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Food Microbiology 27 (2010) 749e756
Contents lists available at ScienceDirect
Food Microbiology
journal homepage: www.elsevier.com/locate/fm
Effect of NaCl reduction and replacement on the growth of fungi important
to the spoilage of bread
S. Samapundo a, *, N. Deschuyffeleer a, b, D. Van Laere c, I. De Leyn c, F. Devlieghere a
a
Ghent University, Faculty of Bioscience Engineering, Department of Food Safety and Food Quality, Laboratory of Food Microbiology and Food Preservation,
Coupure Links 653, 9000 Ghent, Belgium
b
Hogeschool Gent, Department of Food Science and Technology, Voskenslaan 270, 9000 Ghent, Belgium
c
Hogeschool Gent, Department of Food Science and Technology, Laboratory of Cereal Technology, Voskenslaan 270, 9000 Ghent, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 January 2010
Received in revised form
11 March 2010
Accepted 13 March 2010
Available online 23 March 2010
The effect of NaCl and various NaCl replacers (CaCl2, MgCl2, KCl and MgSO4) on the growth of Penicillium
roqueforti and Aspergillus niger was evaluated at 22 C. In addition, challenge tests were performed on
white bread to determine the consequences of NaCl reduction with or without partial replacement on
the growth of P. roqueforti. From the results obtained it can be concluded that at equivalent water phase
concentrations the isolates exhibited differing sensitivities to the salts evaluated with NaCl and MgCl2
having the greatest inhibitory action on the growth of A. niger and P. roqueforti, respectively. MgSO4 had
the least antifungal activity. At equivalent molalities, CaCl2 had in general the largest antifungal activity.
Although the water activity (aw) lowering effects of the compounds studied play a large role in explaining
the trends observed, at equivalent water phase concentrations MgCl2 was found to have a smaller
inhibitory effect on A. niger than that expected from its aw depressing effect. The challenge tests revealed
that no difference occurred in the growth of P. roqueforti on standard white bread, bread with 30% less
NaCl and bread in which 30% of the NaCl has been partially replaced by a mixture of KCl and Sub4Salt.
These results are of importance in assessing the possible microbiological consequences of NaCl reduction
or replacement in bread and similar bakery products.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
NaCl reduction
NaCl replacement
Moulds
Bread & water activity
1. Introduction
Sodium chloride (NaCl) has long been used for the preservation
of food products and as a condiment (Bidlas and Lambert, 2008).
NaCl is a versatile compound that also contributes to the functional properties of several food products and is the primary
dietary source of the essential mineral Naþ (Mattes and Donnelly,
1991). In many populations the dietary NaCl intakes well exceed
the recommended maximum daily intakes of 5e6 g NaCl
(¼2000e2400 mg Naþ) (FSA, 2009a, WHO, 2003). These include
mean intakes as high as 9 g (3540 mg Naþ) in France (WHO, 2003)
and 11.7 g NaCl (4600 mg of Naþ) per day for men in Canada,
Colombia, Hungary, Ladakh (India), Bassiano (Italy), Poland,
Portugal and the Republic of Korea (INTERSALT, 1988). The wellestablished association of high dietary sodium (Naþ) intake with
the development of hypertension (MacGregor and Sever, 1996;
Shank et al., 1983) has prompted public health and regulatory
* Corresponding author. Tel.: þ32 9 264 9902; fax: þ32 9 225 5510.
E-mail addresses: [email protected], [email protected]
(S. Samapundo).
0740-0020/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fm.2010.03.009
authorities to recommend reduction of the dietary intake of NaCl
(Desmond, 2006; Sofos, 1986). In addition to causing illness
cardiovascular diseases (CVD) are of significant economic consequences worldwide, costing the EU approximately V169 billion
per year (Daniels, 2006). The combined estimated direct and
indirect costs of CVD for the US in 2006 were $403.1 billion (Thom
et al., 2006). In a study on the projected effects of dietary salt
reduction on future CVD Bibbins-Domingo et al. (2010) determined that it is more cost-effective to reduce NaCl intake
compared to treatment of hypertension with medications. These
reasons combined form an important part of the rationale for
reducing salt levels in food products.
Due to the diverse and important roles of NaCl in food products,
the reduction of NaCl levels combined with full or partial replacement may have an impact on the shelf stability (via loss of
preservative) (Bidlas and Lambert, 2008) and functional properties
of a food product. Growth related parameters such as the minimum
aw for growth have also been found to be influenced by the nature
of the solute (Sperber, 1983; Troller, 1980) indicating that different
solutes may have additional antimicrobial or even growth stimulating effects on both bacteria and fungi which are not accounted
for by their aw lowering effects (Stringer and Pin, 2005; Suleman
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S. Samapundo et al. / Food Microbiology 27 (2010) 749e756
et al., 2001). The aw depressing capacities of salts also differ
(Samapundo et al., 2010), implying that the application of NaCl
replacers may also influence the microbial stability and safety of
food products as the aw of the altered products could be different to
those with NaCl alone.
To date most of the studies found in literature on NaCl
reduction and/or replacement have explored the functional and
sensorial consequences in food products with very few considering the impact on the microbiological stability and safety. Mould
spoilage is a serious and costly problem for the bakery industry
(Suhr and Nielsen, 2004). Although studies have been done
regarding salt reduction and/or replacement in bread and bakery
products (Salovaara, 1982; Salovaara et al., 1982; Wyatt and
Ronan, 1982), none of these specifically mention the impact of
salt reduction and/or replacement on the microbial stability or
safety of these products. This study had the major objective
partially addressing this paucity by determining the consequences
of NaCl reduction and NaCl replacers on the growth of moulds
important to the spoilage of bread on a general artificial medium
and on actual white bread via challenge tests. Various points of
view were taken into consideration in evaluating the results
including the effect observed at equivalent water phase concentrations, molalities and aw’s. The results of this study provide
a starting point for determining the potential consequences of
NaCl reduction and/or substitution on the microbial stability of
bread.
2. Methods
2.1. Isolates
The isolates used were Penicillium roqueforti var. carneum, Thom
Frisvad (IHEM 6652) isolated from rye bread and Aspergillus niger
(P1118) isolated from corn. The two moulds are maintained in the
culture collection of the Laboratory of Applied Mycology (Department of Food Science and Technology, Hogeschool Gent, Ghent,
Belgium).
2.3. Preparation of inoculum, inoculation, incubation,
and growth assessment
The inoculum of spores used was created as follows. A sterile
inoculation loop was used to aseptically collect sporulating
mycelia from the surface of PDA slants on which the isolates were
maintained. These mycelia were then used to centrally inoculate
the surface of petri plates (90 mm) containing PDA. The inoculum
was then grown by incubating the petri plates for at least 7 days at
30 C to allow adequate sporulation to take place. After incubation
5 ml of wetting agent (3 g Tween-20 L1 distilled H20) was aseptically added to each plate. The colonies (spores and mycelia) were
then scraped off the surfaces of the PDA plates by means of sterile
plastic inoculation loops. The spores and mycelia were then
separated by passing the suspension created above through sterile
glass wool into a sterile 50 ml capacity Falcon tube (Meus, Piove di
Sacco, Italy). This process was repeated a further three times.
The spores were then further separated from any remaining
debris (mostly mycelia) by centrifuging in a Sigma 4K15 centrifuge
(Sigma, Göttingen, Germany) at 10 000 rpm for 15 min. The
wetting agent was then carefully decanted from the pellet of
spores and replaced by 20 ml of sterile phosphate buffered saline
with Tween-20, pH 7.4 (PBS) (SigmaeAldrich, Steinheim,
Germany). The number of spores per ml of spore suspension was
then determined using a Bürker counting chamber (Superior
Mareinfeld, Lauda-Könisghofen, Germany). A Carl Zeiss Axio
Imager A1 microscope (Carl Zeiss, Göttingen, Germany) was used
to visualize the spores on the counting chamber. In most cases
there were approximately 108 spores ml1 of suspension. The
spore suspension was then diluted appropriately in PBS to achieve
a concentration of z103 spores ml1.
Four replicates were prepared per condition (concentration of
salt) evaluated for each isolate. The plates were inoculated centrally
with 25 ml of the spore solution containing approximately
103 spores ml1. The plates were then placed at 22 C and growth
(assessed as the change in diameter of the growing colony) was
determined by periodically measuring two perpendicular diameters per plate using a digital vernier calipers.
2.4. Challenge tests
2.2. Experimental design e effect of pure components
The effects of the pure salts e NaCl (Fluka, Germany), KCl
(SigmaeAldrich, Germany), MgCl2 (SigmaeAldrich, Germany),
CaCl2 (Hansrode Research, Belgium) and MgSO4 (SigmaeAldrich,
Germany) e on the growth of the isolates was studied at added
water phase concentrations of ca. 0, 2, 4.2 and 6.4% in potato
dextrose agar (PDA) (Oxoid, Hampshire, United Kingdom). The
water phase level was chosen as the primary concentration unit
due to its universal use in the food processing industry. All media
were adjusted to a pH value of 7. The experiments were carried
out in quadruple at 22 C. The exact aw of all the growth media
prepared was determined in duplicate by the AW SPRINT
Novasina Thermoconstanter TH-500 (Pfapfikkon, Switzerland).
The molalities (mol kg1 H2O) of the compounds evaluated in all
the growth media were also calculated. This data was used to
evaluate (i) the effect of NaCl reduction without replacement on
the growth of the two isolates and (ii) to make direct comparisons
of the effects of NaCl on the growth of the two isolates to those of
the NaCl replacers studied at equivalent water phase concentrations or equivalent molalities. The aw data was used to determine
if the differences observed in growth were related to differences
in the aw values of the growth media used or if additional antifungal activity occurred for some components other than their aw
lowering effect.
White bread with 30% less NaCl was developed and optimized in
the pilot plant of the Department of Food Science and Technology
(Hogeschool Gent, Ghent, Belgium). Of the several formulations
investigated (results not shown) optimized products which most
closely resembled the reference white bread on the basis of
sensorial and physico-chemical/functional quality (results not
shown) were selected for evaluation in the challenge tests. These
products were i) white bread in which 30% of the NaCl had been
reduced without partial replacement and ii) white breaded in
which 30% of the NaCl had been reduced and partially replaced by
a mixture of salts [KCl þ Sub4Salt (a mixture of NaCl, KCl and
sodium gluconate, Jungbunzlauer, Switzerland); 0.9 þ 1.1]. Sub4Salt
has 35% less Naþ than the same quantity of NaCl.
The reference white bread (recipe: 2 kg flour, 1.1 kg water, 34 g
NaCl, 0.05 g vitamin C and 20 g yeast) had a NaCl level of ca. 1.29 g
(507 mg Naþ) per 100 g of bread. This was reduced to ca. 0.9 g NaCl
per 100 g of bread during the optimization. With regards to the
final Naþ levels, these were 353 mg Naþ per 100 g of bread in which
30% NaCl was reduced without partial replacement and 413 mg Naþ
per 100 g of bread in which 30% of the NaCl had been partially
replaced by the mixture of KCl and Sub4Salt. The final levels of Naþ
achieved in the optimized bread is comparable or even less than the
current targets that have been set by the FSA (UK) for bread and
rolls of 1.1 g NaCl (430 mg Naþ) per 100 g of product by 2010 and
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S. Samapundo et al. / Food Microbiology 27 (2010) 749e756
1.0 g NaCl (400 mg Naþ) per 100 g of product by 2012 (FSA, 2009b).
After kneading of the dough, it was divided into 400 g pieces and
baked (32 minutes at 230 C and 85% relative humidity), cooled
immediately in a laminar flow chamber and sterilized under a UV
lamp for 20 minutes. The aw values of all the types of bread evaluated were measured at 25 C using the AW SPRINT TH-500. Once
sterilized the bread crumb was cut into circular slices (z8.7 cm in
diameter and 5 mm thick) that could fit into 90 mm diameter
petri plates. The slices were then placed in sterile petri plates and
centrally inoculated with 25 ml of a spore solution of P. roqueforti
with 103 spores ml1. Six plates were prepared per type of bread
prepared. The plates were then incubated at 22 C and the radial
growth of the growing colonies was assessed periodically by
measuring their perpendicular diameters by means of a digital
vernier calipers.
2.5. Data analysis
For the moulds the estimation of the most important growth
parameters was done as described in Samapundo et al. (2005). In
brief, the flexible growth function of Baranyi and Roberts (1994)
was fitted to the growth data, average diameters (mm) at each
time of measurement (days), by means of the non-linear regression
function of SPSSÒ Version 15.0 (SPSS Inc., Chicago, Illinois, U.S.A).
This enabled the estimation of the colony growth rate (m, mm d1)
and the lag phase duration (l, d) for each replicate of each experimental condition studied. Average estimated colony growth rates
and lag phase durations, their standard deviations and 95% CI’s
were then calculated in Microsoft Excel 2007 (Microsoft, Redmond,
WA, U.S.A.) from the four replicates. Significant differences
between the effects of NaCl concentration or between the effects of
NaCl and the salt replacers studied on the estimated growth
parameters at equivalent water phase concentrations were determined by comparing their 95% CI’s for overlap or lack thereof.
The relationship between the measured aw of the media and the
estimated colony growth rates and lag phase durations of both
moulds was modeled using Eq. (1).
In m or l ¼ C0 þ C1 bw þ C2 b2w
(1)
2
bw
¼ 1 aw,
where
a transformation proposed by Gibson et al.
(1994) to stabilize variance. C0, C1 and C2 are model coefficients
which were estimated using the non-linear regression function of
SPSS. From Eq. (1) the optimum aw (awopt) for the colony growth
rates of A. niger in the media with the salts evaluated was then
calculated using the estimated coefficients of Eq. (1) as is shown in
Eq. (2). This was only done for the colony growth rates of A. niger as
751
plots of the lag phase durations of A. niger and P. roqueforti and
those of the colony growth rates of P. roqueforti as a function of the
aw of the growth media did not show any obvious asymptotes
(optima or minima).
awopt ¼ 1 bwopt
2
¼ 1 ðC1 = 2C2 Þ2
(2)
3. Results and discussion
3.1. Effect of NaCl reduction and replacement on growth
of the moulds
The effect of NaCl and the NaCl replacers studied on the growth
of the two fungal isolates was analysed and discussed on the basis
of the effect observed at (i) equivalent water phase concentrations
(%) and (ii) equivalent molalities (mol kg1 H2O). For both
comparisons the effect of the aw of the media was also considered
in describing the trends observed. These two comparisons (inclusive of the effect of the realized aw) are of importance as the effect
of NaCl is ambiguously reported in literature on the basis of either
the water phase concentration or the molality.
3.1.1. Effect of NaCl and salt replacers on the basis of the water
phase concentration of the growth media
3.1.1.1. Effect of NaCl. The estimated colony growth rates (m, mm d1)
and lag phase durations (l, d) of P. roqueforti are shown in Tables 1
and 2, respectively, whereas those of A. niger are shown in
Tables 3 and 4, respectively. It can be seen from these tables that
a reduction in the level of the reference compound (NaCl) within
the concentration range studied generally results in an increase in
the colony growth rate and a decrease in the lag phase duration of
P. roqueforti. As can be seen from Table 1, the fastest radial growth
rate of P. roqueforti was observed at a NaCl water phase concentration of 2% water phase (9.86 mm d1), this is however insignificantly different (p > 0.05) from the radial growth rate observed
at 0% (9.72 mm d1). The influence of NaCl concentration was
observed to become significant (p < 0.05) on both the colony
growth rate and lag phase duration of P. roqueforti at water phase
concentrations > 2%. This implies that the consequences of NaCl
reduction on microbial stability (when determined by P. roqueforti)
should only be of importance when the initial water phase
concentration of NaCl is relatively high (> 2%). A plot of the aw of
PDA supplemented with the components evaluated is shown in
Fig. 1a. It can be observed in Fig. 1a that the aw decreased linearly
from approximately 0.998 to 0.957 with an increase in the water
Table 1
Estimated colony growth rate (mm d1) of P. roqueforti on potato dextrose agar supplemented with different water phase concentrations of NaCl and selected salt replacers.
Concentration
NaCl
0%
Estimate
95% CI
9.721
8.968e10.475a*
2%
Estimate
95% CI
9.863
9.663e10.062a
4.2%
Estimate
95% CI
8.275
8.121e8.429a
6.4%
Estimate
95% CI
5.429
5.223e5.634a
KCl
MgSO4
1**
9.721
8.968e10.475a
1
MgCl2
CaCl2
9.721
8.968e10.475a
1
9.721
8.968e10.475a
1
9.956
9.475e10.437ab
9.246
8.918e9.574b
1
9.565
9.167e9.964ab
2
9.057
8.815e9.298b
1
9.306
9.031e9.581b
1
8.895
8.044e9.746ab
3
9.113
8.916e9.311b
1
8.100
7.975e8.224c
2
5.524
5.019e6.029a
1
1
1
2
1
9.721
8.968e10.475a
1
9.455
9.172e9.738ab
1
8.985
8.658e9.312b
1
6.618
6.333e6.903d
2
*Different superscript letters indicate where significant differences (p < 0.05) occur between the components investigated at the same water phase concentration.
**Different superscript numbers indicate where significant differences (p < 0.05) occur between the effects of the same component at different concentrations.
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S. Samapundo et al. / Food Microbiology 27 (2010) 749e756
Table 2
Estimated lag phase durations (d) of P. roqueforti on potato dextrose agar supplemented with different water phase concentrations of NaCl and selected salt replacers.
Concentration
NaCl
0%
Estimate
95% CI
1.043
0.676e1.410a*
2%
Estimate
95% CI
1.272
1.173e1.371a
4.2%
Estimate
95% CI
6.4%
Estimate
95% CI
KCl
MgSO4
MgCl2
CaCl2
1.043
0.676e1.410a
1
1.043
0.676e1.410a
1
1.043
0.676e1.410a
1
1.043
0.676e1.410a
1
1
1.489
1.266e1.711a
1
1.101
0.909e1.292a
1
1.489
1.266e1.711a
12
1.181
1.054e1.309a
1
1.699
1.615e1.783a
2
1.195
1.059e1.332b
1
1.426
1.288e1.564ab
2.077
1.682e2.471a
2
1.609
1.444e1.774a
2
2.482
2.283e2.681a
3
1.339
1.235e1.442b
1
3.351
2.957e3.746c
3.351
2.957e3.746c
3
2.070
1.811e2.330a
3
1**
1
2
*Different superscript letters indicate where significant differences (p < 0.05) occur between the components investigated at the same water phase concentration.
**Different superscript numbers indicate where significant differences (p < 0.05) occur between the effects of the same component at different concentrations.
phase concentration of NaCl from 0 to 6.4%, respectively. This
reduction in aw was correlated to the changes observed in the
colony growth rates and lag phase durations of P. roqueforti.
With regards to the colony growth rates, it can be seen in Table 3
that A. niger responded to an increase in the water phase concentration of NaCl in a more pronounced manner than that observed
for P. roqueforti. The colony growth rate of A. niger increased by two
fold from 0% (7.54 mm d1) to 2% (15.0 mm d1) after which it
decreased steadily with a further increase in the NaCl level. The
colony growth rate of A. niger was determined to be significantly
faster (p < 0.05) at 2% and 4.2% (10.9 mm d1) than it was at 0 and
6% (7.04 mm d1), with no significant differences (p > 0.05) being
observed between the growth rates at 0 and 6%. Only a few studies
were found in literature for comparison to our findings. Cuppers
et al. (1997) developed a model to describe the combined effects
of temperature and NaCl concentration (0 to 7% w/v) on the growth
of food spoilage moulds including A. niger and P. roqueforti. They
observed similar trends to ours for both A. niger and P. roqueforti,
with maximum growth rates at 3.47 and 1.25% NaCl, respectively. In
a study on the NaCl tolerance of 975 species of fungi, Tresner and
Hayes (1971) determined that all 9 isolates of A. niger could grow
at 15% NaCl and four of these could still grow at concentrations 20%. They, however, did not provide any details on the
influence NaCl concentration had on the colony growth rates and
lag phase durations of the isolates. It is evident from our results
that the consequences of NaCl reduction without partial or full
replacement will largely depend on (i) the initial NaCl level and
(ii) the fungal species contaminating the product.
3.1.1.2. Effect of NaCl replacers on the basis of the water phase
concentration in the growth media. As can be seen in Tables 1e4, the
effect of the NaCl replacers evaluated on the estimated colony
growth rates and lag phase durations followed a similar trend to
that of NaCl on the basis of the water phase concentration. The only
exception being MgSO4 which did not significantly affect the
growth of A. niger at concentrations 2% and did not significantly
affect the growth of P. roqueforti across the entire concentration
range studied. This may be due to the fact that unlike the other
components, MgSO4 had a relatively smaller impact on the aw of
the growth media (see Fig. 1a).
From Tables 1e4 it is evident that although the general trends
were similar to those obtained in growth media supplemented
with NaCl, A. niger and P. roqueforti exhibited different sensitivities
to the NaCl replacers evaluated. As the differences between the
magnitude of the effects of the components studied were observed
to become larger and significant at the higher water phase
concentrations studied (where differences in aw also became larger,
see Fig. 1a), the discussion of the results mostly pertains to the
observations made in this range. For A. niger it was observed that at
equivalent water phase concentrations, NaCl gave rise to the
slowest colony growth rates and the longest lag phase durations for
A. niger (see Tables 3 and 4). For P. roqueforti, MgCl2 was determined
to be as inhibitive as NaCl with regards to the colony growth rate
and even more inhibitory than NaCl with regards to the lag phase
duration. In direct contradiction to its large inhibitory effects on the
colony growth rate of P. roqueforti, MgCl2 was (besides MgSO4)
the least inhibitory component on the growth rate of A. niger. MgCl2
Table 3
Estimated colony growth rate (mm d1) of A. niger on potato dextrose agar supplemented with different water phase concentrations of NaCl and selected salt replacers.
KCl
Concentration
NaCl
0%
Estimate
95% CI
MgSO4
7.542
7.030e8.054a*
2%
Estimate
95% CI
15.023
14.943e15.103a
2
14.089
11.407e16.771ab
4.2%
Estimate
95% CI
10.867
10.241e11.493a
3
15.334
14.377e16.290b
6.4%
Estimate
95% CI
7.044
6.336e7.752a
14.895
13.544e16.246b
1**
1
7.542
7.030e8.054a
7.542
7.030e8.054a
1
MgCl2
7.542
7.030e8.054a
1
CaCl2
7.542
7.030e8.054a
1
1
16.262
15.672e16.852b
2
16.517
15.850e17.185b
2
13.452
12.879e14.025c
2
2
13.760
13.275e14.244c
3
15.125
14.817e15.432b
2
12.367
12.265e12.469d
3
2
11.084
10.849e11.320c
4
12.310
12.066e12.553d
3
9.683
9.331e10.034e
2
*Different superscript letters indicate where significant differences (p < 0.05) occur between the components investigated at the same water phase concentration.
**Different superscript numbers indicate where significant differences (p < 0.05) occur between the effects of the same component at different concentrations.
4
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753
Table 4
Estimated lag phase durations (d1) of A. niger on potato dextrose agar supplemented with different water phase concentrations of NaCl and selected salt replacers.
KCl
MgSO4
MgCl2
Concentration
NaCl
0%
Estimate
95% CI
2.913
2.516e3.309a*
2%
Estimate
95% CI
3.327
3.300e3.353a
1
3.289
2.972e3.607ab
4.2%
Estimate
95% CI
3.892
3.761e4.022a
2
3.559
2.529e4.588a
1
3.501
3.066e3.935a
6.4%
Estimate
95% CI
6.117
4.882e7.352a
3
3.573
3.100e4.047b
1
4.193
3.602e4.784bc
2.913
2.516e3.309a
1**
2.913
2.516e3.309a
1
2.913
2.516e3.309a
1
2.913
2.516e3.309a
1
3.117
3.033e3.200b
12
3.168
2.956e3.380ab
12
3.693
3.322e4.065a
2
3.624
2.930e4.316a
12
4.548
4.334e4.763c
2
1
3.192
3.016e3.367ab
1
CaCl2
4.831
4.171e5.490ac
2
3
1
1
*Different superscript letters indicate where significant differences (p < 0.05) occur between the components investigated at the same water phase concentration.
**Different superscript numbers indicate where significant differences (p < 0.05) occur between the effects of the same component at different concentrations.
For P. roqueforti it can be seen in Fig. 2 that although no large
differences can be observed in the estimated colony growth rates at
the same aw, MgCl2 clearly gives rise to the longest lag phase
durations at equivalent aw values. It is clear from these findings that
in addition to the influence of the solute, isolate specific responses
may occur which have to be considered in the selection of an
appropriate salt replacer. The influence of the solute responsible for
aw depression on the growth of bacteria has been reported by
Troller (1980), Sperber (1983) and Stringer and Pin (2005). This
implies that different solutes may have additional antimicrobial or
even growth stimulating effects which are not accounted for by
their aw lowering effects. This has also been observed for fungi by
Suleman et al. (2001) who determined that at equivalent solute
potentials within the range 1.15 to 4.25 MPa, NaCl had the greater
effects than either KCl or glycerol on the radial growth of Chalara
radicicola and Chalara paradoxa, fungi associated with disease in
date palms.
The differences between the responses of A. niger and
P. roqueforti are largely due to differences in the water relations of
the isolates, with A. niger isolates generally having optimal aw for
growth which are lower than those of P. roqueforti isolates
(Cuppers et al., 1997). The estimated coefficients of Eq. (1) and the
awopt based on the colony growth rates of A. niger are listed in
Table 5. It can be seen in Table 5 that the awopt for the colony
growth rate of A. niger was influenced by the solute responsible for
aw depression. The lowest awopt of 0.985 occurred for growth in
1
1
0,99
0,99
0,98
0,98
water activity
water activity
was however second in inhibitory activity on the lag phase of
A. niger. For both isolates it was clear that the inhibitory effects of
CaCl2, KCl and MgSO4 were consistently less than those of NaCl at
equivalent water phase concentrations, with decrease in inhibitory
activity following the same order.
From Fig. 1a and t-tests (results not shown) performed on the aw
data it could be seen that NaCl gave rise to media which had slightly
but non-significantly (p < 0.05) lower aw values than those with
MgCl2. These two were then followed by CaCl2, KCl and MgSO4 in
terms of decreasing capacity to lower the aw. It would therefore be
expected that, in the absence of component specific additional
antifungal or stimulatory activities, NaCl and MgCl2 would have the
largest antimicrobial activities at a given water phase concentration
followed by CaCl2, KCl and then MgSO4 in decreasing order of
magnitude. This trend is largely followed as discussed above with
the exception of the effect of MgCl2. This is confirmed in Fig. 2 (plots
of the estimated growth parameters as a function of the aw of the
growth media) were it can be seen that although aw depression
largely explains the trends observed when comparisons are made
at equivalent water phase concentrations, MgCl2 appears to have
additional effects to those that can be explained by its aw
depressing effects. For example this can be seen for A. niger were
the plots show that although no large differences appear in the
estimated lag phase durations at equivalent aw values, when the
colony growth rate is considered MgCl2 has a much lower antimicrobial activity than the other components.
0,97
a
0,96
0,97
b
0,96
0,95
0,95
0
1
2
3
4
5
6
water phase concentration (g /100 g H2O)
7
0,0
0,2
0,4
0,6
0,8
1,0
1,2
molal (moles/kg H20)
Fig. 1. Plots of the relationship between the a) water phase concentration and b) molality of NaCl (:), KCl (,), CaCl2 (-), MgCl2 (>) and MgSO4 (6) on the water activity of potato
dextrose agar.
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S. Samapundo et al. / Food Microbiology 27 (2010) 749e756
6.5
a
16
a
6
5.5
14
lag phase (d)
radial growth rate (mm d-1)
18
12
10
8
5
4.5
4
3.5
6
3
4
0.95
0.96
0.97
0.98
0.99
2.5
0.95
1
0.96
11
0.98
0.99
1
3.5
b
10
b
3
9
lag phase (d)
radial growth rate (mm d-1)
0.97
water activity
water activity
8
7
6
5
2.5
2
1.5
1
4
0.95
0.96
0.97
0.98
0.99
1
water ac tiv ity
0.5
0.95
0.96
0.97
0.98
0.99
1
water activity
Fig. 2. Plots of the effect of the water activity of growth media adjusted by NaCl (6), KCl (-), CaCl2 (,), MgCl2 (A) and MgSO4 (>) on the growth parameters of (a) A. niger and (b)
P. roqueforti.
media with MgCl2. Growth on media with NaCl and CaCl2 gave rise
to similar awopt’s of 0.990 and 0.989, respectively, whereas growth
on media with KCl and MgSO4 was characterized by the same awopt
of 0.993. The influence of the compound responsible for aw
depression on the growth parameters of A. niger has also been
reported by Parra et al. (2004). They reported that A. niger had an
optimum aw for growth of 0.965 at 35 C on malt extract agar
when glycerol was used to adjust the aw and 0.990 when NaCl was
used (Parra et al., 2004). Parra and Magan (2004) also reported an
awopt of 0.97 for two A. niger strains on malt extract agar whose aw
had been adjusted by glycerol. The awopt reported by Parra et al.
(2004) for growth on media with NaCl is equal to the awopt we
Table 5
2
) and the awopt for
Estimated coefficients of Eq. (1) (in m or l ¼ C0 þ C1bw þ C2bw
A. niger.
awopt r2
Compound Parameter Coefficient
C0
C1
C2
NaCl
ma
lb
2.03 0.05 13.27 1.17
1.08 0.11 1.94 2.48
66.91 5.63 0.990 0.993
25.94 11.94
0.963
MgCl2
m
l
2.03 0.05 13.12 1.10
1.07 0.03 1.33 0.74
54.42 5.49
19.31 3.72
0.985 0.994
0.994
CaCl2
m
l
2.02 0.01 11.28 0.28
1.07 0.03 0.84 0.69
54.68 1.54
17.78 3.78
0.989 0.999
0.994
KCl
m
l
2.04 0.10 17.59 3.22 103.33 21.16 0.993 0.969
1.07 0.04 0.08 1.19
15.21 7.85
0.981
MgSO4
m
l
2.03 0.06 17.60 2.50 104.21 21.24 0.993 0.989
1.07 0.01 3.62 0.49 15.57 4.22
0.995
a
b
Colony growth rate (mm d1).
Lag phase duration (d).
estimated in this study. A similar awopt values of 0.994 at 30 C has
also been reported by Marín et al. (1998) for A. niger.
3.1.1.3. Comparison of the effect of NaCl and NaCl replacers at
equivalent molalities. The relationship between the estimated
growth parameters and the molality of the growth media is shown
in Fig. 3. The relationship between measured aw and molality of
the growth media is shown in Fig. 1b. Some differences could be
observed to the relative effects observed at equivalent water phase
concentrations. It can be seen in Fig. 3 that at equivalent molalities
CaCl2 generally gave rise to the slowest colony growth rates of
A. niger. NaCl and the other salt replacers studied gave rise to similar
colony growth rates at equivalent molalities. For P. roqueforti,
CaCl2 and MgCl2 gave rise to the slowest colony growth rates,
with NaCl and KCl having similar effects.
A clearer trend can be seen with regards to the effect of the
components on the lag phase durations of A. niger and P. roqueforti.
CaCl2 and MgCl2 resulted in the longest lag phases at equivalent
molalities whereas NaCl, KCl and MgSO4 all gave rise to similar lag
phases. It can be deduced from Fig. 1b and Fig. 3 that the overall
trends observed for the antifungal activity at equivalent molalities
are largely in agreement to that for the aw lowering capacities of the
compounds. This may be due to the fact that at equivalent
molalities the differences in the ionic strengths of the media are
better (but not fully) accounted for in comparison to evaluations
based on equivalent water phase concentrations. The only exception to this trend was observed for A. niger, where (as also observed
for comparisons at equivalent water phase concentrations) despite
the fact that at equivalent molalities the growth media with MgCl2
had aw values equal to those of growth media with CaCl2, growth
occurred at a much faster rate on growth media with MgCl2.
Furthermore, although at equivalent molalities growth media with
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S. Samapundo et al. / Food Microbiology 27 (2010) 749e756
6.5
a
16
a
6
5.5
14
lag phase (d)
radial growth rate (mm d-1)
18
12
10
5
4.5
4
8
3.5
6
3
4
0.0
0.2
0.4
0.6
0.8
1.0
2.5
1.2
0.0
0.2
molal (moles/kg H20)
0.4
0.6
0.8
1.0
1.2
molal (moles/kg H20)
3.5
11
b
10
b
3
9
lag phase (d)
radial growth rate (mm d-1)
755
8
7
6
2.5
2
1.5
1
5
0.5
4
0.0
0.5
1.0
1.5
0.0
0.2
0.4
0.6
0.8
1.0
1.2
molal (moles/kg H20)
molal (moles/kg H20)
Fig. 3. Plots of the effects of the molalities of NaCl (6), KCl (-), CaCl2 (,), MgCl2 (A) and MgSO4 (>) on the growth of (a) A. niger and (b) P. roqueforti.
MgCl2 have lower aw values than those with NaCl the colony
growth rates are faster in the presence of MgCl2. The generally
greater antimicrobial activities of the divalent chloride salts may be
due to the fact that for the same molality, the divalent chloride salts
have twice as much toxic anion (Cl1) in solution as NaCl. This has
been demonstrated by Holm and Sherman (1921) who showed that
when the concentrations of MgCl2 and NaCl are adjusted so as to
have equal quantities of Cl1 ions in solution, 0.1 M and 0.2 M,
respectively, their effects on the growth of Bacterium coli are
40
colony diameter (mm)
3.2. Challenge tests
Fig. 4 shows the growth curves of P. roqueforti on the three
different types of white bread. White bread made from the standard recipe, with 30% less NaCl, and that with 30% less NaCl
partially replaced by a mixture of KCl and Sub4Salt had aw values of
0.938 0.007, 0.954 0.003 and 0.944 0.002, respectively. No
significant difference occurred in the estimated colony growth rates
and lag phase durations (results not shown) of P. roqueforti on the
three different types of bread. This indicates that it is possible to
produce acceptable white bread with a NaCl reduction of 30%
without affecting its stability to P. roqueforti. This may be due to the
fact that the initial NaCl level in the standard recipe was low
(z2.01% in the water phase on the basis of an average moisture
content of 51.5% in the final baked bread). As demonstrated in this
study on artificial growth media, reduction or complete replacement in this concentration range would not be expected to result in
a significant difference to the products stability to P. roqueforti.
45
35
30
25
20
15
10
comparable. In addition, it has been determined by measuring
ammonification in soil that chloride anions are more toxic than
sulphate (Greaves, 1916), which would further partially explain
why the chloride based NaCl replacers had a greater antifungal
activity than MgSO4. The exception mentioned above of faster
growth of A. niger in the presence may indicate that Mg2þ might
have a growth stimulating effect on this isolate.
0
1
2
3
4
5
6
7
time (days)
Fig. 4. Growth curves P. roqueforti on reference white bread (,), white bread with 30%
less NaCl (6) and white bread in which NaCl had been reduced by 30% and partially
replaced by a mixture of KCl and Sub4Salt (A).
4. Conclusions
From these results it can be concluded that of the components
studied, NaCl and MgCl2 have in general the largest antimicrobial
activities on A. niger and P. roqueforti, respectively, at equivalent
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S. Samapundo et al. / Food Microbiology 27 (2010) 749e756
water phase concentrations as they gave rise to the slowest colony
growth rates and the longest lag phase durations. This would imply
that the replacement of NaCl with the salt replacers studied would
probably give rise to products of reduced microbial stability with
regards to A. niger. Whilst this is also true for P. roqueforti for the
majority of the salt replacers studied, replacement of NaCl by MgCl2
would imply a product even more stable to P. roqueforti. When
comparisons were made on an equivalent molality basis CaCl2 was
found to generally have a consistently large antimicrobial activity
on the growth of both fungi, whereas MgCl2 had the largest effects
on the growth of P. roqueforti. MgSO4 was determined to have the
least antifungal activity from both an equivalent water phase
concentration or molality point of view. Therefore its use as a NaCl
replacer will also most likely results in products of reduced stability.
Although aw plays a very large role in the trends observed the
differing sensitivities of the fungal isolates to MgCl2 (in particular)
partially highlights the occurrence of species specific additional
effects of the molecule itself other than its aw lowering effects. The
results obtained in this study imply that the microbial consequences of reduction will also largely depend on the initial NaCl
level, the nature of the replacer and the fungal species encountered. At low initial NaCl levels (< 2%) such as those encountered
in the challenge tests performed in this study, NaCl reduction with
or without partial replacement did not affect the stability of white
bread to P. roqueforti. Future experiments should preferentially be
in the form of challenge tests which help to provide important
data on the real life consequences of NaCl reduction and/or partial
replacement on the microbiological stability and safety of food
products.
Acknowledgements
The authors are grateful to Flanders’FOOD (Kunstlaan 43, 1000,
Brussels, Belgium) for their financial support.
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