International Journal of Food Microbiology 215 (2015) 86

94

Contents lists available at ScienceDirect

International Journal of Food Microbiology

j o u r n a l h o me p a g e : w w w . e l s e v i e r . c o m / l o c a t e / i j f o o
dmicro

Review

Inuence of fat addition on the antimicrobial activity of sodium

lactate, lauric arginate and methylparaben in minced meat
Nria Magriny a, Nino Terjung b,c, Myriam Loefer b, Monika Gibis b, Ricard Bou a,d, Jochen Weiss
a
b
c
d

b,

Nutrition and Food Science Department, XaRTA-INSA, Faculty of Pharmacy, University of Barcelona, Av. Joan XXIII s/n, 08028 Barcelona, Spain
Department of Food Physics and Meat Science, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstrasse 21/25, 70599 Stuttgart, Germany
German Institute of Food Technologies, Prof.-von-Klitzing-Str. 7, 49610 Quakenbrck, Germany
Institut de Recerca i Tecnologia Agroalimentries (IRTA), Finca Camps i Armet, 17121, Monells (Girona), Spain

a r t i c l e

i n f o

Article history:
Received 10 March 2015
Received in revised form 17 August
2015
Accepted 23 August 2015
Available online 29 August 2015
Keywords: Fat
addition
Minced meat
Sodium lactate
Lauric arginate
Methylparaben

a b s t r a c t
A minced meat model system containing three different fat levels (0, 15, and 50 wt.%) was used to evaluate
the antimicrobial efcacy of three antimicrobials with different aqueous solubilities (sodium lactate N lauric
arginate (N-lauroyl-L-arginine ethyl ester, LAE) N methylparaben). Various concentrations of sodium lactate
(20, 40, and
60 mg/g), lauric arginate (0.5, 1, 1.5, 2.0, and 2.5 mg/g) and methylparaben (0.1, 0.5, 1.0, and 2.0 mg/g) were
used to evaluate the antimicrobial activity against natural meat microbiota (total aerobic mesophilic colony
counts, co- liform bacteria, and lactic acid bacteria). The results indicate that the three antimicrobials tested are
inuenced at different strengths by the changes of the fat addition of the minced meat. The antimicrobial
efcacy of LAE and methylparaben is increased by a higher fat content in the meat batter, whereas for lactate
no clear lactate propor- tionality relationship can be seen. This structure sensitivity is most strongly
pronounced with lauric arginate, which we attributed to the amphiphilic character of the molecule.
2015 Elsevier B.V. All rights reserved.

Contents
1.
2.

Introduction . . . . . . . . . . . . . . . .
Materials and methods . . . . . . . . . . .
2.1.
Materials . . . . . . . . . . . . . .
2.1.1.
Antimicrobials . . . . . . .
2.1.2.
Microbiological media . . . .
2.2.
Methods . . . . . . . . . . . . . .
2.2.1.
Minced meat preparation
. .
2.2.2.
Microbial growth . . . . . .
2.2.3.
Calculation of relative growth
behavior
2.2.4.
Statistical analysis . . . . . .
3.
Results and discussion . . . . . . . . . . .
3.1.
Relative growth behavior5 . . . . . .
3.1.1.
LAE . . . . . . . . . . . .
3.1.2.
Methylparaben . . . . . . .
3.1.3.
Sodium lactate . . . . . . .
3.2.
Mechanistic insights . . . . . . . . .
4.
Conclusions . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . .
Appendix A.
Supplementary data . . . . . . . .
References . . . . . . . . . . . . . . . . . . .

Corresponding author.
E-mail address: j.weiss@uni-hohenheim.de (J. Weiss).

http://dx.doi.org/10.1016/j.ijfoodmicro.2015.08.017
0168-1605/ 2015 Elsevier B.V. All rights reserved.

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N. Magriny et al. / International Journal of Food Microbiology 215 (2015) 86

94

1. Introduction
Meat is a large part of the human diet and in many countries a
great part of the food industry. Consequently, the safety of meat is of
major concern to consumers, processors, retailers, food service
industries, gov- ernment agencies, educational institutions, public
health professionals, researchers, and the general public locally,
regionally, nationally, and internationally (Fung et al., 2001). The
spoilage of raw meat is largely dependent on initial bacterial ora,
the amount and types of microor- ganisms (pathogenic and/or
spoilage), as well as on the meat package and storage conditions
(Farid et al., 2001). Not surprisingly minced meat is, compared to
larger pieces of meat or processed meat, highly perishable and has a
shortened shelf life (Fik and Leszczyska-Fik,
2007; Michalczyk et al., 2012). This fact is based on an increased
surface area (due to the size reduction) that facilitates the access of
oxygen and promotes microbial growth (Blte and Zens, 2003; Fik and
Leszczyska- Fik, 2007). Nevertheless the shelf life, safety, and quality
of minced can be enhanced by minimizing the initial microbial load,
compliance pro- cess hygiene and the addition of antimicrobial
agents (Fik et al., 2008). The antimicrobial efcacy of these
antimicrobial agents has been shown to depend on the surrounding
product matrix, for instance, the concentrations of fats, proteins,
carbohydrates, and salts, as well as the pH of the product (Zhang et
al., 2009). Additional factors inuencing the antimicrobial efcacy
are: the time of addition of the antimicrobial (Terjung et al., 2014a,b)
and if suitable, carrier systems which can not only enhance the
activity of structure-sensitive antimicrobials (Loefer et al., 2014;
Terjung et al., 2014a), but also decrease the antimicrobial ef- cacy
(Terjung et al., 2014a,b) if these systems are not used correctly.
In detail, ingredients such as polysaccharides can form complexes
with added antimicrobials (Loefer et al., 2014), that may interact
with proteins, rendering them less available for interactions with
micro- organisms (Terjung et al., 2014a) or ingredients such as fat
protect mi- croorganisms from antimicrobials, apparently reducing
their sensitivity to antimicrobials (Gutierrez et al., 2009).
Nevertheless, all microorgan- isms survive only in a water phase of
the product. The concentration of a preservative in the water phase is
directly related to the inhibition
of microbial growth in the food system, whereas the amount of
preser- vative dissolved in the lipid phase could be treated as a loss
of antimi- crobial (Davidson et al., 2005).
To date, it has not yet been fully understood which molecular
prop- erties truly govern these interactions that then render
antimicrobials less effective. It has been suggested that one of the
key properties that play a dominant role is solubility, causing
compounds
to be more or less heterogeneously distributed
throughout a food matrix. Compounds may preferentially partition
into certain phases (e.g. the fat particles), which then contain higher
concentrations of antimicrobials than other
phases. A specic structurefunction relationship exists that is
currently
still not well understood. We hypothesize, therefore, that
antimicrobials with different properties are affected at different
strengths by changes in the structural composition of the food
matrix. To this purpose, we evaluated the antimicrobial effectiveness
of LAE, methylparaben and so- dium lactate, at various
concentrations, against the initial bacterial ora (mesophilic
bacteria, coliform bacteria, and lactic acid bacteria) in minced meat
while varying the addition of fat. Minced meat is both a model
system and a food product, since ground meat is sold as such in the
supermarket and is used in the manufacture of ground meat steaks,
burger patties. The used antimicrobials used for the investigation will
now shortly be introduced.

N -Lauroyl-L-arginine ethyl ester monohydrochloride (Lauric

arginate; LAE), a recently developed ester of arginine and lauric acid,
is able to rapidly reduce the number of microorganisms intrinsically
present upon addition. LAE is an amphiphilic antimicrobial that has
been shown to possess very high antimicrobial activities in model
mi- crobiological systems, i.e. the minimum inhibitory concentration

87

for Listeria monocytogenes has been reported to be as low as 8

g/g (Deutsche Sammlung von Mikroorganismen und Zellkulturen
GmbH

88

N. Magriny et al. / International Journal of Food Microbiology 215 (2015) 86

94
(DSMZ) 20649) (Becerril et al., 2013).
In prior
studies, the
antimicrobial effectiveness of LAE has been assessed alone or in
combination with other antimicrobials to prolong the shelf life of
meat products (Martin et al., 2009; Muriel-Galet et al., 2012;
Stopforth et al., 2010; Taormina and Dorsa, 2009; Terjung et al.,
2014a, 2014b).
Lipophilic antimicrobials, such as parabens, have been used as
anti- microbials in cosmetics, pharmaceutics, and foods for more
than
50 years (Soni et al., 2002). Methyl- and propylparaben have been
de- clared to be generally recognized as safe (GRAS) by the US
FDA in the United States, whereas in the European Union use of the
compounds is more restricted (Bedzka et al., 2014). For example,
according to EU regulations, methylparaben may only be used as a
surface application on dry, cured hams. Although methylparaben is
only slightly soluble
in water, this solubility can be sufcient to produce the effective
antimi- crobial concentration in the aqueous phase (Soni et al., 2002).
Sodium lactate, the sodium salt of lactic acid, is a water-soluble
food additive that has been successfully used to inhibit several
microorgan- isms, such as Salmonella species, Pseudomonas spp., or
lactic acid bacteria (Doores, 2005). The antimicrobial mechanism of
lactic acid and many other antimicrobially active organic acids is
based on lowering the pH in the
bacterial cytoplasm after
permeation of the non-dissociated forms through the bacterial
membranes (Crozier-Dodson et al., 2005). Since compounds are
predominantly present in the aqueous phase, this mass transport
occurs rapidly, making the compounds quite effec- tive (depending
of the pH) (Fung et al., 2001; Gill and Newton, 1982).
We expect that the emulsier LAE, based on its chemical
structure, will be more inuenced by changes of the fat addition,
since methylparaben has the slight ability to be present in both
phases (fat and water). By contrast, we expect that sodium lactate
will not be inu- enced by changes in the matrix structure
composition, since sodium lac- tate is only water-soluble.

2.
Materials
methods

and

2.1. Materials
2.1.1.
Antimicrobials
Mirenat NSM (85.5 g/100 g maltodextrin and 14.5 g/100 g LAE)
(permitted value: 0.2 mg/g) and
methyl 4-hydroxybenzoate
(methylparaben) (permitted value: 1 mg/g) were purchased from
Meat Cracks Technology GmbH (Mhlen, Germany). Sodium lactate
so- lution (50% in H2O) (permitted value: quantum satis) was
purchased
from
Sigma
Aldrich
(Steinheim,
Germany).1
2.1.2.
Microbiological
media
Total aerobic mesophilic counts were determined on Standard I
nu- trient agar (Merck, Darmstadt, Germany). Violet red bile lactose
agar (Carl-Roth, Karlsruhe, Germany) was used for coliforms. Lactic
acid bac- teria were determined on MRS agar (Merck, Darmstadt,
Germany) and incubated in an anaerobic jar (Merck, Darmstadt,
Germany). Double- distilled water was used in the preparation of all
solutions.
2.2. Methods
2.2.1.
Minced
meat
preparation
The minced meat was prepared in the meat pilot plant facilities
at the University of Hohenheim. Fresh lean pork meat and pork back
fat were purchased locally (Mega, Stuttgart, Germany) and stored at
2 C prior to production. Meat (pork shoulder) and fat (pork back
fat) were individually minced using a meat grinder (W114T82 487-1

Seydelmann
1

Chemical characterization of LAE, methylparaben and sodium lactate: aqueous

solu- bility 247, 2.5, and N 1500 (g/l), partitioning coefcient (logarithmic ratio of
concentrations between octanol and water (log P)) 4.03 0.46 (SD), 1.87 0.22 (SD),
and 0.85 0.27 (SD), molecular weights (g/mol) 421, 152, and 112, and pKa
values 3.91(lactic acid), respectively.

KG, Aalen, Germany) to a particle size of 3 mm. After mincing, meat

and fat were mixed together in different ratios to obtain the fat
additions de- sired: 0 wt.% (aw:0.97 0.003(SD) and pH: 5.46
0.01), 15 wt.% (aw:0.96 0.002(SD) and pH: 5.60 0.01) and 50
wt.% (aw:0.97
0.004(SD) and pH: 6.0 0.03).2 The fat levels were chosen to
demonstrate the inuence of fat on the antimicrobials, even fat levels N 20%
are not allowed in minced meat (EU regulation No 1169/2011). The
re- spective antimicrobial preparations were added at various
concentra- tions (LAE 0.5, 1, 1.5, 2, 2.5, methylparaben 1, 5, 10, 20 and
lactate 20,
40, 60 mg/g). The amount of antimicrobials added was adjusted to
the respective weight of the meat portion to achieve the
concentrations de- sired. Differences in the volumes of antimicrobial
solution were com- pensated with sterile distilled water. A control
was prepared for each fat addition using the same volume of sterile
distilled water instead of the antimicrobial solution. After the
addition of the antimicrobial agent, the meat batter was mixed for 2
min with a rotor stator paddle mixer (no further particle size
reduction occurred) (KitchenAid, type Artisan). Subsequently, 20
portions (10 g) of each combination (antimi- crobial and fat addition)
were packed into vacuum bags (PA/PE, 90 my,
135 180 mm; MEGA eG, Stuttgart, Germany). Modied atmosphere
(70% O2:30% CO2)3 was added to the bags using a vacuum sealer
(BOSS RC 63, Bad Homburg, Germany). The samples were stored at
12 C (the increased temperature was selected to accelerate the
growth of the intrinsic ora) for eight days and two samples of each
treatment were randomly selected each day and analyzed as follows.
2.2.2.
Microbial
growth
In order to determine the microbial growth, a new package was
opened each day and the meat was transferred to sterile stomacher
bags (Blender Bag, lateral lter bag for Blender, ionized, Topac Inc.,
USA) and diluted with 90 mL sterile peptone water. The bacteria
were extracted via homogenization at 100 rpm for 60 s using a
Stomacher
400 Circulator (Seward, UK). The extraction liquid (100 L) was
spiral plated on agar plates in duplicate (Don Whitely Scientic
Limited, West Yorkshire, UK). When low colony counts were
assumed 1 mL of the extraction liquid was poured directly on the
plates (detection limit: 1 log CFU/g). Total aerobic mesophilic counts
were determined on Standard I nutrient agar. Violet red bile lactose
agar was used for co- liforms. Lactic acid bacteria were determined on
MRS agar. Standard I nutrient and violet red bile lactose agar plates
were aerobically incubat- ed at 37 C for 24 h, and MRS agar plates
were incubated at 35 C for 72 h in an anaerobic jar containing
Anaerocult (Merck, Darmstadt, Germany). Colony forming units
(CFU/g) were then counted using an aCOLyte spiral plate reader
(Synbiosis, UK).
2.2.3. Calculation
of relative
growth
behavior
In order to determine the relative growth behavior, the number
of CFUs from the total aerobic mesophilic counts, coliform bacteria,
and lactic acid bacteria was plotted over time (Fig. 1). A schematic
illustra- tion of the evaluation is shown in Fig. 1 for further
1
explanation. The area A (log CFU t g ) was dened as the integral
of the log CFUs over time (over eight days). Areas were calculated
using Peakt (Systat Software Inc., San Jose, California). The ratio
between the area below the curve of the minced meat without
antimicrobial (control) and treat- ed with LAE methylparaben and
lactate was carried out to standardize the results and compare the
treatments (modied according (Terjung et al., 2012)) (Eq. (1)).
A

Ao An
100
A0

A0 is the area under the growth curve of the control and An is the
area under the growth curve after treatment with antimicrobials.
The evaluation leads to a value describing the relative growth
behavior.
2.2.4. Statistical analysis
All measurements were repeated at a minimum of three times
using duplicate samples. A pairwise multiple comparison procedure
was per- formed using
Tukey's
test (p b 0.05) on
the
antimicrobial activity expressed as an evaluation of the growth
behavior A for the compar- ison factor fat (0 vs. 15, 0 vs. 50, and
15 vs. 50 wt%) using Sigmaplot
12 (Systat Software Inc., San Jose, CA,
USA).
3.
Results
discussion

and

In this study, the antimicrobial efcacy of antimicrobials with

different solubilities applied to minced meat with different ratios of fat
(0, 15, and
50 wt.%) was evaluated. We hypothesized that the antimicrobial
efcacy of the antimicrobials lauric arginate, methylparaben, and
sodium lactate is pronounced at different strengths by changes in the
addition of fat in the minced meat batter. We based the hypothesis on
the chemical character of the antimicrobials, among others. We
assessed the antimicrobial efca- cy of the three antimicrobials in
minced meat against the microbial ora occurring naturally.
Therefore, the growth of the total aerobic mesophilic bacteria,
coliform bacteria, and
lactic acid bacteria over time was
determined.
Figs. 24 demonstrate the growth behavior (log CFU over time) of
the
aerobic mesophilic bacteria (Fig. 2), the coliform bacteria (Fig. 3), and
the lactic acid bacteria (Fig. 4) in minced meat containing 0 wt.% fat
(A),
15 wt.% fat (B), and 50 wt.% fat (C), and different concentrations of
4
LAE (0, 0.5, 1, 1.5, 2.0, and 2.5 mg/g). The meat was stored at 12 C
under modied atmosphere for eight days. Sodium lactate and
methylparaben components are not shown in the growth curves to
avoid recurrence. Re- gardless of the type of bacteria, a more or less
pronounced impact of the fat addition on the antimicrobial efcacy is
visible, as well as an inuence of the antimicrobial concentration
when comparing A, B, and C in Figs. 24. In detail, it seems that the
antimicrobial efcacy of LAE is re- duced with an increasing fat
addition from 0 to 15 wt.%, and then further
increased from 15 to 50 wt.%. Furthermore, the antimicrobial efcacy
is a lot more pronounced against lactic acid bacteria than against
mesophilic and coliform bacteria. The higher antimicrobial efcacy
against lactic acid bacteria was already noticed in other investigations
(Terjung et al.,
2014a,b), furthermore this effect may become pronounced due to the
ox- ygen input in the meat batter in the mixing step. It should be
noted that the initial microbial ora in samples with 15 wt.% was
generally higher, due to uctuations in the raw material from the
supplier.
Rapid growth of surviving cells causing counts to again increase
after a few days (Figs. 24) has also been reported in other studies
with LAE (Luchansky et al., 2005; Martin et al., 2009; Porto-Fett et
al., 2010; Stopforth et al., 2010; Taormina and Dorsa, 2009). This may
be ascribed to two
different mechanisms. First, LAE may be
hydrolyzed by intrinsic enzymes being present in the meat system.
Second, cells may adapt to the presence of antimicrobials e.g. by
altering their membrane fatty acid prole and therefore are able to
proliferate (Terjung et al., 2012).
Furthermore, it is possible that LAE is bound in e.g. dead cell or with
pro- teins so that a multiplication of surviving cells cannot be
suppressed.

3.1.
Relative
5
behavior

growth

Between the different fat levels, pH-values are signicant different (p b 0.05; n =
6), aw-values not.
3
Storage conditions are standard for fresh meat in retail stores, whereas 70% N2 and
30% CO2 are conditions typically chosen for turkey breast or mortadella.

So far, we could prove that the hypothesis the amount of fat

addi- tion in minced meat has an impact on the antimicrobial efcacy
is cor- rect. In order to assess and compare the effect of fat on the
efcacy of the
4

The growth curves of all microorganism and all antimicrobial treatments are
provided as Supplementary data.
5
The antimicrobial efcacy of all antimicrobials against the different microorganism
as
inuenced by the fat content is provided as Supplementary data.

Fig. 1. Schematic diagram showing the calculation of a relative growth behavior A, based on the integration of the growth curves over time where A1 net growth over time;
A2 net static growth/kill and A3 net kill over time. Relative growth behavior has the maximum inhibition at 80%, due to the detection limit.

antimicrobial, the growth curves were mathematically analyzed using

a simple integration procedure, as described above.
The
characteristic growth behavior was evaluated as described above and
schematically drawn in Fig. 1 to provide a suitable comparison of the
results
The inuence of the fat addition on the antimicrobial efcacy
deter- mined as relative growth behavior was signicant for the three
antimicro- bials with a p value of b 0.05. for all comparisons (0 vs. 15, 0
vs. 50, and 15 vs. 50 wt.% fat) with the exception of two comparisons:
the differences between 0 and 15 wt.% fat when methylparaben was
added against the total aerobic mesophilic bacteria (p value: 1) and 0
and 50 wt.% fat, and when sodium lactate was added against lactic acid
bacteria (p value: 0.39).

3.1.1. LAE
The evaluation of the relative growth behavior, as outlined above,
of aerobic mesophilic (A), coliform (B), and lactic acid bacteria (C)
under the inuence of different LAE concentrations and fat additions
is shown in Fig. 5. LAE showed an antimicrobial activity against
mesophilic micro- organisms, coliform bacteria, and lactic acid bacteria.
The highest antimi- crobial activity was determine against lactic acid
bacteria, with a relative growth behavior of 80% equaling a total
inhibition (see Methods section) (Figs. 4C and 5C). Fig. 5A shows that
only at LAE concentration N 0.5 mg/g that total aerobic mesophilic
bacterial counts were affected. Growth inhi- bition of test
microorganisms signicantly increased when 50 wt.% fat was present
in minced meat compared to minced meat containing 0 or
15 wt.% fat (Fig. 5A
C).
In contrast to the observed behavior of LAE to changes in the fat
con- tent Soni and coauthors reported that when LAE was added to
tryptic soy broth, skim milk (0.0% fat), or whole milk (3.4% fat) at 4 C

for 15 days, 0.2,

0.4 or 0.8 mg/g was needed respectively to reduce L. monocytogenes
levels

to below the detection limit. (Soni et al., 2010). Woodcock and

coauthors obtained similar results (Woodcock et al., 2009). There, 0.2
103 g/g LAE was needed to reduce L. monocytogenes in skim milk
(2% fat) to below the detection limit, while no effect was observed in
full fat milk stored at 6 C for 21 days. The fact that no such fat induced
losses were ob- served in the investigated minced meat may be due
to the fact that the lean minced meat is a compositionally complex
soft matter system that may provide less optimal growth conditions
than a uid system such as milk or tryptic soy broth. While water
activity changes little with increas- ing fat content, less water is
generally available. Similarly, oxygen avail- ability may be reduced
putting additional stress on microorganisms.
The circumstance that in the literature an increasing complexity
of the surrounding media leads to a loss of antimicrobial efcacy but
not in the investigated minced meat may be due to the fact that the
lean minced meat is itself a complex medium and that the addition of
a por- tion fat does not lead to higher complexity nor in more side
reactions of the antimicrobial. In this investigation the opposite
seems to be the case, the reason for the higher antimicrobial efcacy,
is more likely due to de- teriorated living conditions like higher
concentration of antimicrobial in the water phase and a reduced
mound of oxygen.
Lactic acid bacteria seemed to be more susceptible to LAE than
the other species studied since the higher concentration of LAE used
was able to inhibit the proliferation of lactic acid bacteria in all minced
meat systems during storage time (Fig. 4). Lower concentrations of the
antimi- crobial also recorded initial growth reductions of lactic acid
bacteria.
3.1.2.
Methylparaben
The evaluation of the relative growth behavior, as outlined above,
of aerobic mesophilic (A), coliform (B), and lactic acid bacteria (C)
under

Fig. 2. Characteristic growth behavior of aerobic mesophilic bacteria over time in minced meat containing 0 wt.% fat (A), 15 wt.% fat (B), and 50 wt.% fat (C) stored at 12 C for
eight days under modied atmosphere, in the presence of lauric arginate (LAE) at different concentrations (0, 0.5, 1, 1.5, 2,0, and 2.5 mg/g).

the inuence of different methylparaben concentrations and fat

additions is shown in Fig. 6. In accordance with the relative growth
behavior of LAE, the efciency of methylparaben was higher when
minced meat contained
50% fat, with the exception of coliform bacteria. Methylparaben
showed a similar antimicrobial activity regardless of the amount of
fat and antimicrobial concentration (Fig. 6B). With
higher
concentrations of methylparaben, the inuence of fat becomes more
pronounced, even
for the
increase from 0 to 15 wt.% fat.
Methylparaben was highly
effective against coliform bacteria
regardless of the concentrations (Fig. 6B). How- ever, the maximal
concentration of methylparaben used (20 mg/g) was not able to
reduce the growth of the aerobic mesophilic and lactic acid bacteria
above 50% in any of the minced meat systems (Fig. 6A and C).
Regarding the antimicrobial activity of methylparaben, Grampositive bacteria are generally more susceptible to non-polar phenolic
compounds than Gram-negative bacteria, because the latter have the
ability to screen that antimicrobial due to the outer membrane
lipopolysaccharide layer (Davidson, 2005). Methylparaben has been
generally recognized as safe (GRAS) up to a concentration of 1 mg/g
when used as a preservative in foods. An initial lethality (i.e.
microorganisms were rst inactivated, but survivors then adapted
and began to grow again) of methylparaben against bacteria in food
products has been reported, but only at concen- trations signicantly
higher than the value permitted (Soni et al., 2002). Fyfe and Stoddart
(1997) investigated the effect of methylparaben against L.
monocytogenes and Salmonella enterica serotype Enteritidis. Concentrations of 1 mg/g were not effective in inhibiting the growth of either
of the two species in microbiological test media (Fyfe and Stoddart,
1997). Other studies reported that the antimicrobial efcacy of
methylparaben is even lower in food systems than in laboratory
media (Asker et al.,
2008).

3.1.3. Sodium lactate

The evaluation of the relative growth behavior, as outlined above,
of aerobic mesophilic (A), coliform (B), and lactic acid bacteria (C),
under the inuence of different sodium lactate concentrations and fat
addi- tions is shown in Fig. 7. In contrast to the other antimicrobials,
sodium lactate had the lowest antimicrobial efcacy when applied
to the minced meat with a fat addition of 50 wt.% (in comparison
to the other fat additions) (Fig. 7). Sodium lactate had a more
pronounced growth reduction for the three bacteria assessed when
15 wt.% fat was added to the minced meat, in comparison to lean
meat (0 wt.% fat added) and minced meat with 50 wt.% fat.
Several studies carried out on meat products described the efciency of sodium lactate against mesophilic species, such as
L. monocytogenes (Barmpalia et al., 2004; Martin et al., 2009;
Mbandi and Shelef, 2002). Barmpalia et al. (2004) reported that the
growth of L. monocytogenes in Frankfurter type sausages was slowed
down by 18 mg/g sodium lactate, but was not totally inhibited. In
comparison to the control (8.2 log CFU/cm 2), the bacterial counts
2
after storage at 10 C for 20 days were about 3 log CFU/cm lower
(Barmpalia et al., 2005). These ndings are only in accordance with
our results for very high concentrations of sodium lactate (60 mg/
g) which were needed to inhibit the growth to a relative growth behavior of 50% (Fig. 7A).
Regarding coliform bacteria, the addition of sodium lactate was
highly efcient in controlling the bacterial growth, even when the
low- est concentration of the antimicrobial was used (Fig. 7B). These
results are in agreement with other authors who reported a limited
prolifera- tion of Escherichia coli when 30 mg/g sodium lactate was
added to ground beef (Hwang and Juneja, 2011), cooked beef (Miller
and Acuff,

Fig. 3. Characteristic growth behavior of coliform bacteria over time in minced meat containing 0 wt.% fat (A), 15 wt.% fat (B), and 50 wt.% fat (C) stored at 12 C for eight days under

mod- ied atmosphere, in the presence of lauric arginate (LAE) at different concentrations (0, 0.5, 1, 1.5, 2.0, and 2.5 mg/g).

Fig. 4. Characteristic growth behavior of lactic acid bacteria over time in minced meat containing 0 wt.% fat (A), 15 wt.% fat (B), and 50 wt.% fat (C) stored at 12 C for eight days
under modied atmosphere, in the presence of lauric arginate (LAE) at different concentrations (0, 0.5, 1, 1.5, 2.00, and 2.5 mg/g).

1994), or liver sausages (Miller and Acuff, 1994; Shelef and Potluri,
1995).
Overall, it seems that sodium lactate is more efcient when either
no fat or 15 wt.% fat was added to minced meat. A similar behavior
was ob- served by Hu and Shelef (1996) in beaker sausages (Hui et
al., 2001). These authors concluded that the inhibitory activity of
sodium lactate at a concentration of 1.8% was
higher with
increasing fat additions. This could be caused by the increase of
water-soluble salts in the water phase of the food matrix. Therefore,
the fat addition could have a signicant effect on the antimicrobial
activity in minced meat. Alterna- tively, the high fat addition may
counteract the antimicrobial activity of sodium lactate. Fat addition
in meat may exert a physical protection of the bacterial cell or cause
some interactions between the fat in the

meat and bacterial cell wall lipids and, as a result, it allows

microorgan- isms to grow (Mehta and Tatini, 1994) as well as
increasing the pH.
At this point, we know that the fat addition in minced meat has
an impact on the antimicrobial efcacy of all antimicrobials
examined, and that the effect is more or less pronounced
depending of the antimicrobial.
3.2. Mechanistic insights
We suggest that the changes in the antimicrobial efcacy of
methylparaben and sodium lactate are based on the increasing
concen- tration of the particular antimicrobial in the reduced water
phase of the meat batter due to an increase of the fat addition. In
order to draw out

Fig. 5. 3D surface plot of the calculated relative growth behavior A over a period of eight days as a function of the fat (0, 15, 50 wt.%) and LAE (0.5, 1, 1.5, 2.0, and 2.5 mg/g)
concentration in the meat batter. For mesophilic bacteria (A), coliform bacteria (B), and lactic acid bacteria (C).

Fig. 6. 3D surface plot of the calculated relative growth behavior A over a period of eight days as a function of the fat (0, 15, 50 wt.%) and methylparaben (1, 5, 10, and 20 mg/g)
con- centration in the meat batter. For mesophilic bacteria (A), coliform bacteria (B), and lactic acid bacteria (C).

Fig. 7. 3D surface plot of the calculated relative growth behavior A over a period of eight days as a function of the fat (0, 15, 50 wt.%) and sodium lactate (20, 40, and 60 mg/g)
concen- tration in the meat batter. For mesophilic bacteria (A), coliform bacteria (B), and lactic acid bacteria (C).

Fig. 8. Suggested structural interaction behavior of antimicrobials, LAE, methylparaben, and sodium lactate with components (fat, water, and protein) of a simplied dispersed
meat sys- tem containing various amounts of fat. (A) lean meat, (B) 15 wt.% fat addition, and (C) 50 wt.% fat addition.

the mechanistic suggestions of the observations, we prepared a

simpli- ed schematic diagram of a dispersed meat system consisting
of meat and fat particles, and water to illustrate both the changes in
the meat batter due to an increasing fat addition and the structural
organization of antimicrobial compounds within the sausage matrix
(Fig. 8). The mi- croorganisms generally reside in the aqueous phase
while antimicro- bials preferentially associate with one or more of
the dispersed meat phases according to their molecular properties.
I. (A) The positively charged LAE binds to negatively charged
meat particles in turn lowering its antimicrobial efcacy.
Moreover, due to its molecular properties, it is likely that the
amphiphilic LAE molecules preferentially adsorb to the
interface between water and fat. Alternatively, they may be
present as micelles in the aqueous phase. (B) A reduction of
the antimicrobial efcacy occurs because the increased fat
content causes more LAE to be present at the interface and,
therefore, a smaller amount of LAE can be active against
microorganisms. (C) At the highest amount of fat added (50
wt.%), more LAE may again be present in the aqueous phase.
This is because there exists an equilibrium be- tween the
amount of LAE adsorbed at the interface and LAE in the water
phase. Although much LAE is adsorbed at interfaces, the lower
amount of water causes the remaining LAE concentra- tions in
the aqueous phase to be quite high.
II. Methylparaben, as a fat-soluble preservative, is mainly present
in the fat phase and is only slightly soluble in water.
Nevertheless, methylparaben can also only show antimicrobial
activity when partitioning in the water phase. (AC) The
actual concentration of methylparaben increases in the water
phase, since, with an in- creasing fat addition, a lower amount
of overall water is in the system.
III. Sodium lactate is more or less deprotonated depending on the
pH, while only the undissociated form is the microbially active
one. A higher amount of fat in the system results in a higher
pH value. At pH 6 (50% fat), only 0.8% of undissociated lactic
acid is present, at pH 5.6 (15% fat), 2% of undissociated lactic
acid is present, and at pH 5.46 (0% fat), 2.74% of
undissociated and
97.26% of the dissociated form of lactic acid are present. (AB)
With an increasing fat concentration, the concentration of
sodi- um lactate will increase in the water phase, resulting in a
higher antimicrobial efcacy. (BC) With a further increase of
the fat addition, the antimicrobial concentration still increases,
similar to that in the case of mehtylparaben. The pH value
counteracts this effect, since the value of the meat batter
increased to 6
and, therefore, most of the lactate is dissociated, and thus,
cannot diffuse into the bacteria cells.

4. Conclusions
The study has shown that antimicrobial efcacy depends mostly
on the type of organism and mode of action of the antimicrobial. The
inu- ence of fat addition on the antimicrobial efcacy was, thereby,
generally equal for all types of organisms assessed. We could show
that the anti- microbial efcacy of LAE decreased with increasing fat
addition (0 to 15 wt.%), and then increased again with a further
increase of fat addition (15 to 50 wt.%), while for methylparaben, the
antimicrobial efcacy in- creased with increasing fat addition over all
concentrations. For sodium lactate, the efcacy increased with
increasing fat addition (0 to 15 wt%) and then decreased with a
further increase of the fat addition (15 to 50 wt.%).
We conclude that even when the results suggest that all three
anti- microbials tested (LAE, methylparaben and sodium lactate) are
inu- enced by the composition of the meat batter, there are
some signicant differences in the manner. LAE is a sensitive,
structured com- ponent and is, therefore, inuenced by the added

fat, whereas the

antimicrobial efcacy of methylparaben and sodium lactate is inuenced by side-effects of the compositional changes of the meat
batter.
Results should be of interest to food and specically meat
product manufacturers, when changes in the formulations are
planned the anti- microbial formulation could be reconsidered as
well. Finally, additional studies will be needed to enable
understanding in detail where reaction between antimicrobial and
the surrounding matrix takes place.
Acknowledgments
This research project was supported by the German Ministry of
Eco- nomics and Energy (via AiF) and the FEI (Forschungskreis
der Ernhrungsindustrie e.V., Bonn): Project AiF 16969N. We would
like to thank Kurt Herrmann for his technical advice during
preparation of the minced meat samples.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
http://dx. doi.org/10.1016/j.ijfoodmicro.2015.08.017.
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