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Food Control
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a r t i c l e i n f o
Article history:
Received 7 July 2015
Received in revised form
22 October 2015
Accepted 31 October 2015
Available online 14 November 2015
Keywords:
Na -lauroyl arginate ethylester
LAE
Antimicrobial activity
Biocides
Surfactants
Yersinia enterocolitica
Lactobacillus plantarum
Flow cytometry
Electron microscopy
a b s t r a c t
Na -lauroyl arginate ethylester, LAE, which was approved as GRAS by the US Food and Drug Administration (FDA) and by the European Food Safety Authority (EFSA) in 2007, is a surfactant that exhibits
antimicrobial activity. To assess its antimicrobial effect, treated cell suspensions of Yersinia enterocolitica
and Lactobacillus plantarum were analysed for reduction of cell viability. Membrane dysfunction was
determined by staining with bis-oxonol to detect the loss of membrane potential, and with propidium
iodide to detect permeabilized membranes by ow cytometry. LAE treatment for 30 min induced a 4
log10 reduction in cell viability in both bacteria; different subpopulations with variable degrees of cellular
damage were observed by ow cytometry. Permeabilized membranes suggested the leakage of cellular
material; this was also indicated by the loss of potassium ion(s), which was higher in L. plantarum than in
Y. enterocolitica. Structural changes involving collapse of the cytosol and alterations of the cellular envelopes, mainly in Y. enterocolitica, and the formation of mesosomes in L. plantarum were observed by
transmission electron microscopy.
2015 Published by Elsevier Ltd.
1. Introduction
Two approaches to microbiological assessment in food are microbial food safety and microbial food spoilage. The challenge in
microbial food safety is to protect public health by minimizing the
microbiological risk, an increasingly complex task in the new global
market, characterized by new food vehicles, trade and technologies
as well as population migrations and climate change. Over the last
decades, microbial food security has attracted considerable attention and their control is mainly based on a preventive approach,
* Corresponding author.
n), alopezsev@ub.edu
E-mail addresses: jonaco_08@hotmail.com (J. Coronel-Leo
pez), espuny.tomas@cofb.net (M.J. Espuny), mbletran@lamirsa.com
(A. Lo
mez), vedlam@lamirsa.com
(M.T. Beltran), amolinos@lamirsa.com (A. Molinos-Go
(X. Rocabayera), amanresa@ub.edu (A. Manresa).
1
cnica del Litoral, ESPOL, Facultad de
Current address: Escuela Superior Polite
nica y Ciencias de la Produccio
n (ESPOL-FIMCP) Campus Gustavo
Ingeniera eca
Galindo, Guayaquil-Ecuador, Ecuador.
http://dx.doi.org/10.1016/j.foodcont.2015.10.050
0956-7135/ 2015 Published by Elsevier Ltd.
(1NT/NC) 100
where NT is the bacterial count in the treated sample and Nc is the
bacterial count in the control sample.
2.6. Flow cytometry
The staining protocols for FC experiments were as follows: 10 mL
of a 1 mg ml1 stock solution of propidium iodide (PI) in distilled
water was added to 1 ml of the bacterial suspension, containing
107e108 CFU/mL prepared as described earlier in ltered buffered
peptone water. Staining was carried out at room temperature
before the FC analysis. To evaluate membrane potential, 2 mL of a
250 mmol/L stock solution of bis-oxonol in ethanol was added to
1 ml of the bacterial suspension. Heat-killed cells (30 min at 70 C)
were used as a positive control for PI and bis-oxonol staining protocols. Experiments were performed in duplicate.
Flow cytometry experiments were carried out using a Cytomics
FC500 MPL ow cytometer (Beckman Coulter, Inc., Fullerton, CA,
USA). Samples were excited using a 488-nm air-cooled argon-ion
Table 1
Bacterial counts of Yersinia enterocolitica and L. plantarum treated at MIC concentration of LAE.
Time (min)
Y. enterocolitica
0
5
10
30
60
90
120
1.0
2.8
1.1
1.7
7.0
e
e
107
104
104
103
100
L. plantarum
2.1
3.1
1.3
3.5
3.0
e
e
107
104
104
103
100
3. Results
3.1. Antimicrobial activity of LAE
The MIC of LAE was 8 mg ml1 against Y. enterocolitica and
32 mg/mL against L. plantarum. The effect of LAE over time on
bacterial suspensions in buffered peptone water is presented in
Fig. 1. After 5 min of exposure, Y. enterocolitica suffered 3 log10 units
loss of viability which reached 4 log10 units, at 30 min. In
Fig. 2. Dual parameter of Y. enterocolitica stained cells (bis-oxonol and PI) at different LAE concentration a) positive control, untreated population; b) negative thermal treated
population; c) population treated with LAE at 2/3 MIC concentration (5.33 mg ml1); d) population treated with LAE at MIC concentration (8 mg ml1).
Fig. 3. Dual parameter of L. plantarum stained cells (bis-oxonol and PI) at different LAE concentration a) positive control, untreated population; b) negative thermal treated
population; c) population treated with LAE at 2/3 MIC concentration (21.33 mg ml1); d) population treated with LAE at MIC concentration (32 mg ml1).
Table 2
Percentage of stained cells measured by ow cytometry analysis and viability reduction in Yersinia enterocolitica treated with LAE.
positive control
LAE
negative control
Treatment
Cell number
none
5.3 mg/mL
8.0 mg/mL
70 C
5.0
2.0
2.0
2.0
104
104
104
104
98.7
0.4
0.5
0.4
% Of stained cells
Bis-oxonol
PI
0.60
3.70
1.80
6.6
0.7
95.80
97.80
93. 0
0
98.33
100
0
Table 3
Percentage of stained cells measured by ow cytometry analysis and viability reduction in Lactobacillus plantarum treated with LAE.
Treatment
positive control
LAE
negative control
none
21.3 mg/mL
32.0 mg/mL
70 C
Cell number
2.89
5.0
4.5
2.0
104
104
104
104
99.86
72.5
0.25
0.1
% Of stained cells
Bis-oxonol
PI
0.10
15.10
0.30
3.0
0.01
12.4
99.60
96.8
0
98.36
100
0
subpopulations were observed with two levels of uorescence intensity, suggesting different degrees of alteration: those that
retained PI and bis-oxonol (Fig. 2c, D2), and those that retained PI
and were severely damaged (Fig. 2c, D1). These results were
consistent with the 98.33% reduction in cell viability (Table 2). As
expected, at the higher LAE concentration, 8 mg/mL (Table 2 and
Fig. 2d), the depolarization of the membrane (Fig 2d, D4) was
reduced to 1.8% (bis-oxonol-stained cells), while at the same time
the percentage of severely damaged cells increased to 97.8% (Fig 2d
D1, D2). In other words, almost the entire population died, as in the
thermally treated culture (Fig. 2b, D1, D2), which is consistent with
the 100% of reduction in viability (Table 2).
A different effect was found in the gram-positive L. plantarum
(Table 3, Fig. 3). When exposed to LAE at 21.33 mg ml1, 72.51%
remained unstained after 30 min of contact, indicating that most of
the population was intact (Fig. 3b, D3). Up to 15.1% of the population was stained with bis-oxonol (Fig. 3c, D4), indicating a depolarized membrane, and only 12.4% were stained with PI (Fig 3b, D1,
D2), indicating a permeabilized membrane. Despite this, there was
a 98.35% reduction in viability (Table 2). After increasing the biocide
concentration to the MIC, almost the entire population (99.6%)
retained PI (Fig 2d, D2,D1), indicating dead cells, which accounts for
the 100% inhibition of growth (Table 3), as in the thermally treated
culture (Fig. 3b).
3.3. Potassium ion(s)leakage
Fig. 4. Potassium leakage of the cell suspension of (a) L. plantarum and (b)
Y. enterocolitica: control population (C); thermal shock treated population ( ); bacterial population treated with LAE at MIC concentration (D). The amount of extracellular potassium of untreated cells was subtracted at each time point from that of
treated cells. Each point represents the mean of three experiments.
MIC and 2/3 of the MIC of biocide were applied to each cellular
suspension and the damage in the corresponding population was
assessed. In the case of Y. enterocolitica, 8m/mL and 5.33 mg/mL of
LAE were added, whereas for L. plantarum, 32 mg/mL and 21.33 mg/
mL of LAE were added.
The effect of LAE on cell damage is presented in Figs. 2 and 3.
Three subpopulations of cells were observed: i) an unstained subpopulation (D3) of intact cells; ii) a subpopulation corresponding to
depolarized cells, stained with bis-oxonol (D4); and iii) a subpopulation with different degrees of damage, stained with PI, indicating permeabilized cells (D1, D2).
The evolution of cellular damage increased with LAE concentration. As shown (Table 2, and Fig. 2), when 5.3 mg ml1 of LAE
was added to Y. enterocolitica culture, only 3.7% of cells retained bisoxonol (Fig. 2c D4) and thus had a depolarized membrane. Most of
the population (Fig. 2c D1, D2) was stained with PI (95.8%), but two
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