia and Fava 1997)
may prove insufficient for obtaining the desired self life.
Two different methods of introducing the gas mixtures into the
package are used in MAP: (1) “flow-pack,” in which the gas is
flushed in a continuous flow into the package/film tube before
sealing in a form-fill-and-seal machine, which forms pillow packages, or (2) “compensated vacuum” in which a vacuum on the package is broken by the gas, for example, in a thermoforming machine,
which forms a tray for the product and a top/“lidding” film is sealed
onto the package inside the vacuum chamber. The latter method is
advantageous for obtaining the lowest residual air content, especially for porous structured products, while the 1st is recognized for
high rate of production (Saunders 1988).
The enhanced effect of combining MAP with other preservative
measures as for example, nisin (Cabo and others 2001), ethanol
(Daifas and others 2000), or weak acid preservatives in reduced
doses (El Halouat and others 1998) has been demonstrated and is
often the preferred alternative to, for example, actively removing all
residual O2 with O2-absorbers. Besides adding to the costs, commercial use of O2 absorbers in sachets has initially met reluctance (outside Japan) because of the risk of creating favorable conditions for
anaerobic pathogens and the risk of accidental ingestion of the
sachets by consumers (Idol 1997).
Active packaging (AP) can be defined as “a mode of packaging in
which the package, the product, and the environment interact to
prolong shelf life or enhance safety or sensory properties, while
maintaining the quality of the product” (Suppakul and others
2003). An antimicrobial agent with potential use in AP is the essenVol. 70, Nr. 1, 2005—JOURNAL OF FOOD SCIENCE M37
Published on Web 1/11/2005
M: Food Microbiology & Safety
ABSTRA
CT
y e br
ead ar
tificially inoculated with molds w
er
e packed in modified atmospher
es of 0%,
ABSTRACT
CT:: Wheat and rry
bread
artificially
wer
ere
atmospheres
50%, 75%, or 100% C
O 2 balanced with N2, and 3 lev
els of rresidual
esidual O2, 1%, 0.03%, or <0.01%/O2-absorber
ed
CO
levels
-absorber,, and stor
stored
for 30 to 35 d. M
odified atmospher
e packaging (MAP) was quantitativ
ely mor
e effectiv
e for rry
ye br
ead because few
er
Modified
atmosphere
quantitatively
more
effective
bread
fewer
mold species gr
ew at elev
ated C
O 2. H
owev
er
ye br
ead contaminant, Penicillium rroquefor
oquefor
ti
ver
grew
elevated
CO
Ho
ever
er,, the major rry
bread
oqueforti
ti,, was the o
ov
er-esistant mold and only the use of O2-absorber could pr
ev
ent gr
owth of this species
n wheat br
ead,
all most C
O 2-r
-resistant
prev
event
gro
species.. O
On
bread,
CO
the most C
O 2-toler
ant mold was Penicillium commune
owing in 99% C
O 2 (with high rresidual
esidual O2), and A spergillus
CO
-tolerant
commune,, gr
gro
CO
flavus was the mold species that gr
ew at lo
west O2 in 75% C
O2 tr
eatment. The spoilage yyeast/“
east/“
chalk mold
grew
low
CO
treatment.
east/“chalk
mold”” Endomyces
fibuliger was less affected b
y the differ
ent O2 lev
els than the tr
ue filamentous molds
by
different
levels
true
molds,, and none of the tested MAP
tr
eatments could pr
ev
ent gr
owth, but lag-phase was incr
eased with O2-absorber on wheat br
ead and decr
eased with
treatments
prev
event
gro
increased
bread
decreased
1% rresidual
esidual O2 on rry
ye br
ead. E
xper
iments with vvolatile
olatile mustar
d oil sho
wed that A. flavus and Eur
otium rrepens
epens w
er
e
bread.
Exper
xperiments
mustard
show
urotium
wer
ere
the most mustar
d oil–r
esistant species on wheat and rry
ye br
ead, rrespectiv
espectiv
ely
ategy with MAP and
mustard
oil–resistant
bread,
espectively
ely.. A combination str
strategy
mustar
d oil pr
oved most optimal, and total inhibition was achiev
ed with 2 L mustar
d oil/r
ye br
ead slice and bepro
achieved
mustard
oil/ry
mustard
bread
tw
een 2 and 3 L/wheat br
ead. R
esults indicated that the natur
e and sur
face ar
ea of the pr
oduct influences effectiv
etween
bread.
Results
nature
surface
area
product
effectiveness of activ
e packaging with mustar
d oil.
active
mustard
Keywor
ds: modified atmospher
e packaging, activ
ead spoilage
d oil, allyl isothiocyante
eywords:
atmosphere
active
bread
spoilage,, mustar
mustard
e packaging, br
MAP and use of volatile mustard oil for bread . . .
Table 1—Modified atmosphere packaging (MAP) treatments used in packaging of wheat and rye bread. The O 2/CO2
equilibrium inside the package measured on d 2 is shown.a
Day 2
Wheat bread
Packaging composition
% O2
% CO2
abs a
0
0
0
1
0
abs
50
0
50
1
49.5
abs
75
0
75
1
74.5
abs
100
0
100
1
99
Control/Atmospheric air
% N2
100
100
99
50
50
49.5
25
25
24.5
0
0
0
% O2
0.00
0.00
1.060·02
0.00
0.060·03
0.970·01
0.00
0.030·05
0.630·01
0.00
0.030·02
0.950·03
19.240·01
% CO2
0.80·4
0.00·3
0.30·2
44.30·6
48.40·1
48.50·8
67.61·7
70.22·1
67.80·1
100.00·0
100.00·0
100.00·0
1.00·1
Day 2
Rye bread
% O2
0.00
0.020·01
1.000·01
0.00
0.020·01
0.840·04
0.00
0.030·01
0.900·01
0.00
0.030·03
1.040·04
19.510·78
% CO2
0.60·6
0.00·0
1.20·9
47.21·0
49.70·1
47.00·1
72.01·1
73.60·6
72.40·6
100.00·0
100.00·0
99.50·7
0.40·1
aabs = oxygen absorber inserted in package.
M: Food Microbiology & Safety
tial oil of mustard (Nielsen and Rios 2000). The active compound of
the oil is allyl isothiocyante and it has proved to be a 100 to 1000
times more inhibitory when added as a volatile than through direct
addition in media (Sekiyama and others 1996; Suhr and Nielsen
2002). Several packaging film materials have been tested for their
allyl isothiocyante permeability (Lim and Tung 1997; Lim and others 1998), and allyl isothiocyante emitters have also been commercialized in Japan (WasaOuro® system from the Green Cross Corp.,
Osaka, Japan) (Worfel and others 1997).
Some investigations of MAP for bakery products have been
conducted with uninoculated products, which makes them subject to the particular hygienic standard and difficult to compare
(Black and others 1993; Rodriguez and others 2000). Other studies
on specific spoilage fungi, on the other hand, have been conducted on laboratory media (Ellis and others 1993a, 1993b; Smith and
others 1986) making them difficult to interpret directly into real
life situations. The objective of this study was to test the effect of
carbon dioxide and residual oxygen contents in MAP of wheat and
rye bread inoculated with their common spoilage molds. An active
packaging concept combining volatile mustard oil with MAP was
furthermore explored to pursue the goal of a 30 d spoilage-free
shelf life.
Materials and Methods
Fungal isolates and preparation of inoculum
Common spoilage fungi of wheat bread, Penicillium commune
(IBT 21314), P. solitum (IBT 21313), P. polonicum (IBT 21312), Aspergillus flavus (IBT 21323), and of rye bread, P. roqueforti (IBT
18687), P. corylophilum (IBT 13995), Eurotium repens (IBT 9B), and
Endomyces fibuliger (IBT 605) were used for inoculation. All cultures
originated from spoiled bread and were kept in the IBT culture
collection at Biocentrum-DTU, Technical Univ. of Denmark.
Cultures were inoculated on Czapek Yeast autolysate extract
Agar (Samson and others 2002), except Eurotium repens, which
was inoculated on Dichloran 18% Glycerol agar (Samson and others 2002), to check purity and identity. Plates were incubated for
7 d at 25 °C in the dark, after which colonies were transferred to
fresh media and reincubated for 7 d under the same conditions to
generate inoculums. Suspensions of spores for inoculation were
made as 106 to 107 spores/mL in double distilled water with 0.5%
agar and 0.5% Tween-80, and 10 L suspensions were used for inoculations.
M38
JOURNAL OF FOOD SCIENCE—Vol. 70, Nr. 1, 2005
Inoculation of bread and colony measurements
In all experiments, fresh wheat bread (approximately 20.5 cm;
radius 4.2 cm) French baguette type (‘Fransk hot-dog brød,’ Cerealia Bakeries, Hatting, Denmark) and sliced rye bread (approximately 8.8 × 9.9 cm; 0.8 cm thick, “Mørkt rugbrød,” Møllens Brød,
Helsingør, Denmark) was used. No preservatives had been added
to the bread. The bread was stored at 25 °C in the dark, and colony
diameter was measured on day 2 or 3, 7, 14, 21, and 30 or 35 in all experiments.
In the MAP experiment, rye bread slices were punched out into
9-cm Petri dishes before inoculations in 3 points as described by
Gams and others (1998). Whole wheat bread was inoculated in 2
points in the crust. All fungi were inoculated separately and all treatments were done in 5 replicates.
For the 1st active packaging (AP) experiment, 2 cocktails of spores
([1] P. commune, P. solitum, A. flavus, E. fibuliger and [2] P. roqueforti,
P. corylophilum, E. repens, E. fibuliger) were prepared for wheat and
rye bread, respectively, by pooling equal amounts of the single spore
suspensions. Wheat bread was inoculated in 1 point and rye bread
in 2 points. One unit of rye bread equaled 3 stacked slices (approximately 8.8 × 9.9 × 2.4 cm). Recording of colony characteristics/
dominating species supplemented colony diameter readings.
Subsequently, an AP experiment with wheat bread was conducted with A. flavus and E. fibuliger inoculated separately on the same
bread. Additionally, to investigate the effect of inoculation level, A.
flavus was inoculated in 102, 103, 104, 105, and 106 spores/mL, 3
points per bread. For the 3rd AP experiment; P. roqueforti, P. corylophilum, E. fibuliger, E. repens, and A. flavus was inoculated separately on rye bread, 4 points per bread.
Modified atmosphere packaging
Three oxygen levels (1.0% O2; “no”/0.03% O2 [contained in the
bread]; O2-absorber added) were examined at 4 different levels of
carbon dioxide (0%; 50%; 75%; 100%) resulting in a total of 13 different packaging atmospheres, including a control with atmospheric air,
as summarized in Table 1. The inoculated bread was placed in 20- ×
35-cm-high barrier plastic bags (Ecotop 20/50, OPP20/PELD-EVOHPELD4720, Åkerlund & Rausing AB, Lund, Sweden). The volume of
air inside the package (headspace) was more than 3 times the volume
of the product therein. The film laminate was 70 m thick and had
an O2 permeability of 3 mL/m2/24 h/atm at 23 °C and 50% RH, and
water vapor permeability of 1 g/m2/24 h at 25 °C and 75% RH. O2absorbers (ATCO, Atmosphere Controle SA, Caen Cedex, France)
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MAP and use of volatile mustard oil for bread . . .
Table 2—Gas compositions used in active packaging (AP) experiment with volatile mustard oil at a bread factory; both
wheat and rye bread were packed. The O2/CO2 equilibrium measured inside the package just after packaging and on
d 3 is shown.
Packaginga
Day 3b
Day 3b
Wheat bread
Rye bread
% O2
Flow-pack
100% CO2
Control/Atmospheric air
% CO 2
4.75 0·67
0.20 0·07
% O2
79.7 3·2
100.0 0·0
18.65 0 ·77
% CO 2
3.710·42
0.23 0 ·11
0.9 1·1
76.3 1·3
96.1 0·5
18.47 0·40
% O2
% CO2
3.07 0·10
0.00 0·00
0.9 1·0
78.8 0·7
97.1 0·3
a n = 4, b n = 6.
Table 3—Species growing on wheat and rye bread with or without volatile mustard oil (1 mL/package) at different
atmospheric compositions
Wheat bread
Rye bread
Atmospheric air
Flow-pack
100% CO2
Atmospheric air
Flow-pack
100% CO2
Control
Penicillium spp.
Aspergillus flavus
E. fibuliger
E. fibuligera
Penicillium spp.
E. fibuliger
Eurotium repens
E. fibuliger
E. fibuligera
Mustard oil
A. flavusb
E. fibuliger
E. fibuligera
E. fibuliger
E. repens
E. fibuliger
E. fibuliger a
a Weak mycelia formation.
b 1 to 5 L mustard oil/package.
Active packaging (AP) with volatile mustard oil
First AP experiment was conducted at a bread factory and the
packaging atmospheres were as follows: 100% CO2; 80% CO2/5% O2
(simulating flow-pack conditions); or atmospheric air (Table 2).
Additionally, mustard oil (Extract Mex, San Luis Potosi, Mexico) in
concentrations of 0, 1, 3, 5, or 10 L/bread was added to a piece of
1- × 1-cm sterile filter paper (Whatman type 1, Struers Kebo Lab,
Albertslund, Denmark) contained in a Petri dish and packed along
with the bread. All treatments were done in triplicate for wheat
bread and replicate for rye bread. Compensated vacuum on a thermoforming packaging machine (Multivac Type R530) was used
with a 160-m-thick PA/EVOH/PE film with O2 permeability of 2 mL/
m2/24 h/atm, and water vapor permeability of 7 g/m2/24 h. Package dimensions were 19 × 29 × 4.7 cm. Thus, headspace volume was
more than 3 times greater than product volume.
In the 2nd AP experiment, wheat bread was packed in 50%, 75%,
100% CO2 with no or 5% O2 (Table 4) and added 2 L mustard oil/
bread. Packaging was performed as previously described for MAP
packaging and the same bags were used. Testing of inoculum level
effect with A. flavus was done for the 75% CO2/5% O2 treatment only.
In the 3rd AP experiment, rye bread was packed in a simulated
“flow-pack” condition (80% CO2/1% O2) or atmospheric air with
same methodology as described for MAP packaging. After packaging of bread, mustard oil diluted in 96% ethanol was added to the
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filter paper containing Petri dish inside the bag with a syringe
through a gas-tight membrane (TORAY Rubber Seal, Toray, Tokyo,
Japan) fastened on the bags. The total volume added to each package was 100 L, mustard oil content was 0, 1, 2, 3, 4, or 5 L. The
bags were same size as for previous MAP experiments and the film
was “Transobar 12/60” (P-OPET12/PELD-EVOH-PELD55) from Åkerlund & Rausing AB (Lund, Sweden), 62 m thick, and O2 permeability of 3 mL/m2/24 h/atm (23 °C, 50% RH), and water vapor permeability of 1.5 g/m2/24 h (25 °C, 75% RH).
All treatments were done in triplicate, unless otherwise stated.
Sensory evaluation of AP bread
One rye bread slice (‘Mørkt rugbrød’, Møllens Brød, Helsingør,
Denmark) or 1 wheat bread (‘Miniflûtes,’ Cerealia Bakeries, Hatting,
Denmark) was packed with a filter paper containing Petri dish with
0 (control), 1, 2, 3, 4, or 5 L mustard oil in atmospheric air or MAP
(55% CO2/35% N2/10% O2). The same packaging procedure as previously described for MAP experiment, and same type and size of
bags, were used. The packed bread was stored for 2 d (room temperature, in the dark) before sensory evaluation by ranking test
(Soerensen et al. 1986) preformed by 11 untrained persons (4 male,
7 female) in rooms especially designed for the purpose. The descriptor used was mustard taste, since this was evaluated as the
most important attribute from preliminary studies. The mustard
smell disappeared/evaporated very quickly from the bread once
unpacked. Consequently, to avoid unnecessary confusion among
the untrained judges, they were asked to concentrate only on the
taste. The ranking of samples were done by marking on a line (14.2
cm) ranging from (1) “nothing unusual” to (6) “unacceptable disagreeable mustard taste.” Additionally, the judges were asked to
tick off 1 of 2 boxes indicating the bread sample as “acceptable” or
“not acceptable” according to their taste.
Results and Discussion
MAP experiment
Treatments packed with no oxygen had on average 0.03% oxyVol. 70, Nr. 1, 2005—JOURNAL OF FOOD SCIENCE
M39
M: Food Microbiology & Safety
had a capacity of 50 mL. Packaging atmospheres were mixed with a
MAP MIX 9000 gas mixer (PBI-Dansensor A/S, Ringsted, Denmark)
connected to a Multivac “chamber-type” packaging machine (Multivac A 300/42 MC, Wolfertschwenden, Germany) using compensated vacuum technique (Drulhe-Aleman 1996). For treatments without O2, food-grade quality CO2 and N2 with purity 99.7% and
99.9%, respectively, were used. Premixed gasses with 1.0% (vol/vol)
O2 in CO2 and N2 (analytical tolerance ± 2% and blend tolerance 10%
of set point) were used for treatments with O2. The gasses were supplied by AGA Gas GmbH & Co. KG, Bottrop, Germany. O2 and CO2
concentration in packed bags were measured with a Checkmate 9900
gas-analyzing device (PBI Dansensor, Ringsted, Denmark).
MAP and use of volatile mustard oil for bread . . .
Table 4—Gas compositions used in active packaging (with
2 mL mustard oil) experiment with wheat bread. The O 2/
CO2 equilibrium inside the package on d 2 is shown.a
Packaging composition
% O2
% CO2
% N2
0
50
50
0
75
25
0
100
0
5
50
45
5
75
20
5
95
0
Control/Atmospheric air 1
Day 2
% O2
0.28 0·07
0.20 0·03
0.55 0·13
5.09 0·30
4.37 0·00
5.92 0·06
9.25 0·04
% CO2
44.2 1·0
67.5 0·1
96.5 0·9
46.7 0·2
65.7 0·4
91.9 0·1
0.4 0·1
an = 3.
gen in headspace the 2nd day (Table 1). Packages with oxygen absorbers had no measurable oxygen content, and no mold growth
was observed within the testing period on either wheat bread (Figure 1) or rye bread (Figure 2). However, the spoilage yeast Endomyces fibuliger was not inhibited by any of the MAP treatments, albeit
oxygen absorbers delayed onset of growth on wheat bread (Figure
1). Early mold growth—day 2—occurred only in atmospheric air
(both bread types). However, on rye bread, E. fibuliger showed
growth on day 2 in all 1% O2 treatments (Figure 2).
On wheat bread, the inhibitory effect of CO2 was clear (Figure 1),
and decreasing tolerance of low O2 at increasing CO2 for the molds
was particular notable for Penicillium polonicum, P. solitum and P.
commune, which showed growth of both O2 residual treatments in
50% CO2 but only 1% O2 sustained growth in 75% CO2. P. commune
showed the highest CO2 tolerance with growth at 1% residual O2 in
99% CO2. No significant difference in colony diameter was seen with
1% O2 in N2 compared with atmospheric air, but P. commune was also
the only fungus that showed no growth on day 2 in atmospheric air.
The lack of effect when reducing O2 from 21% to 1% has also been
reported for other spoilage fungi (Agar and others 1990). Aspergillius
flavus showed the highest capacity to grow at low residual O2 level at
increasing CO2 on wheat bread as it was the only fungus growing
when no O2 was added (0.03% O2) at 75% CO2. Miller and Golding
(1949) did also find that A. flavus required less O2 than other molds
(A. niger, P. expansum, P. notatum, P. roqueforti) on malt agar.
The decrease in A. flavus lag-phase for “no”/0.03% oxygen at
75% CO2 compared with 50% CO2 was however peculiar (Figure 1).
Growth might have escaped the eye as the mycelium was extremely
weakened in CO2 and thorough examination was difficult through
the film barrier. Also, minor differences in O2 caused by package
differences could explain the peculiarity. The 1st explanation, however, is likely, as the “spot-test” methodology applied requires a
relatively large change in growth to occur before an increase in diameter (>2 mm) is recorded. Both A. flavus and P. polonicum showed
no significant difference between the 2 O2 residual levels in N2. E.
fibuliger was generally little affected by O2 levels on wheat bread
without oxygen-absorber inserted. El Halouat and Debevere (1996)
also found no difference between atmospheric air and various CO2
(20% to 80%) combinations in N2 on growth of a spoilage yeast (Zygosaccharomyces rouxii).
The main spoiler of rye bread, P. roqueforti, differed from the
M: Food Microbiology & Safety
Figure 1—Growth of spoilage molds on wheat bread at different modified atmosphere packaging (MAP) compositions
with 3 residual O2 (1%, 0.03%, or O2-absorber) levels. Controls (atmospheric air) are shown in 100% N2 plots.
M40
JOURNAL OF FOOD SCIENCE—Vol. 70, Nr. 1, 2005
Figure 2—Growth of spoilage molds on rye bread at different modified atmosphere packaging (MAP) compositions with
3 residual O2 (1%, 0.03%, or O2-absorber) levels. Controls
(atmospheric air) are shown in 100% N2 plots.
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MAP and use of volatile mustard oil for bread . . .
Table 5—Sensory evaluation of wheat and rye bread packed in volatile mustard oil containing atmospheres. The percentage of judges finding the bread samples acceptable is shown. a
Mustard essential oil/ package
(a)
(b)
(c)
(d)
Rye bread
Rye bread
Wheat bread
Wheat bread
Air
MAP
Air
MAP
Control
1 L
2 L
3 L
4 L
5 L
100%
100%
100%
100%
73%
91%
100%
100%
73%
36%
91%
82%
36%
0%
64%
18%
27%
36%
55%
27%
45%
9%
36%
0%
a n = 11. MAP = modified atmosphere packaging.
(a)
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(b)
be mediated by water-dissolved CO2, which can make up to 2% of
the water phase (Daniels and others 1985). Overall, strong CO2 inhibitory effect on molds—with the exception of P. roqueforti—was
seen on rye bread compared with wheat bread.
The different natures of the bread types posed a problem regarding quantification of growth. The wheat bread had a more
porous and fragile structure compared with rye bread. Disintegration or collapse of wheat bread structure with microbial activity was
observed in some cases (that is, E. fibuliger in atmospheric air made
holes in the wheat bread), and colonies were spreading inward into
the bread instead of horizontally. This explains why, for example,
the pattern of larger colonies in atmospheric air compared with MAP
on rye bread was not paralleled by wheat bread.
Fungi are known to stop their growth when O2 becomes limited
(Guynot and others 2003a), and this was also the case in the present
study. For example, in atmospheric air packed rye bread P. roqueforti
and E. fibuliger had no measurable O2 in headspace on day 7, and
on day 14, <0.002% O2 was measured for E. repens and P. commune,
which correlated with their growth stagnations.
The ability of some aerobic molds to grow at limited O2 concentrations (between 0% to 1%) must rely on specific regulatory functions, perhaps linked to an efficient O2 molecular bounding property,
or some other kind of stored reserves from the prior growth in an O2rich environment. Oxygen-absorbers worked efficiently removing all
oxygen for the whole testing period of 35 d and thus preventing mold
growth. However, the inhibition of spoilage yeasts needed further
actions, which impelled the experiments with active packaging.
AP experiments
In modified atmospheres, volatile mustard oil inhibited growth
(c)
Figure 3—Mold growth
on wheat bread in
atmospheric air (a),
“flow- pack” /80%
CO2/5% O 2 (b), and
100% CO2/0.2% O2 (c),
with volatile mustard
oil (0, 1, 3, or 5 ␮L)
added inside the
package immediately
before packaging
Vol. 70, Nr. 1, 2005—JOURNAL OF FOOD SCIENCE
M41
M: Food Microbiology & Safety
other molds by growth in all but oxygen-absorber treatments (Figure 2). This extraordinary tolerance to high CO2 and low O2, or even
stimulated growth (Magan and Lacey 1984; Haasum and Nielsen
1998), has also been reported on other substrates (Petersson and
Schnürer 1999; van den Tempel and Nielsen 2000). Eurotium repens
was not able to grow on rye bread at any of the tested MAP conditions. P. commune was inhibited at the lowest (“no”/0.03%) O2 level
in N2 and by all CO2-containing atmospheres.
Contrary to our results that showed significant differences between different mold species, Smith and others (1986) found no difference between growth of A. niger and Penicillium spores (on PDA
media) in MAP with 60% CO2/40% N2 and a wide range of residual O2
values (0.1%, 0.2%, 0.4%, 0.6%, 0.8%, 1.0%, 2.0%, 10%). In another
MAP experiment (CO2 20% to 50%/N2 50% to 100%) with uninoculated wheat bread, Rodríguez and others (2000) found that Aspergillus
spp. were more sensitive to CO2 than Penicillium spp. Unfortunately,
they did not identify the contaminants to species level, which would
have added further value to the results. In our study, A. flavus was
less tolerant to CO2 than P. commune, but more tolerant than P. solitum and P. polonicum. Growth of A. flavus was observed on day 13 to
15 on peanuts packed in high barrier film and 65% CO2/35% N2 (Ellis
and others 1994), similar to our results on wheat bread.
This study showed that rye bread was a better substrate for E.
fibuliger, as growth occurred on day 2 in all 1% O2 treatments and
atmospheric air, compared with only atmospheric air on wheat
bread, and “no” oxygen and oxygen absorber treatments were like
each other on rye bread. P. commune proved to grow less well on rye
bread compared with wheat bread, probably because of the low pH
of rye bread (4.2 to 4.5) and the relatively higher water content
(about 10%) in rye bread. Antimicrobial effect of CO2 is believed to
MAP and use of volatile mustard oil for bread . . .
in doses of >1 L/ wheat bread, whereas 3 and 5 L doses in air
showed growth on 1/3 of the triplicate samples (Figure 3). Growth
of Endomyces fibuliger dominated in MAP, which particularly for
wheat bread was in accordance with the 1st experiments results. In
air, Aspergillus flavus proved to be the most mustard oil–resistant
fungi (Table 3). For rye bread, no qualitative difference (that is, if
measured as “growth” versus “no-growth”) was seen between the 3
packaging atmospheres; 3 L mustard oil/rye bread or more inhibited growth in all gaseous compositions (Figure 4), although growth
was weaker in MAP conditions and delayed in 100% CO2. Distribution of species, however, was distinctive because Penicillium species were highly sensitive to the mustard oil and no sporulation of
molds occurred in rye bread in MAP treatments similar to the wheat
bread observations (Table 3).
In the next AP experiments, fungi were inoculated separately to
avoid interaction effects between the species. Testing of the intermediate dose (2 L/wheat bread) was from a preliminary sensory
evaluation at the bread factory also considered interesting. Growth
of E. fibuliger was delayed by mustard oil and high CO2, but within 1 wk, growth was observed on all treatments with a colony diameter on day 7 of 3.1 1.5 and 4.8 0.8 mm with and without mustard oil, respectively (data not shown). Growth of A. flavus was
prevented by the additive effect of mustard oil in 95% CO2 with 5%
O2 (Figure 5b). At lower CO2 levels, total inhibition was not obtained
within the testing period. Mustard oil addition stimulated growth
at 50% CO2 and low residual O2 (Figure 5a), although lag-phase was
increased initially. Growth stimulation by antimicrobials in subinhibitory concentrations has also been encountered in other studies (Marin and others 2002; Suhr and Nielsen 2004).
Ellis and others (1993a, 1993b) found that optimization of environmental factors (including gaseous atmospheres) inhibiting
growth of A. flavus depended on the inoculum concentration, as
conditions inhibiting 101 spores/g were not effective for higher
inoculum levels. However, they also found that mold growth generally was higher at 5% headspace O 2 when inoculum level was
lower (102 spores compared with 104 spores) while opposite at higher 10% to 15% O2 levels (Ellis and others 1993b). In our study, testing
of A. flavus inoculum levels showed that higher level gave earlier
growth at 5% O2/80% CO2 with mustard oil, as 106 spore/mL inoculums showed growth on wheat bread on day 7, whereas 102 to 105
had no growth. This indicates that increased inhibition would have
M: Food Microbiology & Safety
(a)
M42
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JOURNAL OF FOOD SCIENCE—Vol. 70, Nr. 1, 2005
been achieved if lower inoculation levels had been used in the
present work. In accordance with our results, Mari and others
(2002) also showed that allyl isothiocyanate vapor on P. expansum
inoculated pears had decreasing inhibitory effect with increasing
inoculum level.
A dosing of 2 L mustard oil was sufficient to inhibit growth of P.
roqueforti and E. fibuliger on rye bread (Figure 6). However, additional effect of MAP was necessary to obtain inhibition of Eurotium
repens, since this fungus showed tolerance to 3 L mustard oil in air
but no growth at 1 L in MAP. E. repens growth occurred between
day 21 and 28 in the MAP control treatment (85% CO2/1% O2) opposing the 1st MAP experiment in which no growth was observed
in any MAP. However, packaging films with different water permeabilities, but the same specifications for O2 permeability, and a different batch of rye bread were used, which could have affected the
result. Decreased tolerance to mustard oil was seen when E. repens
was inoculated in a mixed spore solution (Figure 4a), compared with
the separately inoculated experiment (Figure 6b), which could be
due to microbial interactions or the lower number of spores inoculated. A. flavus was inhibited on rye bread by 2 L mustard oil in
air, but showed no tolerance to mustard oil in 85% CO2/1% O2 (Figure 6e). Thus, wheat bread was a better substrate for the mold than
rye bread. The common rye bread spoilage mold, P. corylophilum,
was inhibited by all mustard and MAP conditions (Figure 6d). This
species was also found to be most susceptible to unfavorable environmental factors in a MAP study by Guynot and others (2003b).
The ethanol used for dilution of mustard oil did not affect growth
(Figure 6) with exception of minor extended lag-phases for E. repens
and A. flavus in (ethanol) flow-pack conditions. For achieving antifungal effect by ethanol vapor, higher dosages are required.
The minimal inhibitory concentration of vaporous mustard oil
above agar against fungi has been reported in the range 0.016 to
0.062 g/mL (Isshiki and others 1992), 1.8 to 3.5 g/mL (Nielsen and
Rios 2000), and 3.8 to 118 g/mL gas-phase (Tsunoda 1994). Bread
packages from the chamber-packaging machine had a volume of
925 143 mL, which rendered a concentration of 1.3 to 1.8 g/mL
gas-phase (with 2 L mustard oil), comparable with the above in
vitro results. However, further tests in our laboratory have shown
that the mustard oil should be dosed according to the surface area
of the bread, and headspace volume is less important for the antifungal effect. This indicates furthermore that the specific product
(c)
Figure 4—Mold
growth on rye bread
in atmospheric air
(a), “flow-pack”/
80% CO2/5% O2 (b),
and 100% CO2/
0.2% O2 (c), with
volatile mustard oil
(0, 1, or 3 L) added inside the package immediately
before packaging
URLs and E-mail addresses are active links at www.ift.org
MAP and use of volatile mustard oil for bread . . .
characteristics will be important for the applicability of AP with
volatile mustard oil.
The sensory evaluation showed that 1 L mustard oil/bread package was generally acceptable (Table 5), and significantly better (less
mustard taste) than higher doses (Figure 7). MAP affected the mustard taste negatively, particularly for rye bread, where only 36%
found that the taste of 2 L mustard oil/rye bread slice was acceptable compared with 73% acceptance in atmospheric air (Table 5).
Wheat bread seemed overall most compatible with mustard oil, and
in atmospheric air the ranking order of low level treatments (0 to 3 L)
varied to such an extend that no significance was obtained.
Even though untrained persons performed the testing, the mustard oil was detected in relatively small quantities, which could suggest that volatile mustard oil for preservative purposes is better
suited for less bland products than bread. However, evaporation
out of the package with time will lessen and eventually eliminate the
sensory effects.
AP packed wheat bread with “no” oxygen contained an average of
0.3% residual O2 on day 2 (Table 4). This indicated that less vacuum-
(a)
(b)
ing was used during packaging compared with the 1st MAP experiment (average 0.03% O2; Table 1). The level, however, was comparable with that obtained at the factory with a commercial thermoforming machine (Table 2). Measurements of the O2/CO2 gas equilibrium
inside the packages from the factory experiment
(Table 2) confirmed the tendency from the MAP experiment (Table
1) with (1) greater residual O2 variability in wheat bread compared
with rye and (2) generally higher O2 and lower CO2 headspace levels
in wheat bread compared with rye bread packages. There was an initial 3% to 4% loss of CO2 in treatments packed in 100% CO2 atmospheres both at the factory (Table 2) and laboratory chamber machine (Table 4). This, however, was not monitored for the MAP
experiment. Collapse of package—known as “snog-down” effect—
due to CO2 diffusion into bread or out of package was observed for
some high CO2 packages in various degrees in all experiments. These
different changes in headspace volume and thus gas composition
inside the packages were an unwanted effect and source of error but
did not seem to affect colony diameter greatly, perhaps because the
snog-down effect appeared relatively late (day 21 to 30).
Maintaining low O2 is pivotal in MAP and adequate sealing properties must be required of the high gas-barrier materials. Thermoforming technology has good gas-tight sealing, but leakage problems may occur (Black and others 1993; Smith 1994). This was not
observed in our experiment, but a couple of the manually sealed
bags used in the laboratory chamber-packaging machine showed
leakage and were discarded.
Conclusions
T
(a)
(b)
(c)
(d)
M: Food Microbiology & Safety
Figure 5—Aspergillus flavus growth at modified atmosphere
packaging (MAP) with different CO 2 concentrations with
(+m) or without 2 L volatile mustard oil/package and “no”
residual O2 (a) or 5% O2 (b).
he importance of testing a range of relevant spoilage organisms
was seen in this study as their capacity to circumvent preservative measures differed. Thus, a shift in dominating spoilage organisms from molds to yeasts might be induced by MAP technology.
The level of residual O2 in MAP had a detrimental effect on growth
of spoilage molds, and the high effectiveness of O2-absorbers, regardless of the MAP-gas-composition, was demonstrated since no
mold growth (besides the “chalk mold”/spoilage yeast E. fibuliger)
occurred in packages containing O2-absorbers. Combining MAP
with vaporous mustard oil lessened the critical demand for low residual O2, and furthermore, a combination strategy was found to
inhibit all types of spoilage organisms in massive inoculation loads
for the desired period of 30 d. For wheat bread, however, a higher
(e)
Figure 6—Growth of spoilage molds Penicillium roqueforti (a), Eurotium repens (b), Endomyces fibuliger
(c), Penicillium corylophilum (d), and Aspergillus flavus (e) on rye bread in atmosphere of 85% CO 2 /1% O 2
(“Flow”) or atmospheric air (“Atm”) and with volatile mustard oil (0/control [“no addition” or “ethanol only”],
1, 2, or 3 ␮L) added to the package. The mustard oil was diluted in ethanol.
URLs and E-mail addresses are active links at www.ift.org
Vol. 70, Nr. 1, 2005—JOURNAL OF FOOD SCIENCE
M43
MAP and use of volatile mustard oil for bread . . .
Figure 7—Ranking-test of volatile mustard oil treated rye
bread packed in air (a), rye bread in modified atmosphere
packaging (MAP) (b), wheat bread in air (c), and wheat bread
in MAP (d) according to mustard taste after 2 d storage. The
ranking values from 1 to 6 represents from “no unusual taste”
to “unacceptable disagreeable mustard taste.” Significant
less taste than the other samples at 1% level (a), 5% level
(b). Significant more taste than the other samples at 1% level (c), 5% level (d).
mustard oil dosing was required than for rye bread, to inhibit E.
fibuliger (>2 L/bread) with possible sensory quality implications.
Acknowledgments
M: Food Microbiology & Safety
This work was carried out as a part of Center for Packaging funded
by the Danish Natl. Agency for Enterprise. Cerealia Bakeries, Møllens Brød, AGA A/S, and AB Åkerlund & Rausing were supportive
providing the materials used. The excellent technical assistance of
Anne Winter Hindsby is also gratefully acknowledged.
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