toxins

Article
Detection of the Potential Inactivation of Tetrodotoxin
by Lactic Acid Bacterial Exopolysaccharide
Nguyen Hoang Khue Tu * ID
, Nghe Van Dat, Le Van Canh ID
and Doan Thi Thanh Vinh
Department of Biotechnology, School of Biotechnology, Hochiminh City International University, Vietnam
National University-Hochiminh City, Quarter 6, Linh Trung Ward, Thu Duc District, Hochiminh 700000,
Vietnam; nghevandat@gmail.com (N.V.D.); vancanh105@gmail.com (L.V.C.); dttvinh@hcmiu.edu.vn (D.T.T.V.)
* Correspondence: nhktu@hcmiu.edu.vn




Received: 8 June 2018; Accepted: 2 July 2018; Published: 12 July 2018


Abstract: Screening for compounds that can neutralize the toxicity of tetrodotoxin (TTX) or reduce its
negative effects is necessary. Our study tested the TTX detoxification capacity of exopolysaccharide
(EPS) extracted from lactic acid bacteria. EPS of Leuconostoc mesenteroides N3 isolated from the Vung
Tau sea (Vietnam), Lactobacillus plantarum PN05, and Lactobacillus rhamnosus PN04 were used in
the study. To more completely evaluate the importance of EPS in detoxification, EPS samples of
Leuconostoc mesenteroides N3, Lactobacillus plantarum PN05 and Lactobacillus rhamnosus PN04 were
also tested. The majority of EPS of these bacteria contained glucose; this was observed using
thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC) analysis.
As observed with FTIR analysis, only EPS of Lactobacillus plantarum PN05 contained methyl groups.
The results indicated that detoxification of TTX in mice could be obtained at an optimal dose
of 248 µg EPS from Leuconostoc mesenteroides incubated with 54 µg cuprous oxide for 40 min or
148 µg EPS Lactobacillus rhamnosus incubated with 55 µg cuprous oxide for 40 min, while EPS from
Lactobacillus plantarum showed TTX detoxification capacity without cuprous oxide combination.
Consequently, EPS from Lactobacillus plantarum PN05 can be used in TTX prevention. This is the first
report on the importance of lactic acid bacteria in TTX detoxification.

Keywords: lactic acid bacteria; tetrodotoxin; exopolysaccharide; detoxification; cuprous oxide

Key Contribution: Inactivation of Tetrodotoxin by Lactic Acid Bacterial Exopolysaccharide.

1. Introduction
Up to now, an antidote for tetrodotoxin (TTX) poisoning has not been developed. It is necessary
to find an effective method to minimize the risks associated with clinical trials of TTX.
Many researchers proved that TTX was produced by symbiotic bacteria, and then accumulated
in different organs of puffer fishes [1–4]. TTX was also found commonly in various types of animal
including the California newt [5], the goby [6], the gastropod mollusks [7], the xanthid crab [8], and the
blue-ringed octopus [9]. TTX inhibits the initiation and conduction of potential action by selectively
blocking sodium channels on the nerve and muscle membrane at extremely low concentrations [10].
Furthermore, TTX-containing puffer fish meat was traditionally fermented before use in Japan [11].
Lactic acid bacteria (LAB) are used as probiotics. Lactic acid bacteria can be divided into
homopolysaccharides or heteropolysaccharides to protect themselves from unfavorable culture
conditions such as desiccation, osmotic stress, antibiotics or toxic compounds, predation by protozoans,
phagocytosis and phage attack [12,13]. There are varieties of heteropolysaccharide categorized
according to their structure, composition, molecular mass, and functionalities. Exopolysaccharide (EPS)
production from LAB is strongly influenced by culture conditions, resulting in different structures

Toxins 2018, 10, 288; doi:10.3390/toxins10070288 www.mdpi.com/journal/toxins

Toxins 2018, 10, 288 2 of 11

and functions [14]. The EPS from Streptococcus thermophilus CRL 1190 was found to be effective for
preventing chronic gastritis [15]. The EPS of Lactobacillus paracasei subsp. paracasei NTU 101 and
Lactobacillus plantarum NTU 102 demonstrated potential antioxidant properties [16]. EPS can also
interact with copper forming a complex structure and conferring specific activities in EPS [17].
As the above stated above, the study investigated TTX detoxification using Lactobacilli
exopolysaccharide.

2. Results

2.1. Identification of Leuconostoc mesenteroides N3

After checking the bacterium showing short chain of coccal shape under the microscope, bacterium
was identified by biochemical tests and 16S rRNA sequencing analysis. As a result, the bacterium was
found to be a facultative anaerobe. It can utilize and produce acid by fermenting D-glucose, mannitol,
rhamnose, D-sorbitol, mannose, D-galactose, D-fructose, and 10% lactose. In addition, this bacterium
appeared positive for indole, urease, amylolysis, phenylalanine deaminase, utilization of malonate, and
methyl red tests, but negative for sucrose, maltose and L-arabinose, Voges–Proskauer reaction, gelatin
liquefaction, tryptophan deaminase, ornithine decarboxylase, phenylalanine deaminase, and arginine
decarboxylase. The partial 16S rRNA sequence was 425 bp deposited in DDBJ (accession number:
LC066674). The partial 16S rRNA gene sequence was analyzed using NCBI BLAST, showing 99% of the
homology to Leuconostoc mesenteroides. The isolated strain was identified as Leuconostoc mesenteroides N3.

2.2. EPS Isolation

The EPS yield of Leuconostoc mesenteroides, Lactobacillus plantarum, and Lactobacillus rhamnosus
was 140 mg, 150 mg, and 140 mg per 106 cfu/mL, respectively. However, the EPS solubility in water
of isolated Leuconostoc mesenteroides, Lactobacillus plantarum, and Lactobacillus rhamnosus was 9.88%,
67.73%, and 9.26%, respectively (Table 1).

Table 1. Characteristics of exopolysaccharide (EPS) obtained from lactic acid bacteria.

Crude EPS Water Solubilizing EPS Molecular

Selected LAB Percentage (%)
(mg/0.5 L Culture) (mg/0.5 L Culture) Weight (Da)
Leuconostoc mesenteroides 140 13.83 9.88 3.8 × 104
Lactobacillus plantarum 150 101.6 67.73 4 × 104
Lactobacillus rhamnosus 140 12.96 9.26 4.4 × 104

2.3. Monomer Detection

By thin layer chromatography (TLC) analysis, the EPS of Leuconostoc mesenteroides,
Lactobacillus plantarum, and Lactobacillus rhamnosus was constituted mostly by glucose. The Rf
value of standard D-glucose was 0.73, similarly to the spots of monomer from the EPS of
Leuconostoc mesenteroides, Lactobacillus plantarum, and Lactobacillus rhamnosus (Figure 1).
The monomer detection was also confirmed by high-performance liquid chromatography (HPLC).
The results of the HPLC showed that EPS from Leuconostoc mesenteroides, Lactobacillus plantarum, and
Lactobacillus rhamnosus consisted of D-glucose and one unknown monomer. All hydrolyzed peaks
appear at the same retention time of standard D-glucose (Table 2). Moreover, the ratio between
D -glucose and the unknown monomer was highest in the EPS of Lactobacillus plantarum, then the EPS
of Leuconostoc mesenteroides, and finally lowest in the EPS of Lactobacillus rhamnosus. These differences
could result in a different TTX detoxification capacity.
Toxins 2018, 10, 288 3 of 11
Toxins 2018, 10, x FOR PEER REVIEW 3 of 11

Figure 1.1.The
Figure Thehydrolyzed
hydrolyzed exopolysaccharide
exopolysaccharide (EPS)
(EPS) of of Leuconostoc
Leuconostoc mesenteroides
mesenteroides (1), plantarum
(1), Lactobacillus Lactobacillus
(2),
plantarum
and (2), and
Lactobacillus Lactobacillus
rhamnosus rhamnosus
(3) were (3)thin
detected by were detected
layer by thin layer
chromatography chromatography
(TLC). Standard glucose (TLC).
(4).
Standard glucose (4).
Table 2. The retention time and peak area of monomer revealed by high-performance liquid
The monomer (HPLC).
chromatography detection was also confirmed by high-performance liquid chromatography
(HPLC). The results of the HPLC showed that EPS from Leuconostoc mesenteroides, Lactobacillus
plantarum, andEPS
Hydrolyzed Lactobacillus
from Selectedrhamnosus
Lactic Acidconsisted of D-glucose
Bacteria (LAB) Peakand
Areaone unknown
Retentionmonomer.
Time (min) All
hydrolyzed peaks appear at the same retention time of standard D -glucose
143,843 (Table 2). Moreover,
13.133 the
Leuconostoc mesenteroides
ratio between D-glucose and the unknown monomer was highest87,661 in the EPS of Lactobacillus
15.599 plantarum,
then the EPS of Leuconostoc mesenteroides, and finally lowest in82,310 the EPS of Lactobacillus
13.125 rhamnosus.
Lactobacillus plantarum
These differences could result in a different TTX detoxification capacity.
53,584 15.575
120,313 13.152
Lactobacillus
Table 2. The retention timerhamnosus
and peak area of monomer revealed
34,456 by high-performance
15.620 liquid
chromatography (HPLC).
Standard D-glucose 579,056 15.512
Hydrolyzed EPS from Selected Lactic Acid Bacteria (LAB) Peak Area Retention Time (min)
2.4. Functional Group Detection 143,843 13.133
Leuconostoc mesenteroides
87,661 15.599
FTIR was performed for more clarification on the structure82,310
of EPS and summarized
13.125 in Table 3.
Lactobacillus plantarum
The polymers had group frequencies including OH, CH2 , CH, C–C, C=O, C–O
53,584 at 3425 cm−1 ,
15.575
2934 cm−1 , 1457 cm−1 , 1224 cm−1 , 1645 cm−1 , and 1056 cm−1 ,120,313
respectively. Significantly,
13.152 the EPS
Lactobacillus rhamnosus −
of Lactobacillus plantarum contained a different frequency at 1380.734,456 cm 1 1456.3 cm−1 and
while15.620
− 1
1457.4 cm in the EPS of Standard D-glucose
Lactobacillus 579,056
rhamnosus and Leuconostoc 15.512
mesenteroides, respectively. It was
revealed that the EPS of Lactobacillus plantarum contained a dimethyl group in its monomer. This
2.4. Functional
difference Group
could makeDetection
the EPS of Lactobacillus plantarum have a different activity in comparison with
that of the EPS of Lactobacillus
FTIR was performed for morerhamnosus and Leuconostoc
clarification mesenteroides.
on the structure of EPS and summarized in Table 3.
The polymers had group frequencies including OH, CH2, CH, C–C, C=O, C–O at 3425 cm−1, 2934 cm−1,
Table 3. The Wavenumber (cm−1 ) corresponding to chemical bonds from FTIR spectrum.
1457 cm , 1224 cm , 1645 cm , and 1056 cm , respectively. Significantly, the EPS of Lactobacillus
−1 −1 −1 −1

plantarum contained a different frequency at 1380.7 cm−1 while 1456.3

Wavenumber cm
(cm 1 ) and 1457.4 cm−1 in the EPS
−−1
EPS from Selected LAB
of Lactobacillus rhamnosus and Leuconostoc mesenteroides, respectively. It was revealed that the EPS of
OH C–H C=O N–H C–H C–C C–O C–H
Lactobacillus plantarum contained a dimethyl group in its monomer. This difference could make the
Leuconostoc mesenteroides 3427.5 2934.4 1645.1 1542.5 1457.4 1224.6 1056.2 813.4
EPS of Lactobacillus plantarum have a different activity in comparison with that of the EPS of
Lactobacillus plantarum 3438.2 2936.8 1654.2 1550.9 1380.7 1223.4 1052.4 814.8
Lactobacillus rhamnosus
Lactobacillus rhamnosusand Leuconostoc
3421.9 mesenteroides.
2934.1 1652.1 1539.6 1456.3 1221.6 1057.2 813.6

Table 3. The Wavenumber (cm−1) corresponding to chemical bonds from FTIR spectrum.
2.5. Mice Assay for TTX Detoxification Capacity of EPS
Wavenumber (cm−1)
EPS
TTXfrom
wasSelected LAB with the EPS of Leuconostoc mesenteroides, Lactobacillus plantarum, and
incubated
OH C–H C=O N–H C–H C–C C–O C–H
Lactobacillus rhamnosus together3427.5
Leuconostoc mesenteroides
with cuprous
2934.4
oxide at room1542.5
1645.1
temperature for 20
1457.4
to 60 min.
1224.6
On the813.4
1056.2
basis
of the lethal dose (0.22
Lactobacillus plantarumµg TTX/mouse
3438.2 unit)
2936.8 that caused
1654.2 100%
1550.9of mice to
1380.7 die within
1223.4 30 min,
1052.4 the TTX
814.8
doseLactobacillus
applied torhamnosus
the mice depending
3421.9 on 2934.1
their weight is presented
1652.1 1539.6 in1456.3
Table 4. 1221.6 1057.2 813.6
2.5. Mice Assay for TTX Detoxification Capacity of EPS
TTX was incubated with the EPS of Leuconostoc mesenteroides, Lactobacillus plantarum, and
Lactobacillus rhamnosus together with cuprous oxide at room temperature for 20 to 60 min. On the
Toxins of
basis the10,lethal
2018, 288 dose (0.22 µg TTX/mouse unit) that caused 100% of mice to die within 30 min,4 of
the11
TTX dose applied to the mice depending on their weight is presented in Table 4.
Table 4. The dead time of mouse treated with different dose of TTX.
Table 4. The dead time of mouse treated with different dose of TTX.

MiceWeight
Mice Weight of Mouse
of Mouse (g) (g) Dose
Dose
of of Tetrodotoxin
Tetrodotoxin (TTX)(µg/Mouse
(TTX) (µg/MouseUnit)
Unit) Time Until
Time Until Death
Death(min)
(min)
1 1 19.53
19.53 0.20
0.20 57
57
2 2 20.52
20.52 0.20
0.20 65
3 3 21.02
21.02 0.20
0.20 49
4 4 19 19 0.22
0.22 28
5 5 20.81
20.81 0.22
0.22 30
30
6 6 21 21 0.22
0.22 26
26
7 7 19.95
19.95 0.24
0.24 18
18
8 20.30 0.24 22
8 20.30 0.24 22
9 20.86 0.24 15
9 20.86 0.24 15

2.6.TTX
2.6. TTXDetoxification
DetoxificationCapacity
CapacityofofEPS
EPSofofLactobacillus
Lactobacillus rhamnosus
rhamnosus
Experimentally, the
Experimentally, the optimal
optimal combination
combination of of EPS
EPS of Lactobacillus rhamnosus
of Lactobacillus rhamnosus (147.946
(147.946 µg) with
µg) with
cuprous oxide (55.325 µg) incubated for 40.415 min showed the highest TTX detoxification
cuprous oxide (55.325 µg) incubated for 40.415 min showed the highest TTX detoxification capacity, capacity,
resultingin
resulting in all
all mice
mice recovering
recovering after
after injection
injection (Figure
(Figure 2).
2). EPS
EPSororcuprous
cuprousoxide
oxidealone
alonedid
didnot
nothelp
help
mice recover from TTX. There was no significant difference in the incubation time
mice recover from TTX. There was no significant difference in the incubation time from 20 to 60 minfrom 20 to 60 min
inthe
in theTTX
TTXdetoxification
detoxificationcapacity.
capacity. Table
Table 55 showed
showed thethe reduction
reduction percentage
percentageof of TTX
TTX incubated
incubatedwith
with
EPS (28.99%) or both EPS and cuprous oxide (37.81%). The interaction between the EPS
EPS (28.99%) or both EPS and cuprous oxide (37.81%). The interaction between the EPS of Lactobacillus of Lactobacillus
rhamnosusand
rhamnosus and cuprous
cuprous oxide
oxide in
in TTX
TTX detoxification
detoxification maymay occur
occur in
in aa shorter
shorter time,
time, less
less than
than 20
20 min.
min.

Figure 2. Surface and contour plot of combination of EPS of Lactobacillus rhamnosus, cuprous oxide,
Figure 2. Surface and contour plot of combination of EPS of Lactobacillus rhamnosus, cuprous oxide, and
and time treatment for TTX detoxification capacity on mice.
time treatment for TTX detoxification capacity on mice.
Table 5. The reduction percentage of TTX by EPS in combination with Cu2 O reveal by HPLC.
2.7. TTX Detoxification Capacity of EPS of Lactobacillus plantarum
EPS from TTX Dose EPS Dose Cuprous Incubation Retention %
In the LAB
Selected Figure 3,(µ/mu)
the mixture(µ/mu)
of the EPS of Lactobacillus
Oxide (µ/mu) Timeplantarum
(min) and
Peak cuprous
Area
Timeoxide
(min) didReduction
not show
any Leuconostoc
TTX detoxification 0.5 in mice.
250 However,55 the EPS 40of Lactobacillus
30,026 plantarum
15.038 showed
37.54 TTX
detoxification
mesenteroides capacity
0.5in mice. Actually,
250 EPS0 showed the40higher reduction
45,212 percentage
15.046 of TTX
5.96 than
EPS Lactobacillus
incubated with cuprous
0.5 oxide
150 when analyzed
55 by HPLC40 (Table 5).
29,899 15.068 37.81
rhamnosus 0.5 150 0 40 34,139 15.064 28.99
Lactobacillus 0.5 100 55 40 44,618 15.063 7.19
plantarum 0.5 100 0 40 40,968 15.063 14.78
Cuprous oxide 0.5 0 55 40 31,547 15.083 34.38
Standard TTX 0.5 0 0 40 48,077 15.121 0
 Toxins 2018, 10, 288 5 of 11

2.7. TTX Detoxification Capacity of EPS of Lactobacillus plantarum

In the Figure 3, the mixture of the EPS of Lactobacillus plantarum and cuprous oxide did not show
any TTX detoxification in mice. However, the EPS of Lactobacillus plantarum showed TTX detoxification
capacity in mice. Actually, EPS showed the higher reduction percentage of TTX than EPS incubated
Toxinswith
2018,cuprous
10, x FOR oxide when analyzed by HPLC (Table 5).
PEER REVIEW 5 of 11

Figure 3. Surface and contour plot of combination of EPS of Lactobacillus plantarum, Cu2O, and time
treatment for TTX detoxification capacity on mice.

Table 5. The reduction percentage of TTX by EPS in combination with Cu2O reveal by HPLC.

TTX
EPS from EPS Dose Cuprous Incubation Peak Retention %
Dose
Selected LAB (µ/mu) Oxide (µ/mu) Time (min) Area Time (min) Reduction
(µ/mu)
Leuconostoc 0.5 250 55 40 30,026 15.038 37.54
mesenteroides 0.5 250 0 40 45,212 15046 5.96
Lactobacillus 0.5 150 55 40 29,899 15.068 37.81
rhamnosus 0.5 150 0 40 34,139 15.064 28.99
Lactobacillus 0.5 100 55 40 44,618 15.063 7.19
plantarum 0.5 100 0 40 40,968 15.063 14.78
Cuprous Figure
Figureoxide 3. Surface
3. Surface and contour
0.5 contour
and 0 plot
plot of combination
55 of EPS
of combination of EPS 40Lactobacillus
of
of Lactobacillus plantarum,
31,547
plantarum, Cuand
2 O, time
Cu215.083
O, and time
34.38
Standardtreatment
TTX for TTX
0.5 detoxification
0 capacity
treatment for TTX detoxification capacity on mice. on
0 mice. 40 48,077 15.121 0

2.8.
2.8.TTX
TTXDetoxification
TableDetoxificationCapacity
Capacity
5. The reduction ofofEPS
percentage ofof
EPS ofLeuconostoc
TTX by EPS in mesenteroides
Leuconostoc mesenteroides
combination with Cu2O reveal by HPLC.

The
Thecombination
combination
TTX ofofthe
theEPS
EPSofofLeuconostoc
Leuconostocmesenteroides
mesenteroidesand
andcuprous
cuprousoxide
oxidesignificantly
significantlyaffected
affected
EPS from EPS Dose Cuprous Incubation Peak Retention %
the
thesurvival
survivalinin mice
mice(Figure
Dose (Figure4). 4).InInexperiment,
experiment,the
theoptimal
optimalincubation
incubationtime
timewas
was40.04
40.04min,
min,the
theoptimal
optimal
Selected LAB (µ/mu) Oxide (µ/mu) Time (min) Area Time (min) Reduction
EPS
EPSdosedosewas (µ/mu)
was 248.449
248.449µg,µg,andandcuprous
cuprousoxide
oxidedose
dosewas
was54.204
54.204µg
µgfor
formice
micesurvival
survivalafter
afterinjection.
injection.InIn
Leuconostoc
the
thestudy,
study,thethe0.5
incubation250
incubation times
timesofofEPS 55
EPSand
andcuprous
cuprousoxide40 were
oxide 30,026
werechanged
changedfrom 15.038
from 2020toto60
60min 37.54
min which
which
mesenteroides
insignificantly 0.5
insignificantlyaffected
affectedthe 250
thesurvival 0
survivalininmice.
mice.Table 40
Table5 5showed
showedthat 45,212
thatthe 15046
thereduction
reductionpercentage
percentage 5.96
ofofTTX
TTX
Lactobacillus 0.5 150 55 40 29,899 15.068 37.81
incubated
incubatedwith withEPSEPS(5.96%)
(5.96%)was waslowerlowerthan
thanthe
theEPS
EPSincubated
incubatedwith
with cuprous
cuprous oxide
oxide (37.54%).
(37.54%). TheThe
rhamnosus 0.5 150 0 40 34,139 15.064 28.99
interaction
interactionbetween
betweenthetheEPS
EPSofofLeuconostoc
Leuconostocmesenteroides
mesenteroidesand
andcuprous
cuprousoxide
oxideininTTX
TTXdetoxification
detoxificationmay may
Lactobacillus 0.5 100 55 40 44,618 15.063 7.19
occur
occur
plantarum
ininaashorter
shorter
0.5
time,
time,less
less
100
than
than 2020min.
min.0 40 40,968 15.063 14.78
Cuprous oxide 0.5 0 55 40 31,547 15.083 34.38
Standard TTX 0.5 0 0 40 48,077 15.121 0

2.8. TTX Detoxification Capacity of EPS of Leuconostoc mesenteroides

The combination of the EPS of Leuconostoc mesenteroides and cuprous oxide significantly affected
the survival in mice (Figure 4). In experiment, the optimal incubation time was 40.04 min, the optimal
EPS dose was 248.449 µg, and cuprous oxide dose was 54.204 µg for mice survival after injection. In
the study, the incubation times of EPS and cuprous oxide were changed from 20 to 60 min which
insignificantly affected the survival in mice. Table 5 showed that the reduction percentage of TTX
incubated with EPS (5.96%) was lower than the EPS incubated with cuprous oxide (37.54%). The
interaction between the EPS of Leuconostoc mesenteroides and cuprous oxide in TTX detoxification may
occur in a shorter time, less than 20 min.

Figure 4. Surface and contour plot of combination of EPS of Leuconostoc mesenteroides, cuprous oxide,
and time treatment for TTX detoxification capacity on mice.
ToxinsToxins 10, 288
2018, 2018, 10, x FOR PEER REVIEW 6 of 11 6 of 11

Figure 4. Surface and contour plot of combination of EPS of Leuconostoc mesenteroides, cuprous oxide, and
3. Discussion
time treatment for TTX detoxification capacity on mice.
Lactobacillus plantarum PN05, Leuconostoc mesenteroides N3, and Lactobacillus rhamnosus PN04 were
3. Discussion
chosen to clarify TTX detoxification. EPS is a countering compound produced by lactic acid bacteria in
response Lactobacillus
to disadvantageous
plantarumconditions, hypothesized
PN05, Leuconostoc as a TTX
mesenteroides N3,detoxification
and Lactobacillus compound
rhamnosusinPN04this study.
Different
were bacteria
chosen tomay produce
clarify EPS differently
TTX detoxification. EPS inis response
a counteringto hard conditions,
compound produced resulting
by lacticin acid
different
bacteria
activities [18].in EPS
response
mayto disadvantageous
directly bind and conditions,
reduce the hypothesized as a TTX or
toxicity of antibiotic detoxification compound
prevent antibiotic binding
in this study. Different bacteria may produce EPS differently in response to hard
to active sites. Other studies revealed that TTX can be removed from puffer fish meat by traditional conditions, resulting
in different
fermentation inactivities [18]. acid
which lactic EPS may directly
bacteria playbind
the and
main reduce
roles the toxicity
[11]. of antibiotic
According or prevent
to researched results,
antibiotic binding to active sites. Other studies revealed that TTX can be removed from puffer fish
EPS was chosen and scanned for its TTX detoxification capacity.
meat by traditional fermentation in which lactic acid bacteria play the main roles [11]. According to
Lactobacillus plantarum, Leuconostoc mesenteroides, and Lactobacillus rhamnosus had an equal
researched results, EPS was chosen and scanned for its TTX detoxification capacity.
productivity of EPS, but
Lactobacillus varied Leuconostoc
plantarum, their solubility in water and
mesenteroides, largely because of
Lactobacillus the difference
rhamnosus had an of equal
functional
groups detectedofbyEPS,
productivity FTIR,
butleading to the
varied their different
solubility interaction
in water largely with water
because of theand other compounds.
difference of functional
From the
groups Box–Behnken
detected design,
by FTIR, leading thedifferent
to the experimentally
interactionoptimal
with waterresults indicated
and other that the EPS of
compounds.
LeuconostocFrommesenteroides and Lactobacillus
the Box–Behnken rhamnosus canoptimal
design, the experimentally detoxify TTX and
results help that
indicated micethe survive
EPS ofwhen
Leuconostoc mesenteroides and Lactobacillus rhamnosus can detoxify TTX and help
combined with cuprous oxide, while the EPS of Lactobacillus plantarum alone showed TTX detoxification mice survive when
combined with cuprous oxide, while the EPS of Lactobacillus plantarum
capacity. This variation may be due to the different structure of EPS of the three strains. The alone showed TTXstudy
detoxification capacity. This variation may be due to the different structure of EPS of the three strains.
suggested that the EPS of Lactobacillus plantarum alone could be used in prevent TTX contamination.
The study suggested that the EPS of Lactobacillus plantarum alone could be used in prevent TTX
In the cases of the EPS of Leuconostoc mesenteroides and Lactobacillus rhamnosus, the EPS showed
contamination.
their TTXIndetoxification capacity when combined with copper ion. EPS is a long chain polymer while
the cases of the EPS of Leuconostoc mesenteroides and Lactobacillus rhamnosus, the EPS showed
tetrodotoxin
their TTXcontains manycapacity
detoxification strong functional
when combined groups withincopper
a compact structure.
ion. EPS is a longIn the presence
chain polymer whileof copper,
both tetrodotoxin
EPS structures of Leuconostoc mesenteroides and Lactobacillus rhamnosus contain
contains many strong functional groups in a compact structure. In the presence of functional groups
(O–H, N–H) that interact with copper forming a complexly compact structure that
copper, both EPS structures of Leuconostoc mesenteroides and Lactobacillus rhamnosus contain functional can bind to TTX
groups
through (O–H,
many N–H) that
hydrogen interact
bonds with copper
(Figure 5), then forming
TTX was a complexly
not enough compact
amountstructure that can
to cause TTX bind
toxicity
to TTX
in mice. through many
According to thehydrogen bonds
FTIR result, the(Figure
EPS of5), then TTX was
Lactobacillus not enough
plantarum wasamount to cause
methylated. TTXgroup
This
makestoxicity in mice.density
the electric According to the FTIR
in oxygen result,more
become the EPS of Lactobacillus
negative in charge plantarum
allowing wasthem
methylated. This with
to interact
group makes the electric density in oxygen become more negative in charge allowing them to interact
hydrogen of the hydroxyl groups of TTX (Figure 6). These interactions might happen faster than the
with hydrogen of the hydroxyl groups of TTX (Figure 6). These interactions might happen faster than
interaction of copper with nitrogen in nitro moiety of TTX leading to TTX detoxification without the
the interaction of copper with nitrogen in nitro moiety of TTX leading to TTX detoxification without
presence of copper. Further investigations must be conducted to optimize the way EPS originated in
the presence of copper. Further investigations must be conducted to optimize the way EPS originated
manyinsources in TTX
many sources indetoxification.
TTX detoxification.

Figure 5. The interaction model of TTX and EPS from Leuconostoc mesenteroides N3 and Lactobacillus
rhamnosus PN04 through Cu bridges.
Toxins 2018, 10, x FOR PEER REVIEW 7 of 11

Toxins Figure
2018, 10,5.288
The interaction model of TTX and EPS from Leuconostoc mesenteroides N3 and Lactobacillus7 of 11
rhamnosus PN04 through Cu bridges.

Figure 6. The interaction model of TTX and EPS isolated from Lactobacillus plantarum PN05.
Figure 6. The interaction model of TTX and EPS isolated from Lactobacillus plantarum PN05.

4. Conclusions
4. Conclusions
EPS from Leuconostoc mesenteroides N3 and Lactobacillus rhamnosus PN04 combined with cuprous
EPS from Leuconostoc mesenteroides N3 and Lactobacillus rhamnosus PN04 combined with cuprous
oxide can detoxify TTX in mouse. However, because cuprous oxide is toxic, these combinations
oxide can detoxify TTX in mouse. However, because cuprous oxide is toxic, these combinations
should be considered well. Fortunately, EPS from Lactobacillus plantarum PN05 alone showed TTX
should be considered well. Fortunately, EPS from Lactobacillus plantarum PN05 alone showed TTX
detoxification capacity in mice. Therefore, the study may bring out the potential way in TTX
detoxification capacity in mice. Therefore, the study may bring out the potential way in TTX prevention.
prevention.
5. Materials and Methods
5. Materials and Methods
5.1. Isolation and Identification of Leuconostoc mesenteroides N3
5.1. Isolation and Identification of Leuconostoc mesenteroides N3
The seawater samples were collected at different places in the Vung Tau sea in Vietnam. These
samples Thewere
seawater
handled samples werecondition.
in sterile collected Theat different
isolationplaces in the
for lactic acid Vung Tau was
bacteria sea in Vietnam.
done These
according to
samples were
Schillinger [19]handled in sterile
and Rodriguez condition.
et al. [15]. TheThe isolation
media for lactic
including the deacid bacteria was done(MRS)
Man–Rogosa–Sharpe according
agar
to Schillinger
or broth (Merck, [19]Darmstadt,
and Rodriguez et al. [15].
Germany) were The media
used. including
Briefly, a 10 gthe de Man–Rogosa–Sharpe
of sample was mixed with 90 (MRS)
mL
agar orsolution
saline broth (Merck,
(0.9%) Darmstadt,
to get a ratio Germany)
of 1/10. were used. Briefly,
The solutions wereashaken
10 g of forsample
10 minwastomixed with 90
homogenize.
mL
A saline solution
volume of 1 mL was (0.9%) to getona MRS
plated ratio of 1/10.
agar andThe solutionsinwere
incubated CO2shaken ◦ C for 48 h.
for 10 minatto45homogenize.
(5%) conditions
A volume of 1 mL was plated on MRS agar and incubated in CO (5%)
About ten colonies were grown at the incubated condition. All colonies were taken for identification.
2 conditions at 45 °C for 48 h.
About
The ten colonies
isolated colonieswereweregrown
testedat bythe incubatedexamination
microscopic condition. All colonies
with gram stainwere and
taken for identification.
catalase production.
The isolated colonies were tested by microscopic examination with
The pure colonies were also characterized using an uronic acid test. Then, the strains were identified gram stain and catalase
production.
by biochemical Thecharacterization
pure colonies were basedalso
on thecharacterized
ability of the using an uronic
isolates to utilizeacid test. Then,
different carbonthesources,
strains
were identified
which by biochemical
were determined by API CHL characterization
50 (bioMerieux, based on the
Lyon, ability
France) and of16S
the rRNA
isolates to utilize different
sequencing analysis.
carbon
The sources,
forward primerwhichwaswere 0 -GCAAGTCGAACGCACAGCGA-3
f1 (5determined by API CHL 50 (bioMerieux, 0 ) and
Lyon,
the France)
reverse andprimer16Swas
rRNA f2
(5 0
sequencing analysis. The forward primer
-CACGTATTTAGCCGTCCCTTTC-3 0 ). Thewas
PCRf1reaction
(5′-GCAAGTCGAACGCACAGCGA-3′)
was performed as follows: 95 C for ◦ and the
5 min;
reverse
30 of 95 ◦was
cyclesprimer s, 50 ◦ C for 20 s, and 72 ◦ C for 3 min; andThe
C forf220(5′-CACGTATTTAGCCGTCCCTTTC-3′). a final PCR reactionatwas
extension 72 ◦performed
C for 10 min. as
follows: 95 °C for 5 min; 30 cycles ◦ of 95 °C for 20 s, 50 °C for 20 s,
The PCR product was stored at 4 C. The purified PCR product was then sequenced. The homology and 72 °C for 3 min; and a final
extension atof
comparison 7216S°C rRNA
for 10 gene
min. sequence
The PCR with product
the was
othersstored at 4 °C. Theusing
was performed purified
BLAST PCR product was
(NCBI).
then sequenced. The homology comparison of 16S rRNA gene sequence with the others was
5.2. Cultivation
performed usingof Lactic
BLAST Acid Bacteria for EPS Isolation
(NCBI).
Lactic acid bacteria strains used in this study including Leuconostoc mesenteroides N3,
5.2. Cultivation of Lactic Acid Bacteria for EPS Isolation
Lactobacillus plantarum PN05 isolated from Coriandrum sativum [20], and Lactobacillus rhamnosus PN04
isolated from
Lactic Hottuynia
acid cordataused
bacteria strains Thunb [21].study
in this All these strains
including were cultured
Leuconostoc in MRSN3,
mesenteroides broth at room
Lactobacillus
temperature
plantarum PN05for 48 h.
isolated from Coriandrum sativum [20], and Lactobacillus rhamnosus PN04 isolated from
Hottuynia cordata Thunb [21]. All these strains were cultured in MRS broth at room temperature for
5.3.
48 h.Mice
Mice (20 ± 1 g) selected for this experiment were male, provided by the cell
reprograming laboratory, Hochiminh City International University (HCM-IU), Vietnam National
University—Hochiminh city. All the procedures were done according to the rules of HCM-IU (Ethical
approval code: 302/QD-DHQT-QLKH; Renewed date of approval: 2 July 2018).
Toxins 2018, 10, 288 8 of 11

5.4. Isolation of EPS

All supernatant was harvested after centrifugation at 10,000 rpm, 4 ◦ C for 30 min. The procedure
was done according to Wei’s group [22]. The dried EPS was used for quantification, further purification
for structure determination, and testing for TTX neutralization.

5.5. EPS Determination

The EPS concentration was determined by the phenol-sulphuric acid method [23]. The absorbance
was measured at 490 nm. The concentration of EPS was determined in triplicate. Distilled water was
used as blank. The EPS content of each sample was calculated based on the standard curve.

5.6. Molecular Weight Determination

The extracted EPS was purified according to the method [24]. The precipitated EPS was dissolved
in water, dialyzed in water, and lyophilized. The UV spectrum of the purified EPS was investigated for
the presence of protein or nucleic acid. The molecular weight of EPS was determined by performing gel
permeation chromatography (GPC). The EPS was eluted with 0.1 NaNO3 at a flow rate of 0.6 mL/min.
Dextran was used as standard to estimate the molecular weight of EPS [25].

5.7. Monomer Detection

Monomers of EPS were preliminary detected by thin layer chromatography [26]. After that,
high performance liquid chromatography was performed to thoroughly detect for monomers. The
hydrolyzed EPS was dissolved in Milli-Q water and applied into the monosaccharide Ca2+ column.
Milli-Q water was used as mobile phase. The flow rate was 0.6 mL/min. The monosaccharides such
as D-glucose, D-galactose, fructose, D-mannose, D-xylose, and L-rhamnose were used as standards.
Refractive Index Detector (RID) was used in the study [26].

5.8. FT-IR
FTIR was performed to detect functional groups or chemical bonds in the compound according
to the previous method [27]. To detect functional group present in the EPS, FTIR was carried out by
micro-KBr pellet technique. Lyophilized EPS powder was ground with potassium bromide powder
finely. A spectrum was recorded using FTIR spectrophotometer (Nicolet-5700 Thermo Electron
Corporation, Madison, WI, USA) in the frequency range of 400–4000 cm−1 . Potassium bromide was
used as a background reference.

5.9. Test for TTX Neutralization Capacity of EPS

TTX in a range of 0.1–0.5 µg was screened on mice via intraperitoneal injection to determine
minimal dose at which 100% mice died within 30 min. After determination of exact lethal dose, TTX
was combined with EPS of Leuconostoc mesenteroides, Lactobacillus plantarum, and Lactobacillus rhamnosus
with and without cuprous oxide. The mixtures were incubated for 20–60 min, then intraperitoneally
injected in mice. The symptoms and time of death of injected mice were recorded carefully. In parallel,
we intraperitoneally injected mice with TTX, and then injected EPS or EPS combined with cuprous
oxide. All the symptoms and time of death were then recorded. Box–Behnken design was used to set
up the doses for TTX detoxification in mice.

5.10. Statistical Analysis and Box–Behnken Design

In order to maximize the TTX detoxification capacity of EPS, the different concentrations of EPS
and Cu2 O as well as incubated time were screened. A set of factors was studied at three levels (−1, 0,
+1) those are standing for the lower extreme, central point and the higher extreme of each factor in the
design respectively (Tables 6–8). These factors were symbolized as A, B, and C, respectively.
Toxins 2018, 10, 288 9 of 11

Table 6. Code level of Plackett–Burman design for Lactobacillus plantarum.

Code Levels
Income Variables
−1 0 1
EPS (µg/mouse unit) (A) 100 200 300
Cuprous oxide (µg/mouse unit) (B) 10 55 100
Incubated time (min) (C) 20 40 60

Table 7. Code level of Plackett–Burman design for Lactobacillus rhamnosus.

Code Levels
Income Variables
−1 0 1
EPS (µg/mouse unit) (A) 100 150 200
Cuprous oxide (µg/mouse unit) (B) 10 55 100
Incubated time (min) (C) 20 40 60

Table 8. Code level of Plackett–Burman design for Leuconostoc mesenteroides.

Code Levels
Income Variables
−1 0 1
EPS (µg/mouse unit) (A) 100 250 400
Cuprous oxide (µg/mouse unit) (B) 10 55 100
Incubated time (min) (C) 20 40 60

The experimental matrix was designed by using the Design-Expert@ version 8.06 (Stat-Ease Inc,
East Hennepin Avenue, Minneapolis, MN, USA) design was arranged in Table 9.

Table 9. Plackett–Burman experimental design matrix.

Factors
Experiment
A B C
1 −1 −1 0
2 −1 1 0
3 1 −1 0
4 1 1 0
5 −1 0 −1
6 −1 0 1
7 1 0 −1
8 1 0 1
9 0 −1 −1
10 0 −1 1
11 0 1 −1
12 0 1 1
13 0 0 0
14 0 0 0
15 0 0 0

On the basis of Box–Behnken design, the experiments for determination of optimal combination of
EPS and cuprous oxide in a suitable incubation time were done. All the results were statistically analyzed.

Author Contributions: Data curation, N.H.K.T., N.V.D. and L.V.C.; Formal analysis, N.H.K.T., N.V.D., L.V.C. and
D.T.T.V.; Investigation, N.H.K.T.; Methodology, N.H.K.T. and N.V.D.; Supervision, N.H.K.T.; Validation, N.H.K.T.
and N.V.D.; Writing—original draft, N.V.D., L.V.C. and D.T.T.V.; Writing—review & editing, N.H.K.T.
Funding: This research received no external funding.
Toxins 2018, 10, 288 10 of 11

Conflicts of Interest: The authors declare no conflicts of interest.

References
1. Campbell, S.; Harada, R.M.; DeFelice, S.V.; Bienfang, P.K.; Li, Q.X. Bacterial production of tetrodotoxin in the
pufferfish Arothron hispidus. Nat. Prod. Res. 2009, 23, 1630–1640. [CrossRef] [PubMed]
2. Yu, V.C.; Yu, P.H.; Ho, K.C.; Lee, F.W. Isolation and identification of a new tetrodotoxin-producing bacterial
species, Raoultella terrigena, from Hong Kong marine puffer fish Takifugu niphobles. Mar. Drugs 2011, 9,
2384–2396. [CrossRef] [PubMed]
3. Tu, N.; Tu, Q.; Tung, H.; Hieu, D.; Romero-Jovel, S. Detection of tetrodotoxin-producing Providencia rettgeri
T892 in Lagocephalus pufferfish. World J. Microbiol. Biotechnol. 2014, 30, 1829–1835. [CrossRef] [PubMed]
4. Nguyen, T.H.; Nguyen, H.N.; Nghe, D.V.; Nguyen, K.H. Biological activities of tetrodotoxin-producing
Enterococcus faecium AD1 isolated from puffer fishes. BioMed Res. Int. 2015, 2015, 8. [CrossRef] [PubMed]
5. Bucciarelli, G.M.; Li, A.; Zimmer, R.K.; Kats, L.B.; Green, D.B. Quantifying tetrodotoxin levels in the California
newt using a non-destructive sampling method. Toxicon 2014, 80, 87–93. [CrossRef] [PubMed]
6. Noguchi, T.; Hashimoto, Y. Isolation of tetrodotoxin from a goby Gobius criniger. Toxicon 1973, 11, 305–307.
[CrossRef]
7. Yang, C.C.; Han, K.C.; Lin, T.J.; Tsai, W.J.; Deng, J.F. An outbreak of tetrodotoxin poisoning following
gastropod mollusc consumption. Hum. Exp. Toxicol. 1995, 14, 446–450. [CrossRef] [PubMed]
8. Noguchi, T.; Arakawa, O.; Daigo, K.; Hashimoto, K. Local differences in toxin composition of a xanthid crab
Atergatis floridus inhabiting Ishigaki Island, Okinawa. Toxicon 1986, 24, 705–711. [CrossRef]
9. Yotsu-Yamashita, M.; Mebs, D.; Flachsenberger, W. Distribution of tetrodotoxin in the body of the blue-ringed
octopus (Hapalochlaena maculosa). Toxicon 2007, 49, 410–412. [CrossRef] [PubMed]
10. Narahashi, T. Pharmacology of tetrodotoxin. J. Toxicol. 2001, 20, 67–84. [CrossRef]
11. Anraku, K.; Nonaka, K.; Yamaga, T.; Yamamoto, T.; Shin, M.C.; Wakita, M.; Hamamoto, A.; Akaike, N.
Removal of toxin (Tetrodotoxin) from puffer ovary by traditional fermentation. Toxins 2013, 5, 193–202.
[CrossRef] [PubMed]
12. Patel, A.; Prajapat, J.B. Food and health applications of exopolysaccharides produced by lactic acid bacteria.
Adv. Dairy Res. 2013, 1, 1–8.
13. Rodriguez, J.M.; Martinez, M.I.; Horn, N.; Dodd, H.M. Heterologous production of bacteriocins by lactic
acid bacteria. Int. J. Food Microbiol. 2003, 80, 101–116. [CrossRef]
14. Iliev, I.; Ivanova, I.; Ignatova, C. Glucansucrases from lactic acid bacteria (Lab). Biotechnol. Biotechnol. Equip.
2006, 20, 15–20. [CrossRef]
15. Rodriguez, C.; Medici, M.; Rodriguez, A.V.; Mozzi, F.; Font de Valdez, G. Prevention of chronic gastritis by
fermented milks made with exopolysaccharide-producing Streptococcus thermophilus strains. J. Dairy Sci.
2009, 92, 2423–2434. [CrossRef] [PubMed]
16. Liu, C.F.; Tseng, K.C.; Chiang, S.S.; Lee, B.H.; Hsu, W.H.; Pan, T.M. Immunomodulatory and antioxidant
potential of lactobacillus exopolysaccharides. J. Sci. Food Agric. 2011, 91, 2284–2291. [CrossRef] [PubMed]
17. Jamshidi, Z.; Hossien, F.; Zahra, A.T. Glucose interaction with Au, Ag, and Cu clusters: Theoretical
investigation. Int. J. Quantum Chem. 2013, 113, 1062–1070. [CrossRef]
18. Hall-Stoodley, L.; Costerton, J.W.; Stoodley, P. Bacterial biofilms: From the natural environment to infectious
diseases. Nat. Rev. Microbiol. 2004, 2, 95–108. [CrossRef] [PubMed]
19. Schillinger, U. Isolation and identification of lactobacilli from novel-type probiotic and mild yoghurts and
their stability during refrigerated storage. Int. J. Food Microbiol. 1999, 47, 79–87. [CrossRef]
20. Nguyen, T.; Le, C.; Tran, N. The adjunct therapy of study on antibiotic and Lactobacillus plantarum PN05
isolated in Coriandrum sativum. Wulfenia J. 2015, 22, 195–221.
21. Nguyen, T.H.K.; Doan, V.T.T.; Ha, L.D.; Nguyen, H.N. Molecular cloning, expression of minD gene from
Lactobacillus acidophilus VTCC-B-871 and analyses to identify Lactobacillus rhamnosus PN04 from Vietnam
Hottuynia cordata thumb. Indian J. Microbiol. 2013, 53, 385–390. [CrossRef] [PubMed]
22. Wei, X.; Fang, L.; Cai, P.; Huang, Q.; Chen, H.; Liang, W.; Rong, X. Influence of extracellular polymeric
substances (EPS) on Cd adsorption by bacteria. Environ. Pollut. 2011, 15, 1369–1374. [CrossRef] [PubMed]
23. Zhang, C.-L.; Li, J.-Q.; Guo, H.-T.; Wang, J.; Xu, R.-H. Selection of exopolysaccharide-producing lactic acid
bacteria isolates from Inner Mongolian traditional yoghurt. Mljekarstvo 2014, 64, 254–260. [CrossRef]
Toxins 2018, 10, 288 11 of 11

24. Ismail, B.; Nampoothiri, K.M. Production, purification and structural characterization of an
exopolysaccharide produced by a probiotic Lactobacillus plantarum MTCC 9510. Arch. Microbiol. 2010,
192, 1049–1057. [CrossRef] [PubMed]
25. Grosu-Tudor, S.S.; Zamfir, M. Exopolysaccharide production by selected lactic acid bacteria isolated from
fermented vegetables. Sci. Bull. Ser. F Biotechnol. 2014, 18, 107–114.
26. Ying, C.; Liping, S.; Yong, Z.; Lei, W.; Liguo, A. The production-influencing factors of extracellular
polysaccharide (EPS) from a strain of lactic acid bacteria and EPS extraction. Front. Biol. China 2006,
1, 236–240. [CrossRef]
27. Yadav, V.; Prappulla, S.G.; Jha, A.; Poonia, A. A novel exopolysaccharide from probiotic Lactobacillus fermentum
CFR 2195: Production, purification and characterization. Biotechnol. Bioinf. Bioeng. 2011, 1, 415–421.

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).